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

Tyrosinase and a family of tyrosinase-related proteins (TRPs) are melanocyt e differentiation gene products involved in melanin pigmentation. Members o f 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 hel ix-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 cl ear. We showed earlier that in human melanoma cells TRP-1 can be regulated independently of tyrosinase and pigmentation. To investigate the role of MI TF in TRP-1 regulation, we studied the effect of pharmacological agents tha t modulate transcription of tyrosinase and TRP-1 on MITF. In melanoma cells treated with hexamethylene bisacetamide (HMBA), transcription of TRP-1 gen e was selectively and completely inhibited while steady state levels of tyr osinase, TRP-8, MITF mRNA and melanin content showed a modest increase. HMB A 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 i n the presence of cholera toxin which up-regulates TRP-1 by cAMP-mediated p athways. These data show that TRP-1 gene can be down-regulated independentl y of MITF by de novo activation of negative regulatory factors. Thus, both activation of positive factors such as MITF and inactivation of negative re gulatory factors may be required for TRP-1 gene expression during melanocyt ic differentiation. (C) 1999 Academic Press.