Expression of microphthalmia-associated transcription factor (MITF), which is critical for melanoma progression, is inhibited by both transcription factor GLI2 and transforming growth factor-β.
ABSTRACT The melanocyte-specific transcription factor M-MITF is involved in numerous aspects of melanoblast lineage biology including pigmentation, survival, and migration. It plays complex roles at all stages of melanoma progression and metastasis. We established previously that GLI2, a Kruppel-like transcription factor that acts downstream of Hedgehog signaling, is a direct transcriptional target of the TGF-β/SMAD pathway and contributes to melanoma progression, exerting antagonistic activities against M-MITF to control melanoma cell invasiveness. Herein, we dissected the molecular mechanisms underlying both TGF-β and GLI2-driven M-MITF gene repression. Using transient cell transfection experiments with M-MITF promoter constructs, chromatin immunoprecipitation, site-directed mutagenesis, and electrophoretic mobility shift assays, we identified a GLI2 binding site within the -334/-296 region of the M-MITF promoter, critical for GLI2-driven transcriptional repression. This region is, however, not needed for inhibition of M-MITF promoter activity by TGF-β. We determined that TGF-β rapidly repressed protein kinase A activity, thus reducing both phospho-cAMP-response element-binding protein (CREB) levels and CREB-dependent transcription of the M-MITF promoter. Increased GLI2 binding to its cognate cis-element, associated with reduced CREB-dependent transcription, allowed maximal inhibition of the M-MITF promoter via two distinct mechanisms.
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ABSTRACT: The diversity of functional phenotypes observed within a tumor does not exclusively result from intratumoral genetic heterogeneity but also from the response of cancer cells to the microenvironment. We have previously demonstrated that the morphological and functional phenotypes of melanoma can be dynamically altered upon external stimuli. In the present study, transcriptome profiles were generated to explore the molecules governing phenotypes of melanospheres grown in the bFGF(+)EGF(+) serum-free cultures and monolayers maintained in the serum-containing medium. Higher expression levels of MITF-dependent genes that are responsible for differentiation, e.g., TYR and MLANA, and stemness-related genes, e.g., ALDH1A1, were detected in melanospheres. These results were supported by the observation that the melanospheres contained more pigmented cells and cells exerting the self-renewal capacity than the monolayers. In addition, the expression of the anti-apoptotic, MITF-dependent genes e.g., BCL2A1 was also higher in the melanospheres. The enhanced activity of MITF in melanospheres, as illustrated by the increased expression of 74 MITF-dependent genes, identified MITF as a central transcriptional regulator in melanospheres. Importantly, several genes including MITF-dependent ones were expressed in melanospheres and original tumors at similar levels. The reduced MITF level in monolayers might be partially explained by suppression of the Wnt/β-catenin pathway, and DKK1, a secreted inhibitor of this pathway, was highly up-regulated in monolayers in comparison to melanospheres and original tumors. Furthermore, the silencing of DKK1 in monolayers increased the percentage of cells with self-renewing capacity. Our study indicates that melanospheres can be used to unravel the molecular pathways that sustain intratumoral phenotypic heterogeneity. Melanospheres directly derived from tumor specimens more accurately mirrored the morphology and gene expression profiles of the original tumors compared to monolayers. Therefore, melanospheres represent a relevant preclinical tool to study new anticancer treatment strategies.PLoS ONE 04/2014; 9(4):e95157. · 3.53 Impact Factor
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ABSTRACT: Microphthalmia-associated transcription factor (MITF) is a transcription factor that is expressed in limited types of cells, including osteoclasts, but the expression and role of MITF during osteoclastogenesis have not been fully elucidated. The expression of the MITF-E isoform but not that of the MITF-A isoform was induced in response to differentiation stimulation towards osteoclasts by receptor activator of NF-κB ligand (RANKL) in both RAW264.7 cells and primary bone marrow cells. The RANKL-induced formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells was inhibited in RAW264.7 cells expressing siRNA for MITF-E. Transforming growth factor-β (TGF-β) enhanced RANKL-induced MITF-E expression and -TRAP positive multinucleated cell formation. In particular, TGF-β potentiated the formation of larger osteoclasts. The expression levels of NFATc1, TRAP and CtsK, genes related to osteoclast development and activity, were concurrently enhanced by TGF-β in the presence of RANKL. Furthermore, the expression of dendritic cell-specific transmembrane protein (DC-STAMP), Itgav, Itga2, Itga5, Itgb1, Itgb3 and Itgb5, genes related to cell adhesion and fusion, were up-regulated by co-treatment with TGF-β. In particular, the regulatory expression of Itgav and Itgb5 in response to RANKL with or without TGF-β resembled that of MITF-E. Because MITF is involved in cell fusion in some cell systems, these results imply a role for MITF-E as an enhancer of osteoclastogenesis and that RANKL-induced levels of both MITF-E mRNA and of MITF-dependent gene expression are enhanced by treatment with TGF-β. Copyright © 2014 John Wiley & Sons, Ltd.Cell Biochemistry and Function 02/2014; · 2.13 Impact Factor
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ABSTRACT: NADPH oxidase 4 (NOX4) is a member of the NADPH oxidase gene family that regulates cellular differentiation, innate immunity and tissue fibrosis. Transforming growth factor-β (TGF-β1) is known to induce expression of NOX4 mRNA in mesenchymal cells. However, the mechanisms of transcriptional regulation of NOX4 are not well understood. In this study, we examined the transcriptional regulation of NOX4 in human lung fibroblasts by TGF-β1. Five promoter-reporter constructs containing DNA fragments of 0.74kb, 1.35kb, 1.84kb, 3.97kb and 4.76kb upstream from the transcriptional start site (TSS) of the human NOX4 gene were generated and their relative responsiveness to TGF-β1 analyzed. TGF-β1-induced NOX4 gene promoter activation requires a region between -3.97kb and -4.76kb. Bioinformatics analysis revealed a 15bp AP-1/Smad binding element in this region. Mutation or deletion of either the AP-1 or the Smad element attenuated TGF-β1 responsiveness of the -4.76kb NOX4 promoter. Furthermore, insertion of this AP-1/Smad box conferred TGF-β1 inducibility to the non-responsive -3.97kb NOX4 promoter construct. Chromatin immunoprecipitation analysis indicated that phospho-Smad3 and cJun associate with this element in a TGF-β1-inducible manner. These results demonstrate that the AP-1/Smad box located between 3.97kb and 4.76kb upstream of the TSS site of the NOX4 promoter is essential for NOX4 gene transcription induced by TGF-β1 in human lung fibroblasts. Our study provides insights into the molecular mechanisms of NOX4 gene expression, informing novel therapeutic approaches to interfere with upregulation of NOX4 in diseases characterized by activation of the TGF-β1/NOX4 pathway.Gene 02/2014; · 2.20 Impact Factor