Microphthalmia Gene Product as a Signal Transducer in cAMP-Induced Differentiation of Melanocytes

Abstract

Melanocyte differentiation characterized by an increased melanogenesis, is stimulated by α-melanocyte–stimulating hormone through activation of the cAMP pathway. During this process, the expression of tyrosinase, the enzyme that controls melanin synthesis is upregulated. We previously showed that cAMP regulates transcription of the tyrosinase gene through a CATGTG motif that binds microphthalmia a transcription factor involved in melanocyte survival. Further, microphthalmia stimulates the transcriptional activity of the tyrosinase promoter and cAMP increases the binding of microphthalmia to the CATGTG motif. These observations led us to hypothesize that microphthalmia mediates the effect of cAMP on the expression of tyrosinase. The present study was designed to elucidate the mechanism by which cAMP regulates microphthalmia function and to prove our former hypothesis, suggesting that microphthalmia is a key component in cAMP-induced melanogenesis. First, we showed that cAMP upregulates the transcription of microphthalmia gene through a classical cAMP response element that is functional only in melanocytes. Then, using a dominant-negative mutant of microphthalmia, we demonstrated that microphthalmia is required for the cAMP effect on tyrosinase promoter. These findings disclose the mechanism by which cAMP stimulates tyrosinase expression and melanogenesis and emphasize the critical role of microphthalmia as signal transducer in cAMP-induced melanogenesis and pigment cell differentiation.

The melanocyte inducing factor MITF is stably expressed in cell lines from human clear cell sarcoma

“…In Mel28 cells, the C5 antibody detects four polypeptides within the reported size range of Mitf (55 -75 kDa, Figure 3B) and these polypeptides are absent or expressed at very low levels in nonmelanoma cells (HeLa and Jeg3). The above pattern is consistent with (but more complex than) other studies using melanoma cells and the C5 antibody (Bertolotto et al, 1998;King et al, 1999;Wu et al, 2000) or another antibody (D5) in which an B55 -60 kDa Mitf-M doublet is detected. Several considerations indicate that in Mel28 cells, either the fastest migrating polypeptide (indicated by an open box in Figure 3B) or the slower migrating doublet (open circles, Figure 3B) or both of the above, are Mitf-M. First, Mitf-M is significantly smaller than all other Mitf isoforms ( Figure 3A) and migrates faster in SDS gels at around 55 -60 kDa (Fuse et al, 1999).…”

“…Similar to the above result in CCS cells, exogenous EWS/ATF1 (expressed from pD287C) is unable to activate the Mitf-M promoter in either Jeg3 cells ( Figure 4B) or the melanoma cell line Mel28 ( Figure 4C) in which the Mitf-M promoter is normally active. As a positive control for the integrity of the Mitf-M reporter, pMITF-MCAT is able to exhibit a melanocyte-specific cAMP-response in Mel28 cells ( Figure 4B and C) as expected (Bertolotto et al, 1998). Finally, we tested the ability of the ATF binding site in the Mitf-M promoter to confer EWS/ATF1 responsiveness to a heterologous promoter in Jeg3 cells ( Figure 4C).…”

“…We used a monoclonal antibody (C5) that detects all Mitf isoforms ( Figure 3A) and that has been extensively used for studies of Mitf-M (Bertolotto et al, 1998;King et al, 1999;King et al, 2001;Wu et al, 2000). In Mel28 cells, the C5 antibody detects four polypeptides within the reported size range of Mitf (55 -75 kDa, Figure 3B) and these polypeptides are absent or expressed at very low levels in nonmelanoma cells (HeLa and Jeg3).…”

“…Microphthalmia-associated transcription factor acts in part via direct transcriptional activation of genes (tyrosinase, TYRP1 and TYRP2) required for pigment synthesis (Bentley et al, 1994;Hemesath et al, 1994;Yavuzer et al, 1995;Yasumoto et al, 1997) and also has a role in melanocyte survival and differentiation (Lerner et al, 1986;Opdecamp et al, 1997;Hemesath et al, 1998). *Correspondence: Dr CR Goding; E-mail: c.goding@mcri.ac.uk With respect to CCS, it is significant that transcription of the Mitf-M isoform is controlled by a melanocyte-specific promoter (Fuse et al, 1996) that includes a cAMP-response element (CRE) comprising a single ATF1/CREB binding site (Bertolotto et al, 1998). Thus, the Mitf-M promoter is a potential target for EWS/ ATF1.…”

Tyrp1 and Oculocutaneous Albinism Type 3

“…The differences in catalytic function between murine Tyrp1 and human TYPR1 is however not surprising due to differences in a) the placement of cis-regulatory regions between murine Tyrp1 and human TYRP1 (12, 21, 75), b) amino acid composition [reviewed in Boissy et al, 1998 (66)] and c) transcriptional activities, e.g. Mitf (76)(77)(78), i.e. the amino acid residues between mouse and humans are not conserved in these regions.…”