Reduced WIF-1 Expression Stimulates Skin Hyperpigmentation in Patients with Melasma

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1 See relted commentry on pg ORIGINAL ARTICLE Reduced WIF- Expression Stimultes Skin Hyperpigmenttion in Ptients with Melsm Ji-Young Kim, Te-Ryong Lee nd Ai-Young Lee The expression of Wnt inhiitory fctor- (WIF-) gene, which ws detected y microrry nlysis of hyperpigmented nd normlly pigmented skin sets of melsm ptients, ws significntly reduced in the hyperpigmented skin from melsm ptients, ut not in helthy controls, regrdless of UV irrdition. Wnt signls regulte skin pigmenttion; however, WIF- is expressed in cultured skin kertinocytes nd firolsts, ut not in melnocytes. Therefore, we exmined whether WIF- knockdown in neighoring kertinocytes nd firolsts plys role in melsm. Additionlly, the effect of WIF- overexpression on the meliortion of hyperpigmenttion ws exmined. WIF- knockdown, either in firolsts or in kertinocytes, significntly stimulted tyrosinse expression nd melnosome trnsfer, wheres melnocytes with WIF- overexpression significntly reduced those prmeters. The WIF- knockdown decresed glycogen synthse kinse-3 (GSK-3), -ctenin, nd NFATc (nucler fctor of ctivted T cells, cytoplsmic, clcineurin-dependent ) phosphoryltion nd incresed microphthlmi-ssocited trnscription fctor () expression s in melnocytes with Wnt- overexpression, wheres the WIF- overexpression reversed the results. Expression of Wnts, oth cnonicl nd noncnonicl, ws incresed in the hyperpigmented skin of melsm ptients. Collectively, WIF- downregultion, which my occur in epiderml kertinocytes nd in derml firolsts, is involved in melsm development ecuse of the stimultion of melnogenesis nd melnosome trnsfer through upregultion of the cnonicl nd the noncnonicl Wnt signling pthwy. Journl of Investigtive Dermtology (3) 33, 9 ; doi:.38/jid..7; pulished online 6 Septemer INTRODUCTION Melsm is one of the most common ptterns of hyperpigmenttion tht ffects skin on the upper lip, cheeks, forehed, nd chin, prticulrly during the reproductive lifespn of women. In terms of pigmenttion, melsm does not differ from other conditions with hyperpigmenttion; however, the outcome is different from the hyperpigmenttion cused y inflmmtion or y UV exposure. If regultory mechnisms, which re involved in melnogenesis of these conditions, do not differ, then their outcome should not e different. Recently, we performed microrry nlysis to compre hyperpigmented with normlly pigmented skin specimens Deprtment of Dermtology, Dongguk University Ilsn Hospitl, Goyng-si, Gyeonggi-do, South Kore nd R&D Center, AmorePcific, Yongin-si, Gyeonggi-do, South Kore Correspondence: Ai-Young Lee, Deprtment of Dermtology, Dongguk University Ilsn Hospitl, 84 Siks-dong, Ilsndong-gu, Goyng-si, Gyeonggi-do 4-773, South Kore. E-mil: ly564@nver.com Arevitions: GSK-3, glycogen synthse kinse-3; JNK, c-jun N-terminl kinse; K4, cytokertin 4;, microphthlmi-ssocited trnscription fctor; NFAT, nucler fctor of ctivted T cells; NFATc, nucler fctor of ctivted T cells, cytoplsmic, clcineurin-dependent ; P, protein kinse C; rhwif-, recominnt humn WIF-; sfrp, secreted Frizzled-relted protein; sirna, smll interfering RNA; TYRP-, tyrosinse-relted protein-; WIF-, Wnt inhiitory fctor- Received 6 Septemer ; revised 5 June ; ccepted 6 June ; pulished online 6 Septemer from ptients with melsm to identify specific fctors ssocited with the hyperpigmenttion of melsm. Downregultion of H9 RNA ws one of the identified fctors ssocited with melsm (Kim et l.,, ). There ws lso X-fold downregultion in the expression of the Wnt inhiitory fctor- (WIF-) gene in the hyperpigmented skin of melsm ptients (dt not shown). Wnts, fmily of secreted glycoproteins, regulte vst rry of iologicl processes, including emryonic development, cell fte, cell prolifertion, cell migrtion, stem cell mintennce, tumor suppression, nd oncogenesis. In terms of pigmenttion, Wnt signls hve een shown to e involved in directing neurl crest cells to dopt pigment cell ftes (Dorsky et l., 998) nd in pigment cell differentition (Dorsky et l., ), through either the cnonicl (Ymguchi et l., 8) or the noncnonicl pthwys (Kim et l.,, ). In the cnonicl pthwy, -ctenin plys criticl role, which is lso clled the Wnt/-ctenin pthwy (Miller et l., 999); without the inding of cnonicl Wnts, such s Wnt-, Wnt-3A, nd Wnt-8, -ctenin is degrded y the destruction complex while keeping the levels of - ctenin low. The destruction complex is composed of four proteins, nd glycogen synthse kinse-3 (GSK-3) is the centrl plyer with phosphoryltion of -ctenin (Yost et l., 996). With the inding to their receptor, Frizzled, these cnonicl Wnts prevent the degrdtion, which leds to & 3 The Society for Investigtive Dermtology 9

2 genertion of signling pool of -ctenin, nd trnslocte -ctenin to the nucleus. Therey, it forms complex with the lymphocyte enhncer-inding fctor /T-cell fctor trnscription fctors (Estmn nd Grosschedl, 999; Novk nd Dedhr, 999) to stimulte trnscription of trget genes. In ddition, microphthlmi-ssocited trnscription fctor () intercts with -ctenin to ctivte gene expression (Schepsky et l., 6). On the other hnd, noncnonicl Wnt pthwy is -ctenin independent nd involves diverse signl trnsduction, such s clcium flux, which is clled the Wnt/clcium pthwy, or c-jun N-terminl kinse (JNK). In the Wnt/clcium pthwy, noncnonicl Wnts, such s Wnt- 4, Wnt-5A, nd Wnt-, ctivte clcium-sensitive kinse, protein kinse C (P) (Sheldhl et l., 999), nd clciumresponsive trnscription fctor, which is the nucler fctor of ctivted T cells (NFAT) (Murphy nd Hughes, ; Sneyoshi et l., ). The extrcellulr ntgonists of the Wnt signling pthwy cn e divided into two clsses (Kwno nd Kypt, 3). WIF-, which is secreted ntgonist of Wnt signling (Hsieh et l., 999), elongs to the secreted Frizzled-relted protein (sfrp) clss, which inds directly to Wnts in the extrcellulr spce, long with the sfrp fmily. The Dickkopf clss ntgonists include the memer of certin Dickkopf fmily, which inhiit Wnt signling y inding to the low-densitylipoprotein-receptor-relted protein 5 nd 6 component of the Wnt receptor complex. Antgonists of the sfrp clss hve een considered to inhiit oth cnonicl nd noncnonicl Wnt pthwys, wheres those of the Dickkopf clss inhiit only the cnonicl Wnt pthwy, despite mny of the unresolved issues. Although Wnt signls regulte skin pigmenttion, the expression of WIF- hs not een previously reported in melnocytes. On the other hnd, WIF- expression hs een found in kertinocytes (Sshr et l., 9; Gudjonsson et l., ) nd in firolsts (Cho et l., 9). Melnocyte growth nd differentition is ffected y neighoring firolsts (Ymguchi et l., 4) s well s y kertinocytes, nd in this study we show n ssocition etween downregultion of the WIF- gene in those neighoring skin cells nd melsm long with its function t the moleculr level. As the functionl significnce of WIF- downregultion in cncers hs een identified with WIF- upregultion (Elston et l., 8; Kwkmi et l., 9; Ruin et l., ; Yee et l., ), we used tht pproch to demonstrte tht the upregultion of WIF- using recominnt humn WIF- reduces pigmenttion. RESULTS WIF- expression in the skin of ptients with melsm Levels of WIF- mrna expression were nlyzed using reltime PCR in oth hyperpigmented nd normlly pigmented skin specimens iopsied from 3 ptients with melsm. Levels of WIF- mrna in the hyperpigmented skin were significntly (Po.) lower thn in the normlly pigmented skin (Figure ). However, the WIF- mrna levels did not reduce in the lterl cheeks or the forehed skin compred with the posturiculr skin from the three norml individuls (Figure ). Additionlly, no decrese in the WIF- mrna ws oserved t UV-induced hyperpigmented compred with nonexposed control skin from the domen of three norml individuls (Figure c). Doule stining with ntiodies to WIF- nd cytokertin 4 (K4; kertinocyte mrker), which ws performed in 7 of 3 ptients, suggested in ll cses weker stining of WIF- in the epiderml kertinocytes nd in the cells tht re scttered in the dermis of the hyperpigmented when compred with the normlly pigmented skin specimens (Figure d). WIF- expression in cultured skin cells Immunohistochemistry suggested tht the expression of WIF- decresed in oth kertinocytes nd derml cells. Melnocyte growth nd differentition is ffected y neighoring kertinocytes nd firolsts (Ymguchi et l., 4). Therefore, expression of WIF- ws exmined not only in melnocytes, ut lso in kertinocytes nd in firolsts cultured from norml humn skin. The expression of WIF- mrna nd protein ws detected in oth kertinocytes nd firolsts, ut not in melnocytes regrdless of -Otetrdecnoylphorol-3-cette tretment (Figure nd ). Effect of WIF- downregultion in kertinocytes on hyperpigmenttion After identifying tht trnsfection of WIF- smll interfering RNA (sirna) significntly (Po.) reduced the levels of oth WIF- mrna nd protein expression in cultured norml kertinocytes (Figure 3), their effect ws exmined in kertinocyte/melnocyte co-cultures, without the purifiction of melnocytes. The decresed expression of WIF- in the kertinocytes significntly (Po.5) stimulted tyrosinse expression (Figure 3). Concerning the signling pthwys, the WIF- knockdown significntly (Po.5) decresed the phosphoryltion of GSK-3 nd -ctenin, with incresed expression. The knockdown lso reduced the phosphoryltion of NFAT, cytoplsmic, clcineurin-dependent (NFATc) (Figure 3c). As NFATc nd -ctenin require to trnslocte to the nucleus to exert their ction, western lot nlysis of NFATc nd -ctenin expression performed in nucler frction suggested significnt (Po.5) increse y the WIF- knockdown (Figure 3d). The WIF- knockdown lso significntly (Po.5) incresed the proportion of tyrosinse-relted protein- (TYRP-) nd K4 doule-positive cells, which ws shown in FACS nlysis nd confocl microscopy of co-cultured cells (Figure 3e). Effects of WIF- downregultion in firolsts on tyrosinse expression nd melnosome trnsfer As firolsts re components of the dermis, the effects of WIF- downregultion in cultured firolsts were exmined using mixed-cell three-dimensionl culture system, fter confirming tht WIF- sirna significntly (Po.5) reduced the expression levels of WIF- mrna nd protein in firolsts (Figure 4). To keep the condition the sme s the WIF- downregulted kertinocytes (Figure 3 e), the inserts contining kertinocyte/melnocyte co-cultures were used 9 Journl of Investigtive Dermtology (3), Volume 33

3 . 3 c 3 Reltive rtios of WIF- mrna expression (L/N) Reltive rtios of WIF- mrna expression (C/R).5 Reltive rtios of WIF- mrna expression (UV+/UV ).5 N L Retrouriculr Cheek/forehed UV UV+ d N WIF- K4 Merge BF L WIF- K4 Merge BF N L nd A (rit) nd A (mouse) nd A (rit) nd A (mouse) Figure. Expression of Wnt inhiitory fctor- (WIF-) in the skin of ptients with melsm. ( c) Rel time-pcr nlysis of WIF- mrna expression in () hyperpigmented (L) nd normlly pigmented (N) skin specimens iopsied from 3 ptients with melsm, () in corresponding pired sites, cheeks or forehed (C), nd retrouriculr re (R) from three helthy norml controls, nd (c) in pired sites with (UV þ ) or without UV rdition (UV ) from three helthy norml controls. Po.. (d) Doule stining of hyperpigmented (L) nd normlly pigmented (N) skin of melsm ptients with nti-wif- nd nti-k4 ntiodies, which emit green nd red fluorescence, respectively. Nuclei were counterstined with Hoechst 3358, which emits lue fluorescence. Stining with second ntiodies only is shown for the negtive control. BF, right field; K4, cytokertin 4. Br ¼ 5 mm. for the study. Knockdown of WIF- significntly (Po.5) stimulted the expression levels of tyrosinse (Figure 4). Phosphoryltion of GSK-3, -ctenin, nd NFATc ws significntly (Po.5) reduced, wheres expression incresed (Figure 4c). FACS nlysis to exmine melnosome trnsfer to neighoring kertinocytes suggested tht the percentges of TYRP- nd K4 doule-positive cells were significntly (Po.) incresed following WIF- knockdown (Figure 4d). The effect of WIF- upregultion on melnogenesis nd melnosome trnsfer The incresed expression of WIF- ws directly induced in the monocultures of melnocytes, s well s co-cultures y the tretment with recominnt humn WIF- (rhwif-; Figure 5). Tyrosinse expression ws significntly (Po.) reduced (Figure 5) with expression, wheres the phosphoryltion of GSK-3, -ctenin, nd NFATc ws incresed (Po.5; Figure 5c) in co-cultures. Reduced tyrosinse expression (Figure 5) ws lso identified with decresed -ctenin nd NFATc in the nucler frction (Figure 5d) of melnocyte monocultures. FACS nlysis suggested tht the proportions of TYRP- nd K4 doule-positive cells were significntly decresed (Po.5; Figure 5e). Wnts in the skin of melsm ptients WIF- is secreted ntgonist of Wnt signling (Hsieh et l., 999) tht inhiits oth the cnonicl nd noncnonicl Wnt pthwys. Our results reveled tht the decresed expression of WIF- in either kertinocytes or firolsts ffected oth Wnt pthwys (Figures 3d nd 4c), which were reversed y the WIF- overexpression (Figure 5c nd d). Therefore, the expression of cnonicl Wnts, Wnt- nd Wnt-3A, s well s noncnonicl Wnts, Wnt-5A nd Wnt-, ws compred using rel-time PCR in hyperpigmented nd normlly pigmented skin of the sme 3 ptients with melsm. Levels 93

4 WIF- mrna expression (WIF-/GAPDH) WIF- protein expression (WIF-/β-ctin) Reltive rtios of WIF- mrna expression (TPA/TPA ) WIF- Reltive rtios of WIF- protein expression (TPA/TPA ) WIF FB FB FB TPA TPA TPA TPA TPA TPA FB TPA TPA TPA TPA TPA TPA TPA TPA FB TPA TPA TPA TPA FB Figure. Expression of Wnt inhiitory fctor- (WIF-) in cultured skin cells. Rel-time PCR nd western lot nlysis of WIF- in cultured kertinocytes (s), melnocytes (s), nd firolsts (FBs) using medium () without or () with -O-tetrdecnoylphorol-3- cette (TPA). Dt represent mens±sd of three independent experiments. GAPDH, glycerldehyde-3-phosphte dehydrogense. Po.5. of Wnt- nd Wnt-5A expression were significntly (Po.5) higher in the hyperpigmented skin, wheres levels of other Wnts were not (Figure 6). In fct, tretment with rhwnt- (Figure 6) nd rhwnt-5a (dt not shown) significntly (Po.5) incresed the expression levels of tyrosinse with n ctivtion of the cnonicl nd the noncnonicl pthwys in cultured melnocytes. DISCUSSION This study revels tht decresed expression of WIF- is involved in the hyperpigmented skin of ptients with melsm. As WIF- is n ntgonist of Wnt signling (Hsieh et l., 999), which regultes skin pigmenttion, downregultion of WIF- could e expected to induce hyperpigmenttion of the skin. In fct, WIF- mrna levels re significntly lower in hyperpigmented skin compred with pired normlly pigmented skin specimens from ptients with melsm (Figure ), long with stining intensity of immunohistochemistry, using nti-wif- ntiody (Figure d). Although the iopsied site of normlly pigmented skin is not the forehed or the cheek, ut the retrouriculr re, the effect of sunlight or site difference on the WIF- downregultion could e excluded, ecuse the WIF- mrna levels did not reduce in pired skin specimens of the corresponding sites nd of the sence or presence of UV irrdition from helthy control individuls (Figure nd c). In ddition, UV irrdition on cultured kertinocytes, firolsts, nd melnocytes did not induce ny chnge in WIF- expression levels (dt not shown). Expression of WIF- mrna nd protein, which ws exmined in cellulr components of the epidermis nd the dermis, ws lmost unrecognizle in melnocytes, wheres they were esily detected in kertinocytes nd firolsts (Figure nd ), s previously reported (Cho et l., 9; Sshr et l., 9; Gudjonsson et l., ). The -Otetrdecnoylphorol-3-cette contined in the melnocyte culture medium, which ws lso dded into the medi for kertinocyte/melnocyte co-cultures, could ffect the Wnt signling pthwy through the P ctivtion (Niedel et l., 983). However, the WIF- expression, with or without -O-tetrdecnoylphorol-3-cette, ws not chnged in melnocytes nd kertinocytes, lthough it ws incresed in the firolsts (Figure nd ). Therefore, we hypothesized tht the downregultion of WIF- function in kertinocytes nd/or firolsts, insted of melnocytes, my hve n impct on the skin hyperpigmenttion. In fct, doule stining, which uses ntiodies to WIF- nd K4, suggests weker stining of WIF- in the epiderml kertinocytes of the hyperpigmented compred with tht of the normlly pigmented skin specimens of melsm ptients (Figure d). Purifiction of melnocytes from kertinocyte/melnocyte co-cultures required the tretment of enzymes, lthough tht from co-cultured firolsts did not. Therefore, kertinocyte/ melnocyte co-cultures were used to exmine the effect of WIF- downregulted kertinocytes or firolsts on norml melnocytes. As estlished in the cell-to-cell interctions etween kertinocytes nd melnocytes, the decresed expression of WIF- in kertinocytes stimultes tyrosinse 94 Journl of Investigtive Dermtology (3), Volume 33

5 48 Hours l ro - nt IF o siw sic Reltive rtios of WIF- mrna expression (siwif-/). WIF- l tro IF siw si p-p-δ/θ t-p-δ/θ Tyrosinse p-nfatc p-β-ctenin t-nfatc t-β-ctenin t-jnk Reltive rtios.5 siwif- 8 Hours l tro - IF on siw sic - n Co siwif-.5 siwif- F IT M e 8 Hours l tro si - IF siw K4-FL n Co t-β-ctenin t-nfatc 3 β p- β -3-3 K GS K GS t- p- in ten -c t-β P P θ -δ/ P p- 3.45±.75 Tc FA N p- K JN p- siwif- P P P3 P4 7.95±7.8 P3 9.96±.7 P4 TYRP--FL siwif- in ten c β- K4-FL d Reltive rtios 8 Hours Reltive rtios of tyosinse protein expression (siwif-/) 8 Hours l tro - IF on siw sic c siwif-. Reltive rtios of WIF- protein expression (siwif-/ 3 TYRP--FL 3 siwif-.38±3.8.5 t-β-ctenin t-nfatc Figure 3. The effects of Wnt inhiitory fctor- (WIF-) knockdown in kertinocytes on hyperpigmenttion. () Rel-time PCR nd western lot nlysis for the trnsfection of WIF- sirna (siwif-) nd control sirna () in kertinocytes. Dt represent mens±sd of three nd five independent experiments, respectively. Po. ( d) Western lot nlysis of tyrosinse () nd Wnt signling fctors in totl cell lystes (c) nd in nucler frctions (d) from melnocyte/kertinocyte co-cultures in which the kertinocytes hd een trnsfected with siwif- or. Dt represent mens±sd of five independent experiments. Po.5 nd Po.5 for tyrosinse nd Wnt signling fctors, respectively. (e) FACS nlysis nd immunofluorescence (X Y two-dimensionl (D) imge) study using nti-tyrp- (red) nd nti-k4 ntiodies (green) with or without WIF- knockdown. The cells (white rrow), which contined vrious mount of TYRP--positive prticles round/over the nuclei (lue), indicte positive cells. Dt represent mens±sd of three independent experiments. Po.5. FL (inding ility to fluorochromes) : FITC; FL: phycoerythrin. GSK-3, glycogen synthse kinse-3; JNK, c-jun N-terminl kinse; K4, cytokertin 4;, microphthlmi-ssocited trnscription fctor; NFATc, nucler fctor of ctivted T cells, cytoplsmic, clcineurin-dependent ; p, phospho; P, protein kinse C; sirna, smll interfering RNA; t, totl; TYRP-, tyrosinse-relted protein-. Br ¼ 5 mm. 95

6 Tyrosinse Reltive rtios of WIF- mrna expression (siwif-/). 8 Hours siwif- Reltive rtios of tyrosinse protein expression (siwif-/).5 siwif- WIF- siwif- 48 Hours siwif- c Reltive rtios of WIF- protein expression (siwif-/) p-β-ctenin t-β-ctenin. 8 Hours siwif- p-p-δ/θ t-p-δ/θ p-nfatc t-nfatc siwif- 8 Hours siwif- t-jnk d K4-FL 3 P P3 siwif- P.65±5 3 P P 9.5± P4 K4-FL P3 P4 Reltive rtios.5 siwif- TYRP--FL 3 TYRP--FL 3 p-β-ctenin t-β-ctenin p-p-δ/θ p-nfatc Figure 4. Effects of Wnt inhiitory fctor- (WIF-) knockdown in firolsts on tyrosinse expression nd melnosome trnsfer. () Rel-time PCR nd western lot nlysis for the trnsfection of WIF- sirna (siwif-) nd control sirna () in firolsts. Dt represent mens±sd of three nd five independent experiments, respectively. Po.5. (, c) Western lot nlysis of () tyrosinse nd (c) Wnt signling fctors in kertinocyte/melnocyte cocultures tht were cultured three-dimensionlly with firolsts trnsfected with or without WIF- knockdown. Dt represent mens±sd of five independent experiments.(po.5 nd Po.5 for tyrosinse nd Wnt signling fctors, respectively. (d) FACS nlysis using nti-tyrp- nd nti-k4 ntiodies with or without WIF- knockdown. Dt represent mens±sd of three independent experiments. Po.. FL (inding ility to fluorochromes) : FITC; FL: phycoerythrin. GSK-3, glycogen synthse kinse-3; JNK, c-jun N-terminl kinse; K4, cytokertin 4;, microphthlmi-ssocited trnscription fctor; NFATc, nucler fctor of ctivted T cells, cytoplsmic, clcineurin-dependent ; p, phospho; P, protein kinse C; sirna, smll interfering RNA; t, totl; TYRP-, tyrosinse-relted protein-. expression (Figure 3) nd melnosome trnsfer from cocultured norml melnocytes (Figure 3e). Regultion of melnogenesis could lso result from complex interctions etween melnogenic regultors in melnocytes nd fctors derived from firolsts. Extrcellulr mtrix proteins derived from humn derml firolsts increse tyrosinse ctivity in norml melnocytes (Hedley et l., 996). The expression of high levels of dickkopf (DKK), n inhiitor of the cnonicl Wnt signling pthwy s memer of the Dickkopf clss of Wnt ntgonists, hs lso een reported in plmoplntr firolsts (Ymguchi et l., 4). In our study, the effect of WIF- knockdown in firolsts ws exmined using three-dimensionl collgen gel model (Ymguchi et l., 999) to simulte rel skin conditions. WIF- knockdown in firolsts resulted in increses of oth tyrosinse expression (Figure 4) nd melnosome trnsfer to kertinocytes (Figure 4d). Moreover, the result from the WIF- knockdown in neighoring kertinocytes or firolsts is reversed y tretment with rhwif- in melnocyte monocultures, s well s in kertinocyte/melnocyte co-cultures (Figure 5 nd e), suggesting the significnce of downregulting WIF- function on hyperpigmenttion. WIF- elongs to the sfrp clss of Wnt ntgonists, which cn inhiit oth the cnonicl nd the noncnonicl Wnt pthwys. In this study, the decresed expression of WIF-, either in kertinocytes (Figure 3c) or firolsts (Figure 4c), reduces phosphoryltion of GSK-3 nd -ctenin, which results in trnsloction of -ctenin to nucleus (Figure 3d). These results indicte n involvement of the cnonicl Wnt/ -ctenin pthwy with stimulting trnscription of trget genes. Increse in expression lso supports the involvement of the cnonicl pthwy (Schepsky et l., 96 Journl of Investigtive Dermtology (3), Volume 33

7 WIF- Tyrosinse Reltive rtios of tyrosinse protein expression (rhwif-/) d Reltive rtios. Co-culture t-β-ctenin t-nfatc. Co-culture Co-culture rhwif- rhwif- rhwif- rhwif- rhwif- - hour t-β-ctenin rhwif- control rhwif- t-nfatc control rhwif- c Reltive rtios p-nfatc t-nfatc e K4-FL 3 t-jnk Reltive rtios of WIF- protein expression (rhwif-/).5 P P3 Hour control rhwif rhwif- rhwif- rhwif- Co-culture P 7.45±.35 P4 p-β-ctenin t-β-ctenin p-p-δ/θ t-p-δ/θ P rhwif- p-β-ctenin t-β-ctenin p-p-δ/θ p-nfatc K4-FL 3 P3 rhwif- P.6±.5 P4 3 TYRP--FL TYRP--FL 3 Figure 5. The effect of Wnt inhiitory fctor- (WIF-) overexpression on melnogenesis nd melnosome trnsfer. () Western lot nlysis of WIF- expression following tretment of kertinocytes (s), melnocytes (s), or kertinocyte/melnocyte co-cultures (Co-culture) with recominnt humn WIF- (rhwif-). Dt represent mens±sd of five independent experiments. Po.5. (, d) Western lot nlysis of () tyrosinse nd (c) Wnt signling fctors in totl cell lystes from co-cultures nd/or in (d) nucler frctions from monocultures of melnocytes simultneously treted with or without rhwif-. Dt represent mens±sd of three independent experiments. Po. nd Po.5 for tyrosinse nd Wnt signling fctors, respectively. (e) FACS nlysis using nti-tyrp- nd nti-k4 ntiodies in kertinocyte/melnocyte co-cultures treted with or without rhwif-. Dt represent mens±sd of three independent experiments. Po.5. FL (inding ility to fluorochromes) : FITC; FL: phycoerythrin. GSK-3, glycogen synthse kinse-3; JNK, c-jun N-terminl kinse; K4, cytokertin 4;, microphthlmi-ssocited trnscription fctor; NFATc, nucler fctor of ctivted T cells, cytoplsmic, clcineurin-dependent ; p, phospho; P, protein kinse C; sirna, smll interfering RNA; t, totl; TYRP-, tyrosinse-relted protein-. 6). The WIF- knockdown in either kertinocytes or firolsts lso increses the dephosphoryltion of NFATc, which results in the trnsloction of NFATc to nucleus (Figure 3d) tht indictes the involvement of the noncnonicl pthwy with stimulting trnscription of trget genes. Although P ctivtion responds to clcium signls (Sheldhl et l., 999) s does NFATc (Murphy nd Hughes, ; Sneyoshi et l., ), nd P-z (Sn-Antonio et l., ) nd P-y (Grnj et l., 8) hve intercted with NFATc, the phosphoryltion of P-d/y is not significnt with the WIF- knockdown (Figures 3c nd 4c). The result tht overexpression of WIF- y rhwif- tretment reverses the expression of oth cnonicl nd noncnonicl signling molecules (Figure 5c) supports the notion tht WIF- exerts its ction through oth the cnonicl nd the noncnonicl Wnt pthwys. WIF- overexpression decresed -ctenin nd 97

8 Reltive rtios of Wnt- mrna expression (L/N) Wnt- Tyrosinse p-β-ctenin t-β-ctenin Reltive rtios N L rhwnt- Reltive rtios of Wnt-5A mrna expression (L/N) NFATc in nucler frction of melnocyte monocultures, s well s in totl cell lystes of kertinocyte/melnocyte cocultures (Figure 5c nd d), suggesting the involvement of sme pthwys in melnocytes. WIF- is supposed to inhiit the Wnt ction y direct inding to Wnts. The incresed Wnt- nd Wnt-5A mrna levels in ptients with melsm, who hd reduced WIF- mrna expression levels (Figure 6), hve supported the interction etween WIF- nd Wnts. Although Wnt expression in melnocytes y WIF- knockdown, either in kertinocytes or in firolsts, ws not exmined, the incresed melnogenesis nd the involved signling pthwys y the knockdown were the sme s result of the tretment of cultured melnocytes with rhwnt- (Figure 6) nd rhwnt-5a. Although it is uncertin how the reduced 3.5 p-p-δ/θ t-p-δ/θ p-nfatc t-nfatc t-jnk Wnt- Tyrosinse p-β-ctenin t-β-ctenin p-p-δ/θ N L rhwnt- rhwnt- p-nfatc Figure 6. Expression of Wnts in the skin of melsm ptients. () Rel-time PCR nlysis of Wnt- nd Wnt-5A mrna expression in hyperpigmented (L) nd in normlly pigmented (N) skin specimens from 3 ptients with melsm. Po.5. () Western lot nlysis of tyrosinse nd Wnt signling fctors in cultured melnocytes treted with recominnt humn Wnt- (rhwnt-). Dt represent mens±sd of three independent experiments. Po.5. GSK-3, glycogen synthse kinse-3; JNK, c-jun N-terminl kinse;, microphthlmi-ssocited trnscription fctor; NFATc, nucler fctor of ctivted T cells, cytoplsmic, clcineurin-dependent ; p, phospho; P, protein kinse C; t, totl. WIF- expression in the neighoring kertinocytes or firolsts could ffect the melnogenesis in melnocytes, prcrine effect could e expected, due to n unrecognizle WIF- expression levels in melnocytes. Decresed WIF- expression in the neighoring kertinocytes or firolsts could reduce WIF- inding to Wnts in melnocytes, resulting in n incresed Wnt expression nd exerting the ction through the cnonicl nd the noncnonicl Wnt pthwys. In summry, decresed expression of WIF-, which my occur in epiderml kertinocytes nd derml firolsts, is implicted in melsm development vi the stimultion of melnogenesis nd melnosome trnsfer through the upregultion of Wnt, oth cnonicl nd noncnonicl, signling pthwy. MATERIALS AND METHODS Ptients nd helthy controls A totl of 3 femle ptients dignosed with melsm etween 35 nd 58 yers of ge (men 44 yers) were included in the study. The Institutionl Review Bord of Dongguk University Ilsn Hospitl pproved this study, nd the study ws conducted ccording to the Declrtion of Helsinki Principles. After otining informed written consent from prticipnts, skin specimens were otined y iopsy. Pirs of normlly pigmented nd hyperpigmented smples were tken from ech prticipnts for direct comprisons. As the loction of the hyperpigmented lesions ws on the lterl side of the forehed or the upper cheek nd control skin ws on the retrouriculr re, two corresponding sites were compred in the three ge-mtched helthy femles. Hyperpigmented skin, which ws induced y topicl psorlen ppliction, followed y UVA irrdition twice week for weeks in dominl skin of nother group of three helthy mles, ws lso compred. Norml humn kertinocyte, melnocyte, nd firolst cultures Adult skin specimens otined from Cesren sections nd circumcisions were used to estlish cells in culture. For kertinocyte culture, individul epiderml cells were suspended in EpiLife Medium (ctlog numer M-EPI-5-CA; Invitrogen, Crlsd, CA) supplemented with ovine pituitry extrct, ovine insulin, hydrocortisone, humn EGF, nd ovine trnsferrin (ctlog numer S--5; Invitrogen). Kertinocytes from pssges 3 or 4 were used in these experiments. For melnocytes, individul epiderml cells were suspended in Medium 54 (Invitrogen) supplemented with ovine pituitry extrct, fetl ovine serum, ovine insulin, hydrocortisone, sic firolst growth fctor, ovine trnsferrin, heprin, nd phorol -myristte 3-cette (Invitrogen). Melnocytes t pssges etween 7 nd 5 were used. For firolst culture, individul derml cells were suspended in DMEM (Gico/BRL, Grnd Islnd, NY) supplemented with % fetl ovine serum (Gico/BRL), U ml penicillin (Gico/BRL), nd. mg ml streptomycin (Gico/BRL). Firolsts t pssges etween 5 nd were used. WIF- knockdown For WIF- downregultion, kertinocytes or firolsts were cultured in six-well pltes (NUNC, Roskilde, Denmrk) or mm 98 Journl of Investigtive Dermtology (3), Volume 33

9 culture dishes (NUNC) nd were trnsfected with nm or 5 nm sirna for humn WIF- or negtive control (ON-TARGETplus SMARTpool or Non-trgeting sirna; Dhrmcon Reserch, Lfyette, CO) using the TrnsIT-siQUEST trnsfection regent (Mirus, PnVer, Mdison, WI) ccording to the mnufcturer s protocol. At 4 hours fter the incution, the trnsfected kertinocytes were co-cultured with norml melnocytes for nother 8 hours to exmine the effect of kertinocytes with WIF- knockdown on melnogenesis nd melnosome trnsfer. For the effect of firolst with WIF- knockdown, threedimensionl cultures were performed using the collgen gel model (Ymguchi et l., 999): the trnsfected firolsts were suspended in ml of collgen mtrix (Invitrogen) nd cultured in six-well culture dishes (Corning, Corning, NY) for hour. Then, norml kertinocytes were cultured onto BioCot cell culture inserts (Corning) for 4 hours, efore dding norml melnocytes onto the sme inserts. Tretment with rhwif- or rhwnt- For the overexpression of WIF- or Wnt-, melnocytes nd/or kertinocytes were simultneously treted with or without 5 ng ml rhwif- (R&D Systems, Minnepolis, MN) or Wnt- (BioVision, Milpits, CA) for or 3 hours. Nucler protein extrction Nucler protein frction ws purified from melnocyte monocultures or kertinocyte/melnocyte co-cultures, using the NE-PER Nucler nd Cytoplsmic Extrction Regents kit (Thermo, Rockford, IL). The protein concentrtion of the lyste ws mesured using icinchoninic cid protein ssy kit (Thermo). Rel-time PCR Levels of WIF-, Wnt-, nd Wnt-5A mrnas were quntitted y rel-time PCR using the Light Cycler rel-time PCR (Roche, Mnnheim, Germny). Their reltive mounts were clculted using the rtio of ech mrna reltive to the mount of glycerldehyde-3- phosphte dehydrogense (GAPDH). The primers used nd proes were s follows: WIF- (No. HS8366-m; designed using Applied Biosystems softwre, Foster City, CA); Wnt- 5 -CTCAT GAACCTTCACAACAACGA-3 (forwrd) nd 5 -ATCCCGTGGCAC TTGCA-3 (reverse); Wnt-5A 5 -GACCACATGCAGTACATCGGA GAAG-3 (forwrd) nd 5 -TCCACCTTCGATGTCGGAATTG-3 (reverse); GAPDH 5 -TCCACTGGCGTCTTCACC-3 (forwrd) nd 5 -GGCAGAGATGATGACCCTTT-3 (reverse). Western lot nlysis Equl mounts of extrcted proteins ( mg) were resolved nd trnsferred to nitrocellulose memrnes. The memrnes were incuted with ntiodies to -ctenin, phospho--ctenin, GSK- 3, phospho-gsk3, JNK, phospho-jnk, P-d/y, phospho-p-d/ y, WIF-, nd NFATc (rit polyclonl; Cell Signling Technology, Beverly, MA), phospho-nfatc, (rit polyclonl; Snt Cruz Biotechnology, Snt Cruz, CA), tyrosinse (got polyclonl; Snt Cruz Biotechnology), nd Wnt- (rit polyclonl; Epitomics, Burlingme, CA). After incuting with pproprite nti-mouse or nti-rit horserdish peroxidse conjugted ntiodies (Thermo) or with nti-got horserdish peroxidse conjugted ntiody (Snt Cruz Biotechnology) nd n enhnced chemiluminescence solution (Thermo), the signls were cptured on n Imge Reder (LAS-3; Fuji Photo Film, Tokyo, Jpn). To monitor the mount of protein loded in ech lne, the memrnes were reproed with mouse monoclonl nti--ctin ntiody (Sigm, St Louis, MO) nd were processed s descried ove. The protein nds were then nlyzed y densitometry. Immunohistochemistry Epiderml specimens were fixed in 4% prformldehyde, nd were then dehydrted nd emedded in prffin. After deprffiniztion, the sections were pretreted with citric cid solution ( mm citrte, ph 6.) nd % Triton X-. After locking with 3% BSA, epiderml sections were incuted with nti-wif- (: dilution; rit polyclonl; Snt Cruz Biotechnology) nd K4 (: dilution; mouse monoclonl; Snt Cruz Biotechnology) ntiodies for primry ntiodies. After stining with Alex Fluor leled got nti-rit IgG (: dilution; 488; Moleculr Proes, Eugene, OR) nd Alex Fluor leled got ntimouse IgG (: dilution; 594; Moleculr Proes), the stined cells were oserved, using fluorescence microscope (Dp Mnger.; Olympus Opticl, Tokyo, Jpn). Melnosome trnsfer Flow cytometry nlysis ws performed s descried previously (Lin et l., 8). Briefly, the cells were fixed with % prformldehyde, nd incuted with oth FITC-conjugted got nti-tyrp- ( mg per 6 cells; Snt Cruz Biotechnology) nd phycoerythrin-conjugted mouse nti-k4 (:; BD Biosciences, Sn Jose, CA). Leled cells were nlyzed with Cytomics FC5 flow cytometer (Beckmn Coulter, Hileh, FL) using CXP softwre (Beckmn Coulter). Melnosome trnsfer efficcy ws determined s the numer of K4 nd TYRP- doule-positive cells divided y the totl numer of K4-positive cells. For immunofluorescence study, the cultured cells were fixed with 4% prformldehyde solution nd were pretreted with % of Triton X-. The cells were incuted with nti- TYRP- ntiody (: dilution; got polyclonl; Snt Cruz Biotechnology) nd K4 (: dilution; Snt Cruz Biotechnology), nd then with Alex Fluor leled donkey nti-got IgG (: dilution; Moleculr Proes) s well s Alex Fluor leled got nti-mouse IgG (: dilution; Moleculr Proes). Stined cells were oserved using Zeiss LSM 5 confocl fluorescence microscope (Oerkochen, Germny). For quntittive nlysis of K4 nd TYRP- doule-positive cells, rndom fields were photogrphed. Sttisticl nlysis Sttisticl significnce ws ssessed using nlysis of vrince tests. A P-vlue of o.5 is considered sttisticlly significnt. All results re presented s mens±sd of the comined dt from three or five independent experiments. CONFLICT OF INTEREST The uthors stte no conflict of interest. ACKNOWLEDGMENTS This work ws supported y the Ntionl Reserch Foundtion of Kore (NRF) grnt funded y the Koren government (MEST; -896). 99

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