MITF Protein Research Articles

WS5, a direct target of oncogenic transcription factor Myc, is related to human melanoma glycoprotein genes and has oncogenic potential

We have isolated a gene (WS5) that is specifically expressed at the mRNA and protein level in avian fibroblasts transformed by the v-myc oncogene of avian acute leukemia virus MC29. In a conditional cell transformation system, WS5 gene expression was tightly correlated with v-myc activation. The WS5 gene contains 11 exons, encoding a 733-amino acid protein with a transmembrane region and a polycystic kidney disease (PKD) domain. Near the transcriptional start site, the WS5 promoter contains a cluster of four binding sites for the Myc-Max complex and a binding site for transcription factor C/EBPalpha. Electrophoretic mobility shift assays and chromatin immunoprecipitation showed that Myc, Max and C/EBPalpha bind specifically to these sites. Functional promoter analyses revealed that both the Myc-binding site cluster and the C/EBPalpha-binding site are essential for strong transcriptional activation, and that Myc and C/EBPalpha synergistically activate the WS5 promoter. Ectopic expression of WS5 led to cell transformation documented by anchorage-independent growth. The human melanoma antigen Pmel17, a type I transmembrane glycoprotein, is the mammalian protein with the highest amino acid sequence identity (38%) to WS5. The Pmel17 gene is regulated by the MITF protein, a bHLHZip transcription factor with DNA binding specificities similar to those of Myc/Max. WS5 is also related to human glycoprotein GPNMB expressed in metastatic melanoma cells and implicated in the progression of brain and liver tumors.

963. MITF as a Potential Molecular Target for Gene Therapy for Human Melanoma; a Synergistic Inhibitory Effect of Inactivation of Both MITF and BRAFV600E on Melanoma Growth

Top of pageAbstract MITF (microphthalmia-associated transcription factor) is a family of transcriptional factors controlling melanocyte differentiation. However, the roles of MITF on the cell proliferation have been controversial. Previous works have suggested that MITF negatively regulates cell proliferation or apoptosis in melanocytes, which is mediated by induction of p16INK4a, p21 or Bcl-2. But a recent study showed gene amplification of MITF in melanoma tissues (10-21%) associated with increase of the MITF protein. Furthermore, MITF gene amplification was a prognostic factor for survival, and several in vitro studies suggested that the MITF protein is attributed to increased cell proliferation, suggesting that MITF is an oncogene. Presently, the role of MITF in melanoma and melanomagenesis is not clear. We have attempted to investigate the role of MITF in malignant phenotypes of melanoma cells by using HIV-mediated RNA interference (RNAi) in a panel of melanoma cell lines with different status of the MITF expression to clarify the significance of MITF as a target for gene therapy, and to find any factors affecting sensitivity for the MITF inactivation. Furthermore, we have attempted to evaluate any additional effects by simultaneous suppression of both MITF and BRAFV600E on the melanoma growth, since BRAFV600E, the most frequent form of human BRAF mutation, is attributed to the enhanced MAPK signaling and several malignant phenotypes of melanoma. We have constructed two short hairpin RNA (shRNA) HIV vectors for MITF, and one control shRNA HIV vector (GL3B; for firefly luciferase). An HIV vector expressing two shRNAs for MITF and BRAFV600E was also constructed by tandemly aligning two shRNA expression cassettes in one HIV vector. Seven melanoma cell lines were infected with these shRNA HIV vectors, and the effects of MITF RNAi on the cell growth and apoptosis were evaluated. In six of 7 melanoma cell lines with substantial expression of the MITF protein, the MITF RNAi induced significant inhibition of cell growth, associated with G1 arrest. One melanoma cell line, A375, without any detectable expression of MITF was resistant to the MITF RNAi. One melanoma cell line showed significant apoptotic changes represented by the increase of subG1 population (7.23-7.60%) and increase of cleaved caspase-3 and caspase-9, following the MITF RNAi. Interestingly, the MITF RNAi induced G1 arrest in most melanoma cell lines tested, however, no clear correlation was observed between the protein levels of cdk inhibitors and G1 arrest. Finally, three melanoma cell lines with the simultaneous inhibition of both MITF and BRAFV600E, showed synergistic inhibition of cell growth, which was associated with more G1 arrest. Our results suggest that MITF could be a molecular target for gene therapy for most human melanoma, and the synergistic therapeutic effects of MITF and BRAFV600E indicate the possibly more potent gene therapy for melanoma by simultaneous down-regulation of these molecules.

Editorial: It's back –http://www.pigment.org

Editorial: It's back –http://www.pigment.org

Inhibition of melanoma inhibitory activity (MIA) expression in melanoma cells leads to molecular and phenotypic changes

The secreted protein melanoma inhibitory activity (MIA) is highly expressed in malignant melanoma but not in melanocytes and is associated with tumor progression in vivo. Here, we further investigated the functional role of MIA by inhibiting MIA expression of the human melanoma cell line HMB2 via stable antisense MIA cDNA transfection, and subsequent analysis of the cell clones. MIA-deficient cell clones showed several changes in cell morphology and growth pattern. In monolayer and three-dimensional culture enhanced cell-cell contacts were formed. Furthermore, a re-induction of pigment synthesis in comparison with the amelanotic parental cell line HMB2 was observed. Molecular analyses revealed a re-expression of tyrosinase-related protein 1 (Trp-1) and tyrosinase in the MIA-deficient cell clones necessary for melanin synthesis. In accordance, re-expression of MIA in the MIA-deficient melanoma cell clones resulted in downregulation of Trp-1. To identify the molecular mechanisms of MIA regulating pigmentation, MITF and PAX3, two positive regulators of Trp-1 and tyrosinase transcription, and PIAS3, a negative regulator of MITF activity, were analyzed. Only in MIA-deficient cells, expression of PAX3 mRNA and MITF protein was found. In contrast, strong expression of PIAS3 was detected in HMB2 but not in the MIA-deficient cells. To our knowledge this is the first report demonstrating a correlation between MIA expression and pigmentation and morphology of melanocytic cells.

Modulation of catalase activity and MITF protein in response to oxidative stress in Melanocytes: implications for Vitiligo.

Modulation of catalase activity and MITF protein in response to oxidative stress in Melanocytes: implications for Vitiligo.

The Basic Helix-Loop-Helix Leucine Zipper Transcription Factor Mitf Is Conserved in Drosophila and Functions in Eye Development

The MITF protein is a member of the MYC family of basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factors and is most closely related to the TFE3, TFEC, and TFEB proteins. In the mouse, MITF is required for the development of several different cell types, including the retinal pigment epithelial (RPE) cells of the eye. In Mitf mutant mice, the presumptive RPE cells hyperproliferate, abnormally express the retinal transcriptional regulator Pax6, and form an ectopic neural retina. Here we report the structure of the Mitf gene in Drosophila and demonstrate expression during embryonic development and in the eye-antennal imaginal disc. In vitro, transcriptional regulation by Drosophila Mitf, like its mouse counterpart, is modified by the Eyeless (Drosophila Pax6) transcription factor. In vivo, targeted expression of wild-type or dominant-negative Drosophila Mitf results in developmental abnormalities reminiscent of Mitf function in mouse eye development. Our results suggest that the Mitf gene is the original member of the Mitf-Tfe subfamily of bHLH-Zip proteins and that its developmental function is at least partially conserved between vertebrates and invertebrates. These findings further support the common origin of the vertebrate and invertebrate eyes.

The novel mouse microphthalmia mutations Mitfmi-enu5 and Mitfmi-bcc2 produce dominant negative Mitf proteins

Mutations in the microphthalmia-associated transcription factor ( Mitf) gene affect the development of different cell types, including melanocytes, osteoclasts, and retinal pigmented epithelial cells of the eye. Many different mutations at the locus are known and since they affect the phenotype to different extents they form an allelic series. The Mitf protein is a member of the Mitf-Tfe subfamily of basic helix-loop-helix-leucine zipper (bHLH-Zip) transcription factors and binds the 6-bp canonical CAC/TGTG sequence (E box) as either a homodimer or a heterodimer with related proteins. The many Mitf mutations have provided important insights into the in vivo behavior of a bHLH-Zip protein. Here we describe the phenotype of two new semidominant Mitf mutations recovered in recent mutagenic screens, Mitf mi-enu5 and Mitf mi-bcc2 ; determine the molecular lesions involved; and show that the mutant proteins act in a dominant negative fashion in vitro. The novel mutations are phenotypically distinct from previously known Mitf mutations.

Microphthalmia transcription factor analysis in posterior uveal melanomas

Purpose. The protein encoded by the Microphthalmia gene (MITF) is a transcription factor essential for the development and survival of melanocytes. It serves as a master regulator in modulating extracellular signals. Because of its central role in melanocytes survival and to assess its potential use as a histopathological marker for melanoma, MITF expression was examined in human choroidal melanomas. Methods. Fifty-seven paraffin-embedded sections of choroidal melanoma specimens and 1 choroidal melanoma cell line were analyzed using immunochemistry and RT-PCR. Normal choroids and normal choroidal melanocyte cells were used as control. Results. Sixty-five percent of the tumoral specimens stained positively for MITF with a predominant nuclear pattern of reactivity. MITF-M and MITF-A isoforms were detected by RT-PCR in all specimens examined. Using a chimeric protein resulting from the fusion of each Mitf protein with the GFP, Mitf-M exhibited an exclusive nuclear staining whereas Mitf-A exhibited a mixed nuclear and cytoplasmic staining. No correlation between MITF-positivity and parameters such as cell type, largest tumor diameter, sclera invasion, mitotic figures was observed. In contrast, a significant negative association was found between MITF staining and the pigmentation (p=0.02) and a positive correlation between MITF staining and the proliferative marker Ki67 was found ( p=0.02). Conclusion. MITF may be implicated in choroidal melanoma pigmentation and proliferation. Further analysis should provide new insights into the mechanisms underlying the molecular and cellular changes of choroidal melanomas.

Interallelic complementation at the mouse Mitf locus.

Mutations at the mouse microphthalmia locus (Mitf) affect the development of different cell types, including melanocytes, retinal pigment epithelial cells of the eye, and osteoclasts. The MITF protein is a member of the MYC supergene family of basic-helix-loop-helix-leucine-zipper (bHLHZip) transcription factors and is known to regulate the expression of cell-specific target genes by binding DNA as homodimer or as heterodimer with related proteins. The many mutations isolated at the locus have different effects on the phenotype and can be arranged in an allelic series in which the phenotypes range from near normal to white microphthalmic animals with osteopetrosis. Previous investigations have shown that certain combinations of Mitf alleles complement each other, resulting in a phenotype more normal than that of each homozygote alone. Here we analyze this interallelic complementation in detail and show that it is limited to one particular allele, Mitf(Mi-white) (Mitf(Mi-wh)), a mutation affecting the DNA-binding domain. Both loss- and gain-of-function mutations are complemented, as are other Mitf mutations affecting the DNA-binding domain. Furthermore, this behavior is not restricted to particular cell types: Both eye development and coat color phenotypes are complemented. Our analysis suggests that Mitf(Mi-wh)-associated interallelic complementation is due to the unique biochemical nature of this mutation.

Direct Regulation of the Microphthalmia Promoter by Sox10 Links Waardenburg-Shah Syndrome (WS4)-associated Hypopigmentation and Deafness to WS2

The transcription factor Sox10 is genetically linked with Waardenburg syndrome 4 (WS4) in humans and the Dominant megacolon (Dom) mouse model for this disease. The pigmentary defects observed in the Dom mouse and WS4 are reminiscent of those associated with mutations in the microphthalmia (Mitf) gene, which encodes a transcription factor essential for the development of the melanocyte lineage. We demonstrate here that wild type Sox10 directly binds and activates transcription of the MITF promoter, whereas a mutant form of the Sox10 protein genetically linked with WS4 acts as a dominant-negative repressor of MITF expression and can reduce endogenous MITF protein levels. The ability of Sox10 to activate transcription of the MITF promoter implicates Sox10 in the regulation of melanocyte development and provides a molecular basis for the hypopigmentation and deafness associated with WS4.

Mitf Is Expressed in Osteoclast Progenitors in Vitro

Microphthalmia mutant (mi/mi) mice reveal defects in osteoclastogenesis and exhibit osteopetrosis. However, there have been no studies to test the importance of Mitf in in vitro osteoclastogenesis using the cells derived from mi/mi mice. Therefore, we investigated in vitro osteoclastogenesis using the cells derived from mi/mi mice. We cocultured spleen cells prepared from either wild-type or mi/mi mice with ST2 or TM8 stromal cells and found that formation of TRAP-positive cells was significantly reduced in the cocultures of mi/mi spleen cells compared to wild-type spleen cells in the presence of 1,25(OH)2 vitamin D3 (vitamin D). We further investigated Mitf expression by Northern blot analysis in relation to the differentiation of osteoclasts using the cocultures of bone marrow cells with stromal/osteoblastic cells and found positive correlation in expression levels of c-fms and Mitf. Moreover, osteoclast-progenitor-like C7 cells expressed c-fms as well as Mitf mRNAs when cultured alone. C7 cells also expressed Mitf protein in their nuclei. Similar results were obtained when we used primary spleen cells, which differentiate into osteoclasts cultured in the presence of M-CSF and RANKL/ODF. Mitf expression levels in the cocultures of C7 cells and ST2 cells were not changed by treatment with vitamin D in the presence or absence of dexthamethasone. These results suggest that Mitf is expressed in osteoclast progenitors and its presence facilitates osteoclastogenesis.

The Gene Encoding the T-box Factor Tbx2 Is a Target for the Microphthalmia-associated Transcription Factor in Melanocytes

Commitment to the melanocyte lineage is characterized by the onset of microphthalmia-associated transcription factor (Mitf) expression. Mitf plays a fundamental role in melanocyte development, with mice lacking Mitf being entirely devoid of pigment cells. In the absence of functional Mitf protein, melanoblasts expressing Mitf mRNA disappear around 2 days after their first appearance either by apoptosis or by losing their identity and adopting an alternative cell fate. The role of Mitf must therefore be to regulate genes required for melanoblast survival, proliferation, or the maintenance of melanoblast identity. Yet to date, Mitf has been shown to regulate genes such as Tyrosinase, Tyrp-1, and Dct, which are required for pigmentation, a differentiation-specific process. Because expression of these genes cannot account for the complete absence of pigment cells in Mitf-negative mice, Mitf must regulate the expression of other as yet uncharacterized genes. Here we provide several lines of evidence to suggest that Mitf may regulate the expression of the Tbx2 transcription factor, a member of the T-box family of proteins implicated in the maintenance of cell identity. First, isolation and sequencing of the entire murine Tbx2 gene revealed that the Tbx2 promoter contains a full consensus Mitf recognition element; second, Mitf could bind the promoter in vitro and activate Tbx2 expression in vivo in an E box-dependent fashion; and third, Tbx2 is expressed in melanoma cell lines expressing Mitf, but not in a line in which Mitf expression was not detectable. Taken together, with the fact that Tbx2 is expressed in Mitf-positive melanoblasts and melanocytes, but not in Mitf-negative melanoblast precursor cells, the evidence suggests that the Tbx2 gene may represent one of the first known targets for Mitf that is not a gene involved directly in the manufacture of pigment.

Microphthalmia Transcription Factor: A Sensitive and Specific Melanocyte Marker for Melanoma Diagnosis

Malignant melanomas do not uniformly retain expression of melanocytic gene products-an observation associated with diagnostic dilemmas. Microphthalmia transcription factor (Mitf) is a melanocytic nuclear protein critical for the embryonic development and postnatal viability of melanocytes. It serves as a master regulator in modulating extracellular signals, such as those triggered by alpha-MSH and c-Kit ligand. Because of its central role in melanocyte survival and to assess its potential use as a histopathological marker for melanoma, Mitf expression was examined in histologically confirmed human melanoma specimens. Western blot analysis of melanoma cell lines revealed consistent expression of two Mitf protein isoforms differing by MAP kinase-mediated phosphorylation. In a series of 76 consecutive human melanoma surgical specimens, 100% stained positively for Mitf with a nuclear pattern of reactivity. In a side-by-side comparison, Mitf staining was positive in melanomas that failed to stain for either HMB-45 or S-100, the most common currently used melanoma markers. Of 60 non-melanoma tumors, none displayed nuclear Mitf staining and two displayed cytoplasmic staining. Although Mitf does not distinguish benign from malignant melanocytic lesions, for invasive neoplasms it appears to be a highly sensitive and specific histopathological melanocyte marker for melanoma.

Melanocyte development in vivo and in neural crest cell cultures: crucial dependence on the Mitf basic-helix-loop-helix-zipper transcription factor.

The more than 20 different Mitf mutations in the mouse are all associated with deficiencies in neural crest-derived melanocytes that range from minor functional disturbances with some alleles to complete absence of mature melanocytes with others. In the trunk region of wild-type embryos, Mitf-expressing cells that coexpressed the melanoblast marker Dct and the tyrosine kinase receptor Kit were found in the dorsolateral neural crest migration pathway. In contrast, in embryos homozygous for an Mitf allele encoding a non-functional Mitf protein, Mitf-expressing cells were extremely rare, no Dct expression was ever found, and the number of Kit-expressing cells was much reduced. Wild-type neural crest cell cultures rapidly gave rise to cells that expressed Mitf and coexpressed Kit and Dct. With time in culture, Kit expression was increased, and pigmented, dendritic cells developed. Addition of the Kit ligand Mgf or endothelin 3 or a combination of these factors all rapidly increased the number of Dct-positive cells. Cultures from Mitf mutant embryos initially displayed Mitf-positive cells similar in numbers and Kit-expression as did wild-type cultures. However, Kit expression did not increase with time in culture and the mutant cells never responded to Mgf or endothelin 3, did not express Dct, and never showed pigment. In fact, even Mitf expression was rapidly lost. The results suggest that Mitf first plays a role in promoting the transition of precursor cells to melanoblasts and subsequently, by influencing Kit expression, melanoblast survival.