g q -trpc6-mediated ca entry induces rhoa activation and

G�q-TRPC6-mediated Ca2� Entry Induces RhoA Activationand Resultant Endothelial Cell Shape Changein Response to Thrombin*

Received for publication, August 30, 2006, and in revised form, December 11, 2006 Published, JBC Papers in Press, December 29, 2006, DOI 10.1074/jbc.M608288200

Itender Singh, Nebojsa Knezevic, Gias U. Ahmmed, Vidisha Kini, Asrar B. Malik, and Dolly Mehta1

From the Department of Pharmacology and Center for Lung and Vascular Biology, College of Medicine, University of Illinois,Chicago, Illinois 60612

RhoA activation and increased intracellular Ca2� concentra-tion mediated by the activation of transient receptor potentialchannels (TRPC) both contribute to the thrombin-inducedincrease in endothelial cell contraction, cell shape change, andconsequently to themechanismof increased endothelial perme-ability. Herein, we addressed the possibility that TRPC signalsRhoA activation and thereby contributes in actinomyosin-me-diated endothelial cell contraction and increased endothelialpermeability. Transduction of a constitutively active G�qmutant in human pulmonary arterial endothelial cells inducedRhoA activity. Preventing the increase in intracellular Ca2�

concentration by the inhibitor of G�q or phospholipase C andthe Ca2� chelator, BAPTA-AM, abrogated thrombin-inducedRhoA activation. Depletion of extracellular Ca2� also inhibitedRhoA activation, indicating the requirement of Ca2� entry inthe response. RhoA activation could not be ascribed to store-operated Ca2� (SOC) entry because SOC entry induced withthapsigargin or small interfering RNA-mediated inhibition ofTRPC1 expression, the predominant SOC channel in theseendothelial cells, failed to alter RhoA activity. However, activa-tion of receptor-operatedCa2� entry by oleoyl-2-acetyl-sn-glyc-erol, the membrane permeable analogue of the G�q-phospho-lipase C product diacylglycerol, induced RhoA activity.Receptor-operated Ca2� activation was mediated by TRPC6because small interferingRNA-inducedTRPC6knockdown sig-nificantly reduced Ca2� entry. TRPC6 knockdown also pre-vented RhoA activation, myosin light chain phosphorylation,and actin stress fiber formation aswell as inter-endothelial junc-tional gap formation in response to either oleoyl-2-acetyl-sn-glycerol or thrombin. TRPC6-mediated RhoA activity wasshown to be dependent on PKC� activation. Our results dem-onstrate that G�q activation of TRPC6 signals the activation ofPKC�, and thereby induces RhoA activity and endothelial cellcontraction.

The continuous vascular endothelium lining the intima ofthe blood vessels regulates vascular smooth muscle tone, host-defense reactions, woundhealing, angiogenesis, and function ofthe semi-permeable endothelial barrier (1). Thrombin by bind-ing to the endothelial cell surface protease-activated receptor-1(PAR-1)2 induces a signaling cascade resulting in the develop-ment of minute inter-endothelial junctional gaps that lead toincreased endothelial permeability, the hallmark of tissueinflammation (1). Formation of these gaps occurs as the resultof cell shape change induced by actinomyosin-mediated endo-thelial cell contraction (1).Activation of the monomeric GTPase, RhoA, is crucial in

signaling endothelial cell shape change; i.e. the “rounding up”response of endothelial cells (1–5). Evidence from several celltypes, including endothelial cells, indicated that G-protein-coupled receptors activate RhoA via the � subunit of the het-erotrimeric GTP-binding protein Gq (5–10). For example,RhoA was not activated in response to thrombin in plateletslacking G�q (6, 9). G�q was also required for growth factor-induced activation of RhoA in endothelial cells (7). The G�qpathway is known to induce an increase in intracellular Ca2�

(11–14), which occurs secondary to the mobilization of Ca2�

from endoplasmic reticulum stores and increase in Ca2� entryvia plasmamembrane non-selective cation channels. The latterresponse,mediated by activation of store-operatedCa2� (SOC)and receptor-operated Ca2� (ROC) channels, is crucial for sus-taining the increase in intracellular Ca2� concentration inendothelial cells (11–14). In the present study,we surmised thatthe G�q-mediated increase in intracellular Ca2� concentrationwas crucial in regulating RhoA activity downstream of G-pro-tein-coupled receptors. We had previously shown that activa-tion of PKC�, a downstream effector of G�q and a Ca2�- anddiacylglycerol (DAG)-dependent enzyme (15, 16), was requiredfor RhoA activation (4, 5). Thus, we addressed the possibilitythat themechanism of RhoA activation involves G�q-PLC-me-diated activation of Ca2� entry.

* This work was supported in part by National Institutes of Health Grants HL45638 (to A. B. M.) and 71794 and 084153 (to D. M.) and National ScientificDevelopment Grant from the American Heart Association (to G. U. A.). Thecosts of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked “advertise-ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by American Physiological Society for Giles F. FilleyMemorial Award. To whom correspondence should be addressed: 835 S.Wolcott Ave., Chicago, IL 60612. Tel.: 312-355-0236; Fax: 312-996-1225;E-mail: [email protected].

2 The abbreviations used are: PAR-1, protease-activated receptor-1; TRPC,transient receptor potential channel; PLC, phospholipase C; MLC, myosinlight chain; HPAEC, human pulmonary arterial endothelial cells; siRNA,small interference RNA; RGS2, regulator of G protein signaling 2; GST, glu-tathione S-transferase; OAG, oleoyl-2-acetyl-sn-glycerol; DAG, diacylglyc-erol; SOC, store-operated Ca2�; ROC, receptor-operated Ca2�; dn, domi-nant negative; GEF, GTP exchange factor; GDI, GDP dissociation inhibitor;ER, endoplasmic reticulum; IP3, inositol 1,4,5-trisphosphate; PKC, proteinkinase C; TER, transendothelial electrical resistance; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N�,N�-tetraacetic acid.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 11, pp. 7833–7843, March 16, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

MARCH 16, 2007 • VOLUME 282 • NUMBER 11 JOURNAL OF BIOLOGICAL CHEMISTRY 7833

by guest on April 10, 2018

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Members of themammalian homologues ofDrosophila tran-sient receptor potential channels (TRPC) family form the ROCand SOC channels in many cell types (11–14). TRPC1, TRPC4,and TRPC5 form constituents of SOC because they are acti-vated upon depletion of intracellular Ca2� store by IP3 bindingto IP3R (11–14). TRPC3, TRPC6, and TRPC7 represent ROCconstituents as they are activated by DAG and do not requirestore depletion (11–14). TRPC1, TRPC4, and TRPC6 wereshown to regulate Ca2� entry and the increased endothelialpermeability response (17–25). Because at the mRNA levelTRPC1 and TRPC6 are more abundantly expressed in humanendothelial cells than other cell types (11, 18, 23, 26), in thepresent study we addressed the roles of TRPC1 and TRPC6 inregulating RhoA activation.We demonstrate here the essentialinvolvement of TRPC6 in mediating RhoA activity and endo-thelial cell contraction downstream of activation of PKC� inresponse to thrombin. These results for the first time establisha causal link between the G�q-mediated increase in cytosolicCa2� via TRPC6 and activation of RhoA, which in turn medi-ates endothelial cell contraction and the increase in endothelialpermeability.

EXPERIMENTAL PROCEDURES

Materials—Human �-thrombin was obtained from EnzymeResearch Laboratories (South Bend, IN). Human pulmonaryarterial endothelial cells (HPAEC) and endothelial growthmedium 2 were obtained from Clonetics (San Diego, CA). Fura2-AM and Alexa-phalloidin, were purchased from MolecularProbes (Eugene, OR). U73122, OAG, and thapsigargin wereobtained from Calbiochem (La Jolla, CA). Trypsin was pur-chased from Invitrogen. Electrodes for endothelial monolayerelectrical resistance measurements were from Applied Bio-physics (Troy, NY). Constitutively active G�q (G�qQ209L) andRGS2 (HA-tagged) mutants were obtained from UMR cDNAResource Center (Rolla, MO). Transfection reagents for siRNA(Nucleofector HCAEC kit) and the electroporation systemwere fromAmaxa (Gaithersburg, MD). TRPC6 (M-004192-02-0005 NM_004621), TRPC1 (M-004191-01-0005 NM_003304),and control siRNA (D-001206-13-20) sequences were obtainedfrom Dharmacon (Lafayette, CO). Anti-RhoA, anti-TRPC1, anti-TRPC6, anti-G�q, anti-actin, and anti-PKC� antibodies andsiRNA transfection reagent were purchased fromSanta Cruz Bio-technology (San Diego, CA), whereas phospho-PKC� antibodywas purchased from Upstate (Lake Placid, NY). Rho activity wasdetermined usingGST-rhotekin-Rho binding domain beads fromCytoskeleton (Denver, CO). Anti-phospho-MLC antibody was agift fromDr. Jerold Turner (University of Chicago).Endothelial Cell Culture—HPAEC were cultured in T-75

flasks coated with 0.1% gelatin in endothelial growthmedium 2supplemented with 10% fetal bovine serum. Cells were main-tained at 37 °C in a humidified atmosphere of 5% CO2 and 95%air until confluent. Cells from each primary flask were detachedwith 0.025% trypsin/EDTA and plated on either 60-mm dishesfor the Rho pulldown assay or coverslips for calcium and con-focal imaging studies. In all experiments, HPAEC between pas-sages 6 and 8 were used.Transfection of siRNA or cDNA—siRNA or cDNA were

transduced into cells by electroporation or using transfection

reagents. HPAE cells grown up to 70% confluency weretrypsinized, mixed with 2.8 �g of siRNA or 3.0 �g of cDNAalong with 100 �l of nucleofector solution. Cells were rapidlyelectroporated by the Amaxa nucleofector device using themanufacturer’s recommended program (S-05) dedicated forhuman coronary arterial endothelial cells. The cells wereremoved,mixed in endothelial growthmedium2, and plated oneither 60-mm dishes. HPAE cells plated on coverslips or gold-plated 10-well electrodes were transfected with 100 nM siRNAusing transfection reagent following manufacturer’s protocol.Western Blotting—HPAEC monolayers were washed with

phosphate-buffered saline and lysed with SDS sample buffer.Proteins from each lysate was separated by electrophoresis on a7 or 12.5% polyacrylamide gel, and transferred to nitrocellulosemembrane for Western blotting with the indicated antibodies(17, 19).Cytosolic Ca2� Measurements—An increase in intracellular

Ca2� was measured using the Ca2�-sensitive fluorescent dyeFura 2-AM as described (17, 19). Briefly, cells grown on 25-mmcoverslips were incubated with 3 �M Fura 2-AM for 15 min at37 °C. Cells were washed three to four times with Hank’s bal-anced salt solution and imaged using an Attofluor Ratio Visiondigital fluorescencemicroscopy system (Atto Instruments, Inc.,Rockville, MD) equipped with a Zeiss Axiovert S100 invertedmicroscope and F-Fluor �40 1.3-numerical aperture oilimmersion objective. Regions of interests in individual cellswere marked and excited at 334 and 380 nM with emission at520 nM. The 340/380 nM excitation ratio, which increases as afunction of intracellular Ca2�, was captured at 5-s intervals.RhoA Activity—RhoA activity was measured using the GST-

rhotekin-Rho binding domain that specifically pulls down acti-vated RhoA as described (4, 17). HPAE cell monolayers werestimulated for the indicated timeswith 50 nM thrombin, 100�MOAG, or 2 �M thapsigargin. Cells were quickly washed withice-cold Tris-buffered saline, and lysed with 200 �l of lysisbuffer (50mMTris, pH7.5, 10mMMgCl2, 0.5MNaCl, 1%TritonX-100, 0.5% sodium deoxycholate, 0.1% SDS, and 10 �g/mleach of aprotinin and leupeptin). Cell lysates were clarified bycentrifugation at 14,000� g at 4 °C for 2min and equal volumesof cell lysates were incubated with GST-Rho binding domainbeads (15�g) at 4 °C for 2 h. The beadswerewashed three timeswithwash buffer (25mMTris, pH 7.5, 30mMMgCl2, and 40mMNaCl), and bound RhoA was eluted by boiling each sample inLaemmli sample buffer. Eluted samples from the beads andtotal cell lysate were then electrophoresed on 12.5% SDS-poly-acrylamide gels and Western blotted with rabbit polyclonalanti-RhoA antibody.PKC� Translocation—HPAEC monolayer stimulated with

50 nM thrombin for the indicated times were quickly washedwith ice-cold phosphate-buffered saline and cells werescrapped using Tris buffer (pH 7.5 containing in mM; 10 Tris, 1MgCl2, 5 EDTA, 10 EGTA, 1 Na3VO4) and a mixture of prote-ase inhibitors. Cell lysateswere sonicated for 10 s and an aliquotof the lysates was saved for determination of total PKC�.Lysates were then centrifuged at 100,000 � g for 1 h at 4 °C toseparate cytosolic fraction. The pellets were suspended in theabove lysis buffer plus 1% Triton X-100, sonicated, and incu-bated for 30 min at 4 °C followed by centrifugation at 14,000 �

TRPC6 Regulates RhoA Activation

7834 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 11 • MARCH 16, 2007


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

FIGURE 1. G�q-PLC pathway mediates thrombin-induced RhoA activation. A, constitutively active G�q mutant induces RhoA activity. After 36 h, HPAE cellstransducing control vector (pCMV) or constitutively active G�q (G�q) mutant were lysed to determine the RhoA activity using Rhotekin-bound beads. RhoAactivation is evident by the increased amount of GTP-bound RhoA (top panel; RhoA-GTP). Western blotting with the anti-G�q antibody shows G�q expression(middle panel), whereas it shows equal protein loading with anti-actin antibody (bottom panel). Blot is representative of results from three experiments. B, RhoAactivity in response to thrombin stimulation in HPAE cells transducing control vector or HA-tagged RGS2 mutant. RhoA activation is evident by the increasedamount of GTP-bound RhoA (top panel) compared with the total amount of RhoA in whole cell lysates (bottom panel). Inset, immunoblot of cell lysates withanti-HA antibody shows RGS2 expression (top panel), whereas it shows equal protein loading with anti-actin antibody (bottom panel). C and D, RhoA activity inresponse to thrombin stimulation in HPAE cells pretreated without or with 10 �M U73122 (C) or 25 �M BAPTA-AM (D) for 30 min. RhoA activity was determinedfollowing 1 min after thrombin stimulation. RhoA activation is evident by the increased amount of GTP-bound RhoA (A, top) compared with total amount ofRhoA in whole cell lysates (A, bottom). E, ratiometric measurements of intracellular Ca2� in response to 50 nM thrombin were made after loading the HPAE cellmonolayer with Fura 2-AM for 15 min in RGS2 transducing cells or following a 15-min pretreatment with 10 �M U73122 or 25 �M BAPTA-AM. Each represent-ative tracing is the average response of 30 – 40 cells. Inset, plot shows mean � S.D. of the steady state ratiometric increase in intracellular Ca2� in response tothrombin as described under the experimental conditions (n � 3– 4). *, indicates significant decrease in intracellular Ca2� compared with control cells(p � 0.05). F, RhoA activity after a 1-min thrombin stimulation of HPAE cell monolayer incubated in 1.3 mM Ca2� containing or nominally Ca2�-free buffer. RhoAactivation is evident by the increased amount of GTP-bound RhoA (top panel) compared with the total amount of RhoA in whole cell lysates (bottom panel). G,plot shows mean � S.D. of RhoA activity in response to thrombin from multiple experiments calculated as the -fold increase over the basal value under theindicated experimental conditions (n � 3– 4). *, indicates a significant increase in RhoA activity compared with unstimulated cells, cells transducing RGS2mutant, or cells pretreated with various inhibitors (p � 0.05).




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

g at 4 °C to separate membrane fractions as described (27).Total cell lysates, cytosol, andmembrane fractions were immu-noblotted with PKC� antibody to determine PKC� transloca-tion in themembrane fraction following thrombin stimulation.PKC� Activity—We used GST-GDI-1 fusion protein as a

substrate to assess PKC� activity (4). Briefly, HPAEC mono-layer stimulated with 50 nM thrombin for 5 min was quicklywashed with phosphate-buffered saline and lysed using immu-noprecipitation assay buffer (1% Triton X-100, 150 mM NaCl,10 mM Tris, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mMphenylmethylsulfonyl fluoride, 0.5%Nonidet P-40, and 2�g/mleach of pepstatin A, leupeptin, and aprotinin). Cell lysates werecleared by centrifugation at 4 °C at 14,000 � g for 10 min andimmunoprecipitated with anti-rabbit polyclonal PKC� anti-body. PKC� immunocomplexes were used to phosphorylateGST-GDI-1 fusion protein.Immunofluorescence—Cells were stimulated with 50 nM

thrombin for the indicated times, rinsed quickly with ice-coldHank’s balanced salt solution, and fixed with 2% paraformalde-hyde. Cells were permeabilized for 3 min with 0.1% TritonX-100 in Hank’s balanced salt solution followed by incubationfor 40 min with 1% ovalbumin. Cells were then incubated withanti-PKC� antibody followed by incubation with Alexa-labeledsecondary antibody for another 1 h. Cells were then washedthree times with Hank’s balanced salt solution and mountedwith anti-fade media. For determining actin stress fiber forma-tion, cells were rinsed and incubated with Alexa-labeled phal-

loidin to label actin stress fibers.Cells were viewed using a 63� 1.2NA objective and appropriate filtersusing a Zeiss LSM510 confocalmicroscope.MLC Phosphorylation—HPAEC

monolayer was stimulated withthrombin for the indicated times.Endothelial cells were scraped offand mixed with Laemmli samplebuffer and Western blotted withantibodies for phosphorylated-MLC or pan-MLC antibodies todetermine MLC phosphorylation.Transendothelial Resistance Mea-

surement—The time course of endo-thelial cell retraction in real time, ameasure of increased endothelialpermeability, wasmeasured accord-ing to the procedure describedpreviously (4).Statistical Analysis—Two-tailed

Student’s t test andone-way analysisof variance with the Bonferroni posthoc test were used for statisticalcomparisons. Differences were con-sidered significant at p � 0.05.

RESULTS

Activation of G�q-PLC PathwayInduces RhoA Activity—Stimula-

tion of PAR-1 receptor by thrombin increases intracellularCa2� by the G�q-PLC pathway (11, 14). In the present experi-ments, we sought to determine the contribution of G�q and thePLC-mediated increase in intracellular Ca2� in thrombin-in-duced RhoA activation. We transduced the constitutivelyactive G�q mutant in endothelial cells and determined RhoAactivity using rhotekin-bound fusion proteins. HPAEC trans-ducing the active mutant of G�q showed a 3.8 � 0.4-foldincrease in RhoA activity (Fig. 1A) (p � 0.05). To corroboratethese findings, we inhibited G�q function using RGS2, whichpredominantly increases the intrinsic rate of G�q to hydrolyzeGTP to GDP, thereby inhibiting G�q function (28–30). Weobserved that expression of RGS2 inhibited RhoA activation inresponse to thrombin (Fig. 1, B and G) (p � 0.05). Next, wepretreated HPAEC with U73122, an inhibitor of PLC (31), toassess the requirement of PLC activity in thrombin-inducedactivation of RhoA.We observed that U73122 prevented RhoAactivation in response to thrombin (Fig. 1, C andG) (p � 0.05).To determine whether the thrombin-activated increase inintracellular Ca2� and RhoA activity is causally related, intra-cellular Ca2� was chelated with the membrane permeant Ca2�

chelator BAPTA-2AM. We observed that thrombin failed toinduce RhoA activation in BAPTA-pretreated cells (Fig. 1, Dand G) (p � 0.05). Simultaneous measurement of intracellularCa2� using Fura 2-AM confirmed that these interventions sig-nificantly suppressed thrombin-induced intracellular Ca2� rise(Fig. 1E). In other studies, we depleted extracellular Ca2� to

FIGURE 2. Activation of SOC by thapsigargin fails to induce RhoA activation. A, ratiometric measurementsof intracellular Ca2� in response to 2 �M thapsigargin (Thap) were made after loading the HPAE cell monolayerwith Fura 2-AM for 15 min. Each representative tracing is the average response of 30 – 40 cells, and experimentswere repeated four times. B and C, RhoA activity in response to 50 nM thrombin or 2 �M thapsigargin stimula-tion of HPAE cells for the indicated times. RhoA activation is evident by the increased amount of GTP-boundRhoA (top) compared with total amount of RhoA in whole cell lysates (bottom). C, plot shows mean � S.D. ofRhoA activity from multiple experiments calculated as the -fold increase over the basal value. *, significantlydifferent from unstimulated cells or cells stimulated with thapsigargin (n � 3).




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

assess the contribution of intracellular Ca2� release and Ca2�

entry in signaling RhoA activity. As shown in Fig. 1, F and G,thrombin-induced increase in RhoA activity was significantlyreduced in the absence of extracellular Ca2� (p � 0.05). Thesefindings indicated that the increase in intracellular Ca2�

induced by the G�q-PLC pathway mediated Ca2� entry con-tributed to RhoA activation.Receptor-operated Ca2� Entry Induces RhoA Activation—

G�q upon activation with thrombin stimulates PLC, which inturn activates Ca2� entry through SOC and ROC channels (11,14). Thus, we determined the role of Ca2� entry mediated bySOC and ROC channels in inducing RhoA activation inresponse to thrombin. We used thapsigargin because it acti-vates SOC channels independently of ligand-receptor-G pro-tein-coupled receptors (12). Thapsigargin increased intracellu-lar Ca2� (Fig. 2A); however, it failed to induce RhoA activation(Fig. 2, B and C), indicating SOC channels are not sufficient toactivate RhoA. To determine the role of ROCchannels, we usedOAG, a membrane-permeable analogue of DAG, known toactivate ROC channels (13, 14). OAG in a dose-dependentmanner induced sustained Ca2� entry in endothelial cells (Fig.3A). We also observed that OAG significantly increased RhoAactivity, a response sustained up to 20 min (Fig. 3, B and C).

OAG failed to induce Ca2� entry (Fig. 3A) as well as RhoAactivation (Fig. 3D) in the absence of extracellular Ca2�, indi-cating thatOAG increases the intracellular Ca2� concentrationand RhoA activity by activating ROC channels and does notrequire ER store depletion. To address the possibility that OAGeffects onCa2� entry are the result of PKC� activation byOAG,we overexpressed the dominant negative (dn) mutant of PKC�by infecting endothelial cells with adenoviral vector containingdnPKC� (5, 19). Cells infected with adenoviral vector contain-ing the�-galactosidasemutant served as controls.We observedthat expression of dnPKC� had no effect on the OAG-inducedCa2� entry (0.29 � 0.02 in �-galactosidase expressing cells ver-sus 0.33� 0.02 in dnPKC� expressing cells) (Fig. 3E). However,in the same experiments expression of dnPKC� inhibited thethapsigargin-induced Ca2� entry (0.38 � 0.07 in �-galactosid-ase expressing cells versus 0.12 � 0.07 in dnPKC�-expressingcells; p � 0.05) (Fig. 3F), a finding consistent with our previousreport (19). Thus, the OAG-induced increase in intracellularCa2� concentration occurred independently of PKC� but sec-ondary to Ca2� entry via G�q-induced activation of ROC chan-nels, which in turn signaled RhoA activation.TRPC6 Is Required for RhoA Activation—As TRPC6, a con-

stituent of ROC channels, is abundantly expressed in endothe-

FIGURE 3. ROC activation by OAG induces RhoA activity. A, ratiometric measurements of intracellular Ca2� in response to OAG, a permeable DAG analogue,were made after loading HPAE cell monolayer with Fura 2-AM for 15 min. Each representative tracing is the average response of 30 – 40 cells, and experimentswere repeated five times. OAG in a dose-dependent manner induced Ca2� entry in the presence of extracellular Ca2�. However, OAG fails to induce entry ofCa2� in the absence of extracellular Ca2� (�[Ca2�]o, dotted line). B and C, RhoA activity in response to OAG stimulation of HPAE cells for the indicated times.RhoA activation is evident by the increased amount of GTP-bound RhoA (top panel) compared with total amount of RhoA in whole cell lysates (bottom panel).C, plot shows mean � S.D. of RhoA activity from multiple experiments calculated as the -fold increase over the 0-min value (n � 4). *, indicates significantincrease in RhoA activity (p � 0.05). D, RhoA activity in response to 100 �M OAG stimulation of HPAE cells in the presence or absence of extracellular Ca2�. RhoAactivation is evident by the increased amount of GTP-bound RhoA (top panel) compared with the total amount of RhoA in whole cell lysates (bottom panel). Arepresentative blot from two experiments is shown. E and F, ratiometric measurements of intracellular Ca2� in response to 100 �M OAG or 2 �M thapsigarginwere made after loading HPAE cell monolayer with Fura 2-AM for 15 min after 32 h of infection with adenovirus vector containing �-galactosidase or dnPKC�mutant. Each representative tracing is the average response of 30 – 40 cells, and experiments were repeated three times. Inset in E, Western blot with anti-PKC�antibody shows increased PKC� expression in monolayers infected with adenovirus vector containing dnPKC� mutant (top panel) while it shows equal proteinloading with anti-actin antibody (bottom panel).




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

lial cells (18, 25, 32–34) and enables Ca2� entry, we suppressedTRPC6 expression using siRNA to address its role in regulatingRhoA activation. In addition, we knocked down endogenousTRPC1, a subunit of SOC channels in endothelial cells (17, 19,20, 23, 26), to compare the role of Ca2� entry mediated by SOCchannels in inducing RhoA activation. As a procedural controlwe used cells transfectedwith control siRNA.We observed thatTRPC6 siRNA significantly reduced endogenous TRPC6

expression at 60 h post-transfection(Fig. 4A). The reduction in TRPC6expression had no effect on theexpression of TRPC1 (Fig. 4A,inset). Inhibition of TRPC6 expres-sion prevented OAG-induced Ca2�

entry as compared with cells trans-fected with control siRNA (Fig. 4, Band C). We also observed that OAGfailed to induce RhoA activation inTRPC6 siRNA-transfected cells(Fig. 4, D and E).Next, we determined Ca2� entry

and RhoA activation in response tothrombin in TRPC6-siRNA-trans-fected cells to address the role ofTRPC6 in regulating RhoA activa-tion. Fig. 5, A and B, shows thatknockdown of TRPC6 expressionsignificantly reduced the thrombin-induced rise in intracellular Ca2�

concentration. Thrombin-inducedRhoA activation was also signifi-cantly reduced in cells transfectedwith TRPC6 siRNA (Fig. 5, C andD). However, suppression of TRPC1expression (Fig. 5E), whereas reduc-ing SOC entry (Fig. 5, F and G),failed to alter the thrombin-inducedRhoA activation (3.22 � 0.25-foldincrease in Sc-transfected cells ver-sus 3.36� 0.6-fold increase in SiT1-transfected cells; p� 0.05) (Fig. 5H).These results demonstrate the cru-cial role of TRPC6 in mediatingRhoA activation.TRPC6 Signals RhoA Activation

Downstream of PKC�—Becausethrombin can induce RhoA activa-tion secondary to PKC� activation(4, 5), we addressed the possibilitythat TRPC6 may signal RhoA acti-vation by stimulating PKC�. Trans-location to plasma membrane andphosphorylation of PKC� and invitro phosphorylation of target pro-teins by PKC� have been used asindices of PKC� activation (15, 35).We previously showed that GDI-1,an inhibitor of Rho-GTPases, is a

PKC� substrate (4). Thus, we used these approaches to deter-mine whether TRPC6 knockdown alters PKC� activity. Weobserved that thrombin induced significant translocation ofPKC� to the membrane fraction within 1 min, whereas thisresponse was not seen in membranes isolated from TRPC6knockdown cells (Fig. 6, A and B). Confocal imaging alsoshowed diminished translocation of PKC� to the plasmamem-brane in TRPC6 knockdown cells in response to thrombin

FIGURE 4. Role of TRPC6-mediated ROC activity in signaling RhoA activation in response to OAG. A, TRPC6siRNA suppressed endogenous expression of TRPC6 but has no effect on TRPC1 expression. After 60 h, cellstransfected with TRPC6 siRNA (SiT6) or nonspecific siRNA (Sc) were lysed and Western blotted using anti-TRPC6(inset, top), anti-TRPC1 antibody (inset, middle), or anti-actin (inset, bottom) antibodies to detect protein expres-sion. Plot shows mean � S.D. of percent reduction in TRPC6 expression after TRPC6 knockdown from multipleexperiments taking the value in cells transfected with nonspecific siRNA as 100%. B, ratiometric measurementsof intracellular Ca2� in response to OAG in the presence of extracellular Ca2� in cells transfected with nonspe-cific (Sc) or TRPC6 (SiT6) siRNA. Measurements were made 60 h post-transfection after loading HPAE cellmonolayer with Fura 2-AM for 15 min. Each representative tracing is the average response of 30 – 40 cells, andexperiments were repeated five times. C, plot shows mean � S.D. of steady state ratiometric intracellular Ca2�

values before (�) and after (�) application of OAG in two groups (n � 5). *, indicates a significant increase inintracellular Ca2� (p � 0.05). D and E, RhoA activity in response to a 4-min OAG stimulation in HPAE cellstransfected with nonspecific (Sc) or TRPC6 (SiT6) siRNA. RhoA activation is evident by the increased amount ofGTP-bound RhoA (D, top) compared with total amount of RhoA in whole cell lysates (D, bottom). E, plot showsmean � S.D. of RhoA activity from multiple experiments calculated as the -fold increase over the 0-min value(n � 4). *, indicates significant increase in RhoA activity (p � 0.05).




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

FIGURE 5. TRPC6-mediated Ca2� entry induces RhoA activation in response to thrombin. A, ratiometric measurements of intracellular Ca2� in response tothrombin in the presence of extracellular Ca2� in cells transfected with nonspecific siRNA (Sc) or TRPC6 (SiT6) siRNA. Measurements were made 60 h post-transfection after loading HPAE cell monolayer with Fura 2-AM for 15 min. B, plot shows mean � S.D. of a ratiometric increase in intracellular Ca2� concentra-tion calculated as the area under the curve after application of thrombin in two groups (n � 5). *, indicates significant reduction in intracellular Ca2� in TRPC6knockdown cells compared with cells transfected with nonspecific siRNA (p � 0.05). C and D, RhoA activity in response to a 1-min thrombin stimulation in HPAEcells transfected with nonspecific (Sc) or TRPC6 siRNA (SiT6). RhoA activation is evident by the increased amount of GTP-bound RhoA (C, top) compared withtotal amount of RhoA in whole cell lysates (C, bottom). D, plot shows a mean � S.D. for the thrombin-induced increase in RhoA activity in two groups frommultiple experiments calculated as the -fold increase over the 0-min value (n � 4). *, indicates increased RhoA activity compared with unstimulated cells or cellstransfected with siT6 (p � 0.05). E, knockdown of endogenous expression of TRPC1 with TRPC1 siRNA. After 60 h, cells transfected with TRPC1 (SiT1) ornonspecific (Sc) siRNA were lysed and Western blotted using anti-TRPC1 (top), anti-TRPC6 antibody (middle), or anti-actin (bottom) antibodies to detect proteinexpression. F, ratiometric measurements of [Ca2�]i during extracellular Ca2� depletion-repletion conditions in cells transfected with nonspecific siRNA (Sc) orTRPC1 (SiT1) siRNA. In nominally Ca2�-free media, thrombin induced an initial increase in endothelial [Ca2�]i representing intracellular store release, and asecondary rise in [Ca2�]i on re-addition of 2 mM extracellular Ca2� representing plasmalemmal Ca2� entry. TRPC1 knockdown reduced Ca2� entry uponre-addition of extracellular Ca2� without affecting store release. Measurements were made 60 h post-transfection after loading the HPAE cell monolayer withFura 2-AM for 15 min. Each representative tracing is the average response of 30 – 40 cells, and experiments were repeated five times. G, plot shows mean � S.D.of the -fold increase in intracellular Ca2� by thrombin between two groups (n � 5). *, indicates reduced Ca2� entry in TRPC1 knockdown cells compared withcells transfected with nonspecific siRNA (p � 0.05). H, RhoA activity in response to a 1-min thrombin stimulation in HPAE cells transfected with nonspecific (Sc)or TRPC1 (SiT1) siRNA. RhoA activation is evident by the increased amount of GTP-bound RhoA (top) compared with total amount of RhoA in whole cell lysates(bottom).




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

(Fig. 6C). Using Ser657 phosphospecific-PKC� antibody (36),we observed a significant increase in PKC� phosphorylationfollowing a 5-min thrombin stimulation; however, thrombinfailed to promote phosphorylation at this site in TRPC6 knock-down cells (p � 0.05) (Fig. 6, D and E). To determine whethersuppression of endogenous TRPC6 expression altered PKC�kinase activity required for the phosphorylation of RhoGDI-1,an inhibitor of RhoA activity (4), lysates from endothelial cellstransfected with either control siRNA or TRPC6 siRNA afterthrombin stimulation were immunoprecipitated with PKC�antibodies and used for in vitro kinase assay. We observed thatimmunoprecipitated PKC� from control siRNA-transfectedcells phosphorylated GDI-1 (Fig. 6, F and G). However, PKC�immunoprecipitated from TRPC6 knockdown cells failed toinduce GDI-1 phosphorylation (Fig. 6, F and G). Thus, thesefindings demonstrate that the TRCP6-mediated increase inintracellular Ca2� downstream of G�q is needed, in addition toDAG, for PKC� activation.TRPC6 Knockdown Fails to Alter TRPC1 Activity—We pre-

viously showed that both PKC� and RhoA activation arerequired for TRPC1-induced Ca2� entry upon store depletion

(17, 19). We therefore determinedwhether impairment of PKC� andRhoA activity by TRPC6 knock-down has an effect on activation ofSOC channels. Thus, we experi-mentally separated the two phasesof the rise in intracellular Ca2� con-centration using a Ca2� add-backprotocol (Fig. 7A). Under Ca2�-freebath conditions, TRPC6 knock-down had no effect on thrombin-in-duced mobilization of Ca2� fromstores (Fig. 7, A and B). However,suppression of TRPC6 expressionsignificantly reduced theCa2� entry(Fig. 7, A and B). In TRPC6 knock-down cells, we observed that thapsi-gargin-induced SOC entry was notsignificantly reduced relative tocontrol cells (p � 0.05) (Fig. 7, Cand D).TRPC6 Regulates Endothelial

Cell Contraction—Because RhoAactivation increases endothelialpermeability by inducing cell shapechange via actinomyosin-mediatedendothelial cell contraction (1), wedetermined the functional role ofTRPC6 on thrombin-induced actinstress fiber formation, MLC phos-phorylation, and cell shape change.As shown in Fig. 8, A and B, throm-bin induced MLC phosphorylationin cells transfected with controlsiRNA. However, knockdown ofendogenous TRPC6 with siRNAsignificantly inhibited the phospho-

rylation (p � 0.05). TRPC6 knockdown also inhibited actinstress fiber formation in response to thrombin (Fig. 8C). Tocorroborate the role of TRPC6 in regulating thrombin-inducedcell shape change, we also determined transendothelial electri-cal resistance (TER) in endothelial monolayers. We observedthat TRPC6 knockdown did not altered basal TER. However,knockdown of TRPC6 significantly reduced a thrombin-in-duced decrease in TER (p � 0.05) (Fig. 8D). Thus, TRPC6 acti-vation induced endothelial contraction and subsequentincrease in endothelial permeability occur secondary to RhoA-inducedMLCphosphorylation and actin stress fiber formation.

DISCUSSION

Thrombin binds to and cleaves PAR-1 in endothelial cellsleading to RhoA activation (1, 37). Although the �-subunit ofG12/13 is known to induce RhoA activity by the RhoA-specificGEF, p115 RhoGEF, studies have also shown a requirement ofG�q in themechanism of RhoA activation (6, 7, 9, 10, 38). How-ever, little is known about the signaling pathwaymediatingG�qactivation of RhoA downstream of GPCRs. We previouslyshowed that PKC� activity was required for PAR-1-induced

FIGURE 6. Effect of TRPC6 knockdown on PKC� activity. A and B, PKC� translocation in the membranefraction in response to thrombin in cells transfected with nonspecific siRNA (Sc) or TRPC6 siRNA (SiT6). Cellswere stimulated with thrombin 60 h post-transfection and lysates were separated into cytosol or membranefractions as described under “Experimental Procedures” followed by Western blotting with anti-PKC� antibodyto determine PKC� translocation. B, plot shows mean � S.D. of -fold increase in PKC� translocation at themembrane fraction over that in cytosol fraction without or with thrombin stimulation between two groups(n � 3). *, indicates a significant increase in PKC� translocation compared with unstimulated cells or TRPC6knockdown cells (p � 0.05). C, image showing PKC� translocation (indicated by arrow) in cells transfected withnonspecific or TRPC6 siRNA after stimulation with thrombin. Cells were stimulated 60 h post-transfection,fixed, and stained with anti-PKC� antibodies to determine the translocation using confocal microscope. D,thrombin-induced PKC� phosphorylation (P-PKC�) in cells transfected with nonspecific (Sc) or TRPC6 (SiT6)siRNA. Cells were stimulated with thrombin 60 h post-transfection and lysed. Lysates were Western blottedwith phospho-PKC� or pan-PKC� antibodies to determine PKC phosphorylation. E, plot shows mean � S.D. of-fold increase in PKC� phosphorylation over that at the 0-min value between two groups (n � 3). *, indicates asignificant increase in PKC� phosphorylation compared with unstimulated cells or TRPC6 knockdown cells(p � 0.05). F, GDI phosphorylation by PKC�. After 60 h of transfection with nonspecific siRNA (Sc) or TRPC6siRNA (SiT6), cells were left unstimulated or stimulated with thrombin for 5 min, and lysates were immunopre-cipitated with pan-PKC� antibodies. Immunoprecipitated PKC� complex was used to phosphorylate GDI-1 invitro (top panel). Western blot with pan-PKC� antibodies shows equal protein loading in all lanes (bottompanel). FL, full length. G, plot shows mean � S.D. of the -fold increase in GDI-1 phosphorylation induced byPKC� in two groups (n � 3). *, indicates a significant increase in GDI-1 phosphorylation compared withunstimulated cells or TRPC6 knockdown cells (p � 0.05).




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

RhoA activation (4, 5). Because PKC� is a downstream effectorof G�q and requires an increase in intracellular Ca2� concen-tration and production of DAG for activation (15, 16), weaddressed the possibility that a route of RhoA activation byG�qmay involve a rise in intracellular Ca2� concentration via aDAG-linked pathway. In the present study, we observed thattransduction of the constitutively active G�q mutant inducedRhoA activation in endothelial cells, consistent with previousreports (6, 9, 10). Furthermore, our results established thecausal link between the G�q-mediated increase in cytosolicCa2� via the TRPC6, a subunit of the ROC channel, in themechanism of RhoA activation and the crucial role of this path-way in inducing endothelial contraction.G�q activation triggers an increase in intracellular Ca2� con-

centration by stimulating PLC activity (11–14). PLC inducesthe generation of 2 secondmessengers, IP3 and DAG. IP3 bindsto its receptor on ER and mobilizes Ca2� by releasing Ca2�

from ER stores, which in turn activates SOC channels. Also,DAG by activating Ca2� entry via ROC channels can addition-ally contribute to the increase in intracellular Ca2� (11–14).Our results demonstrated that an increase in intracellular Ca2�

mediated by the ROC channel downstream of the G�q-PLCpathway was required for thrombin-induced RhoA activation.We demonstrated that RhoA activation occurred by a G�q-PLC-mediated increase in the intracellular Ca2� concentra-tion. This conclusion was based on the findings that G�q orPLC inhibition or chelation of intracellular Ca2� prevented thethrombin-induced RhoA activation. OAG, a permeant ana-

logue of DAG, was also shown toinduce RhoA activation. However,OAG itself failed to induce RhoAactivity in the absence of extracellu-lar Ca2�, suggesting that the effectof DAG occurred independently ofSOC. Furthermore, thapsigargin,which directly activates SOC chan-nels (11–14), failed to activateRhoA. These findings raise the pos-sibility that activation of RhoA isdependent on the ROC channel, theother importantCa2� entry channelpresent in endothelial cells (1,11–14).Proteins of the TRPC family form

ROC and SOC channels in endothe-lial cells (1, 11–14). We demon-strated that the ROC-induced Ca2�

entry was mediated by TRPC6,which in turn induced RhoA activa-tion. A recent study in rat aorticsmooth muscle cells has shown thatTRPC6 knockdown had no effect onthe DAG-induced increase in intra-cellular Ca2� concentration (39).The present result showing that60–70% suppression of endogenousTRPC6 expression by siRNA mark-edly reduced the DAG-induced

Ca2� entry is consistent with observations that DAG activatesCa2� entry via TRPC6 (25, 32–34, 40). As the expression ofkinase-defective PKC� had no effect on the OAG-inducedCa2� entry results of the present study rules out the involve-ment of PKC� activation per se in mediating the Ca2� entry inresponse to DAG. The finding that TRPC6-induced Ca2� entrydid not require PKC� activity (41–43) lends further credenceto our contention that PKC� does not regulate the activation ofTRPC6 function. Interestingly, we observed that TRPC6knockdown suppressed PKC� activity, and thereby preventedRhoA activation, MLC phosphorylation, and actin stress fiberformation as well as the increase in endothelial permeability (asreflected by measurement of TER) in response to thrombin.Thus, our data support the model in which TRPC6 activationregulates PKC� activity, which plays a crucial role in themech-anism of RhoA activation and thereby contraction of endothe-lial cells.Deletion of TRPC6 in mice resulted in the up-regulation of

TRPC3 expression in vascular smooth muscle cells (44), whichcould interfere with the interpretation of our data. Humanendothelial cells were shown to abundantly express TRPC6mRNA and TRPC3 to a lesser extent (18). However, we wereunable to detect TRPC3 protein expression in human pulmo-nary arterial endothelial cells even after a significant reductionin TRPC6 expression (data not shown). Moreover, we wouldhave observed enhancement of ROC-induced Ca2� entry, andconsequently of RhoA activation, had TRPC3 been a functionalROC in the TRPC6-knockdown endothelial cells. Therefore, it

FIGURE 7. Effect of TRPC6 knockdown on SOC-induced Ca2� entry. Ratiometric measurements of intracel-lular Ca2� during extracellular Ca2� depletion-repletion conditions after depletion of stores with thrombin(Thr) (A and B) or thapsigargin (Thap) (C and D) in cells transfected with nonspecific (Sc) or TRPC6 (SiT6) siRNA.Measurements were made 60 h post-transfection after loading HPAE cell monolayer with Fura 2-AM for 15 min.Each representative tracing is the average response of 30 – 40 cells, and experiments were repeated five times.B and D, plot shows mean � S.D. of intracellular Ca2� increase following store depletion or Ca2� entry betweentwo groups (n � 5). *, indicates reduced Ca2� entry in TRPC6 knockdown cells compared with cells transfectedwith nonspecific siRNA (p � 0.05).




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

is unlikely that TRPC3 has animportant role in regulating ROC-activated Ca2� entry and subse-quent RhoA activation in endothe-lial cells.The activation of RhoA requires

its dissociation from the RhoA�GDP�GDI-1 complex followed byGTP exchangemediated by guaninenucleotide exchange factors (GEFs)(1). It is known that G�12/13 regu-lates RhoA activation downstreamof PAR-1 by stimulating p115 Rho-GEF, a specific RhoGEF for RhoA(38, 45). We previously showed thatthe activation of p115 RhoGEFrequired not only G�12/13 but alsoPKC�-mediated phosphorylationof p115 RhoGEF (5). The result ofthe present study showed thatdownstream of G�q only TRPC6was responsible for signaling RhoAactivation in response to thrombinstimulation of endothelial cells andthat the TRPC6 effect on RhoA wasmediated by PKC�.

Studies have demonstrated thatinteraction of PKC� with DAG inthe membrane is crucial for activat-ing downstream effectors (46, 47).The increase in the subplasmamembraneCa2� concentration par-ticipates in signaling the DAG-PKC� interaction (46, 47). Studies

of localization of GFP-tagged TRPC6 showed that TRPC6 wasprimarily localized at the plasma membrane, whereas TRPC1was found in intracellular membranes (48). Therefore, theplasma membrane-localized TRPC6 is appropriately situatedto increase the subplasmamembraneCa2� level thereby induc-ing the interaction of DAG with PKC�.We showed previously that inhibition of either RhoA or

PKC� reduced SOC-induced Ca2� entry as well as the SOCcurrent (17, 19). We also showed that thrombin phosphoryla-ted TRPC1 in a PKC�-dependent manner (19). In response tothrombin, RhoA interacted with both TRPC1 and IP3 receptor,and RhoA activation was required for insertion of TRPC1 intoplasmamembrane to mediate Ca2� entry upon ER store deple-tion (17). Thus, it is possible that impairment of TRPC6 func-tion by suppressing PKC� and RhoA activation could interferewithTRPC1-induced SOCactivation.Weobserved that knock-down of TRPC6 prevented Ca2� entry after the ER store wasdepleted by thrombin activation of PAR-1, a finding consistentwith the hypothesis that both PKC� and RhoA can regulateTRPC1 function. However, the knockdown of TRPC6 failed toaffect the thapsigargin-induced SOC entry, indicating thatTRPC6 operates independently of TRPC1. Nevertheless, wecannot completely rule out the possibility that a thresholdreduction of TRPC6 function required for down-regulation of

FIGURE 8. Effects of TRPC6 knockdown on MLC phosphorylation, actin stress fiber formation, and endo-thelial barrier dysfunction in response to thrombin. A, MLC phosphorylation in response to thrombin incells transfected with nonspecific (Sc) or TRPC6 (SiT6) siRNA. Cells were stimulated with thrombin 60 h post-transfection and lysed. Lysates were Western blotted with phospho-MLC (p-MLC) or pan-MLC antibodies todetermine MLC phosphorylation. B, plot shows mean � S.D. of the -fold increase in MLC phosphorylationfollowing thrombin challenge between two groups (n � 3). *, indicates decreased phosphorylation in TRPC6knockdown cells compared with cells transfected with nonspecific siRNA (p � 0.05). C, actin stress fiber forma-tion in response to thrombin in cells transfected with nonspecific (Sc) or TRPC6 (SiT6) siRNA. Cells were stimu-lated with thrombin 60 h post-transfection and fixed followed by staining with Alexa-labeled phalloidin todetermine stress fiber formation by confocal imaging. D, changes in TER in response to thrombin in cellstransfected with nonspecific (Sc) or TRPC6 (SiT6) siRNA. *, indicates decreased TER in TRPC6 knockdown cellscompared with cells transfected with nonspecific siRNA (p � 0.05).

FIGURE 9. Model of thrombin-induced RhoA activation and increased endo-thelial permeability. Thrombin ligation of PAR1 stimulates G�12/13 and G�q.G�12/13 via p115 RhoGEF activates RhoA. G�q-PLC by generating DAG activatesTRPC6 to induce Ca2� entry that by stimulating PKC� translocation to the plasmamembrane enables its interaction with DAG leading to PKC� activation. Acti-vated PKC� induces the phosphorylation of GDI-1 (not shown) and p115 RhoGEFphosphorylation thereby resulting in RhoA activation. Upon activation, RhoApromotes the generation of contractile force through actinomyosin cross-bridg-ing resulting in increased endothelial monolayer permeability.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

the TRPC1 function may not have been achieved with theknockdown of TRPC6. Another possibility is that SOC entrymay not be subject to regulation by TRPC6 in the presence ofthapsigargin because activation of TRPC6 requires a ligand-GPCR interaction.Mutations on chromosome 11q encoding TRPC6 have been

implicated as a predisposing factor for focal and segmental glo-merulosclerosis leading to proteinuria, hypertension, and renalinsufficiency (49, 50). TRPC6 is also up-regulated in idiopathicpulmonary hypertension (51). Thus, it is possible that alteredendothelial cell function in these conditions is a commondenominator responsible for hypertension, vascular prolifera-tive disorders, and renal failure. The present findings suggest alink between the up-regulation of TRPC6 activity and theseclinical manifestations that needs to be explored.We propose a model that helps to explain the mechanism of

PAR-1 signaling of RhoA activation downstream of G�q, whichthereby results in increased endothelial permeability (Fig. 9). Inthis context, our results demonstrate the novel role of TRPC6, asubunit of the ROC channel prominent in endothelial cells, insignaling RhoA activation secondary to the activation of PKC�.

REFERENCES1. Mehta, D., and Malik, A. B. (2006) Physiol. Rev. 86, 279–3672. Carbajal, J. M., and Schaeffer, R. C., Jr. (1999) Am. J. Physiol. 277,

C955–C9643. Dudek, S. M., and Garcia, J. G. (2001) J. Appl. Physiol. 91, 1487–15004. Mehta, D., Rahman, A., and Malik, A. B. (2001) J. Biol. Chem. 276,

22614–226205. Holinstat, M., Mehta, D., Kozasa, T., Minshall, R. D., and Malik, A. B.

(2003) J. Biol. Chem. 278, 28793–287986. Moers, A., Wettschureck, N., Gruner, S., Nieswandt, B., and Offermanns,

S. (2004) J. Biol. Chem. 279, 45354–453597. Zeng, H., Zhao, D., and Mukhopadhyay, D. (2002) J. Biol. Chem. 277,

46791–467988. Lutz, S., Freichel-Blomquist, A., Yang, Y., Rumenapp, U., Jakobs, K. H.,

Schmidt, M., and Wieland, T. (2005) J. Biol. Chem. 280, 11134–111399. Vogt, S., Grosse, R., Schultz, G., and Offermanns, S. (2003) J. Biol. Chem.

278, 28743–2874910. Chikumi, H., Vazquez-Prado, J., Servitja, J. M., Miyazaki, H., and Gutkind,

J. S. (2002) J. Biol. Chem. 277, 27130–2713411. Tiruppathi, C., Minshall, R. D., Paria, B. C., Vogel, S. M., and Malik, A. B.

(2002) Vascul. Pharmacol. 39, 173–18512. Nilius, B., and Droogmans, G. (2001) Physiol. Rev. 81, 1415–145913. Yao, X., and Garland, C. J. (2005) Circ. Res. 97, 853–86314. Ahmmed, G. U., and Malik, A. B. (2005) Pflugers Arch. 451, 131–14215. Newton, A. C. (2001) Chem. Rev. 101, 2353–236416. Nishizuka, Y. (1992) Science 258, 607–61417. Mehta, D., Ahmmed, G. U., Paria, B. C., Holinstat, M., Voyno-

Yasenetskaya, T., Tiruppathi, C., Minshall, R. D., and Malik, A. B. (2003)J. Biol. Chem. 278, 33492–33500

18. Paria, B. C., Vogel, S. M., Ahmmed, G. U., Alamgir, S., Shroff, J., Malik,A. B., and Tiruppathi, C. (2004) Am. J. Physiol. 287, L1303–L1313

19. Ahmmed, G. U., Mehta, D., Vogel, S., Holinstat, M., Paria, B. C., Tirup-pathi, C., and Malik, A. B. (2004) J. Biol. Chem. 279, 20941–20949

20. Brough, G. H., Wu, S., Cioffi, D., Moore, T. M., Li, M., Dean, N., andStevens, T. (2001) FASEB J. 15, 1727–1738

21. Cioffi, D. L., Wu, S., Alexeyev, M., Goodman, S. R., Zhu, M. X., andStevens, T. (2005) Circ. Res. 97, 1164–1172

22. Moore, T., Brough, G., Kelly, J., Babal, P., Li, M., and Stevens, T. (1998)Chest 114, Suppl. 1, 36S–38S

23. Moore, T. M., Brough, G. H., Babal, P., Kelly, J. J., Li, M., and Stevens, T.(1998) Am. J. Physiol. 275, L574–L582

24. Moore, T. M., Norwood, N. R., Creighton, J. R., Babal, P., Brough, G. H.,Shasby, D. M., and Stevens, T. (2000) Am. J. Physiol. 279, L691–L698

25. Pocock, T. M., Foster, R. R., and Bates, D. O. (2004) Am. J. Physiol. 286,H1015–H1026

26. Paria, B. C.,Malik, A. B., Kwiatek, A.M., Rahman,A.,May,M. J., Ghosh, S.,and Tiruppathi, C. (2003) J. Biol. Chem. 278, 37195–37203

27. Javaid, K., Rahman, A., Anwar, K.N., Frey, R. S.,Minshall, R. D., andMalik,A. B. (2003) Circ. Res. 92, 1089–1097

28. Heximer, S. P., Knutsen, R. H., Sun, X., Kaltenbronn, K. M., Rhee, M. H.,Peng, N., Oliveira-dos-Santos, A., Penninger, J. M., Muslin, A. J., Stein-berg, T. H., Wyss, J. M., Mecham, R. P., and Blumer, K. J. (2003) J. Clin.Investig. 111, 445–452

29. Heximer, S. P., Srinivasa, S. P., Bernstein, L. S., Bernard, J. L., Linder,M. E.,Hepler, J. R., and Blumer, K. J. (1999) J. Biol. Chem. 274, 34253–34259

30. Heximer, S. P., Watson, N., Linder, M. E., Blumer, K. J., and Hepler, J. R.(1997) Proc. Natl. Acad. Sci. U. S. A. 94, 14389–14393

31. Smith, R. J., Sam, L. M., Justen, J. M., Bundy, G. L., Bala, G. A., and Bleas-dale, J. E. (1990) J. Pharmacol. Exp. Ther. 253, 688–697

32. Cheng, H. W., James, A. F., Foster, R. R., Hancox, J. C., and Bates, D. O.(2006) Arterioscler. Thromb. Vasc. Biol. 26, 1768–1776

33. Leung, P. C., Cheng, K. T., Liu, C., Cheung, W. T., Kwan, H. Y., Lau, K. L.,Huang, Y., and Yao, X. (2006) J. Vasc. Res. 43, 367–376

34. Yip, H., Chan,W. Y., Leung, P. C., Kwan, H. Y., Liu, C., Huang, Y., Michel,V., Yew, D. T., and Yao, X. (2004) Histochem. Cell Biol. 122, 553–561

35. Siflinger-Birnboim, A., and Johnson, A. (2003) Am. J. Physiol. 284,L435–L451

36. Parekh, D. B., Ziegler, W., and Parker, P. J. (2000) EMBO J. 19, 496–50337. Coughlin, S. R. (2000) Nature 407, 258–26438. Kozasa, T., Jiang, X., Hart, M. J., Sternweis, P. M., Singer, W. D., Gilman,

A. G., Bollag, G., and Sternweis, P. C. (1998) Science 280, 2109–211139. Soboloff, J., Spassova, M., Xu, W., He, L. P., Cuesta, N., and Gill, D. L.

(2005) J. Biol. Chem. 280, 39786–3979440. Gamberucci, A., Giurisato, E., Pizzo, P., Tassi, M., Giunti, R., McIntosh,

D. P., and Benedetti, A. (2002) Biochem. J. 364, 245–25441. Thebault, S., Zholos, A., Enfissi, A., Slomianny, C., Dewailly, E., Roud-

baraki, M., Parys, J., and Prevarskaya, N. (2005) J. Cell. Physiol. 204,320–328

42. Albert, A. P., and Large, W. A. (2003) J. Physiol. 552, 789–79543. Kim, J. Y., and Saffen, D. (2005) J. Biol. Chem. 280, 32035–3204744. Dietrich, A., Kalwa, H., Rost, B. R., and Gudermann, T. (2005) Pflugers

Arch. 451, 72–8045. Hart, M. J., Jiang, X., Kozasa, T., Roscoe,W., Singer, W. D., Gilman, A. G.,

Sternweis, P. C., and Bollag, G. (1998) Science 280, 2112–211446. Reither, G., Schaefer, M., and Lipp, P. (2006) J. Cell Biol. 174, 521–53347. Medkova, M., and Cho, W. (1999) J. Biol. Chem. 274, 19852–1986148. Hofmann, T., Schaefer, M., Schultz, G., and Gudermann, T. (2002) Proc.

Natl. Acad. Sci. U. S. A. 99, 7461–746649. Winn, M. P., Conlon, P. J., Lynn, K. L., Farrington, M. K., Creazzo, T.,

Hawkins, A. F., Daskalakis, N., Kwan, S. Y., Ebersviller, S., Burchette, J. L.,Pericak-Vance, M. A., Howell, D. N., Vance, J. M., and Rosenberg, P. B.(2005) Science 308, 1801–1804

50. Reiser, J., Polu, K. R., Moller, C. C., Kenlan, P., Altintas, M. M., Wei, C.,Faul, C., Herbert, S., Villegas, I., Avila-Casado, C., McGee, M., Sugimoto,H., Brown, D., Kalluri, R., Mundel, P., Smith, P. L., Clapham, D. E., andPollak, M. R. (2005) Nat. Genet. 37, 739–744

51. Yu, Y., Fantozzi, I., Remillard, C. V., Landsberg, J. W., Kunichika, N., Pla-toshyn, O., Tigno, D. D., Thistlethwaite, P. A., Rubin, L. J., and Yuan, J. X.(2004) Proc. Natl. Acad. Sci. U. S. A. 101, 13861–13866




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Dolly MehtaItender Singh, Nebojsa Knezevic, Gias U. Ahmmed, Vidisha Kini, Asrar B. Malik and

Endothelial Cell Shape Change in Response to Thrombin Entry Induces RhoA Activation and Resultant2+-TRPC6-mediated CaqαG

doi: 10.1074/jbc.M608288200 originally published online December 29, 20062007, 282:7833-7843.J. Biol. Chem.

10.1074/jbc.M608288200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

http://www.jbc.org/content/282/11/7833.full.html#ref-list-1

This article cites 44 references, 29 of which can be accessed free at


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M608288200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;282/11/7833&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/282/11/7833

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=282/11/7833&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/282/11/7833

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/282/11/7833.full.html#ref-list-1

http://www.jbc.org/

g q -trpc6-mediated ca entry induces rhoa activation and

Documents