INTRODUCTION

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Melanocytes are specialized cells located at the basal layer of the epidermis that synthesize and transfer melanin pigments to surrounding keratinocytes leading thereby to a uniform skin pigmentation. In vivo, melanin pigments play a key photoprotective role against the carcinogenic effects of solar ultraviolet light, which is in other respects the physiologic stimulus of melanogenesis (1, 2). UV radiation can act directly on melanocytes or indirectly through the release of keratinocyte-derived factors that regulate melanogenesis (3, 4). Among the agents secreted by keratinocytes upon UV-B treatment, -melanocyte-stimulating hormone (-MSH)¹ is one of the most potent activators of melanogenesis. Indeed, addition of -MSH in cultured human melanocytes (5) or in melanoma cells (6) stimulates melanization. Further, subcutaneous injection of this hormone causes a strong stimulation of the local pigmentation in humans (7). -MSH binds to a G protein-coupled heptahelical receptor leading to the activation of G_s protein and to an increase in intracellular cAMP content. In cultured melanoma cells, the melanogenic effect of -MSH can be mimicked by other cAMP-elevating agents such as cholera toxin, forskolin, and isobutylmethylxanthine (8-10). These observations emphasize the pivotal role of cAMP in the regulation of melanogenesis, but the cellular signaling events connecting the rise in cAMP to the stimulation of melanin synthesis are still incompletely clarified.

Melanin biosynthesis or melanogenesis consists in a cascade of enzymatic and spontaneous reactions that converts tyrosine to melanin pigments. The initial and rate-limiting step in melanin synthesis, the hydroxylation of tyrosine to L-DOPA, is controlled by tyrosinase that is the key enzyme in this process. Stimulation of melanogenesis by cAMP-elevating agents, as well as by other melanogenic agents, implies an increase in tyrosinase protein amount as the consequence of the stimulation of the tyrosinase gene transcription (11). Concerning the early events induced by cAMP increase, we have recently demonstrated that cAMP-elevating agents inhibit the phosphatidylinositol 3-kinase and p70^S6 kinase activities (12). Further, the inhibition of these activities by pharmacological inhibitors mimics the melanogenic effect of -MSH or forskolin, suggesting that phosphatidylinositol 3-kinase and p70^S6 kinase inhibition by cAMP-elevating agents is a key event in the regulation of melanogenesis by these agents.

The MAP kinases ERK1 and ERK2 are serine/threonine kinases that are activated upon phosphorylation by the dual specificity MAP kinase kinase or MEK. MEK is phosphorylated and activated by Raf-1, which is itself activated by p21^ras (13). Upon activation, MAP kinases translocate to the nucleus where they phosphorylate and activate many transcription factors of which ternary complex factor/serum response factor and activator protein-1 are the most studied (14, 15). Although MAP kinases have been clearly shown to play a crucial role in growth control (16), they are also involved in the differentiation process of several cell systems (17, 18).

In a previous report, we have shown the activation of ERK1 during cAMP-induced melanogenesis in B16 melanoma cells (10). We have also observed a translocation of ERK1 to the nucleus, with a concomitant stimulation of activator protein-1 DNA binding activity. Further, stimulation of melanogenesis, which is associated with morphological changes characterized by an increased cell dendricity, reflects the differentiation of melanocytes and melanoma cells. These observations led us to hypothesize that the MAP kinase pathway could be involved in the regulation of melanoma cell differentiation and, more precisely, that the activation of ERKs would be a required event in the induction of melanogenesis by cAMP-elevating agents in B16 melanoma cells.

In the present report, using a pharmacological approach with a specific inhibitor of MEK (PD 98059) (19) and a molecular approach with constitutively active or dominant negative mutants of MEK and p21^ras, we clearly demonstrated that cAMP-elevating agents can stimulate melanogenesis in the absence of MAP kinase activation, thereby invalidating our former hypothesis. Further, we showed that a sustained activation of the Ras/MAP kinase pathway led to a down-regulation of melanogenesis, demonstrating the involvement of this pathway in the control of B16 melanoma cell differentiation.

	EXPERIMENTAL PROCEDURES

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Materials-- Forskolin, TPA, L-DOPA, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), aprotinin, and leupeptin were purchased from Sigma. Dulbecco's modified Eagle's medium, LipofectAMINE reagent, and Optimem medium were from Life Technologies, Inc. PD 98059 was from Santa Cruz Biotechnology. Polyclonal rabbit antiserum to human tyrosinase (PEP-7) was provided by Dr. V. Hearing (Bethesda, MD). Antiserum to ERK1 was a generous gift from Dr. E. Van Obberghen (Nice, France). Expression vectors coding for the dominant positive and negative mutants of p21^ras, respectively Val-12 p21 and Asn-17 p21 (20, 21), were provided by Dr. J.C. Chambard (Center de biochimie, Nice, France). Expression vector coding for constitutively active MEK (S222D) and constitutively inactive MEK (S222A) mutants were previously described (22).

Cell Cultures-- B16/F10 murine melanoma cells were cultured in Dulbecco's modified Eagle's medium with 7% fetal calf serum (HyClone Laboratories) and penicillin/streptomycin (100 IU/50 µg/ml) in a humidified atmosphere containing 5% CO₂ in air at 37 °C.

Kinase Assay-- Serum-starved cells were treated as indicated, rinsed, and solubilized in ice-cold lysis buffer (50 mM Hepes, pH 7.4, 150 mM NaCl, 100 mM NaF, 10 mM EDTA, 10 mM Na₄P₂O₇, 2 mM Na₃VO₄, 1% Triton X-100, supplemented with protease inhibitors, aprotinin (2 µg/ml), leupeptin (10 µM), and AEBSF (1 mM)). Then extracts were clarified by centrifugation and incubated for 2 h at 4 °C with ERK1 antibody preadsorbed to protein A-Sepharose. Immune complexes were washed twice with lysis buffer and twice with HNTG buffer (50 mM Hepes, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, and 0.2 mM Na₃VO₄). Pellets were resuspended in 50 µl of HNTG buffer supplemented with 30 mM magnesium acetate, 15 µM cold ATP, and 0.2 mg/ml myelin basic protein (MBP) final concentrations. Reactions were initiated at room temperature by the addition of 3 µCi of [-³²P]ATP (3000 Ci/mmol) and stopped after 45 min by adding Laemmli sample buffer. Samples were then boiled for 5 min, and proteins were separated by SDS-polyacrylamide gel electrophoresis on a 12.5% acrylamide gel. The incorporation of ³²P was visualized by autoradiography. Then film was scanned and bands were quantitated by densitometry using MacBas software.

Determination of Tyrosinase Activity-- Tyrosinase activity was estimated by measuring the rate of oxidation of L-DOPA (23). Cells grown in 6-well dishes were treated as indicated for 48 h in Dulbecco's modified Eagle's medium, 2% fetal calf serum. Then cells were washed in ice-cold phosphate-buffered saline and lysed in 100 µl of phosphate buffer (0.1 M), pH 6.8, containing 1% (w/v) Triton X-100, 2 µg/ml aprotinin, 10 µM leupeptin, and 1 mM AEBSF. Cellular extracts were clarified by centrifugation at 13,000 × g for 5 min. The tyrosinase substrate L-DOPA (2 mg/ml) was prepared in the same lysis phosphate buffer (without Triton). 40 µl of each extract were put in a 96-well plate, and the enzymatic assay was started by adding 100 µl of L-DOPA solution at 37 °C. Control wells contained 40 µl of lysis buffer. Absorbance at 570 nm was read every 10 min for at least 1 h at 37 °C using a microplate reader (Dynatech Laboratories). The blank was removed from each absorbance value, and a plot of absorbance against time was represented for each condition. The final activity was expressed in OD/min/µg of protein for each condition.

Western Blot Analysis-- Cellular extracts were prepared as described above, and an equal amount of protein was separated by SDS-polyacrylamide gel electrophoresis in a 10% acrylamide gel. Proteins were transferred to a Hybond-C extra membrane (Amersham Corp.), and the blot was probed with the PEP-7 polyclonal antibody (directed against tyrosinase) and with an anti-ERK1 polyclonal antibody (to verify that each lane was evenly loaded). Then proteins were visualized by the Amersham ECL system.

Transfection and Luciferase Assays-- B16 melanoma cells, plated in 24-well dishes, were transfected using the LipofectAMINE system according to the recommendations of the manufacturer (Life Technologies, Inc.). The reporter plasmid used consists in a 2.2-kilobase pair fragment of the mouse tyrosinase gene promoter cloned upstream of the luciferase gene and named 2.2 pMT-Luc (11). For determination of the PD 98059 effect, 0.25 µg/well of the reporter plasmid was transfected with 0.05 µg of pCMVGal (Promega) to control the variability in transfection efficiency. In the experiments with mutants of MEK and Ras, the transfection was performed in the same conditions using, 0.25 µg of the reporter plasmid, 0.1 µg of the expression vector, empty or containing the coding sequence of the different mutants, and 0.05 µg of pCMVGal. Forty-eight hours after transfection, cells were washed with a saline phosphate buffer and lysed with 25 mM Tris-phosphate (pH 7.8) buffer containing 1% Triton X-100, 2 mM EDTA, and 2 mM dithiothreitol. Soluble extracts were harvested and assayed for luciferase and -galactosidase activity. All transfections were repeated at least five times using different plasmid preparations and gave similar results.

	RESULTS

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PD 98059 Inhibits cAMP-induced Activation of ERK1-- PD 98059 has been shown to be a specific inhibitor of MEK activity, the kinase functioning directly upstream of MAP kinases (19). We first verified whether this inhibitor was effectively able to inhibit ERK1 activity in B16 melanoma cells. ERK1 was immunoprecipitated from cells treated with TPA or forskolin and preincubated or not with PD 98059. The kinase activity was monitored with MBP as substrate (Fig. 1). Forskolin and TPA caused, respectively, about 2- and 8-fold induction of ERK1 activity. Preincubation of cells with 10 µM PD 98059 resulted in an almost complete inhibition of this induced kinase activity (respectively, 0.7- and 1.2-fold). Identical results were obtained on ERK2 (data not shown). Thus, PD 98059 inhibits the activation of MAP kinases in B16 melanoma cells.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   PD 98059 inhibits the activation of ERK1 by TPA and forskolin. B16 cells were treated with 16 nM TPA or 20 µM forskolin (FK) for 10 min. When indicated, PD 98059 at 10 µM final concentration was added to the cells 30 min before the treatment with TPA and forskolin. Then ERK1 was immunoprecipitated, and its kinase activity was measured in the presence of [-32P]ATP using MBP as a substrate. The obtained autoradiography was scanned, and the bands were quantitated by using the MacBas software. The activation (fold) relative to the basal activity present in untreated cells (BASAL) is indicated.

PD 98059 Induces B16 Melanoma Cell Morphological Differentiation-- Then, we analyzed the effect of PD 98059 on B16 cell dendricity, which is the first observable parameter of melanoma cell differentiation (Fig. 2). Forskolin, which is a potent activator of melanogenesis, strongly stimulated dendritogenesis as compared with untreated cells. Addition of 10 µM PD 98059 in the culture medium was followed by an acquisition of the dendritic phenotype. Further, when cells were treated with forskolin plus PD 98059, dendricity was more pronounced than with forskolin or PD 98059 alone. It is worth remarking that -MSH, at the concentration of 10⁶ M, exerted the same effect of forskolin by inducing dendritogenesis in B16 cells (data not shown). Hence, in our cell system, inhibition of MAP kinases by PD 98059 induces dendrite outgrowth and potentiates the morphological changes induced by cAMP-elevating agents.

View larger version (199K): [in this window] [in a new window]   Fig. 2.   Inhibition of MEK activity induces B16 cell morphological differentiation. B16 cells, grown in 2% FCS-containing medium (BASAL), were incubated for 48 h with 20 µM forskolin (FK) and/or with 10 µM PD 98059. Cells were photographed under phase-contrast microscopy. Bar represents 40 µm.

Inhibition of MAP Kinases by PD98059 Induces Melanogenesis-- Induction of melanogenesis in B16 melanoma cells is characterized by the stimulation of tyrosinase activity resulting from an increase in the tyrosinase protein expression. We therefore studied the effect of PD 98059 on these parameters. First, we measured tyrosinase activity in response to PD 98059. Cells were treated for 48 h with the MEK inhibitor and with forskolin. We then measured, in a cell-free system, the DOPA-oxidase activity that is the second specific activity of tyrosinase. As shown in Fig. 3A, forskolin stimulated about 8-fold the tyrosinase activity, and we observed a 6-fold stimulation with PD 98059. Addition of forskolin together with PD 98059 allowed us to achieve almost a 10-fold stimulation of tyrosinase activity, suggesting that inhibition of MAP kinases potentiates the effect of forskolin on tyrosinase activity.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   PD 98059 stimulates the tyrosinase activity and protein expression. A, B16 cells, grown in serum-containing medium (BASAL), were incubated for 48 h with 20 µM forskolin (FK) and/or 10 µM PD 98059. Cells were then lysed and the DOPA-oxidase activity of tyrosinase was assayed using a solution of L-DOPA as a substrate. Absorbance at 570 nm was read every 10 min, and the activity was corrected by the amount of protein. Data are means ± S.E. of three experiments performed in duplicate. B, the same cellular extracts were subjected to Western blot analysis using the PEP-7 antibody for detection of tyrosinase. The blot was also incubated with an anti-ERK1 antibody to control for the equal loading of the gel. Molecular masses, indicated on the left, are expressed in kilodaltons.

Inhibition of MAP Kinases by PD98059 Stimulates the Tyrosinase Promoter Activity-- In a previous report we have shown that the effect of cAMP on melanogenesis is mediated through a stimulation of tyrosinase promoter activity, reflecting an activation of the tyrosinase gene transcription (11). Using a plasmid containing a 2.2-kilobase pair fragment of the tyrosinase promoter cloned upstream of the luciferase coding sequence as a reporter gene, we investigated whether the inhibition of MAP kinase led also to a stimulation of tyrosinase gene expression (Fig. 4). After transfection with the reporter plasmid, cells were incubated with forskolin, PD 98059, or forskolin plus PD 98059 for 48 h. We observed that PD 98059 alone stimulated luciferase activity 4-fold above the basal level. When forskolin was added to PD 98059, the luciferase activity reached a 9-10-fold stimulation, while forskolin alone induced a 8-fold stimulation. Hence, inhibition of the MAP kinase pathway by PD 98059 triggered an increase in tyrosinase gene expression and potentiate the effect of forskolin. It should be noted that the maximal effect on cell dendricity, melanogenesis, and tyrosinase promoter activity was observed with 10 µM PD 98059. 

View larger version (39K): [in this window] [in a new window]   Fig. 4.   Stimulation of tyrosinase promoter activity by PD 98059. B16 cells were transiently transfected with 2.2 pMT-Luc luciferase reporter plasmid containing a 2.2-kb fragment 5' of the transcriptional start site of the mouse tyrosinase gene. After transfection, cells were incubated with 20 µM forskolin and/or 10 µM PD 98059 for 48 h. Then cells were solubilized and luciferase activity was assayed. Luciferase activity was normalized by the -galactosidase activity, and the results were expressed as fold stimulation of the basal luciferase activity from unstimulated cells (BASAL). Data are means ± S.E. of three experiments performed in triplicate.

Expression of Dominant Negative Mutants of MEK and p21^ras Increases Basal and cAMP-induced Tyrosinase Promoter Activities-- To confirm the results obtained with the pharmacological inhibitor of MEK we have investigated the effect of dominant negative mutants of MEK and p21^ras on the tyrosinase gene expression. p21^ras is a small GTPase that activates Raf-1, the kinase that function upstream of MEK. These two mutants, that have respectively the ability to repress the endogenous activity of MEK and Ras, were cotransfected with the reporter plasmid (Fig. 5). In cells expressing the dominant negative mutants of MEK (MEK), both basal and forskolin-stimulated tyrosinase promoter activities were increased about 3-fold compared with control conditions. The dominant negative mutant of Ras (Ras) induced a 2-fold augmentation of the tyrosinase gene expression in basal and forskolin-treated cells. Thus, inhibition of the Ras/MAP kinase pathway by dominant negative mutants of Ras and MEK stimulates basal and cAMP-induced tyrosinase promoter activities.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Dominant negative mutants of MEK and Ras stimulate the tyrosinase promoter activity. B16 cells were co-transfected with 2.2 pMT luciferase reporter plasmid and an expression vector coding for dominant negative mutants of MEK (MEK) and Ras (Ras). As a control, 2.2 pMT luciferase reporter plasmid was co-transfected with empty expression vector (vector). After transfection cells were left untreated or incubated with 20 µM forskolin as indicated for 48 h. Then cells were solubilized and luciferase activity was assayed. The results are expressed as fold stimulation of the basal luciferase activity from unstimulated cells. Data are means ± S.E. of three experiments performed in triplicate.

Expression of Dominant Positive Mutants of MEK and Ras Inhibits Basal and cAMP-induced Tyrosinase Promoter Activities-- Since the inhibition of MAP kinases induces an augmentation of the tyrosinase gene expression, we wondered whether the expression of dominant positive mutants of p21^ras (Ras⁺) and MEK (MEK⁺) would lead to an inhibition the tyrosinase gene promoter activity (Fig. 6). In cells co-expressing MEK⁺ and the reporter plasmid, we observed about a 2-fold decrease in luciferase activity in both basal and forskolin conditions. Expression of the dominant positive mutant of Ras (Ras⁺) appeared to be more potent to inhibit the forskolin-induced tyrosinase promoter activity. Thus, constitutively activation of the Ras/MAP kinase pathway leads to an inhibition of basal and cAMP-induced tyrosinase gene transcription.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Dominant positive mutants of MEK and Ras inhibit the tyrosinase promoter activity. B16 cells were co-transfected with 2.2 pMT luciferase reporter plasmid and the expression vector coding for constitutively active mutants of MEK (MEK+) and Ras (Ras+). After transfection cells were left untreated or incubated with 20 µM forskolin as indicated for 48 h. Then cells were solubilized and luciferase activity was assayed. The results are expressed as fold stimulation of the basal luciferase activity from unstimulated cells. Data are means ± S.E. of three experiments performed in triplicate.

	DISCUSSION

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The different approaches used in this report clearly demonstrate that the inhibition of the MAP kinase pathway by a pharmacological inhibitor of MEK or by dominant negative mutants of Ras and MEK does not prevent the effects of forskolin on different parameters reflecting the stimulation of melanogenesis. On the contrary, inhibition of the MAP kinase pathway is sufficient to induce dendrite outgrowth and to increase tyrosinase expression and activity. Further, sustained activation of this pathway, by over expression of constitutively active Ras and MEK, inhibits basal and cAMP-induced tyrosinase promoter activities. These findings invalidated our initial hypothesis suggesting that the activation of ERK1 by forskolin in B16 melanoma cells was a key event in cAMP-induced melanogenesis. However, our present results disclose the link between the MAP kinase pathway and the regulation of melanoma cell differentiation and demonstrate that the activation of the MAP kinase pathway leads to the inhibition of melanogenesis and differentiation.

At variance with our results, previous works have demonstrated that MAP kinases activation is required for the megakaryocytic differentiation of K562 cells (24, 25), the neuronal differentiation of PC12 (17) and the adipogenic differentiation of 3T3-L1 cells (18). However, concerning the rat pheochromocytoma cells PC12, that share with melanocytes and melanoma cells the same embryonic origin (neural crest), some other reports have shown that MAP kinases activation is dispensable for neurite outgrowth and differentiation (26, 27). Interestingly, infection by the v-Ha-ras oncogene was reported to inhibited melanogenesis in murine melanocytes (28). Additionally, neurofibromin that stimulates the intrinsic GTPase activity of Ras, thereby leading to a sustained inactivation of Ras, was previously shown to increase the tyrosinase gene expression (29). Thus, consistently with our results, the inhibition of Ras would lead to a stimulation of melanogenesis while its activation would suppress melanogenesis.

It is generally accepted that growth and differentiation are antinomic effects; cells cannot proliferate and differentiate at the same time. This general notion was supported by a recent report demonstrating that the differentiation of the neuroblastoma cell line NE1-115 characterized by the induction of neurite outgrowth is allowed when cells are arrested at the G₁ phase of the cell cycle (30). In addition, Callus et al. (31) have reported that treatment of J2E cells with amiloride suppressed the erythropoietin-induced proliferation and MAP kinase activity, and favored the differentiation of these cells. Thus inhibition of cell growth and interruption of cell cycle would commit the cell in a differentiation program. Our findings are more consistent with this notion. Keeping in mind the mitogenic role of MAP kinases that are required for cell proliferation and reentry in cell cycle after serum starvation (16), we could expect that the inhibition of MAP kinase pathway would lead to the stimulation of B16 melanoma cell differentiation and of melanogenesis. Conversely, activation of the MAP kinase pathway and stimulation of cell growth would inhibit melanogenesis. This hypothesis is supported by the inverse correlation between cell growth and melanogenesis observed in numerous reports. Indeed, UV-B irradiation that is a potent melanogenic stimulus inhibits growth of cultured human melanocytes (32). Growth factors such as basic fibroblast growth factor or TPA inhibit melanogenesis and stimulate growth of melanocytes (33). However, it should be noted that the stimulation of melanogenesis, by the inhibition of MAP kinases, cannot be solely explained by an inhibition of cell growth. Indeed, 10 µM PD98059 has only a very moderate inhibitory effect on growth of B16 cells, while inhibition of cell growth by serum depletion leads to faint increase in melanogenesis.

Additionally, it should be noted that down-regulation of the MAP kinase pathway is thought to trigger cell cycle arrest in the G₁ phase (16). During this phase, a number of cyclin-dependent kinases including Cdk2, 4, 5, and 6 are accumulated (34). Interestingly, it has been recently shown that Cdk5 and its associated regulator protein p39 plays a critical role in neurite outgrowth during neuronal differentiation (35). We can therefore envision that the inhibition of the MAP kinase pathway would result in the accumulation of Cdk5, thereby explaining the stimulation of dendrite outgrowth by PD98059. Consistently, UV-B (32) and cAMP (36), two potent melanogenic agents that stimulate dendricity, were also shown to block cells in G₁ phase. The involvement of Cdk5 in the formation of dendrites induced by cAMP-elevating agents or by the MAP kinase pathway inhibition is an appealing hypothesis that needs to be confirmed.

It seems surprising that the activation of MAP kinase leads to the inhibition of melanogenesis, since forskolin and -MSH, two strong stimulators of melanogenesis, have been shown to activate MAP kinases (10). Hence, cAMP-elevating agents seem to trigger at least two different cascades of molecular events, one ending in the stimulation of melanin synthesis, and a second one decreasing melanogenesis through the activation of the MAP kinase pathway. Induction of antagonistic effects, by a single agent, has already been described in the literature. For instance, EGF activates MAP kinases which phosphorylate the EGF receptor, leading thereby to a decrease in the EGF binding (37). Further, tumor necrosis factor- induces apoptosis and the activation of NFB that plays a key protective role against apoptosis (38). We can envision that these opposite effects elicited by one agent can be involved in a fine tuning of a final biological effect. This retrocontrol would avoid overgrowth, overapoptosis, and over-melanin production that could be noxious for the cell.

In summary, we clearly establish in this report that activation of the MAP kinase pathway is not a required event in the induction of melanogenesis. Furthermore, the activation of this pathway leads to an inhibition of melanoma cell differentiation demonstrating that the up-regulation of melanogenesis by cAMP-elevating agents is the consequence of the activation of two distinct and antagonistic pathways that might allow a precise control of melanocyte and melanoma cell differentiation.