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.

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.

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.

Model for the mechanisms of cAMP induction of HIF1α expression. α-MSH, by up-regulating the cAMP pathway, increases MITF expression. MITF binds and transactivates the Hif1a promoter, thereby increasing the expression of Hif1a gene and protein in a melanocyte cell–specific manner. HIF1α dimerizes with HIF1β to constitute a functional HIF1 transcription factor that is capable of activating target genes such as Vegf and up-regulate other genes involved in melanocyte cell survival and angiogenesis to participate in melanoma development and behavior.

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⁺).

MITF is required for cAMP-dependent Hif1a expression. (A) Hif1a promoter activity assays on B16 cells cotransfected with either an empty vector (pCDNA3) or a construct encoding a dominant-negative mutant of MITF (MITF-DNT) and treated (or not) (NS) with the cAMP-elevating agent forskolin (FK). Results are expressed as the fold stimulation of the luciferase activity compared with the pcDNA3-transfected nonstimulated condition. (B) The same experiment was performed by cotransfecting the Hif1a promoter with either an siRNA-targeting MITF (siRNA-MITF) or a nonrelevant siRNA (siRNA-control).

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.