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.
Hara
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Division of Dermatology & Cutaneous Sciences, Faculty of Medicine, University of Alberta, Edmonton, Canada.
Lee
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MH
Chen
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Luo
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Jimbow
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D015703:Antigens, CD / Q000235:genetics*; D001483:Base Sequence; D015870:Gene Expression / Q000502:physiology*; D006801:Humans; D051907:Lysosomal Membrane Proteins; D008543:Melanins / Q000096:biosynthesis*; D008544:Melanocytes / Q000528:radiation effects / Q000648:ultrastructure; D018328:Melanoma, Amelanotic; D008546:Melanoma, Experimental; D008562:Membrane Glycoproteins / Q000235:genetics*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000235:genetics*; D010088:Oxidoreductases; D010980:Platelet Membrane Glycoproteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics*; D060149:Tetraspanin 30; D014407:Tumor Cells, Cultured / Q000528:radiation effects; D014466:Ultraviolet Rays
D015703:Antigens, CD; C554586:CD63 protein, human; D051907:Lysosomal Membrane Proteins; D008543:Melanins; D008562:Membrane Glycoproteins; D010980:Platelet Membrane Glycoproteins; D011506:Proteins; D060149:Tetraspanin 30; D010088:Oxidoreductases; C489799:TYRP1 protein, human; C064056:tyrosinase-related protein-1; D014442:Monophenol Monooxygenase