CHARACTERIZATION OF DENSITY-DEPENDENT REGULATION OF THE TYROSINASE GENE PROMOTER - ROLE OF PROTEIN-KINASE-C

The rate-limiting step in melanogenesis is catalyzed by tyrosinase, a multifunctional enzyme encoded by the albino locus. We have previously reported that depletion of protein kinase C by long-term treatment of B16 mouse melanoma cells with phorbol dibutyrate (PDBu) prevented cel l density-dependent melanogenesis. This was accompanied by a lack of i nduction of tyrosinase protein and mRNA. We report here the effect of PDBu on the functional activity of the mouse tyrosinase promoter by re porter gene assay and its effect on the binding of nuclear proteins fr om B16 cells to the ''M-box'' region of the mouse tyrosinase promoter. Short-term PDBu treatment of B16 cells transfected with a mouse tyros inase promoter-luciferase construct resulted in increased reporter gen e activity, while long-term PDBu treatment inhibited reporter gene act ivity. Using an oligonucleotide containing the RI-box and its flanking residues in electrophoretic mobility shift assays, we found a density -dependent change in the pattern of DNA-protein complexes. One complex was found to be negatively regulated by longterm PDBu treatment. Comp etition experiments with various mutated oligonucleotides demonstrated that both the RI-box and flanking residues are important for nuclear protein binding. The complex whose formation was inhibited by long-ter m PDBu treatment was shown to contain the basic helix-loop-helix leuci ne zipper protein microphthalmia-associated transcription factor (MITF ). These results suggest that chronic PDBu treatment might inhibit tyr osinase expression (and subsequent melanogenesis) by affecting the amo unt or function of MITF. (C) 1997 Academic Press.