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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 binds to the Hif1a promoter and induces Hif1α gene expression. (A) Schematic representation of the Hif1a promoter. Black arrow denotes the ubiquitously expressed 1.2 exon (Wenger et al., 1998). Vertical rectangles and the associated numbers represent the several E-boxes found in this promoter fragment and their localization within the sequence. Note that E2 and E7 match perfectly with the previously described MITF binding sequence (CACGTG) (Weilbaecher et al., 1998). (B) Chromatin immunoprecipitations were performed on extracts from nonstimulated (NS) and 5-h forskolin-stimulated (FK) B16 melanoma cells as described in Materials and methods. Immunoprecipitation was performed using a specific anti-MITF antibody and primers spanning the Hif1a or the tyrosinase promoter region were used for the PCR amplification. A control of PCR amplification was performed using mouse genomic DNA, which showed a 300-bp and a 900-bp band corresponding to the amplification of the Hif1a and tyrosinase promoter regions, respectively. Another control was performed using a primer pair to the mouse GAPDH (150-bp amplicon). (C) Hif1a promoter activity after cotransfection of B16 cells with the Hif1a promoter fragment together with an empty plasmid (pcDNA3), a vector encoding MITF transcription factor, and two other constructs encoding USF and MYC. As a control of the promoter activation, cells were stimulated with forskolin (FK). Results are shown as the fold stimulation compared with the basal nonstimulated promoter activity (NS). (D) Real-time quantitative PCR analysis to detect Mitf and Hif1a mRNA levels after infection of Mewo (human melanoma cells) with either an empty adenovirus (Adeno-pCDNA3) or an adenovirus encoding MITF. (E) Hif1a promoter activity after cotransfection of NIH-3T3 fibroblasts with an empty vector (pCDNA3) or a construct encoding MITF. Cells were next stimulated (or not) (NS) with forskolin (FK) for 24 h.
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fig3: MITF binds to the Hif1a promoter and induces Hif1α gene expression. (A) Schematic representation of the Hif1a promoter. Black arrow denotes the ubiquitously expressed 1.2 exon (Wenger et al., 1998). Vertical rectangles and the associated numbers represent the several E-boxes found in this promoter fragment and their localization within the sequence. Note that E2 and E7 match perfectly with the previously described MITF binding sequence (CACGTG) (Weilbaecher et al., 1998). (B) Chromatin immunoprecipitations were performed on extracts from nonstimulated (NS) and 5-h forskolin-stimulated (FK) B16 melanoma cells as described in Materials and methods. Immunoprecipitation was performed using a specific anti-MITF antibody and primers spanning the Hif1a or the tyrosinase promoter region were used for the PCR amplification. A control of PCR amplification was performed using mouse genomic DNA, which showed a 300-bp and a 900-bp band corresponding to the amplification of the Hif1a and tyrosinase promoter regions, respectively. Another control was performed using a primer pair to the mouse GAPDH (150-bp amplicon). (C) Hif1a promoter activity after cotransfection of B16 cells with the Hif1a promoter fragment together with an empty plasmid (pcDNA3), a vector encoding MITF transcription factor, and two other constructs encoding USF and MYC. As a control of the promoter activation, cells were stimulated with forskolin (FK). Results are shown as the fold stimulation compared with the basal nonstimulated promoter activity (NS). (D) Real-time quantitative PCR analysis to detect Mitf and Hif1a mRNA levels after infection of Mewo (human melanoma cells) with either an empty adenovirus (Adeno-pCDNA3) or an adenovirus encoding MITF. (E) Hif1a promoter activity after cotransfection of NIH-3T3 fibroblasts with an empty vector (pCDNA3) or a construct encoding MITF. Cells were next stimulated (or not) (NS) with forskolin (FK) for 24 h.

Mentions: The cell specificity of HIF1α induction by cAMP prompted us to hypothesize the existence of specific molecular mechanisms responsible for HIF1α regulation in melanocytes and melanoma cells. Sequence analysis of the Hif1a promoter (Wenger et al., 1998) revealed 10 core E-box consensus sequences composed by the CANNTG motif (Fig. 3 A). Noteworthy, among these 10 E-boxes, 2 perfectly match with the motif of MITF binding (CACGTG) (Aksan and Goding, 1998). We therefore investigated whether MITF could account for the cell-specific cAMP regulation of HIF1α expression. To evaluate the MITF binding to the Hif1a promoter region in the endogenous chromatin context, we performed chromatin immunoprecipitation assays (Fig. 3 B). B16 cells were stimulated with the cAMP-elevating agent forskolin for 5 h to maximally increase MITF protein expression (Bertolotto et al., 1998a). Chromatin complexes were immunoprecipitated with an anti-MITF antibody and a PCR was performed using specific primers to the mouse Hif1a promoter region. As shown in Fig. 3 B, we detected a specific amplification of the Hif1a promoter region that increased after forskolin treatment. As a positive control, we used specific primers spanning the tyrosinase promoter region, which is well known to bind MITF. As expected, the tyrosinase promoter amplification also increased upon cAMP exposure. A specific band corresponding to the Hif1a or tyrosinase promoter was detected when using mouse genomic DNA. In the absence of antibody, no specific amplification was observed. Another negative control was performed by applying 40 cycles of PCR to the MITF immunoprecipitates using a primer pair to the mouse GAPDH. As expected for a sequence that does not present MITF binding sites, no GAPDH amplification was observed while a clear PCR band was detected using the genomic DNA as a template (Fig. 3 B, bottom panel). We therefore conclude that MITF binds to the Hif1a promoter in the chromatin context of B16 melanoma cells.


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 binds to the Hif1a promoter and induces Hif1α gene expression. (A) Schematic representation of the Hif1a promoter. Black arrow denotes the ubiquitously expressed 1.2 exon (Wenger et al., 1998). Vertical rectangles and the associated numbers represent the several E-boxes found in this promoter fragment and their localization within the sequence. Note that E2 and E7 match perfectly with the previously described MITF binding sequence (CACGTG) (Weilbaecher et al., 1998). (B) Chromatin immunoprecipitations were performed on extracts from nonstimulated (NS) and 5-h forskolin-stimulated (FK) B16 melanoma cells as described in Materials and methods. Immunoprecipitation was performed using a specific anti-MITF antibody and primers spanning the Hif1a or the tyrosinase promoter region were used for the PCR amplification. A control of PCR amplification was performed using mouse genomic DNA, which showed a 300-bp and a 900-bp band corresponding to the amplification of the Hif1a and tyrosinase promoter regions, respectively. Another control was performed using a primer pair to the mouse GAPDH (150-bp amplicon). (C) Hif1a promoter activity after cotransfection of B16 cells with the Hif1a promoter fragment together with an empty plasmid (pcDNA3), a vector encoding MITF transcription factor, and two other constructs encoding USF and MYC. As a control of the promoter activation, cells were stimulated with forskolin (FK). Results are shown as the fold stimulation compared with the basal nonstimulated promoter activity (NS). (D) Real-time quantitative PCR analysis to detect Mitf and Hif1a mRNA levels after infection of Mewo (human melanoma cells) with either an empty adenovirus (Adeno-pCDNA3) or an adenovirus encoding MITF. (E) Hif1a promoter activity after cotransfection of NIH-3T3 fibroblasts with an empty vector (pCDNA3) or a construct encoding MITF. Cells were next stimulated (or not) (NS) with forskolin (FK) for 24 h.
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fig3: MITF binds to the Hif1a promoter and induces Hif1α gene expression. (A) Schematic representation of the Hif1a promoter. Black arrow denotes the ubiquitously expressed 1.2 exon (Wenger et al., 1998). Vertical rectangles and the associated numbers represent the several E-boxes found in this promoter fragment and their localization within the sequence. Note that E2 and E7 match perfectly with the previously described MITF binding sequence (CACGTG) (Weilbaecher et al., 1998). (B) Chromatin immunoprecipitations were performed on extracts from nonstimulated (NS) and 5-h forskolin-stimulated (FK) B16 melanoma cells as described in Materials and methods. Immunoprecipitation was performed using a specific anti-MITF antibody and primers spanning the Hif1a or the tyrosinase promoter region were used for the PCR amplification. A control of PCR amplification was performed using mouse genomic DNA, which showed a 300-bp and a 900-bp band corresponding to the amplification of the Hif1a and tyrosinase promoter regions, respectively. Another control was performed using a primer pair to the mouse GAPDH (150-bp amplicon). (C) Hif1a promoter activity after cotransfection of B16 cells with the Hif1a promoter fragment together with an empty plasmid (pcDNA3), a vector encoding MITF transcription factor, and two other constructs encoding USF and MYC. As a control of the promoter activation, cells were stimulated with forskolin (FK). Results are shown as the fold stimulation compared with the basal nonstimulated promoter activity (NS). (D) Real-time quantitative PCR analysis to detect Mitf and Hif1a mRNA levels after infection of Mewo (human melanoma cells) with either an empty adenovirus (Adeno-pCDNA3) or an adenovirus encoding MITF. (E) Hif1a promoter activity after cotransfection of NIH-3T3 fibroblasts with an empty vector (pCDNA3) or a construct encoding MITF. Cells were next stimulated (or not) (NS) with forskolin (FK) for 24 h.
Mentions: The cell specificity of HIF1α induction by cAMP prompted us to hypothesize the existence of specific molecular mechanisms responsible for HIF1α regulation in melanocytes and melanoma cells. Sequence analysis of the Hif1a promoter (Wenger et al., 1998) revealed 10 core E-box consensus sequences composed by the CANNTG motif (Fig. 3 A). Noteworthy, among these 10 E-boxes, 2 perfectly match with the motif of MITF binding (CACGTG) (Aksan and Goding, 1998). We therefore investigated whether MITF could account for the cell-specific cAMP regulation of HIF1α expression. To evaluate the MITF binding to the Hif1a promoter region in the endogenous chromatin context, we performed chromatin immunoprecipitation assays (Fig. 3 B). B16 cells were stimulated with the cAMP-elevating agent forskolin for 5 h to maximally increase MITF protein expression (Bertolotto et al., 1998a). Chromatin complexes were immunoprecipitated with an anti-MITF antibody and a PCR was performed using specific primers to the mouse Hif1a promoter region. As shown in Fig. 3 B, we detected a specific amplification of the Hif1a promoter region that increased after forskolin treatment. As a positive control, we used specific primers spanning the tyrosinase promoter region, which is well known to bind MITF. As expected, the tyrosinase promoter amplification also increased upon cAMP exposure. A specific band corresponding to the Hif1a or tyrosinase promoter was detected when using mouse genomic DNA. In the absence of antibody, no specific amplification was observed. Another negative control was performed by applying 40 cycles of PCR to the MITF immunoprecipitates using a primer pair to the mouse GAPDH. As expected for a sequence that does not present MITF binding sites, no GAPDH amplification was observed while a clear PCR band was detected using the genomic DNA as a template (Fig. 3 B, bottom panel). We therefore conclude that MITF binds to the Hif1a promoter in the chromatin context of B16 melanoma cells.

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