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Role of microphthalmia transcription factor in regulation of melanocyte differentiation marker TRP-1
1Departments of Dermatology and Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, North Carolina, 27157, USA.
Abstract:
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.
Insights
Hexamethylene bisacetamide (HMBA) selectively inhibits tyrosinase-related protein 1 (TRP-1) gene expression in melanoma cells, independent of the microphthalmia transcription factor (MITF). This suggests TRP-1 regulation involves both positive and negative factors during melanocyte differentiation.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Tyrosinase and tyrosinase-related proteins (TRPs) are key regulators of melanin production.
- Microphthalmia transcription factor (MITF) is a melanocyte-specific regulator known to activate tyrosinase and TRP-1 genes.
- Previous studies indicated TRP-1 regulation can be independent of tyrosinase and pigmentation in melanoma cells.
Purpose of the Study:
- To investigate the role of MITF in the in vivo regulation of TRP-1.
- To explore the mechanisms of TRP-1 gene regulation independent of MITF.
Main Methods:
- Treatment of human melanoma cells with hexamethylene bisacetamide (HMBA).
- Analysis of gene expression levels for tyrosinase, TRP-1, TRP-2, and MITF mRNA.
- Assessment of melanin content and MITF cellular localization.
- Investigation of TRP-1 regulation in the presence of cholera toxin.
Main Results:
- HMBA selectively and completely inhibited TRP-1 gene transcription.
- Tyrosinase, TRP-2, MITF mRNA, and melanin content showed a modest increase with HMBA treatment.
- HMBA did not alter cellular MITF levels or nuclear localization.
- TRP-1 down-regulation by HMBA was MITF-independent and required ongoing RNA and protein synthesis.
- Selective TRP-1 down-regulation occurred even with cholera toxin, which normally up-regulates TRP-1.
Conclusions:
- TRP-1 gene expression can be regulated independently of MITF.
- De novo activation of negative regulatory factors contributes to TRP-1 down-regulation.
- Melanocytic differentiation requires both activation of positive factors (like MITF) and inactivation of negative regulatory factors for TRP-1 expression.