Regulation of tyrosinase expression and activity in cultured human retinalpigment epithelial cells

The purpose of this study was to investigate the regulation of tyrosinase g ene expression and activity in cultured human retinal pigment epithelial (R PE) cells. The tyrosinase promoter (Ty.prom) region (400 bp) was PCR amplif ied and cloned into a modified mammalian expression vector (pcDNA3.1) upstr eam 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 mammalia n expression plasmid (pRL-TK) containing a herpes simplex virus thymidine k inase promoter region upstream of Renilla Luc in a protocol designated the 'dual luciferase assay' (DLA). After co-transfection, cells were treated wi th a range of potential melanogenic agents; basic fibroblast growth factor (bFGF), methyl methane sulphonate, alpha -melanocyte stimulating hormone, v erapamil, phorbol myristate acetate, cholera toxin (CT), pigment epithelium derived factor (PEDF), and L-tyrosine. The expression of tyrosinase promot er and enzymatic activities were determined 48 hr post-transfection using t he DLA and DOPA oxidase assays, respectively. Tyrosinase activity could not he detected in RPE cells with any of the treatments. Tyrosinase promoter a ctivity,vas significantly up-regulated in RPE cells treated with bFGF, PEDF , verapamil, CT and tyrosine compared with control cells. In conclusion, th e 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 modif ication point(s), which are necessary for tyrosinase enzymic activity.