INTRODUCTION

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Melanocytes are present in the skin and hair follicles as a dispersed population of differentiated cells, after they migrated along the dorsolateral pathway from the neural crest, as nonpigmented precursors, the melanoblasts (1). In mature melanocytes, melanin synthesis involves three specifically expressed enzymes: tyrosinase (2), tyrosinase-related protein 1 (TRP1)¹ (3), and tyrosinase-related protein 2 (TRP2) (4) that control the quantity and the type of melanins. Indeed, two types of melanins are synthesized during melanogenesis; pheomelanins that are yellow to red pigments and eumelanins that are black to brown. Tyrosinase, which catalyzes the first and rate-limiting step of this process, is required for synthesis of both melanin types, while TRP1 and TRP2 appear to be involved mainly in eumelanin synthesis (5). Recently, a transcription factor, belonging to the basic helix-loop-helix family, named microphthalmia (Mi in mouse and MITF in human) and expressed in a limited number of tissues such as heart, mast cells, osteoclast precursors, and melanocytes, has been involved in melanocyte survival, development, and differentiation (6, 7). Indeed, mutations at the mouse mi locus lead to coat color dilution, white spotting or complete loss of pigmentation (6, 8). Similarly, mutation in the human homologue of the mouse microphthalmia gene has been linked to abnormal pigmentation observed in Waardenburg syndrome type II-A (9, 10). Further, studies have shown that Mi through binding to the M box (a highly conserved 10-base pair motif, GTCATGTGCT) strongly stimulates tyrosinase (11), TRP1 (12), and TRP2 (13) promoter activities, suggesting that Mi is involved in the tissue-specific expression of the melanogenic genes.

The relative amount of eumelanin and pheomelanin pigments in mammals is controlled by the genetic loci agouti and extension; extension encodes the receptor for -melanocyte-stimulating hormone (MSH), also called the melanocortin 1 receptor (MC1R) and agouti encodes a 131-amino acid protein containing a signal sequence, the agouti-signal protein (ASP) (14-18). ASP, which is produced in the dermal papilla cells within the hair follicle, acts on follicular melanocytes to switch them from eumelanin to pheomelanin production (19). In humans, the mechanism that controls the class of melanin synthesized (eumelanin or pheomelanin) has not been elucidated. It is well established that MSH increases the synthesis of eumelanin in human melanocytes (18, 20, 21). A human homologue for the mouse agouti locus has been cloned and its product functions similarly to the mouse protein in vivo and in vitro (22, 23); however, its physiological function in humans remains to be elucidated.

ASP seems to act as an antagonist of MSH signaling mediated by the mouse melanocortin-1 receptor (MC1R). However, its mechanism of action is still controversial. This effect appears to be mediated in part by the ability of ASP to act as an inhibitor of MSH binding to the MC1R (24-26). On the other hand, some studies suggest that agouti protein may act through a receptor distinct from the MC1R (27-30). Indeed ASP also inhibits the melanogenic activity of agents such as forskolin and dibutyryl cAMP, which mimic the effects of MSH but act downstream of the MC1R (31).

In vivo, it has been clearly shown that ASP decreases eumelanin synthesis due to a slight inhibition of the tyrosinase activity and to an almost complete loss of TRP1 and TRP2 expression (32). In human or murine cultured cells, ASP inhibits eumelanin synthesis, cell proliferation, tyrosinase activity and reduces the level of TRP1 without significantly altering the level of tyrosinase (30, 31, 33).

To further understand the molecular mechanisms involved in the inhibition of melanogenesis by agouti protein, we studied its effect on tyrosinase, TRP1, and TRP2 expression and on the transcription activities of the corresponding promoters in B16 melanoma cells. Further, we investigated the effect of ASP on microphthalmia, a transcription factor that mediates, through its binding to an M box motif upstream of the TATA box, the activation of tyrosinase, TRP1 and TRP2 promoter by cAMP elevating agents (13, 34). In addition, we address the question of whether ASP inhibits the cAMP- or MSH-induced differentiation of melanoblasts into functional melanocytes. Using melb-a cells, a cloned immortal line of murine melanoblasts inducible to differentiate to melanocytes, we show that MSH and forskolin promote the differentiation of melanoblasts into melanocytes, a process that can be partially prevented by agouti signal protein.

	EXPERIMENTAL PROCEDURES

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Materials

Forskolin,  melanocyte-stimulating hormone ([Nle⁴, D-Phe⁷]-MSH), phorbol 12-myristate 13-acetate, p-nitrophenyl phosphate, sodium fluoride, sodium orthovanadate, -glycerophosphate, 4-(2-aminoethyl)benzenesulfonyl fluoride, aprotinin, and leupeptin were from Sigma. Basic fibroblast growth factor (bFGF) was from Promega. RPMI medium, Dulbecco's modified Eagle's medium, trypsin, and LipofectAMINE reagent were from Life Technologies, Inc., and fetal calf serum was from Hyclone. The C5 monoclonal antibody was raised against a histidine fusion protein expressed from the amino-terminal Taq-sac fragment of human MITF cDNA and produces a specific gel mobility super shift with microphthalmia, but not with the related proteins TFE3, TFEB, and TFEC (35). The PEP7, PEP1, and PEP8 polyclonal antibodies were raised against the carboxyl termini of, respectively, mouse tyrosinase, TRP1, and TRP2 proteins. Peroxidase-conjugated anti-mouse and rabbit antibodies were from Dakopatts. Mouse ASP was purified from the medium of a T. ni baculovirus expression system (36).

Methods

Cell Culture-- The melb-a line was grown in RPMI 1640 medium supplemented with 12-O-tetradecanoylphorbol-13-acetate (20 nM), bFGF (1 ng/ml), fetal calf serum (5%), and glutamine (2 mM) without feeder cells. To induce differentiation, 12-O-tetradecanoylphorbol-13-acetate and bFGF were replaced by forskolin (20 µM) or MSH (10 nM) and fetal calf serum was 10%. Melanoblasts were treated 24 h after plating and every 48 h for 6 days (a total of three treatments). In the conditions where ASP was added with forskolin or MSH, a 15-min preincubation period was performed. B16-F10 melanoma cells were grown in Dulbecco's modified Eagle's medium supplemented with 7% fetal calf serum.

Western Blot Analysis-- Melb-a cells were treated with 10 nM MSH or 20 µM forskolin in the presence or absence of 1 µM ASP for a total of 6 days as described above. For B16 melanoma cells, the treatment period was 48 h. Cell lysates were prepared in 0.1 M phosphate buffer containing 1% Triton X-100, 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 5 µg/ml leupeptin, and 10 µg/ml aprotinin. Equal amounts of protein (10 µg/lane) were separated on a 10% polyacrylamide gel by electrophoresis. Following transblotting onto nitrocellulose membranes (1 h at 100 V) and blocking in 5% nonfat milk in saline buffer, the membranes were incubated with PEP7, PEP1, and PEP8, each at 1:3000 dilution. Microphthalmia protein was detected with the C5 monoclonal antibody at 1:10 dilution. To check equivalent loading and transfer of the gels, anti-ERK1 antibodies were used (1:3000). The membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit or mouse IgG (1:4000). The immunoreactive bands were detected by chemiluminescence, using the ECL Amersham kit.

Tyrosinase Activity Stain-- L-Dopa colorimetric stain for tyrosinase was performed as described previously (37). Briefly, 5 µg of total protein extracts were mixed with Laemmli sample buffer without -mercaptoethanol, boiling was avoided, and the samples were separated on a 12% polyacrylamide gel. Gels were equilibrated in 50 mM phosphate buffer (pH 6). Colorimetric staining was carried out by incubating the gels for about 15 min at 37 °C in a solution of 1.5 mM L-dopa and 4 mM 3-methyl-2-benzothiazolinone hydrazone in 10 mM phosphate buffer (pH 6.8).

Transfections and Luciferase Assays-- B16 melanoma cells were seeded in 24-well dishes, and transient transfections were performed the following day using 2 µl of LipofectAMINE and 0.35 µg of total plasmid DNA in a 200-µl final volume as indicated in the figure legends. After transfection, cells were incubated with 3 nM MSH, in the presence or in the absence of 0.6 µM ASP. pCMV--galactosidase was transfected with the test plasmids to control the variability in transfection efficiency. Twenty-four hours after transfection, soluble extracts were harvested in 50 µl of lysis buffer and assayed for luciferase and -galactosidase activities. The reporter plasmids, containing the 2.2-kilobase pair fragment of the mouse tyrosinase promoter, the 1.1-kilobase pair fragment of the mouse TRP1 promoter, and the 0.6-kilobase pair fragment of the human TRP2 promoter upstream from the luciferase reporter gene, were previously described (13, 34). The reporter plasmid containing the 2.1-kilobase pair fragment 5' of the transcription start site of the microphthalmia-associated transcription factor (MITF) gene was isolated from SpMITF 1, kindly provided by Dr. Shibahara (38).

Nuclear Extracts and Gel Mobility Shift Assays-- Melb-a cells were stimulated for six days with 10 nM MSH or 20 µM forskolin in the presence or absence of 1 µM ASP, and nuclear extracts were prepared essentially as described previously (39), except that phosphatase inhibitors (1 mM NaVO₄, 5 mM NaF, 20 mM -glycerophosphate, and 10 mM p-nitrophenyl phosphate) were added to the nuclear extraction buffer. A double-stranded synthetic tyrosinase M box probe (5'-GAAAAAGTCATGTGCTTTGCAGAAGA-3') was -³²P-end-labeled with T4 polynucleotide kinase. One µg of nuclear protein was preincubated in binding buffer containing 10 mM Tris, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 4% glycerol, 80 µg/ml salmon sperm DNA, 0.1 µg poly(dI·dC), 0.2% bovine serum albumin, 2 mM MgCl₂, and 2 mM spermidine for 15 min on ice. Then 200,000 cpm of ³²P-labeled probe was added to the binding reaction for 10 min at room temperature. For antibody supershift assay, nuclear extracts were preincubated with 0.3 µl of the C5 monoclonal antibody directed against microphthalmia. DNA-protein complexes were resolved by electrophoresis on a 4% polyacrylamide gel (37.5:1 acrylamide/bisacrylamide) in 0.5% TBE buffer (22.5 mM Tris borate, 0.5 mM EDTA, pH 8) for 2 h at 100 V. After fixation in 7% acetic acid, gels were dried and autoradiographed. Quantitation of the bands were performed using the NIH Image 1.54 software.

	RESULTS

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Molecular Mechanisms Involved in the Inhibition of Melanogenesis in B16 Cells by ASP

ASP Inhibits the Expression of Tyrosinase, TRP1, and TRP2-- In B16 melanoma cells, it was shown that ASP inhibits tyrosinase activity and production of melanin (33). However, regulation concerning the three melanogenic enzymes has not been investigated. Therefore, we performed Western blot experiments on B16 cells exposed for 48 h to 10 nM MSH in the presence or absence of 1 µM ASP (Fig. 1). ASP almost completely abolished the MSH-induced expression of tyrosinase. Potential inhibition of tyrosinase expression by ASP was not detectable under basal conditions owing to its low level of expression in B16 melanoma cells. In addition, ASP reduced both basal and MSH-stimulated expressions of TRP1 and TRP2.

View larger version (52K): [in this window] [in a new window]   Fig. 1.   ASP inhibits expression of tyrosinase, TRP1, and TRP2 in B16 melanoma cells. B16 cells were treated for 48 h with 10 nM MSH (M), 1 µM murine ASP (A), or both (M+A). 15 µg of solubilized total protein were electrophoresed in 10% SDS-PAGE gels and transferred to nitrocellulose membrane. Specific detection of tyrosinase, TRP1 and TRP2 was performed with the antibodies PEP7, PEP1, and PEP8. ERK1 detection was used as an internal control for comparable loading and transfer in all lanes. Detection was carried out by means of a peroxidase labeled secondary antibody and by chemiluminescence. Molecular masses are indicated on the left in kilodaltons. C, untreated control. Similar results were observed in three independent experiments.

ASP Inhibits Transcription Activity of the Tyrosinase, TRP1, and TRP2 Promoters-- To gain more insight into the mechanisms by which ASP modulates the expression of the three melanogenic enzymes, we studied the transcription regulation of their promoters. For this purpose, B16 cells were transiently transfected with the corresponding promoters and exposed to MSH in the presence or absence of ASP. Tyrosinase, TRP1, and TRP2 promoter activities were stimulated upon MSH treatment (between 4- and 7-fold over basal levels). ASP partially inhibited basal activities (about 50% for all three promoters) and completely prevented the effect of MSH on these promoter activities (Fig. 2).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   MSH stimulates tyrosinase, TRP1, and TRP2 promoter activities in B16 cells; reversal of this effect by ASP. B16 cells were transfected with 0.3 µg of luciferase reporter plasmids pTYRO, pTRP1, or pTRP2 and 0.05 µg of pCMV--galactosidase. Cells were treated for 24 h with 3 nM MSH, 0.6 µM murine ASP or both. Luciferase activity was normalized to -galactosidase activity and the results were expressed as fold stimulation of the basal luciferase activity from unstimulated cells. Data are means ± S.E. of three experiments performed in triplicate.

ASP Inhibits Microphthalmia Protein Expression and Promoter Activity-- We have recently proposed that microphthalmia, a transcription factor of the basic helix-loop-helix family, is involved in the regulation of tyrosinase, TRP1, and TRP2 expression by cAMP-elevating agents (13). Hence, we studied the effects of ASP treatment on the expression of microphthalmia and on its promoter activity. As seen by Western blot analysis, a 4-h incubation with MSH-stimulated microphthalmia expression but this was completely inhibited upon ASP addition (Fig. 3, left). Further, microphthalmia promoter activity was up-regulated by MSH (about 3-fold over basal), and this effect was dramatically blocked in the presence of ASP (Fig. 3, right).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   ASP inhibits expression and promoter activity of microphthalmia in B16 melanoma cells. Left, B16 cells were treated for 4 h with 10 nM MSH (M), 1 µM murine ASP (A), or both (M+A). 15 µg of solubilized total proteins were electrophoresed in 10% SDS-PAGE gels and transferred to nitrocellulose membrane. Specific detection of microphthalmia was performed with the monoclonal antibody C5 at 1:10 dilution. C, untreated control. Right, B16 cells were transfected with 0.3 µg of luciferase reporter plasmid pMi and 0.05 µg of pCMV--galactosidase. Cells were treated for 24 h with 3 nM MSH, 0.6 µM murine ASP, or both. Luciferase activity was normalized to -galactosidase activity, and the results were expressed as fold stimulation of the basal luciferase activity from unstimulated cells. Data are means ± S.E. of three experiments performed in triplicate.

Effects of ASP on Differentiation of Murine Melanoblasts into Melanocytes

Inhibition of Tyrosinase Activity and Expression of the Melanogenic Enzymes by ASP-- Melb-a cells underwent differentiation in the presence of 20 µM forskolin or 10 nM MSH for 1 week. We then evaluated tyrosinase activity by carrying out tyrosinase activity stains in polyacrylamide electrophoresis gel (Fig. 4). We observed an increased activity following MSH and forskolin treatments that was significantly reduced by ASP. ASP also inhibited basal activity of tyrosinase.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   ASP inhibits dopa oxidase activity in melb-a cells. Melb-a cells were treated for 6 days with 10 nM MSH (M), 20 µM forskolin (F), 1 µM murine ASP (A), or combined treatments (M+A and F+A). C, untreated cells. After solubilization, 10 µg of total protein were electrophoresed under nonreducing conditions and stained in the presence of L-dopa and 3-methyl-2-benzothiazolinone hydrazone as described under "Methods." Similar results were observed in five independent experiments.

View larger version (64K): [in this window] [in a new window]   Fig. 5.   ASP inhibits expression of tyrosinase, TRP1, and TRP2 in melb-a cells. Melb-a cells were treated for 6 days with 10 nM MSH (M), 20 µM forskolin (F), 1 µM murine ASP (A), or combined treatments (M+A and F+A). 15 µg of total protein were electrophoresed in 10% SDS-PAGE gels and transferred to nitrocellulose membrane. Specific detection of tyrosinase, TRP1, and TRP2 was performed with the antibodies PEP7, PEP1, and PEP8, respectively. ERK1 detection was used as an internal control for comparable loading and transfer in all lanes. C, untreated cells. Similar results were observed in five independent experiments.

Microphthalmia Expression Is Up-regulated during cAMP-induced Melanoblast Differentiation; Its Binding to the M Box Is Inhibited by ASP-- Next we studied the effects of MSH and ASP treatment on the expression of microphthalmia and its binding to the M box. Western blot experiments showed that microphthalmia is constitutively expressed in melanoblasts and that its expression is transiently enhanced by MSH treatment with a maximum at 3 h and noticeably less stimulation by 18 h (Fig. 6).

View larger version (55K): [in this window] [in a new window]   Fig. 6.   Stimulation of the expression of microphthalmia in MSH-treated melb-a cells. Melb-a cells were treated for 6 days with 10 nM MSH and harvested at the time indicated on top of each lane after the last treatment. 0, control cells harvested 3 h following medium change. 15 µg of solubilized total protein were electrophoresed in 10% SDS-PAGE gels and transferred to nitrocellulose membrane. Specific detection of microphthalmia was performed with the monoclonal antibody C5 at 1:10 dilution.

View larger version (121K): [in this window] [in a new window]   Fig. 7.   ASP inhibits the increase of microphthalmia binding to the M box element following forskolin and MSH treatments. Melb-a cells were treated for 6 days with 20 µM forskolin (F), 10 nM MSH (M), 1 µM ASP (A), or combined treatments (F+A and M+A) and harvested 3 h after the last treatment. C, control cells. Gel shift assays were performed as described under "Methods" with a tyrosinase M box probe and 1 µg of nuclear proteins from each condition. MSH and MSH + ASP nuclear extracts were also incubated in the presence of anti-microphthalmia antibody (C5). The arrow indicates the supershifted complexes.

	DISCUSSION

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Skin pigmentation involves a set of complex developmental mechanisms occurring outside as well as inside the embryonic skin, such as (i) emergence of the melanocyte lineage among neural crest cells, (ii) the migration of melanocyte precursors from the neural primordium to the epidermis and hair follicles, and (iii) cell proliferation and final maturation of melanoblasts into melanin-producing cells, as reviewed in Bennett (40). MSH is thought to play an important role in the differentiation of mouse melanocytes in the epidermis and hair bulb by inducing tyrosinase activity, melanin formation, increased dendricity and transfer of melanosomes. It is now well established that structure and function of melanocytes are controlled by both genetic factors and the local tissue environment. In vivo, MSH promotes the production of eumelanin, while expression of the agouti gene promotes the production of pheomelanin. In vitro studies have shown that ASP could induce pheomelanogenesis in eumelanic melanocytes (30, 33) demonstrating that ASP alone is sufficient to elicit such changes. It has also been shown that ASP could down-regulate the stimulation of melanogenesis induced by forskolin and MSH (30, 31). In this report, we show that ASP can by itself inhibit spontaneous and induced-differentiation of melanoblasts into melanocytes. Syntheses of tyrosinase, TRP1, and TRP2 are down-regulated and total tyrosinase activity is decreased in basal-, forskolin-, and MSH-stimulated conditions by ASP. We have also shown that in B16 melanoma cells, used as a model of partially differentiated melanocytes, ASP almost completely inhibited the expression of tyrosinase stimulated by MSH. TRP1 and TRP2 expressions were down-regulated under these conditions. It is interesting that inhibition by ASP of melanoblast differentiation induced by MSH involves a weak effect on tyrosinase expression as compared with the inhibition exerted on TRP1 and TRP2 expression in the same conditions. This observation is consistent with the fact that tyrosinase expression is indispensable for pheomelanogenesis while decrease in TRP1 and TRP2 is a requisite. Thus in a long term process such as differentiation, ASP preserves the acquisition of tyrosinase enzyme and prevents or strongly reduces TRP1 expression which may lead to a population of melanocytes producing pheomelanins instead of eumelanins. This observation is to be compared with the marked inhibition exerted by ASP on MSH-induced expression of tyrosinase in B16 melanoma cells. This suggests the existence of different mechanisms between inhibition of melanoblast differentiation and inhibition of melanogenesis in differentiated melanocytes. Moreover, the fact that ASP does inhibit basal and forskolin-induced expression of tyrosinase in melanoblasts suggests a specific and different mechanism for ASP inhibition of the cAMP-induced events.

The mechanisms of ASP action are only partially elucidated. Competitive inhibition of melanocortin binding has been proposed as a first hypothesis since ASP antagonizes the effects of MSH and inhibits ¹²⁵I-labeled nucleoside diphosphate-MSH binding (24). A classic antagonist used at concentrations 100 times above the ligand concentrations as we did, should have completely blocked the effects of MSH. However, in some of our experiments, this was not the case. These results suggest that in certain conditions, ASP does not antagonize completely the effect of MSH through the MC1R. Moreover, ASP can inhibit basal melanogenesis and spontaneous differentiation as well as forskolin-induced melanogenic events; this observation weakens the antagonist-only hypothesis. The effects of ASP on basal and forskolin-induced differentiation fit better with the hypothesis that ASP would be an inverse agonist of the MC1 receptor. Inverse agonists, ligands that suppress spontaneous receptor signaling activity, have been described for a growing number of G-protein coupled receptors: the ₂-adrenoreceptor of myocardial cells (41), the 5-hydroxytryptamine receptors in NIH-3T3 fibroblasts (42), and the calcitonin receptors (43). Hence, ASP appears likely to act both as an antagonist of MSH binding on MC1R and an inverse agonist to block both the effects induced by cAMP elevating agents and basal effects.

We show that down-regulation of differentiation in melb-a cells and of melanogenesis in B16 cells involve inhibition of tyrosinase, TRP1 and TRP2 expression to different extents. In B16 cells, we also demonstrate that this down-regulation results from inhibition of the tyrosinase, TRP1, and TRP2 promoters. Besides the fact that we described for the first time an inhibitory effect on the transcription activity of genes for the enzymes involved in melanogenesis, it is interesting that, in melanoma cells, ASP exhibited a strong inhibition on both basal and MSH-stimulated activities of the corresponding promoters.

Recently, we suggested that microphthalmia, through the binding to M box, mediates the effect of cAMP on tyrosinase, TRP1, and TRP2 promoter activities (13). In B16 cells, ASP inhibits the activity of the microphthalmia promoter and markedly reduces the expression of the protein. Since microphthalmia has been shown to transactivate tyrosinase (11), TRP1 (12), and TRP2 (13) promoters, we propose that the inhibition of microphthalmia expression by ASP is responsible for the decrease in melanogenic enzyme expression. Beside its putative role in cAMP-induced melanogenesis, the microphthalmia gene plays a crucial role in development and survival of melanocytes and controls the tissue-specific expression of the melanogenic genes. Therefore, we focused our attention on the expression of microphthalmia in melanoblasts during differentiation and the effect of ASP on this process. We observed that microphthalmia is constitutively expressed in melanoblasts, and that MSH and forskolin increase its expression as well as its binding to the M box. ASP inhibited the MSH and forskolin-stimulated expression and binding of microphthalmia to the M box. Our results suggest that MSH and forskolin promote differentiation of melanoblasts into melanocytes and that ASP partially blocks this cAMP-stimulated differentiation and also spontaneous (basal) differentiation, probably through an inhibition of microphthalmia gene expression.

Taken together, our studies indicate a key regulatory role for agouti signal protein in melanocyte differentiation and in melanogenesis, and point to the involvement of microphthalmia in these processes.