SOX9 Is a Key Player in Ultraviolet B-Induced Melanocyte Differentiation and Pigmentation

SOX9 is a key player in ultraviolet B-induced melanocyte differentiation and pigmentation

Thierry Passeron*, Julio C. Valencia*, Corine Bertolotto†, Toshihiko Hoashi*, Elodie Le Pape*, Kaoruko Takahashi*, Robert Ballotti†, and Vincent J. Hearing*‡

*Pigment Cell Biology Section, Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20814; and †Unite´597, Institut National de la Sante´et de la Recherche Me´dicale, Faculte´deMe´ decine, Universite´de Nice Sophia–Antipolis, 06103 Nice, France

Edited by Gertrud M. Schu¨pbach, Princeton University, Princeton, NJ, and approved July 2, 2007 (received for review May 31, 2007) SOX (SRY type HMG box) proteins are transcription factors that are (13–15). Interestingly, ectopic SOX9 expression in the neural crest predominantly known for their roles during development. During is sufficient to promote melanocytic differentiation (16), which melanocyte development from the neural crest, SOX10 regulates suggests a role for SOX9 in melanocytic development. An active microphthalmia-associated transcription factor, which controls a set transcription of SOX9 protein in melanocytes within the skin also of genes critical for pigment cell development and pigmentation, has been indirectly suggested by the presence of antibodies against including dopachrome tautomerase and tyrosinase. We report here SOX9 in the sera of vitiligo patients (17). Interestingly, the cAMP that another SOX factor, SOX9, is expressed by melanocytes in pathway, which plays a key role in melanocyte differentiation, also neonatal and adult human skin and is up-regulated by UVB exposure. has been involved in the control of SOX9 function in chondrocytes We demonstrate that this regulation is mediated by cAMP and protein (18, 19). These observations suggest that SOX9 might participate in kinase. We also show that agouti signal protein, a secreted factor the regulation of melanocyte differentiation by cAMP-elevating known to decrease pigmentation, down-regulates SOX9 expression. agents such as ␣-melanocyte-stimulating hormone (␣-MSH) or UV In adult and neonatal melanocytes, SOX9 regulates microphthal- radiation, that act, at least in part, through the cAMP pathway. In mia-associated transcription factor, dopachrome tautomerase, and the present study, we show the presence of SOX9 at the RNA and tyrosinase promoters, leading to an increase in the expression of protein levels in normal human melanocytes in vitro and in vivo.We these key melanogenic proteins and ﬁnally to a stimulation of show the up-regulation of SOX9 after UVB exposure and its pigmentation. SOX9 completes the complex and tightly regulated increased nuclear localization. We demonstrate that the effects of process leading to the production of melanin by acting at a very UVB act at least through the cAMP pathway and increased levels upstream level. This role of SOX9 in pigmentation emphasizes the of protein kinase A (PKA). We also show that agouti signal protein poorly understood impact of SOX proteins in adult tissues. (ASP), a secreted factor known to decrease pigmentation and to antagonize the signaling pathway of ␣-MSH, down-regulates SOX9 microphthalmia-associated transcription factor ͉ tyrosinase ͉ protein expression. Moreover, we demonstrate the key role of SOX9 in kinase A ͉ melanocyte-stimulating hormone ͉ agouti signal protein regulating pigmentation. Indeed, we show that SOX9 regulates MITF and DCT promoters. Overexpression of SOX9 induces an ox (SRY type HMG box) proteins are transcription factors that increase of MITF, DCT, and tyrosinase proteins, which leads to an Sbelong to the HMG box superfamily of DNA-binding proteins increased production of melanin within the cells. and play a key role during development. SOX9 belongs to the SOX-E subgroup, which includes SOX8, SOX9, and SOX10. The Results structures of these proteins show a high conservation and similar SOX9 Is Expressed in Melanocytes in Vivo. To determine whether positions of their HMG boxes (1). SOX10 has been shown to play SOX9 mRNA is expressed in human skin, we designed a specific a key role in the regulation of melanocyte differentiation (2), and probe directed against SOX9 and performed a tissue in situ mutations in SOX10 lead to Waardenburg syndrome type 4, a hybridization (TISH) study. We were able to detect a strong genetic hypomelanosis with deafness and megacolon (3). During positive staining in the epidermis with the antisense probe melanocyte development from the neural crest, SOX10 regulates whereas staining with the sense probe was negative (Fig. 1A). To the expression of microphthalmia-associated transcription factor differentiate melanocytes from keratinocytes, we coupled the (MITF), which in turn controls a set of genes critical for pigment cell TISH protocol with standard immunohistochemistry performed development and pigmentation (4). Indeed, in conjunction with with antibodies directed against MART1, a specific marker of other transcription factors, MITF regulates dopachrome tautomer- melanocytes. Melanocytes identified by MART1 also were ase (DCT), tyrosinase (the limiting enzyme for melanogenesis), and stained by the SOX9 TISH probe (Fig. 1B), demonstrating that tyrosinase-related protein 1 (TYRP1). All of these proteins are essential for the full differentiation of melanocytes and are directly Author contributions: T.P., R.B., and V.J.H. designed research; T.P. and J.C.V. performed involved in melanin synthesis. SOX10 also acts as a critical trans- research; C.B., T.H., E.L.P., and K.T. contributed new reagents/analytic tools; T.P., R.B., and activator of DCT, which MITF, on its own, is insufficient to V.J.H. analyzed data; and T.P., R.B., and V.J.H. wrote the paper. stimulate (5–7). The authors declare no conﬂict of interest. SOX9 has a key role in sexual determination and chondrogenesis, This article is a PNAS Direct Submission. and mutations in SOX9 can lead to campomelic dysplasia, a skeletal Abbreviations: a-LP, adult lightly pigmented; ␣-MSH, ␣-melanocyte-stimulating hormone; dysmorphology associated in most XY cases with sex reversal (8, 9). ASP, agouti signal protein; CRE, cAMP response element; CREB, CRE-binding protein; DCT, During embryonic development, the SOX9 gene becomes active in dopachrome tautomerase; MITF, microphthalmia-associated transcription factor; n-LP, all prechondrocytic mesenchymal condensations, and its expression neonatal lightly pigmented; n-DP, neonatal darkly pigmented; NHM, normal human epidermal melanocytes; n-MP, neonatal moderately pigmented; TISH, tissue in situ hybrid- is maintained at high levels in fully differentiated chondrocytes. The ization; TYRP1, tyrosinase-related protein 1; PKA, protein kinase A. direct target for SOX9 is a chondrocyte-specific enhancer in the ‡To whom correspondence may be addressed. Laboratory of Cell Biology, National Cancer gene for collagen type II (10, 11). With aging, the loss of expression Institute, National Institutes of Health, Building 37, Room 2132, MSC 4256, Bethesda, of SOX9 in some disk cells may play a role in disk degeneration by MD 20892. E-mail: [email protected]. resulting in decreased expression and production of collagen type This article contains supporting information online at www.pnas.org/cgi/content/full/ II (12). There have been increased numbers of reports showing the 0705117104/DC1. active role of SOX9 in other tissues such as heart, kidney, or brain © 2007 by The National Academy of Sciences of the USA

13984–13989 ͉ PNAS ͉ August 28, 2007 ͉ vol. 104 ͉ no. 35 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0705117104 Downloaded by guest on September 27, 2021 21 mJ/cm2 UVB and compared them with nonirradiated controls. Using RT-PCR, we showed that the expression level of SOX9 was increased1hafterUVBexposure compared with nonirradiated cells (Fig. 2A). At the protein level, an increase in SOX9 was observed starting 2 h after the UVB exposure, increased further until 8 h, and then decreased at 24 h (Fig. 2B). The stimulation of SOX9 expression was confirmed by immunoprecipitation of 35S- labeled SOX9. Finally, we compared the basal expression of SOX9 in neonatal lightly pigmented (n-LP) skin and in neonatal darkly pigmented (n-DP) skin NHM by using immunoblotting. The darker melanocytes expressed more SOX9 than did the lightly pigmented melanocytes (Fig. 2C).

The Action of UVB on SOX9 Is Mediated by cAMP and PKA. The cAMP pathway plays a key role in regulating pigmentation and mediates most of the effects of UV on melanogenesis (20), so we next investigated the effect of cAMP on SOX9. After 35S metabolic labeling, NHM were exposed to 21 mJ/cm2 UVBortoforskolin,a cAMP-stimulating agent, and then SOX9 was immunoprecipitated. Treatment with either UVB or forskolin led to increased expression of SOX9 (Fig. 3A). PKA, the most important downstream target of cAMP, can be inhibited by H89. The expressions of SOX9 and SOX9 phosphorylated at S181 were both increased after UVB exposure, and in both cases, pretreatment with H89 prevented the increased expression of SOX9 by UVB (Fig. 3B). The up-regulation of SOX9 after forskolin treatment also was observed in NHM by using immunocytochemistry. Four hours after forskolin treatment, the cells were fixed and stained with SOX9 antibodies. An increased expression of SOX9 was noted in NHM Fig. 1. TISH analysis of SOX9 expression in the skin in vivo.(A) Antisense probes directed against SOX9 are present in the skin with strong nuclear staining, treated with forskolin (Fig. 4A). We also used a reconstructed skin whereas staining with the sense probe against SOX9 is negative and shows the model (MelanoDerm; MatTek, Ashland, MA) to assess the effect presence of SOX9 RNA in the skin. (B) Coupling of TISH with antisense probes of UVB on SOX9 expression in the skin. After 4 days of growth, against SOX9 and immunohistochemistry with MART1 antibody as a melanocytic MelanoDerms were exposed or not to 21 mJ/cm2 UVB. The marker shows that SOX9 is expressed in keratinocytes and in melanocytes. samples were fixed 8 h after the UV irradiation and were then double-stained with SOX9 and MART1 antibodies to identify melanocytes. The results confirmed the increased SOX9 staining in melanocytes express SOX9 in human skin in vivo. The presence melanocytes with a predominant nuclear localization after UVB of SOX9-positive melanocytes also was detected at the protein exposure (Fig. 4B). level with immunohistochemistry by using antibodies directed against SOX9 and MART1 (data not shown). SOX9 Is Down-Regulated by ASP. ASP is an antagonist of the melanocortin 1 receptor, which plays a key role in regulating SOX9 Is Up-Regulated by UVB Exposure. To investigate whether pigmentation (21). ASP inhibits melanin formation in mouse

SOX9 is regulated during melanocyte differentiation, we exposed melanocytes and in melanoma cells as well as in NHM (22–24). We CELL BIOLOGY melanocytes to their most common external stimulus, UV radia- treated NHM obtained from neonatal moderately pigmented (n- tion. We irradiated normal human epidermal melanocytes (NHM) MP) skin with ASP. RNAs then were extracted, and with RT-PCR, obtained from adult lightly pigmented (a-LP) skin in culture with we showed that the expression level of SOX9 was decreased 4 h

Fig. 2. SOX9 expression is up- regulated after UVB exposure. (A) Ex- pression levels of SOX9 mRNA using RT-PCR in untreated NHM (basal) or 1 h after 21 mJ/cm2 UVB irradiation. ␤-actin expression served as a control. The histogram shows quantiﬁcation of the data with the means Ϯ SD of three independent experiments. Re- sults are expressed as a ratio compared with the basal condition. (B) Protein expression levels of SOX9 using immunoblotting in a-LP NHM without treatment (basal) and after irradiation with 21 mJ/cm2 UVB at 0, 1, 2, 4, 6, 8, and 24 h. (C) Expression levels of SOX9 by immunoblotting in n-DP NHM without treatment (basal) and after irradiation with 21 mJ/cm2 UVB at 1, 2, and 4 h. Numbers above the gels indicate levels of intensity compared with GAPDH. (D) Comparison of the basal level expression of SOX9 in n-LP cells compared with n-DP cells using immunoblotting. The histogram shows quantiﬁcation of the data with means Ϯ SD in three independent experiments. Results are expressed as a ratio compared with the basal condition. SOX9 is expressed at higher levels in the more pigmented melanocytes.

Passeron et al. PNAS ͉ August 28, 2007 ͉ vol. 104 ͉ no. 35 ͉ 13985 Downloaded by guest on September 27, 2021 Fig. 3. The action of UVB on SOX9 is mediated through the cAMP pathway. (A) 35S labeling of a-LP NHM treated for 3 h with 20 ␮M forskolin or 21 mJ/cm2 UVB or untreated (Basal). Labeled proteins were immunoprecipitated with SOX9 or GAPDH antibodies. (B) UVB acts through the activation of PKA to up-regulate SOX9. 35S labeling of a-LP NHM without treatment (Basal) or with 21 mJ/cm2 UVB irradiation for 3 h with or without a PKA inhibitor (5 ␮M H89). Fig. 5. SOX9 is down-regulated by ASP. (A) Expression levels of SOX9 gene using Proteins were immunoprecipitated with antibodies against SOX9 and GAPDH. RT-PCR in n-MP NHM untreated (basal) or treated with ASP (10 nM) for 4 h. ␤-actin expression served as a control. The histogram shows quantification of the data with the means Ϯ SD of three independent experiments. Results are expressed as a ratio compared with the basal condition. The expression of SOX9 is down- after ASP treatment compared with nontreated cells (Fig. 5A). regulated in melanocytes treated by ASP. (B) Proteins from n-MP NHM treated Proteins were extracted from n-MP NHM treated for 48 h with ASP 48 h with 10 nM ASP were extracted and analyzed by immunoblotting. The and were analyzed by immunoblotting. Concordantly with the histogram shows quantification of the data with the means Ϯ SD of three RT-PCR results, SOX9 expression was decreased significantly in independent experiments. Results are expressed as a ratio compared with the NHM after treatment with ASP (Fig. 5B). basal condition. SOX9 protein expression was decreased significantly in NHM after treatment with ASP. SOX9 Induces Transcription of MITF, DCT, and TYR. As SOX9 was up-regulated after UVB exposure, we then investigated the effects B16 melanoma cells. Overexpression of SOX9, or treatment with of SOX9 on the activities of MITF, TYR, and DCT promoters in forskolin to a lesser extent, induced a strong activation of the MITF, DCT, and TYR promoters [supporting information (SI) Fig. 11]. Conversely, SOX9 silencing using siRNA decreased the MITF, DCT, and TYR promoter activities and impaired forskolin-induced stimulation (SI Fig. 11B). As a control, we showed that the stimulation of a cAMP response element (CRE)-containing promoter by forskolin was not affected by SOX9 silencing (SI Fig. 12). Finally, in HeLa cells, which, in contrast to B16 cells, do not express the protein MITF, overexpression of SOX9 stimulated MITF and DCT promoter activities but not the TYR promoter activity, and forskolin had no effect on the TYR and DCT promoter activities (data not shown). The sum of these data supports the direct activation of the MITF and DCT promoters by SOX9, whereas the TYR promoter seems not to be directly activated by SOX9. To assess whether SOX9 directly regulates MITF expression, we performed ChIP assays on NHM before and4haftertreatment with forskolin. The ChIP assays clearly showed that SOX9 directly occupies the MITF promoter and that the binding was significantly increased after treatment with forskolin (Fig. 6). To determine the relative roles of SOX9 and CRE-binding protein (CREB) on the activation of the MITF promoter, we studied the effects of SOX9 overexpression and of stimulation with forskolin on several trun- cated (in the 5Ј flanking region) or mutated luciferase constructs of the MITF promoter (SI Fig. 13). The 217-bp construct, which has the CRE but lacks all putative SOX9 binding sites, showed no significant activation after SOX9 overexpression or forskolin treat-

Fig. 4. UVB and cAMP up-regulate SOX9 and increase its nuclear concentration. (A) Immunocytochemical staining of a-LP NHM. Four hours after treatment without (Control) or with 20 ␮M forskolin (FSK), the cells were fixed and stained with SOX9 antibody (red). Counterstaining was performed with DAPI (blue) to visualize nuclei. An increased expression of SOX9 was noted in cells treated with forskolin. The transversal cuts shown beneath under the Control and FSK images confirmed the nuclear localization of SOX9. (B) Immunohistochemistry with MelanoDerm 8 h after 21 mJ/cm2 UVB irradiation or without UVB (Control). Samples were stained with antibodies to SOX9 (green) and MART1 (red; as a Fig. 6. SOX9 directly regulates the MITF promoter. ChIP of NHM was melanocytic marker). An increased staining of SOX9 was observed after UVB performed with SOX9 or IgG antibodies with or without 4-h treatment with 20 irradiation both in keratinocytes and in melanocytes, with a stronger increase in ␮M forskolin. The DNA recovered was subjected to amplification by PCR (30 melanocytes and a clear nuclear localization. cycles) using primers for human MITF promoter and human GAPDH promoter.

13986 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0705117104 Passeron et al. Downloaded by guest on September 27, 2021 Fig. 7. SOX9 regulates MITF expression. n-MP NHM were silenced for SOX9 using siRNA. After 24 h, they were ﬁxed, and an immunocytochemistry was performed using SOX9 (red) and MITF (green) antibodies. DAPI (blue) served as a counterstaining. Cells silenced for SOX9 (white arrow) showed no or slight expression of MITF.

ment. As previously reported, mutation of the CRE prevents activation of the MITF promoter by forskolin (25). This mutation significantly decreased activation of the MITF promoter after Fig. 9. SOX9 regulates proteins involved in melanogenesis and increases SOX9 overexpression without completely preventing it. Finally, Ϫ pigmentation of melanocytes. (A) SOX9 was silenced in n-LP NHM by using siRNA selectively mutating the SOX9 element (at residues 266 and for 24 h; then, proteins were extracted and analyzed by immunoblotting. (Left) Ϫ260) showed significant but incomplete decreases in cAMP or Concomitantly with the silencing of SOX9, the expression of DCT and tyrosinase SOX9 responsiveness. This incomplete effect could be due to the proteins was decreased. SOX9 was overexpressed for 24 h in n-LP NHM. Again, action of several other putative SOX9 binding sites present on the after extraction, proteins were analyzed by immunoblotting. (Right) A parallel MITF promoter. increase of DCT and tyrosinase proteins was observed when SOX9 was overex- To confirm the role of SOX9 on MITF expression, we used pressed. (B) n-MP NHM were silenced or overexpressed for SOX9 for 24 and 48 h. immunocytochemistry to examine the effects of silencing SOX9. After protein extraction, the amount of melanin for each condition was calcu- lated and adjusted against the total protein concentration. The histogram shows NHM were transfected with SOX9 siRNA, and, after 24 h, the cells quantification of the data with the means Ϯ SD of three independent experi- were fixed and stained with SOX9 and MITF antibodies. Cells ments. Results are expressed as a ratio compared with the basal condition. Except silenced for SOX9 showed a marked decrease in the expression of after 24 h of SOX9 silencing, there was a significant decrease of melanin after 48 h MITF (Fig. 7). of SOX9 silencing and a significant increase after overexpression of SOX9.

Effects of SOX9 Are Independent of SOX10 Activation. SOX10 has been shown to interact with the MITF and DCT promoters, at least n-LP NHM, whereas control n-LP NHM were transfected with during embryogenesis (7). Thus, some effects of SOX9 could be siRNA scrambles or overexpression mocks. Proteins then were mediated by SOX10. Therefore, we investigated the putative action extracted and analyzed with Western blot. The expression of DCT of SOX9 on SOX10 expression. After silencing SOX9 in NHM, we and tyrosinase proteins was increased when SOX9 was overex- extracted proteins 48 h later and analyzed them by Western blotting. pressed. Concomitantly, when siRNA was used to silence SOX9, The decrease of SOX9 did not affect the level of SOX10 protein NHM expressed less DCT and tyrosinase proteins compared with

(Fig. 8 Left). We then overexpressed SOX9 in NHM and extracted the controls (Fig. 9A). As a final target, we analyzed the amount of CELL BIOLOGY proteins 24 h later and then analyzed them by Western blotting. melanin produced in n-MP NHM after silencing or overexpressing Again, the increase of SOX9 showed no effect on SOX10 protein SOX9 for 24 and 48 h. We found that the amount of melanin was levels (Fig. 8 Right). These results show that, in NHM, the effects increased by the overexpression of SOX9 and was decreased by its of SOX9 on DCT and MITF targets are not mediated or facilitated silencing (Fig. 9B). through SOX10 regulation. Discussion SOX9 Increases Expression of Melanogenic Proteins and Enhances Acquired pigmentation, so called ‘‘tanning,’’ is a complex process Pigmentation. To investigate the action of SOX9 on proteins that leads to an increase in the production of melanin within involved in pigmentation, we overexpressed or silenced SOX9 in specialized organelles called melanosomes. These melanosomes are produced within melanocytes and then are transported to the extremities of their dendrites and are finally transferred to surrounding keratinocytes. This physiological phenomenon is induced by UV exposure and helps protect the skin from the carcinogenic action of UV irradiation. The activation of melanocortin I receptor receptors at the surface of melanocytes by ␣-MSH secreted by surrounding keratinocytes leads to the activation of the cAMP pathway, which plays a key role not only in the process of melanogenesis but also in the transport of melanosomes (20, 26). The downstream activation of CRE, then MITF, and finally the DCT Fig. 8. SOX9 does not regulate SOX10. SOX9 was silenced in n-DP NHM by and tyrosinase promoters is considered the main pathway leading using siRNA for 48 h, after which proteins were extracted and analyzed by immunoblotting. (Left) The silencing of SOX9 does not affect the expression to the production of melanin (27, 28). However, although MITF is of SOX10. SOX9 was overexpressed for 24 h in n-DP NHM, and again, after required, it does not seem to be sufficient to induce the expression extraction, proteins were analyzed by immunoblotting. (Right) The expression of melanogenic enzymes (29). Concomitantly, it has been demon- of SOX10 was not affected when SOX9 was overexpressed. strated that SOX10 is able to bind the MITF promoter and acts with

Passeron et al. PNAS ͉ August 28, 2007 ͉ vol. 104 ͉ no. 35 ͉ 13987 Downloaded by guest on September 27, 2021 in early childhood, which occupies the attention of the clinicians. A possible compensation by SOX10 probably explains why SOX9 was never previously investigated for its role(s) in the pigmentary process. Interestingly, inappropriate expression of SOX9 during embryogenesis in mice leads to microphthalmia and pigmentary defects (36). Thus, it cannot be ruled out that some Waardenburg- like syndrome features in humans could be due to not-yet- recognized mutations in the SOX9 gene. We have shown in this study that the regulation of SOX9 does not affect the expression of SOX10 in neonatal and adult NHM and that the effects of SOX9 on differentiation are not mediated through an increase of SOX10. Although SOX10 plays a key role in regulating the development of melanocytes, its expression decreases as they differentiate, whereas SOX9 expression increases (30). Thus, in contrast to SOX10, SOX9 Fig. 10. Schematic representation of the role of SOX9 in regulating pigmen- could have a minor role in early stages of melanocyte development tation. UVB radiation, at least through the activation of cAMP via PKA, increases but could then play an increasing role in regulating differentiation. SOX9 and CREB expression. Those two transcription factors regulate the MITF The hypothesis already has been suggested that, once MITF promoter. SOX9 and MITF then act together to regulate the DCT promoter, expression is established, there may not be a continued requirement whereas MITF also will act on the TYR promoter, ﬁnally leading to increased for the same factors later in development and that the role of production of melanin within melanosomes. SOX10 might well be undertaken by other members of the SOX family later in development (37). Indeed, we show that SOX9 CREB to activate its transcription (25). The importance of SOX10 directly binds and regulates the MITF promoter, increases the in pigmentation already was known because mutations in that gene expression of DCT and tyrosinase, and, as a result, increases the lead to a genetic hypomelanosis disorder, the Waardenburg syn- pigmentation of melanocytic cells. This finding was corroborated by drome (3). Recently, it has been shown that as melanoblasts the results obtained when SOX9 was silenced, showing very slight differentiate to melanocytes, the expression of SOX10 decreases, or no activation of MITF, DCT, or TYR promoters after forskolin but, at the same time, the expression of SOX9 increases (30). Of stimulation. These results emphasize the key roles of both MITF interest, the authors of ref. 30 report that SOX9 was expressed at and SOX9 in the melanogenic process in neonatal and adult higher levels in pigmented melanoma cell lines compared with melanocytes. Moreover, the relative importance of SOX9/SOX10 after embryogenesis and their potential synergistic action required nonpigmented ones. further investigations. The schematic role of SOX9 in UV-induced In this study, we demonstrate that SOX9 is expressed by NHM pigmentation is summarized in Fig. 10. in culture as well in adult human skin. We also show that the UVB radiation, through at least the activation of the cAMP expression of SOX9 is up-regulated in melanocytes after UVB pathway by ␣-MSH, increases SOX9 expression in melanocytes. irradiation. As SOX9 has no UV-responsive element in its pro- However, we also found a decrease in SOX9 expression after moter, the action of UV should act through an intermediate to treatment with ASP. Interestingly, ASP is known to decrease activate SOX9. It has been recently demonstrated that the SOX9 pigmentation in mouse and human melanocytes (22–25). Trans- promoter is regulated directly by CREB and sp1 in chondrocytes fection of the ASP gene in the skin and hair follicles of rats (31). The increase of CREB through the cAMP pathway could decreases tyrosinase expression and pigmentation (38). However, explain, at least in part, the effect of UV on SOX9 levels in little is known about the mechanism of action of ASP on melano- melanocytes. However, it is highly probable that the regulation of cytes. It has been demonstrated that MITF is inhibited by ASP (39). SOX9 is more complex and that other factors might act concom- The basic helix–loop–helix transcription factor (ITF2) has been itantly with CREB. Indeed, a putative binding site for LEF1 also is shown to be involved in the action of ASP on pigmentation (40). present in the SOX9 promoter. LEF1 binding sites also are present ␤ Indeed, ITF2 is up-regulated by ASP and is down-regulated by on the MITF promoter (32). Indeed, the Wnt/ -catenin pathway ␣-MSH. The overexpression of ITF2 leads to the decreased ex- could be involved in SOX9 regulation. This pathway already is pression of MITF, DCT, and tyrosinase, whereas its silencing known to be involved in pigmentation through MITF activation induces opposite effects (41). Here, we show that SOX9 is up- ␤ (32). Moreover, Wnt/ -catenin also appears to be a key pathway for regulated by ␣-MSH but is down-regulated by ASP in human melanoma (33) and could link SOX9 not only to differentiation but melanocytes. According to our results on the effects of SOX9 on also to proliferation. We showed that, as for most of the key players MITF, DCT, and tyrosinase expression, the down-regulation of in melanogenesis, the up-regulation of SOX9 is mediated by the SOX9 after ASP treatment could explain, at least in part, the cAMP pathway. The transcription factor Usf-1 also has been shown mechanism used by ASP to decrease pigmentation. to mediate the UV response in melanocytes by binding the con- Recently, SOX9 has been shown to be expressed in keratinocytes served E-Box elements in the tyrosinase promoter (34). We show and in the sebaceous glands of adult skin (42). These data are in this study the activation and role of SOX9, another transcription concordant with the results obtained in our study that demonstrate factor, in response to UV stimulation. Indeed, response to UV the expression of SOX9 at protein and RNA levels in keratinocytes. radiation appears to be tightly regulated and involves several actors, The previously unrecognized role of SOX9 in pigmentation in including USF-1 and SOX9, which regulate differentiation in neonatal and in adult melanocytes emphasizes the poor under- melanocytic cells. standing of the roles of SOX proteins in adult tissues. In melano- The regulation observed in melanocytes of adult skin suggests an cytic cells, SOX9 completes the complex and tightly regulated active role of SOX9 after embryogenesis. However, although process leading to the production of melanin by acting at a very mutations of SOX10 induce strong pigmentary defects, such as upstream level. those observed in Waardenburg syndrome, mutations of SOX9 lead to phenotypes in which pigmentation defects are slight (pigmented Materials and Methods ears and light coat pigmentation) (35). To the best of our knowl- For full details regarding reagents, cell lines, culture conditions, edge, pigmentary disorders have not been reported in patients plasmid construction, siRNA, transfection, immunoblotting, im- suffering from campomelic dysplasia. This could be due to the munohistochemistry, and immunocytochemical staining, see SI severity of the other abnormalities that lead in most cases to death Materials and Methods.

13988 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0705117104 Passeron et al. Downloaded by guest on September 27, 2021 Reconstructed Skin. The epidermal equivalent MelanoDerm was reporter plasmid and 0.05 ␮g of pCMV␤-Gal (Promega, Madison, obtained from MatTek (Ashland, MA), and normal human kera- WI) to control the variability of transfection efficiency. In some tinocytes and melanocytes were obtained from Asian neonatal experiments, SOX9 was overexpressed by using 0.1 ␮gofthe foreskin tissues. MelanoDerms were grown at the air/liquid inter- pCDNA3 encoding SOX9 or was silenced with 100 nM SOX9 face of the maintenance medium MEL–NHM-113 (MatTek), and siRNA. Empty pCDNA3 or siRNA scrambles were used as controls culture medium was renewed every 2 days. depending on the experiments. All transfections were made with Lipofectamine (Invitrogen) according to the manufacturer’s in- RT-PCR. Total RNA was reverse-transcribed by using SuperScript III structions. Thirty-six hours later, cells were washed with a saline (Invitrogen). PCRs consisted of 25 cycles for ␤-actin and 35 cycles phosphate buffer and lysed with reporter lysis buffer (Promega). for SOX9 using TaqDNA polymerase (Invitrogen, Carlsbad, CA). Soluble extracts were harvested and assayed for luciferase and ␤-gal PCR products for ␤-actin and SOX9 were 838 and 288 bp, activities. All transfections were repeated at least three times with respectively, and were electrophoresed in parallel with DNA mo- different plasmid preparations and gave similar results. The pMITF lecular mass markers (Invitrogen). Full details can be found in SI wild-type and mutant pDCT, pTYR, and pCRE constructs used Materials and Methods. have been described previously (4, 27, 44).

TISH. Oligonucleotide probes specific for human SOX9 were de- ChIP. ChIP assays were performed as previously described (46) with ␮ ␮ signed. Target sites were selected based on the analysis of sequence 4 g of SOX9 antibody (Chemicon, Billerica, MA) or 4 gof matches and mismatches BLAST (GenBank). Probes showed no nonspecific IgG (Invitrogen). The DNA recovered was subjected to evidence of cross-reaction with sequences of other genes, including amplification by PCR before analysis with agarose gel electrophore- sis. The primers used for the PCRs were the human MITF promoter other SOX family genes. Full details can be found in SI Materials Ј Ј Ј and Methods. region (5 -GATGATGTCTCCTCCAAAGG-3 and 5 -AGC- CCTACGAGTTTGGTCTT-3Ј) and the GAPDH promoter (5Ј- CGGTGCGTGCCCAGTTG-3Ј and 5Ј-GCGACGCAAAA- Metabolic Labeling. Radioactive metabolic labeling and immuno- GAAGATG-3Ј). precipitation was performed as described previously (43). Where indicated, cells were irradiated with 21 mJ/cm2 UVB, and/or Melanin Content Assay. Melanin content was determined as de- chemical agents were added from the ‘‘pulse’’ period to the scribed previously (45). Melanin contents are expressed as nano- ‘‘chase’’ period. Cell extracts were incubated with normal rabbit grams of melanin divided by the total protein in micograms. Values serum (Vector, Burlingame, CA) and then were incubated with are reported compared with values obtained in controls and are protein G beads (GE Healthcare, Buckinghamshire, U.K.). The reported as ratios. Each experiment was repeated at least three supernatants were incubated with SOX9 (Abcam, Cambridge, times. MA) or GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies. The immune complexes were separated by incuba- Statistical Analysis. Data are presented as means Ϯ SD. Student’s tion with beads and were washed further with immunoprecipi- t test was used to analyze differences. Values of P Ͻ 0.05 were tate lysis buffer. The pellets were eluted, electrophoresed, and considered significant. visualized by autoradiography. We thank Karine Bille for her technical support. This research was Luciferase Assay. B16 melanoma cells or HeLa cells were seeded in supported by the Intramural Research Program of the National Cancer 24-well dishes and were transfected with 0.3 ␮g of the luciferase Institute.

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