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 vasc ular endothelial cells that has been implicated in tumor neovasculariz ation. 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 c ells with serum. CCL39 derivatives, transformed with Polyoma virus or with active members of the p42/p44 mitogen-activated protein (MAP) kin ase pathway, Gly/Val point mutant of Ras at position 12 (Ras-Val(12)), MKK1 in which Ser(218) and Ser(222) were mutated to Asp (MKK1-SS/DD)) , express very high levels of VEGF mRNA, To analyze the contribution o f the p42/p44MAP kinase in this induction, we used the CCL39 derived c ell line (Raf-1:ER) expressing an estradiol-activable Raf-l. We show a time and an estradiol dose-dependent up-regulation of VEGF mRNA clear ly detectable after 2 h of stimulation. The induction of VEGF mRNA in response to conditioned activation of Raf-l is reverted by an inhibito r of MKK1, PD 098059, highlighting a specific role for the p42/p44 MAP kinase pathway in VEGF expression. Interestingly, hypoxia has an addi tive effect on VEGF induction in CCL39 cells stimulated by serum or in Raf-l:ER cells stimulated by estradiol, In contrast to VEGF, the isof orms VEGF-B and VEGF-C are poorly regulated by growth and oncogenic fa ctors. We have identified a GC-rich region of the VEGF promoter betwee n -88 and -66 base pairs which contains all the elements responsible o f its up-regulation by constitutive active Ras or MKK1-SS/DD. By mutat ion of the putative binding sites and electrophoretic mobility supersh ift experiments, we showed that the GC-rich region constitutively bind s Spl and AP-2 transcription factors. Furthermore, following activatio n of the p42/p44 MAP kinase module, the binding of Spl and AP-2 is inc reased in the complexes formed in this region of the promoter. Altoget her, these data suggest that hypoxia and p42/p44 MAP kinase independen tly play a key role in the regulation of the VEGF expression.