IN B16 MELANOMA-CELLS, THE INHIBITION OF MELANOGENESIS BY TPA RESULTSFROM PKC ACTIVATION AND DIMINUTION OF MICROPHTHALMIA BINDING TO THE M-BOX OF THE TYROSINASE PROMOTER

In B16 melanoma cells, cAMP-induced melanogenesis is inhibited by the tumor promoting phorbol ester, TPA, However, the role of PKC activatio n or depletion in the inhibition of melanogenesis by TPA remains contr oversial, In this report, using specific PKC inhibitors, ne demonstrat ed that PKC inhibition does not impair cAMP-induced melanin synthesis and tyrosinase expression, Further, the inhibition of melanogenesis by TPA results from a decrease of the tyrosinase promoter transcriptiona l activity and this effect is mimicked by over-expression of a constit utively active form of PKC alpha, These findings clearly demonstrate t hat PKC activation accounts for the inhibition of melanin synthesis by TPA, Additional experiments were undertaken to elucidate the mechanis m by which TPA inhibits the tyrosinase gene transcription, Deletions a nd mutation in the tyrosinase promoter showed that TPA acts on a M-box which is involved in tissue-specific expression and regulation ba cAM P of the tyrosinase gene, We showed that TPA decreases the binding of microphthalmia, a basic helix-loop-helix transcription factor, to the M-box. Since microphthalmia, strongly stimulates the transcriptional a ctivity of the promoter we propose that TPA, through PKC activation, d ecreases microphthalmia binding to the M-box of the tyrosinase promote r, thereby leading to a reduced tyrosinase expression and melanogenesi s inhibition.