JOURNAL OF CELLULAR PHYSIOLOGY 190:238±250 (2002)DOI 10.1002/JCP.10059

Induction of KDR Expression in Bovine ArterialEndothelial Cells by Thrombin: Involvement of

Nitric Oxide

JIE WANG, IKUO MORITA,* MITSUE ONODERA, AND SEI-ITSU MUROTA

Section of Cellular Physiological Chemistry, Graduate School,Tokyo Medical and Dental University, Tokyo, Japan

Thrombin, a multifunctional serine protease, is generated at the site with vascularinjuries. It not only participates in the coagulation cascade, but also can induce alot of events related to cell mitogenesis and migration. In this study, weinvestigated the effect of thrombin on endothelial cell proliferation induced byvascular endothelial growth factor (VEGF). Thrombin promoted proliferation ofcultured bovine carotid endothelial cells in a time- and dose-dependent manner.Moreover, it drastically enhanced the cell growth stimulated by VEGF. Thisstimulatory effect was reduced by inhibitors of either protein kinase C (PKC) ormitogen-activated protein kinase kinase (MAPKK). Thrombin induced a signi®cantincrease in the level of mRNA of the kinase domain-containing receptor (KDR),but not tms-like tyrosine kinase (Flt-1), in a time-dependent manner, whichreached the maximum after 24 h of stimulation. This increase coincides well withthe KDR protein expression. The luciferase assay showed that thrombin inducedan about 7.5-fold increase in the KDR promoter activity compared with thecontrol. This enhanced KDR promoter activity was also abolished by inhibitors ofeither PKC or MAPKK. The deletion analyses indicated that the region betweenÿ115 and ÿ97 (containing Sp1 binding region) within the KDR promoter genewas required for the enhanced KDR expression induced by thrombin and VEGF.Moreover, the nitric oxide synthase (NOS) inhibitor abolished both theaccelerated cell proliferation and the increased KDR expression induced bythrombin and VEGF. This inhibition was abrogated by DETA NONOate, a NOdonor with long half-life. These ®ndings suggest that thrombin might potentiatethe VEGF-induced angiogenic activity through increasing the level of the VEGFreceptor KDR, in which production of NO is involved. J. Cell. Physiol. 190:238±250, 2002. ß 2002 Wiley-Liss, Inc.

Angiogenesis, the formation of new blood capillaries,plays a key role in the process of development, woundhealing, pathological conditions such as solid tumorgrowth and metastasis, rheumatoid arthritis, diabeticretinopathy, and atherosclerosis (Folkman, 1986; Gos-podarowicz et al., 1987; Klagsbrun and D'Amore, 1991).Several angiogenic growth factors such as aFGF, bFGF,PDGF, and vascular endothelial growth factor (VEGF)have been identi®ed. Now, it is widely recognized thatthrombin, a multifunctional serine protease, is also animportant growth factor for embryogenesis, angiogen-esis, and tumor cell metastasis (Chen and Buchanan,1975; Berk et al., 1991; Nierodzik et al., 1992; Mcnamaraet al., 1993; Nishino et al., 1993). Highly puri®edthrombin stimulated proliferation of cultured humanvascular endothelial cells (Chen and Buchanan, 1975),epithelial cells (Mcnamara et al., 1993), various ®bro-blast cells (Berk et al., 1991), mouse embryo cells(Nishino et al., 1993), vascular smooth muscle cells(Nierodzik et al., 1992), neuronal cells (Mohle et al.,

1997), and tumor cells (Pinedo et al., 1998), all of whichinvolved a speci®c membrane receptor of thrombin.

Thrombin is generated from prothrombin in placeswith injury or in¯ammation. Thrombin exhibits itseffects through activation of the G protein-coupledreceptor PAR-1 that belongs to a new family of proteaseactivated receptor (Vu et al., 1991). Binding to itsreceptor results in intracellular signal transduction,including the G protein-stimulated phosphatidylinosi-tol metabolism via phospholipase C-b (PLC-b) (Van

ß 2002 WILEY-LISS, INC.

*Correspondence to: Ikuo Morita, Section of Cellular PhysiologicalChemistry, Graduate School, Tokyo Medical and Dental Uni-versity, 1-5-45 Yushima, Bunkyo-Ku, Tokyo 113-8549, Japan.E-mail: morita.cell@tmd.ac.jp

Received 22 May 2001; Accepted 19 September 2001

Obberghen-Schilling and Pouyssegur, 1993), proteinkinase C (PKC) activation (Berk et al., 1990; Webb et al.,1993), intracellular calcium (Ca2�) mobilization, andcalcium-dependent protein kinase activation (Berket al., 1990, 1991). Intracellular protein tyrosine kinasesare also activated, thereafter leading to the tyrosinephosphorylation. Downstream components of thrombin-activated signaling cascades include Raf-1 and MAPkinase (Molloy et al., 1996; Ellis et al., 1999). Thesesignaling events lead to nuclear protooncogene tran-scription activation (c-fos, c-jun, c-myc) (Bobik andCampbell, 1993; Ross, 1993) and increased expressionof endogenous mitogenic factors, which subsequentlycause cell proliferation and migration.

VEGF, a disulphide-linked 46 kD glycoprotein,related to platelet-derived growth factor, is a potentangiogenic factor and also termed as vascular perme-ability factor (VPF) (Keck et al., 1989; Conn et al., 1990).It is widely expressed during the fetal velopment andadulthood. Recent studies have shown that VEGF ismost abundant in heart, spinal cord, cerebral cortex, andbrown fat. Vast lines of evidence indicate that VEGFinduces proliferation of endothelial cells. The biologicaleffects of VEGF on endothelial cells are mediated by thespeci®c membrane receptors kinase domain-containingreceptor (KDR) and ¯t-1, both are of the class III tyrosinekinases (Plouet and Moukadiri, 1990; Vaisman et al.,1990; Olander et al., 1991). KDR is only expressed inendothelial cells, whereas ¯t-1 is expressed both inendothelial cells and in monocytes. VEGF binds to KDRand ¯t-1 with high af®nity, and then initiates autopho-sphorylation of tyrosine residues in the cytoplasmicdomain of these receptors, which subsequently inducesintrinsic tyrosine kinase activation. The tyrosine-phos-phorylated receptors also stimulate cellular responsesinvolving PLC-g (Takahashi and Shibuya, 1997), PKC(Xia et al., 1996), phosphatidylinostial 3-kinase (PI3kinase) (Wu and Patterson, 1999), and MAPK activation(D'Angelo et al., 1995). The roles of VEGF, ¯t-1, andKDR in developmental angiogenesis are proved by theresults of the target disruption of these genes in mice.Mice only lacking VEGF, died in utero at days 11±12,and those lacking either ¯t-1 or ¯k-1 (the mousehomology of KDR) also died in utero at around days8.5±9.5. Flk-1 null mice failed to produce hematopoieticand endothelial precursors (Shalaby et al., 1995),whereas those lacking ¯t-1 had disorganized embryonicvasculature (Fong et al., 1995). Most interestingly, boththe neutralized antibodies against VEGF and expres-sion of the antisense to VEGF sequences can inhibittumor growth in vivo by suppressing the tumor angio-genesis (Prewett et al., 1999). These results provideevidence that the VEGF/KDR system plays a centralrole in angiogenesis.

Thrombosis has long been recognized to be associatedwith angiogenesis-related diseases such as cancer. First,thrombin itself can be detected in a variety type oftumors (Zacharski et al., 1995). Second, thrombin stimu-lated angiogenesis in both the in vivo and the in vitromodel systems (Tsopanoglou et al., 1993). Thrombin is apotent stimulus that induces the release of VEGFfrom stores within the platelet (Mohle et al., 1997).Thrombin also upregulates expression of the VEGFreceptors to potentiate VEGF activity on endothelial

cells (Tsopanoglou and Maragoudakis, 1999). On theother hand, VEGF accelerates thrombin generation(Zucker et al., 1998), suggesting these two factors mayconsist of a positive feedback loop to accelerate theangiogenic process.

Here we show evidence from another aspect thatthrombin augmented the VEGF-induced cell prolifera-tion, up-regulated the KDR gene transcription, and in-creased KDR expression in cultured BAE cells; further,all events were dependent on the NO production.Moreover, we identi®ed for the ®rst time that the DNAsequence containing a Sp1 site that was essential formodulating the effects of thrombin and VEGF in thepromoter region of the KDR gene. Our ®nding mayreveal a physiologically important positive feedbackmechanism for the ampli®cation of the angiogenicresponse in vitro.

MATERIALS AND METHODSCell culture

Bovine arterial endothelial cells (BAE cells) wereisolated from bovine carotid artery and maintained aspreviously described (Morita et al., 1984). The cells wereseeded in 100-mm culture dishes with MEM Medium(Life Technologies, Grand Island, NY) containing 10%fetal bovine serum (FBS) (JRH Biosciences, Lenexia,KS) and sub-cultured with 5% FBS±MEM. At con-¯uence, BAE cells were harvested with 0.05% trypin and0.05% EDTA in PBS and resuspended in 5% FBS±MEMfor further experiments. All cells were incultured at378C in 5% CO2, and 95% air. The medium were changedevery 2±3 days. BAE cells between passages 10±20 wereused for this study.

Cell proliferation assay

For cell proliferation assay, BAE cells were seeded in48-well multi-culture plate with 10% FBS±MEM, andallowed to attach for 24 h. Medium was then replacedwith 0.5% FBS±MEM containing test substancesand incubated for four days. To assess the effect of nitricoxide synthase (NOS) inhibition, Nitro-L-Arginine(Sigma) was added to the media at a ®nal concentrationof 2 mM one day before the test substances were added.In the experiments in which a NO donor was used,cells were treated with DETA NONOate (CaymanChemical Company, Ann Arbor, MI) at the ®nalconcentration of 0.01 mM from the beginning of theexperiment. To test the effects of reagents on mitogensignaling, BAE cells were treated with 10 mMGF109203X (Sigma) or 0.2 mM PD98059 (Sigma) forone day, then mitogens were added. To determinewhether the effect of thrombin was mediated via pro-teolytic activation of the thrombin receptor, a thrombinantagonist Hirudin (Sigma) was added to the mediumat a ®nal concentration of 5 U/ml for one day. Thecell numbers were counted by a Coulter Counter ZM(Counter Electronics, Luton Beds, UK).

Extraction of total cellular RNA andRT-PCR of VEGF receptors

When cells reached subcon¯uency, they were washedonce with PBS and cultured in the absence or presenceof 5 U/ml of thrombin combined with 10 ng/ml of

EFFECTS OF THROMBIN ON KDR EXPRESSION 239

VEGF for indicated times. For the isolation of RNA, cellswere washed once with PBS, and RNA was isolated byusing the ISOGENE reagent (Clontic) according to theprotocol. RT-PCR was performed with the SuperScriptOne-Step RT-PCR system (GIBCO-BRL) according tothe manufacturer's instructions. To detect the VEGFreceptor 1 (¯t-1) and 2 (KDR/¯k-1), the followingprimers were used as previously reported: sense primer50-CAGCGGCTTTTGTGGAAGACTCAC-30 and anti-sense primer 50-ACTTCTCGGTGTCACTTCTTGGAC-30 for ¯t-1, and sense primer 50-CAACAAAGTCGGGA-GAGGAG-30 and antisense primer 50-ATGACGATGGA-CAAGTAGCC-30 for KDR (Witzenbichler et al., 1998).As an internal control, mRNA of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was ampli®ed byusing the sense primer 50-CCACCCATGGCAAATTC-CATGGCA-30 and the antisense primer 50-TCTA-GACGGCAGGTCAGGTCCACC-30 (Slater et al., 1999).PCR was performed with 35 cycles of the followingampli®cation protocol: 948C for 1 min, 488C for 1.5 min,and 728C for 1 min. Ten microliter of the ampli®ed PCRproducts were electrophoresed on a 1% agarose gel(Sigma), stained with ethidium bromide and photo-graphed with a Polaroid camera under UV light.

Immuno¯uorescence staining of KDRand image analysis

BAE cells (1� 104) were seeded into 24-well cultureplates, VEGF was added at the indicated days treatedwith or without thrombin in 0.5% FBS±MEM andcultured for 2 days. Cells were washed twice with PBS,®xed with 1.9% formaldehyde, and rinsed three timeswith PBS. After blocking with PBS containing 0.2%bovine serum albumin (blocking buffer), the cells wereincubated with af®nity-puri®ed anti-KDR antibody(Santa Cruz Biotechnology, Santa Cruz, CA) (1:20diluted in blocking solution) for 45 min at roomtemperature and then with the FITC-conjugated goatantirabbit IgG (Sigma) (1:40 solution) for 45 min. The¯uorescence image analysis was performed using aninteractive laser cytometer ACAS-570 (Meridian, Oke-mos, MI) and the ¯uorescence intensities of individualcells were measured. For each condition, three wellswere prepared and four areas per well (cell number;15±20/each area) were assayed.

Plasmids

The pGL3 Basic plasmid and pGL3 Control (PromegaCorp, Madison, WI) contained the ®rely luciferase gene.The pGL3 Basic had no promoter, whereas the pGL3Control was driven by the SV40 promoter and enhancer.The pSVb-Gal control vector (Promega Corp) containedthe b-galactosidase gene driven by the SV40 promoterand enhancer.

Five fragments were ampli®ed from human genomicDNA isolated from human placenta (Clinic, PaloAlto, CA) by polymerase chain reation (PCR) usingspeci®c oligonucleotides designed according to a pub-lished KDR promoter sequence (Gerber et al., 1997).The fragments were cloned into pGL3 Bisic plasmid,after 50-BgI II (Promega Corp) and 30-Hind III(Promega Corp) digestion. All constructs were se-quenced from the 50 and the 30 end to con®rm orienta-

tion, and the sequence was compared with previouslypublished data.

Transfection and luciferase assay

Transient transfections were performed with Fu-GENE 6 transfection reagent (Boehringer) accordingto the manufacturer's instructions. Cells (1� 104) wereseeded in 24-well tissue-culture plates one day prior totransfection. The pSVb-Gal and pGL3 were co-trans-fected as negative control. Cells were incubated intransfection mixture (containing 0.18 mg of the appro-priate reporter constructs, 0.02 mg pSVb-Gal to correctthe variability in transfection ef®ciency), and 0.4 mlFuGENE 6 transfection reagent in a total volume of 1 mlMEM per well for 48 h. Luciferase assays wereperformed using a Luciferase Assay System (PromegaCorp), and determined with the AML3000 microtiterplate. The transfection ef®ciency was normalized to theb-galactosidase production detected by the AURORATM

GAL-XE system (Chemiluminescent Receptor Assayfor b-Galactosidase, ICN Pharmaceuticals, Inc. CostaMesa, CA) according to the protocol.

Statistical analysis

All experiments were performed three times intriplicate. All values are expressed as mean�SE, unlessotherwise indicated. Comparison of the effects of dif-ferent inhibitor and time points were performed byANOVA. Multigroup comparisons were carried outusing Bonferroni-modi®ed t-test. P< 0.05 is consideredto be statistically signi®cant.

RESULTSThrombin accelerates the VEGF-induced

cell growth of BAE cells

Because thrombin and VEGF are reported to induceproliferation of various tumor cell lines, so we ®rstinvestigated their effects on the growth of BAE cells. Thegrowth-arrested BAE cells were treated with thrombinat concentrations ranging from 0.5 to 5 U/ml for 4 days.As shown in Figure 1A, thrombin induced a small butsigni®cant increase in the cell proliferation. Interest-ingly, in the presence of VEGF, thrombin induced afurther increase in the cell proliferation in a dose-dependent manner, with the maximal effect of about3.9� 0.5-fold increase at the concentration of 5 U/ml.Then we examined the time course of the thrombin-induced cell proliferation. As shown in Figure 1B,during the ®rst 3 days, the cell proliferation in thecontrol, VEGF, and thrombin-treated cells was nearlythe same. After cultured for four days, stimulation with10 ng/ml VEGF or 5 U/ml thrombin alone increased thecell proliferation by 1.3�0.2-fold (P< 0.05) or1.92� 0.1-fold (P<0.05) compared with the control,respectively. However, when BAE cells were treatedwith thrombin and VEGF, the cell proliferation wassigni®cantly enhanced in a time-dependent manner,with about a three-fold increase compared with VEGFalone at the fourth day. These data show that thrombinnot only induces cell proliferation, but it also acceleratesthe VEGF-induced growth of BAE cells, suggesting thatthrombin and VEGF may exert a cooperative effect onthe BAE cell proliferation.

240 WANG ET AL.

Effects of PKC and MAP kinase downregulationon the thrombin-induced cell proliferation

Since PAR-1 has been suggested to act as thethrombin receptor in many types of cells (Vergnolleet al., 1999), we examined whether PAR-1 mediatedthe thrombin-induced cell proliferation or not. Forthis purpose, we examined the effects of Hirudin, anantagonist of PAR-1. Pretreatment with Hirudin (5 U/ml) resulted in inhibition of cell proliferation inducedby thrombin, VEGF, and thrombin combined withVEGF to 53� 3% and 55�8%, respectively (Fig. 2A),suggesting that the effect of thrombin on the cellproliferation was mediated through the cell surfacereceptor.

Since thrombin has been reported to activate PKC andMAPK (He et al., 1992), we asked whether PKC orMAPK was involved in the thrombin-induced cellproliferation or not. The BAE cells were pretreated witha highly selective PKC inhibitor GFX for one day.Increasing the concentration of GFX from 1 to 10 mMcaused a dose-dependent inhibition in the cell prolifera-tion induced by thrombin and thrombin with VEGF.The maximal growth inhibition obtained at 10 mM was51 and 49%, respectively (data not shown). Similarly,PD98059, the speci®c MAP kinase kinase inhibitor,also reduced the cell proliferation induced by thrombinand thrombin with VEGF in a dose-dependent manner,and the maximal growth inhibition was obtained at0.2mMby55�1.2%,or43�1.9%,respectively (Fig.2C).In contrast, these inhibitors alone had no effect onthe cell proliferation. These data indicate that thePKC and MAP kinase signal pathways were necessaryfor BAE cell proliferation induced by thrombin andVEGF.

Thrombin upregulated KDR mRNAexpression, protein synthesis, and

promoter activity induced by VEGF

To understand whether thrombin potentiates theVEGF action by regulation of expression of the VEGFreceptors, we ®rst performed RT-PCR to examine geneexpression of the VEGF receptors KDR and Flt-1. Asshown in Figure 3A, PCR products of KDR, Flt-1, andGAPDH were obtained with the expected size of 819,735, and 452 bp, respectively. The control cells showedclear expression of Flt-1 mRNA, whereas expression ofthe KDR mRNA was hardly detected. However, stimu-lation with thrombin and VEGF induced a time-dependent increase in the level of KDR mRNA, withthe maximal effect after 24 h of treatment. In contrast,no apparent difference in the Flt-1 mRNA level wasobserved between control and the treated groups.Neither VEGF nor thrombin alone had effects on theKDR mRNA level (data not shown). Then we measuredthe KDR protein expression using a immuno¯uores-cence staining assay. The data in Figure 3B showed asimilar increase in the level of KDR proteins as that ofthe KDR mRNA, and it is quite clear that thrombincaused an enhancement in the expression of KDRproteins induced by VEGF.

Next, to understand whether the thrombin-inducedincrease in the KDR mRNA and proteins was mediatedthrough transcriptional regulation or not, we examined

Fig. 1. Effects of thrombin and VEGF on proliferation of BAE cells.A: Dose dependency. BAE cells (4� 104) were seeded into 24-wellculture plates and treated with various concentrations of thrombin forfour days with or without VEGF (10 ng/ml). B: Time dependency. BAEcells (4� 104) cultured with 0.5% FBS±MEM were treated with 5 U/mlthrombin with or without 10 ng/ml VEGF for the indicated timeperiods. The data are presented as means�SE from four samples.Similar results were obtained from three separate experiments.Statistical analysis was performed using Bonferroni-modi®ed t-test.*P< 0.05 or **P<0.01 vs. the unstimulated control.

EFFECTS OF THROMBIN ON KDR EXPRESSION 241

the effect of thrombin on the KDR promoter activityusing a reporter gene assay. A smallest constructcontaining the full KDR promoter activity (ÿ226 to�268) (Patterson et al., 1995) inserted upstream of aluciferase reporter gene (pGL3 (ÿ226 to �268)-luc) wastransfected into BAE cells, and the luciferase activitieswere evaluated. VEGF increased the KDR promoteractivity, and this increase was more signi®cant in thepresence of thrombin, which changed in a time- anddose-dependent manner (Fig. 4A,B). These data indicatethat thrombin enhanced the effect of VEGF on cellproliferation through up-regulation of the KDR promo-ter activity.

Involvement of PKC and MAP kinase in thetranscriptional activation of the KDR promoter

induced by thrombin and VEGF

To determine if the PAR-1, PKC, and MAP kinasesignal transduction pathways are also involved in thetranscriptional activation of the KDR gene promoterinduced by thrombin and VEGF, the effects of Hirudin,GFX, and PD98059 on the KDR promoter activity wereexamined. The BAE cells were pretreated with Hirudin,GFX, or PD98059 for 1 h followed by addition ofthrombin with or without VEGF. The data clearlyshowed that pretreatment with Hirudin, GFX, orPD98059 drastically inhibited the transcriptional activ-ity of the KDR promoter gene increased by thrombin andVEGF at the same manner as that on the cell prolifera-tion (Fig. 5A,B,C). On the other hand, these inhibitors

alone did not affect the KDR promoter activity. Thesedata argue again that the effect of thrombin wasmediated via proteolytic activation of the receptorsand the PKC and MAP kinase signal transductionpathways are involved in the up-regulation of KDR bythrombin and VEGF.

Deletion analysis of the human KDR promoter

To identify the essential regions for the promoteractivity increased by thrombin and VEGF, we generatedvarious mutants, in which the 50-end from ÿ226 to ÿ40was gradually deleted, while the common 30-end fromÿ40 to �268 was kept unchanged. Then they wereinserted into the luciferase reporter plasmid pGL3 andtransfected into BAE cells together with pSVb-Gal (tocorrect the differences derived from transfection ef®-ciency). The luciferase activity was normalized to thatof the pGL3 Control vector. As shown in Figure 6, thepromoter region from ÿ226 to �268 showed a 7.5-foldinduction of the luciferase activity upon thrombin andVEGF stimulation as compared with the untreatedcells. Deletion of the region from ÿ226 to ÿ115 did notaffect both the basal and the thrombin and VEGFinduced transcriptional activity of the KDR promoter.However, when the construct was further deleted toÿ97, an about 80% reduction in the transcriptionalactivity was observed, and the basal promoter activitywas also reduced. This suggests that the region betweenÿ115 and ÿ97 is required for the full response of theKDR promoter gene to thrombin and VEGF; what's

Fig. 2. Effects of the thrombin antagonist, PKC inhibitor andMAPKK inhibitor on cell proliferation induced by thrombin andVEGF. BAE cells cultured in 0.5% FBS±MEM were treated withHirudin, GFX, or PD98059 one day earlier than treatment withthrombin with or without VEGF. A: 5 U/ml Hirudin, (B) 10 mM GFX,and (C) 0.2 mM PD98059. The cell number was determined on the

fourth day. The data represent means�SE from four samples. Similarresults were obtained from three separate experiments. Statisticalanalysis was performed using Bonferroni-modi®ed t-test. *P< 0.05 vs.the unstimulated group; **P<0.05 vs. treated with thrombin orVEGF alone.

242 WANG ET AL.

more, it is important for the basal transcription of theKDR gene.

Contribution of NOS to the human KDRexpression and cell proliferation induced by

thrombin and VEGF

Since studies have implicated a role of NO in theangiogenic process (Ziche et al., 1994; Jenkins et al.,1995), and thrombin induced NO release from culturedHUVEC (Tsukahara et al., 1993), so we examinedwhether NO contributed to the thrombin-enhancedKDR promoter activity or not. As shown in Figure 7,increases in the KDR promoter activity induced bythrombin, VEGF, or thrombin with VEGF were blockedby pretreatment with Nitro-L-Arginine, a NOS inhibi-tor, by 56� 2%, 70� 1.5%, and 89�5%, respectively.However, this inhibitory effect of Nitro-L-Arginine wasabrogated by the simultaneous addition of DETANONOate, a NO donor with long half-life (Fig. 8A).The similar results were also observed in the KDRprotein expression (Fig. 8B). These data suggest thatNO was involved in the thrombin-induced KDR genetranscription and protein expression.

Then we asked whether NO participated in thethrombin-induced cell proliferation or not. Pretreat-ment of BAE cells with Nitro-L-Arginine greatly reduced

the cell proliferation induced by thrombin and/or VEGF(Fig. 9A); however, this inhibition was repressed bythe simultaneous addition of DETA NONOate (Fig. 9B).These data indicate that the endogenous NO productionis necessary for the cell proliferation induced bythrombin and/or VEGF in BAE cells.

Because the present data showed that NO, PKC, andMAPK, all played important roles in the thrombin-induced cell proliferation, we subsequently investigatedthe relationship among these three pathways. As shownin Figure 10, the MAPK inhibitor PD98059 abolishedthe NO-induced KDR promoter activity and cell pro-liferation, whereas the PKC inhibitor failed to inhibitthe increase in the KDR promoter activity and cellproliferation induced by the NO donor, suggesting thatthe effect of NO requires the activation of MAPK, but notPKC cascade.

DISCUSSION

It has been realized that thrombin has many functionsdistinct from the activation of the coagulation cascade. Itis capable of transmitting intracellular signals andappears to participate in metastasis and the tumor-associated angiogenesis (Nierodzik et al., 1992; Even-Ram et al., 1998). Involvement of thrombin in angiogen-esis has been reported to be associated with VEGF

Fig. 3. Regulation of the VEGF receptors KDR and Flt-1. A: RT-PCRanalysis of the KDR mRNA level from BAE cells. BAE cells wereseeded in 10% FBS±MEM for one day and cultured with 0.5% FBS±MEM for another one day, and then treated with VEGF and thrombinfor the indicated times. Treatment with thrombin and VEGF was thesame as that in Figure 2. RT-PCR was performed with the samesamples using the KDR, Flt-1, and GAPDH primer sets as described inMaterials and Methods. The data represent a typical result from three

experiments, and all had the similar results. B: BAE cells weresubjected to indirect immuno¯uorescence staining using af®nity-puri®ed rabbit antibody against KDR as described in Materials andMethods. The ®nal concentration of the primary antibody used was 5ng/ml. A: Background staining without the KDR antibody. B: KDRexpression in cells treated with VEGF. C: KDR expression in cellstreated with VEGF and thrombin. Magni®cation: 100�.

EFFECTS OF THROMBIN ON KDR EXPRESSION 243

production in the endothelial cells. For this reason, wewere attempting to investigate the molecular mechan-isms by which thrombin led to angiogenesis in endothe-lial cells. In the present study, we investigated theeffects of thrombin on the VEGF-induced angiogenicactivity and the VEGF receptor expression, which isthought to be a major pathological change in tumor cells.

The results demonstrated that thrombin potentiatedcell growth induced by VEGF and increased expressionof the VEGF receptor KDR in cultured BAE cells.

VEGF mediates its effects through the endothelialcell-speci®c receptors. Two of these receptors have beenidenti®ed: namely, the phosphotyrosine kinase recep-tors Flt-1 and KDR/Flk-1. Both of these receptors are

Fig. 4. Effects of thrombin and VEGF on the KDR gene promoteractivity in BAE cells. BAE cells were transiently transfected with 0.18mg KDR gene promoter luciferase reporter containing the KDRpromoter from ÿ226 to �268 relative to the transcriptional start site,and 0.02 mg pSVb-Gal to correct for variability in transfectionef®ciency. The cells were then treated for 48 h with thrombin andVEGF. The relative promoter activities were determined as describedin the Materials and Methods. The promoter activity of the control

transfection was arbitrarily set to 1. *P<0.05 vs. the unstimulatedgroup. A: Dose-dependent changes of the luciferase activity. Thetransfected BAE cells were treated with various concentrations ofthrombin combined with or without VEGF in MEM containing 0.5%FBS. B: Time-dependent changes of the luciferase activity. Thetransfected BAE cells were treated with 5 U/ml thrombin for theindicated time periods, with or without 10 ng/ml VEGF.

244 WANG ET AL.

expressed in BAE cells. The KDR/Flk-1 receptor isinvolved primarily in mitogenesis, whereas the Flt-1receptor is required for the endothelial cell morphogen-esis (Ferrara and Davis, 1997). Although BAE cellsexpress both KDR/Flk-1 and Flt-1 receptors, it ispresently unclear which one is responsible for mediatingthe thrombin-induced effects. The ®nding that thrombinand VEGF did not in¯uence the Flt-1 mRNA suggeststhat thrombin exhibited its effects through induction ofKDR, but not of Flt-1.

As the receptor for VEGF, KDR plays an importantrole in angiogenesis and endothelial growth. But themechanisms underlying its expression are not wellunderstood. As the best we know, this is the ®rst reportto characterize the KDR promoter activity induced bythrombin and VEGF. We identi®ed the regions contain-ing a positive regulatory element within the 50-¯ankingregion of the human KDR gene. By deletion analysis, a18-bp fragment within the 50-¯anking region of the KDRpromoter was identi®ed, which appears to be essentialfor the KDR expression induced by thrombin and VEGF.No signi®cant changes were noted after deletion of theelement from ÿ226 to ÿ164, whereas deletion betweenÿ164 and ÿ115 reduced the promoter activities to 77%that of the whole promoter fragment. However, thepromoter activity was reduced signi®cantly when ele-ments fromÿ115 toÿ97 was removed. This region of the

KDR promoter only contains a putative Sp1 binding site.Sp1 belongs to a zinc ®nger family of transcriptionfactors that can activate transcription of a subset ofgenes containing Sp1 sites, including human tissuefactor (Cui et al., 1996), human VEGF gene (Finkenzel-ler et al., 1997), and human matrix metalloproteinase-2(Qin et al., 1999). The present data suggest that Sp1within ÿ115 to ÿ97 may be necessary for up-regulationof the KDR promoter activity induced by thrombin andVEGF. Thrombin is an important growth factor andimmuno-regulator for the tissue injury. Highly puri®edthrombin stimulates proliferation of chick embryo andmammalian ®broblasts under serum-free culture con-dition, and it activates monocytes, NK cells, T cells, andendothelial cells. Early studies showed that action at cellsurface is suf®cient for thrombin to stimulate cellproliferation by initiating transmembrane signals.Several laboratories have cloned members of a proteo-lytically active seven-transmembrane G-protein-linkedreceptor family that include PAR1, PAR2, PAR3, andPAR4 (Vu et al., 1991; Nystedt et al., 1994; Ishiharaet al., 1997; Kahn et al., 1998). These receptors areproteolytically activated by thrombin to generate a newNH2 terminus, which acts as a tethered ligand andpromotes the interaction between the receptor and theG-proteins at the intracellular side of the membrane.Activation of these G-protein-linked receptors appears

Fig. 5. Effects of the thrombin antagonist Hirudin, PKC, andMAPKK inhibitors on the increased KDR promoter activity inducedby thrombin and VEGF. Transfection was the same as in Figure 4. A:The transfected BAE cells were pretreated with Hirudin (5 U/ml) for 1h before addition of thrombin with or without VEGF, and incubatedfor 48 h. B: The transfected BAE cells were pretreated with GFX (10mM) for 1 h before addition of thrombin with or without VEGF, and

incubated for 48 h. C: The transfected BAE cells were pretreated withPD98059 (0.2 mM) for 1 h before addition of thrombin combined withor without VEGF, and were incubated for 48 h. Relative promoteractivities were determined as described in the section of Materials andMethods. The promoter activity of the control transfection wasarbitrarily set to 1. *P<0.05 vs. the untreated group.

EFFECTS OF THROMBIN ON KDR EXPRESSION 245

to be responsible for all of the proteolytic cleavage-dependent signals initiated by thrombin (Vu et al.,1991). The signaling cascade that follows thrombininteraction with receptor tyrosine kinases on endothe-lial cells is only partially understood that is to causeactivation of tyrosine phosphorylation of receptors, andinitiation of series cellular signaling events includingPLC-g, PKC, phosphatidylinositol 3-kinase, and MAPkinase. To understand whether the thrombin-inducedKDR up-regulation was also through these pathways,we investigated effects of various inhibitors of thethrombin receptor, PKC, and MAP kinase kinase. Here,we show that induction of KDR by thrombin and VEGFwas inhibited by pretreatment with Hirudin, a thrombinreceptor antagonist. This suggests that thrombin actsvia activation of the receptor. We also show here that thethrombin-induced KDR increase was abolished bypretreatment with GFX, a highly selective PKC inhi-bitor, or PD98059, a speci®c mitogen-activated proteinkinase kinase (MAPKK) inhibitor at a concentrationreported to completely block PKC and MAPKK activa-tion in BAE cells. These data suggest that PKC andMAPKK activation was an essential step in the KDRinduction by thrombin and VEGF.

NO, which is formed from the guanidine-nitrogenterminal of L-arginine by the NOS, is activated byreceptor-dependent and -independent agonists in acalcium-dependent manner. It has been identi®ed asan important mediator of endothelial functions regulat-ing the vascular tone and proliferation of vascular cells(Ziche et al., 1993, 1994; Morbidelli et al., 1996).Increased NO levels have been found in human tumors

Fig. 6. Deletion analysis of the increased human KDR promoteractivity by thrombin and VEGF. The pGL3 deletion constructs con-taining variable length of the human KDR promoter DNA sequencesranging from ÿ226 to �268 bp were introduced into BAE cells. The

transfected BAE cells were treated with thrombin with or withoutVEGF, and incubated for 48 h. The relative promoter activities weredetermined as described in the section of Materials and Methods. Thepromoter activity of the control cell was arbitrarily set to 1.

Fig. 7. Effects of the NOS inhibitor on the KDR promoter activityinduced by thrombin and VEGF. Serum-starved BAE cells weretreated with thrombin and VEGF in MEM containing 0.5% FBS andincubated for four days. In some plates, cells were pretreated with theNOS inhibitor Nitro-L-Arginine (2 mM) for one day. The relativepromoter activities were determined as described in the Materials andMethods section. The promoter activity of the control transfection wasarbitrarily set to 1. *P< 0.01 vs. the NOS inhibitor-untreated group.

246 WANG ET AL.

(Thosen et al., 1994; Cobbs et al., 1995). Transfection ofthe inducible NOS into a colon adenocarcinoma cell linepromoted the tumor growth and the transgenic cellswere more vascularized than the parental cells (Jenkinset al., 1995). Other observations that agree that NO is aspeci®c signal for tumor vascularization show thatblocking the NOS activity retards the growth of tumors(Oruceive and Lala, 1996) and excessive production ofNO sustains tumor growth (Doi et al., 1996). Thesedemonstrate that NO is an essential mediator forangiogenesis. Our data show that inhibition of NOS

Fig. 8. Effects of the NOS inhibitor and NO donor on the KDRpromoter activity and KDR expression induced by thrombin andVEGF. The transfected BAE cells were treated with thrombin, VEGF,or a combination of thrombin and VEGF in MEM containing the NOdonor (DETA NONOate, 0.01 mM) for 48 h. In some plates, cells were®rst pretreated with the NOS inhibitor Nitro-L-Arginine for one day.A: Luciferase assay. The relative promoter activities were determinedas described in the Materials and Methods section. The promoteractivity of the control transfection was arbitrarily set to 1. *P<0.01vs. NOS inhibitor-untreated group. **P< 0.05 vs. the NOS inhibitor-treated group. B: KDR protein expression. The quanti®cation of KDRprotein expression was described in Materials and Methods. *P<0.05vs. VEGF and thrombin-treated group, **P<0.05 vs. the NOSinhibitor-treated group.

Fig. 9. Effects of NOS inhibitor Nitro-L-Arginine and the NO donorDETA on thrombin- and VEGF-induced cell proliferation. A: Serum-starved BAE cells were treated with thrombin and VEGF in MEMcontaining 0.5% FBS for four days. In some plates, cells were ®rstpretreated with the NOS inhibitor Nitro-L-Arginine (2 mM) for oneday. The columns and bars represent means�SE of four samples.Similar results were obtained from three separate experiments.*P<0.05 vs. untreated group; **P<0.05 vs. the NOS inhibitor-untreated group, respectively. B: Serum-starved BAE cells weretreated with thrombin and VEGF in MEM containing 0.5% FBS withor without NO donor DETA (0.01 mM) for four days. In some plates,cells were ®rst pretreated with the NOS inhibitor Nitro-L-Arginine (2mM) for one day. The columns and bars represent means�SE of foursamples. Similar results were obtained from three separate experi-ments. *P< 0.01 vs. the thrombin�VEGF-stimulated group;**P<0.05 vs. the NOS inhibitor-treated group. The statisticalanalysis was performed using Bonferroni-modi®ed t-test.

EFFECTS OF THROMBIN ON KDR EXPRESSION 247

reduced thrombin-induced KDR up-regulation, whereasaddiminastration of NO donor suppressed the inhibitoryeffect of the NOS inhibitor, indirectly suggesting thatNO contributes to the thrombin-induced KDR up-regulation in endothelial cells. However, it was also

reported that SNAP downregulated VEGF expression insmooth muscle cells (Tsurumi et al., 1997), and NOinhibited proliferation and migration of endothelial cells(Yang et al., 1994; Lau and Ma, 1996). These contra-dictory data indicate that NO has both inhibitory andactivating effects on angiogenesis, depending upon thecellular environment and the types of cells in which theassay is performed.

It is known that endothelial NOS was induced by basic®broblast via activation of MAP kinase cascade, sug-gesting that MAPK cascase lies upstream of NO.However, in this study, the expression of KDR enhancedby NO was suppressed by MAPKK inhibitor, suggestingthat MAPK cascade lies downstream of NO. Thepossibility is supported by the paper that the guanylatecyclase inhibitor, blocked the activation of ERK1/2induced by VEGF (Parenti et al., 1998). In this study,the up-regulation of KDR was abrogated by thrombinand VEGF was also abrogated by either MAPKKinhibitor or PKC inhibitor. In contrast, that by NOdonor was abrogated by MAPKK inhibitor, but not byPKC. These data suggest that NO produced by thrombinand VEGF affects the MAPKK activity and the PKC liesupstream of the MAPK cascade. Recent study hasdemonstrated that VEGF-induced activation of Raf-MAPK-MAP kinase and DNA synthesis are mainlymediated by PKC-dependent pathway (Takahashi et al.,1999). More precise relationship between the PKC, andMAPK cascades and NO is not yet clear.

In summary, our study ®rstly demonstrate thatthrombin potentiated the endothelial cell growthinduced by VEGF, which is dependent on up-regulationof KDR via a transcriptional level mechanism. Wefurther demonstrated the critical role of the region fromÿ115 to ÿ97 of the KDR promoter in mediating up-regulation of KDR induced by thrombin and VEGF, andin the basic KDR promoter activity. We also show thatNO is required for thrombin to potentiate endothelialgrowth induced by VEGF. The exact mechanisms bywhich the region fromÿ115 toÿ97 exerts its activity onthe expression of KDR remains unclear and is, there-fore, the focus of our future study.

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