d

,

Letters to the Editor / Journal of Dermatological Science 68 (2012) 194–204 197

[4] Messenger A, Elliott K, Temple A, Randall VA. Expression of basement mem-brane proteins and interstitial collagens in dermal papillae of human hairfollicles. J Invest Dermatol 1991;96:93–7.

[5] Varani J, Spearman D, Perone P, Fligiel SEG, Datta SC, Wang ZQ, et al.Inhibition of type I procollagen synthesis by damaged collagen in photoagedskin and by collagenase-degraded collagen in vitro. Am J Pathol 2001;158:931.

[6] Kwon OS, Oh JK, Kim MH, Park SH, Pyo HK, Kim KH, et al. Human hair growth exvivo is correlated with in vivo hair growth: selective categorization of hairfollicles for more reliable hair follicle organ culture. Arch Dermatol Res2006;297:367–71.

[7] Muller-Rover S, Handjiski B, van der Veen C, Eichmuller S, Foitzik K, McKay IA,et al. A comprehensive guide for the accurate classification of murine hairfollicles in distinct hair cycle stages. J Invest Dermatol 2001;117:3–15.

[8] Paus R, Stenn K, Link R. Telogen skin contains an inhibitor of hair growth. Br J

aDepartment of Dermatology, Seoul National University College of

Medicine, Seoul, Republic of Korea; bLaboratory of Cutaneous Aging an

Hair Research, Clinical Research Institute, Seoul National University

Hospital, Seoul, Republic of Korea; cInstitute of Dermatological Science

Seoul National University, Seoul, Republic of Korea

*Corresponding author at: Department of Dermatology, SeoulNational University College of Medicine, 101 Daehak-ro,Chongno-Gu, Seoul 110-744, Republic of Korea.Tel.: +822 2072 2414; fax: +822 742 7344E-mail address: jhchung@snu.ac.kr

gsny].

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Dermatol 1990;122:777–84.[9] Varani J, Warner RL, Gharaee-Kermani M, Phan SH, Kang S, Chung JH, et al.

Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collagen accumula-tion in naturally aged human skin. J Invest Dermatol 2000;114:480–6.

[10] Griffiths C. The role of retinoids in the prevention and repair of aged andphotoaged skin. Clin Exp Dermatol 2001;26:613–8.

Jun Kyu Oha,1,2, Oh Sang Kwona,b,c,1, Mi Hyang Kima, Seong Jin Joa,c,Ji Hyun Hana,b,c, Kyu Han Kima,b,c, Hee Chul Euna,b,c, Jin Ho Chunga,b,c,

*

Letter to the Editor

Multipotential functions of Hic-5 in growth, differentiation,migration and adhesion of human keratinocytes

Hic-5, a paxillin homologue, was initially identified as a TGF-b1- and hydrogen peroxide-inducible gene. Its expression

Fig. 1. Intracellular distribution of Hic-5 and cyclin D1 during differentiation in no

coverslips and cultured until around 50% confluent in KGM at 0.1 mM Ca2+. Then, the

48 h later, cells were fixed and permeabilized with 4% paraformaldehyde and 0.2

Probes, OR, US) for 30 min at room temperature and thereafter incubated sequentially

polyclonal rabbit IgG against human cyclin D1 (M-20, sc-718) (Santa Cruz Biotechno

mouse IgG (green, 1:200) and Alexa-Fluor1 594 conjugated gout anti-rabbit IgG (red, 1

(J.H. Chung)

1These two authors contributed equally.2Current address: Rich Hair Clinic, Seoul, Republic of Korea.

16 April 2012

http://dx.doi.org/10.1016/j.jdermsci.2012.09.006

increases during cellular senescence and decreases durinimmortalization. Furthermore, its forced expression inducegrowth retardation and senescence cell-like morphology ifibroblasts [1]. Hic-5 inhibits cell spreading in NIH 3T3 cells bcompeting with paxillin for binding to focal adhesion kinase [2

al human keratinocytes in vitro. Normal human keratinocytes were seeded onto glas

lture media were changed to KGM at 0.1 mM Ca2+ or 1.8 mM Ca2+ for differentiation. A

riton X-100, followed by incubation with Image-iTTM FX signal enhancer (Molecula

with anti-Hic-5 mouse monoclonal IgG (clone 34) (BD Biosciences, CA, US) (1:200) and

logy, Inc, CA, US) (1:200) for 60 min and then Alexa-Fluor1 488 conjugated gout anti-

:200) (Molecular Probes, OR, US) at room temperature. The scale bars represent 20 mm.

Hic-5 was cloned as a nuclear androgen receptor coactivator from acDNA library of human prostate tissue [3]. Furthermore, it islocalized in the nucleus and cell membrane and can shuttlebetween them [4,5]. Hic-5 potentially exerts multiple physiologi-cal and pathological effects in various cells types. We reported thatin the skin Hic-5 is present in mesenchymal components includinghair follicle dermal papilla cells [6] and fibroblastic cells in keloids[7]. However, until date the expression and roles of Hic-5 inepidermal keratinocytes remain unknown.

In this study, the intracellular distribution of Hic-5 and cyclinD1 in normal human keratinocytes (NHKs) was examined byimmunocytochemistry. Stable knockdown HaCat cell line (Hic-5KD-HaCat) and control HaCat cell line (cHaCat) were obtained bystable transfection with the human Hic-5 shRNA plasmid (sc-37685-SH) and the control shRNA plasmid (sc-108060) (Santa CruzBiotechnology, Inc, CA, US) using Fugene1-6 (Roche DiagnosticsCorp., Indianapolis, IN, US). This was followed by selection with8 mg/ml puromycin and the limiting dilution method. A prolifera-tion assay for HaCat keratinocytes was performed using the DNA

IdU labeling and detection kit. An in vitro migration assay forHic-5KD-HaCat and cHaCat keratinocytes was performed by in

vitro wounding of the monolayer cells by scraping with a yellowpipette tip. HaCat keratinocyte differentiation was induced byculturing the cells on poly-2-hydroxyethyl methacrylate-coateddishes under floating conditions. Cell adhesion was examinedusing a cell adhesion assay kit.

We examined the intracellular distribution of Hic-5 in NHKs in

vitro and found cytosolic staining in undifferentiated keratinocytes(Fig. 1, left upper: low Ca, Hic-5). After terminal differentiationunder high-calcium conditions, we detected Hic-5 localization onfocal adhesion kinases (right upper: high Ca, Hic-5) possibly owingto shift and/or de novo synthesis of Hic-5. Furthermore, in view of arecent report of the competitive nuclear export of Hic-5 and cyclinD1 [8], we performed double staining. Cyclin D1 was positivelystained in the nucleus of both undifferentiated (left lower: low Ca,Hic-5, and cyclin D1) and differentiated keratinocytes (right lower:high Ca, Hic-5 and cyclin D1), suggesting that the distribution ofHic-5 does not markedly influence cyclin D1 localization. The

Fig. 2. Effect of Hic-5 knockdown on HaCat keratinocyte proliferation migration, differentiation and adhesion. (a) Human Hic-5 shRNA plasmids (sc-37685-SH) and control

shRNA plasmids (sc-108060) (Santa Cruz Biotechnology, Inc, CA, US) were transfected into subconfluent HaCat keratinocytes using Fugene1-6 (Roche Diagnostics Corp.,

Indianapolis, IN, US). At two days after transfection, 8 mg/ml puromycin was added into the culture, followed by incubation for three days. By limiting dilution method under

selection with puromycin, we obtained three cell lines. Among them, by Western blotting we confirmed the stable lines of knockdown HaCat (Hic-5KD-HaCat, lane 1) and

control HaCat (cHaCat, lane 2) keratinocytes, stably transfected with control vectors. (b) One hundred Hic-5KD-HaCat and cHaCat keratinocytes were seeded on a 96-well

plate and incubated one overnight and then IdU labeling proliferation assay was performed using DNA IdU labeling and detection kit (Takara Biomedical, Osaka, Japan)

according to the manufacturer’s instruction. Data are indicated as the mean � SD. *p < 0.05, significantly different (n = 4) by Mann–Whitney U test. (c) The Hic-5KD-HaCat (left

panels) and cHaCat (right panels) keratinocytes were cultured on the plastic dishes until subconfluency in DMEM containing 10% fetal calf serum, then starved for 24 h, treated with

10 mg/ml mitomycin C for 2 h and subjected to in vitro migration assay. A cell-free area was introduced by scraping the monolayer with a yellow pipette tip. At 0 (uppermost), 24

(middle) and 48 h later (lower), cell migration to the cell-free area is shown. (d) In the in vitro migration assay shown in (c) the migration rates were calculated using migration

distance of Hic-5KD-HaCat and cHacat for 24 h. Data are indicated as the mean � SD. *p < 0.05, significantly different (n = 3) by Mann–Whitney U test. (e) Effect of Hic-5 knockdown

on early and terminal differentiation of HaCat keratinocytes. Hic-5KD-HaCat and cHaCat keratinocytes were grown on poly-HEMA coated dishes in a floating condition to induce

differentiation. At 0, 72 and 168 h later the cells were harvested and pelleted by centrifugation and lysed in 2% sodium dodecyl sulfate. The lysate was sonicated and 8 mg/lane were

subjected to Western blotting with anti-keratin 1 (K1) rabbit antibodies (1:250) (upper panel) or anti-involucrin mouse IgG (sc-21748) (1:1000) (lower panel). (f) The adhesion

ability of Hic-5KD-HaCat and cHaCat keratinocytes was assayed using a cell adhesion assay kit, CytoSelectTM 48-Well Cell Adhesion Assay (Collagen IV-Coated, Colorimetric Format).

Briefly, the 7.5 � 104 cells were seeded on the collagen IV-coated plates and incubated for 30 min and then cells were stained and the OD 560 nm was measured in a plate reader.

Data are indicated as the mean � SD. *p < 0.05, significantly different (n = 5) by Mann–Whitney U test.

Letters to the Editor / Journal of Dermatological Science 68 (2012) 194–204198

change in the intracellular distribution in NHKs prompted us toinvestigate the physiological function of Hic-5 in human kerati-nocytes. For this purpose, we decided to generate stable HaCatkeratinocytes, given that these show the capacity for normalepidermal differentiation [9]. By the limiting dilution methodunder selection with puromycin, we obtained stable Hic-5KD-HaCat and cHaCat keratinocytes, which were stably transfectedwith control vectors. Western blotting showed that the amount ofHic-5 was reduced to 17.6% in Hic-5KD-HaCat keratinocytes(Fig. 2a). To compare the proliferative activities of these cell lines,we performed a proliferation assay using IdU labeling. After DNAIdU labeling for 3 h, the incorporation of IdU into Hic-5KD-HaCatand cHaCat cells indicated that Hic-5 knockdown suppressed DNAsynthesis by 35.7% (Fig. 2b), suggesting a stimulatory function ofendogenous Hic-5 in human keratinocyte proliferation. When weperformed DNA IdU labeling for 6 h, the results were similar. Toexamine the effect of Hic-5 knockdown on migration activities ofHaCat keratinocytes, we performed an in vitro migration assay.Following in vitro wounding of the subconfluent Hic-5KD-HaCatand cHaCat cells (Fig. 2c, uppermost picture panel), we observedclosure of the cell-free area by migrating cells. After 24 h, Hic-5KD-HaCat keratinocytes migrated more than cHaCat (Fig. 2c, middlepicture panel). In particular, Hic-5KD-HaCat keratinocytes showedconsiderable direct invasion into the cell-free area, preserving cell–cell adhesion. At 48 h after in vitro wounding, Hic-5KD-HaCatkeratinocytes closed the wound, whereas a wide wound persistedin the cHaCat culture (Fig. 2c, lower picture panel). The migrationrate of Hic-5KD-HaCat was considerably faster than that ofcHacat (Fig. 2d). These results indicated that knockdown ofHic-5 accelerated cell migration, suggesting that endogenous Hic-5suppresses keratinocyte migration. To examine the effect of Hic-5knockdown on early and terminal differentiation of HaCatkeratinocytes, we induced differentiation of HaCat keratinocytesby anchorage depletion [10]. Next, at 0 and 72 h after floating, weharvested Hic-5KD-HaCat and cHaCat keratinocytes and subjectedthem to Western blotting for K1, an early differentiation marker.This assay showed that while K1 expression is increased in cHaCatkeratinocytes after anchorage depletion, it is not altered in Hic-5KD-HaCat keratinocytes (Fig. 2e, upper panel). In contrast, at168 h after floating culture, the expression level of involucrin, aterminal differentiation marker, was similarly increased in thesecell lines (Fig. 2e, lower panel). These results indicate that Hic-5knockdown suppresses early differentiation but does not alterterminal differentiation, suggesting that endogenous Hic-5 plays arole in early and not terminal differentiation. Finally, to examinethe effect of Hic-5 knockdown on adhesion of keratinocytes, weassayed cell adhesion on collagen IV-coated plates after incubatingfor 30 min using a cell adhesion assay kit and found that celladhesion was suppressed to 54.9% in Hic-5KD-HaCat cells (Fig. 2f).Hic-5 can potentially exercise multiple functions in growth,differentiation, migration and adhesion of human keratinocytes,partially via nuclear-cell membrane shuttling, and our findings

represent a novel contribution to keratinocyte biology. Thesefindings indicate Hic-5 to be a new potential therapeutic target forskin diseases with keratinocyte abnormality such as psoriasis,ichthyosis, or epidermal tumors. Moreover, the reduction ofadhesion and enhancement of migration by Hic-5 knockdownmay promote wound healing.

Acknowledgement

This work was supported by Grants-in-Aid for ScientificResearch (KAKENHI).

References

[1] Shibanuma M, Mochizuki E, Maniwa R, Mashimo J, Nishiya N, Imai S, et al.Induction of senescence-like phenotypes by forced expression of hic-5, whichencodes a novel LIM motif protein, in immortalized human fibroblasts. MolCell Biol 1997;17:1224–35.

[2] Nishiya N, Tachibana K, Shibanuma M, Mashimo JI, Nose K. Hic-5-reduced cellspreading on fibronectin: competitive effects between paxillin and Hic-5through interaction with focal adhesion kinase. Mol Cell Biol 2001;21: 5332–45.

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

[4] Shibanuma M, Kim-Kaneyama JR, Ishino K, Sakamoto N, Hishiki T, YamaguchiK, et al. Hic-5 communicates between focal adhesions and the nucleus throughoxidant-sensitive nuclear export signal. Mol Biol Cell 2003;14:1158–71.

[5] Heitzer MD, DeFranco DB. Hic-5/ARA55, a LIM domain-containing nuclearreceptor coactivator expressed in prostate stromal cells. Cancer Res 2006;66:7326–33.

[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] Inui S, Shono F, Noguchi F, Nakajima T, Hosokawa K, Itami S. In vitro and in vivoevidence of pathogenic roles of Hic-5/ARA55 in keloids through Smad pathwayand profibrotic transcription. J Dermatol Sci 2010;58:152–4.

[8] Mori K, Hirao E, Toya Y, Oshima Y, Ishikawa F, Nose K, et al. Competitivenuclear export of cyclin D1 and Hic-5 regulates anchorage dependence of cellgrowth and survival. Mol Biol Cell 2009;20:218–32.

[9] Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A, FusenigNE. Normal keratinization in a spontaneously immortalized aneuploid humankeratinocyte cell line. J Cell Biol 1988;106:761–71.

[10] Nishi K, Inoue H, Schnier JB, Rice RH. Cyclin D1 downregulation is importantfor permanent cell cycle exit and initiation of differentiation induced byanchorage-deprivation in human keratinocytes. J Cell Biochem 2009;106:63–72.

Shigeki Inui*, Fumihito Noguchi, Aki Nishiyama, Satoshi ItamiDepartment of Regenerative Dermatology, Graduate School of

Medicine, Osaka University, 2-2, G2, Yamada-oka, Suita-shi,

Osaka 565-0871, Japan

*Corresponding author. Tel.: +81 6 6879 3031;fax: +81 6 6879 3039E-mail address: inui@r-derma.med.osaka-u.ac.jp (S. Inui)

2 July 2012

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Letters to the Editor / Journal of Dermatological Science 68 (2012) 194–204 199

Letter to the Editor

Epidermal-type FABP is a predictive marker of clinical response

to systemic treatment and ultraviolet therapy in psoriatic skinlesions

Psoriasis vulgaris is a chronic skin disease. The psoriasis area andseverity index (PASI) is useful as a marker of therapeutic efficacy butrelies on a subjective assessment of clinical symptoms. New reliablebiomarkers that better reflect clinical condition are needed.

Special attention has been given to the relationship betwee

fatty acids and psoriasis vulgaris. A high intake of dietary fat hasbeen reported to correlate positively with the occurrence ofpsoriasis vulgaris [1], and the consumption of n-3 fatty acids canimprove the symptoms of psoriasis [2]. Because of their insolubili-ty, fatty acids rely on specific processes for intracellular transport.A family of fatty acid-binding proteins (FABPs) plays a role in theseprocesses. Epidermal-type FABP (E-FABP), also known as FABP5, is