Regulation of tyrosinase expression and activity in cultured human retinal pigment epithelial cells.
The purpose of this study was to investigate the regulation of tyrosinase gene expression and activity in cultured human retinal pigment epithelial (RPE) cells. The tyrosinase promoter (Ty.prom) region (400 bp) was PCR amplified and cloned into a modified mammalian expression vector (pcDNA3.1) upstream of a firefly luciferase (Luc) cDNA and was designated 'pcDNA3.1-Ty.prom.Luc'. The plasmid was co-transfected into RPE cells with a second mammalian expression plasmid (pRL-TK) containing a herpes simplex virus thymidine kinase promoter region upstream of Renilla Luc in a protocol designated the 'dual luciferase assay' (DLA). After co-transfection, cells were treated with a range of potential melanogenic agents; basic fibroblast growth factor (bFGF), methyl methane sulphonate, alpha-melanocyte stimulating hormone, verapamil, phorbol myristate acetate, cholera toxin (CT), pigment epithelium derived factor (PEDF), and L-tyrosine. The expression of tyrosinase promoter and enzymatic activities were determined 48 hr post-transfection using the DLA and DOPA oxidase assays, respectively. Tyrosinase activity could not be detected in RPE cells with any of the treatments. Tyrosinase promoter activity was significantly up-regulated in RPE cells treated with bFGF, PEDF, verapamil, CT and tyrosine compared with control cells. In conclusion, the tyrosinase gene is not only expressed but can be regulated in response to different chemicals in cultured human RPE cells. However, it appears that RPE cells in culture lack a post-transcriptional and/or translational modification point(s), which are necessary for tyrosinase enzymic activity.
Abul-Hassan
K
K
Department of Optometry and Vision Sciences, Cardiff University, United Kingdom.
Walmsley
R
R
Tombran-Tink
J
J
Boulton
M
M
D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000368:Aged; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002273:Carcinogens; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism*; D005136:Eye Proteins; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008544:Melanocytes / Q000378:metabolism; D008741:Methyl Methanesulfonate; D008875:Middle Aged; D014442:Monophenol Monooxygenase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D009153:Mutagens; D009414:Nerve Growth Factors; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000494:pharmacology; D012323:RNA Processing, Post-Transcriptional; D015843:Serpins / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000494:pharmacology; D015854:Up-Regulation; D014602:Uvea / Q000166:cytology; D014700:Verapamil / Q000494:pharmacology; D000521:alpha-MSH / Q000494:pharmacology; D000099174:Pigment Epithelium-Derived Factor
D000276:Adjuvants, Immunologic; D002121:Calcium Channel Blockers; D002273:Carcinogens; D002772:Cholera Toxin; D018076:DNA, Complementary; D005136:Eye Proteins; D016222:Fibroblast Growth Factor 2; D008156:Luciferases; D008741:Methyl Methanesulfonate; D014442:Monophenol Monooxygenase; D009153:Mutagens; D009414:Nerve Growth Factors; D011506:Proteins; D015843:Serpins; D013755:Tetradecanoylphorbol Acetate; D014443:Tyrosine; D014700:Verapamil; D000521:alpha-MSH; D000099174:Pigment Epithelium-Derived Factor