p42/p44 MAP Kinase Module Plays a Key Role in the Transcriptional Regulation of the Vascular Endothelial Growth Factor Gene in Fibroblasts
1998; Elsevier BV; Volume: 273; Issue: 29 Linguagem: Inglês
10.1074/jbc.273.29.18165
ISSN1083-351X
AutoresJulie Milanini, Francesc Viñals, Jacques Pouysségur, Gilles Pagès,
Tópico(s)Vascular Tumors and Angiosarcomas
ResumoVascular 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. 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. Angiogenesis is a fundamental physiological process by which new blood vessels are formed (1Folkman J. Merler E. Abernathy C. William G. J. Exp. Med. 1971; 133: 275-288Crossref PubMed Scopus (1362) Google Scholar). One of the most widely described mechanisms controlling neovascularization associated with pathological processes (2Hanahan D. Folkman J. Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6140) Google Scholar) is the increased secretion by the "stressed cells" (inflammation, psoriasis) or nutrient-deprived tumor cells of multiple growth factors (3Basilico C. Moscatelii D. Adv. Cancer Res. 1992; 59: 115-165Crossref PubMed Scopus (1057) Google Scholar, 4Folkman J. Shing Y. J. Biol. Chem. 1992; 267: 10931-10934Abstract Full Text PDF PubMed Google Scholar, 5Leung D.W. Cachianes G. Kuang W.J. Goeddel D.V. Ferrara N. Science. 1989; 246: 1306-1309Crossref PubMed Scopus (4478) Google Scholar, 6Breier G. Risau W. Trends Cell Biol. 1996; 6: 454-456Abstract Full Text PDF PubMed Scopus (147) Google Scholar, 7Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4891) Google Scholar) and cytokines (8Cozzolino F. Torcia M. Lucibello M. Morbidelli L. Ziche M. Platt J. Fabiani S. Brett J. Stern D. J. Clin. Invest. 1993; 91: 2504-2512Crossref PubMed Scopus (80) Google Scholar, 9Toi M. Harris A.L. Bicknell R. Biochem. Biophys. Res. Commun. 1991; 174: 1287-1293Crossref PubMed Scopus (83) Google Scholar). Among growth factors, two major classes have been characterized: acid and basic FGF of the FGF family (3Basilico C. Moscatelii D. Adv. Cancer Res. 1992; 59: 115-165Crossref PubMed Scopus (1057) Google Scholar, 4Folkman J. Shing Y. J. Biol. Chem. 1992; 267: 10931-10934Abstract Full Text PDF PubMed Google Scholar) and Vascular Endothelial Growth Factor, VEGF, 1The 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. a new family of secreted growth factors structurally related to PDGF (40% homology at the amino acid level) (5Leung D.W. Cachianes G. Kuang W.J. Goeddel D.V. Ferrara N. Science. 1989; 246: 1306-1309Crossref PubMed Scopus (4478) Google Scholar, 6Breier G. Risau W. Trends Cell Biol. 1996; 6: 454-456Abstract Full Text PDF PubMed Scopus (147) Google Scholar, 7Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4891) Google Scholar). VEGF, also described as a permeability factor, stimulates endothelial cell migration and proliferationin vitro and has angiogenic activity in vivo (10Ferrara N. Henzel W.J. Biochem. Biophys. Res. Commun. 1989; 161: 851-858Crossref PubMed Scopus (2031) Google Scholar,11Shweiki D. Itin A. Soffer D. Keshet E. Nature. 1992; 359: 843-945Crossref PubMed Scopus (4186) Google Scholar). Different isoforms of 121, 165, 189, and 206 amino acids resulted from alternative splicing of the same gene (12Tischer E. Mitchell R. Hartman T. Silva M. Gospodarowicz D. Fiddes J.C. Abraham J.A. J. Biol. Chem. 1991; 266: 11947-11954Abstract Full Text PDF PubMed Google Scholar). Many tissues and cell types express VEGF mRNA, especially tissues which are highly vascularized in addition to tumor-derived cell lines (13Berse B. Brown L.F. Van de Water L. Dvorak H.F. Senger D.R. Mol. Biol. Cell. 1992; 3: 211-220Crossref PubMed Scopus (864) Google Scholar). Stimulation of serum-deprived NIH 3T3 cells by PDGF also results in VEGF induction in a Ras- and Raf-dependent manner (14Grugel S. Finkenzeller G. Weindel K. Barleon B. Marmé D. J. Biol. Chem. 1995; 270: 25915-25919Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Deprivation of oxygen during cell culture, which mimics the necrotic hypoxic regions in solid tumors, induces VEGF mRNA expression by both an increase in the rate of transcription but also by stabilization of its mRNA (15Finkenzeller G. Technau A. Marme D. Biochem. Biophys. Res. Commun. 1995; 208: 432-439Crossref PubMed Scopus (119) Google Scholar, 16Ikeda E. Achen M.G. Breier G. Risau W. J. Biol. Chem. 1995; 270: 19761-19766Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar, 17Levy A.P. Levy N.S. Goldberg M.A. J. Biol. Chem. 1996; 271: 2746-2753Abstract Full Text Full Text PDF PubMed Scopus (560) Google Scholar). Considering the key role played by VEGF in the control of neovascularization (6Breier G. Risau W. Trends Cell Biol. 1996; 6: 454-456Abstract Full Text PDF PubMed Scopus (147) Google Scholar, 7Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4891) Google Scholar), it is of primary importance to decipher the growth factor-activated signaling pathways involved in controlling its expression. In the present report, we have compared the expression of VEGF in resting, serum-stimulated, or oncogenically transformed CCL39 fibroblasts (18Seuwen K. Lagarde A. Pouysségur J. EMBO J. 1988; 7: 161-168Crossref PubMed Scopus (101) Google Scholar, 19Brunet A. Pagès G. Pouysségur J. Oncogene. 1994; 9: 3379-3387PubMed Google Scholar). Exploiting a CCL39-derived cell line in which Raf-1 can be rapidly activated by estradiol (Raf-1:ER) (20Samuels M.L. Weber J.M. McMahon M. Mol. Biol. Cell. 1993; 13: 6241-6252Crossref Scopus (324) Google Scholar, 21Samuels M.L. McMahon M. Mol. Biol. Cell. 1994; 14: 7855-7866Crossref Google Scholar, 22Lenormand P. McMahon M. Pouysségur J. J. Biol. Chem. 1996; 271: 15762-15768Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), we demonstrated that the p42/p44 MAP kinase cascade is critical in the control of VEGF expression. To further characterize the effect of constitutively active Ras or MKK1 on the VEGF expression, we have assayed different constructs of the VEGF promoter in order to define cis-active regions sufficient to promote regulation of VEGF transcription by members of the p42/p44 MAP kinase module. By electrophoretic mobility assays (EMSAs) and supershift assays, we also defined transcription factors whose binding on the VEGF promoter is regulated through p42/p44 MAP kinase cascade. The established Chinese hamster lung fibroblast line CCL39 (American Type Culture Collection), their derivatives PS120 and PS200, which lack NHE1 antiporter activity (23Pouysségur J. Sardet C. Franchi A. L'Allemain G. Paris S. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4833-4837Crossref PubMed Scopus (440) Google Scholar), and corresponding transfected cells were cultivated in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 7.5% fetal calf serum, penicillin (50 units/ml), and streptomycin sulfate (50 μg/ml). Growth-arrested cells were obtained by total deprivation of serum for 16–24 h. Raf-1:ER cells (clone 18 or 19) are a derivative of CCL39, and they stably expressed a fusion protein between the catalytic domain of Raf-1 and the hormone binding domain of the estrogen receptor (20Samuels M.L. Weber J.M. McMahon M. Mol. Biol. Cell. 1993; 13: 6241-6252Crossref Scopus (324) Google Scholar, 21Samuels M.L. McMahon M. Mol. Biol. Cell. 1994; 14: 7855-7866Crossref Google Scholar, 22Lenormand P. McMahon M. Pouysségur J. J. Biol. Chem. 1996; 271: 15762-15768Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). These cells were cultivated in the same medium described above without phenol red to reduce the basal activity of the Raf-1:ER construct. Hypoxia was generated by placing the cells in hermetic jars together with the Gas Pak Plus system from Becton Dickinson. In this system, hydrogen generated from sodium borohydride following the addition of water combines with the oxygen present in the jar in the presence of palladium catalyst to form water. Oxygen deprivation is almost complete after 1 h of incubation. Approximately 4–10% carbon dioxide is generated during this oxygen removal. Thus, cells are cultivated in the same bicarbonate-buffered medium. Restriction and DNA modifying enzymes were obtained from New England Biolabs or from Eurogentec, Liège, Belgium. [α-32P]dCTP, [α-33P]dATP were from ICN. Synthetic oligonucleotides were from Eurogentec, Liège, Belgium. First strand cDNA was synthesized from 1 μg of CCL39 poly(A)+ RNA using avian myeloblastosis virus reverse transcriptase with oligo(dT) primer. This material was used as template for polymerase chain reaction (PCR) amplification. The following oligonucleotides derived, respectively, from human VEGF, mouse VEGF-B (24Olofsson B. Pajulosa K. Kaipainen A. Von Euler G. Joukov V. Saksela O. Orpana A. Pettersson R.F. Alitalo K. Eriksson U. Proc. Natl. Acad. Sci U. S. A. 1996; 93: 2576-2581Crossref PubMed Scopus (630) Google Scholar), human VEGF-C (25Joukov V. Pajulosa K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1167) Google Scholar) sequences, were synthesized and used as primers for the PCR reaction: 5′-ATGAACTTTCTGCTGTCTTGGG-3′ and 5′-CCGCCTCGGCTTGTCACATCTGC-3′; 5′-ATGAGCCCCCTGCTCCGTCGCCTG-3′ and 5′-CTTTCGCGGCTTCCGGCACC-3′; and 5′-ATGACTGTACTCTACCCAGAATATTG-3′ and 5′-GCTCATTTGTGGTCTTTTC-3′. An aliquot of cDNA was amplified in a 50-μl reaction volume with 200 ng of each primer, 200 μm dNTPs, and 2.5 units of GoldstarTaq DNA polymerase (Eurogentec) or ampli-Taq from Boehringer Mannheim in a buffer containing 10 mm Tris-HCl, pH 8.3, 50 mm KCl, 1.5 mm MgCl2, and 0.001% gelatin. The PCR amplification was performed in a DNA thermal cycler (Biotechnia) using the following parameters: 30 s at 95 °C, 1 min at 55 °C, and 1 min at 72 °C for 30 cycles followed by an extra cycle with a 10-min extension step at 72 °C. Expected fragments of approximately 600 and 550 bp for VEGF, 600 bp for VEGF-B, and 1200 bp for VEGF-C were obtained. These fragments were purified on agarose gels and used as probes for Northern analysis. The fragments were also cloned in the pTAG vector using the manufacturer protocol (R & D Systems Europe Ltd.). The different inserts were sequenced using a Universal or T7 primer or specific oligonucleotides for each sequence. No specific problems related to low abundance of mRNA were encountered during the cloning of VEGF-B and VEGF-C from Chinese hamster lung fibroblasts even though lung is a tissue where they represent poorly abundant mRNA species (24Olofsson B. Pajulosa K. Kaipainen A. Von Euler G. Joukov V. Saksela O. Orpana A. Pettersson R.F. Alitalo K. Eriksson U. Proc. Natl. Acad. Sci U. S. A. 1996; 93: 2576-2581Crossref PubMed Scopus (630) Google Scholar, 25Joukov V. Pajulosa K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1167) Google Scholar). The high percentage of homology (98%) with Chinese hamster, mouse, or human homologs has allowed us to use both mouse and hamster VEGF probes for Northern experiments. CCL39 cells in 12-well dishes (105 cells/well) were transiently transfected by CaPO4 precipitation with the indicated plasmids (250 ng/well of the reporter plasmid, 200 ng of expression vector, and 100 ng of CMV β-galactosidase as a control of transfection efficiency). Sixteen hours after addition of DNA, the cells were washed twice with PBS and incubated with Dulbecco's modified Eagle's medium with or without 7.5% fetal calf serum. Two days later, the cells were washed with cold PBS, and luciferase assays were performed as follows (Promega protocols and applications guide). Cells were lysed in lysis buffer (25 mm Tris-phosphate, pH 7.8, 2 mm DTT, 2 mm 1, 2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, 10% glycerol, and 1% Triton X-100) for 15 min at room temperature, and the lysate was cleared by centrifugation. The assay of luciferase activity was performed in a chemioluminometer in a buffer containing 20 mm Tricine, 1.07 mm(MgCO3)Mg(OH)2, 5H2O, 2.67 mm MgSO4, 0.1 mm EDTA, 33.3 mm DTT, 270 mm coenzyme A, 470 mmluciferine, and 530 μm ATP. Protein concentration was measured using the bicinchonic acid (BCA) protein assay kit (Pierce) with bovine serum albumin as standard. Cells were washed in ice-cold PBS and lysed in the "RNA Insta-Pure" buffer from Eurogentec. The supernatant was cleared by centrifugation, ethanol precipitated, and resuspended in sterile water. Dr. Werner Risau kindly provided the human VEGF promoter gene construct (−1176/+54) (16Ikeda E. Achen M.G. Breier G. Risau W. J. Biol. Chem. 1995; 270: 19761-19766Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar) cloned in the pGL2 basic vector from Promega. Construct −88/+54 was obtained by cutting the above vector bySmaI (one site in the vector and one site at position −88 of the promoter) and re-ligating. Construct −27/+54 was obtained by subcloning a DraI/NheI fragment within theSmaI/NheI sites of pGL2 basic. The −66/+54 and −52/+54 constructs were generated by PCR by using oligo 1, 5′-GCGGGTACC(T)CCCGGCGGGGCGG-3′; and oligo 2, 5′-GCGGGTACC(A)GCCATGCGCCCCC-3′, respectively. Bases shown in bold correspond to positions −66 and −52, respectively. At their 5′ ends, both oligos contain the KpnI restriction site. They were used in a PCR reaction with oligo 3, 5′-CTTTATGTTTTTGGCGTCTTCCA-3′, which corresponds to a sequence within the vector at the 3′ end of the promoter. The amplified fragments were digested with KpnI and NheI and inserted into the pGL2 basic vector (Promega). We mutated the AP-2 site, both Sp1 sites or all three binding sites by the PCR method (26Higushi R. Krummel B. Saiki R.K. Nucleic Acids Res. 1988; 16: 7351-7367Crossref PubMed Scopus (2107) Google Scholar, 27Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene. 1989; 77: 51-59Crossref PubMed Scopus (6861) Google Scholar). The following oligonucleotides were chosen: oligo 4, 5′-TGTATCTTATGGTACTGTAACTG-3′; oligo 5, 5′-GGGGCGGGCC(TA)GGGCGGGG-3′; oligo 6, 5′-CCCCGCCC(TA)GGCCCGCCCC-3′; oligo 7, 5′-GCCCCCCGGG(AA)GGGCCGGGG(AA)GGGGTCCG-3′; oligo 8, 5′-CGGACCCC(TT)CCCCGGCCC(TT)CCCGGGGGGC-3′; oligo 9, 5′-GGG(AA)GGCC(TA)GG(AA)GGGGTC-3′; and oligo 10, 5′-GAACCCC(TT)CC(TA)GGCCC(TT)CCC-3′. Oligo 4 corresponds to a sequence within the vector at the 5′ end of the promoter. For oligos 5, 6, 7, 8, 9, and 10, bases shown in bold indicate those modified in relation to the wild type sequence (oligos 5 and 6 for AP-2 mutation; oligos 7 and 8 for mutation of both Sp1 sites; oligos 9 and 10 for mutation of the AP-2 site and both Sp1 sites; see also Fig. 6 a). After obtention of the –1176/+54 mutated constructs, we digested them with SmaI to obtain the corresponding −88/+54 constructs. For the triple mutant, we used the construct −1176/+54 that was mutated for both Sp1 binding sites and oligos 9 and 10 for mutation of the remaining AP-2 binding site before digestion with SmaI to obtain the −88/+54 construct. The presence of the mutations were verified by gel sequencing. Confluent Raf-1:ER cells cultures were serum-starved overnight followed by stimulation with or without estradiol (1 μm) for 3 h. Nuclei were isolated by the isotonic/Nonidet P-40 procedure: cells were resuspended in HNB (0.5m sucrose, 15 mm Tris, pH 7.5, 60 mm KCl, 0.25 mm EDTA, pH 8, 0.125 mm EGTA, 0.5 mm spermidine, 1 mmDTT, 0.1 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 5 μg/ml pepstatin, 5 μg/ml leupeptin, 50 mmNaF, 40 mm β-glycerophosphate, 200 mmparanitrophenylphosphate, 0.2 mm orthovanadate) and homogenized in HNB containing 0.2% Nonidet P-40. Nuclei were recovered by centrifugation at 3000 rpm and rinsed in HNB alone. Nuclear extracts were then prepared by the method described by Dignam et al.(28Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9168) Google Scholar). The probe was synthesized to span the region of the human VEGF promoter comprised between the −88 and −66 bp: 5′-TTTCCGGGGCGGGCCGGGGGCGGGGTAT-3′ (random sequences added to the wild type sequence are shown in italic letters). Protruding 5′ ends were filled in with (exo-) Klenow fragment (from Stratagene) and [α-32P]-dCTP and dATP. The DNA binding reaction was performed for 15 min at room temperature in a final volume of 15 μl. A first volume of 7.5 μl was prepared containing 5 μg of nuclear extracts, 0.75 mg/ml poly(dI:dC) (Sigma), dialysis buffer (20 mm Hepes, pH 7.9, 60 mm KCl, 20% glycerol, 0.25 mm EDTA, 0.125 mm EGTA, 1 mmDTT), protease and phosphatase inhibitors) (see HNB). The residual 7.5 μl was comprised of 90 fmol of labeled probe (1–2.105cpm), with or without excess (60–600-fold) of unlabeled probe, and with or without excess (100-fold) of Sp1 or AP-2 consensus oligonucleotides (Promega): Sp1, 5′-ATTCGATCGGGGCGGGGCGAGC-3′ and 3′-TAAGCTAGCCCCGCCCCGCTCG-5′; AP-2, 5′-GATCGAACTGACCGCCCGCGGCCCGT-3′ and 3′-CTAGCTTGACTGGCGGGCGCCGGGCA-5′. The competitors were added 15 min before addition of the probe. Supershift assays were performed with 2.5–3 μg of nuclear extracts. The Sp1 (PEP-2) and AP-2α (C-18) antibodies were purchased from Santa Cruz Biotechnology. 0.2 μg of Sp1 antibody was incubated 30 min at 4 °C after addition of the probe, whereas 1 μg of AP-2α antibody was added 30 min before the addition of the probe. Electrophoresis was performed on a 4% polyacrylamide (30 acrylamide, 0.8 bisacrylamide) gel, in 0.5× TBE (44.5 mm Tris, 44.5 mm boric acid, 1 mm EDTA), for 4–5 h at 280–300 V/10–12 mA. Under normoxic conditions, exponentially growing CCL39 or its derivative PS 200 Chinese hamster lung fibroblasts express barely detectable levels of VEGF mRNA (right lanes of Fig. 1). Serum stimulation of growth-arrested CCL39 (data not shown) or PS 200 cells (left lanes of Fig. 1) triggers the induction of VEGF mRNA. However, this expression is strongly elevated in cells transformed either with Polyoma virus, Ha-Ras (Ras-Val12) (18Seuwen K. Lagarde A. Pouysségur J. EMBO J. 1988; 7: 161-168Crossref PubMed Scopus (101) Google Scholar) or a constitutive active form of MAP kinase kinase (MKK1-SS/DD) (19Brunet A. Pagès G. Pouysségur J. Oncogene. 1994; 9: 3379-3387PubMed Google Scholar). At least four isoforms that correspond to the spliced variants described (12Tischer E. Mitchell R. Hartman T. Silva M. Gospodarowicz D. Fiddes J.C. Abraham J.A. J. Biol. Chem. 1991; 266: 11947-11954Abstract Full Text PDF PubMed Google Scholar) detectably hybridize to a mouse VEGF probe. Fig. 1 shows that, in the Polyoma virus, Ha-Ras and MKK1-SS/DD transformed cells, the different VEGF mRNA isoforms are expressed at a level approximately 10-fold superior to that of control cells. This overexpression is particularly prominent for the clone 5c that overexpressed Ha-Ras (18Seuwen K. Lagarde A. Pouysségur J. EMBO J. 1988; 7: 161-168Crossref PubMed Scopus (101) Google Scholar). For each of the cell lines tested, FCS was able to increase the amount of VEGF mRNA, although in transformed cells the basal level was extremely elevated. However, this is not the case for cells expressing MKK1-SS/DD and isolated from a tumor produced in nude mice (T.MKK1-SS/DD). Interestingly, these cells were shown to be fully independent of serum growth factors (19Brunet A. Pagès G. Pouysségur J. Oncogene. 1994; 9: 3379-3387PubMed Google Scholar). This could explain the inability of serum to further modify the elevated level of VEGF mRNA in these tumor cells. In the different cell lines tested, the other members of the VEGF family, VEGF-B and VEGF-C, are constitutively expressed showing that both genes are not tightly regulated via growth or oncogenic factors even if VEGF-C seems to be up-regulated in MKK1-SS/DD transformed cells. To further examine the contribution of the Ras/p42/p44 MAP kinase pathway in VEGF expression, we have chosen a cell line expressing an estradiol-inducible Raf-1 (Raf-1:ER cells) (20Samuels M.L. Weber J.M. McMahon M. Mol. Biol. Cell. 1993; 13: 6241-6252Crossref Scopus (324) Google Scholar, 21Samuels M.L. McMahon M. Mol. Biol. Cell. 1994; 14: 7855-7866Crossref Google Scholar, 22Lenormand P. McMahon M. Pouysségur J. J. Biol. Chem. 1996; 271: 15762-15768Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). In this case, the p42/p44 MAP kinase activity is rapidly activated by estradiol, eliminating the contribution of SAP kinase cascade (p38MAPK/JNK) (29Han J. Lee J.D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2425) Google Scholar, 30Galcheva-Gargova Z. Derijard B. Wu I.H. Davis R.J. Science. 1994; 265: 806-808Crossref PubMed Scopus (532) Google Scholar, 31Derijard B. Raingeaud J. Barrett T. Wu I.H. Han J. Ulevitch J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1417) Google Scholar) and phosphatidylinositol 3-kinase cascade (32Rodriguez-Viciana P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1733) Google Scholar, 33Kodaki T. Woscholski R. Hallberg B. Rodriguez-Viciana P. Downward J. Parker P.J. Curr. Biol. 1994; 4: 798-806Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar) that are generally activated by serum or constitutively active Ras. Raf-1:ER-expressing cells (see "Experimental Procedures") were serum-deprived for 16 h and then stimulated by the addition of estradiol for the times indicated. Fig. 2 a shows that VEGF transcripts are expressed at a detectable level after 2 h and are maximally expressed after 3 h of estradiol stimulation, the expression being sustained for up to 5 h. A longer exposure of the blot shows detectable transcripts after only 30 min of stimulation (data not shown), and the expression of the three other spliced variants are revealed in Fig. 1. This rapid induction is compatible with the kinetics of activation of p42/p44 MAP kinases in these cells (22Lenormand P. McMahon M. Pouysségur J. J. Biol. Chem. 1996; 271: 15762-15768Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The expression of VEGF-B and VEGF-C mRNA species are not modified by estradiol treatment, confirming that activation of the p42/p44 MAP kinase pathway does not play any role in controlling their expression in these cells. Induction of VEGF mRNA in response to estradiol was dose-dependent, with 70% of the maximal induction obtained after a stimulation with 10 nm estradiol (Fig. 2 b, lane 7), and maximal induction obtained with 100 nm estradiol (Fig. 2 b, lane 9). This dose-response activity reflects the magnitude of activation of p42/p44 MAP kinase in these cells (22Lenormand P. McMahon M. Pouysségur J. J. Biol. Chem. 1996; 271: 15762-15768Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). If the activity of MKK1, the kinase directly downstream of Raf, is blocked by the specific inhibitor PD 098059 (34Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2597) Google Scholar) (Fig. 2 c, lane 7), the p42/p44 MAP kinase pathway-dependent induction of VEGF is inhibited by 50% after 4 h of stimulation by estradiol (compare lanes 3 and 7 of Fig. 2 c). This result supports the hypothesis that the p42/p44 MAP kinase cascade plays a key role in VEGF gene induction. The partial inhibition of MKK1 by PD 098059 (60–70% inhibition at this concentration) 2F. R. McKenzie, J. M. Brondello, and A. Brunet, unpublished results. explains the residual VEGF mRNA amount in Fig. 2 c. To test whether induction of VEGF mRNA by hypoxia is dependent or not on the action of growth factors, we submitted quiescent or exponentially growing CCL39 cells to hypoxia for four hours. Fig. 3 shows that oxygen deprivation on its own is able to induce VEGF mRNA induction in quiescent cells to a level that is comparable with the basal level present in exponentially growing cells. When serum is present, VEGF mRNA levels reached a level superior to that present in quiescent cells following hypoxia. There is no discrepancy between the level observed in this experiment and the results presented in Fig. 1. In Fig. 1, the blot was underexposed to compare the high levels of mRNA in transformed cells. We routinely observed a basal level of mRNA in exponentially growing cells. However, the amounts of mRNA obtained after serum stimulation of quiescent cells is at least five times more elevated. Again, to analyze more directly the contribution of p42/p44 MAP kinase, we used the Raf-1:ER cells stimulated by estradiol in the presence or absence of oxygen (see Fig. 2 b). At suboptimal concentrations of estradiol (0.1 and 1 nm), which do not maximally activate p42/p44 MAP kinase (22Lenormand P. McMahon M. Pouysségur J. J. Biol. Chem. 1996; 271: 15762-15768Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), we observed the induction of VEGF by hypoxia. Estradiol and hypoxia exert additive effects on VEGF mRNA induction at a dose of estradiol (10 nm) that induces half of the p42/p44 MAP kinase activity (see lane 8 of Fig. 2 b). This situation is comparable with that observed in Fig. 3 where exponentially growing cells are submitted to hypoxia. When a maximal dose of estradiol is used (100 nm or 1 μm), a small additive effect with hypoxia persists, but it is less detectable than that observed with 10 nm estradiol. In the presence of PD 09
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