p42/p44 MAP kinase module plays a key role in the transcriptional regulation of the vascular endothelial growth factor gene in fibroblasts.
Vascular Endothelial Growth Factor (VEGF) is a potent mitogen for vascular endothelial cells that has been implicated in tumor neovascularization. We show that, in hamster fibroblasts (CCL39 cells), VEGF mRNAs are expressed at low levels in serum-deprived or exponentially growing cells, whereas it is rapidly induced after stimulation of quiescent cells with serum. CCL39 derivatives, transformed with Polyoma virus or with active members of the p42/p44 mitogen-activated protein (MAP) kinase pathway, Gly/Val point mutant of Ras at position 12 (Ras-Val12), MKK1 in which Ser218 and Ser222 were mutated to Asp (MKK1-SS/DD)), express very high levels of VEGF mRNA. To analyze the contribution of the p42/p44MAP kinase in this induction, we used the CCL39-derived cell line (Raf-1:ER) expressing an estradiol-activable Raf-1. We show a time and an estradiol dose-dependent up-regulation of VEGF mRNA clearly detectable after 2 h of stimulation. The induction of VEGF mRNA in response to conditioned activation of Raf-1 is reverted by an inhibitor of MKK1, PD 098059, highlighting a specific role for the p42/p44 MAP kinase pathway in VEGF expression. Interestingly, hypoxia has an additive effect on VEGF induction in CCL39 cells stimulated by serum or in Raf-1:ER cells stimulated by estradiol. In contrast to VEGF, the isoforms VEGF-B and VEGF-C are poorly regulated by growth and oncogenic factors. We have identified a GC-rich region of the VEGF promoter between -88 and -66 base pairs which contains all the elements responsible of its up-regulation by constitutive active Ras or MKK1-SS/DD. By mutation of the putative binding sites and electrophoretic mobility supershift experiments, we showed that the GC-rich region constitutively binds Sp1 and AP-2 transcription factors. Furthermore, following activation of the p42/p44 MAP kinase module, the binding of Sp1 and AP-2 is increased in the complexes formed in this region of the promoter. Altogether, these data suggest that hypoxia and p42/p44 MAP kinase independently play a key role in the regulation of the VEGF expression.
Milanini
J
J
Centre de Biochimie, CNRS-UMR 6543, Université de Nice, Parc Valrose, 06108 Nice, France.
Viñals
F
F
Pouysségur
J
J
Pagès
G
G
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000378:metabolism; D016228:Endothelial Growth Factors / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation; D008222:Lymphokines / Q000235:genetics*; D051379:Mice; D008819:Mice, Nude; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D011401:Promoter Regions, Genetic; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D042461:Vascular Endothelial Growth Factor A; D042561:Vascular Endothelial Growth Factor B; D042582:Vascular Endothelial Growth Factor C; D042442:Vascular Endothelial Growth Factors
D050659:Core Binding Factor Alpha 1 Subunit; D004268:DNA-Binding Proteins; D016228:Endothelial Growth Factors; D008222:Lymphokines; D012333:RNA, Messenger; C493716:Runx2 protein, mouse; D017923:Sodium-Hydrogen Exchangers; D016329:Sp1 Transcription Factor; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D042561:Vascular Endothelial Growth Factor B; D042582:Vascular Endothelial Growth Factor C; D042442:Vascular Endothelial Growth Factors; C069139:growth factor-activatable Na-H exchanger NHE-1; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases