Glucosamine abrogates the stem cell factor + endothelin-1-induced stimulation of melanogenesis via a deficiency in MITF expression due to the proteolytic degradation of CREB in human melanocytes.

Abstract

We have already reported that glucosamine (GlcN) distinctly abrogates the pigmentation of human epidermal equivalents stimulated by stem cell factor + endothelin-1 (SE). In this study, we characterized the molecular mechanism involved in the anti-melanogenic effects of GlcN using normal human melanocytes (NHMs) in culture. The SE-stimulated gene (12h) and protein (24h) expression levels of melanocyte-specific proteins (at the indicated times post-stimulation) were significantly abrogated by pretreatment with GlcN for 72h. Western blotting analysis of the phosphorylation of intracellular signaling molecules in the MAPK pathway revealed that despite the significantly decreased level of total CREB protein at all times post-stimulation, the SE-stimulated phosphorylation of ERK, CREB and MITF is not attenuated at 15min post-stimulation in GlcN-treated NHMs. However, the SE-stimulated protein expression level of total MITF at 2 and 6h post-stimulation was significantly abrogated by 72h pretreatment with GlcN. Consistently, pretreatment with GlcN for 72h abrogated the stimulated gene and protein expression levels of MITF at 1h and 2h post-stimulation, respectively. Analysis of gene and protein expression levels also demonstrated that pretreatment with GlcN for 72h significantly reduced the protein levels of CREB and MITF without affecting their gene expression levels prior to the SE stimulation. Silencing with a CREB siRNA distinctly abrogated the SE-stimulated expression of MITF (at 2h post-stimulation) and melanocyte-specific proteins (at 24h post-stimulation). Similarly, transfection of MITF siRNA markedly abrogated the SE-stimulated expression of MITF protein and melanocyte-specific proteins at 2 and 24h post-stimulation, respectively. Finally, the decreased levels of CREB and MITF proteins induced by 72h pretreatment with GlcN were abrogated by the co-addition of the proteosomal degradation inhibitor MG132. These findings suggest that the anti-melanogenic effect elicited by GlcN is mediated via the decreased expression of MITF which results from the attenuated transcriptional activity of CREB due to proteolytic degradation.