In humans, stimulation of skin pigmentation over the basal constitutive level, which we commonly call tanning, is physiologically stimulated by ultraviolet (UV) radiation of the solar light. UV-induced skin darkening involves an increase in the melanocyte number as well as a stimulation of melanin neosynthesis and melanocyte dendricity, a crucial morphological feature required for melanin transfer to keratinocytes. These events, corresponding to the final steps of melanocyte differentiation, play a central role in the tanning response and are subjected to an accurate research which aims to understand the precise mechanisms governing their regulation. In the present review, we will mainly focus our attention on the molecular processes involved in the regulation of melanogenesis.

In melanocytes and in melanoma cells, cyclic AMP (cAMP)-elevating agents stimulate melanogenesis and increase the transcription of tyrosinase, the rate-limiting enzyme in melanin synthesis. However, two other enzymes, tyrosinase-related protein 1 (TRP1) and TRP2, are required for a normal melanization process leading to eumelanin synthesis. In B16 melanoma cells, we demonstrated that stimulation of melanogenesis by cAMP-elevating agents results in an increase in tyrosinase, TRP1, and TRP2 expression. cAMP, through a cAMP-dependent protein kinase pathway, stimulates TRP1 and TRP2 promoter activities in both B16 mouse melanoma cells and normal human melanocytes. Regulation of the TRP1 and TRP2 promoters by cAMP involves a M box and an E box. Further, a classical cAMP response element-like motif participates in the cAMP responsiveness of the TRP2 promoter, demonstrating that the TRP2 gene is subjected to different regulatory processes, which could account for its different expression patterns during embryonic development or under specific physiological and pathological conditions. We also found that microphthalmia, a basic helix-loop-helix transcription factor, strongly stimulates the transcriptional activities of the TRP1 and TRP2 promoters, mainly through binding to the M boxes. Additionally, we demonstrated that cAMP increases microphthalmia expression and thereby its binding to TRP1 and TRP2 M boxes. These convergent and compelling results disclose at least a part of the molecular mechanism involved in the regulation of melanogenic gene expression by cAMP and emphasize the pivotal role of microphthalmia in this process.In mammals, pigmentation results from the synthesis and distribution of melanin in the skin, hair bulbs, and eyes. Melanin synthesis (melanogenesis) takes place in the melanocyte after differentiation of the nonpigmented precursor, the melanoblast (27). Three melanocyte-specific enzymes, tyrosinase, tyrosinase-related protein 1 (TRP1), and TRP2, are involved in this enzymatic process that converts tyrosine to melanin pigments. Although these proteins have similar structures and features, they are expressed by different genes and possess distinct enzymatic activities. Tyrosinase, encoded by the albino locus of the mouse, catalyzes the conversion of tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and of DOPA to DOPA quinone (14,25,31). TRP2, encoded by the mouse slaty locus, possesses a Dopachrome tautomerase activity, converting the Dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) (3,19,42). TRP1, which has been mapped in mouse to the brown locus, catalyzes the oxidation of DHICA to indole-5,6-quinone-2-carboxylic acid (21, 24).In vivo, melanogenesis is regulated by UVB radiation that can act either directly on melanocytes or indirectly through the release of keratinocyte-derived factors such as interleukins, prostaglandins, and alpha melanocyte-stimulating hormone (␣-MSH) (1,12,22,35). Interestingly, ␣-MSH, one of the most potent activators of melanogenesis, binds to an ␣ s -coupled rece...

In melanocytes and melanoma cells, cAMP activates extracellular signal-regulated kinases (ERKs) and MEK-1 by an unknown mechanism. We demonstrate that B-Raf is activated by cAMP in melanocytes. A dominant-negative mutant of B-Raf, but not of Raf-1, blocked the cAMP-induced activation of ERK, indicating that B-Raf is the MEK-1 upstream regulator mediating this cAMP effect. Studies using Clostridium sordelii lethal toxin and Clostridium difficile toxin B have suggested that Rap-1 or Ras might transduce cAMP action. We show that Ras, but not Rap-1, is activated cell-specifically and mediates the cAMP-dependent activation of ERKs, while Rap-1 is not involved in this process in melanocytes. Our results suggest a novel, cell-specific mechanism involving Ras small GTPase and B-Raf kinase as mediators of ERK activation by cAMP. Also, in melanocytes, Ras or ERK activation by cAMP is not mediated through protein kinase A activation. Neither the Ras exchange factor, Son of sevenless (SOS), nor the cAMP-responsive Rap-1 exchange factor, Epac, participate in the cAMP-dependent activation of Ras. These findings suggest the existence of a melanocyte-specific Ras exchange factor directly regulated by cAMP.

␣-Melanocyte-stimulating hormone and cAMP-elevating agents are known to induce B16 cell differentiation, characterized by increased melanin synthesis and dendrite outgrowth. In order to elucidate intracellular signaling pathways involved in this differentiation process, we focused our interest on the phosphatidylinositol 3-kinase/p70 S6 -kinase pathway. The specific inhibition of phosphatidylinositol 3-kinase by LY294002 markedly stimulated dendrite outgrowth, thus mimicking the action of cAMP-elevating agents on B16 cell morphology. In addition, LY294002 and rapamycin, a specific p70 S6 -kinase inhibitor, were found to independently stimulate tyrosinase expression, thus increasing melanin synthesis. In an attempt to better dissect the molecular mechanisms triggered by cAMP to induce melanoma cell differentiation, we examined the effects of a cAMPelevating agent forskolin, on both phosphatidylinositol 3-kinase and p70 S6 -kinase activities. Specific kinase assays revealed that forskolin partially inhibited phosphatidylinositol 3-kinase activity and completely blocked p70 S6-kinase activity and phosphorylation. In conclusion, our results clearly demonstrate that the inhibition of phosphatidylinositol 3-kinase and p70 S6 -kinase is involved in the regulation of B16 cell differentiation. Furthermore, we provide evidence which suggests that cAMP-induced melanogenesis and dendricity are, at least partially, mediated by the cAMP inhibition of the phosphatidylinositol 3-kinase/p70 S6 -kinase signaling pathway.In the epidermis, melanocytes synthesize melanin, which is responsible for skin pigmentation. Melanin synthesis is carried out by a cell-specific enzymatic pathway controlled by tyrosinase (EC 1.14.18.1), the enzyme that catalyzes the initial two rate-limiting reactions of this process, the hydroxylation of tyrosine to dopa and its subsequent oxidation to dopaquinone (1-4). In vivo, melanogenesis is induced mainly by ultraviolet A and B radiation of sunlight and ␣-melanocyte-stimulating hormone (␣-MSH) 1 (5) which binds to a specific G proteincoupled receptor. In cultured melanocytes or in melanoma cells, melanogenesis can be induced by ultraviolet A and B radiation and by a large array of effectors including ␣-MSH (4) and pharmacological agents such as forskolin, cholera toxin, or isobutylmethylxanthine (6 -9). These agents increase the intracellular cAMP content, thereby indicating the importance of the cAMP pathway in melanogenesis. The stimulation of melanogenesis by cAMP-elevating agents seems to occur through the induction of tyrosinase expression and stimulation of its intrinsic enzymatic activity ensuing post-translational modifications (10). However, few data are available concerning molecular mechanisms that connect the cAMP signaling pathway and tyrosinase regulation. Recently, we have shown in B16 melanoma cells that cAMP-elevating agents stimulate ERK1 activity and induce its translocation to the nucleus (9), whereas in the majority of cell systems, cAMP has been described to inhibit this kinase (11)....

The MAP kinase signaling cascade Ras/Raf/MEK/ERK has been involved in a large variety of cellular and physiological processes that are crucial for life. Many pathological situations have been associated to this pathway. More than one isoform has been described at each level of the cascade. In this review we devoted our attention to ERK1 and ERK2, which are the effector kinases of the pathway. Whether ERK1 and ERK2 specify functional differences or are in contrast functionally redundant, constitutes an ongoing debate despite the huge amount of studies performed to date. In this review we compiled data on ERK1 vs. ERK2 gene structures, protein sequences, expression levels, structural and molecular mechanisms of activation and substrate recognition. We have also attempted to perform a rigorous analysis of studies regarding the individual roles of ERK1 and ERK2 by the means of morpholinos, siRNA, and shRNA silencing as well as gene disruption or gene replacement in mice. Finally, we comment on a recent study of gene and protein evolution of ERK isoforms as a distinct approach to address the same question. Our review permits the evaluation of the relevance of published studies in the field especially when measurements of global ERK activation are taken into account. Our analysis favors the hypothesis of ERK1 and ERK2 exhibiting functional redundancy and points to the concept of the global ERK quantity, and not isoform specificity, as being the essential determinant to achieve ERK function.

In B16 melanoma cells, mitogen-activated protein (MAP) kinases are activated during cAMP-induced melanogenesis (Englaro, W., Rezzonico, R., Durand-Clé ment, M., Lallemand, D., Ortonne, J. P., and Ballotti, R. (1995) J. Biol. Chem. 270, 24315-24320). To establish the role of the MAP kinases in melanogenesis, we studied the effects of a specific MAP kinase kinase (MEK) inhibitor PD 98059 on different melanogenic parameters. We showed that PD 98059 inhibits the activation of MAP kinase extracellular signal-regulated kinase 1 by cAMP, but does not impair the effects of cAMP either on the morphological differentiation, characterized by an increase in dendrite outgrowth, or on the up-regulation of tyrosinase that is the key enzyme in melanogenesis. On the contrary, PD 98059 promotes by itself cell dendricity and increases the tyrosinase amount and activity. Moreover, down-regulation of the MAP kinase pathway by PD 98059, or with dominant negative mutants of p21 ras and MEK, triggers a stimulation of the tyrosinase promoter activity and enhances the effect of cAMP on this parameter. Conversely, activation of the MAP kinase pathway, using constitutive active mutants of p21 ras and MEK, leads to an inhibition of basal and cAMP-induced tyrosinase gene transcription. These results demonstrate that the MAP kinase pathway activation is not required for cAMP-induced melanogenesis. Furthermore, the inhibition of this pathway induces B16 melanoma cell differentiation, while a sustained activation impairs the melanogenic effect of cAMP-elevating agents.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, ␣-melanocytestimulating 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 ...

Covering denuded dermal surfaces after injury requires migration, proliferation, and differentiation of skin keratinocytes. To clarify the major traits controlling these intermingled biological events, we surveyed the genomic modifications occurring during the course of a scratch wound closure of cultured human keratinocytes. Using a DNA microarray approach, we report the identification of 161 new markers of epidermal repair. Expression data, combined with functional analysis performed with specific inhibitors of ERK, p38MAPK and phosphatidylinositol 3-kinase (PI3K), demonstrate that kinase pathways exert very selective functions by precisely controlling the expression of specific genes. Inhibition of the ERK pathway totally blocks the wound closure and inactivates many early transcription factors and EGF-type growth factors. p38 MAPK inhibition only delays "healing," probably in line with the control of genes involved in the propagation of injury-initiated signaling. In contrast, PI3K inhibition accelerates the scratch closure and potentiates the scratch-dependent stimulation of three genes related to epithelial cell transformation, namely HAS3, HBEGF, and ETS1. Our results define in vitro human keratinocyte wound closure as a repair process resulting from a fine balance between positive signals controlled by ERK and p38 MAPK and negative ones triggered by PI3K. The perturbation of any of these pathways might lead to dysfunction in the healing process, similar to those observed in pathological wounding phenotypes, such as hypertrophic scars or keloids.

In melanocytes and melanoma cells α-melanocyte stimulating hormone (α-MSH), via the cAMP pathway, elicits a large array of biological responses that control melanocyte differentiation and influence melanoma development or susceptibility. In this work, we show that cAMP transcriptionally activates Hif1a gene in a melanocyte cell–specific manner and increases the expression of a functional hypoxia-inducible factor 1α (HIF1α) protein resulting in a stimulation of Vegf expression. Interestingly, we report that the melanocyte-specific transcription factor, microphthalmia-associated transcription factor (MITF), binds to the Hif1a promoter and strongly stimulates its transcriptional activity. Further, MITF “silencing” abrogates the cAMP effect on Hif1a expression, and overexpression of MITF in human melanoma cells is sufficient to stimulate HIF1A mRNA. Our data demonstrate that Hif1a is a new MITF target gene and that MITF mediates the cAMP stimulation of Hif1a in melanocytes and melanoma cells. Importantly, we provide results demonstrating that HIF1 plays a pro-survival role in this cell system. We therefore conclude that the α-MSH/cAMP pathway, using MITF as a signal transducer and HIF1α as a target, might contribute to melanoma progression.