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Biochemical and Biophysical Research Communications 286, 923–928 (2001)

doi:10.1006/bbrc.2001.5494, available online at http://www.idealibrary.com on

GE2 Stimulates VEGF Expression in Endothelial Cellsia ERK2/JNK1 Signaling Pathways

ama Pai, Imre L. Szabo, Brian A. Soreghan, Songul Atay,irofumi Kawanaka, and Andrzej S. Tarnawski1

edical Service, Department of Veterans Affairs Medical Center, Long Beach, California; andepartment of Medicine, University of California at Irvine, Irvine, California 92717

eceived July 31, 2001

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Vascular endothelial growth factor (VEGF) plays anssential role in the initiation and regulation ofngiogenesis—a crucial component of wound healingnd cancer growth. Prostaglandins (PGs) stimulatengiogenesis but the precise mechanisms of their pro-ngiogenic actions remain unexplained. We investi-ated whether prostaglandin E2 (PGE2) can induceEGF expression in rat gastric microvascular endo-

helial cells (RGMEC) and the signaling pathway(s)nvolved. We demonstrated that PGE2 significantly in-reased ERK2 and JNK1 activation and VEGF mRNAnd protein expression. Incubation of RGMEC withD 98059 (MEK kinase inhibitor) significantly reducedGE2-induced ERK2 activity, VEGF mRNA and pro-

ein expression. Furthermore, PD 98059 treatment al-ost completely abolished JNK1 activation. Our data

uggest that PGE2-stimulates VEGF expression inGMEC via transactivation of JNK1 by ERK2. Oneotential implication of this finding is that increasedG levels in cancers could facilitate tumor growth bytimulating VEGF synthesis and angiogenesis. © 2001

cademic Press

Key Words: angiogenesis; prostaglandins; endothe-ial cells; VEGF; MAPK.

Angiogenesis, formation of new capillary blood ves-els, which enables delivery of oxygen and nutrients, isssential for wound and ulcer healing and growth andetastasis of solid tumors (1, 2). Vascular endothelial

rowth factor (VEGF) is the most potent inducer ofngiogenesis, endothelial cell proliferation and capil-ary permeability (3, 4). VEGF is distinct from other

Supported by the Department of Veterans Affairs, Medical Re-earch Service, Merit Review and Research Enhancement Awardrogram to A.S.T.

1 To whom correspondence should be addressed at Gastroenterol-gy Section (111G), DVA Medical Center, 5901 East Seventh Street,ong Beach, CA 90822. Fax: (562) 494-5675. E-mail: [email protected].

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ial cell-specific mitogen in vitro, and the only knownrowth factor with vascular permeability-inducing ac-ivity (5, 6). Activation of VEGF during healing of gas-ric mucosal injury has previously been established (7).

Prostaglandins (PGs) are arachidonic acid metabo-ites with a wide range of biological actions. They areroduced by cyclooxygenases (Cox-1 and Cox-2) in aide variety of tissues and function as lipid mediators.rostaglandin E2 (PGE2) can cause vasodilation andtimulate angiogenesis (8–10). Recent studies haveemonstrated that inhibition of Cox-2 activity and thusesulting prostaglandin generation, significantly down-egulates VEGF expression, inhibits angiogenesis anduppresses tumor growth (11, 12). Previous studiesave shown that PGE2 stimulates VEGF expression inultured osteoblasts (13), synovial fibroblasts (14) andat Muller cells (15). In contrast, in retinal epithelialells, choroidal fibroblasts and vascular endothelialells elevation of intracellular cAMP did not have anyignificant effect on VEGF expression suggesting thatn endothelial cells VEGF expression could be modu-ated by different signaling systems. Whether PGE2

an regulate VEGF expression in endothelial cells re-ains unknown.Mitogen-activated protein (MAP) kinases are serine-

hreonine kinases that are rapidly activated in re-ponse to a variety of growth stimuli (16–18). Arachi-onic acid and its metabolites (e.g., hydroxyeicosa-etranoic acids, prostaglandins) activate ERK1, ERK2nd JNK1 kinases in vascular smooth muscle cells (19,0). Whether PGE2 can induce VEGF expression inndothelial cells and the signaling pathway(s) involvedemain unknown forming the basis of this study.

ATERIALS AND METHODS

Endothelial-SFM medium and heparin were obtained from GibcoRL (Grand Island, NY). Antibiotic:antimycotic supplement andther tissue culture reagents were obtained from Fisher Scientific

0006-291X/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

(Tustin, CA). Fetal bovine serum (FBS) was obtained from AtlantaBat((

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Vol. 286, No. 5, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

iologicals (Norcross, GA). Rabbit polyclonal anti-ERK2, anti-VEGF,nti-JNK1 antibody, GST-c-Jun was obtained from Santa Cruz Bio-echnology (Santa Cruz, CA). Prostaglandin E2, myelin basic proteinMBP), and other chemicals were obtained from Sigma Chemical Co.St. Louis, MO).

Cell culture. Rat primary gastric microvascular endothelial cellsere isolated following the modified procedure previously described

21). Isolated cells were routinely grown in endothelial–SFM me-ium supplemented with 20% FBS, 100 mg/ml heparin and antibiot-cs in an atmosphere of 5% CO2 and 95% air at 37°C in a humidifiedncubator. Subcultures were made from confluent stock cultures byrypsinization in PBS containing 0.5 mM EDTA and 0.25% trypsin.

Kinase activity assay. Kinase activity assays were performed fol-owing the modified procedure previously described (22). In brief,erum starved cells were treated with either vehicle, PD 98059nd/or PGE2. After incubation, cells were washed in cold PBS andysed in lysis buffer containing 20 mM Hepes (pH 7.4), 50 mM-glycerophosphate, 1% Triton X-100, 10% glycerol, 2 mM EGTA, 1M DTT, 10 mM NaF, 1 mM sodium orthovanadate, 1 mM PMSF, 1g/ml aprotinin and 1 mg/ml leupeptin. The cell lysates were clarifiedy centrifugation at 14,500 rpm for 10 min at 4°C. JNK1 and ERK2ere immunoprecipitated from protein normalized cell lysates usingrotein A Sepharose–antibody complex. Beads were washed twiceith lysis buffer and twice with kinase assay buffer (25 mM Hepes,0 mM MgCl2, 20 mM b-glycerophosphate, 20 mM para-nitrophenylhosphate, 2 mM DTT). Kinase reactions were performed in 30 ml ofinase assay buffer containing 5.0 mCi/tube [g-32P]ATP, 30 mM ATP,nd 2 mg of GST-c-Jun for JNK1 and 30 mg of MBP for ERK2 andncubated for 30 min at 30°C. Reactions were terminated by theddition of SDS sample buffer and boiling for 5 min. The proteinsere resolved by SDS–PAGE. The gels were stained with Coomassielue R250, dried and autoradiographed. Individual bands were cutut and counted by liquid scintillation spectrometry.

RNA isolation and RT-PCR. Total RNA was isolated using theuanidinium isothiocyanate-phenol–chloroform method (23). Re-erse transcription and polymerize chain reaction (RT/PCR) wereerformed using a GeneAmp RNA PCR kit and a DNA thermal cyclerApplied Biosystems, Foster City, CA) as previously described (7).riefly, RT was performed at 42°C for 15 min, at 99°C for 5 min, andt 5°C for 5 min. Resulting cDNA was amplified by using primershat recognize all four isoforms of VEGF mRNA. The nucleotideequence of primers utilized were 59-CCTGGTGGACATCTTCCAG-AGTACC-39 (sense) and 59-GAAGCTCATCTCTCCTATGTGCT-GC-39 (antisense). The primers for b-actin were 59-TTGTAACC-ACTGGGACGATATGG-39 (sense) and 59-GATCTTGATCTTCAT-GTGCTAGG-39 (antisense). The primers for b-actin were purchased

rom Clontech, Palo Alto, CA. The PCR amplification was performedor 28 cycles of 1 min at 94°C for denaturing, 1 min at 55°C fornnealing and 2 min at 72°C for extension. Ten microliter aliquots ofhe products were subjected to electrophoresis on a 1.25% agarose gelnd DNA was visualized by ethidium bromide staining. Location ofhe products and their sizes were determined by using a 100-bpadder (GIBCO, Gaithersburg, MD). The gel was photographed un-er ultraviolet illumination. For quantitative assessment of the PCRroducts, a video image analysis system (Image-1/FL, Universalmaging Corp., Westchester, PA) was used. The image system canistinguish density on a scale of 0–255 units. Each measurementas standardized by subtracting the background intensity in average.

Western blot analysis. Western blot analysis was performed iden-ically as in our previous paper (24). In brief, cells were washed withce-cold PBS and lysed with a lysis buffer. The protein content of thelarified lysate was determined using a BCA protein assay kit (Piercehemical Co., Rockford, IL). Cell lysates containing equal amountsf proteins were subjected to SDS–PAGE and transferred onto aitrocellulose membrane. Blots were stained with Ponceau Red tonsure equal loading and complete transfer of proteins. The blot was

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ncubated with a blocking buffer, and probed with specific primaryntibodies (listed under Materials). Blots were washed and incu-ated with specific peroxidase-conjugated secondary antibodies. Af-er washing, bound antibody was visualized by an ECL detectionystem (Amersham Corp., Arlington Heights, IL) according to theanufacturer’s instructions. The density of the protein bands was

nalyzed using a video image analysis system (Image-1/FL, Univer-al Imaging Corp., Westchester, PA).

Statistical analysis. All data are reported as mean 6 SD. Statis-ical significance of differences between mean values was assessed bytudent’s t test for unpaired data. A P value of ,0.05 was consideredtatistically significant.

ESULTS

Effect of PGE2 on VEGF mRNA expression. Ratastric microvascular endothelial cells (RGMEC) werereated with varying concentrations (100 nM–10 mM)f PGE2 for 1 and 3 h. Exposure of endothelial cells toGE2 for 3 h caused dose-dependent increase in VEGFRNA expression (Fig. 1).

Effect of PGE2 on activation of ERK2, JNK1 and p38inases. Because arachidonic acid and its metabolite5-hydroxyeicosatetraenoic acid have been shown toctivate MAP kinase in vascular smooth muscle cells19, 20), we investigated whether PGE2-mediated in-rease in VEGF expression involves activation of MAPinases including ERK2, JNK1 and p38. Treatment ofGMEC with PGE2 (100 nM to 10 mM, 5 min) signifi-

antly increased ERK2 activity (Fig. 2) in a dose-ependent manner. To determine whether this activa-

FIG. 1. Prostaglandin E2 dose-dependently increases VEGFRNA expression in rat gastric microvascular endothelial cells.erum-starved RGMEC were treated with varying concentrations ofGE2 (100 nM to 10 mM) for 3 h. RT-PCR using specific primers forEGF was performed as described under Materials and Methods.uantitative assessments of the PCR products were performed usingideo image analysis system. Values are mean intensity 6 SD ofhree separate experiments.

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ion is mediated via MEK, RGMEC were pretreatedith 20 mM PD 98059 (a MEK inhibitor) for 50 min and

ollowed by PGE2 (10 mM, 5 min) treatment. Inhibitionf MEK significantly inhibited PGE2-induced ERK2ctivity (Fig. 3). Furthermore, treatment of RGMECsith PGE2 significantly increased JNK1 activity (Fig.) but not p38 kinase (data not shown). Inhibition ofRK2 activation by PD 98059 treatment almost com-letely abolished JNK1 activation (Fig. 4), clearly in-icating that ERK2 transactivates JNK1.

Effect of PD 98059 on VEGF mRNA expression. Be-ause PGE2 significantly increased both VEGF mRNAxpression and ERK activity, we sought to determinehether PGE2-induced VEGF mRNA expression in-olves the ERK2 pathway. Pretreatment of RGMECith MEK inhibitor (PD 98059, 20 mM) for 50 min

ignificantly reduced PGE2-induced VEGF mRNA (Fig.), indicating involvement of ERK2 in PGE2-mediatedpregulation of VEGF mRNA expression.

Effect of PGE2 on VEGF protein expression. To as-ertain whether PGE2-induced VEGF mRNA expres-ion accompanies increase in VEGF protein expres-ion, RGMEC were treated with PGE2 (10 mM) for 3, 6

FIG. 2. PGE2 dose-dependently increases ERK2 activity in ratastric microvascular endothelial cells. Serum-starved RGMEC werereated with varying concentrations of PGE2 (100 nM to 10 mM) formin. Cell lysates containing equal amounts of protein after treat-ents were immunoprecipitated with anti-ERK2 and subjected to in

itro kinase reaction using [g-32P]ATP and myelin basic protein as aubstrate as described under Materials and Methods. (Top) Repre-entative autoradiograph showing myelin basic protein phosphory-ated by ERK2 immunoprecipitated from RGMEC. (Middle) Westernlot analysis using equal amounts of total protein from the corre-ponding lysates used to determine ERK2 protein levels. (Bottom)uantitative analysis of ERK2 activity (6SD) from three separatexperiments each performed in triplicate.

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ncreased at all the time points studied with a maximalesponse at 3 h. Pretreatment of RGMEC with PD8059 significantly reduced PGE2-induced increase ofEGF protein expression (Fig. 6), suggesting involve-ent of the ERK2 pathway.

ISCUSSION

Prostaglandins stimulate angiogenesis in vitro andn vivo (9–11, 25, 26). Prostaglandins concentrationnd expression of cyclooxygenase (PGs synthesizingnzymes) are increased in a variety of cancers (27–29),hus implicating PGs in pathological angiogenesis (11,2). However, the precise mechanism(s) by which PGstimulate angiogenesis is not explained.Vascular endothelial growth factor (VEGF) is known

o induce angiogenesis by increasing endothelial cellroliferation, migration and microvascular hyperper-eability (30). Besides normal angiogenesis (e.g., dur-

ng wound healing) (7, 30), VEGF also stimulatesathological angiogenesis including cancer growth (1,1, 32). Although previous studies have reported thatrostaglandins increase VEGF expression in other

FIG. 3. MEK inhibitor (PD 98059) reduces PGE2-induced ERK2ctivation in rat gastric microvascular endothelial cells. Serum-tarved RGMEC were treated with either (a) vehicle, (b) PD 9805920 mM, 50 min), (c) PGE2 (10 mM, 5 min), or (d) PD 98059 plus PGE2.ell lysates containing equal amounts of protein after various treat-ents were immunoprecipitated with anti-ERK2 and subjected to in

itro kinase reaction using [g-32P]ATP and myelin basic protein as aubstrate as described under Materials and Methods. (Top) Repre-entative autoradiograph showing myelin basic protein phosphory-ated by ERK2 immunoprecipitated from RGMEC after variousreatments. (Middle) Western blot analysis using equal amounts ofotal protein from the corresponding lysates used to determine ERK2rotein levels. (Bottom) Quantitative analysis of ERK2 activitymean 6 SD) from three separate experiments each performed inriplicate.

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ells (osteoblasts, synovial fibroblasts and Muller cells)13–15), the effect of PGE2 on the ultimate target cellsor angiogenesis—endothelial cells—remain unknown.he present study demonstrates for the first time that

n rat gastric microvascular endothelial cells PGE2 in-reases VEGF mRNA and protein expression viaRK2/JNK1 signaling pathway thus providing a newolecular mechanism for the pro-angiogenic action

f PGs.Earlier studies have shown important role of ERK1

nd ERK2 MAP kinases in the regulation of angiogen-sis, endothelial cell proliferation, reduction of apopto-is, and stimulation of VEGF expression by activatingts transcription via recruitment of the AP-2/Sp1 (acti-ator protein-2) complex to the VEGF promoter (33). Inmore recent study, Pages and co-workers demon-

trated using a Chinese hamster lung fibroblast lineCCL39) and its derivatives (PS120, PS200) thattress-activated protein kinases (JNK and p38/HOG)re essential for VEGF mRNA stability (34). Our find-ng that PGE2-induced ERK2 transactivates JNK1 inastric microvascular endothelial cells suggests thatNK1 activation is likely to play a role in increasedEGF expression by stabilizing the VEGF transcript.

FIG. 4. PGE2-induced ERK2 transactivates JNK1 in rat gastricicrovascular endothelial cells. Serum-starved RGMEC were

reated with either (a) vehicle, (b) PD 98059 (20 mM, 50 min), (c)GE2 (10 mM, 5 min), or (d) PD 98059 plus PGE2. Cell lysatesontaining equal amounts of protein after various treatments weremmunoprecipitated with anti-JNK1 and subjected to in vitro kinaseeaction using [g-32P]ATP and GST-c-Jun as a substrate as describednder Materials and Methods. (Top) Representative autoradiographhowing GST-c-Jun phosphorylated by JNK1 immunoprecipitatedrom RGMEC after various treatments. (Middle) Western blot anal-sis using equal amounts of total protein from the correspondingysates used to determine JNK1 protein levels. (Bottom) Quantita-ive analysis of JNK1 activity (mean 6 SD) from three separatexperiments each performed in triplicate.

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Several studies have demonstrated that PGE2 in-uces VEGF expression in rat Muller cells, lung cancerells and in a human monocytic (THP-1) cell line (15,

FIG. 5. Inhibition of ERK2 activation reduces PGE-induced VEGFRNA expression in rat gastric microvascular endothelial cells. Serum

tarved RGMEC were treated with either (a) vehicle, (b) PD 98059 (20M, 50 min), (c) PGE2 (10 mM, 3 h), or (d) PD 98059 plus PGE2. RT-PCRsing specific primers for VEGF was performed as described underaterials and Methods. Quantitative assessments of the PCR productsere performed using video image analysis system. Values are mean

ntensity 6 SD of three separate experiments.

FIG. 6. PGE2 induces VEGF protein expression in rat gas-ric microvascular endothelial cells. Serum-starved RGMEC werereated with either (a) vehicle, (b) PD 98059 (20 mM, 50 min), (c)GE2 (10 mM, 3 h), or (d) PD plus PGE2. Detergent-solubilized cell

ysates (150 mg protein per lane) were subjected to Western blotnalysis using a specific polyclonal antibody against VEGF. Theelative densities of protein bands were analyzed using a video imagenalysis system. Values are mean relative density 6 SD of threeeparate experiments.

35, 36) via cAMP. In contrast, Kvanta demonstratedtbcpdMev

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hat in retinal pigment epithelial cells, choroidal fibro-lasts and vascular endothelial cells, elevation ofAMP had no significant effect on VEGF mRNA ex-ression (37). Furthermore, activation of cAMP in en-othelial cells has been shown to inhibit activation ofAPK (38). In the present study we found that in

ndothelial cells PGE2-induced VEGF expression in-olves ERK2/JNK1 activation.

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pge2 stimulates vegf expression in endothelial cells via erk2/jnk1 signaling pathways

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