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Letters to the Editor / Journal of Dermatological Science 64 (2011) 142–151 149

Naoki OisoDepartment of Dermatology, Kinki University

School of Medicine, Osaka, Japan

Atsushi TanemuraDepartment of Dermatology, Osaka University Graduate School of

Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan

Akira KawadaDepartment of Dermatology, Kinki University

School of Medicine, Osaka, Japan

Ichiro KatayamDepartment of Dermatology, Osaka University Graduat

School of Medicine, 2-2 Yamadaoka, Suita

Osaka 565-0871, Japa

*Corresponding author. Tel.: +81 6 6879 303E-mail address: mkaneda@derma.med.osaka-u.ac.j

(M. Wataya-Kaneda

Received 4 April 201Revised 17 August 201

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Tamio SuzukiDepartment of Dermatology, Yamagata University

School of Medicine, Yamagata, Japan

Letter to the Editor

Androgen receptor transactivity is potentiated by TGF-b1through Smad3 but checked by its coactivator Hic-5/ARA55 inbalding dermal papilla cells

We previously reported that TGF-b1 is a paracrine mediatorfrom dermal papilla to hair follicle epithelium in the pathogenesisof androgenetic alopecia (AGA) [1,2]. Because TGF-b1 can inducecatagen in hair cycling [3], it has been suggested that it functionsas a paracrine pathogenic mediator from dermal papilla in AGA.On the other hand, TGF-b1 reportedly modulates androgenreceptor (AR) transactivation in the monkey kidney cell line CV-1as well as the human prostate cell lines PC-3 and DU145 cells [4,5].However, it depends on the cell type or conditions whether TGF-b1 potentiates [5] or represses AR [4]. It is therefore ofconsiderable interest to examine potential modulation by TGF-b1 and its downstream signaling for AR transcriptional activity inbalding dermal papilla cells (bald DPCs). To address this issue, weused mouse mammary tumor virus long-terminal repeat(MMTV)-luciferase assays to examine whether TGF-b1 can alterAR transactivation in bald DPCs. The DPCs obtained at passages 4–6 from an AGA bald frontal scalp were cultured on a 12-well platein Dulbecco’s modified Eagle’s medium (DMEM) (Nissui Pharma-ceutical, Tokyo, Japan) supplemented with 10% charcoal-treatedfetal calf serum (FCS) (JRH Biosciences, Lenexa, KS, USA), penicillin(50 units/ml) and streptomycin (50 mg/ml) at 37 8C in a humidi-fied atmosphere of 95% O2 and 5% CO2. At subconfluency, cellswere transiently transfected by means of Fugene 6 (RocheDiagnostic Corp., Indianapolis, IN) with 0.1 mg pSG5-AR, 0.3 mgMMTV-luciferase reporter plasmid and 0.1 mg pRL-CMV vector asan internal control and 24 h later the medium was refreshed and1 nM R1881 and 0.2 or 2.0 ng/ml of human recombinant TGF-b1(R&D Systems Inc., Minneapolis, MN) or the corresponding mockswere added to the culture. After incubation for 24 h, the cells wereharvested and subjected to luciferase assays using the Dual-Luciferase reporter assay system (Promega, Madison, WI). Theresults showed that 0.2 or 2.0 ng/ml of TGF-b1 can significantlyenhance AR activity by a factor of 1.9 or 2.3 (Fig. 1), respectively,indicating that TGF-b1 signaling positively stimulates ARtransactivation in bald DPCs. Next, to investigate the need forSmad3 to obtain this effect by TGF-b1, we examined the effect ofSmad3 knockdown by siRNA on TGF-b1-induced AR transactiva-tion. The bald DPCs were transiently transfected with 0.1 mgpSG5-AR, 0.3 mg MMTV-luciferase reporter plasmid, 0.1 mg pRL-

CMV, and 100 pg/ml siRNA against Smad3 (siTrio, NM_005902; B-Bridge International, Inc., Cupertino, CA) or control RNA (siTrio

Accepted 21 August 201

doi:10.1016/j.jdermsci.2011.08.006

negative control). Twenty hours later, the medium was refresheand 1 nM R1881, 0.2 ng/ml human recombinant TGF-b1 or one othe corresponding mocks was added to the culture. Afteincubation for 24 h, the cells were harvested and subjected tluciferase assays. The results demonstrated that knockdown oSmad3 eliminated the effect of TGF-b1 on MMTV-luciferasactivity (Fig. 2A, upper panel), indicating that Smad3 is necessarfor TGF-b1 to exert its effect. The successful knockdown of Smadby siRNA was confirmed in this experiment (Fig. 2A, lower panelIn addition, because interaction of Smad3 and Hic-5/ARA55which we previously reported is an androgen sensitivity regulatoin DPCs [6], has been proven [7,8], we studied the effect of TGF-bon AR activity by using the MMTV-luciferase assays for bald DPC

Fig. 1. Effect of TGF-b1 on transfected androgen receptor transactivity in baldin

dermal papilla cells (DPCs). The DPCs from AGA bald frontal scalp at subconfluenc

in a 12-well plate were transiently transfected at passages 4–6 with 0.1 mg pSG5

AR, 0.3 mg MMTV-luciferase reporter plasmid and 0.1 mg pRL-CMV vector usin

Fugene 6 as an internal control. At 24 h after transfection, 1 nM R1881 (lanes 2–4

synthetic androgen, or an ethanol mock solution (lane 1), and TGF-b1 at th

indicated concentration (lanes 3 and 4) or a corresponding mock solution (4 mM

HCl/0.1% BSA) (lanes 1 and 2) was added to the culture. After incubation for 24 h, th

cells were harvested and subjected to luciferase assays. Each luciferase activit

(relative LUC) is shown relative to the mean transactivation observed in the absenc

of TGF-b1 and the presence of R1881 (lane 2). Bars represent the mean � standard

deviations of three independent experiments. *p < 0.05; n.s., not significant (p > 0.05);

Mann–Whitney’s U test.

overexpressing Hic-5/ARA55 as a result of transient transfectionwith its expression vector. The assay results showed that TGF-b1did not have any significant effect on AR transcription (Fig. 2B,upper panel), suggesting that Hic-5/ARA55 impeded the enhance-ment of AR activity by TGF-b1. The successful overexpression ofHic-5/ARA55 generated by its expression vector was confirmed inthis experiment (Fig. 2B, lower panel).

Our data presented here suggest that TGF-b1 can enhanceandrogen sensitivity through Smad3 in the dermal papilla of AGAin an autocrine manner. Because TGF-b1 from bald DPCs inhibitshab1pathseoust5/oninexcoinw

between the AR-Hic-5/ARA55 and TGF-b-Smad pathways dependson the cell type or organ [5,8], the interaction or crosstalk ofthese and various other molecules supposedly determines whetherHic-5/ARA55 and Smad3 function as positive or negativemodulators. According to a very recent report, normal testisgrowth and maturation require coordinated and interdependentactivin/TGF-b and androgen signaling with tightly regulatedproduction of Smad3 in order to control the balance between cellgrowth, differentiation, and maturation [10]. Putting thesefindings together suggests that a properly balanced crosstalkmpa

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Fig. 2. Effect of Smad3 knockdown and Hic-5/ARA55 overexpression on transfected androgen receptor transactivity in balding dermal papilla cells. (A, upper panel) The bald

DPCs were transiently transfected with pSG5-AR, MMTV-luciferase reporter plasmid, pRL-CMV (lanes 1–4), and 100 pg/ml siRNA against Smad3 (lane 4) or control RNA (lanes

1–3), as mentioned in Fig. 1. At 24 h after transfection, 1 nM R1881 (lanes 2–4), synthetic androgen, or an ethanol mock solution (lane 1), and 0.2 ng/ml TGF-b1 (lanes 3 and 4)

or a corresponding mock solution (4 mM HCl/0.1% BSA) (lanes 1 and 2) was added to the culture. After 24 h incubation, the cells were harvested and subjected to the luciferase

assays. (A, lower panel) The knockdown of Smad3 was confirmed by semiquantitative RT-PCR. The following oligonucleotide primers were used: for Smad3 as sense primer:

50-GAGTAGAGACGCCAGTTCTACC-30 and as antisense primer: 50-GGTTTGGAGAACCTGCGTCCAT-30; for glyceraldehyde-3-phosphate dehydrogenase glyceraldehyde-3-

phosphate dehydrogenase (G3PDH) as internal control: 50-CCCATCACCATCTTCCAG-30 and 50-CCTGCTTCACCACCTTCT-30 . PCR amplification was performed as 30 cycles of

denaturation at 94 8C for 15 s, annealing at 62 8C for 30 s and extension at 72 8C for 2 min for Smad3 or 23 cycles of denaturation at 94 8C for 30 s, annealing at 55 8C for 30 s,

and extension at 72 8C for 30 s for G3PDH. (B, upper panel) The bald DPCs were transiently transfected with pSG5-AR, MMTV-luciferase reporter plasmid, pRL-CMV and 0.5 mg

pSG5-ARA55 (lanes 1–3). At 24 h after transfection, 1 nM R1881 (lanes 2 and 3), or mock ethanol (lane 1), and 0.2 ng/ml TGF-b1 (lane 3) or mock solution (4 mM HCl/0.1%

BSA) (lanes 1 and 2) were added to the culture. (B, lower panel) The bald DPCs transiently transfected with 0.5 mg pSG5 mock vector (left lane) or pSG5-ARA55 (right lane)

were lysed in 1% Nonidet P-40, 0.4 M NaCl and aprotinin and 5 mg of cell lysate protein per lane were loaded onto Novex1 4–12% Tris-glycine gel (EC60352box) (Invitrogen,

CA, USA) and transferred to nitrocellulose membranes. The membrane was soaked in 5% skimmed milk in phosphate-buffered saline/0.05% Tween 20 for 2 h at room

temperature and then incubated with anti-Hic-5 monoclonal IgG antibody (BD transduction laboratories, San Jose, CA) at a 1:500 dilution or monoclonal anti-b-actin IgG

antibody (Sigma–Aldrich, St. Louis, MO) at a 1:15,000 dilution in Can get signal1 immunoreaction enhancer solution (Toyobo life science, Tokyo, Japan) for 2 h at room

temperature. After being washed three times at intervals of 10 min with phosphate-buffered saline/0.05% Tween 20, the membranes were incubated with horseradish

peroxidase linked sheep anti-mouse IgG (NA934V and NA931V, GE Healthcare, Piscataway, NJ) at a 1:10,000 dilution for 1 h at room temperature. Each luciferase activity

(relative LUC) is shown relative to the mean transactivation observed in the absence of TGF-b1 and the presence of R1881 (lane 2 in upper panels of A and B). Bars represent

the mean � standard deviation of three independent experiments. *p < 0.05; n.s., not significant (p > 0.05); Mann–Whitney’s U test (upper panels of A and B).

Letters to the Editor / Journal of Dermatological Science 64 (2011) 142–151150

ir follicle epithelial cell growth in a paracrine manner [1], TGF- exerts its pathogenic roles with dual secretion, autocrine andracrine, between epithelium and dermal papilla in AGA. One other hand, although Hic-5/ARA55 upregulates androgennsitivity via coactivation for AR in DPCs [6], the data obtained inr current study indicated that this molecule impedes the AR

imulation by TGF-b1. This may be due to crosstalk between Hic-ARA55 and Smad3 [8] or possibly the attenuated effect of TGF-b1

the high expression of Hic-5/ARA55, which is reportedlycreased by TGF-b1 [9]. Given that Hic-5/ARA55 is highlypressed in the androgen-sensitive DPCs from AGA [6], a complexmpensatory mechanism through reciprocal interaction must be

place between TGF-b-Smad and androgen-AR signaling path-ays in the hair follicles of AGA. Because the function of crosstalk

ay be required for normal physiology of hair cycling as well asthogenesis of AGA.In conclusion, the findings of our in vivo investigation of AGA

dicate that TGF-b1 produced by androgen from bald DPCsppresses epithelial cell growth and simultaneously enhancesdrogen sensitivity in bald DPCs in an autocrine manner.

knowledgement

We thank Dr. Chawnshang Chang at University of Rochester fors kind gift of the plasmids and Mrs. Naoko Yamada for hercellent technical assistance. This work was supported by aant-in-aid for Scientific Research from the Ministry of Education,

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Letters to the Editor / Journal of Dermatological Science 64 (2011) 142–151 151

References

[1] Inui S, Fukuzato Y, Nakajima T, Yoshikawa K, Itami S. Androgen-inducible TGF-beta1 from balding dermal papilla cells inhibits epithelial cell growth: a clue tounderstand paradoxical effects of androgen on human hair growth. FASEB J2002;16:1967–9.

[2] Inui S, Itami S. Molecular basis of androgenetic alopecia: from androgen toparacrine mediators through dermal papilla. J Dermatol Sci 2011;61:1–6.

[3] Foitzik K, Lindner G, Mueller-Roever S, Maurer M, Botchkareva N, BotchkarevV, et al. Control of murine hair follicle regression (catagen) by TGF-beta1 invivo. FASEB J 2000;14:752–60.

[4] Hayes SA, Zarnegar M, Sharma M, Yang F, Peehl DM, ten Dijke P, et al. SMAD3represses androgen receptor-mediated transcription. Cancer Res 2001;61:2112–8.

[5] Kang HY, Lin HK, Hu YC, Yeh S, Huang KE, Chang C. From transforming growthfactor-beta signaling to androgen action: identification of Smad3 as anandrogen receptor coregulator in prostate cancer cells. Proc Natl Acad SciUSA 2001;98:3018–23.

[6] Inui S, Fukuzato Y, Nakajima T, Kurata S, Itami S. Androgen receptor co-activator Hic-5/ARA55 as a molecular regulator of androgen sensitivity indermal papilla cells of human hair follicles. J Invest Dermatol 2007;127:2302–6.

[7] Shibanuma M, Kim-Kaneyama JR, Sato S, Nose K. A LIM protein. Hic-5,

functions as a potential coactivator for Sp1. J Cell Biochem 2004;91:

[8] Wang H, Song K, Sponseller TL, Danielpour D. Novel function of androgereceptor-associated protein 55/Hic-5 as a negative regulator of Smad3 signaing. J Biol Chem 2005;280:5154–62.

[9] Fujimoto N, Yeh S, Kang HY, Inui S, Chang HC, Mizokami A, et al. Cloning ancharacterization of androgen receptor coactivator. ARA55, in human prostateBiol Chem 1999;274:8316–21.

[10] Itman C, Wong C, Hunyadi B, Ernst M, Jans DA, Loveland KL. Smad3 dosagdetermines androgen responsiveness and sets the pace of postnatal testdevelopment. Endocrinology 2011;152:2076–89.

Shigeki Inui*, Satoshi ItamDepartment of Regenerative Dermatolog

Graduate School of Medicine, Osaka University

2-2 Yamada-oka, Suita-shi, Osaka 565-0871, Japa

*Corresponding author. Tel.: +81 6 6879 3031

1

fax: +81 6 6879 303E-mail address: inui@r-derma.med.osaka-u.ac.jp (S. Inui

19 July 201

633–45.

doi:10.1016/j.jdermsci.2011.08.010