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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 mediates the cAMP increase of HIF1α protein. (A) Immunofluorescence study of B16 cells transfected with a nonrelevant siRNA (siRNA control) (left), an siRNA-targeting MITF (middle), and an siRNA to HIF1α (right). Cells were either left nonstimulated (NS) or were stimulated for 24 h with the cAMP-elevating agent forskolin (FK). MITF protein expression was detected with the C5 monoclonal primary antibody and a secondary fluorescein-conjugated antibody (green), HIF1α was labeled with a primary pAb and a secondary Texas red–conjugated antibody (red), and nuclei were stained with bisbenzidine (blue). Bar, 20 μM. (B) Western blot analysis of HIF1α protein from B16 cell extracts treated as in A. Cells were stimulated with Co2+ for 12 h as a positive control. ERK2 detection showed the equal protein loading of each gel lane. (C) Real-time quantitative PCR to detect Vegf mRNA levels on total RNA extracts from B16 cells transfected and treated as described in A. A transfection condition using an siRNA to silence PHD2 (to increase HIF1α protein) was added.
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fig5: MITF mediates the cAMP increase of HIF1α protein. (A) Immunofluorescence study of B16 cells transfected with a nonrelevant siRNA (siRNA control) (left), an siRNA-targeting MITF (middle), and an siRNA to HIF1α (right). Cells were either left nonstimulated (NS) or were stimulated for 24 h with the cAMP-elevating agent forskolin (FK). MITF protein expression was detected with the C5 monoclonal primary antibody and a secondary fluorescein-conjugated antibody (green), HIF1α was labeled with a primary pAb and a secondary Texas red–conjugated antibody (red), and nuclei were stained with bisbenzidine (blue). Bar, 20 μM. (B) Western blot analysis of HIF1α protein from B16 cell extracts treated as in A. Cells were stimulated with Co2+ for 12 h as a positive control. ERK2 detection showed the equal protein loading of each gel lane. (C) Real-time quantitative PCR to detect Vegf mRNA levels on total RNA extracts from B16 cells transfected and treated as described in A. A transfection condition using an siRNA to silence PHD2 (to increase HIF1α protein) was added.

Mentions: To study the impact of MITF on the endogenous HIF1α protein, the same siRNA approach was undertaken. The expression of MITF and HIF1α proteins was then monitored by immunofluorescence using specific antibodies to MITF (green fluorescein labeling) and HIF1α (Texas red labeling). After 24 h of stimulation with the cAMP-elevating agent forskolin we observed an increased nuclear labeling corresponding to MITF expression, as well as a significant increase in HIF1α protein (Fig. 5 A). As expected, MITF silencing led to an important inhibition of cAMP-dependent MITF induction as well as the cAMP-induced HIF1α expression. In the presence of an siRNA to silence HIF1α expression, although cAMP elevating agents expectedly increased MITF expression, HIF1α expression was inhibited in both basal and cAMP conditions. A Western blot analysis of samples treated in parallel (Fig. 5 B) confirmed the same results in a more quantitative way. We therefore demonstrate that MITF controls HIF1α transactivation and constitutes a critical step in the cAMP up-regulation of HIF1α 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 mediates the cAMP increase of HIF1α protein. (A) Immunofluorescence study of B16 cells transfected with a nonrelevant siRNA (siRNA control) (left), an siRNA-targeting MITF (middle), and an siRNA to HIF1α (right). Cells were either left nonstimulated (NS) or were stimulated for 24 h with the cAMP-elevating agent forskolin (FK). MITF protein expression was detected with the C5 monoclonal primary antibody and a secondary fluorescein-conjugated antibody (green), HIF1α was labeled with a primary pAb and a secondary Texas red–conjugated antibody (red), and nuclei were stained with bisbenzidine (blue). Bar, 20 μM. (B) Western blot analysis of HIF1α protein from B16 cell extracts treated as in A. Cells were stimulated with Co2+ for 12 h as a positive control. ERK2 detection showed the equal protein loading of each gel lane. (C) Real-time quantitative PCR to detect Vegf mRNA levels on total RNA extracts from B16 cells transfected and treated as described in A. A transfection condition using an siRNA to silence PHD2 (to increase HIF1α protein) was added.
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fig5: MITF mediates the cAMP increase of HIF1α protein. (A) Immunofluorescence study of B16 cells transfected with a nonrelevant siRNA (siRNA control) (left), an siRNA-targeting MITF (middle), and an siRNA to HIF1α (right). Cells were either left nonstimulated (NS) or were stimulated for 24 h with the cAMP-elevating agent forskolin (FK). MITF protein expression was detected with the C5 monoclonal primary antibody and a secondary fluorescein-conjugated antibody (green), HIF1α was labeled with a primary pAb and a secondary Texas red–conjugated antibody (red), and nuclei were stained with bisbenzidine (blue). Bar, 20 μM. (B) Western blot analysis of HIF1α protein from B16 cell extracts treated as in A. Cells were stimulated with Co2+ for 12 h as a positive control. ERK2 detection showed the equal protein loading of each gel lane. (C) Real-time quantitative PCR to detect Vegf mRNA levels on total RNA extracts from B16 cells transfected and treated as described in A. A transfection condition using an siRNA to silence PHD2 (to increase HIF1α protein) was added.
Mentions: To study the impact of MITF on the endogenous HIF1α protein, the same siRNA approach was undertaken. The expression of MITF and HIF1α proteins was then monitored by immunofluorescence using specific antibodies to MITF (green fluorescein labeling) and HIF1α (Texas red labeling). After 24 h of stimulation with the cAMP-elevating agent forskolin we observed an increased nuclear labeling corresponding to MITF expression, as well as a significant increase in HIF1α protein (Fig. 5 A). As expected, MITF silencing led to an important inhibition of cAMP-dependent MITF induction as well as the cAMP-induced HIF1α expression. In the presence of an siRNA to silence HIF1α expression, although cAMP elevating agents expectedly increased MITF expression, HIF1α expression was inhibited in both basal and cAMP conditions. A Western blot analysis of samples treated in parallel (Fig. 5 B) confirmed the same results in a more quantitative way. We therefore demonstrate that MITF controls HIF1α transactivation and constitutes a critical step in the cAMP up-regulation of HIF1α 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