Role of Gene Expression and Protein Synthesis of Tyrosinase, TRP-1, Lamp-1, and CD63 in UVB-Induced Melanogenesis in Human Melanomas

Using melanotic cells (SK-MEL-23 and G361) and amelanotic cells (C32 and SK-MEL-24) of human melanoma, this study examined whether UV-B irradiation has a direct stimulatory effect on the expression of genes involved in melanogenesis. Our initial screening of methylthiazol tetrazolium (MTT)-formazan formation assay indicated a low dose of ultraviolet (UV)-B irradiation, 2.5 and 5.0 mJ/cm2, can metabolically stimulate these cells. Repeated exposure of UV-B at 5.0 mJ/cm2 for seven consecutive days resulted in increased tyrosinase activity and melanin synthesis in SK- MEL-23 and G361 cells, but not in C32 and SK-MEL-24 cells. On reverse-transcription – polymerase chain reaction and immunoprecipitation studies, the two melanotic cell lines exhibited upregulated expression of mRNA and antigenic epitopes of tyrosinase, tyrosinase-related protein (TRP-1; gp75/HMSA-5), and lysosomal membrane associated protein (Lamp-1). The amelanotic cell line, C32, expressed the tyrosinase gene and protein constitutively but revealed no active tyrosinase or melanin synthesis even after UV-B exposure. Another amelanotic cell line, SK-MEL-24, exhibited no expression of tyrosinase gene and protein before and after UV-B exposure and, therefore, no melanin synthesis. Both C32 and SK-MEL-24 showed no gene or protein expression of TRP-1 before or after UV exposure, but upregulation of the Lamp-1 gene and protein expressions after exposure.We conclude that tyrosinase is the key enzyme responsible for UVB-induced melanogenesis. Both TRP-1 and Lamp-1 act together in melanogenesis, TRP-1 being essential and necessary. There is no change in the expression of CD63 lysosomal membrane protein at either the mRNA or protein level