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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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cAMP induces HIF1α expression in a cell-specific manner. (A) B16 cells and NIH-3T3 cells were transfected with a fragment of the Hif1a gene promoter cloned upstream of the luciferase reporter gene, and were then stimulated (or not) (NS) with forskolin (FK). Luciferase activity was normalized by the β-galactosidase activity and results are represented as the fold stimulation of the cAMP-activated promoter compared with the basal value. (B) Real-time quantitative PCR to detect Hif1a mRNA levels was performed on total RNAs from NIH-3T3 fibroblasts nonstimulated (NS) or treated for 24 h with forskolin (FK). (C) NIH-3T3 extracts were subjected to Western blot analysis to detect HIF1α protein levels after cell stimulation with forskolin (FK) for 24 h, or cobalt (Co2+) for 12 h. ERK2 levels show a control of the gel protein loading. White lines indicate that intervening lanes have been spliced out. (D) 3-HRE-LUC reporter assay on B16 and NIH-3T3 cells nonstimulated (NS) of stimulated with forskolin (FK) or cobalt (Co2+).
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fig2: cAMP induces HIF1α expression in a cell-specific manner. (A) B16 cells and NIH-3T3 cells were transfected with a fragment of the Hif1a gene promoter cloned upstream of the luciferase reporter gene, and were then stimulated (or not) (NS) with forskolin (FK). Luciferase activity was normalized by the β-galactosidase activity and results are represented as the fold stimulation of the cAMP-activated promoter compared with the basal value. (B) Real-time quantitative PCR to detect Hif1a mRNA levels was performed on total RNAs from NIH-3T3 fibroblasts nonstimulated (NS) or treated for 24 h with forskolin (FK). (C) NIH-3T3 extracts were subjected to Western blot analysis to detect HIF1α protein levels after cell stimulation with forskolin (FK) for 24 h, or cobalt (Co2+) for 12 h. ERK2 levels show a control of the gel protein loading. White lines indicate that intervening lanes have been spliced out. (D) 3-HRE-LUC reporter assay on B16 and NIH-3T3 cells nonstimulated (NS) of stimulated with forskolin (FK) or cobalt (Co2+).

Mentions: To further elucidate the mechanisms by which cAMP regulates HIF1α expression, we performed Hif1a promoter activity assays. An Hif1a promoter fragment cloned upstream of the luciferase reporter gene (Wenger et al., 1998) was transfected in B16 melanoma cells. The promoter activity, monitored by the luciferase levels, increased upon cAMP treatment (Fig. 2 A). The same experiment was performed in NIH-3T3 fibroblasts, and we observed that forskolin had no impact on Hif1a promoter activity (Fig. 2 A). Furthermore, in these cells cAMP decreased Hif1a mRNA amount (Fig. 2 B). At the protein level, cAMP treatment did not affect HIF1α expression in NIH-3T3 fibroblasts (Fig. 2 C), whereas the control Co2+ induced the protein expression. A comparison of the functional HIF1α protein levels between B16 melanocyte cells and NIH-3T3 nonmelanocyte cells was performed using the 3-HRE-LUC reporter system (Fig. 2 D). We observed that although forskolin significantly increased the reporter transactivation in B16 cells, this did not occur in NIH-3T3 fibroblasts. In contrast, Co2+ treatment increased luciferase expression in both cell lines. These results indicate that cAMP increases Hif1α gene transcription in a melanocyte cell–specific manner.


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)

cAMP induces HIF1α expression in a cell-specific manner. (A) B16 cells and NIH-3T3 cells were transfected with a fragment of the Hif1a gene promoter cloned upstream of the luciferase reporter gene, and were then stimulated (or not) (NS) with forskolin (FK). Luciferase activity was normalized by the β-galactosidase activity and results are represented as the fold stimulation of the cAMP-activated promoter compared with the basal value. (B) Real-time quantitative PCR to detect Hif1a mRNA levels was performed on total RNAs from NIH-3T3 fibroblasts nonstimulated (NS) or treated for 24 h with forskolin (FK). (C) NIH-3T3 extracts were subjected to Western blot analysis to detect HIF1α protein levels after cell stimulation with forskolin (FK) for 24 h, or cobalt (Co2+) for 12 h. ERK2 levels show a control of the gel protein loading. White lines indicate that intervening lanes have been spliced out. (D) 3-HRE-LUC reporter assay on B16 and NIH-3T3 cells nonstimulated (NS) of stimulated with forskolin (FK) or cobalt (Co2+).
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fig2: cAMP induces HIF1α expression in a cell-specific manner. (A) B16 cells and NIH-3T3 cells were transfected with a fragment of the Hif1a gene promoter cloned upstream of the luciferase reporter gene, and were then stimulated (or not) (NS) with forskolin (FK). Luciferase activity was normalized by the β-galactosidase activity and results are represented as the fold stimulation of the cAMP-activated promoter compared with the basal value. (B) Real-time quantitative PCR to detect Hif1a mRNA levels was performed on total RNAs from NIH-3T3 fibroblasts nonstimulated (NS) or treated for 24 h with forskolin (FK). (C) NIH-3T3 extracts were subjected to Western blot analysis to detect HIF1α protein levels after cell stimulation with forskolin (FK) for 24 h, or cobalt (Co2+) for 12 h. ERK2 levels show a control of the gel protein loading. White lines indicate that intervening lanes have been spliced out. (D) 3-HRE-LUC reporter assay on B16 and NIH-3T3 cells nonstimulated (NS) of stimulated with forskolin (FK) or cobalt (Co2+).
Mentions: To further elucidate the mechanisms by which cAMP regulates HIF1α expression, we performed Hif1a promoter activity assays. An Hif1a promoter fragment cloned upstream of the luciferase reporter gene (Wenger et al., 1998) was transfected in B16 melanoma cells. The promoter activity, monitored by the luciferase levels, increased upon cAMP treatment (Fig. 2 A). The same experiment was performed in NIH-3T3 fibroblasts, and we observed that forskolin had no impact on Hif1a promoter activity (Fig. 2 A). Furthermore, in these cells cAMP decreased Hif1a mRNA amount (Fig. 2 B). At the protein level, cAMP treatment did not affect HIF1α expression in NIH-3T3 fibroblasts (Fig. 2 C), whereas the control Co2+ induced the protein expression. A comparison of the functional HIF1α protein levels between B16 melanocyte cells and NIH-3T3 nonmelanocyte cells was performed using the 3-HRE-LUC reporter system (Fig. 2 D). We observed that although forskolin significantly increased the reporter transactivation in B16 cells, this did not occur in NIH-3T3 fibroblasts. In contrast, Co2+ treatment increased luciferase expression in both cell lines. These results indicate that cAMP increases Hif1α gene transcription in a melanocyte cell–specific manner.

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