p42/p44 MAP Kinase Module Plays a Key Role in the Transcriptional Regulation of the Vascular Endothelial Growth Factor Gene in Fibroblasts

doi:10.1074/jbc.273.29.18165

p42/p44 MAP Kinase Module Plays a Key Role in the Transcriptional Regulation of the Vascular Endothelial Growth Factor Gene in Fibroblasts*

From the Centre de Biochimie, CNRS-UMR 6543, Université de Nice, Parc Valrose, 06108 Nice, France

Abstract

Vascular EndothelialGrowth 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.

Footnotes

↵* This work was supported by the CNRS (UMR 6543), the Université de Nice, the Ministère de la Recherche (ACC-SV9), the Ligue Nationale contre le Cancer, the Association pour la Recherche Contre le Cancer (ARC), the Ministerio de Educacion y Cultura (Spain),and the Fédération Nationale des Groupements des Entreprises Françaises et Monégasques dans la Lutte Contre le Cancer (FEGEFLUC).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed: Centre de Biochimie, CNRS-UMR 6543, Université de Nice, Parc Valrose, 06108 Nice, France. Tel.: 33-492 07 64 30; Fax: 33-492 07 64 32; E-mail:gpages{at}unice.fr.
↵1 The abbreviations used are: VEGF, vascular endothelial growth factor; Ras-Val¹², Gly/Val point mutant of Ras at position 12; p38/HOG, protein kinase ofM _r 38 activated by osmotic shock (mammalian homolog of the yeast kinase HOG); p42/p44 MAPK, mitogen-activated protein kinases of 42 and 44 kDa, respectively; JNK, c-Jun N-terminal kinase; MKK1 or MEK1, MAP kinase kinase 1; MKK1-SS/DD, MKK1 in which Ser²¹⁸ and Ser²²² were mutated to Asp; Raf-1:ER cells, cells stably expressing an estradiol-inducible Raf-1; AP-1, activator protein 1; AP-2, activator protein 2; EMSA, electrophoretic mobility shift assay; PDGF, platelet-derived growth factor; PCR, polymerase chain reaction; bp, base pair(s); PBS, phosphate-buffered saline; DTT, dithiothreitol; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; FCS, fetal calf serum; TGF, transforming growth factor.
↵2 F. R. McKenzie, J. M. Brondello, and A. Brunet, unpublished results.
- Received April 9, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.