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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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Related in: MedlinePlus

HIF1α plays a pro-survival role in B16 melanoma cells. (A) Immunofluorescence study to detect the presence of cleaved caspase-3 in B16 cells nonstimulated (NS) or stimulated for 24 with forskolin (FK), and nontreated (−St) or treated with staurosporin (+St) for 6 h. Bar, 40 μM. (B) Immunofluorescence analysis of B16 cells transfected with a nonrelevant siRNA (siRNA-Ctrol) or an siRNA to invalidate HIF1α. Cleaved caspase-3 was labeled with a secondary Texas red–conjugated antibody (red) and HIF1α protein was detected by an mAb and a secondary FITC-conjugated secondary one (green). Nuclei were labeled with bisbenzidine (blue). Bar, 20 μM.
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fig6: HIF1α plays a pro-survival role in B16 melanoma cells. (A) Immunofluorescence study to detect the presence of cleaved caspase-3 in B16 cells nonstimulated (NS) or stimulated for 24 with forskolin (FK), and nontreated (−St) or treated with staurosporin (+St) for 6 h. Bar, 40 μM. (B) Immunofluorescence analysis of B16 cells transfected with a nonrelevant siRNA (siRNA-Ctrol) or an siRNA to invalidate HIF1α. Cleaved caspase-3 was labeled with a secondary Texas red–conjugated antibody (red) and HIF1α protein was detected by an mAb and a secondary FITC-conjugated secondary one (green). Nuclei were labeled with bisbenzidine (blue). Bar, 20 μM.

Mentions: For that we treated B16 melanoma cells with staurosporin, considered a strong cell death inducing agent, and we used an immunofluorescence analysis to detect the presence of cleaved caspase-3 as a marker of cell death. We observed that treatment with staurosporin for 6 h greatly induced caspase-3 cleavage in our cell system (Fig. 6 A). We next pretreated B16 cells for 24 h with forskolin to increase HIF1α. We interestingly observed that cAMP strongly reduced the cleaved caspase-3 labeling thereby indicating that cAMP inhibits the staurosporin-induced cell death (Fig. 6 A).


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)

HIF1α plays a pro-survival role in B16 melanoma cells. (A) Immunofluorescence study to detect the presence of cleaved caspase-3 in B16 cells nonstimulated (NS) or stimulated for 24 with forskolin (FK), and nontreated (−St) or treated with staurosporin (+St) for 6 h. Bar, 40 μM. (B) Immunofluorescence analysis of B16 cells transfected with a nonrelevant siRNA (siRNA-Ctrol) or an siRNA to invalidate HIF1α. Cleaved caspase-3 was labeled with a secondary Texas red–conjugated antibody (red) and HIF1α protein was detected by an mAb and a secondary FITC-conjugated secondary one (green). Nuclei were labeled with bisbenzidine (blue). Bar, 20 μM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171372&req=5

fig6: HIF1α plays a pro-survival role in B16 melanoma cells. (A) Immunofluorescence study to detect the presence of cleaved caspase-3 in B16 cells nonstimulated (NS) or stimulated for 24 with forskolin (FK), and nontreated (−St) or treated with staurosporin (+St) for 6 h. Bar, 40 μM. (B) Immunofluorescence analysis of B16 cells transfected with a nonrelevant siRNA (siRNA-Ctrol) or an siRNA to invalidate HIF1α. Cleaved caspase-3 was labeled with a secondary Texas red–conjugated antibody (red) and HIF1α protein was detected by an mAb and a secondary FITC-conjugated secondary one (green). Nuclei were labeled with bisbenzidine (blue). Bar, 20 μM.
Mentions: For that we treated B16 melanoma cells with staurosporin, considered a strong cell death inducing agent, and we used an immunofluorescence analysis to detect the presence of cleaved caspase-3 as a marker of cell death. We observed that treatment with staurosporin for 6 h greatly induced caspase-3 cleavage in our cell system (Fig. 6 A). We next pretreated B16 cells for 24 h with forskolin to increase HIF1α. We interestingly observed that cAMP strongly reduced the cleaved caspase-3 labeling thereby indicating that cAMP inhibits the staurosporin-induced cell death (Fig. 6 A).

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