TGF-β signaling cooperates with at motif-bindingfactor-1 for repression of the α-fetoproteinpromoterNobuo Sakata Showa Pharmaceutical UniversitySatoshi Kaneko Showa Pharmaceutical UniversitySouichi Ikeno Showa Pharmaceutical UniversityYutaka Miura Nagoya City University Graduate School of Medical SciencesHidekazu Nakabayashi Hokkaido Information UniversityXueyuan Dong Emory UniversityJin-Tang Dong Emory UniversityTaiki Tamaoki University of CalgaryNaoko Nakano Showa Pharmaceutical UniversitySusumu Itoh Showa Pharmaceutical University

Journal Title Journal of Signal TransductionVolume Volume 2014Publisher Hindawi Publishing Corporation | 2014-07-03 Pages970346-970346Type of Work Article | Final Publisher PDFPublisher DOI 1011552014970346Permanent URL httpspidemoryeduark25593s2thg

Final published version httpdxdoiorg1011552014970346

Copyright informationcopy 2014 Nobuo Sakata et alThis is an Open Access work distributed under the terms of the CreativeCommons Attribution 30 Unported License(httpcreativecommonsorglicensesby30)

Accessed January 13 2022 849 PM EST

Research ArticleTGF-120573 Signaling Cooperates with AT Motif-Binding Factor-1 forRepression of the 120572-Fetoprotein Promoter

Nobuo Sakata1 Satoshi Kaneko1 Souichi Ikeno1 Yutaka Miura2 Hidekazu Nakabayashi3

Xue-Yuan Dong4 Jin-Tang Dong4 Taiki Tamaoki5 Naoko Nakano1 and Susumu Itoh1

1 Laboratory of Biochemistry Showa Pharmaceutical University 3-3165 Higashi-Tamagawagakuen Machida Tokyo 194-8543 Japan2Department of Molecular Neurobiology Nagoya City University Graduate School of Medical Sciences Nagoya 467-8601 Japan3Department of Medical Management amp Information Hokkaido Information University Ebetsu Hokkaido 069-8585 Japan4Department of Hematology and Medical Oncology and Winship Cancer Institute Emory University School of MedicineAtlanta GA 30322 USA

5Department of Biochemistry and Molecular Biology University of Calgary Calgary AB Canada T2N 4N1

Correspondence should be addressed to Susumu Itoh sitohacshoyakuacjp

Received 18 April 2014 Accepted 23 May 2014 Published 3 July 2014

Academic Editor Shoukat Dedhar

Copyright copy 2014 Nobuo Sakata et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

120572-Fetoprotein (AFP) is known to be highly produced in fetal liver despite its barely detectable level in normal adult liver Onthe other hand hepatocellular carcinoma often shows high expression of AFP Thus AFP seems to be an oncogenic markerIn our present study we investigated how TGF-120573 signaling cooperates with AT motif-binding factor-1 (ATBF1) to inhibit AFPtranscription Indeed the expression of AFP mRNA in HuH-7 cells was negatively regulated by TGF-120573 signaling To furtherunderstand how TGF-120573 suppresses the transcription of theAFP gene we analyzed the activity of theAFP promoter in the presenceof TGF-120573 We found that the TGF-120573 signaling and ATBF1 suppressed AFP transcription through two ATBF1 binding elements(AT-motifs) Using a heterologous reporter system both AT-motifs were required for transcriptional repression upon TGF-120573stimulation Furthermore Smads were found to interact with ATBF1 at both its N-terminal and C-terminal regions Since the N-terminal (ATBF1N) and C-terminal regions of ATBF1 (ATBF1C) lack the ability of DNA binding both truncated mutants rescuedthe cooperative inhibitory action by the TGF-120573 signaling and ATBF1 in a dose-dependent manner Taken together these findingsindicate that TGF-120573 signaling can act in concert with ATBF1 to suppress the activity of theAFP promoter through direct interactionof ATBF1 with Smads

1 Introduction

The oncofetal glycoprotein 120572-fetoprotein (AFP) is a majorserumprotein expressed at high levels in the yolk sac and liverduring embryonic development [1 2] However AFP in adultserum is undetectable except in patients who suffer fromhepatocellular carcinoma (HCC)Thus AFP is a useful tumormarker for measuring the malignancy grade of HCC [3] Thehuman AFP gene is activated by hepatocyte nuclear factor-1(HNF-1) which can bind to an AT-motif in the proximalandor distal promoter region of AFP [4ndash6]

AT motif-binding factor-1 (ATBF1) encodes a proteincomprising multiple zinc fingers and homeodomains [7 8]ATBF1 was originally discovered as a negative transcriptional

regulator of the human AFP gene which competes withHNF-1 for binding to the AT-motifs [4] Thus ATBF1 seemsto act as a transcriptional repressor of the AFP gene [4 9 10]Besides the AFP gene ATBF1 can also negatively regulate thetranscription of the Myb gene [11] When ATBF1 was over-expressed in C2C12 cells the expression of Id3 and cyclin Dincreased whereas that of MyoD and myogenin did not [12]Thus ATBF1 seems to play a key role not only as a negativebut also as a positive transcriptional regulator RecentlyATBF1was suspected to be a candidate tumor suppressor genebecause it is frequently mutated or deleted in prostate breastand gastric tumors and its expression is also suppressed insome tumors [13ndash17]

Hindawi Publishing CorporationJournal of Signal TransductionVolume 2014 Article ID 970346 11 pageshttpdxdoiorg1011552014970346

2 Journal of Signal Transduction

Transforming growth factor-120573 (TGF-120573) regulates a greatnumber of cellular responses including proliferation differ-entiation apoptosis extracellular matrix production motil-ity and immunosuppression TGF-120573 elicits its cellular effectsby making a heteromeric complex between TGF-120573 type I(T120573RI also termed activin receptor-like kinase 5 [ALK5])and TGF-120573 type II receptors (T120573RII) After TGF-120573 bindsto T120573RII T120573RI is recruited by T120573RII to be phosphorylatedwithin its GS domain by the constitutively active T120573RIIkinase The activated T120573RI kinase initiates signaling throughphosphorylation of specific receptor-regulated Smads (R-Smads ie Smad2 and Smad3) Subsequently two activatedR-Smads form ternary complexes with a common-partnerSmad (Co-Smad) Smad4 These complexes can regulateTGF-120573-responsive genes either directly via their binding tothe promoters of these genes or indirectly via their associationwith a large number of transcription factors coactivatorsandor corepressors Of interest Co-Smad and R-Smadsexcept for Smad2 possess the ability to bind to specific DNAsequences whereas Smad2 has 30-aa insertion immediatelyprior to obvious DNA binding region in its MH1 domainwhich prevents Smad2 from binding to DNA [18ndash20]

TGF-120573 is known to negatively regulate AFP expression[21 22] Furthermore TGF-120573-mediated repression of AFPinvolves recruitment of Smad to p53 together with SnoN andmSin3A in the AFP promoter [22 23] Although ATBF1 alsocontributes to negative regulation of AFP how TGF-120573 sig-naling cooperates with ATBF1 to inhibit AFP transcriptionremains veiled In this study we could confirm that the TGF-120573Smad signaling pathway plays an inhibitory role in theAFPtranscription via the interaction of Smads with ATBF1

2 Materials and Methods

21 Cell Culture COS7 cells monkey kidney fibroblast-likecells were cultured in Dulbeccorsquos modified Eaglersquos medium(Nacalai) containing 10 fetal calf serum (FCS Invitrogen)HepG2 andHuH-7 cells hepatocellular carcinoma cells weremaintained in Eaglersquos minimum essential medium (Wako)containing 10 FCS 1 nonessential amino acids and 1sodium pyruvate All media were supplemented with100 IUmL penicillin and 100 120583gmL streptomycin

22 Plasmid Constructions The expression constructs forHA-ATBF1 Myc-ATBF1 HA-ATBF1N HA-ATBF1M andHA-ATBF1C have been previously described [24 25] TheAFP luciferase reporters and Flag-Smad2 Flag-Smad3 andFlag-Smad4 have been documented [6 26 27] Variouslengths of the 51015840promoter region of human AFP were clonedby PCR and inserted into the luciferase gene with either TK(TKLuc) or CMV minimal promoter (CMV Luc) Mutationin the AFP promoter was performed using a KOD-Plus-Mutagenesis kit (TOYOBO) Adenoviral ALK5ca was gener-ously provided by Fujii et al [28]

23 Luciferase Reporter Assay HepG2 cells were seeded at15 times 105 cellswell in a 12-well plate one day before transfec-tionThe cells were transfectedwith reporter constructs using

polyethyleneimine (PEI) After 24 h of transfection cells werestimulated with 5 ngmL TGF-120573 for 18 h In all luciferaseassays120573-galactosidase activity in cells transfectedwith 100 ngof pCH110 (GE Healthcare) was measured to normalize thetransfection efficiency The results were the averages of threeindependent transfections and were repeated at least twiceThe representative data were shown All values representmeanplusmn SD Each 119905-test between two columnswas performedlowast

119875lt 005 lowastlowast119875lt 001 and lowastlowastlowast119875lt 0001

24 RT-PCR Analysis Total RNAs from HuH-7 cells wereextracted using an RNeasy Plus Mini kit (Qiagen) Reversetranscription was carried out using a High-Capacity RNA tocDNA kit (Applied Biosystems) PCR was performed usingGoTaq (Promega) according to the manufacturerrsquos instruc-tions

25 Immunoprecipitation and Western Blot Analysis Todetect interactions among the proteins plasmids were trans-fected into COS7 cells (5 times 105 cells6 cm dish) using PEIForty hours after the transfection cells were lysed in 500120583L ofTNE buffer (10mM Tris (pH 74) 150mM NaCl 1mMethylenediamine-N1015840N1015840N1015840N1015840-tetraacetic acid (EDTA) 1NP-40 1mM phenylmethylsulfonyl-l-fluoride (PMSF)5 120583gmL leupeptin 100 UmL aprotinin 2mM sodiumvanadate 40mMNaF and 20mM 120573-glycerophosphate) Thecell lysates were precleared with protein G-Sepharose beads(GE Healthcare) for 30min at 4∘C and then incubated withanti-Myc or anti-Flag antibody (Sigma) for 2 h at 4∘C Theprotein complexes were immunoprecipitated by incubationwith proteinG-Sepharose beads for 30min at 4∘C followed bythree washes with TNE buffer The immunoprecipitatedproteins and aliquots of the total cell lysates were boiled for5min in sample buffer separated by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred to a Hybond-CExtra membrane (GE Healthcare) The membranes wereprobed with primary antibodies The primary antibodieswere detected with horseradish peroxidase-conjugated sec-ondary antibodies and chemiluminescent substrate (ThermoScientific) Protein expression in the total cell lysates wasevaluated by Western blot analysis

26 DNA Affinity Precipitation (DNAP) COS7 cells wereseeded at 15 times 106 cellswell in a 10 cm dish one day beforetransfection Tenmicrograms of indicated expression vectorswere transfected into COS7 cells using PEI Forty hours afterthe transfection the cells were lysed in 1mL of TNE bufferThen each cell lysate was divided and mixed for DNAP Thecombined cell lysates were precleared with 12 120583gmL poly(dI sdot dC) and streptavidin agarose (Sigma) for 30min andthen incubated with 24120583M biotinylated (AT-motif)

3for 2 h

at 4∘C Subsequently streptavidin agarose was added to thereaction mixture and incubated for 30min at 4∘C After theprecipitates had been washed with TNE buffer three timesthe precipitates and aliquots of the total lysates were separatedby SDS-PAGE The proteins were then transferred to themembrane The membrane was incubated with the indicatedprimary antibodies The primary antibodies were detected

Journal of Signal Transduction 3

Ad-A

LK5c

a

AFP

TMEPAI

Ad-L

acZ

120573-Actin

(a)

Ad-A

LK5c

a

p-Smad2

ALK5ca

Ad-L

acZ

120573-Actin

IB PS2

IB ALK5

IB 120573-actin

(b)

12 240 2 6

ATBF1

AFP

(h)

120573-Actin

(c)

Figure 1 TGF-120573 represses AFP mRNA (a) AFP mRNA expression was inhibited by ALK5ca in HuH-7 cells The cells were infected withLacZ (left lane) or ALK5ca-expressing adenoviruses (right lane) at a dose of 250 multiplicity of infection After 42 hours of infection totalRNA and cell lysates were prepared for RT-PCR analysis (a) andWestern blot analysis (IB) (b) respectively (a) Expression of AFP TMEPAIand 120573-actin mRNA was detected by RT-PCR TMEPAI which is a direct target gene of TGF-120573 signal [29ndash31] was used as a positive control(b) For IB antiphosphorylated Smad2 (PS2) anti-ALK5 and anti-120573-actin antibodies were used (c) Expression of AFP mRNA upon TGF-120573stimulation HuH-7 cells were treated with 5 ngmL TGF-120573 for the indicated times Then RT-PCR was performed as described in (a) EachPCR condition is described in Table 1

as described above The sequences of the biotinylated(AT-motif)

3were as follows 51015840-biotinylated CTCGAGGCT-

GTTAATTATTGGGGCTGTTAATTATTGGGGCTGTTAA-TTATTGAATTC-3101584031015840-GAATTCAATAATTAACAGCCCC-AATAATTAACAGCCCCAATAATTAACAGCCTCGAG-51015840

3 Results

31 AFP mRNA Is Inhibited upon TGF-120573 Stimulation It hasbeen reported that TGF-120573 contributes to the suppression ofthe activity of the AFP promoter [21 22] Indeed we exam-ined if the expression of AFP mRNA was affected in HuH-7cells upon ALK5 activation AFP mRNA was considerablyreduced in cells infected with constitutively active ALK5(ALK5ca) expressing adenovirus whereas TMEPAI mRNAwhich has been known to be a direct target gene of TGF-120573Smad signal [29ndash31] was remarkably induced (Figure 1(a))Furthermore AFP mRNA slightly decreased with time whenHuH-7 cells were stimulated with TGF-120573 On the otherhand ATBF1mRNAdid not change upon TGF-120573 stimulation(Figure 1(c))

32 TGF-120573Smad Signaling Inhibits the Activity of the AFPPromoter Together with ATBF1 It has already been demon-strated that enhancer elements (119864

119860 119864119861) silencer elements

(119878119863 119878119875) and a promoter (P) are present in the 51015840 flanking

region of AFP gene [5 32ndash34] ATBF1 is known to bind to119864119861and P to repress the activity of the AFP promoter [4

6 26] To evaluate the involvement of these elements inTGF-120573-mediated repression of the activity of the AFP pro-moter each luciferase reporter shown in Figure 2(a) wastransfected into HepG2 cells The cells were then stimulatedwith TGF-120573 As shown in Figure 2(b) both 49Luc andΔ27Luc which possessed a high basal level of the reporteractivity obviously showed TGF-120573-mediated repression of

the reporter activity 3Luc 16Luc and 09Luc contain Smadbinding elementp53 response element (SBEp53RE) whichhas been reported to confer TGF-120573-mediated repression ofthe AFP promoter [22 23] Indeed these reporter activitieswere also reduced upon TGF-120573 stimulation in spite of a verylow basal activity of their luciferase reporters Like thesethree reporters 02Luc which possesses one AT-motif couldexhibit TGF-120573-mediated repression of the luciferase activityas well (Figure 2(b)) These results supported the notion thatthe region between minus4995 and minus2953 concededly includescis-element(s) for TGF-120573 to suppress the activity of the AFPpromoter To test the possibility that ATBF1 can suppressthe activity of the AFP promoter upon TGF-120573 stimulationΔ27Luc was cotransfected with or without ATBF1 Subse-quently the cells were stimulated with TGF-120573 As seen inFigure 2(c) ATBF1 could further reduce the luciferase activ-ity thatwas repressed byTGF-120573 Since Smad3 and Smad4 playkey roles in the canonical TGF-120573 signaling pathway [18 20]we investigated whether Smad3 andor Smad4 are implicatedin TGF-120573-mediated repression of theAFP promoter togetherwith ATBF1 As shown in Figure 2(d) Smad3 alone or thecombination of Smad3 with Smad4 suppressed the reporteractivity Moreover ATBF1 further diminished the reporteractivity that was restrained by both Smad3 and Smad4 uponTGF-120573 stimulation

33 Requirement of ATBF1 Binding Sites for TranscriptionalRepression of the AFP Gene by TGF-120573 Because the regionbetween minus4995 and minus2953 is important for the TGF-120573Smadsignaling to cooperate with ATBF1 for the suppression of theactivity of the AFP promoter we speculated that 119864

119861 which

includes the ATBF1 binding site is involved in an inhibitoryaction via the TGF-120573Smad signaling pathway To test thispossibility the region between minus4990 and minus2968 was dividedinto three piecesThen each fragment was ligated to the luci-ferase reporter driven by the herpes virus thymidine kinase

4 Journal of Signal Transduction

Table 1 PCR primers to amplify human cDNAs

Human cDNA Sequence 119871 AT Cycle

AFP (+) ATCCAGGAGAGCCAAGCATT 434 58∘C 20(minus) TTCATCGTTTGCAGCGCTAC

ATBF1 (+) TGGCATCAAGTACAGCGCTC 392 53∘C 35(minus) GAACAGTTGTGCTGGGCAGA

TMEPAI (+) GATCATCATCATCGTGGTGG 455 60∘C 35(minus) CACTGTCGAAGATGGTTCTG

120573-actin (+) CACCCACACTGTGCCCATCTACGA 410 63∘C 22(minus) TGGCGTACAGGTCTTTGCGGATGT

119871 length of PCR fragment AT annealing temperature

AFP

Luc

Luc

Luc

Luc

Luc

Luc

minus4995

minus4995

EA EB SD SP P

ATBF1 ATBF1

49Luc

Δ27Luc

3Luc

16Luc

09Luc

02Luc+45

+45

+45

+45

+45

+45

minus178

minus178

minus2953

minus2953

minus1653

minus871

(a)

00

10

20

30

40

50

60

Relat

ive l

ucife

rase

activ

ity

lowast lowast lowastlowast

lowastlowast

lowastlowastlowast

lowastlowast

49Luc Δ27Luc 3Luc 16Luc 09Luc 02Luc

TGF-120573 (minus)TGF-120573 (+)

(b)

00

02

04

06

08

10

12

ATBF1

Rela

tive l

ucife

rase

activ

ity

lowastlowastlowast

lowast

minusΔ27Luc

(c)

00

02

04

06

08

10

12

Relat

ive l

ucife

rase

activ

ity

ATBF1 minus + minus minus+minusminus

minusminus

minus

minus+

minus+minus

++

++

+minus

+ minus + minus minus+minusminus

minusminus

minus+

minus+minus

++

++

+minus

+Smad3Smad4

+

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowast

TGF-120573

(d)

Figure 2 TGF-120573Smad signaling inhibits the activity of the AFP promoter together with ATBF1 (a) Schematic presentation of deletionmutants of the AFP promoter 119864

119860between minus4120 and minus3756 and 119864

119861between minus3492 and minus3300 are enhancer elements 119878

119863between minus1807

and minus1725 and 119878119875between minus317 and minus300 are silencer elements P indicates a promoter (b) Effect of TGF-120573 on the activity of the AFP

promoterThe examined luciferase reporters (500 ng) were transfected into HepG2 cellsThe cells were then stimulated with 5 ngmL TGF-120573(c) Cooperation of ATBF1 with the TGF-120573 signaling for suppression of the activity of the AFP promoter ATBF1 expression plasmid (200 ng)was transfected into HepG2 cells with Δ27Luc (500 ng) The cells were then stimulated with 5 ngmL TGF-120573 (d) Effect of Smads and ATBF1on the transcriptional activity of Δ27Luc HepG2 cells were transfected with Smads (100 ng) ATBF1 (200 ng) or their combination togetherwith Δ27Luc (500 ng) The cells were then stimulated with 5 ngmL TGF-120573

Journal of Signal Transduction 5

TK promoter

Luc

Luc

Luc

Luc

Luc

EAminus4995 minus2953

minus4990

minus4990 minus4309

minus4363 minus3601

minus2968

minus2968minus3658

Δ27Luc+45minus178

fTKLuc

5998400TKLuc

mTKLuc

3998400TKLuc

EB

(a)

00

02

04

06

08

10

12

14

Relat

ive l

ucife

rase

activ

ity

lowast

lowast

fTKLuc 5998400TKLuc mTKLuc 3998400TKLuc

(b)

AT-motif AT-motif

EB

Luc

Luc

Luc

Luc

Luc

Luc

39984005Luc

399840051Luc

39984003Luc

02Luc

399840052Luc

39984001Luc+45minus178

+45minus178

+45minus178

+45minus178

+45minus178

+45minus178

minus3148

minus3148

minus3445

minus3422

minus3492 minus3300

minus3422

minus3658

minus3658 minus3563

minus3562

minus3658

(c)

00

05

10

15

20

25

30

35

40

Rela

tive l

ucife

rase

activ

ity

lowastlowastlowast

lowastlowastlowast

lowastlowast

39984005Luc 39984003Luc39984001Luc

(d)

00

02

04

06

08

10

12

14

Rela

tive l

ucife

rase

activ

ity

lowastlowastlowast

399840051Luc 02Luc399840052Luc

(e)

00

02

04

06

08

10

12

14

ATBF1

Rela

tive l

ucife

rase

activ

ity

lowast

lowast

399840052Luc

TGF-120573 (minus)TGF-120573 (+)

minus

(f)

Figure 3 Requirement of a distal AT-motif for TGF-120573-mediated repression of the activity of the AFP promoter (a) Schematic presentationof TKLuc conjugated with several fragments of the AFP promoter between minus4990 and minus2968 (b) Effect of TGF-120573 on the activity of TKLucreporters conjugated with several truncated AFP promoter regions Each reporter (200 ng) was transfected into HepG2 cells The cells werethen stimulated with 5 ngmL TGF-120573 (c) Schematic presentation of 02Luc conjugated with several fragments of the AFP promoter betweenminus3658 andminus3148 (d) Effect of TGF-120573 on the activity of the luciferase reporters described in Figure 3(c) Each reporter (200 ng)was transfectedinto HepG2 cellsThe cells were then stimulated with 5 ngmL TGF-120573 (e) Requirement of the fragment between minus3562 and minus3422 for TGF-120573-mediated suppression of theAFP promoter HepG2 cells were transfected with the indicated plasmids (200 ng)The cells were then stimulatedwith 5 ngmL TGF-120573 (f) Cooperation of ATBF1 with the TGF-120573 signaling for 3101584052Luc activity HepG2 cells were transfected with either352Luc reporter (200 ng) in the presence or absence of ATBF1 (200 ng) The cells were then stimulated with 5 ngmL TGF-120573

6 Journal of Signal Transduction

AT-motif AT-motif

AT-motifAT-motif

AT-motifCMV promoter

CMV promoter

65CMVLucAT-motif

CMV promoter

minus3562 minus3422

minus3562 minus3422 minus155 minus92

minus155 minus92

minus3562 minus3422

minus178Luc

Luc

Luc

Luc

+45

+1399840052Luc

399840052CMVLuc

399840052 65CMVLuc

(a)

00

02

04

06

08

10

12

Rela

tive l

ucife

rase

activ

ity

65CMVLuc399840052CMVLuc399840052 65CMVLuc

TGF-120573 (minus)TGF-120573 (+)

lowast

(b)

Figure 4 Importance of both distal and proximal AT-motifs for TGF-120573-mediated repression of theAFP promoter (a) Schematic presentationof CMVLuc conjugatedwith distal and proximal AT-motifs (b)HepG2 cells were transfectedwith each reporter (200 ng) and then stimulatedwith 5 ngmL TGF-120573

promoter (TKLuc) (Figure 3(a)) As shown in Figure 3(b)TGF-120573 suppressed the reporter activity of fTKLuc Thusthe region between minus4990 and minus2968 possesses an inhibitorycis-element(s) upon TGF-120573 stimulation Within this regionthe fragment between minus3658 and minus2968 (termed 31015840TKLuc)revealed a remarkably inhibitory cis-element by TGF-120573although TGF-120573-mediated repression of the reporter activitycould also be seen in mTKLuc Intriguingly 119864

119861contain-

ing the AT-motif is present between minus3492 and minus3300(Figure 3(c)) To further narrow down a TGF-120573-responsiveinhibitory cis-element(s) we made four luciferase reportersthat were linked to the AFPminimal promoter (Figure 3(c))TGF-120573 dramatically suppressed the luciferase activity of310158401Luc 310158405Luc and 3101584052Luc although 310158403Luc activity was alsoinhibited by TGF-120573 to some extent in spite of its low basalluciferase activity indicating that the region from minus3562 tominus3422 possesses a TGF-120573-mediated inhibitory cis-element ifnot more (Figures 3(d) and 3(e)) Thus an AT-motif andorits adjacent sequences in 119864

119861were highly suggestive of a TGF-

120573-mediated inhibitory cis-element In addition ATBF1 couldfurther inhibit the 3101584052Luc activity suppressed by TGF-120573(Figure 3(f)) Therefore it is possible that the TGF-120573Smadpathway interferes via the region between minus3562 and minus3422

34 A Role of the Proximal AT-Motif in the AFP PromoterSince there are two functional AT-motifs within the AFPpromoter between minus4995 and +45 (Figure 2(a)) it is possiblethat the proximal AT-motif also influences TGF-120573-mediatedinhibition of the activity of theAFP promoter To confirm thatthe proximal AT-motif can also contribute to TGF-120573-mediated repression of the activity of the AFP promoter the141nt-length fragment from minus3562 to minus3422 was ligated to the

64nt-length region between minus155 and minus92 of the AFP pro-moter Then this fragment was further placed at the 51015840portof CMV Luc (3101584052 65CMVLuc) because the CMV promotershows the high level of luciferase activity Figure 4(b) demon-strates that both the distal and the proximal AT-motifs areneeded for TGF-120573 to suppress the activity of the AFPpromoter

35 Association of Smad Proteins with ATBF1 Since ATBF1cooperates with Smad3 for inhibition of the activity of theAFP promoter we tried to determine whether ATBF1 couldinteract with R-Smads Expectedly ATBF1 could associatewith Smad2 and Smad3 although ALK5 activation was notinvolved in its interaction (Figure 5(a) and data not shown)In these experiments we could observe multiple bands cor-responding to ATBF1 These multiple bands might be due tocalpain-1-dependent proteolysis in COS7 cells [35] To iden-tify the domain(s) of ATBF1 that interact with Smads wedivided ATBF1 into three pieces (Figure 5(b)) ATBF1N aswell as ATBF1C had the ability to interact with Smad2 andSmad3 although ATBF1M no longer possessed the ability tobind to either Smad2 or Smad3 (Figures 5(c)ndash5(e)) It hasbeen reported that DNA-binding domains are present inATBF1M including zinc finger domains and homeodomains[12]When either ATBF1NorATBF1Cwas cotransfectedwithATBF1 TGF-120573-mediated inhibition of the activity of theAFPpromoter was improved owing to their competition withATBF1 for Smad binding (Figures 6(a) and 6(b)) On theother hand ATBF1M could inhibit the reporter activity as adominant negative formofATBF1 becauseATBF1Mpossessesthe ability to bind to AT-motif via its DNA binding domain(data not shown) These results indicate that the N-terminal

Journal of Signal Transduction 7

Flag-Smad3

Smad3

ATBF1

Smad3

ALK5ca

minus +minus

++ +

Myc-ATBF1ALK5caV5

IP MycIB flag

IB Myc

IB flag

IB V5

(a)

3703

ATBF1

1

ATBF1N

8951

ATBF1M

3293894

ATBF1C

37033290

Zinc fingerHomeodomain

(b)

HA-ATBF1NFlag-Smad2Flag-Smad3

IP flagIB HA

IB HA

IB flag

minus + minusminus

Smad2Smad3

ATBF1N

ATBF1N

+minus

(c)

HA-ATBF1MFlag-Smad2Flag-Smad3

minus +minus +minus

IP flagIB HA

IB HA

IB flag

minus

ATBF1M

ATBF1M

Smad2Smad3

(d)

HA-ATBF1CFlag-Smad2Flag-Smad3

minus +minus +minus

IP flagIB HA

IB HA

IB flag

minus

ATBF1C

ATBF1C

Smad2Smad3

(e)

Figure 5 Association of Smads with ATBF1 (a) Interaction of Smad3 with ATBF1 COS7 cells were transfected with Flag-Smad3 (1120583g) withMyc-ATBF1 (2120583g) in combination with ALK5caV5 (05 120583g) or without it Cell lysates were subjected to immunoprecipitation (IP) with anti-Myc antibody followed by IB with anti-Flag antibody to show the interaction of Flag-Smad3 with Myc-ATBF1 (b) Schematic presentation ofATBF1 mutants (cndashe) Interaction of each ATBF1 mutant with Smad2 or Smad3 COS7 cells were transfected with Flag-Smad2 or Flag-Smad3(15120583g) with HA-ATBF1N (2120583g) (c) HA-ATBF1M (2120583g) (d) or HA-ATBF1C (2120583g) (e) Cell lysates were subjected to IP with anti-Flagantibody followed by IB with anti-HA antibody to show the interaction of Smad2 and Smad3 with ATBF1 mutants

and C-terminal domains are needed for ATBF1 to interactwith R-Smads

36 Indirect Binding of Smads to the AFP Promoter via ATBF1We could find only one Smad binding element (SBE) [36 37]in 3101584052Luc (Figure 7(a))Thus we examinedwhether this SBEis necessary for TGF-120573-mediated repression of the AFP pro-moter For that purpose wemade one reporter construct thathad mutations within the SBE of 3101584052Luc (3101584052mSBELuc)

(Figure 7(a)) However 3101584052mSBELuc activity was still inhib-ited by TGF-120573 although its basal level became weaker thanthat of 3101584052Luc (Figure 7(b)) Thus the SBE in the AFP pro-moter is not essential for TGF-120573Smad signaling to repressthe activity of the AFP promoter Indeed a DNAP assayshowed that Smad3 can be detected in the presence of ATBF1using three repeats of an AT-motif as the probe (Figure 7(c))Thus Smad3 probably binds to the promoter region of theAFP gene indirectly through its interaction with ATBF1

8 Journal of Signal Transduction

00

02

04

06

08

10

12

14

16

ATBF1ATBF1N

Rela

tive l

ucife

rase

activ

ity

+ + ++minus

minus minus

TGF-120573 (minus)TGF-120573 (+)

lowast

lowast

lowastlowastlowast

(a)

00

02

04

06

08

10

12

14

ATBF1ATBF1C

Rela

tive l

ucife

rase

activ

ity

+ + ++minus

minus minus

TGF-120573 (minus)TGF-120573 (+)

lowast

lowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

(b)

Figure 6 ATBF1N and ATBF1C perturb ATBF1-mediated suppression of the activity of theAFP promoter upon TGF-120573 stimulation ATBF1N(a) and ATNF1C (b) rescue ATBF1-mediated repression of the activity of the AFP promoter HepG2 cells were transfected with increasedamounts of either ATBF1N (50 100 or 200 ng) (a) or ATBF1C (50 100 or 200 ng) (b) together with ATBF1 (200 ng) and Δ27Luc (200 ng)The cells were then stimulated with 5 ngmL TGF-120573

4 Discussion

The hepatoma cells HuH-7 and HepG2 cells are known tosecrete a large and a low amount of AFP respectively [38] Agrowing body of evidence indicates that the level of AFP inserum from patients is linked to HCC tumorigenicity [3] p53has been reported to cooperate with the TGF-120573Smad path-way to repress AFP expression via induction of SnoN [22]However it remains veiled whether ATBF1 which is knownto blockAFP transcription [4 10] can also regulate the activ-ity of the AFP promoter in consort with the TGF-120573Smadpathway because both ATBF1 and TGF-120573Smad might pos-sess an antioncogenic function [13ndash17 20 39 40] ATBF1could inhibit the activity of the AFP promoter via its regionsfrom minus4995 to minus2953 andor from minus178 to +45 [4 26] SinceΔ27Luc had a remarkably basal promoter activity comparedwith 02Luc (Figure 2(b)) we initially focused on the distalregion of the AFP promoter for involvement of the TGF-120573Smad signaling with ATBF1 As expected the TGF-120573-mediated repressive element in the AFP promoter lay near119864119861 which contains a distal AT-motif The luciferase reporter

conjugated with the inhibitory element including a distalAT-motif (352Luc) showed coordinated inhibitory actionbetween the TGF-120573 signaling and ATBF1 (Figure 3(f))Therefore we were struck with the notion that ATBF1physically interacts with Smads which play key roles in thecanonical TGF-120573 signaling pathway [18ndash20] Not surpris-ingly ATBF1 could interact with Smad2 and Smad3 althoughC-terminal phosphorylation of R-Smads was dispensable

(Figure 5(a) and data not shown) In addition both the N-terminal (ATBF1N) and the C-terminal mutants of ATBF1(ATBF1C) but not the middle domain of ATBF1 (ATBF1M)which possesses DNA-binding activity [12] could interactwith R-Smads (Figures 5(c)ndash5(e))We hypothesized that bothATBF1N and ATBF1C might compete with wildtype ATBF1for Smad binding to rescueATBF1-mediated repression of theAFP promoter uponTGF-120573 stimulation because both of themhave the ability to bind to R-Smads According to our expec-tation both ATBF1N and ATBF1C improved the reporteractivity decreased by the cooperative ATBF1 and TGF-120573signaling (Figures 6(a) and 6(b)) As shown in Figure 2(b)02Luc activity could also be reduced upon TGF-120573 stimu-lation despite very low basal promoter activity Because ofthe presence of a proximal AT-motif within this region wespeculated that a proximal AT-motif also contributes to TGF-120573-mediated-repression of the activity of the AFP promoterThe deletion of a proximal AT-motif from 3101584052 65CMVLucled us to recognize the importance of a proximal AT-motif aswell as a distal AT-motif for TGF-120573-mediated repression ofthe activity of theAFP promoter (Figure 4(b)) A Smad bind-ing element (SBE) composed of consensus 51015840-AGAC-31015840 isflanked to a proximal AT-motif However themutation of theSBE in 3101584052Luc showed reduced basal promoter activity butstill maintained TGF-120573-mediated repression of the AFP pro-moter (Figure 7(b)) Thus Smad might not be necessary tobind to a promoter element(s) in the AFP gene directly whenATBF1 cooperates with TGF-120573 signaling whereas p53-mediated repression of the AFP promoter activity needs

Journal of Signal Transduction 9

SBEminus3422

minus3422

minus3562

minus3562

LucAT-motif

minus178 +45

+1

LucAT-motif

AT-motif

AT-motifminus178 +45

+1

399840052Luc

399840052mSBELuc

(a)

00

02

04

06

08

10

12

Relat

ive l

ucife

rase

activ

ity

TGF-120573 (minus)TGF-120573 (+)

lowastlowast

lowastlowast

399840052Luc 399840052mSBELuc

(b)

DNAP

Flag-Smad3IB flag

IB HA

Totallysates

HA-ATBF1

HA-ATBF1

Flag-Smad3IB flag

IB HA

p-Smad3IB PS3

IB V5 ALK5caV5

Biotinylated probe minus + + +

Flag-Smad3HA-ATBF1

+ + + minusminus + +minus

ALK5caV5 + + + minus

(AT-motif)3

(c)

Figure 7 SBE in the proximal region of the AFP promoter is not essential for TGF-120573-mediated suppression of the AFP promoter (a)Schematic presentation of 3101584052mSBELuc A putative SBE 51015840-CAGATA-31015840 was mutated to 51015840-TTTTTT-31015840 (b) Effect of TGF-120573 on 3101584052Lucand 3101584052mSBELuc activities HepG2 cells were transfected with each reporter plasmid (200 ng) The cells were then stimulated with 5 ngmLTGF-120573 (c) Indirect binding of Smad3 to an AT-motif via ATBF1 The indicated cell lysates were mixed with biotinylated AT-motifs Thenstreptavidin-agarose was added to the mixture Subsequently a protein(s) bound to streptavidin-agarose was purified After SDS-PAGE IBwas carried out using anti-Flag or anti-HA antibody Using the total lysates each protein was detected with anti-Flag anti-HA anti-V5 oranti-phosphorylated Smad3 (PS3) antibody

direct binding of Smads to DNA [22 23] The requirement ofdirect DNA binding for R-Smads is probably dependent onwhich transcriptional regulator(s) they cooperate or syner-gize with Indeed Smad3 could bind toAT-motifs in the pres-ence of ATBF1 but not in its absence (Figure 7(c)) Althoughwe do not know if the ATBF1Smad complex at a distalAT-motif can physically interact with that at a proximal

AT-motif directly or indirectly through another protein(s)(ldquoXrdquo Figure 8) both AT-motifs are definitely important forcooperation between ATBF1 and the TGF-120573 signaling tosuppress the activity of the AFP promoter

ATBF-1 is known to compete with HNF-1 for binding toAT-motifs in the AFP promoter [4] Thus the following pos-sibility can be speculated upon TGF-120573 stimulation Smads

10 Journal of Signal Transduction

AT-motif

AT-motif

AFP

ATBF1

ATBF1ATBF1Smad23-Smad4

X

Smad23-Smad4

ATBF1

Figure 8 Proposed inhibitory mechanism by which the TGF-120573Smad signaling and ATBF1 cooperatively regulate the activity ofthe AFP promoter The distal and proximal AT-motifs in the AFPpromoter are bound by ATBF1 Upon TGF-120573 signaling the R-SmadsSmad4 complex interacts with ATBF1 to make a repressivecomplex for the activity of the AFP promoter However whetheranother protein(s) (termed ldquoXrdquo) is essential for the R-SmadSmad4ATBF1 complex remains veiled

can not only bind to AT-motifs via ATBF-1 indirectly but alsointeract with HNF-1 to dissociate it from AT-motifs Due todual function of Smads the AFP prompter activity might beinhibited This possibility is interesting to be investigated

p53-mediated repression of the AFP promoter activityneeds direct binding of Smads to SBEp53REWilkinson et alfurther demonstrated that mSin3A and SnoN which is adirect target gene of TGF-120573 signal are recruited by p53Smadscomplex when the transcription of the AFP gene is inhib-ited [22 23] In our current study the region includingSBEp53RE in theAFP promoterwas not investigated becausethe basal reporter activities of 3Luc 16Luc and 09Luc werevery low compared with that of Δ27Luc (Figure 2(b)) Itis possible that ATBF1 might interplay with p53 via Smadcomplex to effectively repress the transcription of the AFPgene Further experiments are needed in the future to confirmthese hypotheses as well

5 Conclusion

In summary we report a novel interaction between ATBF1and Smads The interaction between ATBF1 and Smadsappears to cooperatively inhibit the transcription of AFPgene upon TGF-120573 signaling In particular both proximal anddistal AT-motifs are required for TGF-120573Smad signaling tocounteract with the transcription of the AFP gene

Conflict of Interests

The authors have no competing financial interests to declare

Acknowledgments

This research was supported by a Grant-in-Aid for Explor-atory Research (A) from the Japan Society for the Promotionof Science (JSPS) (to Nobuo Sakata) the MEXT-Supported

Program for the Strategic Research Foundation at PrivateUniversities (2013ndash2017) (to Susumu Itoh) the Takeda Sci-ence Foundation (to Susumu Itoh) the Smoking ResearchFoundation (to Susumu Itoh) the Daiichi-Sankyo Founda-tion of Life Science (to Susumu Itoh) the Naito Founda-tion (to Susumu Itoh) the Vehicle Racing Commemora-tive Foundation (to Susumu Itoh) and a Grant-in-Aid forYoung Scientists of Showa Pharmaceutical University (toSouichi Ikeno) The authors were also supported by the JointUsageResearch Program of the Medical Research Institutethe Tokyo Medical and Dental University and by the Core-to-Core Program ldquoCooperative International Framework inTGF-120573 Family Signalingrdquo of the JSPS They thank Ms FMiyamasu for excellent English proofreading

References

[1] M A Dziadek and G K Andrews ldquoTissue specificity of 120572-fetoprotein messenger RNA expression during mouse embryo-genesisrdquoThe EMBO Journal vol 2 no 4 pp 549ndash554 1983

[2] J L Nahon I Tratner A Poliard et al ldquoAlbumin and 120572-feto-protein gene expression in various nonhepatic rat tissuesrdquo TheJournal of Biological Chemistry vol 263 no 23 pp 11436ndash114421988

[3] A A Terentiev and N T Moldogazieva ldquo120572-fetoprotein a ren-aissancerdquo Tumor Biology vol 34 no 4 pp 2075ndash2091 2013

[4] H Yasuda A Mizuno T Tamaoki and T Morinaga ldquoATBF1 amultiple-homeodomain zinc finger protein selectively down-regulates AT-rich elements of the human 120572-fetoprotein generdquoMolecular and Cellular Biology vol 14 no 2 pp 1395ndash14011994

[5] K Sawadaishi T Morinaga and T Tamaoki ldquoInteraction of ahepatoma-specific nuclear factor with transcription-regulatorysequences of the human 120572-fetoprotein and albumin genesrdquoMolecular andCellular Biology vol 8 no 12 pp 5179ndash5187 1988

[6] H Nakabayashi Y Koyama H Suzuki et al ldquoFunctional map-ping of tissue-specific elements of the human 120572-fetoproteingene enhancerrdquo Biochemical and Biophysical Research Commu-nications vol 318 no 3 pp 773ndash785 2004

[7] T Morinaga H Yasuda T Hashimoto K Higashio and TTamaoki ldquoA human 120572-fetoprotein enhancer-binding proteinATBF1 contains four homeodomains and seventeen zinc fin-gersrdquo Molecular and Cellular Biology vol 11 no 12 pp 6041ndash6049 1991

[8] Y Miura T Tam A Ido et al ldquoCloning and characterization ofan ATBF1 isoform that expresses in a neuronal differentiation-dependent mannerrdquo The Journal of Biological Chemistry vol270 no 45 pp 26840ndash26848 1995

[9] H Kataoka Y Miura T Joh et al ldquo120572-fetoprotein producinggastric cancer lacks transcription factor ATBF1rdquo Oncogene vol20 no 7 pp 869ndash873 2001

[10] T Ninomiya K Mihara K Fushimi Y Hayashi T Hashimoto-Tamaoki and T Tamaoki ldquoRegulation of the 120572-fetoprotein geneby the isoforms of ATBF1 transcription factor in human hep-atomardquo Hepatology vol 35 no 1 pp 82ndash87 2002

[11] P Kaspar M Dvorakova J Kralova P Pajer Z Kozmik andM Dvorak ldquoMyb-interacting protein ATBF1 represses tran-scriptional activity of Myb oncoproteinrdquoThe Journal of Biologi-cal Chemistry vol 274 no 20 pp 14422ndash14428 1999

[12] F B Berry YMiura KMihara et al ldquoPositive and negative reg-ulation of myogenic differentiation of C2C12 cells by isoforms

Journal of Signal Transduction 11

of the multiple homeodomain zinc finger transcription factorATBF1rdquoThe Journal of Biological Chemistry vol 276 no 27 pp25057ndash25065 2001

[13] X Sun H F Frierson C Chen et al ldquoFrequent somaticmutations of the transcription factor ATBF1 in human prostatecancerrdquo Nature Genetics vol 37 no 6 pp 407ndash412 2005

[14] X Sun Y Zhou K B Otto et al ldquoInfrequentmutation of ATBF1in human breast cancerrdquo Journal of Cancer Research and ClinicalOncology vol 133 no 2 pp 103ndash105 2007

[15] Y G Cho J H Song C J Kim et al ldquoGenetic alterations of theATBF1 gene in gastric cancerrdquo Clinical Cancer Research vol 13no 15 pp 4355ndash4359 2007

[16] K Kai Z Zhang H Yamashita Y Yamamoto Y Miura and HIwase ldquoLoss of heterozygosity at the ATBF1-A locus located inthe 16q22 minimal region in breast cancerrdquo BMC Cancer vol 8article 262 2008

[17] A M Cleton-Jansen R van Eijk M Lombaerts et al ldquoATBF1and NQO1 as candidate targets for allelic loss at chromosomearm 16q in breast cancer absence of somatic ATBF1 mutationsand no role for the C609TNQO1 polymorphismrdquo BMCCancervol 8 article 105 2008

[18] J Massague J Seoane and D Wotton ldquoSmad transcriptionfactorsrdquo Genes amp Development vol 19 no 23 pp 2783ndash28102005

[19] AMoustakas and C H Heldin ldquoThe regulation of TGF120573 signaltransductionrdquo Development vol 136 no 22 pp 3699ndash37142009

[20] J Massague ldquoTGF120573 signalling in contextrdquo Nature ReviewsMolecular Cell Biology vol 13 no 10 pp 616ndash630 2012

[21] K Nakao K Nakata S Mitsuoka et al ldquoTransforming growthfactor 120573 1 differentially regulates 120572-fetoprotein and albumin inHuH-7 human hepatoma cellsrdquo Biochemical and BiophysicalResearch Communications vol 174 no 3 pp 1294ndash1299 1991

[22] D SWilkinson S K Ogden S A Stratton et al ldquoA direct inter-section between p53 and transforming growth factor 120573 path-ways targets chromatin modification and transcription repres-sion of the 120572-fetoprotein generdquoMolecular and Cellular Biologyvol 25 no 3 pp 1200ndash1212 2005

[23] D S Wilkinson W W Tsai M A Schumacher and M CBarton ldquoChromatin-bound p53 anchors activated Smads andthe mSin3A corepressor to confer transforming-growth-factor-120573-mediated transcription repressionrdquo Molecular and CellularBiology vol 28 no 6 pp 1988ndash1998 2008

[24] C G Jung H J Kim M Kawaguchi et al ldquoHomeotic factorATBF1 induces the cell cycle arrest associated with neuronaldifferentiationrdquo Development vol 132 no 23 pp 5137ndash51452005

[25] X Y Dong X Sun P Guo et al ldquoATBF1 inhibits estrogenreceptor (ER) function by selectively competing with AIB1 forbinding to the ER in ER-positive breast cancer cellsrdquoThe Journalof Biological Chemistry vol 285 no 43 pp 32801ndash32809 2010

[26] H Kataoka P Bonnefin D Vieyra et al ldquoING1 repressestranscription by direct DNA binding and through effects onp53rdquo Cancer Research vol 63 no 18 pp 5785ndash5792 2003

[27] A Nakao T Imamura S Souchelnytskyi et al ldquoTGF-120573receptor-mediated signalling through Smad2 Smad3 andSmad4rdquoThe EMBO Journal vol 16 no 17 pp 5353ndash5362 1997

[28] M Fujii K Takeda T Imamura et al ldquoRoles of bone morpho-genetic protein type I receptors and Smad proteins in osteoblastand chondroblast differentiationrdquoMolecular Biology of the Cellvol 10 no 11 pp 3801ndash3812 1999

[29] YWatanabe S Itoh T Goto et al ldquoTMEPAI a transmembraneTGF-120573-inducible protein sequesters smad proteins from activeparticipation in TGF-120573 signalingrdquoMolecular Cell vol 37 no 1pp 123ndash134 2010

[30] S Itoh M Thorikay M Kowanetz et al ldquoElucidation of Smadrequirement in transforming growth factor-120573 type I receptor-induced responsesrdquo The Journal of Biological Chemistry vol278 no 6 pp 3751ndash3761 2003

[31] N Nakano S Itoh Y Watanabe K Maeyama F Itoh and MKato ldquoRequirement of TCF7L2 for TGF-120573 dependent transcrip-tional activation of the TMEPAI generdquoThe Journal of BiologicalChemistry vol 285 no 49 pp 38023ndash38033 2010

[32] K Watanabe A Saito and T Tamaoki ldquoCell-specific enhanceractivity in a far upstream region of the human 120572-fetoproteingenerdquo The Journal of Biological Chemistry vol 262 no 10 pp4812ndash4818 1987

[33] H Nakabayashi K Watanabe A Saito A Otsuru KSawadaishi and T Tamaoki ldquoTranscriptional regulation of 120573-fetoprotein expression by dexamethasone in human hepatomacellsrdquo The Journal of Biological Chemistry vol 264 no 1 pp266ndash271 1989

[34] H Nakabayashi T Hashimoto Y Miyao K-K Tjong J Chanand T Tamaoki ldquoA position-dependent silencer plays a majorrole in repressing 120573-fetoprotein expression in human hep-atomardquoMolecular and Cellular Biology vol 11 no 12 pp 5885ndash5893 1991

[35] S Zhang T S Kim Y Dong et al ldquoAT motif binding factor 1(ATBF1) is highly phosphorylated in embryonic brain and pro-tected from cleavage by calpain-1rdquo Biochemical and BiophysicalResearch Communications vol 427 no 3 pp 537ndash541 2012

[36] L J Jonk S Itoh C H Heldin P ten Dijke and W KruijerldquoIdentification and functional characterization of a smad bind-ing element (SBE) in the JunBpromoter that acts as a transform-ing growth factor-120573 activin and bone morphogenetic protein-inducible enhancerrdquo The Journal of Biological Chemistry vol273 no 33 pp 21145ndash21152 1998

[37] S Dennler S Itoh D Vivien P ten Dijke S Huet and J Gau-thier ldquoDirect binding of Smad3 and Smad4 to critical TGF120573-inducible elements in the promoter of human plasminogenactivator inhibitor-type 1 generdquo The EMBO Journal vol 17 no11 pp 3091ndash3100 1998

[38] A Ido K Nakata Y Kato et al ldquoGene therapy for hep-atoma cells using a retrovirus vector carrying herpes simplexvirus thymidine kinase gene under the control of human 120572-fetoprotein gene promoterrdquo Cancer Research vol 55 no 14 pp3105ndash3109 1995

[39] A B Roberts and L M Wakefield ldquoThe two faces of trans-forming growth factor 120573 in carcinogenesisrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 15 pp 8621ndash8623 2003

[40] B Bierie and H L Moses ldquoTumour microenvironment TGF120573the molecular Jekyll and Hyde of cancerrdquo Nature ReviewsCancer vol 6 no 7 pp 506ndash520 2006

Journal of Signal Transduction 3

Ad-A

LK5c

a

AFP

TMEPAI

Ad-L

acZ

120573-Actin

(a)

Ad-A

LK5c

a

p-Smad2

ALK5ca

Ad-L

acZ

120573-Actin

IB PS2

IB ALK5

IB 120573-actin

(b)

12 240 2 6

ATBF1

AFP

(h)

120573-Actin

(c)

Figure 1 TGF-120573 represses AFP mRNA (a) AFP mRNA expression was inhibited by ALK5ca in HuH-7 cells The cells were infected withLacZ (left lane) or ALK5ca-expressing adenoviruses (right lane) at a dose of 250 multiplicity of infection After 42 hours of infection totalRNA and cell lysates were prepared for RT-PCR analysis (a) andWestern blot analysis (IB) (b) respectively (a) Expression of AFP TMEPAIand 120573-actin mRNA was detected by RT-PCR TMEPAI which is a direct target gene of TGF-120573 signal [29ndash31] was used as a positive control(b) For IB antiphosphorylated Smad2 (PS2) anti-ALK5 and anti-120573-actin antibodies were used (c) Expression of AFP mRNA upon TGF-120573stimulation HuH-7 cells were treated with 5 ngmL TGF-120573 for the indicated times Then RT-PCR was performed as described in (a) EachPCR condition is described in Table 1

as described above The sequences of the biotinylated(AT-motif)

3were as follows 51015840-biotinylated CTCGAGGCT-

GTTAATTATTGGGGCTGTTAATTATTGGGGCTGTTAA-TTATTGAATTC-3101584031015840-GAATTCAATAATTAACAGCCCC-AATAATTAACAGCCCCAATAATTAACAGCCTCGAG-51015840

3 Results

31 AFP mRNA Is Inhibited upon TGF-120573 Stimulation It hasbeen reported that TGF-120573 contributes to the suppression ofthe activity of the AFP promoter [21 22] Indeed we exam-ined if the expression of AFP mRNA was affected in HuH-7cells upon ALK5 activation AFP mRNA was considerablyreduced in cells infected with constitutively active ALK5(ALK5ca) expressing adenovirus whereas TMEPAI mRNAwhich has been known to be a direct target gene of TGF-120573Smad signal [29ndash31] was remarkably induced (Figure 1(a))Furthermore AFP mRNA slightly decreased with time whenHuH-7 cells were stimulated with TGF-120573 On the otherhand ATBF1mRNAdid not change upon TGF-120573 stimulation(Figure 1(c))

32 TGF-120573Smad Signaling Inhibits the Activity of the AFPPromoter Together with ATBF1 It has already been demon-strated that enhancer elements (119864

119860 119864119861) silencer elements

(119878119863 119878119875) and a promoter (P) are present in the 51015840 flanking

region of AFP gene [5 32ndash34] ATBF1 is known to bind to119864119861and P to repress the activity of the AFP promoter [4

6 26] To evaluate the involvement of these elements inTGF-120573-mediated repression of the activity of the AFP pro-moter each luciferase reporter shown in Figure 2(a) wastransfected into HepG2 cells The cells were then stimulatedwith TGF-120573 As shown in Figure 2(b) both 49Luc andΔ27Luc which possessed a high basal level of the reporteractivity obviously showed TGF-120573-mediated repression of

the reporter activity 3Luc 16Luc and 09Luc contain Smadbinding elementp53 response element (SBEp53RE) whichhas been reported to confer TGF-120573-mediated repression ofthe AFP promoter [22 23] Indeed these reporter activitieswere also reduced upon TGF-120573 stimulation in spite of a verylow basal activity of their luciferase reporters Like thesethree reporters 02Luc which possesses one AT-motif couldexhibit TGF-120573-mediated repression of the luciferase activityas well (Figure 2(b)) These results supported the notion thatthe region between minus4995 and minus2953 concededly includescis-element(s) for TGF-120573 to suppress the activity of the AFPpromoter To test the possibility that ATBF1 can suppressthe activity of the AFP promoter upon TGF-120573 stimulationΔ27Luc was cotransfected with or without ATBF1 Subse-quently the cells were stimulated with TGF-120573 As seen inFigure 2(c) ATBF1 could further reduce the luciferase activ-ity thatwas repressed byTGF-120573 Since Smad3 and Smad4 playkey roles in the canonical TGF-120573 signaling pathway [18 20]we investigated whether Smad3 andor Smad4 are implicatedin TGF-120573-mediated repression of theAFP promoter togetherwith ATBF1 As shown in Figure 2(d) Smad3 alone or thecombination of Smad3 with Smad4 suppressed the reporteractivity Moreover ATBF1 further diminished the reporteractivity that was restrained by both Smad3 and Smad4 uponTGF-120573 stimulation

33 Requirement of ATBF1 Binding Sites for TranscriptionalRepression of the AFP Gene by TGF-120573 Because the regionbetween minus4995 and minus2953 is important for the TGF-120573Smadsignaling to cooperate with ATBF1 for the suppression of theactivity of the AFP promoter we speculated that 119864

119861 which

includes the ATBF1 binding site is involved in an inhibitoryaction via the TGF-120573Smad signaling pathway To test thispossibility the region between minus4990 and minus2968 was dividedinto three piecesThen each fragment was ligated to the luci-ferase reporter driven by the herpes virus thymidine kinase

4 Journal of Signal Transduction

Table 1 PCR primers to amplify human cDNAs

Human cDNA Sequence 119871 AT Cycle

AFP (+) ATCCAGGAGAGCCAAGCATT 434 58∘C 20(minus) TTCATCGTTTGCAGCGCTAC

ATBF1 (+) TGGCATCAAGTACAGCGCTC 392 53∘C 35(minus) GAACAGTTGTGCTGGGCAGA

TMEPAI (+) GATCATCATCATCGTGGTGG 455 60∘C 35(minus) CACTGTCGAAGATGGTTCTG

120573-actin (+) CACCCACACTGTGCCCATCTACGA 410 63∘C 22(minus) TGGCGTACAGGTCTTTGCGGATGT

119871 length of PCR fragment AT annealing temperature

AFP

Luc

Luc

Luc

Luc

Luc

Luc

minus4995

minus4995

EA EB SD SP P

ATBF1 ATBF1

49Luc

Δ27Luc

3Luc

16Luc

09Luc

02Luc+45

+45

+45

+45

+45

+45

minus178

minus178

minus2953

minus2953

minus1653

minus871

(a)

00

10

20

30

40

50

60

Relat

ive l

ucife

rase

activ

ity

lowast lowast lowastlowast

lowastlowast

lowastlowastlowast

lowastlowast

49Luc Δ27Luc 3Luc 16Luc 09Luc 02Luc

TGF-120573 (minus)TGF-120573 (+)

(b)

00

02

04

06

08

10

12

ATBF1

Rela

tive l

ucife

rase

activ

ity

lowastlowastlowast

lowast

minusΔ27Luc

(c)

00

02

04

06

08

10

12

Relat

ive l

ucife

rase

activ

ity

ATBF1 minus + minus minus+minusminus

minusminus

minus

minus+

minus+minus

++

++

+minus

+ minus + minus minus+minusminus

minusminus

minus+

minus+minus

++

++

+minus

+Smad3Smad4

+

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowast

TGF-120573

(d)

Figure 2 TGF-120573Smad signaling inhibits the activity of the AFP promoter together with ATBF1 (a) Schematic presentation of deletionmutants of the AFP promoter 119864

119860between minus4120 and minus3756 and 119864

119861between minus3492 and minus3300 are enhancer elements 119878

119863between minus1807

and minus1725 and 119878119875between minus317 and minus300 are silencer elements P indicates a promoter (b) Effect of TGF-120573 on the activity of the AFP

promoterThe examined luciferase reporters (500 ng) were transfected into HepG2 cellsThe cells were then stimulated with 5 ngmL TGF-120573(c) Cooperation of ATBF1 with the TGF-120573 signaling for suppression of the activity of the AFP promoter ATBF1 expression plasmid (200 ng)was transfected into HepG2 cells with Δ27Luc (500 ng) The cells were then stimulated with 5 ngmL TGF-120573 (d) Effect of Smads and ATBF1on the transcriptional activity of Δ27Luc HepG2 cells were transfected with Smads (100 ng) ATBF1 (200 ng) or their combination togetherwith Δ27Luc (500 ng) The cells were then stimulated with 5 ngmL TGF-120573

Journal of Signal Transduction 5

TK promoter

Luc

Luc

Luc

Luc

Luc

EAminus4995 minus2953

minus4990

minus4990 minus4309

minus4363 minus3601

minus2968

minus2968minus3658

Δ27Luc+45minus178

fTKLuc

5998400TKLuc

mTKLuc

3998400TKLuc

EB

(a)

00

02

04

06

08

10

12

14

Relat

ive l

ucife

rase

activ

ity

lowast

lowast

fTKLuc 5998400TKLuc mTKLuc 3998400TKLuc

(b)

AT-motif AT-motif

EB

Luc

Luc

Luc

Luc

Luc

Luc

39984005Luc

399840051Luc

39984003Luc

02Luc

399840052Luc

39984001Luc+45minus178

+45minus178

+45minus178

+45minus178

+45minus178

+45minus178

minus3148

minus3148

minus3445

minus3422

minus3492 minus3300

minus3422

minus3658

minus3658 minus3563

minus3562

minus3658

(c)

00

05

10

15

20

25

30

35

40

Rela

tive l

ucife

rase

activ

ity

lowastlowastlowast

lowastlowastlowast

lowastlowast

39984005Luc 39984003Luc39984001Luc

(d)

00

02

04

06

08

10

12

14

Rela

tive l

ucife

rase

activ

ity

lowastlowastlowast

399840051Luc 02Luc399840052Luc

(e)

00

02

04

06

08

10

12

14

ATBF1

Rela

tive l

ucife

rase

activ

ity

lowast

lowast

399840052Luc

TGF-120573 (minus)TGF-120573 (+)

minus

(f)

Figure 3 Requirement of a distal AT-motif for TGF-120573-mediated repression of the activity of the AFP promoter (a) Schematic presentationof TKLuc conjugated with several fragments of the AFP promoter between minus4990 and minus2968 (b) Effect of TGF-120573 on the activity of TKLucreporters conjugated with several truncated AFP promoter regions Each reporter (200 ng) was transfected into HepG2 cells The cells werethen stimulated with 5 ngmL TGF-120573 (c) Schematic presentation of 02Luc conjugated with several fragments of the AFP promoter betweenminus3658 andminus3148 (d) Effect of TGF-120573 on the activity of the luciferase reporters described in Figure 3(c) Each reporter (200 ng)was transfectedinto HepG2 cellsThe cells were then stimulated with 5 ngmL TGF-120573 (e) Requirement of the fragment between minus3562 and minus3422 for TGF-120573-mediated suppression of theAFP promoter HepG2 cells were transfected with the indicated plasmids (200 ng)The cells were then stimulatedwith 5 ngmL TGF-120573 (f) Cooperation of ATBF1 with the TGF-120573 signaling for 3101584052Luc activity HepG2 cells were transfected with either352Luc reporter (200 ng) in the presence or absence of ATBF1 (200 ng) The cells were then stimulated with 5 ngmL TGF-120573

6 Journal of Signal Transduction

AT-motif AT-motif

AT-motifAT-motif

AT-motifCMV promoter

CMV promoter

65CMVLucAT-motif

CMV promoter

minus3562 minus3422

minus3562 minus3422 minus155 minus92

minus155 minus92

minus3562 minus3422

minus178Luc

Luc

Luc

Luc

+45

+1399840052Luc

399840052CMVLuc

399840052 65CMVLuc

(a)

00

02

04

06

08

10

12

Rela

tive l

ucife

rase

activ

ity

65CMVLuc399840052CMVLuc399840052 65CMVLuc

TGF-120573 (minus)TGF-120573 (+)

lowast

(b)

Figure 4 Importance of both distal and proximal AT-motifs for TGF-120573-mediated repression of theAFP promoter (a) Schematic presentationof CMVLuc conjugatedwith distal and proximal AT-motifs (b)HepG2 cells were transfectedwith each reporter (200 ng) and then stimulatedwith 5 ngmL TGF-120573

promoter (TKLuc) (Figure 3(a)) As shown in Figure 3(b)TGF-120573 suppressed the reporter activity of fTKLuc Thusthe region between minus4990 and minus2968 possesses an inhibitorycis-element(s) upon TGF-120573 stimulation Within this regionthe fragment between minus3658 and minus2968 (termed 31015840TKLuc)revealed a remarkably inhibitory cis-element by TGF-120573although TGF-120573-mediated repression of the reporter activitycould also be seen in mTKLuc Intriguingly 119864

119861contain-

ing the AT-motif is present between minus3492 and minus3300(Figure 3(c)) To further narrow down a TGF-120573-responsiveinhibitory cis-element(s) we made four luciferase reportersthat were linked to the AFPminimal promoter (Figure 3(c))TGF-120573 dramatically suppressed the luciferase activity of310158401Luc 310158405Luc and 3101584052Luc although 310158403Luc activity was alsoinhibited by TGF-120573 to some extent in spite of its low basalluciferase activity indicating that the region from minus3562 tominus3422 possesses a TGF-120573-mediated inhibitory cis-element ifnot more (Figures 3(d) and 3(e)) Thus an AT-motif andorits adjacent sequences in 119864

119861were highly suggestive of a TGF-

120573-mediated inhibitory cis-element In addition ATBF1 couldfurther inhibit the 3101584052Luc activity suppressed by TGF-120573(Figure 3(f)) Therefore it is possible that the TGF-120573Smadpathway interferes via the region between minus3562 and minus3422

34 A Role of the Proximal AT-Motif in the AFP PromoterSince there are two functional AT-motifs within the AFPpromoter between minus4995 and +45 (Figure 2(a)) it is possiblethat the proximal AT-motif also influences TGF-120573-mediatedinhibition of the activity of theAFP promoter To confirm thatthe proximal AT-motif can also contribute to TGF-120573-mediated repression of the activity of the AFP promoter the141nt-length fragment from minus3562 to minus3422 was ligated to the

64nt-length region between minus155 and minus92 of the AFP pro-moter Then this fragment was further placed at the 51015840portof CMV Luc (3101584052 65CMVLuc) because the CMV promotershows the high level of luciferase activity Figure 4(b) demon-strates that both the distal and the proximal AT-motifs areneeded for TGF-120573 to suppress the activity of the AFPpromoter

35 Association of Smad Proteins with ATBF1 Since ATBF1cooperates with Smad3 for inhibition of the activity of theAFP promoter we tried to determine whether ATBF1 couldinteract with R-Smads Expectedly ATBF1 could associatewith Smad2 and Smad3 although ALK5 activation was notinvolved in its interaction (Figure 5(a) and data not shown)In these experiments we could observe multiple bands cor-responding to ATBF1 These multiple bands might be due tocalpain-1-dependent proteolysis in COS7 cells [35] To iden-tify the domain(s) of ATBF1 that interact with Smads wedivided ATBF1 into three pieces (Figure 5(b)) ATBF1N aswell as ATBF1C had the ability to interact with Smad2 andSmad3 although ATBF1M no longer possessed the ability tobind to either Smad2 or Smad3 (Figures 5(c)ndash5(e)) It hasbeen reported that DNA-binding domains are present inATBF1M including zinc finger domains and homeodomains[12]When either ATBF1NorATBF1Cwas cotransfectedwithATBF1 TGF-120573-mediated inhibition of the activity of theAFPpromoter was improved owing to their competition withATBF1 for Smad binding (Figures 6(a) and 6(b)) On theother hand ATBF1M could inhibit the reporter activity as adominant negative formofATBF1 becauseATBF1Mpossessesthe ability to bind to AT-motif via its DNA binding domain(data not shown) These results indicate that the N-terminal

Journal of Signal Transduction 7

Flag-Smad3

Smad3

ATBF1

Smad3

ALK5ca

minus +minus

++ +

Myc-ATBF1ALK5caV5

IP MycIB flag

IB Myc

IB flag

IB V5

(a)

3703

ATBF1

1

ATBF1N

8951

ATBF1M

3293894

ATBF1C

37033290

Zinc fingerHomeodomain

(b)

HA-ATBF1NFlag-Smad2Flag-Smad3

IP flagIB HA

IB HA

IB flag

minus + minusminus

Smad2Smad3

ATBF1N

ATBF1N

+minus

(c)

HA-ATBF1MFlag-Smad2Flag-Smad3

minus +minus +minus

IP flagIB HA

IB HA

IB flag

minus

ATBF1M

ATBF1M

Smad2Smad3

(d)

HA-ATBF1CFlag-Smad2Flag-Smad3

minus +minus +minus

IP flagIB HA

IB HA

IB flag

minus

ATBF1C

ATBF1C

Smad2Smad3

(e)

Figure 5 Association of Smads with ATBF1 (a) Interaction of Smad3 with ATBF1 COS7 cells were transfected with Flag-Smad3 (1120583g) withMyc-ATBF1 (2120583g) in combination with ALK5caV5 (05 120583g) or without it Cell lysates were subjected to immunoprecipitation (IP) with anti-Myc antibody followed by IB with anti-Flag antibody to show the interaction of Flag-Smad3 with Myc-ATBF1 (b) Schematic presentation ofATBF1 mutants (cndashe) Interaction of each ATBF1 mutant with Smad2 or Smad3 COS7 cells were transfected with Flag-Smad2 or Flag-Smad3(15120583g) with HA-ATBF1N (2120583g) (c) HA-ATBF1M (2120583g) (d) or HA-ATBF1C (2120583g) (e) Cell lysates were subjected to IP with anti-Flagantibody followed by IB with anti-HA antibody to show the interaction of Smad2 and Smad3 with ATBF1 mutants

and C-terminal domains are needed for ATBF1 to interactwith R-Smads

36 Indirect Binding of Smads to the AFP Promoter via ATBF1We could find only one Smad binding element (SBE) [36 37]in 3101584052Luc (Figure 7(a))Thus we examinedwhether this SBEis necessary for TGF-120573-mediated repression of the AFP pro-moter For that purpose wemade one reporter construct thathad mutations within the SBE of 3101584052Luc (3101584052mSBELuc)

(Figure 7(a)) However 3101584052mSBELuc activity was still inhib-ited by TGF-120573 although its basal level became weaker thanthat of 3101584052Luc (Figure 7(b)) Thus the SBE in the AFP pro-moter is not essential for TGF-120573Smad signaling to repressthe activity of the AFP promoter Indeed a DNAP assayshowed that Smad3 can be detected in the presence of ATBF1using three repeats of an AT-motif as the probe (Figure 7(c))Thus Smad3 probably binds to the promoter region of theAFP gene indirectly through its interaction with ATBF1

8 Journal of Signal Transduction

00

02

04

06

08

10

12

14

16

ATBF1ATBF1N

Rela

tive l

ucife

rase

activ

ity

+ + ++minus

minus minus

TGF-120573 (minus)TGF-120573 (+)

lowast

lowast

lowastlowastlowast

(a)

00

02

04

06

08

10

12

14

ATBF1ATBF1C

Rela

tive l

ucife

rase

activ

ity

+ + ++minus

minus minus

TGF-120573 (minus)TGF-120573 (+)

lowast

lowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

(b)

Figure 6 ATBF1N and ATBF1C perturb ATBF1-mediated suppression of the activity of theAFP promoter upon TGF-120573 stimulation ATBF1N(a) and ATNF1C (b) rescue ATBF1-mediated repression of the activity of the AFP promoter HepG2 cells were transfected with increasedamounts of either ATBF1N (50 100 or 200 ng) (a) or ATBF1C (50 100 or 200 ng) (b) together with ATBF1 (200 ng) and Δ27Luc (200 ng)The cells were then stimulated with 5 ngmL TGF-120573

4 Discussion

The hepatoma cells HuH-7 and HepG2 cells are known tosecrete a large and a low amount of AFP respectively [38] Agrowing body of evidence indicates that the level of AFP inserum from patients is linked to HCC tumorigenicity [3] p53has been reported to cooperate with the TGF-120573Smad path-way to repress AFP expression via induction of SnoN [22]However it remains veiled whether ATBF1 which is knownto blockAFP transcription [4 10] can also regulate the activ-ity of the AFP promoter in consort with the TGF-120573Smadpathway because both ATBF1 and TGF-120573Smad might pos-sess an antioncogenic function [13ndash17 20 39 40] ATBF1could inhibit the activity of the AFP promoter via its regionsfrom minus4995 to minus2953 andor from minus178 to +45 [4 26] SinceΔ27Luc had a remarkably basal promoter activity comparedwith 02Luc (Figure 2(b)) we initially focused on the distalregion of the AFP promoter for involvement of the TGF-120573Smad signaling with ATBF1 As expected the TGF-120573-mediated repressive element in the AFP promoter lay near119864119861 which contains a distal AT-motif The luciferase reporter

conjugated with the inhibitory element including a distalAT-motif (352Luc) showed coordinated inhibitory actionbetween the TGF-120573 signaling and ATBF1 (Figure 3(f))Therefore we were struck with the notion that ATBF1physically interacts with Smads which play key roles in thecanonical TGF-120573 signaling pathway [18ndash20] Not surpris-ingly ATBF1 could interact with Smad2 and Smad3 althoughC-terminal phosphorylation of R-Smads was dispensable

(Figure 5(a) and data not shown) In addition both the N-terminal (ATBF1N) and the C-terminal mutants of ATBF1(ATBF1C) but not the middle domain of ATBF1 (ATBF1M)which possesses DNA-binding activity [12] could interactwith R-Smads (Figures 5(c)ndash5(e))We hypothesized that bothATBF1N and ATBF1C might compete with wildtype ATBF1for Smad binding to rescueATBF1-mediated repression of theAFP promoter uponTGF-120573 stimulation because both of themhave the ability to bind to R-Smads According to our expec-tation both ATBF1N and ATBF1C improved the reporteractivity decreased by the cooperative ATBF1 and TGF-120573signaling (Figures 6(a) and 6(b)) As shown in Figure 2(b)02Luc activity could also be reduced upon TGF-120573 stimu-lation despite very low basal promoter activity Because ofthe presence of a proximal AT-motif within this region wespeculated that a proximal AT-motif also contributes to TGF-120573-mediated-repression of the activity of the AFP promoterThe deletion of a proximal AT-motif from 3101584052 65CMVLucled us to recognize the importance of a proximal AT-motif aswell as a distal AT-motif for TGF-120573-mediated repression ofthe activity of theAFP promoter (Figure 4(b)) A Smad bind-ing element (SBE) composed of consensus 51015840-AGAC-31015840 isflanked to a proximal AT-motif However themutation of theSBE in 3101584052Luc showed reduced basal promoter activity butstill maintained TGF-120573-mediated repression of the AFP pro-moter (Figure 7(b)) Thus Smad might not be necessary tobind to a promoter element(s) in the AFP gene directly whenATBF1 cooperates with TGF-120573 signaling whereas p53-mediated repression of the AFP promoter activity needs

Journal of Signal Transduction 9

SBEminus3422

minus3422

minus3562

minus3562

LucAT-motif

minus178 +45

+1

LucAT-motif

AT-motif

AT-motifminus178 +45

+1

399840052Luc

399840052mSBELuc

(a)

00

02

04

06

08

10

12

Relat

ive l

ucife

rase

activ

ity

TGF-120573 (minus)TGF-120573 (+)

lowastlowast

lowastlowast

399840052Luc 399840052mSBELuc

(b)

DNAP

Flag-Smad3IB flag

IB HA

Totallysates

HA-ATBF1

HA-ATBF1

Flag-Smad3IB flag

IB HA

p-Smad3IB PS3

IB V5 ALK5caV5

Biotinylated probe minus + + +

Flag-Smad3HA-ATBF1

+ + + minusminus + +minus

ALK5caV5 + + + minus

(AT-motif)3

(c)

Figure 7 SBE in the proximal region of the AFP promoter is not essential for TGF-120573-mediated suppression of the AFP promoter (a)Schematic presentation of 3101584052mSBELuc A putative SBE 51015840-CAGATA-31015840 was mutated to 51015840-TTTTTT-31015840 (b) Effect of TGF-120573 on 3101584052Lucand 3101584052mSBELuc activities HepG2 cells were transfected with each reporter plasmid (200 ng) The cells were then stimulated with 5 ngmLTGF-120573 (c) Indirect binding of Smad3 to an AT-motif via ATBF1 The indicated cell lysates were mixed with biotinylated AT-motifs Thenstreptavidin-agarose was added to the mixture Subsequently a protein(s) bound to streptavidin-agarose was purified After SDS-PAGE IBwas carried out using anti-Flag or anti-HA antibody Using the total lysates each protein was detected with anti-Flag anti-HA anti-V5 oranti-phosphorylated Smad3 (PS3) antibody

direct binding of Smads to DNA [22 23] The requirement ofdirect DNA binding for R-Smads is probably dependent onwhich transcriptional regulator(s) they cooperate or syner-gize with Indeed Smad3 could bind toAT-motifs in the pres-ence of ATBF1 but not in its absence (Figure 7(c)) Althoughwe do not know if the ATBF1Smad complex at a distalAT-motif can physically interact with that at a proximal

AT-motif directly or indirectly through another protein(s)(ldquoXrdquo Figure 8) both AT-motifs are definitely important forcooperation between ATBF1 and the TGF-120573 signaling tosuppress the activity of the AFP promoter

ATBF-1 is known to compete with HNF-1 for binding toAT-motifs in the AFP promoter [4] Thus the following pos-sibility can be speculated upon TGF-120573 stimulation Smads

10 Journal of Signal Transduction

AT-motif

AT-motif

AFP

ATBF1

ATBF1ATBF1Smad23-Smad4

X

Smad23-Smad4

ATBF1

Figure 8 Proposed inhibitory mechanism by which the TGF-120573Smad signaling and ATBF1 cooperatively regulate the activity ofthe AFP promoter The distal and proximal AT-motifs in the AFPpromoter are bound by ATBF1 Upon TGF-120573 signaling the R-SmadsSmad4 complex interacts with ATBF1 to make a repressivecomplex for the activity of the AFP promoter However whetheranother protein(s) (termed ldquoXrdquo) is essential for the R-SmadSmad4ATBF1 complex remains veiled

can not only bind to AT-motifs via ATBF-1 indirectly but alsointeract with HNF-1 to dissociate it from AT-motifs Due todual function of Smads the AFP prompter activity might beinhibited This possibility is interesting to be investigated

p53-mediated repression of the AFP promoter activityneeds direct binding of Smads to SBEp53REWilkinson et alfurther demonstrated that mSin3A and SnoN which is adirect target gene of TGF-120573 signal are recruited by p53Smadscomplex when the transcription of the AFP gene is inhib-ited [22 23] In our current study the region includingSBEp53RE in theAFP promoterwas not investigated becausethe basal reporter activities of 3Luc 16Luc and 09Luc werevery low compared with that of Δ27Luc (Figure 2(b)) Itis possible that ATBF1 might interplay with p53 via Smadcomplex to effectively repress the transcription of the AFPgene Further experiments are needed in the future to confirmthese hypotheses as well

5 Conclusion

In summary we report a novel interaction between ATBF1and Smads The interaction between ATBF1 and Smadsappears to cooperatively inhibit the transcription of AFPgene upon TGF-120573 signaling In particular both proximal anddistal AT-motifs are required for TGF-120573Smad signaling tocounteract with the transcription of the AFP gene

Conflict of Interests

The authors have no competing financial interests to declare

Acknowledgments

This research was supported by a Grant-in-Aid for Explor-atory Research (A) from the Japan Society for the Promotionof Science (JSPS) (to Nobuo Sakata) the MEXT-Supported

Program for the Strategic Research Foundation at PrivateUniversities (2013ndash2017) (to Susumu Itoh) the Takeda Sci-ence Foundation (to Susumu Itoh) the Smoking ResearchFoundation (to Susumu Itoh) the Daiichi-Sankyo Founda-tion of Life Science (to Susumu Itoh) the Naito Founda-tion (to Susumu Itoh) the Vehicle Racing Commemora-tive Foundation (to Susumu Itoh) and a Grant-in-Aid forYoung Scientists of Showa Pharmaceutical University (toSouichi Ikeno) The authors were also supported by the JointUsageResearch Program of the Medical Research Institutethe Tokyo Medical and Dental University and by the Core-to-Core Program ldquoCooperative International Framework inTGF-120573 Family Signalingrdquo of the JSPS They thank Ms FMiyamasu for excellent English proofreading

References

[1] M A Dziadek and G K Andrews ldquoTissue specificity of 120572-fetoprotein messenger RNA expression during mouse embryo-genesisrdquoThe EMBO Journal vol 2 no 4 pp 549ndash554 1983

[2] J L Nahon I Tratner A Poliard et al ldquoAlbumin and 120572-feto-protein gene expression in various nonhepatic rat tissuesrdquo TheJournal of Biological Chemistry vol 263 no 23 pp 11436ndash114421988

[3] A A Terentiev and N T Moldogazieva ldquo120572-fetoprotein a ren-aissancerdquo Tumor Biology vol 34 no 4 pp 2075ndash2091 2013

[4] H Yasuda A Mizuno T Tamaoki and T Morinaga ldquoATBF1 amultiple-homeodomain zinc finger protein selectively down-regulates AT-rich elements of the human 120572-fetoprotein generdquoMolecular and Cellular Biology vol 14 no 2 pp 1395ndash14011994

[5] K Sawadaishi T Morinaga and T Tamaoki ldquoInteraction of ahepatoma-specific nuclear factor with transcription-regulatorysequences of the human 120572-fetoprotein and albumin genesrdquoMolecular andCellular Biology vol 8 no 12 pp 5179ndash5187 1988

[6] H Nakabayashi Y Koyama H Suzuki et al ldquoFunctional map-ping of tissue-specific elements of the human 120572-fetoproteingene enhancerrdquo Biochemical and Biophysical Research Commu-nications vol 318 no 3 pp 773ndash785 2004

[7] T Morinaga H Yasuda T Hashimoto K Higashio and TTamaoki ldquoA human 120572-fetoprotein enhancer-binding proteinATBF1 contains four homeodomains and seventeen zinc fin-gersrdquo Molecular and Cellular Biology vol 11 no 12 pp 6041ndash6049 1991

[8] Y Miura T Tam A Ido et al ldquoCloning and characterization ofan ATBF1 isoform that expresses in a neuronal differentiation-dependent mannerrdquo The Journal of Biological Chemistry vol270 no 45 pp 26840ndash26848 1995

[9] H Kataoka Y Miura T Joh et al ldquo120572-fetoprotein producinggastric cancer lacks transcription factor ATBF1rdquo Oncogene vol20 no 7 pp 869ndash873 2001

[10] T Ninomiya K Mihara K Fushimi Y Hayashi T Hashimoto-Tamaoki and T Tamaoki ldquoRegulation of the 120572-fetoprotein geneby the isoforms of ATBF1 transcription factor in human hep-atomardquo Hepatology vol 35 no 1 pp 82ndash87 2002

[11] P Kaspar M Dvorakova J Kralova P Pajer Z Kozmik andM Dvorak ldquoMyb-interacting protein ATBF1 represses tran-scriptional activity of Myb oncoproteinrdquoThe Journal of Biologi-cal Chemistry vol 274 no 20 pp 14422ndash14428 1999

[12] F B Berry YMiura KMihara et al ldquoPositive and negative reg-ulation of myogenic differentiation of C2C12 cells by isoforms

Journal of Signal Transduction 11

of the multiple homeodomain zinc finger transcription factorATBF1rdquoThe Journal of Biological Chemistry vol 276 no 27 pp25057ndash25065 2001

[13] X Sun H F Frierson C Chen et al ldquoFrequent somaticmutations of the transcription factor ATBF1 in human prostatecancerrdquo Nature Genetics vol 37 no 6 pp 407ndash412 2005

[14] X Sun Y Zhou K B Otto et al ldquoInfrequentmutation of ATBF1in human breast cancerrdquo Journal of Cancer Research and ClinicalOncology vol 133 no 2 pp 103ndash105 2007

[15] Y G Cho J H Song C J Kim et al ldquoGenetic alterations of theATBF1 gene in gastric cancerrdquo Clinical Cancer Research vol 13no 15 pp 4355ndash4359 2007

[16] K Kai Z Zhang H Yamashita Y Yamamoto Y Miura and HIwase ldquoLoss of heterozygosity at the ATBF1-A locus located inthe 16q22 minimal region in breast cancerrdquo BMC Cancer vol 8article 262 2008

[17] A M Cleton-Jansen R van Eijk M Lombaerts et al ldquoATBF1and NQO1 as candidate targets for allelic loss at chromosomearm 16q in breast cancer absence of somatic ATBF1 mutationsand no role for the C609TNQO1 polymorphismrdquo BMCCancervol 8 article 105 2008

[18] J Massague J Seoane and D Wotton ldquoSmad transcriptionfactorsrdquo Genes amp Development vol 19 no 23 pp 2783ndash28102005

[19] AMoustakas and C H Heldin ldquoThe regulation of TGF120573 signaltransductionrdquo Development vol 136 no 22 pp 3699ndash37142009

[20] J Massague ldquoTGF120573 signalling in contextrdquo Nature ReviewsMolecular Cell Biology vol 13 no 10 pp 616ndash630 2012

[21] K Nakao K Nakata S Mitsuoka et al ldquoTransforming growthfactor 120573 1 differentially regulates 120572-fetoprotein and albumin inHuH-7 human hepatoma cellsrdquo Biochemical and BiophysicalResearch Communications vol 174 no 3 pp 1294ndash1299 1991

[22] D SWilkinson S K Ogden S A Stratton et al ldquoA direct inter-section between p53 and transforming growth factor 120573 path-ways targets chromatin modification and transcription repres-sion of the 120572-fetoprotein generdquoMolecular and Cellular Biologyvol 25 no 3 pp 1200ndash1212 2005

[23] D S Wilkinson W W Tsai M A Schumacher and M CBarton ldquoChromatin-bound p53 anchors activated Smads andthe mSin3A corepressor to confer transforming-growth-factor-120573-mediated transcription repressionrdquo Molecular and CellularBiology vol 28 no 6 pp 1988ndash1998 2008

[24] C G Jung H J Kim M Kawaguchi et al ldquoHomeotic factorATBF1 induces the cell cycle arrest associated with neuronaldifferentiationrdquo Development vol 132 no 23 pp 5137ndash51452005

[25] X Y Dong X Sun P Guo et al ldquoATBF1 inhibits estrogenreceptor (ER) function by selectively competing with AIB1 forbinding to the ER in ER-positive breast cancer cellsrdquoThe Journalof Biological Chemistry vol 285 no 43 pp 32801ndash32809 2010

[26] H Kataoka P Bonnefin D Vieyra et al ldquoING1 repressestranscription by direct DNA binding and through effects onp53rdquo Cancer Research vol 63 no 18 pp 5785ndash5792 2003

[27] A Nakao T Imamura S Souchelnytskyi et al ldquoTGF-120573receptor-mediated signalling through Smad2 Smad3 andSmad4rdquoThe EMBO Journal vol 16 no 17 pp 5353ndash5362 1997

[28] M Fujii K Takeda T Imamura et al ldquoRoles of bone morpho-genetic protein type I receptors and Smad proteins in osteoblastand chondroblast differentiationrdquoMolecular Biology of the Cellvol 10 no 11 pp 3801ndash3812 1999

[29] YWatanabe S Itoh T Goto et al ldquoTMEPAI a transmembraneTGF-120573-inducible protein sequesters smad proteins from activeparticipation in TGF-120573 signalingrdquoMolecular Cell vol 37 no 1pp 123ndash134 2010

[30] S Itoh M Thorikay M Kowanetz et al ldquoElucidation of Smadrequirement in transforming growth factor-120573 type I receptor-induced responsesrdquo The Journal of Biological Chemistry vol278 no 6 pp 3751ndash3761 2003

[31] N Nakano S Itoh Y Watanabe K Maeyama F Itoh and MKato ldquoRequirement of TCF7L2 for TGF-120573 dependent transcrip-tional activation of the TMEPAI generdquoThe Journal of BiologicalChemistry vol 285 no 49 pp 38023ndash38033 2010

[32] K Watanabe A Saito and T Tamaoki ldquoCell-specific enhanceractivity in a far upstream region of the human 120572-fetoproteingenerdquo The Journal of Biological Chemistry vol 262 no 10 pp4812ndash4818 1987

[33] H Nakabayashi K Watanabe A Saito A Otsuru KSawadaishi and T Tamaoki ldquoTranscriptional regulation of 120573-fetoprotein expression by dexamethasone in human hepatomacellsrdquo The Journal of Biological Chemistry vol 264 no 1 pp266ndash271 1989

[34] H Nakabayashi T Hashimoto Y Miyao K-K Tjong J Chanand T Tamaoki ldquoA position-dependent silencer plays a majorrole in repressing 120573-fetoprotein expression in human hep-atomardquoMolecular and Cellular Biology vol 11 no 12 pp 5885ndash5893 1991

[35] S Zhang T S Kim Y Dong et al ldquoAT motif binding factor 1(ATBF1) is highly phosphorylated in embryonic brain and pro-tected from cleavage by calpain-1rdquo Biochemical and BiophysicalResearch Communications vol 427 no 3 pp 537ndash541 2012

[36] L J Jonk S Itoh C H Heldin P ten Dijke and W KruijerldquoIdentification and functional characterization of a smad bind-ing element (SBE) in the JunBpromoter that acts as a transform-ing growth factor-120573 activin and bone morphogenetic protein-inducible enhancerrdquo The Journal of Biological Chemistry vol273 no 33 pp 21145ndash21152 1998

[37] S Dennler S Itoh D Vivien P ten Dijke S Huet and J Gau-thier ldquoDirect binding of Smad3 and Smad4 to critical TGF120573-inducible elements in the promoter of human plasminogenactivator inhibitor-type 1 generdquo The EMBO Journal vol 17 no11 pp 3091ndash3100 1998

[38] A Ido K Nakata Y Kato et al ldquoGene therapy for hep-atoma cells using a retrovirus vector carrying herpes simplexvirus thymidine kinase gene under the control of human 120572-fetoprotein gene promoterrdquo Cancer Research vol 55 no 14 pp3105ndash3109 1995

[39] A B Roberts and L M Wakefield ldquoThe two faces of trans-forming growth factor 120573 in carcinogenesisrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 15 pp 8621ndash8623 2003

[40] B Bierie and H L Moses ldquoTumour microenvironment TGF120573the molecular Jekyll and Hyde of cancerrdquo Nature ReviewsCancer vol 6 no 7 pp 506ndash520 2006

4 Journal of Signal Transduction

Table 1 PCR primers to amplify human cDNAs

Human cDNA Sequence 119871 AT Cycle

AFP (+) ATCCAGGAGAGCCAAGCATT 434 58∘C 20(minus) TTCATCGTTTGCAGCGCTAC

ATBF1 (+) TGGCATCAAGTACAGCGCTC 392 53∘C 35(minus) GAACAGTTGTGCTGGGCAGA

TMEPAI (+) GATCATCATCATCGTGGTGG 455 60∘C 35(minus) CACTGTCGAAGATGGTTCTG

120573-actin (+) CACCCACACTGTGCCCATCTACGA 410 63∘C 22(minus) TGGCGTACAGGTCTTTGCGGATGT

119871 length of PCR fragment AT annealing temperature

AFP

Luc

Luc

Luc

Luc

Luc

Luc

minus4995

minus4995

EA EB SD SP P

ATBF1 ATBF1

49Luc

Δ27Luc

3Luc

16Luc

09Luc

02Luc+45

+45

+45

+45

+45

+45

minus178

minus178

minus2953

minus2953

minus1653

minus871

(a)

00

10

20

30

40

50

60

Relat

ive l

ucife

rase

activ

ity

lowast lowast lowastlowast

lowastlowast

lowastlowastlowast

lowastlowast

49Luc Δ27Luc 3Luc 16Luc 09Luc 02Luc

TGF-120573 (minus)TGF-120573 (+)

(b)

00

02

04

06

08

10

12

ATBF1

Rela

tive l

ucife

rase

activ

ity

lowastlowastlowast

lowast

minusΔ27Luc

(c)

00

02

04

06

08

10

12

Relat

ive l

ucife

rase

activ

ity

ATBF1 minus + minus minus+minusminus

minusminus

minus

minus+

minus+minus

++

++

+minus

+ minus + minus minus+minusminus

minusminus

minus+

minus+minus

++

++

+minus

+Smad3Smad4

+

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowast

TGF-120573

(d)

Figure 2 TGF-120573Smad signaling inhibits the activity of the AFP promoter together with ATBF1 (a) Schematic presentation of deletionmutants of the AFP promoter 119864

119860between minus4120 and minus3756 and 119864

119861between minus3492 and minus3300 are enhancer elements 119878

119863between minus1807

and minus1725 and 119878119875between minus317 and minus300 are silencer elements P indicates a promoter (b) Effect of TGF-120573 on the activity of the AFP

promoterThe examined luciferase reporters (500 ng) were transfected into HepG2 cellsThe cells were then stimulated with 5 ngmL TGF-120573(c) Cooperation of ATBF1 with the TGF-120573 signaling for suppression of the activity of the AFP promoter ATBF1 expression plasmid (200 ng)was transfected into HepG2 cells with Δ27Luc (500 ng) The cells were then stimulated with 5 ngmL TGF-120573 (d) Effect of Smads and ATBF1on the transcriptional activity of Δ27Luc HepG2 cells were transfected with Smads (100 ng) ATBF1 (200 ng) or their combination togetherwith Δ27Luc (500 ng) The cells were then stimulated with 5 ngmL TGF-120573

Journal of Signal Transduction 5

TK promoter

Luc

Luc

Luc

Luc

Luc

EAminus4995 minus2953

minus4990

minus4990 minus4309

minus4363 minus3601

minus2968

minus2968minus3658

Δ27Luc+45minus178

fTKLuc

5998400TKLuc

mTKLuc

3998400TKLuc

EB

(a)

00

02

04

06

08

10

12

14

Relat

ive l

ucife

rase

activ

ity

lowast

lowast

fTKLuc 5998400TKLuc mTKLuc 3998400TKLuc

(b)

AT-motif AT-motif

EB

Luc

Luc

Luc

Luc

Luc

Luc

39984005Luc

399840051Luc

39984003Luc

02Luc

399840052Luc

39984001Luc+45minus178

+45minus178

+45minus178

+45minus178

+45minus178

+45minus178

minus3148

minus3148

minus3445

minus3422

minus3492 minus3300

minus3422

minus3658

minus3658 minus3563

minus3562

minus3658

(c)

00

05

10

15

20

25

30

35

40

Rela

tive l

ucife

rase

activ

ity

lowastlowastlowast

lowastlowastlowast

lowastlowast

39984005Luc 39984003Luc39984001Luc

(d)

00

02

04

06

08

10

12

14

Rela

tive l

ucife

rase

activ

ity

lowastlowastlowast

399840051Luc 02Luc399840052Luc

(e)

00

02

04

06

08

10

12

14

ATBF1

Rela

tive l

ucife

rase

activ

ity

lowast

lowast

399840052Luc

TGF-120573 (minus)TGF-120573 (+)

minus

(f)

Figure 3 Requirement of a distal AT-motif for TGF-120573-mediated repression of the activity of the AFP promoter (a) Schematic presentationof TKLuc conjugated with several fragments of the AFP promoter between minus4990 and minus2968 (b) Effect of TGF-120573 on the activity of TKLucreporters conjugated with several truncated AFP promoter regions Each reporter (200 ng) was transfected into HepG2 cells The cells werethen stimulated with 5 ngmL TGF-120573 (c) Schematic presentation of 02Luc conjugated with several fragments of the AFP promoter betweenminus3658 andminus3148 (d) Effect of TGF-120573 on the activity of the luciferase reporters described in Figure 3(c) Each reporter (200 ng)was transfectedinto HepG2 cellsThe cells were then stimulated with 5 ngmL TGF-120573 (e) Requirement of the fragment between minus3562 and minus3422 for TGF-120573-mediated suppression of theAFP promoter HepG2 cells were transfected with the indicated plasmids (200 ng)The cells were then stimulatedwith 5 ngmL TGF-120573 (f) Cooperation of ATBF1 with the TGF-120573 signaling for 3101584052Luc activity HepG2 cells were transfected with either352Luc reporter (200 ng) in the presence or absence of ATBF1 (200 ng) The cells were then stimulated with 5 ngmL TGF-120573

6 Journal of Signal Transduction

AT-motif AT-motif

AT-motifAT-motif

AT-motifCMV promoter

CMV promoter

65CMVLucAT-motif

CMV promoter

minus3562 minus3422

minus3562 minus3422 minus155 minus92

minus155 minus92

minus3562 minus3422

minus178Luc

Luc

Luc

Luc

+45

+1399840052Luc

399840052CMVLuc

399840052 65CMVLuc

(a)

00

02

04

06

08

10

12

Rela

tive l

ucife

rase

activ

ity

65CMVLuc399840052CMVLuc399840052 65CMVLuc

TGF-120573 (minus)TGF-120573 (+)

lowast

(b)

Figure 4 Importance of both distal and proximal AT-motifs for TGF-120573-mediated repression of theAFP promoter (a) Schematic presentationof CMVLuc conjugatedwith distal and proximal AT-motifs (b)HepG2 cells were transfectedwith each reporter (200 ng) and then stimulatedwith 5 ngmL TGF-120573

promoter (TKLuc) (Figure 3(a)) As shown in Figure 3(b)TGF-120573 suppressed the reporter activity of fTKLuc Thusthe region between minus4990 and minus2968 possesses an inhibitorycis-element(s) upon TGF-120573 stimulation Within this regionthe fragment between minus3658 and minus2968 (termed 31015840TKLuc)revealed a remarkably inhibitory cis-element by TGF-120573although TGF-120573-mediated repression of the reporter activitycould also be seen in mTKLuc Intriguingly 119864

119861contain-

ing the AT-motif is present between minus3492 and minus3300(Figure 3(c)) To further narrow down a TGF-120573-responsiveinhibitory cis-element(s) we made four luciferase reportersthat were linked to the AFPminimal promoter (Figure 3(c))TGF-120573 dramatically suppressed the luciferase activity of310158401Luc 310158405Luc and 3101584052Luc although 310158403Luc activity was alsoinhibited by TGF-120573 to some extent in spite of its low basalluciferase activity indicating that the region from minus3562 tominus3422 possesses a TGF-120573-mediated inhibitory cis-element ifnot more (Figures 3(d) and 3(e)) Thus an AT-motif andorits adjacent sequences in 119864

119861were highly suggestive of a TGF-

120573-mediated inhibitory cis-element In addition ATBF1 couldfurther inhibit the 3101584052Luc activity suppressed by TGF-120573(Figure 3(f)) Therefore it is possible that the TGF-120573Smadpathway interferes via the region between minus3562 and minus3422

34 A Role of the Proximal AT-Motif in the AFP PromoterSince there are two functional AT-motifs within the AFPpromoter between minus4995 and +45 (Figure 2(a)) it is possiblethat the proximal AT-motif also influences TGF-120573-mediatedinhibition of the activity of theAFP promoter To confirm thatthe proximal AT-motif can also contribute to TGF-120573-mediated repression of the activity of the AFP promoter the141nt-length fragment from minus3562 to minus3422 was ligated to the

64nt-length region between minus155 and minus92 of the AFP pro-moter Then this fragment was further placed at the 51015840portof CMV Luc (3101584052 65CMVLuc) because the CMV promotershows the high level of luciferase activity Figure 4(b) demon-strates that both the distal and the proximal AT-motifs areneeded for TGF-120573 to suppress the activity of the AFPpromoter

35 Association of Smad Proteins with ATBF1 Since ATBF1cooperates with Smad3 for inhibition of the activity of theAFP promoter we tried to determine whether ATBF1 couldinteract with R-Smads Expectedly ATBF1 could associatewith Smad2 and Smad3 although ALK5 activation was notinvolved in its interaction (Figure 5(a) and data not shown)In these experiments we could observe multiple bands cor-responding to ATBF1 These multiple bands might be due tocalpain-1-dependent proteolysis in COS7 cells [35] To iden-tify the domain(s) of ATBF1 that interact with Smads wedivided ATBF1 into three pieces (Figure 5(b)) ATBF1N aswell as ATBF1C had the ability to interact with Smad2 andSmad3 although ATBF1M no longer possessed the ability tobind to either Smad2 or Smad3 (Figures 5(c)ndash5(e)) It hasbeen reported that DNA-binding domains are present inATBF1M including zinc finger domains and homeodomains[12]When either ATBF1NorATBF1Cwas cotransfectedwithATBF1 TGF-120573-mediated inhibition of the activity of theAFPpromoter was improved owing to their competition withATBF1 for Smad binding (Figures 6(a) and 6(b)) On theother hand ATBF1M could inhibit the reporter activity as adominant negative formofATBF1 becauseATBF1Mpossessesthe ability to bind to AT-motif via its DNA binding domain(data not shown) These results indicate that the N-terminal

Journal of Signal Transduction 7

Flag-Smad3

Smad3

ATBF1

Smad3

ALK5ca

minus +minus

++ +

Myc-ATBF1ALK5caV5

IP MycIB flag

IB Myc

IB flag

IB V5

(a)

3703

ATBF1

1

ATBF1N

8951

ATBF1M

3293894

ATBF1C

37033290

Zinc fingerHomeodomain

(b)

HA-ATBF1NFlag-Smad2Flag-Smad3

IP flagIB HA

IB HA

IB flag

minus + minusminus

Smad2Smad3

ATBF1N

ATBF1N

+minus

(c)

HA-ATBF1MFlag-Smad2Flag-Smad3

minus +minus +minus

IP flagIB HA

IB HA

IB flag

minus

ATBF1M

ATBF1M

Smad2Smad3

(d)

HA-ATBF1CFlag-Smad2Flag-Smad3

minus +minus +minus

IP flagIB HA

IB HA

IB flag

minus

ATBF1C

ATBF1C

Smad2Smad3

(e)

Figure 5 Association of Smads with ATBF1 (a) Interaction of Smad3 with ATBF1 COS7 cells were transfected with Flag-Smad3 (1120583g) withMyc-ATBF1 (2120583g) in combination with ALK5caV5 (05 120583g) or without it Cell lysates were subjected to immunoprecipitation (IP) with anti-Myc antibody followed by IB with anti-Flag antibody to show the interaction of Flag-Smad3 with Myc-ATBF1 (b) Schematic presentation ofATBF1 mutants (cndashe) Interaction of each ATBF1 mutant with Smad2 or Smad3 COS7 cells were transfected with Flag-Smad2 or Flag-Smad3(15120583g) with HA-ATBF1N (2120583g) (c) HA-ATBF1M (2120583g) (d) or HA-ATBF1C (2120583g) (e) Cell lysates were subjected to IP with anti-Flagantibody followed by IB with anti-HA antibody to show the interaction of Smad2 and Smad3 with ATBF1 mutants

and C-terminal domains are needed for ATBF1 to interactwith R-Smads

36 Indirect Binding of Smads to the AFP Promoter via ATBF1We could find only one Smad binding element (SBE) [36 37]in 3101584052Luc (Figure 7(a))Thus we examinedwhether this SBEis necessary for TGF-120573-mediated repression of the AFP pro-moter For that purpose wemade one reporter construct thathad mutations within the SBE of 3101584052Luc (3101584052mSBELuc)

(Figure 7(a)) However 3101584052mSBELuc activity was still inhib-ited by TGF-120573 although its basal level became weaker thanthat of 3101584052Luc (Figure 7(b)) Thus the SBE in the AFP pro-moter is not essential for TGF-120573Smad signaling to repressthe activity of the AFP promoter Indeed a DNAP assayshowed that Smad3 can be detected in the presence of ATBF1using three repeats of an AT-motif as the probe (Figure 7(c))Thus Smad3 probably binds to the promoter region of theAFP gene indirectly through its interaction with ATBF1

8 Journal of Signal Transduction

00

02

04

06

08

10

12

14

16

ATBF1ATBF1N

Rela

tive l

ucife

rase

activ

ity

+ + ++minus

minus minus

TGF-120573 (minus)TGF-120573 (+)

lowast

lowast

lowastlowastlowast

(a)

00

02

04

06

08

10

12

14

ATBF1ATBF1C

Rela

tive l

ucife

rase

activ

ity

+ + ++minus

minus minus

TGF-120573 (minus)TGF-120573 (+)

lowast

lowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

(b)

Figure 6 ATBF1N and ATBF1C perturb ATBF1-mediated suppression of the activity of theAFP promoter upon TGF-120573 stimulation ATBF1N(a) and ATNF1C (b) rescue ATBF1-mediated repression of the activity of the AFP promoter HepG2 cells were transfected with increasedamounts of either ATBF1N (50 100 or 200 ng) (a) or ATBF1C (50 100 or 200 ng) (b) together with ATBF1 (200 ng) and Δ27Luc (200 ng)The cells were then stimulated with 5 ngmL TGF-120573

4 Discussion

The hepatoma cells HuH-7 and HepG2 cells are known tosecrete a large and a low amount of AFP respectively [38] Agrowing body of evidence indicates that the level of AFP inserum from patients is linked to HCC tumorigenicity [3] p53has been reported to cooperate with the TGF-120573Smad path-way to repress AFP expression via induction of SnoN [22]However it remains veiled whether ATBF1 which is knownto blockAFP transcription [4 10] can also regulate the activ-ity of the AFP promoter in consort with the TGF-120573Smadpathway because both ATBF1 and TGF-120573Smad might pos-sess an antioncogenic function [13ndash17 20 39 40] ATBF1could inhibit the activity of the AFP promoter via its regionsfrom minus4995 to minus2953 andor from minus178 to +45 [4 26] SinceΔ27Luc had a remarkably basal promoter activity comparedwith 02Luc (Figure 2(b)) we initially focused on the distalregion of the AFP promoter for involvement of the TGF-120573Smad signaling with ATBF1 As expected the TGF-120573-mediated repressive element in the AFP promoter lay near119864119861 which contains a distal AT-motif The luciferase reporter

conjugated with the inhibitory element including a distalAT-motif (352Luc) showed coordinated inhibitory actionbetween the TGF-120573 signaling and ATBF1 (Figure 3(f))Therefore we were struck with the notion that ATBF1physically interacts with Smads which play key roles in thecanonical TGF-120573 signaling pathway [18ndash20] Not surpris-ingly ATBF1 could interact with Smad2 and Smad3 althoughC-terminal phosphorylation of R-Smads was dispensable

(Figure 5(a) and data not shown) In addition both the N-terminal (ATBF1N) and the C-terminal mutants of ATBF1(ATBF1C) but not the middle domain of ATBF1 (ATBF1M)which possesses DNA-binding activity [12] could interactwith R-Smads (Figures 5(c)ndash5(e))We hypothesized that bothATBF1N and ATBF1C might compete with wildtype ATBF1for Smad binding to rescueATBF1-mediated repression of theAFP promoter uponTGF-120573 stimulation because both of themhave the ability to bind to R-Smads According to our expec-tation both ATBF1N and ATBF1C improved the reporteractivity decreased by the cooperative ATBF1 and TGF-120573signaling (Figures 6(a) and 6(b)) As shown in Figure 2(b)02Luc activity could also be reduced upon TGF-120573 stimu-lation despite very low basal promoter activity Because ofthe presence of a proximal AT-motif within this region wespeculated that a proximal AT-motif also contributes to TGF-120573-mediated-repression of the activity of the AFP promoterThe deletion of a proximal AT-motif from 3101584052 65CMVLucled us to recognize the importance of a proximal AT-motif aswell as a distal AT-motif for TGF-120573-mediated repression ofthe activity of theAFP promoter (Figure 4(b)) A Smad bind-ing element (SBE) composed of consensus 51015840-AGAC-31015840 isflanked to a proximal AT-motif However themutation of theSBE in 3101584052Luc showed reduced basal promoter activity butstill maintained TGF-120573-mediated repression of the AFP pro-moter (Figure 7(b)) Thus Smad might not be necessary tobind to a promoter element(s) in the AFP gene directly whenATBF1 cooperates with TGF-120573 signaling whereas p53-mediated repression of the AFP promoter activity needs

Journal of Signal Transduction 9

SBEminus3422

minus3422

minus3562

minus3562

LucAT-motif

minus178 +45

+1

LucAT-motif

AT-motif

AT-motifminus178 +45

+1

399840052Luc

399840052mSBELuc

(a)

00

02

04

06

08

10

12

Relat

ive l

ucife

rase

activ

ity

TGF-120573 (minus)TGF-120573 (+)

lowastlowast

lowastlowast

399840052Luc 399840052mSBELuc

(b)

DNAP

Flag-Smad3IB flag

IB HA

Totallysates

HA-ATBF1

HA-ATBF1

Flag-Smad3IB flag

IB HA

p-Smad3IB PS3

IB V5 ALK5caV5

Biotinylated probe minus + + +

Flag-Smad3HA-ATBF1

+ + + minusminus + +minus

ALK5caV5 + + + minus

(AT-motif)3

(c)

Figure 7 SBE in the proximal region of the AFP promoter is not essential for TGF-120573-mediated suppression of the AFP promoter (a)Schematic presentation of 3101584052mSBELuc A putative SBE 51015840-CAGATA-31015840 was mutated to 51015840-TTTTTT-31015840 (b) Effect of TGF-120573 on 3101584052Lucand 3101584052mSBELuc activities HepG2 cells were transfected with each reporter plasmid (200 ng) The cells were then stimulated with 5 ngmLTGF-120573 (c) Indirect binding of Smad3 to an AT-motif via ATBF1 The indicated cell lysates were mixed with biotinylated AT-motifs Thenstreptavidin-agarose was added to the mixture Subsequently a protein(s) bound to streptavidin-agarose was purified After SDS-PAGE IBwas carried out using anti-Flag or anti-HA antibody Using the total lysates each protein was detected with anti-Flag anti-HA anti-V5 oranti-phosphorylated Smad3 (PS3) antibody

direct binding of Smads to DNA [22 23] The requirement ofdirect DNA binding for R-Smads is probably dependent onwhich transcriptional regulator(s) they cooperate or syner-gize with Indeed Smad3 could bind toAT-motifs in the pres-ence of ATBF1 but not in its absence (Figure 7(c)) Althoughwe do not know if the ATBF1Smad complex at a distalAT-motif can physically interact with that at a proximal

AT-motif directly or indirectly through another protein(s)(ldquoXrdquo Figure 8) both AT-motifs are definitely important forcooperation between ATBF1 and the TGF-120573 signaling tosuppress the activity of the AFP promoter

ATBF-1 is known to compete with HNF-1 for binding toAT-motifs in the AFP promoter [4] Thus the following pos-sibility can be speculated upon TGF-120573 stimulation Smads

10 Journal of Signal Transduction

AT-motif

AT-motif

AFP

ATBF1

ATBF1ATBF1Smad23-Smad4

X

Smad23-Smad4

ATBF1

Figure 8 Proposed inhibitory mechanism by which the TGF-120573Smad signaling and ATBF1 cooperatively regulate the activity ofthe AFP promoter The distal and proximal AT-motifs in the AFPpromoter are bound by ATBF1 Upon TGF-120573 signaling the R-SmadsSmad4 complex interacts with ATBF1 to make a repressivecomplex for the activity of the AFP promoter However whetheranother protein(s) (termed ldquoXrdquo) is essential for the R-SmadSmad4ATBF1 complex remains veiled

can not only bind to AT-motifs via ATBF-1 indirectly but alsointeract with HNF-1 to dissociate it from AT-motifs Due todual function of Smads the AFP prompter activity might beinhibited This possibility is interesting to be investigated

p53-mediated repression of the AFP promoter activityneeds direct binding of Smads to SBEp53REWilkinson et alfurther demonstrated that mSin3A and SnoN which is adirect target gene of TGF-120573 signal are recruited by p53Smadscomplex when the transcription of the AFP gene is inhib-ited [22 23] In our current study the region includingSBEp53RE in theAFP promoterwas not investigated becausethe basal reporter activities of 3Luc 16Luc and 09Luc werevery low compared with that of Δ27Luc (Figure 2(b)) Itis possible that ATBF1 might interplay with p53 via Smadcomplex to effectively repress the transcription of the AFPgene Further experiments are needed in the future to confirmthese hypotheses as well

5 Conclusion

In summary we report a novel interaction between ATBF1and Smads The interaction between ATBF1 and Smadsappears to cooperatively inhibit the transcription of AFPgene upon TGF-120573 signaling In particular both proximal anddistal AT-motifs are required for TGF-120573Smad signaling tocounteract with the transcription of the AFP gene

Conflict of Interests

The authors have no competing financial interests to declare

Acknowledgments

This research was supported by a Grant-in-Aid for Explor-atory Research (A) from the Japan Society for the Promotionof Science (JSPS) (to Nobuo Sakata) the MEXT-Supported

Program for the Strategic Research Foundation at PrivateUniversities (2013ndash2017) (to Susumu Itoh) the Takeda Sci-ence Foundation (to Susumu Itoh) the Smoking ResearchFoundation (to Susumu Itoh) the Daiichi-Sankyo Founda-tion of Life Science (to Susumu Itoh) the Naito Founda-tion (to Susumu Itoh) the Vehicle Racing Commemora-tive Foundation (to Susumu Itoh) and a Grant-in-Aid forYoung Scientists of Showa Pharmaceutical University (toSouichi Ikeno) The authors were also supported by the JointUsageResearch Program of the Medical Research Institutethe Tokyo Medical and Dental University and by the Core-to-Core Program ldquoCooperative International Framework inTGF-120573 Family Signalingrdquo of the JSPS They thank Ms FMiyamasu for excellent English proofreading

References

[1] M A Dziadek and G K Andrews ldquoTissue specificity of 120572-fetoprotein messenger RNA expression during mouse embryo-genesisrdquoThe EMBO Journal vol 2 no 4 pp 549ndash554 1983

[2] J L Nahon I Tratner A Poliard et al ldquoAlbumin and 120572-feto-protein gene expression in various nonhepatic rat tissuesrdquo TheJournal of Biological Chemistry vol 263 no 23 pp 11436ndash114421988

[3] A A Terentiev and N T Moldogazieva ldquo120572-fetoprotein a ren-aissancerdquo Tumor Biology vol 34 no 4 pp 2075ndash2091 2013

[4] H Yasuda A Mizuno T Tamaoki and T Morinaga ldquoATBF1 amultiple-homeodomain zinc finger protein selectively down-regulates AT-rich elements of the human 120572-fetoprotein generdquoMolecular and Cellular Biology vol 14 no 2 pp 1395ndash14011994

[5] K Sawadaishi T Morinaga and T Tamaoki ldquoInteraction of ahepatoma-specific nuclear factor with transcription-regulatorysequences of the human 120572-fetoprotein and albumin genesrdquoMolecular andCellular Biology vol 8 no 12 pp 5179ndash5187 1988

[6] H Nakabayashi Y Koyama H Suzuki et al ldquoFunctional map-ping of tissue-specific elements of the human 120572-fetoproteingene enhancerrdquo Biochemical and Biophysical Research Commu-nications vol 318 no 3 pp 773ndash785 2004

[7] T Morinaga H Yasuda T Hashimoto K Higashio and TTamaoki ldquoA human 120572-fetoprotein enhancer-binding proteinATBF1 contains four homeodomains and seventeen zinc fin-gersrdquo Molecular and Cellular Biology vol 11 no 12 pp 6041ndash6049 1991

[8] Y Miura T Tam A Ido et al ldquoCloning and characterization ofan ATBF1 isoform that expresses in a neuronal differentiation-dependent mannerrdquo The Journal of Biological Chemistry vol270 no 45 pp 26840ndash26848 1995

[9] H Kataoka Y Miura T Joh et al ldquo120572-fetoprotein producinggastric cancer lacks transcription factor ATBF1rdquo Oncogene vol20 no 7 pp 869ndash873 2001

[10] T Ninomiya K Mihara K Fushimi Y Hayashi T Hashimoto-Tamaoki and T Tamaoki ldquoRegulation of the 120572-fetoprotein geneby the isoforms of ATBF1 transcription factor in human hep-atomardquo Hepatology vol 35 no 1 pp 82ndash87 2002

[11] P Kaspar M Dvorakova J Kralova P Pajer Z Kozmik andM Dvorak ldquoMyb-interacting protein ATBF1 represses tran-scriptional activity of Myb oncoproteinrdquoThe Journal of Biologi-cal Chemistry vol 274 no 20 pp 14422ndash14428 1999

[12] F B Berry YMiura KMihara et al ldquoPositive and negative reg-ulation of myogenic differentiation of C2C12 cells by isoforms

Journal of Signal Transduction 11

of the multiple homeodomain zinc finger transcription factorATBF1rdquoThe Journal of Biological Chemistry vol 276 no 27 pp25057ndash25065 2001

[13] X Sun H F Frierson C Chen et al ldquoFrequent somaticmutations of the transcription factor ATBF1 in human prostatecancerrdquo Nature Genetics vol 37 no 6 pp 407ndash412 2005

[14] X Sun Y Zhou K B Otto et al ldquoInfrequentmutation of ATBF1in human breast cancerrdquo Journal of Cancer Research and ClinicalOncology vol 133 no 2 pp 103ndash105 2007

[15] Y G Cho J H Song C J Kim et al ldquoGenetic alterations of theATBF1 gene in gastric cancerrdquo Clinical Cancer Research vol 13no 15 pp 4355ndash4359 2007

[16] K Kai Z Zhang H Yamashita Y Yamamoto Y Miura and HIwase ldquoLoss of heterozygosity at the ATBF1-A locus located inthe 16q22 minimal region in breast cancerrdquo BMC Cancer vol 8article 262 2008

[17] A M Cleton-Jansen R van Eijk M Lombaerts et al ldquoATBF1and NQO1 as candidate targets for allelic loss at chromosomearm 16q in breast cancer absence of somatic ATBF1 mutationsand no role for the C609TNQO1 polymorphismrdquo BMCCancervol 8 article 105 2008

[18] J Massague J Seoane and D Wotton ldquoSmad transcriptionfactorsrdquo Genes amp Development vol 19 no 23 pp 2783ndash28102005

[19] AMoustakas and C H Heldin ldquoThe regulation of TGF120573 signaltransductionrdquo Development vol 136 no 22 pp 3699ndash37142009

[20] J Massague ldquoTGF120573 signalling in contextrdquo Nature ReviewsMolecular Cell Biology vol 13 no 10 pp 616ndash630 2012

[21] K Nakao K Nakata S Mitsuoka et al ldquoTransforming growthfactor 120573 1 differentially regulates 120572-fetoprotein and albumin inHuH-7 human hepatoma cellsrdquo Biochemical and BiophysicalResearch Communications vol 174 no 3 pp 1294ndash1299 1991

[22] D SWilkinson S K Ogden S A Stratton et al ldquoA direct inter-section between p53 and transforming growth factor 120573 path-ways targets chromatin modification and transcription repres-sion of the 120572-fetoprotein generdquoMolecular and Cellular Biologyvol 25 no 3 pp 1200ndash1212 2005

[23] D S Wilkinson W W Tsai M A Schumacher and M CBarton ldquoChromatin-bound p53 anchors activated Smads andthe mSin3A corepressor to confer transforming-growth-factor-120573-mediated transcription repressionrdquo Molecular and CellularBiology vol 28 no 6 pp 1988ndash1998 2008

[24] C G Jung H J Kim M Kawaguchi et al ldquoHomeotic factorATBF1 induces the cell cycle arrest associated with neuronaldifferentiationrdquo Development vol 132 no 23 pp 5137ndash51452005

[25] X Y Dong X Sun P Guo et al ldquoATBF1 inhibits estrogenreceptor (ER) function by selectively competing with AIB1 forbinding to the ER in ER-positive breast cancer cellsrdquoThe Journalof Biological Chemistry vol 285 no 43 pp 32801ndash32809 2010

[26] H Kataoka P Bonnefin D Vieyra et al ldquoING1 repressestranscription by direct DNA binding and through effects onp53rdquo Cancer Research vol 63 no 18 pp 5785ndash5792 2003

[27] A Nakao T Imamura S Souchelnytskyi et al ldquoTGF-120573receptor-mediated signalling through Smad2 Smad3 andSmad4rdquoThe EMBO Journal vol 16 no 17 pp 5353ndash5362 1997

[28] M Fujii K Takeda T Imamura et al ldquoRoles of bone morpho-genetic protein type I receptors and Smad proteins in osteoblastand chondroblast differentiationrdquoMolecular Biology of the Cellvol 10 no 11 pp 3801ndash3812 1999

[29] YWatanabe S Itoh T Goto et al ldquoTMEPAI a transmembraneTGF-120573-inducible protein sequesters smad proteins from activeparticipation in TGF-120573 signalingrdquoMolecular Cell vol 37 no 1pp 123ndash134 2010

[30] S Itoh M Thorikay M Kowanetz et al ldquoElucidation of Smadrequirement in transforming growth factor-120573 type I receptor-induced responsesrdquo The Journal of Biological Chemistry vol278 no 6 pp 3751ndash3761 2003

[31] N Nakano S Itoh Y Watanabe K Maeyama F Itoh and MKato ldquoRequirement of TCF7L2 for TGF-120573 dependent transcrip-tional activation of the TMEPAI generdquoThe Journal of BiologicalChemistry vol 285 no 49 pp 38023ndash38033 2010

[32] K Watanabe A Saito and T Tamaoki ldquoCell-specific enhanceractivity in a far upstream region of the human 120572-fetoproteingenerdquo The Journal of Biological Chemistry vol 262 no 10 pp4812ndash4818 1987

[33] H Nakabayashi K Watanabe A Saito A Otsuru KSawadaishi and T Tamaoki ldquoTranscriptional regulation of 120573-fetoprotein expression by dexamethasone in human hepatomacellsrdquo The Journal of Biological Chemistry vol 264 no 1 pp266ndash271 1989

[34] H Nakabayashi T Hashimoto Y Miyao K-K Tjong J Chanand T Tamaoki ldquoA position-dependent silencer plays a majorrole in repressing 120573-fetoprotein expression in human hep-atomardquoMolecular and Cellular Biology vol 11 no 12 pp 5885ndash5893 1991

[35] S Zhang T S Kim Y Dong et al ldquoAT motif binding factor 1(ATBF1) is highly phosphorylated in embryonic brain and pro-tected from cleavage by calpain-1rdquo Biochemical and BiophysicalResearch Communications vol 427 no 3 pp 537ndash541 2012

[36] L J Jonk S Itoh C H Heldin P ten Dijke and W KruijerldquoIdentification and functional characterization of a smad bind-ing element (SBE) in the JunBpromoter that acts as a transform-ing growth factor-120573 activin and bone morphogenetic protein-inducible enhancerrdquo The Journal of Biological Chemistry vol273 no 33 pp 21145ndash21152 1998

[37] S Dennler S Itoh D Vivien P ten Dijke S Huet and J Gau-thier ldquoDirect binding of Smad3 and Smad4 to critical TGF120573-inducible elements in the promoter of human plasminogenactivator inhibitor-type 1 generdquo The EMBO Journal vol 17 no11 pp 3091ndash3100 1998

[38] A Ido K Nakata Y Kato et al ldquoGene therapy for hep-atoma cells using a retrovirus vector carrying herpes simplexvirus thymidine kinase gene under the control of human 120572-fetoprotein gene promoterrdquo Cancer Research vol 55 no 14 pp3105ndash3109 1995

[39] A B Roberts and L M Wakefield ldquoThe two faces of trans-forming growth factor 120573 in carcinogenesisrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 15 pp 8621ndash8623 2003

[40] B Bierie and H L Moses ldquoTumour microenvironment TGF120573the molecular Jekyll and Hyde of cancerrdquo Nature ReviewsCancer vol 6 no 7 pp 506ndash520 2006

Journal of Signal Transduction 5

TK promoter

Luc

Luc

Luc

Luc

Luc

EAminus4995 minus2953

minus4990

minus4990 minus4309

minus4363 minus3601

minus2968

minus2968minus3658

Δ27Luc+45minus178

fTKLuc

5998400TKLuc

mTKLuc

3998400TKLuc

EB

(a)

00

02

04

06

08

10

12

14

Relat

ive l

ucife

rase

activ

ity

lowast

lowast

fTKLuc 5998400TKLuc mTKLuc 3998400TKLuc

(b)

AT-motif AT-motif

EB

Luc

Luc

Luc

Luc

Luc

Luc

39984005Luc

399840051Luc

39984003Luc

02Luc

399840052Luc

39984001Luc+45minus178

+45minus178

+45minus178

+45minus178

+45minus178

+45minus178

minus3148

minus3148

minus3445

minus3422

minus3492 minus3300

minus3422

minus3658

minus3658 minus3563

minus3562

minus3658

(c)

00

05

10

15

20

25

30

35

40

Rela

tive l

ucife

rase

activ

ity

lowastlowastlowast

lowastlowastlowast

lowastlowast

39984005Luc 39984003Luc39984001Luc

(d)

00

02

04

06

08

10

12

14

Rela

tive l

ucife

rase

activ

ity

lowastlowastlowast

399840051Luc 02Luc399840052Luc

(e)

00

02

04

06

08

10

12

14

ATBF1

Rela

tive l

ucife

rase

activ

ity

lowast

lowast

399840052Luc

TGF-120573 (minus)TGF-120573 (+)

minus

(f)

Figure 3 Requirement of a distal AT-motif for TGF-120573-mediated repression of the activity of the AFP promoter (a) Schematic presentationof TKLuc conjugated with several fragments of the AFP promoter between minus4990 and minus2968 (b) Effect of TGF-120573 on the activity of TKLucreporters conjugated with several truncated AFP promoter regions Each reporter (200 ng) was transfected into HepG2 cells The cells werethen stimulated with 5 ngmL TGF-120573 (c) Schematic presentation of 02Luc conjugated with several fragments of the AFP promoter betweenminus3658 andminus3148 (d) Effect of TGF-120573 on the activity of the luciferase reporters described in Figure 3(c) Each reporter (200 ng)was transfectedinto HepG2 cellsThe cells were then stimulated with 5 ngmL TGF-120573 (e) Requirement of the fragment between minus3562 and minus3422 for TGF-120573-mediated suppression of theAFP promoter HepG2 cells were transfected with the indicated plasmids (200 ng)The cells were then stimulatedwith 5 ngmL TGF-120573 (f) Cooperation of ATBF1 with the TGF-120573 signaling for 3101584052Luc activity HepG2 cells were transfected with either352Luc reporter (200 ng) in the presence or absence of ATBF1 (200 ng) The cells were then stimulated with 5 ngmL TGF-120573

6 Journal of Signal Transduction

AT-motif AT-motif

AT-motifAT-motif

AT-motifCMV promoter

CMV promoter

65CMVLucAT-motif

CMV promoter

minus3562 minus3422

minus3562 minus3422 minus155 minus92

minus155 minus92

minus3562 minus3422

minus178Luc

Luc

Luc

Luc

+45

+1399840052Luc

399840052CMVLuc

399840052 65CMVLuc

(a)

00

02

04

06

08

10

12

Rela

tive l

ucife

rase

activ

ity

65CMVLuc399840052CMVLuc399840052 65CMVLuc

TGF-120573 (minus)TGF-120573 (+)

lowast

(b)

Figure 4 Importance of both distal and proximal AT-motifs for TGF-120573-mediated repression of theAFP promoter (a) Schematic presentationof CMVLuc conjugatedwith distal and proximal AT-motifs (b)HepG2 cells were transfectedwith each reporter (200 ng) and then stimulatedwith 5 ngmL TGF-120573

promoter (TKLuc) (Figure 3(a)) As shown in Figure 3(b)TGF-120573 suppressed the reporter activity of fTKLuc Thusthe region between minus4990 and minus2968 possesses an inhibitorycis-element(s) upon TGF-120573 stimulation Within this regionthe fragment between minus3658 and minus2968 (termed 31015840TKLuc)revealed a remarkably inhibitory cis-element by TGF-120573although TGF-120573-mediated repression of the reporter activitycould also be seen in mTKLuc Intriguingly 119864

119861contain-

ing the AT-motif is present between minus3492 and minus3300(Figure 3(c)) To further narrow down a TGF-120573-responsiveinhibitory cis-element(s) we made four luciferase reportersthat were linked to the AFPminimal promoter (Figure 3(c))TGF-120573 dramatically suppressed the luciferase activity of310158401Luc 310158405Luc and 3101584052Luc although 310158403Luc activity was alsoinhibited by TGF-120573 to some extent in spite of its low basalluciferase activity indicating that the region from minus3562 tominus3422 possesses a TGF-120573-mediated inhibitory cis-element ifnot more (Figures 3(d) and 3(e)) Thus an AT-motif andorits adjacent sequences in 119864

119861were highly suggestive of a TGF-

120573-mediated inhibitory cis-element In addition ATBF1 couldfurther inhibit the 3101584052Luc activity suppressed by TGF-120573(Figure 3(f)) Therefore it is possible that the TGF-120573Smadpathway interferes via the region between minus3562 and minus3422

34 A Role of the Proximal AT-Motif in the AFP PromoterSince there are two functional AT-motifs within the AFPpromoter between minus4995 and +45 (Figure 2(a)) it is possiblethat the proximal AT-motif also influences TGF-120573-mediatedinhibition of the activity of theAFP promoter To confirm thatthe proximal AT-motif can also contribute to TGF-120573-mediated repression of the activity of the AFP promoter the141nt-length fragment from minus3562 to minus3422 was ligated to the

64nt-length region between minus155 and minus92 of the AFP pro-moter Then this fragment was further placed at the 51015840portof CMV Luc (3101584052 65CMVLuc) because the CMV promotershows the high level of luciferase activity Figure 4(b) demon-strates that both the distal and the proximal AT-motifs areneeded for TGF-120573 to suppress the activity of the AFPpromoter

35 Association of Smad Proteins with ATBF1 Since ATBF1cooperates with Smad3 for inhibition of the activity of theAFP promoter we tried to determine whether ATBF1 couldinteract with R-Smads Expectedly ATBF1 could associatewith Smad2 and Smad3 although ALK5 activation was notinvolved in its interaction (Figure 5(a) and data not shown)In these experiments we could observe multiple bands cor-responding to ATBF1 These multiple bands might be due tocalpain-1-dependent proteolysis in COS7 cells [35] To iden-tify the domain(s) of ATBF1 that interact with Smads wedivided ATBF1 into three pieces (Figure 5(b)) ATBF1N aswell as ATBF1C had the ability to interact with Smad2 andSmad3 although ATBF1M no longer possessed the ability tobind to either Smad2 or Smad3 (Figures 5(c)ndash5(e)) It hasbeen reported that DNA-binding domains are present inATBF1M including zinc finger domains and homeodomains[12]When either ATBF1NorATBF1Cwas cotransfectedwithATBF1 TGF-120573-mediated inhibition of the activity of theAFPpromoter was improved owing to their competition withATBF1 for Smad binding (Figures 6(a) and 6(b)) On theother hand ATBF1M could inhibit the reporter activity as adominant negative formofATBF1 becauseATBF1Mpossessesthe ability to bind to AT-motif via its DNA binding domain(data not shown) These results indicate that the N-terminal

Journal of Signal Transduction 7

Flag-Smad3

Smad3

ATBF1

Smad3

ALK5ca

minus +minus

++ +

Myc-ATBF1ALK5caV5

IP MycIB flag

IB Myc

IB flag

IB V5

(a)

3703

ATBF1

1

ATBF1N

8951

ATBF1M

3293894

ATBF1C

37033290

Zinc fingerHomeodomain

(b)

HA-ATBF1NFlag-Smad2Flag-Smad3

IP flagIB HA

IB HA

IB flag

minus + minusminus

Smad2Smad3

ATBF1N

ATBF1N

+minus

(c)

HA-ATBF1MFlag-Smad2Flag-Smad3

minus +minus +minus

IP flagIB HA

IB HA

IB flag

minus

ATBF1M

ATBF1M

Smad2Smad3

(d)

HA-ATBF1CFlag-Smad2Flag-Smad3

minus +minus +minus

IP flagIB HA

IB HA

IB flag

minus

ATBF1C

ATBF1C

Smad2Smad3

(e)

Figure 5 Association of Smads with ATBF1 (a) Interaction of Smad3 with ATBF1 COS7 cells were transfected with Flag-Smad3 (1120583g) withMyc-ATBF1 (2120583g) in combination with ALK5caV5 (05 120583g) or without it Cell lysates were subjected to immunoprecipitation (IP) with anti-Myc antibody followed by IB with anti-Flag antibody to show the interaction of Flag-Smad3 with Myc-ATBF1 (b) Schematic presentation ofATBF1 mutants (cndashe) Interaction of each ATBF1 mutant with Smad2 or Smad3 COS7 cells were transfected with Flag-Smad2 or Flag-Smad3(15120583g) with HA-ATBF1N (2120583g) (c) HA-ATBF1M (2120583g) (d) or HA-ATBF1C (2120583g) (e) Cell lysates were subjected to IP with anti-Flagantibody followed by IB with anti-HA antibody to show the interaction of Smad2 and Smad3 with ATBF1 mutants

and C-terminal domains are needed for ATBF1 to interactwith R-Smads

36 Indirect Binding of Smads to the AFP Promoter via ATBF1We could find only one Smad binding element (SBE) [36 37]in 3101584052Luc (Figure 7(a))Thus we examinedwhether this SBEis necessary for TGF-120573-mediated repression of the AFP pro-moter For that purpose wemade one reporter construct thathad mutations within the SBE of 3101584052Luc (3101584052mSBELuc)

(Figure 7(a)) However 3101584052mSBELuc activity was still inhib-ited by TGF-120573 although its basal level became weaker thanthat of 3101584052Luc (Figure 7(b)) Thus the SBE in the AFP pro-moter is not essential for TGF-120573Smad signaling to repressthe activity of the AFP promoter Indeed a DNAP assayshowed that Smad3 can be detected in the presence of ATBF1using three repeats of an AT-motif as the probe (Figure 7(c))Thus Smad3 probably binds to the promoter region of theAFP gene indirectly through its interaction with ATBF1

8 Journal of Signal Transduction

00

02

04

06

08

10

12

14

16

ATBF1ATBF1N

Rela

tive l

ucife

rase

activ

ity

+ + ++minus

minus minus

TGF-120573 (minus)TGF-120573 (+)

lowast

lowast

lowastlowastlowast

(a)

00

02

04

06

08

10

12

14

ATBF1ATBF1C

Rela

tive l

ucife

rase

activ

ity

+ + ++minus

minus minus

TGF-120573 (minus)TGF-120573 (+)

lowast

lowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

(b)

Figure 6 ATBF1N and ATBF1C perturb ATBF1-mediated suppression of the activity of theAFP promoter upon TGF-120573 stimulation ATBF1N(a) and ATNF1C (b) rescue ATBF1-mediated repression of the activity of the AFP promoter HepG2 cells were transfected with increasedamounts of either ATBF1N (50 100 or 200 ng) (a) or ATBF1C (50 100 or 200 ng) (b) together with ATBF1 (200 ng) and Δ27Luc (200 ng)The cells were then stimulated with 5 ngmL TGF-120573

4 Discussion

The hepatoma cells HuH-7 and HepG2 cells are known tosecrete a large and a low amount of AFP respectively [38] Agrowing body of evidence indicates that the level of AFP inserum from patients is linked to HCC tumorigenicity [3] p53has been reported to cooperate with the TGF-120573Smad path-way to repress AFP expression via induction of SnoN [22]However it remains veiled whether ATBF1 which is knownto blockAFP transcription [4 10] can also regulate the activ-ity of the AFP promoter in consort with the TGF-120573Smadpathway because both ATBF1 and TGF-120573Smad might pos-sess an antioncogenic function [13ndash17 20 39 40] ATBF1could inhibit the activity of the AFP promoter via its regionsfrom minus4995 to minus2953 andor from minus178 to +45 [4 26] SinceΔ27Luc had a remarkably basal promoter activity comparedwith 02Luc (Figure 2(b)) we initially focused on the distalregion of the AFP promoter for involvement of the TGF-120573Smad signaling with ATBF1 As expected the TGF-120573-mediated repressive element in the AFP promoter lay near119864119861 which contains a distal AT-motif The luciferase reporter

conjugated with the inhibitory element including a distalAT-motif (352Luc) showed coordinated inhibitory actionbetween the TGF-120573 signaling and ATBF1 (Figure 3(f))Therefore we were struck with the notion that ATBF1physically interacts with Smads which play key roles in thecanonical TGF-120573 signaling pathway [18ndash20] Not surpris-ingly ATBF1 could interact with Smad2 and Smad3 althoughC-terminal phosphorylation of R-Smads was dispensable

(Figure 5(a) and data not shown) In addition both the N-terminal (ATBF1N) and the C-terminal mutants of ATBF1(ATBF1C) but not the middle domain of ATBF1 (ATBF1M)which possesses DNA-binding activity [12] could interactwith R-Smads (Figures 5(c)ndash5(e))We hypothesized that bothATBF1N and ATBF1C might compete with wildtype ATBF1for Smad binding to rescueATBF1-mediated repression of theAFP promoter uponTGF-120573 stimulation because both of themhave the ability to bind to R-Smads According to our expec-tation both ATBF1N and ATBF1C improved the reporteractivity decreased by the cooperative ATBF1 and TGF-120573signaling (Figures 6(a) and 6(b)) As shown in Figure 2(b)02Luc activity could also be reduced upon TGF-120573 stimu-lation despite very low basal promoter activity Because ofthe presence of a proximal AT-motif within this region wespeculated that a proximal AT-motif also contributes to TGF-120573-mediated-repression of the activity of the AFP promoterThe deletion of a proximal AT-motif from 3101584052 65CMVLucled us to recognize the importance of a proximal AT-motif aswell as a distal AT-motif for TGF-120573-mediated repression ofthe activity of theAFP promoter (Figure 4(b)) A Smad bind-ing element (SBE) composed of consensus 51015840-AGAC-31015840 isflanked to a proximal AT-motif However themutation of theSBE in 3101584052Luc showed reduced basal promoter activity butstill maintained TGF-120573-mediated repression of the AFP pro-moter (Figure 7(b)) Thus Smad might not be necessary tobind to a promoter element(s) in the AFP gene directly whenATBF1 cooperates with TGF-120573 signaling whereas p53-mediated repression of the AFP promoter activity needs

Journal of Signal Transduction 9

SBEminus3422

minus3422

minus3562

minus3562

LucAT-motif

minus178 +45

+1

LucAT-motif

AT-motif

AT-motifminus178 +45

+1

399840052Luc

399840052mSBELuc

(a)

00

02

04

06

08

10

12

Relat

ive l

ucife

rase

activ

ity

TGF-120573 (minus)TGF-120573 (+)

lowastlowast

lowastlowast

399840052Luc 399840052mSBELuc

(b)

DNAP

Flag-Smad3IB flag

IB HA

Totallysates

HA-ATBF1

HA-ATBF1

Flag-Smad3IB flag

IB HA

p-Smad3IB PS3

IB V5 ALK5caV5

Biotinylated probe minus + + +

Flag-Smad3HA-ATBF1

+ + + minusminus + +minus

ALK5caV5 + + + minus

(AT-motif)3

(c)

Figure 7 SBE in the proximal region of the AFP promoter is not essential for TGF-120573-mediated suppression of the AFP promoter (a)Schematic presentation of 3101584052mSBELuc A putative SBE 51015840-CAGATA-31015840 was mutated to 51015840-TTTTTT-31015840 (b) Effect of TGF-120573 on 3101584052Lucand 3101584052mSBELuc activities HepG2 cells were transfected with each reporter plasmid (200 ng) The cells were then stimulated with 5 ngmLTGF-120573 (c) Indirect binding of Smad3 to an AT-motif via ATBF1 The indicated cell lysates were mixed with biotinylated AT-motifs Thenstreptavidin-agarose was added to the mixture Subsequently a protein(s) bound to streptavidin-agarose was purified After SDS-PAGE IBwas carried out using anti-Flag or anti-HA antibody Using the total lysates each protein was detected with anti-Flag anti-HA anti-V5 oranti-phosphorylated Smad3 (PS3) antibody

direct binding of Smads to DNA [22 23] The requirement ofdirect DNA binding for R-Smads is probably dependent onwhich transcriptional regulator(s) they cooperate or syner-gize with Indeed Smad3 could bind toAT-motifs in the pres-ence of ATBF1 but not in its absence (Figure 7(c)) Althoughwe do not know if the ATBF1Smad complex at a distalAT-motif can physically interact with that at a proximal

AT-motif directly or indirectly through another protein(s)(ldquoXrdquo Figure 8) both AT-motifs are definitely important forcooperation between ATBF1 and the TGF-120573 signaling tosuppress the activity of the AFP promoter

ATBF-1 is known to compete with HNF-1 for binding toAT-motifs in the AFP promoter [4] Thus the following pos-sibility can be speculated upon TGF-120573 stimulation Smads

10 Journal of Signal Transduction

AT-motif

AT-motif

AFP

ATBF1

ATBF1ATBF1Smad23-Smad4

X

Smad23-Smad4

ATBF1

Figure 8 Proposed inhibitory mechanism by which the TGF-120573Smad signaling and ATBF1 cooperatively regulate the activity ofthe AFP promoter The distal and proximal AT-motifs in the AFPpromoter are bound by ATBF1 Upon TGF-120573 signaling the R-SmadsSmad4 complex interacts with ATBF1 to make a repressivecomplex for the activity of the AFP promoter However whetheranother protein(s) (termed ldquoXrdquo) is essential for the R-SmadSmad4ATBF1 complex remains veiled

can not only bind to AT-motifs via ATBF-1 indirectly but alsointeract with HNF-1 to dissociate it from AT-motifs Due todual function of Smads the AFP prompter activity might beinhibited This possibility is interesting to be investigated

p53-mediated repression of the AFP promoter activityneeds direct binding of Smads to SBEp53REWilkinson et alfurther demonstrated that mSin3A and SnoN which is adirect target gene of TGF-120573 signal are recruited by p53Smadscomplex when the transcription of the AFP gene is inhib-ited [22 23] In our current study the region includingSBEp53RE in theAFP promoterwas not investigated becausethe basal reporter activities of 3Luc 16Luc and 09Luc werevery low compared with that of Δ27Luc (Figure 2(b)) Itis possible that ATBF1 might interplay with p53 via Smadcomplex to effectively repress the transcription of the AFPgene Further experiments are needed in the future to confirmthese hypotheses as well

5 Conclusion

In summary we report a novel interaction between ATBF1and Smads The interaction between ATBF1 and Smadsappears to cooperatively inhibit the transcription of AFPgene upon TGF-120573 signaling In particular both proximal anddistal AT-motifs are required for TGF-120573Smad signaling tocounteract with the transcription of the AFP gene

Conflict of Interests

The authors have no competing financial interests to declare

Acknowledgments

This research was supported by a Grant-in-Aid for Explor-atory Research (A) from the Japan Society for the Promotionof Science (JSPS) (to Nobuo Sakata) the MEXT-Supported

Program for the Strategic Research Foundation at PrivateUniversities (2013ndash2017) (to Susumu Itoh) the Takeda Sci-ence Foundation (to Susumu Itoh) the Smoking ResearchFoundation (to Susumu Itoh) the Daiichi-Sankyo Founda-tion of Life Science (to Susumu Itoh) the Naito Founda-tion (to Susumu Itoh) the Vehicle Racing Commemora-tive Foundation (to Susumu Itoh) and a Grant-in-Aid forYoung Scientists of Showa Pharmaceutical University (toSouichi Ikeno) The authors were also supported by the JointUsageResearch Program of the Medical Research Institutethe Tokyo Medical and Dental University and by the Core-to-Core Program ldquoCooperative International Framework inTGF-120573 Family Signalingrdquo of the JSPS They thank Ms FMiyamasu for excellent English proofreading

References

[1] M A Dziadek and G K Andrews ldquoTissue specificity of 120572-fetoprotein messenger RNA expression during mouse embryo-genesisrdquoThe EMBO Journal vol 2 no 4 pp 549ndash554 1983

[2] J L Nahon I Tratner A Poliard et al ldquoAlbumin and 120572-feto-protein gene expression in various nonhepatic rat tissuesrdquo TheJournal of Biological Chemistry vol 263 no 23 pp 11436ndash114421988

[3] A A Terentiev and N T Moldogazieva ldquo120572-fetoprotein a ren-aissancerdquo Tumor Biology vol 34 no 4 pp 2075ndash2091 2013

[4] H Yasuda A Mizuno T Tamaoki and T Morinaga ldquoATBF1 amultiple-homeodomain zinc finger protein selectively down-regulates AT-rich elements of the human 120572-fetoprotein generdquoMolecular and Cellular Biology vol 14 no 2 pp 1395ndash14011994

[5] K Sawadaishi T Morinaga and T Tamaoki ldquoInteraction of ahepatoma-specific nuclear factor with transcription-regulatorysequences of the human 120572-fetoprotein and albumin genesrdquoMolecular andCellular Biology vol 8 no 12 pp 5179ndash5187 1988

[6] H Nakabayashi Y Koyama H Suzuki et al ldquoFunctional map-ping of tissue-specific elements of the human 120572-fetoproteingene enhancerrdquo Biochemical and Biophysical Research Commu-nications vol 318 no 3 pp 773ndash785 2004

[7] T Morinaga H Yasuda T Hashimoto K Higashio and TTamaoki ldquoA human 120572-fetoprotein enhancer-binding proteinATBF1 contains four homeodomains and seventeen zinc fin-gersrdquo Molecular and Cellular Biology vol 11 no 12 pp 6041ndash6049 1991

[8] Y Miura T Tam A Ido et al ldquoCloning and characterization ofan ATBF1 isoform that expresses in a neuronal differentiation-dependent mannerrdquo The Journal of Biological Chemistry vol270 no 45 pp 26840ndash26848 1995

[9] H Kataoka Y Miura T Joh et al ldquo120572-fetoprotein producinggastric cancer lacks transcription factor ATBF1rdquo Oncogene vol20 no 7 pp 869ndash873 2001

[10] T Ninomiya K Mihara K Fushimi Y Hayashi T Hashimoto-Tamaoki and T Tamaoki ldquoRegulation of the 120572-fetoprotein geneby the isoforms of ATBF1 transcription factor in human hep-atomardquo Hepatology vol 35 no 1 pp 82ndash87 2002

[11] P Kaspar M Dvorakova J Kralova P Pajer Z Kozmik andM Dvorak ldquoMyb-interacting protein ATBF1 represses tran-scriptional activity of Myb oncoproteinrdquoThe Journal of Biologi-cal Chemistry vol 274 no 20 pp 14422ndash14428 1999

[12] F B Berry YMiura KMihara et al ldquoPositive and negative reg-ulation of myogenic differentiation of C2C12 cells by isoforms

Journal of Signal Transduction 11

of the multiple homeodomain zinc finger transcription factorATBF1rdquoThe Journal of Biological Chemistry vol 276 no 27 pp25057ndash25065 2001

[13] X Sun H F Frierson C Chen et al ldquoFrequent somaticmutations of the transcription factor ATBF1 in human prostatecancerrdquo Nature Genetics vol 37 no 6 pp 407ndash412 2005

[14] X Sun Y Zhou K B Otto et al ldquoInfrequentmutation of ATBF1in human breast cancerrdquo Journal of Cancer Research and ClinicalOncology vol 133 no 2 pp 103ndash105 2007

[15] Y G Cho J H Song C J Kim et al ldquoGenetic alterations of theATBF1 gene in gastric cancerrdquo Clinical Cancer Research vol 13no 15 pp 4355ndash4359 2007

[16] K Kai Z Zhang H Yamashita Y Yamamoto Y Miura and HIwase ldquoLoss of heterozygosity at the ATBF1-A locus located inthe 16q22 minimal region in breast cancerrdquo BMC Cancer vol 8article 262 2008

[17] A M Cleton-Jansen R van Eijk M Lombaerts et al ldquoATBF1and NQO1 as candidate targets for allelic loss at chromosomearm 16q in breast cancer absence of somatic ATBF1 mutationsand no role for the C609TNQO1 polymorphismrdquo BMCCancervol 8 article 105 2008

[18] J Massague J Seoane and D Wotton ldquoSmad transcriptionfactorsrdquo Genes amp Development vol 19 no 23 pp 2783ndash28102005

[19] AMoustakas and C H Heldin ldquoThe regulation of TGF120573 signaltransductionrdquo Development vol 136 no 22 pp 3699ndash37142009

[20] J Massague ldquoTGF120573 signalling in contextrdquo Nature ReviewsMolecular Cell Biology vol 13 no 10 pp 616ndash630 2012

[21] K Nakao K Nakata S Mitsuoka et al ldquoTransforming growthfactor 120573 1 differentially regulates 120572-fetoprotein and albumin inHuH-7 human hepatoma cellsrdquo Biochemical and BiophysicalResearch Communications vol 174 no 3 pp 1294ndash1299 1991

[22] D SWilkinson S K Ogden S A Stratton et al ldquoA direct inter-section between p53 and transforming growth factor 120573 path-ways targets chromatin modification and transcription repres-sion of the 120572-fetoprotein generdquoMolecular and Cellular Biologyvol 25 no 3 pp 1200ndash1212 2005

[23] D S Wilkinson W W Tsai M A Schumacher and M CBarton ldquoChromatin-bound p53 anchors activated Smads andthe mSin3A corepressor to confer transforming-growth-factor-120573-mediated transcription repressionrdquo Molecular and CellularBiology vol 28 no 6 pp 1988ndash1998 2008

[24] C G Jung H J Kim M Kawaguchi et al ldquoHomeotic factorATBF1 induces the cell cycle arrest associated with neuronaldifferentiationrdquo Development vol 132 no 23 pp 5137ndash51452005

[25] X Y Dong X Sun P Guo et al ldquoATBF1 inhibits estrogenreceptor (ER) function by selectively competing with AIB1 forbinding to the ER in ER-positive breast cancer cellsrdquoThe Journalof Biological Chemistry vol 285 no 43 pp 32801ndash32809 2010

[26] H Kataoka P Bonnefin D Vieyra et al ldquoING1 repressestranscription by direct DNA binding and through effects onp53rdquo Cancer Research vol 63 no 18 pp 5785ndash5792 2003

[27] A Nakao T Imamura S Souchelnytskyi et al ldquoTGF-120573receptor-mediated signalling through Smad2 Smad3 andSmad4rdquoThe EMBO Journal vol 16 no 17 pp 5353ndash5362 1997

[28] M Fujii K Takeda T Imamura et al ldquoRoles of bone morpho-genetic protein type I receptors and Smad proteins in osteoblastand chondroblast differentiationrdquoMolecular Biology of the Cellvol 10 no 11 pp 3801ndash3812 1999

[29] YWatanabe S Itoh T Goto et al ldquoTMEPAI a transmembraneTGF-120573-inducible protein sequesters smad proteins from activeparticipation in TGF-120573 signalingrdquoMolecular Cell vol 37 no 1pp 123ndash134 2010

[30] S Itoh M Thorikay M Kowanetz et al ldquoElucidation of Smadrequirement in transforming growth factor-120573 type I receptor-induced responsesrdquo The Journal of Biological Chemistry vol278 no 6 pp 3751ndash3761 2003

[31] N Nakano S Itoh Y Watanabe K Maeyama F Itoh and MKato ldquoRequirement of TCF7L2 for TGF-120573 dependent transcrip-tional activation of the TMEPAI generdquoThe Journal of BiologicalChemistry vol 285 no 49 pp 38023ndash38033 2010

[32] K Watanabe A Saito and T Tamaoki ldquoCell-specific enhanceractivity in a far upstream region of the human 120572-fetoproteingenerdquo The Journal of Biological Chemistry vol 262 no 10 pp4812ndash4818 1987

[33] H Nakabayashi K Watanabe A Saito A Otsuru KSawadaishi and T Tamaoki ldquoTranscriptional regulation of 120573-fetoprotein expression by dexamethasone in human hepatomacellsrdquo The Journal of Biological Chemistry vol 264 no 1 pp266ndash271 1989

[34] H Nakabayashi T Hashimoto Y Miyao K-K Tjong J Chanand T Tamaoki ldquoA position-dependent silencer plays a majorrole in repressing 120573-fetoprotein expression in human hep-atomardquoMolecular and Cellular Biology vol 11 no 12 pp 5885ndash5893 1991

[35] S Zhang T S Kim Y Dong et al ldquoAT motif binding factor 1(ATBF1) is highly phosphorylated in embryonic brain and pro-tected from cleavage by calpain-1rdquo Biochemical and BiophysicalResearch Communications vol 427 no 3 pp 537ndash541 2012

[36] L J Jonk S Itoh C H Heldin P ten Dijke and W KruijerldquoIdentification and functional characterization of a smad bind-ing element (SBE) in the JunBpromoter that acts as a transform-ing growth factor-120573 activin and bone morphogenetic protein-inducible enhancerrdquo The Journal of Biological Chemistry vol273 no 33 pp 21145ndash21152 1998

[37] S Dennler S Itoh D Vivien P ten Dijke S Huet and J Gau-thier ldquoDirect binding of Smad3 and Smad4 to critical TGF120573-inducible elements in the promoter of human plasminogenactivator inhibitor-type 1 generdquo The EMBO Journal vol 17 no11 pp 3091ndash3100 1998

[38] A Ido K Nakata Y Kato et al ldquoGene therapy for hep-atoma cells using a retrovirus vector carrying herpes simplexvirus thymidine kinase gene under the control of human 120572-fetoprotein gene promoterrdquo Cancer Research vol 55 no 14 pp3105ndash3109 1995

[39] A B Roberts and L M Wakefield ldquoThe two faces of trans-forming growth factor 120573 in carcinogenesisrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 15 pp 8621ndash8623 2003

[40] B Bierie and H L Moses ldquoTumour microenvironment TGF120573the molecular Jekyll and Hyde of cancerrdquo Nature ReviewsCancer vol 6 no 7 pp 506ndash520 2006

6 Journal of Signal Transduction

AT-motif AT-motif

AT-motifAT-motif

AT-motifCMV promoter

CMV promoter

65CMVLucAT-motif

CMV promoter

minus3562 minus3422

minus3562 minus3422 minus155 minus92

minus155 minus92

minus3562 minus3422

minus178Luc

Luc

Luc

Luc

+45

+1399840052Luc

399840052CMVLuc

399840052 65CMVLuc

(a)

00

02

04

06

08

10

12

Rela

tive l

ucife

rase

activ

ity

65CMVLuc399840052CMVLuc399840052 65CMVLuc

TGF-120573 (minus)TGF-120573 (+)

lowast

(b)

Figure 4 Importance of both distal and proximal AT-motifs for TGF-120573-mediated repression of theAFP promoter (a) Schematic presentationof CMVLuc conjugatedwith distal and proximal AT-motifs (b)HepG2 cells were transfectedwith each reporter (200 ng) and then stimulatedwith 5 ngmL TGF-120573

promoter (TKLuc) (Figure 3(a)) As shown in Figure 3(b)TGF-120573 suppressed the reporter activity of fTKLuc Thusthe region between minus4990 and minus2968 possesses an inhibitorycis-element(s) upon TGF-120573 stimulation Within this regionthe fragment between minus3658 and minus2968 (termed 31015840TKLuc)revealed a remarkably inhibitory cis-element by TGF-120573although TGF-120573-mediated repression of the reporter activitycould also be seen in mTKLuc Intriguingly 119864

119861contain-

ing the AT-motif is present between minus3492 and minus3300(Figure 3(c)) To further narrow down a TGF-120573-responsiveinhibitory cis-element(s) we made four luciferase reportersthat were linked to the AFPminimal promoter (Figure 3(c))TGF-120573 dramatically suppressed the luciferase activity of310158401Luc 310158405Luc and 3101584052Luc although 310158403Luc activity was alsoinhibited by TGF-120573 to some extent in spite of its low basalluciferase activity indicating that the region from minus3562 tominus3422 possesses a TGF-120573-mediated inhibitory cis-element ifnot more (Figures 3(d) and 3(e)) Thus an AT-motif andorits adjacent sequences in 119864

119861were highly suggestive of a TGF-

120573-mediated inhibitory cis-element In addition ATBF1 couldfurther inhibit the 3101584052Luc activity suppressed by TGF-120573(Figure 3(f)) Therefore it is possible that the TGF-120573Smadpathway interferes via the region between minus3562 and minus3422

34 A Role of the Proximal AT-Motif in the AFP PromoterSince there are two functional AT-motifs within the AFPpromoter between minus4995 and +45 (Figure 2(a)) it is possiblethat the proximal AT-motif also influences TGF-120573-mediatedinhibition of the activity of theAFP promoter To confirm thatthe proximal AT-motif can also contribute to TGF-120573-mediated repression of the activity of the AFP promoter the141nt-length fragment from minus3562 to minus3422 was ligated to the

64nt-length region between minus155 and minus92 of the AFP pro-moter Then this fragment was further placed at the 51015840portof CMV Luc (3101584052 65CMVLuc) because the CMV promotershows the high level of luciferase activity Figure 4(b) demon-strates that both the distal and the proximal AT-motifs areneeded for TGF-120573 to suppress the activity of the AFPpromoter

35 Association of Smad Proteins with ATBF1 Since ATBF1cooperates with Smad3 for inhibition of the activity of theAFP promoter we tried to determine whether ATBF1 couldinteract with R-Smads Expectedly ATBF1 could associatewith Smad2 and Smad3 although ALK5 activation was notinvolved in its interaction (Figure 5(a) and data not shown)In these experiments we could observe multiple bands cor-responding to ATBF1 These multiple bands might be due tocalpain-1-dependent proteolysis in COS7 cells [35] To iden-tify the domain(s) of ATBF1 that interact with Smads wedivided ATBF1 into three pieces (Figure 5(b)) ATBF1N aswell as ATBF1C had the ability to interact with Smad2 andSmad3 although ATBF1M no longer possessed the ability tobind to either Smad2 or Smad3 (Figures 5(c)ndash5(e)) It hasbeen reported that DNA-binding domains are present inATBF1M including zinc finger domains and homeodomains[12]When either ATBF1NorATBF1Cwas cotransfectedwithATBF1 TGF-120573-mediated inhibition of the activity of theAFPpromoter was improved owing to their competition withATBF1 for Smad binding (Figures 6(a) and 6(b)) On theother hand ATBF1M could inhibit the reporter activity as adominant negative formofATBF1 becauseATBF1Mpossessesthe ability to bind to AT-motif via its DNA binding domain(data not shown) These results indicate that the N-terminal

Journal of Signal Transduction 7

Flag-Smad3

Smad3

ATBF1

Smad3

ALK5ca

minus +minus

++ +

Myc-ATBF1ALK5caV5

IP MycIB flag

IB Myc

IB flag

IB V5

(a)

3703

ATBF1

1

ATBF1N

8951

ATBF1M

3293894

ATBF1C

37033290

Zinc fingerHomeodomain

(b)

HA-ATBF1NFlag-Smad2Flag-Smad3

IP flagIB HA

IB HA

IB flag

minus + minusminus

Smad2Smad3

ATBF1N

ATBF1N

+minus

(c)

HA-ATBF1MFlag-Smad2Flag-Smad3

minus +minus +minus

IP flagIB HA

IB HA

IB flag

minus

ATBF1M

ATBF1M

Smad2Smad3

(d)

HA-ATBF1CFlag-Smad2Flag-Smad3

minus +minus +minus

IP flagIB HA

IB HA

IB flag

minus

ATBF1C

ATBF1C

Smad2Smad3

(e)

Figure 5 Association of Smads with ATBF1 (a) Interaction of Smad3 with ATBF1 COS7 cells were transfected with Flag-Smad3 (1120583g) withMyc-ATBF1 (2120583g) in combination with ALK5caV5 (05 120583g) or without it Cell lysates were subjected to immunoprecipitation (IP) with anti-Myc antibody followed by IB with anti-Flag antibody to show the interaction of Flag-Smad3 with Myc-ATBF1 (b) Schematic presentation ofATBF1 mutants (cndashe) Interaction of each ATBF1 mutant with Smad2 or Smad3 COS7 cells were transfected with Flag-Smad2 or Flag-Smad3(15120583g) with HA-ATBF1N (2120583g) (c) HA-ATBF1M (2120583g) (d) or HA-ATBF1C (2120583g) (e) Cell lysates were subjected to IP with anti-Flagantibody followed by IB with anti-HA antibody to show the interaction of Smad2 and Smad3 with ATBF1 mutants

and C-terminal domains are needed for ATBF1 to interactwith R-Smads

36 Indirect Binding of Smads to the AFP Promoter via ATBF1We could find only one Smad binding element (SBE) [36 37]in 3101584052Luc (Figure 7(a))Thus we examinedwhether this SBEis necessary for TGF-120573-mediated repression of the AFP pro-moter For that purpose wemade one reporter construct thathad mutations within the SBE of 3101584052Luc (3101584052mSBELuc)

(Figure 7(a)) However 3101584052mSBELuc activity was still inhib-ited by TGF-120573 although its basal level became weaker thanthat of 3101584052Luc (Figure 7(b)) Thus the SBE in the AFP pro-moter is not essential for TGF-120573Smad signaling to repressthe activity of the AFP promoter Indeed a DNAP assayshowed that Smad3 can be detected in the presence of ATBF1using three repeats of an AT-motif as the probe (Figure 7(c))Thus Smad3 probably binds to the promoter region of theAFP gene indirectly through its interaction with ATBF1

8 Journal of Signal Transduction

00

02

04

06

08

10

12

14

16

ATBF1ATBF1N

Rela

tive l

ucife

rase

activ

ity

+ + ++minus

minus minus

TGF-120573 (minus)TGF-120573 (+)

lowast

lowast

lowastlowastlowast

(a)

00

02

04

06

08

10

12

14

ATBF1ATBF1C

Rela

tive l

ucife

rase

activ

ity

+ + ++minus

minus minus

TGF-120573 (minus)TGF-120573 (+)

lowast

lowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

(b)

Figure 6 ATBF1N and ATBF1C perturb ATBF1-mediated suppression of the activity of theAFP promoter upon TGF-120573 stimulation ATBF1N(a) and ATNF1C (b) rescue ATBF1-mediated repression of the activity of the AFP promoter HepG2 cells were transfected with increasedamounts of either ATBF1N (50 100 or 200 ng) (a) or ATBF1C (50 100 or 200 ng) (b) together with ATBF1 (200 ng) and Δ27Luc (200 ng)The cells were then stimulated with 5 ngmL TGF-120573

4 Discussion

The hepatoma cells HuH-7 and HepG2 cells are known tosecrete a large and a low amount of AFP respectively [38] Agrowing body of evidence indicates that the level of AFP inserum from patients is linked to HCC tumorigenicity [3] p53has been reported to cooperate with the TGF-120573Smad path-way to repress AFP expression via induction of SnoN [22]However it remains veiled whether ATBF1 which is knownto blockAFP transcription [4 10] can also regulate the activ-ity of the AFP promoter in consort with the TGF-120573Smadpathway because both ATBF1 and TGF-120573Smad might pos-sess an antioncogenic function [13ndash17 20 39 40] ATBF1could inhibit the activity of the AFP promoter via its regionsfrom minus4995 to minus2953 andor from minus178 to +45 [4 26] SinceΔ27Luc had a remarkably basal promoter activity comparedwith 02Luc (Figure 2(b)) we initially focused on the distalregion of the AFP promoter for involvement of the TGF-120573Smad signaling with ATBF1 As expected the TGF-120573-mediated repressive element in the AFP promoter lay near119864119861 which contains a distal AT-motif The luciferase reporter

conjugated with the inhibitory element including a distalAT-motif (352Luc) showed coordinated inhibitory actionbetween the TGF-120573 signaling and ATBF1 (Figure 3(f))Therefore we were struck with the notion that ATBF1physically interacts with Smads which play key roles in thecanonical TGF-120573 signaling pathway [18ndash20] Not surpris-ingly ATBF1 could interact with Smad2 and Smad3 althoughC-terminal phosphorylation of R-Smads was dispensable

(Figure 5(a) and data not shown) In addition both the N-terminal (ATBF1N) and the C-terminal mutants of ATBF1(ATBF1C) but not the middle domain of ATBF1 (ATBF1M)which possesses DNA-binding activity [12] could interactwith R-Smads (Figures 5(c)ndash5(e))We hypothesized that bothATBF1N and ATBF1C might compete with wildtype ATBF1for Smad binding to rescueATBF1-mediated repression of theAFP promoter uponTGF-120573 stimulation because both of themhave the ability to bind to R-Smads According to our expec-tation both ATBF1N and ATBF1C improved the reporteractivity decreased by the cooperative ATBF1 and TGF-120573signaling (Figures 6(a) and 6(b)) As shown in Figure 2(b)02Luc activity could also be reduced upon TGF-120573 stimu-lation despite very low basal promoter activity Because ofthe presence of a proximal AT-motif within this region wespeculated that a proximal AT-motif also contributes to TGF-120573-mediated-repression of the activity of the AFP promoterThe deletion of a proximal AT-motif from 3101584052 65CMVLucled us to recognize the importance of a proximal AT-motif aswell as a distal AT-motif for TGF-120573-mediated repression ofthe activity of theAFP promoter (Figure 4(b)) A Smad bind-ing element (SBE) composed of consensus 51015840-AGAC-31015840 isflanked to a proximal AT-motif However themutation of theSBE in 3101584052Luc showed reduced basal promoter activity butstill maintained TGF-120573-mediated repression of the AFP pro-moter (Figure 7(b)) Thus Smad might not be necessary tobind to a promoter element(s) in the AFP gene directly whenATBF1 cooperates with TGF-120573 signaling whereas p53-mediated repression of the AFP promoter activity needs

Journal of Signal Transduction 9

SBEminus3422

minus3422

minus3562

minus3562

LucAT-motif

minus178 +45

+1

LucAT-motif

AT-motif

AT-motifminus178 +45

+1

399840052Luc

399840052mSBELuc

(a)

00

02

04

06

08

10

12

Relat

ive l

ucife

rase

activ

ity

TGF-120573 (minus)TGF-120573 (+)

lowastlowast

lowastlowast

399840052Luc 399840052mSBELuc

(b)

DNAP

Flag-Smad3IB flag

IB HA

Totallysates

HA-ATBF1

HA-ATBF1

Flag-Smad3IB flag

IB HA

p-Smad3IB PS3

IB V5 ALK5caV5

Biotinylated probe minus + + +

Flag-Smad3HA-ATBF1

+ + + minusminus + +minus

ALK5caV5 + + + minus

(AT-motif)3

(c)

Figure 7 SBE in the proximal region of the AFP promoter is not essential for TGF-120573-mediated suppression of the AFP promoter (a)Schematic presentation of 3101584052mSBELuc A putative SBE 51015840-CAGATA-31015840 was mutated to 51015840-TTTTTT-31015840 (b) Effect of TGF-120573 on 3101584052Lucand 3101584052mSBELuc activities HepG2 cells were transfected with each reporter plasmid (200 ng) The cells were then stimulated with 5 ngmLTGF-120573 (c) Indirect binding of Smad3 to an AT-motif via ATBF1 The indicated cell lysates were mixed with biotinylated AT-motifs Thenstreptavidin-agarose was added to the mixture Subsequently a protein(s) bound to streptavidin-agarose was purified After SDS-PAGE IBwas carried out using anti-Flag or anti-HA antibody Using the total lysates each protein was detected with anti-Flag anti-HA anti-V5 oranti-phosphorylated Smad3 (PS3) antibody

direct binding of Smads to DNA [22 23] The requirement ofdirect DNA binding for R-Smads is probably dependent onwhich transcriptional regulator(s) they cooperate or syner-gize with Indeed Smad3 could bind toAT-motifs in the pres-ence of ATBF1 but not in its absence (Figure 7(c)) Althoughwe do not know if the ATBF1Smad complex at a distalAT-motif can physically interact with that at a proximal

AT-motif directly or indirectly through another protein(s)(ldquoXrdquo Figure 8) both AT-motifs are definitely important forcooperation between ATBF1 and the TGF-120573 signaling tosuppress the activity of the AFP promoter

ATBF-1 is known to compete with HNF-1 for binding toAT-motifs in the AFP promoter [4] Thus the following pos-sibility can be speculated upon TGF-120573 stimulation Smads

10 Journal of Signal Transduction

AT-motif

AT-motif

AFP

ATBF1

ATBF1ATBF1Smad23-Smad4

X

Smad23-Smad4

ATBF1

Figure 8 Proposed inhibitory mechanism by which the TGF-120573Smad signaling and ATBF1 cooperatively regulate the activity ofthe AFP promoter The distal and proximal AT-motifs in the AFPpromoter are bound by ATBF1 Upon TGF-120573 signaling the R-SmadsSmad4 complex interacts with ATBF1 to make a repressivecomplex for the activity of the AFP promoter However whetheranother protein(s) (termed ldquoXrdquo) is essential for the R-SmadSmad4ATBF1 complex remains veiled

can not only bind to AT-motifs via ATBF-1 indirectly but alsointeract with HNF-1 to dissociate it from AT-motifs Due todual function of Smads the AFP prompter activity might beinhibited This possibility is interesting to be investigated

p53-mediated repression of the AFP promoter activityneeds direct binding of Smads to SBEp53REWilkinson et alfurther demonstrated that mSin3A and SnoN which is adirect target gene of TGF-120573 signal are recruited by p53Smadscomplex when the transcription of the AFP gene is inhib-ited [22 23] In our current study the region includingSBEp53RE in theAFP promoterwas not investigated becausethe basal reporter activities of 3Luc 16Luc and 09Luc werevery low compared with that of Δ27Luc (Figure 2(b)) Itis possible that ATBF1 might interplay with p53 via Smadcomplex to effectively repress the transcription of the AFPgene Further experiments are needed in the future to confirmthese hypotheses as well

5 Conclusion

In summary we report a novel interaction between ATBF1and Smads The interaction between ATBF1 and Smadsappears to cooperatively inhibit the transcription of AFPgene upon TGF-120573 signaling In particular both proximal anddistal AT-motifs are required for TGF-120573Smad signaling tocounteract with the transcription of the AFP gene

Conflict of Interests

The authors have no competing financial interests to declare

Acknowledgments

This research was supported by a Grant-in-Aid for Explor-atory Research (A) from the Japan Society for the Promotionof Science (JSPS) (to Nobuo Sakata) the MEXT-Supported

Program for the Strategic Research Foundation at PrivateUniversities (2013ndash2017) (to Susumu Itoh) the Takeda Sci-ence Foundation (to Susumu Itoh) the Smoking ResearchFoundation (to Susumu Itoh) the Daiichi-Sankyo Founda-tion of Life Science (to Susumu Itoh) the Naito Founda-tion (to Susumu Itoh) the Vehicle Racing Commemora-tive Foundation (to Susumu Itoh) and a Grant-in-Aid forYoung Scientists of Showa Pharmaceutical University (toSouichi Ikeno) The authors were also supported by the JointUsageResearch Program of the Medical Research Institutethe Tokyo Medical and Dental University and by the Core-to-Core Program ldquoCooperative International Framework inTGF-120573 Family Signalingrdquo of the JSPS They thank Ms FMiyamasu for excellent English proofreading

References

[1] M A Dziadek and G K Andrews ldquoTissue specificity of 120572-fetoprotein messenger RNA expression during mouse embryo-genesisrdquoThe EMBO Journal vol 2 no 4 pp 549ndash554 1983

[2] J L Nahon I Tratner A Poliard et al ldquoAlbumin and 120572-feto-protein gene expression in various nonhepatic rat tissuesrdquo TheJournal of Biological Chemistry vol 263 no 23 pp 11436ndash114421988

[3] A A Terentiev and N T Moldogazieva ldquo120572-fetoprotein a ren-aissancerdquo Tumor Biology vol 34 no 4 pp 2075ndash2091 2013

[4] H Yasuda A Mizuno T Tamaoki and T Morinaga ldquoATBF1 amultiple-homeodomain zinc finger protein selectively down-regulates AT-rich elements of the human 120572-fetoprotein generdquoMolecular and Cellular Biology vol 14 no 2 pp 1395ndash14011994

[5] K Sawadaishi T Morinaga and T Tamaoki ldquoInteraction of ahepatoma-specific nuclear factor with transcription-regulatorysequences of the human 120572-fetoprotein and albumin genesrdquoMolecular andCellular Biology vol 8 no 12 pp 5179ndash5187 1988

[6] H Nakabayashi Y Koyama H Suzuki et al ldquoFunctional map-ping of tissue-specific elements of the human 120572-fetoproteingene enhancerrdquo Biochemical and Biophysical Research Commu-nications vol 318 no 3 pp 773ndash785 2004

[7] T Morinaga H Yasuda T Hashimoto K Higashio and TTamaoki ldquoA human 120572-fetoprotein enhancer-binding proteinATBF1 contains four homeodomains and seventeen zinc fin-gersrdquo Molecular and Cellular Biology vol 11 no 12 pp 6041ndash6049 1991

[8] Y Miura T Tam A Ido et al ldquoCloning and characterization ofan ATBF1 isoform that expresses in a neuronal differentiation-dependent mannerrdquo The Journal of Biological Chemistry vol270 no 45 pp 26840ndash26848 1995

[9] H Kataoka Y Miura T Joh et al ldquo120572-fetoprotein producinggastric cancer lacks transcription factor ATBF1rdquo Oncogene vol20 no 7 pp 869ndash873 2001

[10] T Ninomiya K Mihara K Fushimi Y Hayashi T Hashimoto-Tamaoki and T Tamaoki ldquoRegulation of the 120572-fetoprotein geneby the isoforms of ATBF1 transcription factor in human hep-atomardquo Hepatology vol 35 no 1 pp 82ndash87 2002

[11] P Kaspar M Dvorakova J Kralova P Pajer Z Kozmik andM Dvorak ldquoMyb-interacting protein ATBF1 represses tran-scriptional activity of Myb oncoproteinrdquoThe Journal of Biologi-cal Chemistry vol 274 no 20 pp 14422ndash14428 1999

[12] F B Berry YMiura KMihara et al ldquoPositive and negative reg-ulation of myogenic differentiation of C2C12 cells by isoforms

Journal of Signal Transduction 11

of the multiple homeodomain zinc finger transcription factorATBF1rdquoThe Journal of Biological Chemistry vol 276 no 27 pp25057ndash25065 2001

[13] X Sun H F Frierson C Chen et al ldquoFrequent somaticmutations of the transcription factor ATBF1 in human prostatecancerrdquo Nature Genetics vol 37 no 6 pp 407ndash412 2005

[14] X Sun Y Zhou K B Otto et al ldquoInfrequentmutation of ATBF1in human breast cancerrdquo Journal of Cancer Research and ClinicalOncology vol 133 no 2 pp 103ndash105 2007

[15] Y G Cho J H Song C J Kim et al ldquoGenetic alterations of theATBF1 gene in gastric cancerrdquo Clinical Cancer Research vol 13no 15 pp 4355ndash4359 2007

[16] K Kai Z Zhang H Yamashita Y Yamamoto Y Miura and HIwase ldquoLoss of heterozygosity at the ATBF1-A locus located inthe 16q22 minimal region in breast cancerrdquo BMC Cancer vol 8article 262 2008

[17] A M Cleton-Jansen R van Eijk M Lombaerts et al ldquoATBF1and NQO1 as candidate targets for allelic loss at chromosomearm 16q in breast cancer absence of somatic ATBF1 mutationsand no role for the C609TNQO1 polymorphismrdquo BMCCancervol 8 article 105 2008

[18] J Massague J Seoane and D Wotton ldquoSmad transcriptionfactorsrdquo Genes amp Development vol 19 no 23 pp 2783ndash28102005

[19] AMoustakas and C H Heldin ldquoThe regulation of TGF120573 signaltransductionrdquo Development vol 136 no 22 pp 3699ndash37142009

[20] J Massague ldquoTGF120573 signalling in contextrdquo Nature ReviewsMolecular Cell Biology vol 13 no 10 pp 616ndash630 2012

[21] K Nakao K Nakata S Mitsuoka et al ldquoTransforming growthfactor 120573 1 differentially regulates 120572-fetoprotein and albumin inHuH-7 human hepatoma cellsrdquo Biochemical and BiophysicalResearch Communications vol 174 no 3 pp 1294ndash1299 1991

[22] D SWilkinson S K Ogden S A Stratton et al ldquoA direct inter-section between p53 and transforming growth factor 120573 path-ways targets chromatin modification and transcription repres-sion of the 120572-fetoprotein generdquoMolecular and Cellular Biologyvol 25 no 3 pp 1200ndash1212 2005

[23] D S Wilkinson W W Tsai M A Schumacher and M CBarton ldquoChromatin-bound p53 anchors activated Smads andthe mSin3A corepressor to confer transforming-growth-factor-120573-mediated transcription repressionrdquo Molecular and CellularBiology vol 28 no 6 pp 1988ndash1998 2008

[24] C G Jung H J Kim M Kawaguchi et al ldquoHomeotic factorATBF1 induces the cell cycle arrest associated with neuronaldifferentiationrdquo Development vol 132 no 23 pp 5137ndash51452005

[25] X Y Dong X Sun P Guo et al ldquoATBF1 inhibits estrogenreceptor (ER) function by selectively competing with AIB1 forbinding to the ER in ER-positive breast cancer cellsrdquoThe Journalof Biological Chemistry vol 285 no 43 pp 32801ndash32809 2010

[26] H Kataoka P Bonnefin D Vieyra et al ldquoING1 repressestranscription by direct DNA binding and through effects onp53rdquo Cancer Research vol 63 no 18 pp 5785ndash5792 2003

[27] A Nakao T Imamura S Souchelnytskyi et al ldquoTGF-120573receptor-mediated signalling through Smad2 Smad3 andSmad4rdquoThe EMBO Journal vol 16 no 17 pp 5353ndash5362 1997

[28] M Fujii K Takeda T Imamura et al ldquoRoles of bone morpho-genetic protein type I receptors and Smad proteins in osteoblastand chondroblast differentiationrdquoMolecular Biology of the Cellvol 10 no 11 pp 3801ndash3812 1999

[29] YWatanabe S Itoh T Goto et al ldquoTMEPAI a transmembraneTGF-120573-inducible protein sequesters smad proteins from activeparticipation in TGF-120573 signalingrdquoMolecular Cell vol 37 no 1pp 123ndash134 2010

[30] S Itoh M Thorikay M Kowanetz et al ldquoElucidation of Smadrequirement in transforming growth factor-120573 type I receptor-induced responsesrdquo The Journal of Biological Chemistry vol278 no 6 pp 3751ndash3761 2003

[31] N Nakano S Itoh Y Watanabe K Maeyama F Itoh and MKato ldquoRequirement of TCF7L2 for TGF-120573 dependent transcrip-tional activation of the TMEPAI generdquoThe Journal of BiologicalChemistry vol 285 no 49 pp 38023ndash38033 2010

[32] K Watanabe A Saito and T Tamaoki ldquoCell-specific enhanceractivity in a far upstream region of the human 120572-fetoproteingenerdquo The Journal of Biological Chemistry vol 262 no 10 pp4812ndash4818 1987

[33] H Nakabayashi K Watanabe A Saito A Otsuru KSawadaishi and T Tamaoki ldquoTranscriptional regulation of 120573-fetoprotein expression by dexamethasone in human hepatomacellsrdquo The Journal of Biological Chemistry vol 264 no 1 pp266ndash271 1989

[34] H Nakabayashi T Hashimoto Y Miyao K-K Tjong J Chanand T Tamaoki ldquoA position-dependent silencer plays a majorrole in repressing 120573-fetoprotein expression in human hep-atomardquoMolecular and Cellular Biology vol 11 no 12 pp 5885ndash5893 1991

[35] S Zhang T S Kim Y Dong et al ldquoAT motif binding factor 1(ATBF1) is highly phosphorylated in embryonic brain and pro-tected from cleavage by calpain-1rdquo Biochemical and BiophysicalResearch Communications vol 427 no 3 pp 537ndash541 2012

[36] L J Jonk S Itoh C H Heldin P ten Dijke and W KruijerldquoIdentification and functional characterization of a smad bind-ing element (SBE) in the JunBpromoter that acts as a transform-ing growth factor-120573 activin and bone morphogenetic protein-inducible enhancerrdquo The Journal of Biological Chemistry vol273 no 33 pp 21145ndash21152 1998

[37] S Dennler S Itoh D Vivien P ten Dijke S Huet and J Gau-thier ldquoDirect binding of Smad3 and Smad4 to critical TGF120573-inducible elements in the promoter of human plasminogenactivator inhibitor-type 1 generdquo The EMBO Journal vol 17 no11 pp 3091ndash3100 1998

[38] A Ido K Nakata Y Kato et al ldquoGene therapy for hep-atoma cells using a retrovirus vector carrying herpes simplexvirus thymidine kinase gene under the control of human 120572-fetoprotein gene promoterrdquo Cancer Research vol 55 no 14 pp3105ndash3109 1995

[39] A B Roberts and L M Wakefield ldquoThe two faces of trans-forming growth factor 120573 in carcinogenesisrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 15 pp 8621ndash8623 2003

[40] B Bierie and H L Moses ldquoTumour microenvironment TGF120573the molecular Jekyll and Hyde of cancerrdquo Nature ReviewsCancer vol 6 no 7 pp 506ndash520 2006

Journal of Signal Transduction 7

Flag-Smad3

Smad3

ATBF1

Smad3

ALK5ca

minus +minus

++ +

Myc-ATBF1ALK5caV5

IP MycIB flag

IB Myc

IB flag

IB V5

(a)

3703

ATBF1

1

ATBF1N

8951

ATBF1M

3293894

ATBF1C

37033290

Zinc fingerHomeodomain

(b)

HA-ATBF1NFlag-Smad2Flag-Smad3

IP flagIB HA

IB HA

IB flag

minus + minusminus

Smad2Smad3

ATBF1N

ATBF1N

+minus

(c)

HA-ATBF1MFlag-Smad2Flag-Smad3

minus +minus +minus

IP flagIB HA

IB HA

IB flag

minus

ATBF1M

ATBF1M

Smad2Smad3

(d)

HA-ATBF1CFlag-Smad2Flag-Smad3

minus +minus +minus

IP flagIB HA

IB HA

IB flag

minus

ATBF1C

ATBF1C

Smad2Smad3

(e)

Figure 5 Association of Smads with ATBF1 (a) Interaction of Smad3 with ATBF1 COS7 cells were transfected with Flag-Smad3 (1120583g) withMyc-ATBF1 (2120583g) in combination with ALK5caV5 (05 120583g) or without it Cell lysates were subjected to immunoprecipitation (IP) with anti-Myc antibody followed by IB with anti-Flag antibody to show the interaction of Flag-Smad3 with Myc-ATBF1 (b) Schematic presentation ofATBF1 mutants (cndashe) Interaction of each ATBF1 mutant with Smad2 or Smad3 COS7 cells were transfected with Flag-Smad2 or Flag-Smad3(15120583g) with HA-ATBF1N (2120583g) (c) HA-ATBF1M (2120583g) (d) or HA-ATBF1C (2120583g) (e) Cell lysates were subjected to IP with anti-Flagantibody followed by IB with anti-HA antibody to show the interaction of Smad2 and Smad3 with ATBF1 mutants

and C-terminal domains are needed for ATBF1 to interactwith R-Smads

36 Indirect Binding of Smads to the AFP Promoter via ATBF1We could find only one Smad binding element (SBE) [36 37]in 3101584052Luc (Figure 7(a))Thus we examinedwhether this SBEis necessary for TGF-120573-mediated repression of the AFP pro-moter For that purpose wemade one reporter construct thathad mutations within the SBE of 3101584052Luc (3101584052mSBELuc)

(Figure 7(a)) However 3101584052mSBELuc activity was still inhib-ited by TGF-120573 although its basal level became weaker thanthat of 3101584052Luc (Figure 7(b)) Thus the SBE in the AFP pro-moter is not essential for TGF-120573Smad signaling to repressthe activity of the AFP promoter Indeed a DNAP assayshowed that Smad3 can be detected in the presence of ATBF1using three repeats of an AT-motif as the probe (Figure 7(c))Thus Smad3 probably binds to the promoter region of theAFP gene indirectly through its interaction with ATBF1

8 Journal of Signal Transduction

00

02

04

06

08

10

12

14

16

ATBF1ATBF1N

Rela

tive l

ucife

rase

activ

ity

+ + ++minus

minus minus

TGF-120573 (minus)TGF-120573 (+)

lowast

lowast

lowastlowastlowast

(a)

00

02

04

06

08

10

12

14

ATBF1ATBF1C

Rela

tive l

ucife

rase

activ

ity

+ + ++minus

minus minus

TGF-120573 (minus)TGF-120573 (+)

lowast

lowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

(b)

Figure 6 ATBF1N and ATBF1C perturb ATBF1-mediated suppression of the activity of theAFP promoter upon TGF-120573 stimulation ATBF1N(a) and ATNF1C (b) rescue ATBF1-mediated repression of the activity of the AFP promoter HepG2 cells were transfected with increasedamounts of either ATBF1N (50 100 or 200 ng) (a) or ATBF1C (50 100 or 200 ng) (b) together with ATBF1 (200 ng) and Δ27Luc (200 ng)The cells were then stimulated with 5 ngmL TGF-120573

4 Discussion

The hepatoma cells HuH-7 and HepG2 cells are known tosecrete a large and a low amount of AFP respectively [38] Agrowing body of evidence indicates that the level of AFP inserum from patients is linked to HCC tumorigenicity [3] p53has been reported to cooperate with the TGF-120573Smad path-way to repress AFP expression via induction of SnoN [22]However it remains veiled whether ATBF1 which is knownto blockAFP transcription [4 10] can also regulate the activ-ity of the AFP promoter in consort with the TGF-120573Smadpathway because both ATBF1 and TGF-120573Smad might pos-sess an antioncogenic function [13ndash17 20 39 40] ATBF1could inhibit the activity of the AFP promoter via its regionsfrom minus4995 to minus2953 andor from minus178 to +45 [4 26] SinceΔ27Luc had a remarkably basal promoter activity comparedwith 02Luc (Figure 2(b)) we initially focused on the distalregion of the AFP promoter for involvement of the TGF-120573Smad signaling with ATBF1 As expected the TGF-120573-mediated repressive element in the AFP promoter lay near119864119861 which contains a distal AT-motif The luciferase reporter

conjugated with the inhibitory element including a distalAT-motif (352Luc) showed coordinated inhibitory actionbetween the TGF-120573 signaling and ATBF1 (Figure 3(f))Therefore we were struck with the notion that ATBF1physically interacts with Smads which play key roles in thecanonical TGF-120573 signaling pathway [18ndash20] Not surpris-ingly ATBF1 could interact with Smad2 and Smad3 althoughC-terminal phosphorylation of R-Smads was dispensable

(Figure 5(a) and data not shown) In addition both the N-terminal (ATBF1N) and the C-terminal mutants of ATBF1(ATBF1C) but not the middle domain of ATBF1 (ATBF1M)which possesses DNA-binding activity [12] could interactwith R-Smads (Figures 5(c)ndash5(e))We hypothesized that bothATBF1N and ATBF1C might compete with wildtype ATBF1for Smad binding to rescueATBF1-mediated repression of theAFP promoter uponTGF-120573 stimulation because both of themhave the ability to bind to R-Smads According to our expec-tation both ATBF1N and ATBF1C improved the reporteractivity decreased by the cooperative ATBF1 and TGF-120573signaling (Figures 6(a) and 6(b)) As shown in Figure 2(b)02Luc activity could also be reduced upon TGF-120573 stimu-lation despite very low basal promoter activity Because ofthe presence of a proximal AT-motif within this region wespeculated that a proximal AT-motif also contributes to TGF-120573-mediated-repression of the activity of the AFP promoterThe deletion of a proximal AT-motif from 3101584052 65CMVLucled us to recognize the importance of a proximal AT-motif aswell as a distal AT-motif for TGF-120573-mediated repression ofthe activity of theAFP promoter (Figure 4(b)) A Smad bind-ing element (SBE) composed of consensus 51015840-AGAC-31015840 isflanked to a proximal AT-motif However themutation of theSBE in 3101584052Luc showed reduced basal promoter activity butstill maintained TGF-120573-mediated repression of the AFP pro-moter (Figure 7(b)) Thus Smad might not be necessary tobind to a promoter element(s) in the AFP gene directly whenATBF1 cooperates with TGF-120573 signaling whereas p53-mediated repression of the AFP promoter activity needs

Journal of Signal Transduction 9

SBEminus3422

minus3422

minus3562

minus3562

LucAT-motif

minus178 +45

+1

LucAT-motif

AT-motif

AT-motifminus178 +45

+1

399840052Luc

399840052mSBELuc

(a)

00

02

04

06

08

10

12

Relat

ive l

ucife

rase

activ

ity

TGF-120573 (minus)TGF-120573 (+)

lowastlowast

lowastlowast

399840052Luc 399840052mSBELuc

(b)

DNAP

Flag-Smad3IB flag

IB HA

Totallysates

HA-ATBF1

HA-ATBF1

Flag-Smad3IB flag

IB HA

p-Smad3IB PS3

IB V5 ALK5caV5

Biotinylated probe minus + + +

Flag-Smad3HA-ATBF1

+ + + minusminus + +minus

ALK5caV5 + + + minus

(AT-motif)3

(c)

Figure 7 SBE in the proximal region of the AFP promoter is not essential for TGF-120573-mediated suppression of the AFP promoter (a)Schematic presentation of 3101584052mSBELuc A putative SBE 51015840-CAGATA-31015840 was mutated to 51015840-TTTTTT-31015840 (b) Effect of TGF-120573 on 3101584052Lucand 3101584052mSBELuc activities HepG2 cells were transfected with each reporter plasmid (200 ng) The cells were then stimulated with 5 ngmLTGF-120573 (c) Indirect binding of Smad3 to an AT-motif via ATBF1 The indicated cell lysates were mixed with biotinylated AT-motifs Thenstreptavidin-agarose was added to the mixture Subsequently a protein(s) bound to streptavidin-agarose was purified After SDS-PAGE IBwas carried out using anti-Flag or anti-HA antibody Using the total lysates each protein was detected with anti-Flag anti-HA anti-V5 oranti-phosphorylated Smad3 (PS3) antibody

direct binding of Smads to DNA [22 23] The requirement ofdirect DNA binding for R-Smads is probably dependent onwhich transcriptional regulator(s) they cooperate or syner-gize with Indeed Smad3 could bind toAT-motifs in the pres-ence of ATBF1 but not in its absence (Figure 7(c)) Althoughwe do not know if the ATBF1Smad complex at a distalAT-motif can physically interact with that at a proximal

AT-motif directly or indirectly through another protein(s)(ldquoXrdquo Figure 8) both AT-motifs are definitely important forcooperation between ATBF1 and the TGF-120573 signaling tosuppress the activity of the AFP promoter

ATBF-1 is known to compete with HNF-1 for binding toAT-motifs in the AFP promoter [4] Thus the following pos-sibility can be speculated upon TGF-120573 stimulation Smads

10 Journal of Signal Transduction

AT-motif

AT-motif

AFP

ATBF1

ATBF1ATBF1Smad23-Smad4

X

Smad23-Smad4

ATBF1

Figure 8 Proposed inhibitory mechanism by which the TGF-120573Smad signaling and ATBF1 cooperatively regulate the activity ofthe AFP promoter The distal and proximal AT-motifs in the AFPpromoter are bound by ATBF1 Upon TGF-120573 signaling the R-SmadsSmad4 complex interacts with ATBF1 to make a repressivecomplex for the activity of the AFP promoter However whetheranother protein(s) (termed ldquoXrdquo) is essential for the R-SmadSmad4ATBF1 complex remains veiled

can not only bind to AT-motifs via ATBF-1 indirectly but alsointeract with HNF-1 to dissociate it from AT-motifs Due todual function of Smads the AFP prompter activity might beinhibited This possibility is interesting to be investigated

p53-mediated repression of the AFP promoter activityneeds direct binding of Smads to SBEp53REWilkinson et alfurther demonstrated that mSin3A and SnoN which is adirect target gene of TGF-120573 signal are recruited by p53Smadscomplex when the transcription of the AFP gene is inhib-ited [22 23] In our current study the region includingSBEp53RE in theAFP promoterwas not investigated becausethe basal reporter activities of 3Luc 16Luc and 09Luc werevery low compared with that of Δ27Luc (Figure 2(b)) Itis possible that ATBF1 might interplay with p53 via Smadcomplex to effectively repress the transcription of the AFPgene Further experiments are needed in the future to confirmthese hypotheses as well

5 Conclusion

In summary we report a novel interaction between ATBF1and Smads The interaction between ATBF1 and Smadsappears to cooperatively inhibit the transcription of AFPgene upon TGF-120573 signaling In particular both proximal anddistal AT-motifs are required for TGF-120573Smad signaling tocounteract with the transcription of the AFP gene

Conflict of Interests

The authors have no competing financial interests to declare

Acknowledgments

This research was supported by a Grant-in-Aid for Explor-atory Research (A) from the Japan Society for the Promotionof Science (JSPS) (to Nobuo Sakata) the MEXT-Supported

Program for the Strategic Research Foundation at PrivateUniversities (2013ndash2017) (to Susumu Itoh) the Takeda Sci-ence Foundation (to Susumu Itoh) the Smoking ResearchFoundation (to Susumu Itoh) the Daiichi-Sankyo Founda-tion of Life Science (to Susumu Itoh) the Naito Founda-tion (to Susumu Itoh) the Vehicle Racing Commemora-tive Foundation (to Susumu Itoh) and a Grant-in-Aid forYoung Scientists of Showa Pharmaceutical University (toSouichi Ikeno) The authors were also supported by the JointUsageResearch Program of the Medical Research Institutethe Tokyo Medical and Dental University and by the Core-to-Core Program ldquoCooperative International Framework inTGF-120573 Family Signalingrdquo of the JSPS They thank Ms FMiyamasu for excellent English proofreading

References

[1] M A Dziadek and G K Andrews ldquoTissue specificity of 120572-fetoprotein messenger RNA expression during mouse embryo-genesisrdquoThe EMBO Journal vol 2 no 4 pp 549ndash554 1983

[2] J L Nahon I Tratner A Poliard et al ldquoAlbumin and 120572-feto-protein gene expression in various nonhepatic rat tissuesrdquo TheJournal of Biological Chemistry vol 263 no 23 pp 11436ndash114421988

[3] A A Terentiev and N T Moldogazieva ldquo120572-fetoprotein a ren-aissancerdquo Tumor Biology vol 34 no 4 pp 2075ndash2091 2013

[4] H Yasuda A Mizuno T Tamaoki and T Morinaga ldquoATBF1 amultiple-homeodomain zinc finger protein selectively down-regulates AT-rich elements of the human 120572-fetoprotein generdquoMolecular and Cellular Biology vol 14 no 2 pp 1395ndash14011994

[5] K Sawadaishi T Morinaga and T Tamaoki ldquoInteraction of ahepatoma-specific nuclear factor with transcription-regulatorysequences of the human 120572-fetoprotein and albumin genesrdquoMolecular andCellular Biology vol 8 no 12 pp 5179ndash5187 1988

[6] H Nakabayashi Y Koyama H Suzuki et al ldquoFunctional map-ping of tissue-specific elements of the human 120572-fetoproteingene enhancerrdquo Biochemical and Biophysical Research Commu-nications vol 318 no 3 pp 773ndash785 2004

[7] T Morinaga H Yasuda T Hashimoto K Higashio and TTamaoki ldquoA human 120572-fetoprotein enhancer-binding proteinATBF1 contains four homeodomains and seventeen zinc fin-gersrdquo Molecular and Cellular Biology vol 11 no 12 pp 6041ndash6049 1991

[8] Y Miura T Tam A Ido et al ldquoCloning and characterization ofan ATBF1 isoform that expresses in a neuronal differentiation-dependent mannerrdquo The Journal of Biological Chemistry vol270 no 45 pp 26840ndash26848 1995

[9] H Kataoka Y Miura T Joh et al ldquo120572-fetoprotein producinggastric cancer lacks transcription factor ATBF1rdquo Oncogene vol20 no 7 pp 869ndash873 2001

[10] T Ninomiya K Mihara K Fushimi Y Hayashi T Hashimoto-Tamaoki and T Tamaoki ldquoRegulation of the 120572-fetoprotein geneby the isoforms of ATBF1 transcription factor in human hep-atomardquo Hepatology vol 35 no 1 pp 82ndash87 2002

[11] P Kaspar M Dvorakova J Kralova P Pajer Z Kozmik andM Dvorak ldquoMyb-interacting protein ATBF1 represses tran-scriptional activity of Myb oncoproteinrdquoThe Journal of Biologi-cal Chemistry vol 274 no 20 pp 14422ndash14428 1999

[12] F B Berry YMiura KMihara et al ldquoPositive and negative reg-ulation of myogenic differentiation of C2C12 cells by isoforms

Journal of Signal Transduction 11

of the multiple homeodomain zinc finger transcription factorATBF1rdquoThe Journal of Biological Chemistry vol 276 no 27 pp25057ndash25065 2001

[13] X Sun H F Frierson C Chen et al ldquoFrequent somaticmutations of the transcription factor ATBF1 in human prostatecancerrdquo Nature Genetics vol 37 no 6 pp 407ndash412 2005

[14] X Sun Y Zhou K B Otto et al ldquoInfrequentmutation of ATBF1in human breast cancerrdquo Journal of Cancer Research and ClinicalOncology vol 133 no 2 pp 103ndash105 2007

[15] Y G Cho J H Song C J Kim et al ldquoGenetic alterations of theATBF1 gene in gastric cancerrdquo Clinical Cancer Research vol 13no 15 pp 4355ndash4359 2007

[16] K Kai Z Zhang H Yamashita Y Yamamoto Y Miura and HIwase ldquoLoss of heterozygosity at the ATBF1-A locus located inthe 16q22 minimal region in breast cancerrdquo BMC Cancer vol 8article 262 2008

[17] A M Cleton-Jansen R van Eijk M Lombaerts et al ldquoATBF1and NQO1 as candidate targets for allelic loss at chromosomearm 16q in breast cancer absence of somatic ATBF1 mutationsand no role for the C609TNQO1 polymorphismrdquo BMCCancervol 8 article 105 2008

[18] J Massague J Seoane and D Wotton ldquoSmad transcriptionfactorsrdquo Genes amp Development vol 19 no 23 pp 2783ndash28102005

[19] AMoustakas and C H Heldin ldquoThe regulation of TGF120573 signaltransductionrdquo Development vol 136 no 22 pp 3699ndash37142009

[20] J Massague ldquoTGF120573 signalling in contextrdquo Nature ReviewsMolecular Cell Biology vol 13 no 10 pp 616ndash630 2012

[21] K Nakao K Nakata S Mitsuoka et al ldquoTransforming growthfactor 120573 1 differentially regulates 120572-fetoprotein and albumin inHuH-7 human hepatoma cellsrdquo Biochemical and BiophysicalResearch Communications vol 174 no 3 pp 1294ndash1299 1991

[22] D SWilkinson S K Ogden S A Stratton et al ldquoA direct inter-section between p53 and transforming growth factor 120573 path-ways targets chromatin modification and transcription repres-sion of the 120572-fetoprotein generdquoMolecular and Cellular Biologyvol 25 no 3 pp 1200ndash1212 2005

[23] D S Wilkinson W W Tsai M A Schumacher and M CBarton ldquoChromatin-bound p53 anchors activated Smads andthe mSin3A corepressor to confer transforming-growth-factor-120573-mediated transcription repressionrdquo Molecular and CellularBiology vol 28 no 6 pp 1988ndash1998 2008

[24] C G Jung H J Kim M Kawaguchi et al ldquoHomeotic factorATBF1 induces the cell cycle arrest associated with neuronaldifferentiationrdquo Development vol 132 no 23 pp 5137ndash51452005

[25] X Y Dong X Sun P Guo et al ldquoATBF1 inhibits estrogenreceptor (ER) function by selectively competing with AIB1 forbinding to the ER in ER-positive breast cancer cellsrdquoThe Journalof Biological Chemistry vol 285 no 43 pp 32801ndash32809 2010

[26] H Kataoka P Bonnefin D Vieyra et al ldquoING1 repressestranscription by direct DNA binding and through effects onp53rdquo Cancer Research vol 63 no 18 pp 5785ndash5792 2003

[27] A Nakao T Imamura S Souchelnytskyi et al ldquoTGF-120573receptor-mediated signalling through Smad2 Smad3 andSmad4rdquoThe EMBO Journal vol 16 no 17 pp 5353ndash5362 1997

[28] M Fujii K Takeda T Imamura et al ldquoRoles of bone morpho-genetic protein type I receptors and Smad proteins in osteoblastand chondroblast differentiationrdquoMolecular Biology of the Cellvol 10 no 11 pp 3801ndash3812 1999

[29] YWatanabe S Itoh T Goto et al ldquoTMEPAI a transmembraneTGF-120573-inducible protein sequesters smad proteins from activeparticipation in TGF-120573 signalingrdquoMolecular Cell vol 37 no 1pp 123ndash134 2010

[30] S Itoh M Thorikay M Kowanetz et al ldquoElucidation of Smadrequirement in transforming growth factor-120573 type I receptor-induced responsesrdquo The Journal of Biological Chemistry vol278 no 6 pp 3751ndash3761 2003

[31] N Nakano S Itoh Y Watanabe K Maeyama F Itoh and MKato ldquoRequirement of TCF7L2 for TGF-120573 dependent transcrip-tional activation of the TMEPAI generdquoThe Journal of BiologicalChemistry vol 285 no 49 pp 38023ndash38033 2010

[32] K Watanabe A Saito and T Tamaoki ldquoCell-specific enhanceractivity in a far upstream region of the human 120572-fetoproteingenerdquo The Journal of Biological Chemistry vol 262 no 10 pp4812ndash4818 1987

[33] H Nakabayashi K Watanabe A Saito A Otsuru KSawadaishi and T Tamaoki ldquoTranscriptional regulation of 120573-fetoprotein expression by dexamethasone in human hepatomacellsrdquo The Journal of Biological Chemistry vol 264 no 1 pp266ndash271 1989

[34] H Nakabayashi T Hashimoto Y Miyao K-K Tjong J Chanand T Tamaoki ldquoA position-dependent silencer plays a majorrole in repressing 120573-fetoprotein expression in human hep-atomardquoMolecular and Cellular Biology vol 11 no 12 pp 5885ndash5893 1991

[35] S Zhang T S Kim Y Dong et al ldquoAT motif binding factor 1(ATBF1) is highly phosphorylated in embryonic brain and pro-tected from cleavage by calpain-1rdquo Biochemical and BiophysicalResearch Communications vol 427 no 3 pp 537ndash541 2012

[36] L J Jonk S Itoh C H Heldin P ten Dijke and W KruijerldquoIdentification and functional characterization of a smad bind-ing element (SBE) in the JunBpromoter that acts as a transform-ing growth factor-120573 activin and bone morphogenetic protein-inducible enhancerrdquo The Journal of Biological Chemistry vol273 no 33 pp 21145ndash21152 1998

[37] S Dennler S Itoh D Vivien P ten Dijke S Huet and J Gau-thier ldquoDirect binding of Smad3 and Smad4 to critical TGF120573-inducible elements in the promoter of human plasminogenactivator inhibitor-type 1 generdquo The EMBO Journal vol 17 no11 pp 3091ndash3100 1998

[38] A Ido K Nakata Y Kato et al ldquoGene therapy for hep-atoma cells using a retrovirus vector carrying herpes simplexvirus thymidine kinase gene under the control of human 120572-fetoprotein gene promoterrdquo Cancer Research vol 55 no 14 pp3105ndash3109 1995

[39] A B Roberts and L M Wakefield ldquoThe two faces of trans-forming growth factor 120573 in carcinogenesisrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 15 pp 8621ndash8623 2003

[40] B Bierie and H L Moses ldquoTumour microenvironment TGF120573the molecular Jekyll and Hyde of cancerrdquo Nature ReviewsCancer vol 6 no 7 pp 506ndash520 2006

8 Journal of Signal Transduction

00

02

04

06

08

10

12

14

16

ATBF1ATBF1N

Rela

tive l

ucife

rase

activ

ity

+ + ++minus

minus minus

TGF-120573 (minus)TGF-120573 (+)

lowast

lowast

lowastlowastlowast

(a)

00

02

04

06

08

10

12

14

ATBF1ATBF1C

Rela

tive l

ucife

rase

activ

ity

+ + ++minus

minus minus

TGF-120573 (minus)TGF-120573 (+)

lowast

lowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

(b)

Figure 6 ATBF1N and ATBF1C perturb ATBF1-mediated suppression of the activity of theAFP promoter upon TGF-120573 stimulation ATBF1N(a) and ATNF1C (b) rescue ATBF1-mediated repression of the activity of the AFP promoter HepG2 cells were transfected with increasedamounts of either ATBF1N (50 100 or 200 ng) (a) or ATBF1C (50 100 or 200 ng) (b) together with ATBF1 (200 ng) and Δ27Luc (200 ng)The cells were then stimulated with 5 ngmL TGF-120573

4 Discussion

The hepatoma cells HuH-7 and HepG2 cells are known tosecrete a large and a low amount of AFP respectively [38] Agrowing body of evidence indicates that the level of AFP inserum from patients is linked to HCC tumorigenicity [3] p53has been reported to cooperate with the TGF-120573Smad path-way to repress AFP expression via induction of SnoN [22]However it remains veiled whether ATBF1 which is knownto blockAFP transcription [4 10] can also regulate the activ-ity of the AFP promoter in consort with the TGF-120573Smadpathway because both ATBF1 and TGF-120573Smad might pos-sess an antioncogenic function [13ndash17 20 39 40] ATBF1could inhibit the activity of the AFP promoter via its regionsfrom minus4995 to minus2953 andor from minus178 to +45 [4 26] SinceΔ27Luc had a remarkably basal promoter activity comparedwith 02Luc (Figure 2(b)) we initially focused on the distalregion of the AFP promoter for involvement of the TGF-120573Smad signaling with ATBF1 As expected the TGF-120573-mediated repressive element in the AFP promoter lay near119864119861 which contains a distal AT-motif The luciferase reporter

conjugated with the inhibitory element including a distalAT-motif (352Luc) showed coordinated inhibitory actionbetween the TGF-120573 signaling and ATBF1 (Figure 3(f))Therefore we were struck with the notion that ATBF1physically interacts with Smads which play key roles in thecanonical TGF-120573 signaling pathway [18ndash20] Not surpris-ingly ATBF1 could interact with Smad2 and Smad3 althoughC-terminal phosphorylation of R-Smads was dispensable

(Figure 5(a) and data not shown) In addition both the N-terminal (ATBF1N) and the C-terminal mutants of ATBF1(ATBF1C) but not the middle domain of ATBF1 (ATBF1M)which possesses DNA-binding activity [12] could interactwith R-Smads (Figures 5(c)ndash5(e))We hypothesized that bothATBF1N and ATBF1C might compete with wildtype ATBF1for Smad binding to rescueATBF1-mediated repression of theAFP promoter uponTGF-120573 stimulation because both of themhave the ability to bind to R-Smads According to our expec-tation both ATBF1N and ATBF1C improved the reporteractivity decreased by the cooperative ATBF1 and TGF-120573signaling (Figures 6(a) and 6(b)) As shown in Figure 2(b)02Luc activity could also be reduced upon TGF-120573 stimu-lation despite very low basal promoter activity Because ofthe presence of a proximal AT-motif within this region wespeculated that a proximal AT-motif also contributes to TGF-120573-mediated-repression of the activity of the AFP promoterThe deletion of a proximal AT-motif from 3101584052 65CMVLucled us to recognize the importance of a proximal AT-motif aswell as a distal AT-motif for TGF-120573-mediated repression ofthe activity of theAFP promoter (Figure 4(b)) A Smad bind-ing element (SBE) composed of consensus 51015840-AGAC-31015840 isflanked to a proximal AT-motif However themutation of theSBE in 3101584052Luc showed reduced basal promoter activity butstill maintained TGF-120573-mediated repression of the AFP pro-moter (Figure 7(b)) Thus Smad might not be necessary tobind to a promoter element(s) in the AFP gene directly whenATBF1 cooperates with TGF-120573 signaling whereas p53-mediated repression of the AFP promoter activity needs

Journal of Signal Transduction 9

SBEminus3422

minus3422

minus3562

minus3562

LucAT-motif

minus178 +45

+1

LucAT-motif

AT-motif

AT-motifminus178 +45

+1

399840052Luc

399840052mSBELuc

(a)

00

02

04

06

08

10

12

Relat

ive l

ucife

rase

activ

ity

TGF-120573 (minus)TGF-120573 (+)

lowastlowast

lowastlowast

399840052Luc 399840052mSBELuc

(b)

DNAP

Flag-Smad3IB flag

IB HA

Totallysates

HA-ATBF1

HA-ATBF1

Flag-Smad3IB flag

IB HA

p-Smad3IB PS3

IB V5 ALK5caV5

Biotinylated probe minus + + +

Flag-Smad3HA-ATBF1

+ + + minusminus + +minus

ALK5caV5 + + + minus

(AT-motif)3

(c)

Figure 7 SBE in the proximal region of the AFP promoter is not essential for TGF-120573-mediated suppression of the AFP promoter (a)Schematic presentation of 3101584052mSBELuc A putative SBE 51015840-CAGATA-31015840 was mutated to 51015840-TTTTTT-31015840 (b) Effect of TGF-120573 on 3101584052Lucand 3101584052mSBELuc activities HepG2 cells were transfected with each reporter plasmid (200 ng) The cells were then stimulated with 5 ngmLTGF-120573 (c) Indirect binding of Smad3 to an AT-motif via ATBF1 The indicated cell lysates were mixed with biotinylated AT-motifs Thenstreptavidin-agarose was added to the mixture Subsequently a protein(s) bound to streptavidin-agarose was purified After SDS-PAGE IBwas carried out using anti-Flag or anti-HA antibody Using the total lysates each protein was detected with anti-Flag anti-HA anti-V5 oranti-phosphorylated Smad3 (PS3) antibody

direct binding of Smads to DNA [22 23] The requirement ofdirect DNA binding for R-Smads is probably dependent onwhich transcriptional regulator(s) they cooperate or syner-gize with Indeed Smad3 could bind toAT-motifs in the pres-ence of ATBF1 but not in its absence (Figure 7(c)) Althoughwe do not know if the ATBF1Smad complex at a distalAT-motif can physically interact with that at a proximal

AT-motif directly or indirectly through another protein(s)(ldquoXrdquo Figure 8) both AT-motifs are definitely important forcooperation between ATBF1 and the TGF-120573 signaling tosuppress the activity of the AFP promoter

ATBF-1 is known to compete with HNF-1 for binding toAT-motifs in the AFP promoter [4] Thus the following pos-sibility can be speculated upon TGF-120573 stimulation Smads

10 Journal of Signal Transduction

AT-motif

AT-motif

AFP

ATBF1

ATBF1ATBF1Smad23-Smad4

X

Smad23-Smad4

ATBF1

Figure 8 Proposed inhibitory mechanism by which the TGF-120573Smad signaling and ATBF1 cooperatively regulate the activity ofthe AFP promoter The distal and proximal AT-motifs in the AFPpromoter are bound by ATBF1 Upon TGF-120573 signaling the R-SmadsSmad4 complex interacts with ATBF1 to make a repressivecomplex for the activity of the AFP promoter However whetheranother protein(s) (termed ldquoXrdquo) is essential for the R-SmadSmad4ATBF1 complex remains veiled

can not only bind to AT-motifs via ATBF-1 indirectly but alsointeract with HNF-1 to dissociate it from AT-motifs Due todual function of Smads the AFP prompter activity might beinhibited This possibility is interesting to be investigated

p53-mediated repression of the AFP promoter activityneeds direct binding of Smads to SBEp53REWilkinson et alfurther demonstrated that mSin3A and SnoN which is adirect target gene of TGF-120573 signal are recruited by p53Smadscomplex when the transcription of the AFP gene is inhib-ited [22 23] In our current study the region includingSBEp53RE in theAFP promoterwas not investigated becausethe basal reporter activities of 3Luc 16Luc and 09Luc werevery low compared with that of Δ27Luc (Figure 2(b)) Itis possible that ATBF1 might interplay with p53 via Smadcomplex to effectively repress the transcription of the AFPgene Further experiments are needed in the future to confirmthese hypotheses as well

5 Conclusion

In summary we report a novel interaction between ATBF1and Smads The interaction between ATBF1 and Smadsappears to cooperatively inhibit the transcription of AFPgene upon TGF-120573 signaling In particular both proximal anddistal AT-motifs are required for TGF-120573Smad signaling tocounteract with the transcription of the AFP gene

Conflict of Interests

The authors have no competing financial interests to declare

Acknowledgments

This research was supported by a Grant-in-Aid for Explor-atory Research (A) from the Japan Society for the Promotionof Science (JSPS) (to Nobuo Sakata) the MEXT-Supported

Program for the Strategic Research Foundation at PrivateUniversities (2013ndash2017) (to Susumu Itoh) the Takeda Sci-ence Foundation (to Susumu Itoh) the Smoking ResearchFoundation (to Susumu Itoh) the Daiichi-Sankyo Founda-tion of Life Science (to Susumu Itoh) the Naito Founda-tion (to Susumu Itoh) the Vehicle Racing Commemora-tive Foundation (to Susumu Itoh) and a Grant-in-Aid forYoung Scientists of Showa Pharmaceutical University (toSouichi Ikeno) The authors were also supported by the JointUsageResearch Program of the Medical Research Institutethe Tokyo Medical and Dental University and by the Core-to-Core Program ldquoCooperative International Framework inTGF-120573 Family Signalingrdquo of the JSPS They thank Ms FMiyamasu for excellent English proofreading

References

[1] M A Dziadek and G K Andrews ldquoTissue specificity of 120572-fetoprotein messenger RNA expression during mouse embryo-genesisrdquoThe EMBO Journal vol 2 no 4 pp 549ndash554 1983

[2] J L Nahon I Tratner A Poliard et al ldquoAlbumin and 120572-feto-protein gene expression in various nonhepatic rat tissuesrdquo TheJournal of Biological Chemistry vol 263 no 23 pp 11436ndash114421988

[3] A A Terentiev and N T Moldogazieva ldquo120572-fetoprotein a ren-aissancerdquo Tumor Biology vol 34 no 4 pp 2075ndash2091 2013

[4] H Yasuda A Mizuno T Tamaoki and T Morinaga ldquoATBF1 amultiple-homeodomain zinc finger protein selectively down-regulates AT-rich elements of the human 120572-fetoprotein generdquoMolecular and Cellular Biology vol 14 no 2 pp 1395ndash14011994

[5] K Sawadaishi T Morinaga and T Tamaoki ldquoInteraction of ahepatoma-specific nuclear factor with transcription-regulatorysequences of the human 120572-fetoprotein and albumin genesrdquoMolecular andCellular Biology vol 8 no 12 pp 5179ndash5187 1988

[6] H Nakabayashi Y Koyama H Suzuki et al ldquoFunctional map-ping of tissue-specific elements of the human 120572-fetoproteingene enhancerrdquo Biochemical and Biophysical Research Commu-nications vol 318 no 3 pp 773ndash785 2004

[7] T Morinaga H Yasuda T Hashimoto K Higashio and TTamaoki ldquoA human 120572-fetoprotein enhancer-binding proteinATBF1 contains four homeodomains and seventeen zinc fin-gersrdquo Molecular and Cellular Biology vol 11 no 12 pp 6041ndash6049 1991

[8] Y Miura T Tam A Ido et al ldquoCloning and characterization ofan ATBF1 isoform that expresses in a neuronal differentiation-dependent mannerrdquo The Journal of Biological Chemistry vol270 no 45 pp 26840ndash26848 1995

[9] H Kataoka Y Miura T Joh et al ldquo120572-fetoprotein producinggastric cancer lacks transcription factor ATBF1rdquo Oncogene vol20 no 7 pp 869ndash873 2001

[10] T Ninomiya K Mihara K Fushimi Y Hayashi T Hashimoto-Tamaoki and T Tamaoki ldquoRegulation of the 120572-fetoprotein geneby the isoforms of ATBF1 transcription factor in human hep-atomardquo Hepatology vol 35 no 1 pp 82ndash87 2002

[11] P Kaspar M Dvorakova J Kralova P Pajer Z Kozmik andM Dvorak ldquoMyb-interacting protein ATBF1 represses tran-scriptional activity of Myb oncoproteinrdquoThe Journal of Biologi-cal Chemistry vol 274 no 20 pp 14422ndash14428 1999

[12] F B Berry YMiura KMihara et al ldquoPositive and negative reg-ulation of myogenic differentiation of C2C12 cells by isoforms

Journal of Signal Transduction 11

of the multiple homeodomain zinc finger transcription factorATBF1rdquoThe Journal of Biological Chemistry vol 276 no 27 pp25057ndash25065 2001

[13] X Sun H F Frierson C Chen et al ldquoFrequent somaticmutations of the transcription factor ATBF1 in human prostatecancerrdquo Nature Genetics vol 37 no 6 pp 407ndash412 2005

[14] X Sun Y Zhou K B Otto et al ldquoInfrequentmutation of ATBF1in human breast cancerrdquo Journal of Cancer Research and ClinicalOncology vol 133 no 2 pp 103ndash105 2007

[15] Y G Cho J H Song C J Kim et al ldquoGenetic alterations of theATBF1 gene in gastric cancerrdquo Clinical Cancer Research vol 13no 15 pp 4355ndash4359 2007

[16] K Kai Z Zhang H Yamashita Y Yamamoto Y Miura and HIwase ldquoLoss of heterozygosity at the ATBF1-A locus located inthe 16q22 minimal region in breast cancerrdquo BMC Cancer vol 8article 262 2008

[17] A M Cleton-Jansen R van Eijk M Lombaerts et al ldquoATBF1and NQO1 as candidate targets for allelic loss at chromosomearm 16q in breast cancer absence of somatic ATBF1 mutationsand no role for the C609TNQO1 polymorphismrdquo BMCCancervol 8 article 105 2008

[18] J Massague J Seoane and D Wotton ldquoSmad transcriptionfactorsrdquo Genes amp Development vol 19 no 23 pp 2783ndash28102005

[19] AMoustakas and C H Heldin ldquoThe regulation of TGF120573 signaltransductionrdquo Development vol 136 no 22 pp 3699ndash37142009

[20] J Massague ldquoTGF120573 signalling in contextrdquo Nature ReviewsMolecular Cell Biology vol 13 no 10 pp 616ndash630 2012

[21] K Nakao K Nakata S Mitsuoka et al ldquoTransforming growthfactor 120573 1 differentially regulates 120572-fetoprotein and albumin inHuH-7 human hepatoma cellsrdquo Biochemical and BiophysicalResearch Communications vol 174 no 3 pp 1294ndash1299 1991

[22] D SWilkinson S K Ogden S A Stratton et al ldquoA direct inter-section between p53 and transforming growth factor 120573 path-ways targets chromatin modification and transcription repres-sion of the 120572-fetoprotein generdquoMolecular and Cellular Biologyvol 25 no 3 pp 1200ndash1212 2005

[23] D S Wilkinson W W Tsai M A Schumacher and M CBarton ldquoChromatin-bound p53 anchors activated Smads andthe mSin3A corepressor to confer transforming-growth-factor-120573-mediated transcription repressionrdquo Molecular and CellularBiology vol 28 no 6 pp 1988ndash1998 2008

[24] C G Jung H J Kim M Kawaguchi et al ldquoHomeotic factorATBF1 induces the cell cycle arrest associated with neuronaldifferentiationrdquo Development vol 132 no 23 pp 5137ndash51452005

[25] X Y Dong X Sun P Guo et al ldquoATBF1 inhibits estrogenreceptor (ER) function by selectively competing with AIB1 forbinding to the ER in ER-positive breast cancer cellsrdquoThe Journalof Biological Chemistry vol 285 no 43 pp 32801ndash32809 2010

[26] H Kataoka P Bonnefin D Vieyra et al ldquoING1 repressestranscription by direct DNA binding and through effects onp53rdquo Cancer Research vol 63 no 18 pp 5785ndash5792 2003

[27] A Nakao T Imamura S Souchelnytskyi et al ldquoTGF-120573receptor-mediated signalling through Smad2 Smad3 andSmad4rdquoThe EMBO Journal vol 16 no 17 pp 5353ndash5362 1997

[28] M Fujii K Takeda T Imamura et al ldquoRoles of bone morpho-genetic protein type I receptors and Smad proteins in osteoblastand chondroblast differentiationrdquoMolecular Biology of the Cellvol 10 no 11 pp 3801ndash3812 1999

[29] YWatanabe S Itoh T Goto et al ldquoTMEPAI a transmembraneTGF-120573-inducible protein sequesters smad proteins from activeparticipation in TGF-120573 signalingrdquoMolecular Cell vol 37 no 1pp 123ndash134 2010

[30] S Itoh M Thorikay M Kowanetz et al ldquoElucidation of Smadrequirement in transforming growth factor-120573 type I receptor-induced responsesrdquo The Journal of Biological Chemistry vol278 no 6 pp 3751ndash3761 2003

[31] N Nakano S Itoh Y Watanabe K Maeyama F Itoh and MKato ldquoRequirement of TCF7L2 for TGF-120573 dependent transcrip-tional activation of the TMEPAI generdquoThe Journal of BiologicalChemistry vol 285 no 49 pp 38023ndash38033 2010

[32] K Watanabe A Saito and T Tamaoki ldquoCell-specific enhanceractivity in a far upstream region of the human 120572-fetoproteingenerdquo The Journal of Biological Chemistry vol 262 no 10 pp4812ndash4818 1987

[33] H Nakabayashi K Watanabe A Saito A Otsuru KSawadaishi and T Tamaoki ldquoTranscriptional regulation of 120573-fetoprotein expression by dexamethasone in human hepatomacellsrdquo The Journal of Biological Chemistry vol 264 no 1 pp266ndash271 1989

[34] H Nakabayashi T Hashimoto Y Miyao K-K Tjong J Chanand T Tamaoki ldquoA position-dependent silencer plays a majorrole in repressing 120573-fetoprotein expression in human hep-atomardquoMolecular and Cellular Biology vol 11 no 12 pp 5885ndash5893 1991

[35] S Zhang T S Kim Y Dong et al ldquoAT motif binding factor 1(ATBF1) is highly phosphorylated in embryonic brain and pro-tected from cleavage by calpain-1rdquo Biochemical and BiophysicalResearch Communications vol 427 no 3 pp 537ndash541 2012

[36] L J Jonk S Itoh C H Heldin P ten Dijke and W KruijerldquoIdentification and functional characterization of a smad bind-ing element (SBE) in the JunBpromoter that acts as a transform-ing growth factor-120573 activin and bone morphogenetic protein-inducible enhancerrdquo The Journal of Biological Chemistry vol273 no 33 pp 21145ndash21152 1998

[37] S Dennler S Itoh D Vivien P ten Dijke S Huet and J Gau-thier ldquoDirect binding of Smad3 and Smad4 to critical TGF120573-inducible elements in the promoter of human plasminogenactivator inhibitor-type 1 generdquo The EMBO Journal vol 17 no11 pp 3091ndash3100 1998

[38] A Ido K Nakata Y Kato et al ldquoGene therapy for hep-atoma cells using a retrovirus vector carrying herpes simplexvirus thymidine kinase gene under the control of human 120572-fetoprotein gene promoterrdquo Cancer Research vol 55 no 14 pp3105ndash3109 1995

[39] A B Roberts and L M Wakefield ldquoThe two faces of trans-forming growth factor 120573 in carcinogenesisrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 15 pp 8621ndash8623 2003

[40] B Bierie and H L Moses ldquoTumour microenvironment TGF120573the molecular Jekyll and Hyde of cancerrdquo Nature ReviewsCancer vol 6 no 7 pp 506ndash520 2006

Journal of Signal Transduction 9

SBEminus3422

minus3422

minus3562

minus3562

LucAT-motif

minus178 +45

+1

LucAT-motif

AT-motif

AT-motifminus178 +45

+1

399840052Luc

399840052mSBELuc

(a)

00

02

04

06

08

10

12

Relat

ive l

ucife

rase

activ

ity

TGF-120573 (minus)TGF-120573 (+)

lowastlowast

lowastlowast

399840052Luc 399840052mSBELuc

(b)

DNAP

Flag-Smad3IB flag

IB HA

Totallysates

HA-ATBF1

HA-ATBF1

Flag-Smad3IB flag

IB HA

p-Smad3IB PS3

IB V5 ALK5caV5

Biotinylated probe minus + + +

Flag-Smad3HA-ATBF1

+ + + minusminus + +minus

ALK5caV5 + + + minus

(AT-motif)3

(c)

Figure 7 SBE in the proximal region of the AFP promoter is not essential for TGF-120573-mediated suppression of the AFP promoter (a)Schematic presentation of 3101584052mSBELuc A putative SBE 51015840-CAGATA-31015840 was mutated to 51015840-TTTTTT-31015840 (b) Effect of TGF-120573 on 3101584052Lucand 3101584052mSBELuc activities HepG2 cells were transfected with each reporter plasmid (200 ng) The cells were then stimulated with 5 ngmLTGF-120573 (c) Indirect binding of Smad3 to an AT-motif via ATBF1 The indicated cell lysates were mixed with biotinylated AT-motifs Thenstreptavidin-agarose was added to the mixture Subsequently a protein(s) bound to streptavidin-agarose was purified After SDS-PAGE IBwas carried out using anti-Flag or anti-HA antibody Using the total lysates each protein was detected with anti-Flag anti-HA anti-V5 oranti-phosphorylated Smad3 (PS3) antibody

direct binding of Smads to DNA [22 23] The requirement ofdirect DNA binding for R-Smads is probably dependent onwhich transcriptional regulator(s) they cooperate or syner-gize with Indeed Smad3 could bind toAT-motifs in the pres-ence of ATBF1 but not in its absence (Figure 7(c)) Althoughwe do not know if the ATBF1Smad complex at a distalAT-motif can physically interact with that at a proximal

AT-motif directly or indirectly through another protein(s)(ldquoXrdquo Figure 8) both AT-motifs are definitely important forcooperation between ATBF1 and the TGF-120573 signaling tosuppress the activity of the AFP promoter

ATBF-1 is known to compete with HNF-1 for binding toAT-motifs in the AFP promoter [4] Thus the following pos-sibility can be speculated upon TGF-120573 stimulation Smads

10 Journal of Signal Transduction

AT-motif

AT-motif

AFP

ATBF1

ATBF1ATBF1Smad23-Smad4

X

Smad23-Smad4

ATBF1

Figure 8 Proposed inhibitory mechanism by which the TGF-120573Smad signaling and ATBF1 cooperatively regulate the activity ofthe AFP promoter The distal and proximal AT-motifs in the AFPpromoter are bound by ATBF1 Upon TGF-120573 signaling the R-SmadsSmad4 complex interacts with ATBF1 to make a repressivecomplex for the activity of the AFP promoter However whetheranother protein(s) (termed ldquoXrdquo) is essential for the R-SmadSmad4ATBF1 complex remains veiled

can not only bind to AT-motifs via ATBF-1 indirectly but alsointeract with HNF-1 to dissociate it from AT-motifs Due todual function of Smads the AFP prompter activity might beinhibited This possibility is interesting to be investigated

p53-mediated repression of the AFP promoter activityneeds direct binding of Smads to SBEp53REWilkinson et alfurther demonstrated that mSin3A and SnoN which is adirect target gene of TGF-120573 signal are recruited by p53Smadscomplex when the transcription of the AFP gene is inhib-ited [22 23] In our current study the region includingSBEp53RE in theAFP promoterwas not investigated becausethe basal reporter activities of 3Luc 16Luc and 09Luc werevery low compared with that of Δ27Luc (Figure 2(b)) Itis possible that ATBF1 might interplay with p53 via Smadcomplex to effectively repress the transcription of the AFPgene Further experiments are needed in the future to confirmthese hypotheses as well

5 Conclusion

In summary we report a novel interaction between ATBF1and Smads The interaction between ATBF1 and Smadsappears to cooperatively inhibit the transcription of AFPgene upon TGF-120573 signaling In particular both proximal anddistal AT-motifs are required for TGF-120573Smad signaling tocounteract with the transcription of the AFP gene

Conflict of Interests

The authors have no competing financial interests to declare

Acknowledgments

This research was supported by a Grant-in-Aid for Explor-atory Research (A) from the Japan Society for the Promotionof Science (JSPS) (to Nobuo Sakata) the MEXT-Supported

Program for the Strategic Research Foundation at PrivateUniversities (2013ndash2017) (to Susumu Itoh) the Takeda Sci-ence Foundation (to Susumu Itoh) the Smoking ResearchFoundation (to Susumu Itoh) the Daiichi-Sankyo Founda-tion of Life Science (to Susumu Itoh) the Naito Founda-tion (to Susumu Itoh) the Vehicle Racing Commemora-tive Foundation (to Susumu Itoh) and a Grant-in-Aid forYoung Scientists of Showa Pharmaceutical University (toSouichi Ikeno) The authors were also supported by the JointUsageResearch Program of the Medical Research Institutethe Tokyo Medical and Dental University and by the Core-to-Core Program ldquoCooperative International Framework inTGF-120573 Family Signalingrdquo of the JSPS They thank Ms FMiyamasu for excellent English proofreading

References

[1] M A Dziadek and G K Andrews ldquoTissue specificity of 120572-fetoprotein messenger RNA expression during mouse embryo-genesisrdquoThe EMBO Journal vol 2 no 4 pp 549ndash554 1983

[2] J L Nahon I Tratner A Poliard et al ldquoAlbumin and 120572-feto-protein gene expression in various nonhepatic rat tissuesrdquo TheJournal of Biological Chemistry vol 263 no 23 pp 11436ndash114421988

[3] A A Terentiev and N T Moldogazieva ldquo120572-fetoprotein a ren-aissancerdquo Tumor Biology vol 34 no 4 pp 2075ndash2091 2013

[4] H Yasuda A Mizuno T Tamaoki and T Morinaga ldquoATBF1 amultiple-homeodomain zinc finger protein selectively down-regulates AT-rich elements of the human 120572-fetoprotein generdquoMolecular and Cellular Biology vol 14 no 2 pp 1395ndash14011994

[5] K Sawadaishi T Morinaga and T Tamaoki ldquoInteraction of ahepatoma-specific nuclear factor with transcription-regulatorysequences of the human 120572-fetoprotein and albumin genesrdquoMolecular andCellular Biology vol 8 no 12 pp 5179ndash5187 1988

[6] H Nakabayashi Y Koyama H Suzuki et al ldquoFunctional map-ping of tissue-specific elements of the human 120572-fetoproteingene enhancerrdquo Biochemical and Biophysical Research Commu-nications vol 318 no 3 pp 773ndash785 2004

[7] T Morinaga H Yasuda T Hashimoto K Higashio and TTamaoki ldquoA human 120572-fetoprotein enhancer-binding proteinATBF1 contains four homeodomains and seventeen zinc fin-gersrdquo Molecular and Cellular Biology vol 11 no 12 pp 6041ndash6049 1991

[8] Y Miura T Tam A Ido et al ldquoCloning and characterization ofan ATBF1 isoform that expresses in a neuronal differentiation-dependent mannerrdquo The Journal of Biological Chemistry vol270 no 45 pp 26840ndash26848 1995

[9] H Kataoka Y Miura T Joh et al ldquo120572-fetoprotein producinggastric cancer lacks transcription factor ATBF1rdquo Oncogene vol20 no 7 pp 869ndash873 2001

[10] T Ninomiya K Mihara K Fushimi Y Hayashi T Hashimoto-Tamaoki and T Tamaoki ldquoRegulation of the 120572-fetoprotein geneby the isoforms of ATBF1 transcription factor in human hep-atomardquo Hepatology vol 35 no 1 pp 82ndash87 2002

[11] P Kaspar M Dvorakova J Kralova P Pajer Z Kozmik andM Dvorak ldquoMyb-interacting protein ATBF1 represses tran-scriptional activity of Myb oncoproteinrdquoThe Journal of Biologi-cal Chemistry vol 274 no 20 pp 14422ndash14428 1999

[12] F B Berry YMiura KMihara et al ldquoPositive and negative reg-ulation of myogenic differentiation of C2C12 cells by isoforms

Journal of Signal Transduction 11

of the multiple homeodomain zinc finger transcription factorATBF1rdquoThe Journal of Biological Chemistry vol 276 no 27 pp25057ndash25065 2001

[13] X Sun H F Frierson C Chen et al ldquoFrequent somaticmutations of the transcription factor ATBF1 in human prostatecancerrdquo Nature Genetics vol 37 no 6 pp 407ndash412 2005

[14] X Sun Y Zhou K B Otto et al ldquoInfrequentmutation of ATBF1in human breast cancerrdquo Journal of Cancer Research and ClinicalOncology vol 133 no 2 pp 103ndash105 2007

[15] Y G Cho J H Song C J Kim et al ldquoGenetic alterations of theATBF1 gene in gastric cancerrdquo Clinical Cancer Research vol 13no 15 pp 4355ndash4359 2007

[16] K Kai Z Zhang H Yamashita Y Yamamoto Y Miura and HIwase ldquoLoss of heterozygosity at the ATBF1-A locus located inthe 16q22 minimal region in breast cancerrdquo BMC Cancer vol 8article 262 2008

[17] A M Cleton-Jansen R van Eijk M Lombaerts et al ldquoATBF1and NQO1 as candidate targets for allelic loss at chromosomearm 16q in breast cancer absence of somatic ATBF1 mutationsand no role for the C609TNQO1 polymorphismrdquo BMCCancervol 8 article 105 2008

[18] J Massague J Seoane and D Wotton ldquoSmad transcriptionfactorsrdquo Genes amp Development vol 19 no 23 pp 2783ndash28102005

[19] AMoustakas and C H Heldin ldquoThe regulation of TGF120573 signaltransductionrdquo Development vol 136 no 22 pp 3699ndash37142009

[20] J Massague ldquoTGF120573 signalling in contextrdquo Nature ReviewsMolecular Cell Biology vol 13 no 10 pp 616ndash630 2012

[21] K Nakao K Nakata S Mitsuoka et al ldquoTransforming growthfactor 120573 1 differentially regulates 120572-fetoprotein and albumin inHuH-7 human hepatoma cellsrdquo Biochemical and BiophysicalResearch Communications vol 174 no 3 pp 1294ndash1299 1991

[22] D SWilkinson S K Ogden S A Stratton et al ldquoA direct inter-section between p53 and transforming growth factor 120573 path-ways targets chromatin modification and transcription repres-sion of the 120572-fetoprotein generdquoMolecular and Cellular Biologyvol 25 no 3 pp 1200ndash1212 2005

[23] D S Wilkinson W W Tsai M A Schumacher and M CBarton ldquoChromatin-bound p53 anchors activated Smads andthe mSin3A corepressor to confer transforming-growth-factor-120573-mediated transcription repressionrdquo Molecular and CellularBiology vol 28 no 6 pp 1988ndash1998 2008

[24] C G Jung H J Kim M Kawaguchi et al ldquoHomeotic factorATBF1 induces the cell cycle arrest associated with neuronaldifferentiationrdquo Development vol 132 no 23 pp 5137ndash51452005

[25] X Y Dong X Sun P Guo et al ldquoATBF1 inhibits estrogenreceptor (ER) function by selectively competing with AIB1 forbinding to the ER in ER-positive breast cancer cellsrdquoThe Journalof Biological Chemistry vol 285 no 43 pp 32801ndash32809 2010

[26] H Kataoka P Bonnefin D Vieyra et al ldquoING1 repressestranscription by direct DNA binding and through effects onp53rdquo Cancer Research vol 63 no 18 pp 5785ndash5792 2003

[27] A Nakao T Imamura S Souchelnytskyi et al ldquoTGF-120573receptor-mediated signalling through Smad2 Smad3 andSmad4rdquoThe EMBO Journal vol 16 no 17 pp 5353ndash5362 1997

[28] M Fujii K Takeda T Imamura et al ldquoRoles of bone morpho-genetic protein type I receptors and Smad proteins in osteoblastand chondroblast differentiationrdquoMolecular Biology of the Cellvol 10 no 11 pp 3801ndash3812 1999

[29] YWatanabe S Itoh T Goto et al ldquoTMEPAI a transmembraneTGF-120573-inducible protein sequesters smad proteins from activeparticipation in TGF-120573 signalingrdquoMolecular Cell vol 37 no 1pp 123ndash134 2010

[30] S Itoh M Thorikay M Kowanetz et al ldquoElucidation of Smadrequirement in transforming growth factor-120573 type I receptor-induced responsesrdquo The Journal of Biological Chemistry vol278 no 6 pp 3751ndash3761 2003

[31] N Nakano S Itoh Y Watanabe K Maeyama F Itoh and MKato ldquoRequirement of TCF7L2 for TGF-120573 dependent transcrip-tional activation of the TMEPAI generdquoThe Journal of BiologicalChemistry vol 285 no 49 pp 38023ndash38033 2010

[32] K Watanabe A Saito and T Tamaoki ldquoCell-specific enhanceractivity in a far upstream region of the human 120572-fetoproteingenerdquo The Journal of Biological Chemistry vol 262 no 10 pp4812ndash4818 1987

[33] H Nakabayashi K Watanabe A Saito A Otsuru KSawadaishi and T Tamaoki ldquoTranscriptional regulation of 120573-fetoprotein expression by dexamethasone in human hepatomacellsrdquo The Journal of Biological Chemistry vol 264 no 1 pp266ndash271 1989

[34] H Nakabayashi T Hashimoto Y Miyao K-K Tjong J Chanand T Tamaoki ldquoA position-dependent silencer plays a majorrole in repressing 120573-fetoprotein expression in human hep-atomardquoMolecular and Cellular Biology vol 11 no 12 pp 5885ndash5893 1991

[35] S Zhang T S Kim Y Dong et al ldquoAT motif binding factor 1(ATBF1) is highly phosphorylated in embryonic brain and pro-tected from cleavage by calpain-1rdquo Biochemical and BiophysicalResearch Communications vol 427 no 3 pp 537ndash541 2012

[36] L J Jonk S Itoh C H Heldin P ten Dijke and W KruijerldquoIdentification and functional characterization of a smad bind-ing element (SBE) in the JunBpromoter that acts as a transform-ing growth factor-120573 activin and bone morphogenetic protein-inducible enhancerrdquo The Journal of Biological Chemistry vol273 no 33 pp 21145ndash21152 1998

[37] S Dennler S Itoh D Vivien P ten Dijke S Huet and J Gau-thier ldquoDirect binding of Smad3 and Smad4 to critical TGF120573-inducible elements in the promoter of human plasminogenactivator inhibitor-type 1 generdquo The EMBO Journal vol 17 no11 pp 3091ndash3100 1998

[38] A Ido K Nakata Y Kato et al ldquoGene therapy for hep-atoma cells using a retrovirus vector carrying herpes simplexvirus thymidine kinase gene under the control of human 120572-fetoprotein gene promoterrdquo Cancer Research vol 55 no 14 pp3105ndash3109 1995

[39] A B Roberts and L M Wakefield ldquoThe two faces of trans-forming growth factor 120573 in carcinogenesisrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 15 pp 8621ndash8623 2003

[40] B Bierie and H L Moses ldquoTumour microenvironment TGF120573the molecular Jekyll and Hyde of cancerrdquo Nature ReviewsCancer vol 6 no 7 pp 506ndash520 2006

10 Journal of Signal Transduction

AT-motif

AT-motif

AFP

ATBF1

ATBF1ATBF1Smad23-Smad4

X

Smad23-Smad4

ATBF1

Figure 8 Proposed inhibitory mechanism by which the TGF-120573Smad signaling and ATBF1 cooperatively regulate the activity ofthe AFP promoter The distal and proximal AT-motifs in the AFPpromoter are bound by ATBF1 Upon TGF-120573 signaling the R-SmadsSmad4 complex interacts with ATBF1 to make a repressivecomplex for the activity of the AFP promoter However whetheranother protein(s) (termed ldquoXrdquo) is essential for the R-SmadSmad4ATBF1 complex remains veiled

can not only bind to AT-motifs via ATBF-1 indirectly but alsointeract with HNF-1 to dissociate it from AT-motifs Due todual function of Smads the AFP prompter activity might beinhibited This possibility is interesting to be investigated

p53-mediated repression of the AFP promoter activityneeds direct binding of Smads to SBEp53REWilkinson et alfurther demonstrated that mSin3A and SnoN which is adirect target gene of TGF-120573 signal are recruited by p53Smadscomplex when the transcription of the AFP gene is inhib-ited [22 23] In our current study the region includingSBEp53RE in theAFP promoterwas not investigated becausethe basal reporter activities of 3Luc 16Luc and 09Luc werevery low compared with that of Δ27Luc (Figure 2(b)) Itis possible that ATBF1 might interplay with p53 via Smadcomplex to effectively repress the transcription of the AFPgene Further experiments are needed in the future to confirmthese hypotheses as well

5 Conclusion

In summary we report a novel interaction between ATBF1and Smads The interaction between ATBF1 and Smadsappears to cooperatively inhibit the transcription of AFPgene upon TGF-120573 signaling In particular both proximal anddistal AT-motifs are required for TGF-120573Smad signaling tocounteract with the transcription of the AFP gene

Conflict of Interests

The authors have no competing financial interests to declare

Acknowledgments

This research was supported by a Grant-in-Aid for Explor-atory Research (A) from the Japan Society for the Promotionof Science (JSPS) (to Nobuo Sakata) the MEXT-Supported

Program for the Strategic Research Foundation at PrivateUniversities (2013ndash2017) (to Susumu Itoh) the Takeda Sci-ence Foundation (to Susumu Itoh) the Smoking ResearchFoundation (to Susumu Itoh) the Daiichi-Sankyo Founda-tion of Life Science (to Susumu Itoh) the Naito Founda-tion (to Susumu Itoh) the Vehicle Racing Commemora-tive Foundation (to Susumu Itoh) and a Grant-in-Aid forYoung Scientists of Showa Pharmaceutical University (toSouichi Ikeno) The authors were also supported by the JointUsageResearch Program of the Medical Research Institutethe Tokyo Medical and Dental University and by the Core-to-Core Program ldquoCooperative International Framework inTGF-120573 Family Signalingrdquo of the JSPS They thank Ms FMiyamasu for excellent English proofreading

References

[1] M A Dziadek and G K Andrews ldquoTissue specificity of 120572-fetoprotein messenger RNA expression during mouse embryo-genesisrdquoThe EMBO Journal vol 2 no 4 pp 549ndash554 1983

[2] J L Nahon I Tratner A Poliard et al ldquoAlbumin and 120572-feto-protein gene expression in various nonhepatic rat tissuesrdquo TheJournal of Biological Chemistry vol 263 no 23 pp 11436ndash114421988

[3] A A Terentiev and N T Moldogazieva ldquo120572-fetoprotein a ren-aissancerdquo Tumor Biology vol 34 no 4 pp 2075ndash2091 2013

[4] H Yasuda A Mizuno T Tamaoki and T Morinaga ldquoATBF1 amultiple-homeodomain zinc finger protein selectively down-regulates AT-rich elements of the human 120572-fetoprotein generdquoMolecular and Cellular Biology vol 14 no 2 pp 1395ndash14011994

[5] K Sawadaishi T Morinaga and T Tamaoki ldquoInteraction of ahepatoma-specific nuclear factor with transcription-regulatorysequences of the human 120572-fetoprotein and albumin genesrdquoMolecular andCellular Biology vol 8 no 12 pp 5179ndash5187 1988

[6] H Nakabayashi Y Koyama H Suzuki et al ldquoFunctional map-ping of tissue-specific elements of the human 120572-fetoproteingene enhancerrdquo Biochemical and Biophysical Research Commu-nications vol 318 no 3 pp 773ndash785 2004

[7] T Morinaga H Yasuda T Hashimoto K Higashio and TTamaoki ldquoA human 120572-fetoprotein enhancer-binding proteinATBF1 contains four homeodomains and seventeen zinc fin-gersrdquo Molecular and Cellular Biology vol 11 no 12 pp 6041ndash6049 1991

[8] Y Miura T Tam A Ido et al ldquoCloning and characterization ofan ATBF1 isoform that expresses in a neuronal differentiation-dependent mannerrdquo The Journal of Biological Chemistry vol270 no 45 pp 26840ndash26848 1995

[9] H Kataoka Y Miura T Joh et al ldquo120572-fetoprotein producinggastric cancer lacks transcription factor ATBF1rdquo Oncogene vol20 no 7 pp 869ndash873 2001

[10] T Ninomiya K Mihara K Fushimi Y Hayashi T Hashimoto-Tamaoki and T Tamaoki ldquoRegulation of the 120572-fetoprotein geneby the isoforms of ATBF1 transcription factor in human hep-atomardquo Hepatology vol 35 no 1 pp 82ndash87 2002

[11] P Kaspar M Dvorakova J Kralova P Pajer Z Kozmik andM Dvorak ldquoMyb-interacting protein ATBF1 represses tran-scriptional activity of Myb oncoproteinrdquoThe Journal of Biologi-cal Chemistry vol 274 no 20 pp 14422ndash14428 1999

[12] F B Berry YMiura KMihara et al ldquoPositive and negative reg-ulation of myogenic differentiation of C2C12 cells by isoforms

Journal of Signal Transduction 11

of the multiple homeodomain zinc finger transcription factorATBF1rdquoThe Journal of Biological Chemistry vol 276 no 27 pp25057ndash25065 2001

[13] X Sun H F Frierson C Chen et al ldquoFrequent somaticmutations of the transcription factor ATBF1 in human prostatecancerrdquo Nature Genetics vol 37 no 6 pp 407ndash412 2005

[14] X Sun Y Zhou K B Otto et al ldquoInfrequentmutation of ATBF1in human breast cancerrdquo Journal of Cancer Research and ClinicalOncology vol 133 no 2 pp 103ndash105 2007

[15] Y G Cho J H Song C J Kim et al ldquoGenetic alterations of theATBF1 gene in gastric cancerrdquo Clinical Cancer Research vol 13no 15 pp 4355ndash4359 2007

[16] K Kai Z Zhang H Yamashita Y Yamamoto Y Miura and HIwase ldquoLoss of heterozygosity at the ATBF1-A locus located inthe 16q22 minimal region in breast cancerrdquo BMC Cancer vol 8article 262 2008

[17] A M Cleton-Jansen R van Eijk M Lombaerts et al ldquoATBF1and NQO1 as candidate targets for allelic loss at chromosomearm 16q in breast cancer absence of somatic ATBF1 mutationsand no role for the C609TNQO1 polymorphismrdquo BMCCancervol 8 article 105 2008

[18] J Massague J Seoane and D Wotton ldquoSmad transcriptionfactorsrdquo Genes amp Development vol 19 no 23 pp 2783ndash28102005

[19] AMoustakas and C H Heldin ldquoThe regulation of TGF120573 signaltransductionrdquo Development vol 136 no 22 pp 3699ndash37142009

[20] J Massague ldquoTGF120573 signalling in contextrdquo Nature ReviewsMolecular Cell Biology vol 13 no 10 pp 616ndash630 2012

[21] K Nakao K Nakata S Mitsuoka et al ldquoTransforming growthfactor 120573 1 differentially regulates 120572-fetoprotein and albumin inHuH-7 human hepatoma cellsrdquo Biochemical and BiophysicalResearch Communications vol 174 no 3 pp 1294ndash1299 1991

[22] D SWilkinson S K Ogden S A Stratton et al ldquoA direct inter-section between p53 and transforming growth factor 120573 path-ways targets chromatin modification and transcription repres-sion of the 120572-fetoprotein generdquoMolecular and Cellular Biologyvol 25 no 3 pp 1200ndash1212 2005

[23] D S Wilkinson W W Tsai M A Schumacher and M CBarton ldquoChromatin-bound p53 anchors activated Smads andthe mSin3A corepressor to confer transforming-growth-factor-120573-mediated transcription repressionrdquo Molecular and CellularBiology vol 28 no 6 pp 1988ndash1998 2008

[24] C G Jung H J Kim M Kawaguchi et al ldquoHomeotic factorATBF1 induces the cell cycle arrest associated with neuronaldifferentiationrdquo Development vol 132 no 23 pp 5137ndash51452005

[25] X Y Dong X Sun P Guo et al ldquoATBF1 inhibits estrogenreceptor (ER) function by selectively competing with AIB1 forbinding to the ER in ER-positive breast cancer cellsrdquoThe Journalof Biological Chemistry vol 285 no 43 pp 32801ndash32809 2010

[26] H Kataoka P Bonnefin D Vieyra et al ldquoING1 repressestranscription by direct DNA binding and through effects onp53rdquo Cancer Research vol 63 no 18 pp 5785ndash5792 2003

[27] A Nakao T Imamura S Souchelnytskyi et al ldquoTGF-120573receptor-mediated signalling through Smad2 Smad3 andSmad4rdquoThe EMBO Journal vol 16 no 17 pp 5353ndash5362 1997

[28] M Fujii K Takeda T Imamura et al ldquoRoles of bone morpho-genetic protein type I receptors and Smad proteins in osteoblastand chondroblast differentiationrdquoMolecular Biology of the Cellvol 10 no 11 pp 3801ndash3812 1999

[29] YWatanabe S Itoh T Goto et al ldquoTMEPAI a transmembraneTGF-120573-inducible protein sequesters smad proteins from activeparticipation in TGF-120573 signalingrdquoMolecular Cell vol 37 no 1pp 123ndash134 2010

[30] S Itoh M Thorikay M Kowanetz et al ldquoElucidation of Smadrequirement in transforming growth factor-120573 type I receptor-induced responsesrdquo The Journal of Biological Chemistry vol278 no 6 pp 3751ndash3761 2003

[31] N Nakano S Itoh Y Watanabe K Maeyama F Itoh and MKato ldquoRequirement of TCF7L2 for TGF-120573 dependent transcrip-tional activation of the TMEPAI generdquoThe Journal of BiologicalChemistry vol 285 no 49 pp 38023ndash38033 2010

[32] K Watanabe A Saito and T Tamaoki ldquoCell-specific enhanceractivity in a far upstream region of the human 120572-fetoproteingenerdquo The Journal of Biological Chemistry vol 262 no 10 pp4812ndash4818 1987

[33] H Nakabayashi K Watanabe A Saito A Otsuru KSawadaishi and T Tamaoki ldquoTranscriptional regulation of 120573-fetoprotein expression by dexamethasone in human hepatomacellsrdquo The Journal of Biological Chemistry vol 264 no 1 pp266ndash271 1989

[34] H Nakabayashi T Hashimoto Y Miyao K-K Tjong J Chanand T Tamaoki ldquoA position-dependent silencer plays a majorrole in repressing 120573-fetoprotein expression in human hep-atomardquoMolecular and Cellular Biology vol 11 no 12 pp 5885ndash5893 1991

[35] S Zhang T S Kim Y Dong et al ldquoAT motif binding factor 1(ATBF1) is highly phosphorylated in embryonic brain and pro-tected from cleavage by calpain-1rdquo Biochemical and BiophysicalResearch Communications vol 427 no 3 pp 537ndash541 2012

[36] L J Jonk S Itoh C H Heldin P ten Dijke and W KruijerldquoIdentification and functional characterization of a smad bind-ing element (SBE) in the JunBpromoter that acts as a transform-ing growth factor-120573 activin and bone morphogenetic protein-inducible enhancerrdquo The Journal of Biological Chemistry vol273 no 33 pp 21145ndash21152 1998

[37] S Dennler S Itoh D Vivien P ten Dijke S Huet and J Gau-thier ldquoDirect binding of Smad3 and Smad4 to critical TGF120573-inducible elements in the promoter of human plasminogenactivator inhibitor-type 1 generdquo The EMBO Journal vol 17 no11 pp 3091ndash3100 1998

[38] A Ido K Nakata Y Kato et al ldquoGene therapy for hep-atoma cells using a retrovirus vector carrying herpes simplexvirus thymidine kinase gene under the control of human 120572-fetoprotein gene promoterrdquo Cancer Research vol 55 no 14 pp3105ndash3109 1995

[39] A B Roberts and L M Wakefield ldquoThe two faces of trans-forming growth factor 120573 in carcinogenesisrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 15 pp 8621ndash8623 2003

[40] B Bierie and H L Moses ldquoTumour microenvironment TGF120573the molecular Jekyll and Hyde of cancerrdquo Nature ReviewsCancer vol 6 no 7 pp 506ndash520 2006

Journal of Signal Transduction 11

of the multiple homeodomain zinc finger transcription factorATBF1rdquoThe Journal of Biological Chemistry vol 276 no 27 pp25057ndash25065 2001

[13] X Sun H F Frierson C Chen et al ldquoFrequent somaticmutations of the transcription factor ATBF1 in human prostatecancerrdquo Nature Genetics vol 37 no 6 pp 407ndash412 2005

[14] X Sun Y Zhou K B Otto et al ldquoInfrequentmutation of ATBF1in human breast cancerrdquo Journal of Cancer Research and ClinicalOncology vol 133 no 2 pp 103ndash105 2007

[15] Y G Cho J H Song C J Kim et al ldquoGenetic alterations of theATBF1 gene in gastric cancerrdquo Clinical Cancer Research vol 13no 15 pp 4355ndash4359 2007

[16] K Kai Z Zhang H Yamashita Y Yamamoto Y Miura and HIwase ldquoLoss of heterozygosity at the ATBF1-A locus located inthe 16q22 minimal region in breast cancerrdquo BMC Cancer vol 8article 262 2008

[17] A M Cleton-Jansen R van Eijk M Lombaerts et al ldquoATBF1and NQO1 as candidate targets for allelic loss at chromosomearm 16q in breast cancer absence of somatic ATBF1 mutationsand no role for the C609TNQO1 polymorphismrdquo BMCCancervol 8 article 105 2008

[18] J Massague J Seoane and D Wotton ldquoSmad transcriptionfactorsrdquo Genes amp Development vol 19 no 23 pp 2783ndash28102005

[19] AMoustakas and C H Heldin ldquoThe regulation of TGF120573 signaltransductionrdquo Development vol 136 no 22 pp 3699ndash37142009

[20] J Massague ldquoTGF120573 signalling in contextrdquo Nature ReviewsMolecular Cell Biology vol 13 no 10 pp 616ndash630 2012

[21] K Nakao K Nakata S Mitsuoka et al ldquoTransforming growthfactor 120573 1 differentially regulates 120572-fetoprotein and albumin inHuH-7 human hepatoma cellsrdquo Biochemical and BiophysicalResearch Communications vol 174 no 3 pp 1294ndash1299 1991

[22] D SWilkinson S K Ogden S A Stratton et al ldquoA direct inter-section between p53 and transforming growth factor 120573 path-ways targets chromatin modification and transcription repres-sion of the 120572-fetoprotein generdquoMolecular and Cellular Biologyvol 25 no 3 pp 1200ndash1212 2005

[23] D S Wilkinson W W Tsai M A Schumacher and M CBarton ldquoChromatin-bound p53 anchors activated Smads andthe mSin3A corepressor to confer transforming-growth-factor-120573-mediated transcription repressionrdquo Molecular and CellularBiology vol 28 no 6 pp 1988ndash1998 2008

[24] C G Jung H J Kim M Kawaguchi et al ldquoHomeotic factorATBF1 induces the cell cycle arrest associated with neuronaldifferentiationrdquo Development vol 132 no 23 pp 5137ndash51452005

[25] X Y Dong X Sun P Guo et al ldquoATBF1 inhibits estrogenreceptor (ER) function by selectively competing with AIB1 forbinding to the ER in ER-positive breast cancer cellsrdquoThe Journalof Biological Chemistry vol 285 no 43 pp 32801ndash32809 2010

[26] H Kataoka P Bonnefin D Vieyra et al ldquoING1 repressestranscription by direct DNA binding and through effects onp53rdquo Cancer Research vol 63 no 18 pp 5785ndash5792 2003

[27] A Nakao T Imamura S Souchelnytskyi et al ldquoTGF-120573receptor-mediated signalling through Smad2 Smad3 andSmad4rdquoThe EMBO Journal vol 16 no 17 pp 5353ndash5362 1997

[28] M Fujii K Takeda T Imamura et al ldquoRoles of bone morpho-genetic protein type I receptors and Smad proteins in osteoblastand chondroblast differentiationrdquoMolecular Biology of the Cellvol 10 no 11 pp 3801ndash3812 1999

[29] YWatanabe S Itoh T Goto et al ldquoTMEPAI a transmembraneTGF-120573-inducible protein sequesters smad proteins from activeparticipation in TGF-120573 signalingrdquoMolecular Cell vol 37 no 1pp 123ndash134 2010

[30] S Itoh M Thorikay M Kowanetz et al ldquoElucidation of Smadrequirement in transforming growth factor-120573 type I receptor-induced responsesrdquo The Journal of Biological Chemistry vol278 no 6 pp 3751ndash3761 2003

[31] N Nakano S Itoh Y Watanabe K Maeyama F Itoh and MKato ldquoRequirement of TCF7L2 for TGF-120573 dependent transcrip-tional activation of the TMEPAI generdquoThe Journal of BiologicalChemistry vol 285 no 49 pp 38023ndash38033 2010

[32] K Watanabe A Saito and T Tamaoki ldquoCell-specific enhanceractivity in a far upstream region of the human 120572-fetoproteingenerdquo The Journal of Biological Chemistry vol 262 no 10 pp4812ndash4818 1987

[33] H Nakabayashi K Watanabe A Saito A Otsuru KSawadaishi and T Tamaoki ldquoTranscriptional regulation of 120573-fetoprotein expression by dexamethasone in human hepatomacellsrdquo The Journal of Biological Chemistry vol 264 no 1 pp266ndash271 1989

[34] H Nakabayashi T Hashimoto Y Miyao K-K Tjong J Chanand T Tamaoki ldquoA position-dependent silencer plays a majorrole in repressing 120573-fetoprotein expression in human hep-atomardquoMolecular and Cellular Biology vol 11 no 12 pp 5885ndash5893 1991

[35] S Zhang T S Kim Y Dong et al ldquoAT motif binding factor 1(ATBF1) is highly phosphorylated in embryonic brain and pro-tected from cleavage by calpain-1rdquo Biochemical and BiophysicalResearch Communications vol 427 no 3 pp 537ndash541 2012

[36] L J Jonk S Itoh C H Heldin P ten Dijke and W KruijerldquoIdentification and functional characterization of a smad bind-ing element (SBE) in the JunBpromoter that acts as a transform-ing growth factor-120573 activin and bone morphogenetic protein-inducible enhancerrdquo The Journal of Biological Chemistry vol273 no 33 pp 21145ndash21152 1998

[37] S Dennler S Itoh D Vivien P ten Dijke S Huet and J Gau-thier ldquoDirect binding of Smad3 and Smad4 to critical TGF120573-inducible elements in the promoter of human plasminogenactivator inhibitor-type 1 generdquo The EMBO Journal vol 17 no11 pp 3091ndash3100 1998

[38] A Ido K Nakata Y Kato et al ldquoGene therapy for hep-atoma cells using a retrovirus vector carrying herpes simplexvirus thymidine kinase gene under the control of human 120572-fetoprotein gene promoterrdquo Cancer Research vol 55 no 14 pp3105ndash3109 1995

[39] A B Roberts and L M Wakefield ldquoThe two faces of trans-forming growth factor 120573 in carcinogenesisrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 15 pp 8621ndash8623 2003

[40] B Bierie and H L Moses ldquoTumour microenvironment TGF120573the molecular Jekyll and Hyde of cancerrdquo Nature ReviewsCancer vol 6 no 7 pp 506ndash520 2006