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Microbes regulate a large panel of intracellular signalling events that can promote inflammation and/or enhance tumour progression. Indeed, it has been shown that infection of human intestinal cells with the Afa/Dr diffusely adhering E. coli C1845 strain induces expression of pro-angiogenic and pro-inflammatory genes. Here, we demonstrate that exposure of cryptic-like intestinal epithelial cells to C1845 bacteria induces HIF-1α protein levels. This effect depends on the binding of F1845 adhesin to the membrane-associated DAF receptor that initiates signalling cascades promoting translational mechanisms. Indeed, inhibition of MAPK and PI-3K decreases HIF-1α protein levels and blocks C1845-induced phosphorylation of the ribosomal S6 protein. Using RNA interference we show that bacteria-induced HIF-1α regulates the expression of IL-8, VEGF and Twist1, thereby pointing to a role for HIF-1 in angiogenesis and inflammation. In addition, infection correlates with a loss of E-cadherin and cytokeratin 18 and a rise in fibronectin, suggesting that bacteria may induce an epithelial to mesenchymal transition-like phenotype. Since HIF-1α silencing results in reversion of bacteria-induced EMT markers, we speculate that HIF-1α plays a key role linking bacterial infection to angiogenesis, inflammation and some aspects of cancer initiation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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-l, 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. [ABSTRACT FROM AUTHOR]

Investigates the possible regulation of melanosome transport in mammalian melanocytes by α-melanocyte stimulating hormone. Effect of cyclic adenosine monophosphate (cAMP) activation on proteins involved in the control of melanosome transport; Role of RhoA and p160Rock in the redistribution of melanosomes; Impact of cAMP on the interaction between melanophin/Slac2-a and actin.