From the Centre de Biochimie, CNRS-UMR 6543, Université de Nice, Parc Valrose, 06108 Nice, France
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ABSTRACT |
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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.
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INTRODUCTION |
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Angiogenesis is a fundamental physiological process by which new blood vessels are formed (1). One of the most widely described mechanisms controlling neovascularization associated with pathological processes (2) is the increased secretion by the "stressed cells" (inflammation, psoriasis) or nutrient-deprived tumor cells of multiple growth factors (3-7) and cytokines (8, 9). Among growth factors, two major classes have been characterized: acid and basic FGF of the FGF family (3, 4) and Vascular Endothelial Growth Factor, VEGF,1 a new family of secreted growth factors structurally related to PDGF (40% homology at the amino acid level) (5-7). VEGF, also described as a permeability factor, stimulates endothelial cell migration and proliferation in vitro and has angiogenic activity in vivo (10, 11). Different isoforms of 121, 165, 189, and 206 amino acids resulted from alternative splicing of the same gene (12). Many tissues and cell types express VEGF mRNA, especially tissues which are highly vascularized in addition to tumor-derived cell lines (13). Stimulation of serum-deprived NIH 3T3 cells by PDGF also results in VEGF induction in a Ras- and Raf-dependent manner (14). 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 (15-17). Considering the key role played by VEGF in the control of neovascularization (6, 7), 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 (18, 19). Exploiting a CCL39-derived cell line in which Raf-1 can be rapidly activated by estradiol (Raf-1:ER) (20-22), 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.
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EXPERIMENTAL PROCEDURES |
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Cell Culture-- The established Chinese hamster lung fibroblast line CCL39 (American Type Culture Collection), their derivatives PS120 and PS200, which lack NHE1 antiporter activity (23), 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 (20-22). 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.
Materials--
Restriction and DNA modifying enzymes were
obtained from New England Biolabs or from Eurogentec, Liège,
Belgium. [-32P]dCTP, [
-33P]dATP were
from ICN. Synthetic oligonucleotides were from Eurogentec, Liège,
Belgium.
Production of VEGF, VEGF-B, and VEGF-C Probes-- 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 (24), human VEGF-C (25) 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 (24, 25). 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.
Transient Transfection and Luciferase Assay--
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 -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 mM
luciferine, and 530 µM ATP. Protein concentration was
measured using the bicinchonic acid (BCA) protein assay kit (Pierce)
with bovine serum albumin as standard.
Preparation of RNA-- 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.
Promoter Construction and Mutagenesis Experiments--
Dr.
Werner Risau kindly provided the human VEGF promoter gene construct
(1176/+54) (16) cloned in the pGL2 basic vector from Promega.
Construct
88/+54 was obtained by cutting the above vector by
SmaI (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 the
SmaI/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 (26, 27). 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. 6a). 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.
Preparation of Nuclear Extracts and Gel Mobility Shift
Assays--
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 (0.5 M 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 mM
DTT, 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/ml
aprotinin, 5 µg/ml pepstatin, 5 µg/ml leupeptin, 50 mM
NaF, 40 mM -glycerophosphate, 200 mM
paranitrophenylphosphate, 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.
(28). 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 [
-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 mM
DTT), protease and phosphatase inhibitors) (see HNB). The residual 7.5 µl was comprised of 90 fmol of labeled probe (1-2.105
cpm), 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'.
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RESULTS |
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VEGF mRNA but Not VEGF-B and VEGF-C Are Regulated by Growth and Oncogenic Factors in CCL39 Cells-- 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) (18) or a constitutive active form of MAP kinase kinase (MKK1-SS/DD) (19). At least four isoforms that correspond to the spliced variants described (12) 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 (18). 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 (19). 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.
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p42/p44 MAP Kinase Cascade Specifically Induces VEGF mRNA Expression in Raf-1:ER-expressing 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) (20-22). In this case, the p42/p44 MAP kinase activity is rapidly activated by estradiol, eliminating the contribution of SAP kinase cascade (p38MAPK/JNK) (29, 30, 31) and phosphatidylinositol 3-kinase cascade (32, 33) 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. 2a 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 (22). 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. 2b, lane 7), and maximal induction obtained with 100 nM estradiol (Fig. 2b, lane 9). This dose-response activity reflects the magnitude of activation of p42/p44 MAP kinase in these cells (22). If the activity of MKK1, the kinase directly downstream of Raf, is blocked by the specific inhibitor PD 098059 (34) (Fig. 2c, 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. 2c). 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)2 explains the residual VEGF mRNA amount in Fig. 2c.
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Additive Effect of Hypoxia and Growth Factors on VEGF mRNA Induction-- 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. 2b). At suboptimal concentrations of estradiol (0.1 and 1 nM), which do not maximally activate p42/p44 MAP kinase (22), 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. 2b). 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 098059, the induction of VEGF mRNA by hypoxia still occurs even if the estradiol-mediated induction of VEGF mRNA is inhibited by 50% (see Fig. 2c). This result suggests that the hypoxia-mediated VEGF mRNA increase is independent of p42/p44 MAP kinase activity. This is further emphasized by the fact that hypoxia is not capable of activating p42/p44 MAP kinase activity in CCL39 cells (data not shown).
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Expression of Constitutively Active Members of the p42/p44 MAP
Kinase Pathway Increase VEGF Gene Transcription--
To assess the
mechanism by which p42/p44 MAP kinase cascade stimulates VEGF mRNA
production, we directly analyzed the activation of the VEGF promoter
coupled to the luciferase reporter gene as previously reported (15, 16,
35, 36). Fig. 4 shows that constitutively
active Ras (Ras-Val12) or constitutively active MKK1
(MKK1-SS/DD) can strongly stimulate the VEGF promoter (-1176/+54) in
the absence of FCS when compared with empty vector. Thus, both members
of the p42/p44 MAP kinase pathway can stimulate VEGF gene
transcription. VEGF gene transcription is not significantly increased
when a p44 MAP kinase encoding construct is cotransfected with the
reporter vector in the absence of serum. However, when cells are
transfected with a p44 MAP kinase encoding construct in the presence of
serum, the level of transcription is significantly increased when the
-1176/+54 and the -88/+54 constructs are used. VEGF promoter
constructs (888/+54 (data not shown) and -88/+54) still conserved
the capacity to be induced by constitutively active Ras or MKK1 even if
the response to Ras-Val12 is reduced by 40% for the
-88/+54 construct and the response to MKK1-SS/DD is not modified. This
is surprising since the -1176/-88 region contains putative AP-1
consensus binding sites which are targets of transcription factors
whose activity is up-regulated by the p42/p44 MAP kinase pathway
(Jun/Fos). The difference observed between the stimulatory effects of
Ras and MKK1 is not attributable to the stress-activated kinase
pathways that are also activated by Ras because transfection of c-Jun
N-terminal kinase (JNK) or p38/HOG in the presence of activating agents
(anisomycin, IL-1
) or their constitutively active activating kinases
(MKK3 or MKK4) (31) has no effect on VEGF promoter activity (data not
shown). However, if AP-2 and Sp1 binding sites are deleted (-27/+54), the induction obtained with MKK1-SS/DD is totally abrogated and the
activation obtained with Ras-Val12 is inhibited by
80%.
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Characterization of the p42/p44 MAP Kinase Pathway Responsive Region of the VEGF Promoter-- We next constructed and analyzed the activities of the -66/+54 and -52/+54 constructs in comparison with the -1176, -88 and -27/+54 constructs in the presence or absence of MKK1-SS/DD to localize the p42/p44 MAP kinase pathway responsive element. Fig. 5 shows that while the -1176 and -88/+54 constructs displayed constitutive base-line activity, which was increased by cotransfection with MKK1-SS/DD by a factor of 3.3 and 4.6, respectively, analysis of the -66, -52, and -27/+54 constructs showed a loss of both basal and MKK 1 SS/DD transcriptional activation. These results suggest that sequences between -88 and -66 are absolutely required for basal and p42/p44 MAP kinase-dependent pathway-stimulated promoter activity. The loss of both basal and stimulated transcriptional activity could reflect a truncation of transcription factor binding sites in the -88/+54 construct that can regulate the overall activity of the promoter.
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Cooperative Effects of AP-2 and Sp1 in MKK1-SS/DD Stimulation of
VEGF Promoter--
As shown by Gille et al. (37),
TGF--mediated induction of the VEGF promoter is a mechanism
involving the AP-2 transcription factor but not Egr-1, a member of the
same family. TGF-
acts via the EGF receptor whose signaling is in
part mediated by the p42/p44 MAP kinase pathway (for reviews, see Refs.
38 and 39). Transfection of an expression vector encoding AP-2 can
stimulate the VEGF promoter in CCL39 cells (data not shown), which is
in agreement with the data of Gille et al. (37). Based on
these results, we have investigated whether the p42/p44 MAP kinase
pathway induced VEGF mRNA levels by directly activating AP-2 or
Sp1, whose binding sites are present between -88 and -66. Fig.
6a shows that within this
region there exists two putative binding sites for Sp1 and one for
AP-2. These sites are conserved between human, mouse, and rat promoters
(35, 40-42). Therefore, the intact -88/+54 construct or a
construction with point mutations in the AP-2, both Sp1, or the three
binding sites (Fig. 6a) were transfected in exponentially
growing cells, and activation of the VEGF promoter was analyzed by
luciferase assay. The response to the p42/p44 MAP kinase module was
assessed by co-expression of constitutively active MKK1. Fig.
6b shows that mutations of the AP-2 or both Sp1 putative
binding sites done individually do not significantly modify basal and
MKK1-SS/DD-stimulated promoter activity. However, a combined mutation
of AP-2 and both Sp1 binding sites dramatically decreases basal and
MKK1-SS/DD-dependent transcriptional activation. This
result suggests a cooperative effect of AP-2 and Sp1 for maximal
transcriptional activation of the VEGF promoter.
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p42/p44 MAP Kinase Pathway Controls the Binding Activity of Nuclear
Protein Extracts to the 88/
66 VEGF Promoter Region in Raf-1:ER
Cells--
In order to confirm the specific role of AP-2 and Sp1
transcription factors in the regulation of the VEGF promoter, we
utilized a double-stranded probe encompassing the -88/-66-bp region
in EMSA experiments. Fig. 7 shows four
constitutive DNA binding complexes in resting or in
estradiol-stimulated Raf-1:ER cells (complexes a,
B, c, D; see lanes 1 and
6 of Fig. 7b). To demonstrate that either Sp1 or
AP-2 are present in at least the large complex B, we performed
supershift experiments. Indeed, Sp1 antibodies supershifted part of
complex B formed with extracts from resting cells (data not shown) or
estradiol-stimulated cells (Fig. 7a, left,
compare lanes 1 and 2). Similarly, AP-2
antibodies supershifted part of complex B with extracts from
estradiol-stimulated cells (Fig. 7a, right,
compare lanes 2 and 3). This supershift, however,
is more evident in the presence of Sp1 neutralizing oligonucleotides. The binding specificity of the complexes formed were determined by
exclusive competition with an excess of identical unlabeled DNA (Fig.
7b). Under resting conditions, DNA binding of complexes a,
B, and c are clearly inhibited by either Sp1 or AP-2-specific oligonucleotides (Fig. 7b, compare lane 1 or
5 with lanes 3 and 4). However, a
remarkable change is observed when p42/p44 MAP kinase was specifically
stimulated with estradiol. Nuclear extracts of cells stimulated for
3 h with estradiol show a strong increase in the binding of
complex B. This is seen in Fig. 7b, right panel, where complex B is better resolved and enlarged. Another striking change occurs when binding is inhibited with a 100-fold excess of Sp1
oligonucleotides (Fig. 7b, compare lanes 3 and
8). Under stimulated conditions, complex B resists the
competition with the Sp1 oligonucleotides, reflecting that more
proteins are bound and/or have a higher affinity. The same result is
observed when AP-2 oligonucleotides are used as a competitor, even if
in this case the labeling of the resistant complex B is less intense. Altogether, these data clearly demonstrate that Sp1 and AP-2
transcription factors bind to the -88/-66 region of the VEGF promoter
and that p42/p44 MAP kinase activity plays a key role in controlling
the VEGF promoter activity via these sites.
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DISCUSSION |
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The expression level of the VEGF mRNA is tightly regulated by
both transcriptional and post-transcriptional mechanisms (15-17, 35,
43). A variety of cytokines and growth factors, including epidermal
growth factor, transforming growth factor , transforming growth
factor
, interleukins 1 and 6 (44-47), as well as transforming agents such as v-Ha-Ras and v-Raf (14) were shown to induce VEGF
expression in several cell lines, and Pedram et al. (48) have proposed a role for Erk in the endothelin activation of the VEGF
promoter. However, the signaling cascades involved have not been fully
deciphered. Here, we have analyzed VEGF, VEGF-B (24), and VEGF-C (25)
expression in quiescent, serum-stimulated, or oncogenically transformed
CCL39 cells. In all the conditions where p42/p44 MAP kinase activity is
strongly enhanced, the VEGF mRNA levels are especially increased;
this is particularly notable in Ras-Val12 or MKK1-SS/DD
transformed cells. This first result suggests that regulation of VEGF
gene expression occurs through a p42/p44 MAP kinase-dependent mechanism. To confirm more directly the
role of the p42/p44 MAP kinase pathway in this mechanism, we have used a cell line where p42/p44 MAP kinase activity is tightly regulated by
an estradiol-inducible Raf-1 (Raf-1:ER). The stimulation of p42/p44 MAP
kinase cascade via the estradiol-dependent Raf activity eliminates the contribution of alternative signaling cascades such as
stress-activated protein kinases (p38 HOG/JNK) or phosphatidylinositol 3-kinase activated by serum or oncogenic Ras. Hence, production of VEGF
mRNA is stimulated by estradiol in a time- and a
dose-dependent manner. Furthermore, utilization of the MKK1
inhibitor PD 098059 (34) in Raf-1:ER cells after a maximal activation
of p42/p44 MAP kinase confirmed the importance of the p42/p44 MAP
kinase pathway in VEGF gene induction by growth factors. Also notable is that the two new VEGF-related mRNAs, VEGF-B and VEGF-C, show a
different mode of regulation. Their mRNA levels do not vary or do
not present the same spectacular induction in the transformed cells
tested and are not regulated by estradiol stimulation in Raf-1:ER cells
(compare Figs. 1 and 2a). A possible interpretation of this
data is that the cellular system investigated is not appropriate for
testing the fine regulation of both genes. Enholm et al.
(49) have observed an induction of VEGF and VEGF-C in IMR-90 cells by
serum, PDGF, EGF, and TGF-
but no regulation of VEGF-B after such
stimulations, which is in favor of a specific cell context for such an
induction.
We then analyzed the contribution of members of the p42/p44 MAP kinase
module on VEGF transcription. The human, the mouse, and the rat VEGF
promoter contains binding sites for AP-2, Sp1, or Sp1-related factors
(35, 40-42, 50) in addition to binding sites for AP-1 (51) and HIF-1
(16, 35), which regulate the transcription of the gene during hypoxia
(43). The mouse VEGF promoter also contains additional NFB binding
sites (42). A recent report by Gille et al. (37) described
the AP-2 transcription factor as the major factor implicated in the
TGF-
stimulation of VEGF gene transcription in A431 cells. They also
showed that Sp1 is constitutively bound to the promoter (37). However,
the promoter region responsible for such a regulation can bind AP-2 as
well as Egr-1, but AP-2 only regulates the promoter activity. Furthermore, they showed that promoter activity still remains even
after AP-2 binding site mutation but is reduced by 50%. In the present
study, we have dissected the events responsible for the increase of
VEGF transcription. The proximal promoter elements contained within
construct -88/+54 were found to be sufficient to drive the stimulation
of transcription induced by p42/p44 MAP kinase activation.
Interestingly, the -88/+54 construct can still be activated by
Ras-Val12 and MKK1-SS/DD. This is surprising since the
region comprised between -1176 and -88 contains a consensus AP-1
binding site that is regulated via the p42/p44 MAP kinase module.
However, mutations on the AP-2 or Sp1 binding sites, and in particular
mutations in the three binding sites, strongly reduced basal and
MKK1-SS/DD-stimulated VEGF promoter transcription. This is in accord
with the results of Gille et al. (37) who showed that even
if the AP-2 binding site is mutated, the promoter still displays high
basal activity, possibly driven through Sp1 binding.
We have further analyzed the -88/-66 region by EMSA as well as
supershift experiments. We observed four major DNA-proteins complexes
either with extracts from stimulated or unstimulated Raf-1:ER cells.
With the use of specific antibodies, we confirmed that AP-2 and Sp1 are
components of the more intense complex B. This fundamental role of Sp1
is in agreement with the results of Yoshida et al. (52) who
have shown that Sp1 is required for the stimulation of VEGF
transcription by TNF- and that antisense oligo-nucleotides can
partially inhibit the TNF-
-dependent production of VEGF.
We also show a spectacular increase in the labeling of part of complex
B upon estradiol stimulation, which is resistant to competition with
Sp1 oligonucleotides. Part of complex B is also enhanced upon estradiol
stimulation when AP-2 competitor oligonucleotides are used. This result
confirmed that activation of p42/p44 MAP kinase activation has a direct
effect on AP-2 and Sp1. We are presently investigating whether this
effect is mediated through an increase in binding affinity which could
be regulated by phosphorylation or mediated by an increase in the
amounts of both factors. However, we cannot exclude that p42/p44 MAP
kinase may activate other transcription factors that could be
components of the B complex. Egr-1 is a good candidate for such a
regulation even if it has been shown that the transfection of an Egr-1
encoding construct has no effect on the VEGF promoter activity (37). We
also observed two other complexes in EMSAs (a and c) that are inhibited
by an excess of cold Sp1 or AP-2 oligonucleotides. While complex a is
enhanced by estradiol treatment, complex c is not affected. Complex a
could be the result of an association between Sp1 and AP-2. Complex c
could be the result of Sp1 (41, 53) or AP-2-related factors binding
(54, 55). This is in agreement with previous results which have shown
that Egr-1 can bind to this region of the promoter (37).
Another interesting feature of VEGF regulation is its strong up-regulation upon oxygen deprivation by both transcriptional induction and stabilization of the mRNA by interaction of proteins with the 3'-untranslated region (15-17, 35, 43). Mukhopadhyay et al. have shown that hypoxic induction of VEGF is blocked by genistein and that c-Src is implicated in such an activation. They also showed that the dominant negative form of c-Src or Raf-1 can block hypoxic induction of VEGF (56). In our cell system, we show that such an induction is totally independent on growth factor action but that a combination of growth factor stimulation and oxygen deprivation have additive effects on VEGF mRNA induction. In Raf-1:ER cells, the inhibition of p42/p44 MAP kinase pathway by PD 098059 does not affect induction of VEGF by hypoxia (Fig. 2c), confirming that the induction of VEGF mRNA strictly attributable to p42/p44 MAP kinase activation and induction of VEGF by hypoxia are two independent mechanisms. There is no discrepancy between our results and those of Mukhopadhyay et al. (56) since Raf-1 can signal independently of Erk (22, 33), and dominant negative forms of Raf-1 can also titrate signals emerging from Ras which then activate independent pathways (32, 33). Our results have a strong physiological implication. The role of Ras oncogenes in the pathogenesis of human cancers is well established. Here we show that one of the target genes of the p42/p44 MAP kinase pathway which is activated by Ras is VEGF. As tumors are known to secrete growth factors activating the p42/p44 MAP kinase pathway such as FGF or relatives (3, 4, 57), VEGF expression initiated at least by constitutively active members of the p42/p44 MAP kinase pathway is amplified by a paracrine mechanism via the same transduction pathway. Such a regulation is strictly transcriptional and depends on activation of the VEGF promoter through at least two transcription factors, AP-2 and Sp1. We also show that growth factors and oxygen deprivation have additive effects which contribute to the increase of VEGF expression. We are now deciphering how the signal mediated by oxygen deprivation is sensed by the cells and which transduction pathways are implicated in VEGF induction.
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ACKNOWLEDGEMENTS |
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We thank Drs. W. Risau for providing the VEGF promoter constructs, P. Lenormand for establishing the Raf-1:ER CCL39 cells, F. R. McKenzie and D. E. Richard for critical review of this manuscript, and D. Grall and Y. Fantei for excellent technical assistance.
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FOOTNOTES |
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* 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 of Mr 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.
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