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Ras mediates the cAMP-dependent activation of extracellular signal-regulated kinases (ERKs) in melanocytes

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.

Different cis-Acting Elements Are Involved in the Regulation of TRP1 and TRP2 Promoter Activities by Cyclic AMP: Pivotal Role of M Boxes (GTCATGTGCT) and of Microphthalmia

ABSTRACT 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.

Inhibition of the Phosphatidylinositol 3-Kinase/p70S6-Kinase Pathway Induces B16 Melanoma Cell Differentiation

α-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/p70S6-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 p70S6-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 cAMP-elevating agent forskolin, on both phosphatidylinositol 3-kinase and p70S6-kinase activities. Specific kinase assays revealed that forskolin partially inhibited phosphatidylinositol 3-kinase activity and completely blocked p70S6-kinase activity and phosphorylation. In conclusion, our results clearly demonstrate that the inhibition of phosphatidylinositol 3-kinase and p70S6-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/p70S6-kinase signaling pathway.

Hypoxia-inducible factor 1α is a new target of microphthalmia-associated transcription factor (MITF) in melanoma cells

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.

Microphthalmia-associated transcription factor regulates RAB27A gene expression and controls melanosome transport.

Melanosomes are lysosome-related organelles specialized in melanin synthesis and transport. In this study, we show that microphthalmia-associated transcription factor (MITF) silencing induces melanosome gathering around the nucleus and causes the relocalization of Rab27A, Slac2a-Mlph, and Myo5a that control the transport of melanosomes on the actin network. In an attempt to elucidate the mechanism by which MITF controls melanosome distribution, we identify RAB27A as a new MITF target gene. Indeed, MITF silencing leads to a dramatic decrease in Rab27A expression and blocks the stimulation of Rab27A expression evoked by cAMP. Further, forced expression of MITF increases Rab27A expression, indicating that MITF is required and sufficient for Rab27A expression in melanoma cells. MITF binds to two E-boxes in the proximal region of the Rab27A promoter and stimulates its transcriptional activity. Finally, re-expression of Rab27A, in MITF-depleted cells, restores the transport of melanosomes to the cell periphery. These results show that RAB27A is a new direct transcriptional target of MITF and link MITF to melanosome transport, another key parameter of melanocyte differentiation and skin pigmentation. Interestingly, Rab27A is involved in other fundamental physiological functions, such as the transport of lytic granules and insulin secretion. Thus our results, deciphering the mechanism of Rab27A transcriptional regulation, have an interest that goes beyond the skin pigmentation field.

Characterization of the molecular defects in Rab27a, caused by RAB27A missense mutations found in patients with Griscelli syndrome.

Rab27a plays a pivotal role in the transport of melanosomes to dendrite tips of melanocytes and mutations in RAB27A, which impair melanosome transport cause the pigmentary dilution and the immune deficiency found in several patients with Griscelli syndrome (GS). Interestingly, three GS patients present single homozygous missense mutations in RAB27A, leading to W73G, L130P, and A152P transitions that affect highly conserved residues among Rab proteins. However, the functional consequences of these mutations have not been studied. In the present report, we evaluated the effect of overexpression of these mutants on melanosome, melanophilin, and myosin-Va localization in B16 melanoma cells. Then we studied several key parameters for Rab27a function, including GTP binding and interaction with melanophilin/myosin-Va complex, which links melanosomes to the actin network. Our results showed that Rab27a-L130P cannot bind GTP, does not interact with melanophilin, and consequently cannot allow melanosome transport on the actin filaments. Interestingly, Rab27a-W73G binds GTP but does not interact with melanophilin. Thus, Rab27a-W73G cannot support the actin-dependent melanosome transport. Finally, Rab27a-A152P binds both GTP and melanophilin. However, Rab27a-A152P does not allow melanosome transport and acts as a dominant negative mutant, because its overexpression, in B16 melanoma cells, mimics a GS phenotype. Hence, the interaction of Rab27a with melanophilin/myosin-Va is not sufficient to ensure a correct melanosome transport. Our results pointed to an unexpected complexity of Rab27a function and open the way to the search for new Rab27a effectors or regulators that control the transport of Rab27a-dependent vesicles.

Tumor necrosis factor alpha-mediated inhibition of melanogenesis is dependent on nuclear factor kappa B activation.

Melanogenesis is a physiological process resulting in the synthesis of melanin pigments which play a crucial protective role against skin photocarcinogenesis. In vivo, solar ultraviolet light triggers the secretion of numerous keratinocyte-derived factors that are implicated in the regulation of melanogenesis. Among these, tumor necrosis factor α (TNFα), a cytokine implicated in the pro-inflammatory response, down-regulates pigment synthesis in vitro. In this report, we aimed to determine the molecular mechanisms by which this cytokine inhibits melanogenesis in B16 melanoma cells. First, we show that TNFα inhibits the activity and protein expression of tyrosinase which is the key enzyme of melanogenesis. Further, we demonstrate that this effect is subsequent to a down-regulation of the tyrosinase promoter activity in both basal and cAMP-induced melanogenesis. Finally, we present evidence indicating that the inhibitory effect of TNFα on melanogenesis is dependent on nuclear factor kappa B (NFκB) activation. Indeed, overexpression of this transcription factor in B16 cells is sufficient to inhibit tyrosinase promoter activity. Furthermore, a mutant of inhibitory kappa B (IκB), that prevents NFκB activation, is able to revert the effect of TNFα on the tyrosinase promoter activity. Taken together, our results clarify the mechanisms by which TNFα inhibits pigmentation and point out the key role of NFκB in the regulation of melanogenesis.

Cyclic AMP promotes a peripheral distribution of melanosomes and stimulates melanophilin/Slac2-a and actin association

Melanosomes are melanin‐containing organelles that belong to a recently individualized group of lysosome‐related organelles. Recently, numerous reports have dissected the molecular mechanisms that control melanosome transport, but nothing was known about the possible regulation of melanosome distribution by exogenous physiological stimulus. In the present report, we demonstrate that a physiological melanocyte‐differentiating agent such as α‐melanocyte‐stimulating hormone, through the stimulation of the cAMP pathway, induces a rapid centrifugal transport of melanosomes, leading to their accumulation at the dendrite tips of melanocytes. Interestingly, the small GTP binding proteins of the p21Rho family and one of their effectors, p160 Rho‐associated kinase, but not PKA, play a key role in redistribution of melanosomes at the extremities of the dendrites. Further, we have investigated, at the molecular level, the effect of cAMP on the different proteins involved in the control of melanosome transport. We demonstrate that cAMP stimulates the expression of Rab27a and rapidly increases the interaction of the melanophilin/Slac2‐a with actin. Thus, we propose that the stimulation of the interaction between melanophilin/Slac2‐a and actin would allow the rapid accumulation of melanosomes in the actin‐rich region of the dendrite extremities.

ERK1 and ERK2 Map Kinases: Specific Roles or Functional Redundancy?

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.