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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 increases the expression of a functional HIF1α protein in melanocyte cells. (A) B16 cells were stimulated for 6, 24, and 48 h with forskolin (FK) and cell extracts were subjected to Western blot analysis to detect HIF1α protein levels. A control of the protein loading was performed by detecting ERK2. (B) The same experiment was performed by stimulating B16 cells either with forskolin (FK) or α-MSH (α-M) for 24 h. Cells were incubated, either in normal oxygen conditions (Normoxia, 20% O2) or maintained under hypoxia (1–2% O2). (C) A Western blot to analyze HIF1α protein expression was performed using extracts from normal human melanocytes. Cells were starved and treated with forskolin (FK) for 24 h or with cobalt (Co2+) to mimic hypoxia as a positive control. (D) B16 cells were transfected with the 3-HRE-LUC reporter construct and treated (or not) (NS) with forskolin (FK) for at least 36 h, with cobalt (Co2+) for 12 h, or with both (Co2+ + FK). Luciferase activity was normalized by the β-galactosidase activity and data are expressed in fold stimulation of the basal 3-HRE-LUC activity. Data are means ± SE of five experiments performed in triplicate. (E) Real-time quantitative PCR to detect Vegf mRNA levels on total RNA extracts from B16 cells treated as described in D.
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fig1: cAMP increases the expression of a functional HIF1α protein in melanocyte cells. (A) B16 cells were stimulated for 6, 24, and 48 h with forskolin (FK) and cell extracts were subjected to Western blot analysis to detect HIF1α protein levels. A control of the protein loading was performed by detecting ERK2. (B) The same experiment was performed by stimulating B16 cells either with forskolin (FK) or α-MSH (α-M) for 24 h. Cells were incubated, either in normal oxygen conditions (Normoxia, 20% O2) or maintained under hypoxia (1–2% O2). (C) A Western blot to analyze HIF1α protein expression was performed using extracts from normal human melanocytes. Cells were starved and treated with forskolin (FK) for 24 h or with cobalt (Co2+) to mimic hypoxia as a positive control. (D) B16 cells were transfected with the 3-HRE-LUC reporter construct and treated (or not) (NS) with forskolin (FK) for at least 36 h, with cobalt (Co2+) for 12 h, or with both (Co2+ + FK). Luciferase activity was normalized by the β-galactosidase activity and data are expressed in fold stimulation of the basal 3-HRE-LUC activity. Data are means ± SE of five experiments performed in triplicate. (E) Real-time quantitative PCR to detect Vegf mRNA levels on total RNA extracts from B16 cells treated as described in D.

Mentions: Western blot experiments using a specific anti HIF1α antibody showed that HIF1α protein slightly increased at 6 h and reached its maximal levels upon 24 h of forskolin treatment. After 48 h, HIF1α protein expression remained stable (Fig. 1 A). α-MSH, a physiological activator of the cAMP pathway in melanocytes, also increased HIF1α protein levels similarly to hypoxia (24 h), which was used as a positive control of HIF1α protein induction. In addition, cAMP plus hypoxia appeared to have additive effects (Fig. 1 B). The same results were obtained using normal human melanocytes (NHM). In this case we used Co2+, to mimic hypoxia (Berra et al., 2003), as a control (Fig. 1 C). Therefore, HIF1α protein increased upon cAMP up-regulation in B16 melanoma cells and NHM independently of the oxygen context.


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 increases the expression of a functional HIF1α protein in melanocyte cells. (A) B16 cells were stimulated for 6, 24, and 48 h with forskolin (FK) and cell extracts were subjected to Western blot analysis to detect HIF1α protein levels. A control of the protein loading was performed by detecting ERK2. (B) The same experiment was performed by stimulating B16 cells either with forskolin (FK) or α-MSH (α-M) for 24 h. Cells were incubated, either in normal oxygen conditions (Normoxia, 20% O2) or maintained under hypoxia (1–2% O2). (C) A Western blot to analyze HIF1α protein expression was performed using extracts from normal human melanocytes. Cells were starved and treated with forskolin (FK) for 24 h or with cobalt (Co2+) to mimic hypoxia as a positive control. (D) B16 cells were transfected with the 3-HRE-LUC reporter construct and treated (or not) (NS) with forskolin (FK) for at least 36 h, with cobalt (Co2+) for 12 h, or with both (Co2+ + FK). Luciferase activity was normalized by the β-galactosidase activity and data are expressed in fold stimulation of the basal 3-HRE-LUC activity. Data are means ± SE of five experiments performed in triplicate. (E) Real-time quantitative PCR to detect Vegf mRNA levels on total RNA extracts from B16 cells treated as described in D.
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fig1: cAMP increases the expression of a functional HIF1α protein in melanocyte cells. (A) B16 cells were stimulated for 6, 24, and 48 h with forskolin (FK) and cell extracts were subjected to Western blot analysis to detect HIF1α protein levels. A control of the protein loading was performed by detecting ERK2. (B) The same experiment was performed by stimulating B16 cells either with forskolin (FK) or α-MSH (α-M) for 24 h. Cells were incubated, either in normal oxygen conditions (Normoxia, 20% O2) or maintained under hypoxia (1–2% O2). (C) A Western blot to analyze HIF1α protein expression was performed using extracts from normal human melanocytes. Cells were starved and treated with forskolin (FK) for 24 h or with cobalt (Co2+) to mimic hypoxia as a positive control. (D) B16 cells were transfected with the 3-HRE-LUC reporter construct and treated (or not) (NS) with forskolin (FK) for at least 36 h, with cobalt (Co2+) for 12 h, or with both (Co2+ + FK). Luciferase activity was normalized by the β-galactosidase activity and data are expressed in fold stimulation of the basal 3-HRE-LUC activity. Data are means ± SE of five experiments performed in triplicate. (E) Real-time quantitative PCR to detect Vegf mRNA levels on total RNA extracts from B16 cells treated as described in D.
Mentions: Western blot experiments using a specific anti HIF1α antibody showed that HIF1α protein slightly increased at 6 h and reached its maximal levels upon 24 h of forskolin treatment. After 48 h, HIF1α protein expression remained stable (Fig. 1 A). α-MSH, a physiological activator of the cAMP pathway in melanocytes, also increased HIF1α protein levels similarly to hypoxia (24 h), which was used as a positive control of HIF1α protein induction. In addition, cAMP plus hypoxia appeared to have additive effects (Fig. 1 B). The same results were obtained using normal human melanocytes (NHM). In this case we used Co2+, to mimic hypoxia (Berra et al., 2003), as a control (Fig. 1 C). Therefore, HIF1α protein increased upon cAMP up-regulation in B16 melanoma cells and NHM independently of the oxygen context.

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