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Hypoxia-inducible factor 1{alpha} is a new target of microphthalmia-associated transcription factor (MITF) in melanoma cells.

Buscà R, Berra E, Gaggioli C, Khaled M, Bille K, Marchetti B, Thyss R, Fitsialos G, Larribère L, Bertolotto C, Virolle T, Barbry P, Pouysségur J, Ponzio G, Ballotti R - J. Cell Biol. (2005)

Bottom Line: Interestingly, we report that the melanocyte-specific transcription factor, microphthalmia-associated transcription factor (MITF), binds to the Hif1a promoter and strongly stimulates its transcriptional activity.Importantly, we provide results demonstrating that HIF1 plays a pro-survival role in this cell system.We therefore conclude that the alpha-MSH/cAMP pathway, using MITF as a signal transducer and HIF1alpha as a target, might contribute to melanoma progression.

View Article: PubMed Central - PubMed

Affiliation: INSERM U597, Biologie et physiopathologie des cellules mélanocytaires, Faculty of Medicine, 06107 Nice cedex 2, France. busca@unice.fr

ABSTRACT
In melanocytes and melanoma cells alpha-melanocyte stimulating hormone (alpha-MSH), via the cAMP pathway, elicits a large array of biological responses that control melanocyte differentiation and influence melanoma development or susceptibility. In this work, we show that cAMP transcriptionally activates Hif1a gene in a melanocyte cell-specific manner and increases the expression of a functional hypoxia-inducible factor 1alpha (HIF1alpha) protein resulting in a stimulation of Vegf expression. Interestingly, we report that the melanocyte-specific transcription factor, microphthalmia-associated transcription factor (MITF), binds to the Hif1a promoter and strongly stimulates its transcriptional activity. Further, MITF "silencing" abrogates the cAMP effect on Hif1a expression, and overexpression of MITF in human melanoma cells is sufficient to stimulate HIF1A mRNA. Our data demonstrate that Hif1a is a new MITF target gene and that MITF mediates the cAMP stimulation of Hif1a in melanocytes and melanoma cells. Importantly, we provide results demonstrating that HIF1 plays a pro-survival role in this cell system. We therefore conclude that the alpha-MSH/cAMP pathway, using MITF as a signal transducer and HIF1alpha as a target, might contribute to melanoma progression.

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MITF is required for cAMP-dependent Hif1a expression. (A) Hif1a promoter activity assays on B16 cells cotransfected with either an empty vector (pCDNA3) or a construct encoding a dominant-negative mutant of MITF (MITF-DNT) and treated (or not) (NS) with the cAMP-elevating agent forskolin (FK). Results are expressed as the fold stimulation of the luciferase activity compared with the pcDNA3-transfected nonstimulated condition. (B) The same experiment was performed by cotransfecting the Hif1a promoter with either an siRNA-targeting MITF (siRNA-MITF) or a nonrelevant siRNA (siRNA-control).
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fig4: MITF is required for cAMP-dependent Hif1a expression. (A) Hif1a promoter activity assays on B16 cells cotransfected with either an empty vector (pCDNA3) or a construct encoding a dominant-negative mutant of MITF (MITF-DNT) and treated (or not) (NS) with the cAMP-elevating agent forskolin (FK). Results are expressed as the fold stimulation of the luciferase activity compared with the pcDNA3-transfected nonstimulated condition. (B) The same experiment was performed by cotransfecting the Hif1a promoter with either an siRNA-targeting MITF (siRNA-MITF) or a nonrelevant siRNA (siRNA-control).

Mentions: To evaluate whether MITF was required for the cAMP induction of the Hif1a gene expression, B16 cells were cotransfected with the Hif1a promoter fragment together with a dominant-negative mutant of MITF (MITF DNT) lacking the NH2-terminal transactivation domain, and cells were then stimulated with forskolin. Interestingly, we observed that MITF DNT completely abolished the cAMP stimulation of the Hif1a promoter activity (Fig. 4 A). To further strengthen the MITF specificity of such response, we performed the same experiment by using specific small interference RNAs (siRNAs) targeting MITF. As a control, cells were cotransfected with the Hif1a promoter together with a nonrelevant siRNA (control siRNA). Fig. 4 B shows that MITF silencing clearly inhibited the cAMP-induced Hif1a promoter transactivation, thereby indicating that MITF mediates the cAMP effects on Hif1a gene expression.


Hypoxia-inducible factor 1{alpha} is a new target of microphthalmia-associated transcription factor (MITF) in melanoma cells.

Buscà R, Berra E, Gaggioli C, Khaled M, Bille K, Marchetti B, Thyss R, Fitsialos G, Larribère L, Bertolotto C, Virolle T, Barbry P, Pouysségur J, Ponzio G, Ballotti R - J. Cell Biol. (2005)

MITF is required for cAMP-dependent Hif1a expression. (A) Hif1a promoter activity assays on B16 cells cotransfected with either an empty vector (pCDNA3) or a construct encoding a dominant-negative mutant of MITF (MITF-DNT) and treated (or not) (NS) with the cAMP-elevating agent forskolin (FK). Results are expressed as the fold stimulation of the luciferase activity compared with the pcDNA3-transfected nonstimulated condition. (B) The same experiment was performed by cotransfecting the Hif1a promoter with either an siRNA-targeting MITF (siRNA-MITF) or a nonrelevant siRNA (siRNA-control).
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Related In: Results  -  Collection

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fig4: MITF is required for cAMP-dependent Hif1a expression. (A) Hif1a promoter activity assays on B16 cells cotransfected with either an empty vector (pCDNA3) or a construct encoding a dominant-negative mutant of MITF (MITF-DNT) and treated (or not) (NS) with the cAMP-elevating agent forskolin (FK). Results are expressed as the fold stimulation of the luciferase activity compared with the pcDNA3-transfected nonstimulated condition. (B) The same experiment was performed by cotransfecting the Hif1a promoter with either an siRNA-targeting MITF (siRNA-MITF) or a nonrelevant siRNA (siRNA-control).
Mentions: To evaluate whether MITF was required for the cAMP induction of the Hif1a gene expression, B16 cells were cotransfected with the Hif1a promoter fragment together with a dominant-negative mutant of MITF (MITF DNT) lacking the NH2-terminal transactivation domain, and cells were then stimulated with forskolin. Interestingly, we observed that MITF DNT completely abolished the cAMP stimulation of the Hif1a promoter activity (Fig. 4 A). To further strengthen the MITF specificity of such response, we performed the same experiment by using specific small interference RNAs (siRNAs) targeting MITF. As a control, cells were cotransfected with the Hif1a promoter together with a nonrelevant siRNA (control siRNA). Fig. 4 B shows that MITF silencing clearly inhibited the cAMP-induced Hif1a promoter transactivation, thereby indicating that MITF mediates the cAMP effects on Hif1a gene expression.

Bottom Line: Interestingly, we report that the melanocyte-specific transcription factor, microphthalmia-associated transcription factor (MITF), binds to the Hif1a promoter and strongly stimulates its transcriptional activity.Importantly, we provide results demonstrating that HIF1 plays a pro-survival role in this cell system.We therefore conclude that the alpha-MSH/cAMP pathway, using MITF as a signal transducer and HIF1alpha as a target, might contribute to melanoma progression.

View Article: PubMed Central - PubMed

Affiliation: INSERM U597, Biologie et physiopathologie des cellules mélanocytaires, Faculty of Medicine, 06107 Nice cedex 2, France. busca@unice.fr

ABSTRACT
In melanocytes and melanoma cells alpha-melanocyte stimulating hormone (alpha-MSH), via the cAMP pathway, elicits a large array of biological responses that control melanocyte differentiation and influence melanoma development or susceptibility. In this work, we show that cAMP transcriptionally activates Hif1a gene in a melanocyte cell-specific manner and increases the expression of a functional hypoxia-inducible factor 1alpha (HIF1alpha) protein resulting in a stimulation of Vegf expression. Interestingly, we report that the melanocyte-specific transcription factor, microphthalmia-associated transcription factor (MITF), binds to the Hif1a promoter and strongly stimulates its transcriptional activity. Further, MITF "silencing" abrogates the cAMP effect on Hif1a expression, and overexpression of MITF in human melanoma cells is sufficient to stimulate HIF1A mRNA. Our data demonstrate that Hif1a is a new MITF target gene and that MITF mediates the cAMP stimulation of Hif1a in melanocytes and melanoma cells. Importantly, we provide results demonstrating that HIF1 plays a pro-survival role in this cell system. We therefore conclude that the alpha-MSH/cAMP pathway, using MITF as a signal transducer and HIF1alpha as a target, might contribute to melanoma progression.

Show MeSH
Related in: MedlinePlus