Search

In melanocytes and melanoma cells alpha-melanocyte stimulating hormone (alpha-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 1alpha (HIF1alpha) 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 alpha-MSH/cAMP pathway, using MITF as a signal transducer and HIF1alpha as a target, might contribute to melanoma progression.

Microphthalmia-associated transcription factor (MITF) plays a pivotal role in melanocyte survival and differentiation. Nevertheless, until now it has not been possible to show that MITF regulates the expression of the endogenous tyrosinase or Tyrp1. Further, a direct involvement of MITF in the regulation of melanin synthesis, a key parameter of melanocyte differentiation, remains to be demonstrated. In the present report, using recombinant adenovirus encoding the wild-type or a dominant negative form of MITF, as well as stable cell lines expressing tetracycline inducible wild-type MITF, we reassessed the role of MITF in melanocyte differentiation and in the regulation of melanin synthesis. Immunofluorescence studies, as well as Western blot analyses, show that infection of B16 mouse melanoma cells or human melanocytes with adenovirus encoding wild-type MITF does not increase the expression of the endogenous melanogenic enzymes. However, infection with the MITF dominant negative mutant inhibits the expression of endogenous tyrosinase and Tyrp1 proteins and blocks cAMP-induced melanin synthesis. Thus, MITF is required but does not seem to be sufficient to induce the expression of melanogenic enzymes and we show for the first time a direct involvement of MITF in the regulation of melanin pigment synthesis. As a whole, our data point to the existence of still unknown regulatory mechanisms that co-operate or synergize with MITF to control melanogenic gene expression and melanin synthesis. The identification of such mechanisms will greatly improve our understanding of the melanocyte differentiation processes.

Epidermal wound repair is a complex process involving the fine orchestrated regulation of crucial cell functions, such as proliferation, adhesion and migration. Using an in vitro model that recapitulates central aspects of epidermal wound healing, we demonstrate that the transcription factor HIF1 is strongly stimulated in keratinocyte cultures submitted to mechanical injury. Signals generated by scratch wounding stabilise the HIF1α protein, which requires activation of the PI3K pathway independently of oxygen availability. We further show that upregulation of HIF1α plays an essential role in keratinocyte migration during the in vitro healing process, because HIF1α inhibition dramatically delays the wound closure. In this context, we demonstrate that HIF1α controls the expression of laminin-332, one of the major epithelial cell adhesion ligands involved in cell migration and invasion. Indeed, silencing of HIF1α abrogates injury-induced laminin-332 expression, and we provide evidence that HIF1 directly regulates the promoter activity of the lamininα 3 chain. Our results suggest that HIF1 contributes to keratinocyte migration and thus to the re-epithelialisation process by regulating laminin-332. [ABSTRACT FROM AUTHOR]