In vitro migration of melanoblasts requires matrix metalloproteinase-2: implications to vitiligo therapy by photochemotherapy.

Abstract

It is known that the migration of melanocyte precursors (melanoblasts) from the outer root sheath of hair follicles into clinically depigmented epidermis is crucial to the repigmentation of vitiliginous skin treated with photochemotherapy (PUVA), but such migratory cells must penetrate extracellular matrix tissue barriers in vivo. To test the hypothesis that matrix metalloproteinases (MMPs) are required for this process, we determined whether cultured melb-a cells, an immortal line of melanoblasts isolated from neonatal mouse epidermis, express and secrete MMPs and whether a synthetic metalloproteinase inhibitor, GM6001 (Galardin), inhibits their migratory behavior in vitro. Reverse transcriptase-polymerase chain reaction and Western blotting were used to determine the patterns of MMP expression by melanoblasts at the mRNA and protein levels, respectively. The proteolytic activities of MMPs secreted into the culture medium were assessed by gelatin zymography. The capacity of melanoblasts to migrate on fibronectin, laminin or laminin-5 substrates was estimated using Transwell migration assays. The results show that MMP2, MMP9 and MT1-MMP transcripts are expressed by these melanoblasts, but only MMP2 is secreted and activated in the extracellular environment. Although the therapeutic efficacy of PUVA in stimulating repigmentation of vitiliginous skin might derive from direct effects of UVA and/or 8-methoxypsoralen (8MOP), recent studies have shown that keratinocyte-derived factors induced by ultraviolet radiation, especially alpha-melanocyte stimulating hormone (alpha MSH), play a major role in regulating melanocyte function. Therefore, we also examined whether 8MOP and/or alphaMSH are involved in the up-regulation of MMP2 expression in melanoblasts. Western blotting and zymographic analyses revealed that MMP2 synthesis and secretion were induced by 8MOP and/or by alpha MSH. This induction of MMP2 resulted in significant increases of migration by melanoblasts on laminin or on laminin-5 substrates, while concomitant treatment with GM6001 blocked that induced migration. Taken together, these results suggest the importance of MMP2 in melanoblast migration and in the response to PUVA therapy.