Cotransfection of genes encoding human tyrosinase and tyrosinase-related protein-1 prevents melanocyte death and enhances melanin pigmentation and gene expression of Lamp-1.
We constructed two genes specific to melanogenesis, human tyrosinase (HT) and tyrosinase-related protein-1 (TRP-1) genes, into two separate expression vectors so that the cloned genes were under the control of a human cytomegalovirus promoter and enhancer. Monkey kidney COS-7 cells and human amelanotic and melanotic melanoma cells were then cotransfected by both HT and TRP-1 or transfected individually with each gene. The transfectants were examined for mRNA expression by reverse transcription-mediated RNA-PCR amplification. HT or TRP-1 mRNA was strongly expressed in HT or TRP-1 transfectants and cotransfectants of the two genes. Both light and electron microscopic observations indicated that degeneration and premature death of melanocytes occurred in HT transfectants, but not in TRP-1 transfectants or in HT and TRP-1 cotransfectants. Cotransfected cells from five cell lines revealed numerous granular reaction products with an anti-TRP-1 antibody and lysosomal granules with electron-dense material. Our melanin assay confirmed the new production of melanin pigments in these cells, indicating that the lysosomal granules would contain melanin pigments. The gene expression studies of lysosomal protein (beta-galactosidase, CD63, Lamp-1, and Lamp-2) revealed a dramatically elevated gene expression of Lamp-1, which is associated with the membrane receptor of lysosomal granules, in HT- and TRP-1-cotransfected cells. Conversely, the treatment of melanoma cells with antisense oligodeoxynucleotides against Lamp-1 resulted in a decreased expression of TRP-1 protein by immunoprecipitation, supporting the observations of the HT and TRP-1 cotransfection study regarding the up-regulation of Lamp-1 expression. We conclude that HT, TRP-1, and Lamp-1 gene products may function together, being expressed as a multiprotein complex within the melanosomal compartment. Specifically, HT and TRP-1 may function together via Lamp-1 by stabilizing the enzyme-protein complex within the melanosome and prevent the premature death of melanocytes due to tyrosinase-mediated cytotoxicity.
Faculty of Medicine, University of Alberta, Edmonton, Canada.
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D000818:Animals; D015703:Antigens, CD; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D003587:Cytomegalovirus / Q000235:genetics; D017931:DNA Primers; D004742:Enhancer Elements, Genetic; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors; D006801:Humans; D007668:Kidney; D051907:Lysosomal Membrane Proteins; D008543:Melanins / Q000378:metabolism*; D008545:Melanoma / Q000378:metabolism / Q000648:ultrastructure; D008562:Membrane Glycoproteins / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D016376:Oligonucleotides, Antisense; D015388:Organelles / Q000473:pathology / Q000648:ultrastructure; D010088:Oxidoreductases; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis*; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D017931:DNA Primers; D051907:Lysosomal Membrane Proteins; D008543:Melanins; D008562:Membrane Glycoproteins; D016376:Oligonucleotides, Antisense; D011506:Proteins; D012333:RNA, Messenger; D010088:Oxidoreductases; C489799:TYRP1 protein, human; C064056:tyrosinase-related protein-1; D014442:Monophenol Monooxygenase