Role of microphthalmia transcription factor in regulation of melanocyte differentiation marker TRP-1.

Tyrosinase and a family of tyrosinase-related proteins (TRPs) are melanocyte differentiation gene products involved in melanin pigmentation. Members of the tyrosinase family share upstream transcriptional regulatory elements suggesting that expression of these genes is regulated by shared mechanisms. Microphthalmia transcription factor MITF, a melanocyte-specific basic helix-loop-helix protein, has been shown to transactivate tyrosinase and TRP-1 genes in vitro by binding to a shared regulatory sequence known as M box. The role of MITF in concomitant regulation of these genes in vivo is not clear. We showed earlier that in human melanoma cells TRP-1 can be regulated independently of tyrosinase and pigmentation. To investigate the role of MITF in TRP-1 regulation, we studied the effect of pharmacological agents that modulate transcription of tyrosinase and TRP-1 on MITF. In melanoma cells treated with hexamethylene bisacetamide (HMBA), transcription of TRP-1 gene was selectively and completely inhibited while steady state levels of tyrosinase, TRP-2, MITF mRNA and melanin content showed a modest increase. HMBA caused no detectable change in cellular MITF or its nuclear localization. This MITF-independent regulation of TRP-1 required continued synthesis of RNA and protein. Selective down-regulation of TRP-1 by HMBA occurred even in the presence of cholera toxin which up-regulates TRP-1 by cAMP-mediated pathways. These data show that TRP-1 gene can be down-regulated independently of MITF by de novo activation of negative regulatory factors. Thus, both activation of positive factors such as MITF and inactivation of negative regulatory factors may be required for TRP-1 gene expression during melanocytic differentiation.