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Gαq-TRPC6-mediated Ca2+ Entry Induces RhoA Activation and Resultant Endothelial Cell Shape Change in Response to Thrombin

2006; Elsevier BV; Volume: 282; Issue: 11 Linguagem: Inglês

10.1074/jbc.m608288200

ISSN

1083-351X

Autores

Itender Singh, Nebojša Nick Knežević, Gias U. Ahmmed, Vidisha Kini, Asrar B. Malik, Dolly Mehta,

Tópico(s)

Protein Kinase Regulation and GTPase Signaling

Resumo

RhoA activation and increased intracellular Ca2+ concentration mediated by the activation of transient receptor potential channels (TRPC) both contribute to the thrombin-induced increase in endothelial cell contraction, cell shape change, and consequently to the mechanism of increased endothelial permeability. Herein, we addressed the possibility that TRPC signals RhoA activation and thereby contributes in actinomyosin-mediated endothelial cell contraction and increased endothelial permeability. Transduction of a constitutively active Gαq mutant in human pulmonary arterial endothelial cells induced RhoA activity. Preventing the increase in intracellular Ca2+ concentration by the inhibitor of Gαq or phospholipase C and the Ca2+ chelator, BAPTA-AM, abrogated thrombin-induced RhoA activation. Depletion of extracellular Ca2+ also inhibited RhoA activation, indicating the requirement of Ca2+ entry in the response. RhoA activation could not be ascribed to storeoperated Ca2+ (SOC) entry because SOC entry induced with thapsigargin or small interfering RNA-mediated inhibition of TRPC1 expression, the predominant SOC channel in these endothelial cells, failed to alter RhoA activity. However, activation of receptor-operated Ca2+ entry by oleoyl-2-acetyl-sn-glycerol, the membrane permeable analogue of the Gαq-phospholipase C product diacylglycerol, induced RhoA activity. Receptor-operated Ca2+ activation was mediated by TRPC6 because small interfering RNA-induced TRPC6 knockdown significantly reduced Ca2+ entry. TRPC6 knockdown also prevented RhoA activation, myosin light chain phosphorylation, and actin stress fiber formation as well as inter-endothelial junctional gap formation in response to either oleoyl-2-acetyl-sn-glycerol or thrombin. TRPC6-mediated RhoA activity was shown to be dependent on PKCα activation. Our results demonstrate that Gαq activation of TRPC6 signals the activation of PKCα, and thereby induces RhoA activity and endothelial cell contraction. RhoA activation and increased intracellular Ca2+ concentration mediated by the activation of transient receptor potential channels (TRPC) both contribute to the thrombin-induced increase in endothelial cell contraction, cell shape change, and consequently to the mechanism of increased endothelial permeability. Herein, we addressed the possibility that TRPC signals RhoA activation and thereby contributes in actinomyosin-mediated endothelial cell contraction and increased endothelial permeability. Transduction of a constitutively active Gαq mutant in human pulmonary arterial endothelial cells induced RhoA activity. Preventing the increase in intracellular Ca2+ concentration by the inhibitor of Gαq or phospholipase C and the Ca2+ chelator, BAPTA-AM, abrogated thrombin-induced RhoA activation. Depletion of extracellular Ca2+ also inhibited RhoA activation, indicating the requirement of Ca2+ entry in the response. RhoA activation could not be ascribed to storeoperated Ca2+ (SOC) entry because SOC entry induced with thapsigargin or small interfering RNA-mediated inhibition of TRPC1 expression, the predominant SOC channel in these endothelial cells, failed to alter RhoA activity. However, activation of receptor-operated Ca2+ entry by oleoyl-2-acetyl-sn-glycerol, the membrane permeable analogue of the Gαq-phospholipase C product diacylglycerol, induced RhoA activity. Receptor-operated Ca2+ activation was mediated by TRPC6 because small interfering RNA-induced TRPC6 knockdown significantly reduced Ca2+ entry. TRPC6 knockdown also prevented RhoA activation, myosin light chain phosphorylation, and actin stress fiber formation as well as inter-endothelial junctional gap formation in response to either oleoyl-2-acetyl-sn-glycerol or thrombin. TRPC6-mediated RhoA activity was shown to be dependent on PKCα activation. Our results demonstrate that Gαq activation of TRPC6 signals the activation of PKCα, and thereby induces RhoA activity and endothelial cell contraction. The continuous vascular endothelium lining the intima of the blood vessels regulates vascular smooth muscle tone, host-defense reactions, wound healing, angiogenesis, and function of the semi-permeable endothelial barrier (1Mehta D. Malik A.B. Physiol. Rev. 2006; 86: 279-367Crossref PubMed Scopus (1313) Google Scholar). Thrombin by binding to the endothelial cell surface protease-activated receptor-1 (PAR-1) 2The abbreviations used are: PAR-1, protease-activated receptor-1; TRPC, transient receptor potential channel; PLC, phospholipase C; MLC, myosin light chain; HPAEC, human pulmonary arterial endothelial cells; siRNA, small interference RNA; RGS2, regulator of G protein signaling 2; GST, glutathione S-transferase; OAG, oleoyl-2-acetyl-sn-glycerol; DAG, diacylglycerol; SOC, store-operated Ca2+; ROC, receptor-operated Ca2+; dn, dominant negative; GEF, GTP exchange factor; GDI, GDP dissociation inhibitor; ER, endoplasmic reticulum; IP3, inositol 1,4,5-trisphosphate; PKC, protein kinase C; TER, transendothelial electrical resistance; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. 2The abbreviations used are: PAR-1, protease-activated receptor-1; TRPC, transient receptor potential channel; PLC, phospholipase C; MLC, myosin light chain; HPAEC, human pulmonary arterial endothelial cells; siRNA, small interference RNA; RGS2, regulator of G protein signaling 2; GST, glutathione S-transferase; OAG, oleoyl-2-acetyl-sn-glycerol; DAG, diacylglycerol; SOC, store-operated Ca2+; ROC, receptor-operated Ca2+; dn, dominant negative; GEF, GTP exchange factor; GDI, GDP dissociation inhibitor; ER, endoplasmic reticulum; IP3, inositol 1,4,5-trisphosphate; PKC, protein kinase C; TER, transendothelial electrical resistance; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. induces a signaling cascade resulting in the development of minute inter-endothelial junctional gaps that lead to increased endothelial permeability, the hallmark of tissue inflammation (1Mehta D. Malik A.B. Physiol. Rev. 2006; 86: 279-367Crossref PubMed Scopus (1313) Google Scholar). Formation of these gaps occurs as the result of cell shape change induced by actinomyosin-mediated endothelial cell contraction (1Mehta D. Malik A.B. Physiol. Rev. 2006; 86: 279-367Crossref PubMed Scopus (1313) Google Scholar).Activation of the monomeric GTPase, RhoA, is crucial in signaling endothelial cell shape change; i.e. the “rounding up” response of endothelial cells (1Mehta D. Malik A.B. Physiol. Rev. 2006; 86: 279-367Crossref PubMed Scopus (1313) Google Scholar, 2Carbajal J.M. Schaeffer Jr., R.C. Am. J. Physiol. 1999; 277: C955-C964Crossref PubMed Google Scholar, 3Dudek S.M. Garcia J.G. J. Appl. Physiol. 2001; 91: 1487-1500Crossref PubMed Scopus (830) Google Scholar, 4Mehta D. Rahman A. Malik A.B. J. Biol. Chem. 2001; 276: 22614-22620Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 5Holinstat M. Mehta D. Kozasa T. Minshall R.D. Malik A.B. J. Biol. Chem. 2003; 278: 28793-28798Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Evidence from several cell types, including endothelial cells, indicated that G-protein-coupled receptors activate RhoA via the α subunit of the heterotrimeric GTP-binding protein Gq (5Holinstat M. Mehta D. Kozasa T. Minshall R.D. Malik A.B. J. Biol. Chem. 2003; 278: 28793-28798Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 6Moers A. Wettschureck N. Gruner S. Nieswandt B. Offermanns S. J. Biol. Chem. 2004; 279: 45354-45359Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 7Zeng H. Zhao D. Mukhopadhyay D. J. Biol. Chem. 2002; 277: 46791-46798Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 8Lutz S. Freichel-Blomquist A. Yang Y. Rumenapp U. Jakobs K.H. Schmidt M. Wieland T. J. Biol. Chem. 2005; 280: 11134-11139Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 9Vogt S. Grosse R. Schultz G. Offermanns S. J. Biol. Chem. 2003; 278: 28743-28749Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 10Chikumi H. Vazquez-Prado J. Servitja J.M. Miyazaki H. Gutkind J.S. J. Biol. Chem. 2002; 277: 27130-27134Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). For example, RhoA was not activated in response to thrombin in platelets lacking Gαq (6Moers A. Wettschureck N. Gruner S. Nieswandt B. Offermanns S. J. Biol. Chem. 2004; 279: 45354-45359Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 9Vogt S. Grosse R. Schultz G. Offermanns S. J. Biol. Chem. 2003; 278: 28743-28749Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Gαq was also required for growth factor-induced activation of RhoA in endothelial cells (7Zeng H. Zhao D. Mukhopadhyay D. J. Biol. Chem. 2002; 277: 46791-46798Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The Gαq pathway is known to induce an increase in intracellular Ca2+ (11Tiruppathi C. Minshall R.D. Paria B.C. Vogel S.M. Malik A.B. Vascul. Pharmacol. 2002; 39: 173-185Crossref PubMed Scopus (247) Google Scholar, 12Nilius B. Droogmans G. Physiol. Rev. 2001; 81: 1415-1459Crossref PubMed Scopus (753) Google Scholar, 13Yao X. Garland C.J. Circ. Res. 2005; 97: 853-863Crossref PubMed Scopus (228) Google Scholar, 14Ahmmed G.U. Malik A.B. Pflugers Arch. 2005; 451: 131-142Crossref PubMed Scopus (83) Google Scholar), which occurs secondary to the mobilization of Ca2+ from endoplasmic reticulum stores and increase in Ca2+ entry via plasma membrane non-selective cation channels. The latter response, mediated by activation of store-operated Ca2+ (SOC) and receptor-operated Ca2+ (ROC) channels, is crucial for sustaining the increase in intracellular Ca2+ concentration in endothelial cells (11Tiruppathi C. Minshall R.D. Paria B.C. Vogel S.M. Malik A.B. Vascul. Pharmacol. 2002; 39: 173-185Crossref PubMed Scopus (247) Google Scholar, 12Nilius B. Droogmans G. Physiol. Rev. 2001; 81: 1415-1459Crossref PubMed Scopus (753) Google Scholar, 13Yao X. Garland C.J. Circ. Res. 2005; 97: 853-863Crossref PubMed Scopus (228) Google Scholar, 14Ahmmed G.U. Malik A.B. Pflugers Arch. 2005; 451: 131-142Crossref PubMed Scopus (83) Google Scholar). In the present study, we surmised that the Gαq-mediated increase in intracellular Ca2+ concentration was crucial in regulating RhoA activity downstream of G-protein-coupled receptors. We had previously shown that activation of PKCα, a downstream effector of Gαq and a Ca2+- and diacylglycerol (DAG)-dependent enzyme (15Newton A.C. Chem. Rev. 2001; 101: 2353-2364Crossref PubMed Scopus (827) Google Scholar, 16Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4215) Google Scholar), was required for RhoA activation (4Mehta D. Rahman A. Malik A.B. J. Biol. Chem. 2001; 276: 22614-22620Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 5Holinstat M. Mehta D. Kozasa T. Minshall R.D. Malik A.B. J. Biol. Chem. 2003; 278: 28793-28798Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Thus, we addressed the possibility that the mechanism of RhoA activation involves Gαq-PLC-mediated activation of Ca2+ entry.Members of the mammalian homologues of Drosophila transient receptor potential channels (TRPC) family form the ROC and SOC channels in many cell types (11Tiruppathi C. Minshall R.D. Paria B.C. Vogel S.M. Malik A.B. Vascul. Pharmacol. 2002; 39: 173-185Crossref PubMed Scopus (247) Google Scholar, 12Nilius B. Droogmans G. Physiol. Rev. 2001; 81: 1415-1459Crossref PubMed Scopus (753) Google Scholar, 13Yao X. Garland C.J. Circ. Res. 2005; 97: 853-863Crossref PubMed Scopus (228) Google Scholar, 14Ahmmed G.U. Malik A.B. Pflugers Arch. 2005; 451: 131-142Crossref PubMed Scopus (83) Google Scholar). TRPC1, TRPC4, and TRPC5 form constituents of SOC because they are activated upon depletion of intracellular Ca2+ store by IP3 binding to IP3R (11Tiruppathi C. Minshall R.D. Paria B.C. Vogel S.M. Malik A.B. Vascul. Pharmacol. 2002; 39: 173-185Crossref PubMed Scopus (247) Google Scholar, 12Nilius B. Droogmans G. Physiol. Rev. 2001; 81: 1415-1459Crossref PubMed Scopus (753) Google Scholar, 13Yao X. Garland C.J. Circ. Res. 2005; 97: 853-863Crossref PubMed Scopus (228) Google Scholar, 14Ahmmed G.U. Malik A.B. Pflugers Arch. 2005; 451: 131-142Crossref PubMed Scopus (83) Google Scholar). TRPC3, TRPC6, and TRPC7 represent ROC constituents as they are activated by DAG and do not require store depletion (11Tiruppathi C. Minshall R.D. Paria B.C. Vogel S.M. Malik A.B. Vascul. Pharmacol. 2002; 39: 173-185Crossref PubMed Scopus (247) Google Scholar, 12Nilius B. Droogmans G. Physiol. Rev. 2001; 81: 1415-1459Crossref PubMed Scopus (753) Google Scholar, 13Yao X. Garland C.J. Circ. Res. 2005; 97: 853-863Crossref PubMed Scopus (228) Google Scholar, 14Ahmmed G.U. Malik A.B. Pflugers Arch. 2005; 451: 131-142Crossref PubMed Scopus (83) Google Scholar). TRPC1, TRPC4, and TRPC6 were shown to regulate Ca2+ entry and the increased endothelial permeability response (17Mehta D. Ahmmed G.U. Paria B.C. Holinstat M. Voyno-Yasenetskaya T. Tiruppathi C. Minshall R.D. Malik A.B. J. Biol. Chem. 2003; 278: 33492-33500Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 18Paria B.C. Vogel S.M. Ahmmed G.U. Alamgir S. Shroff J. Malik A.B. Tiruppathi C. Am. J. Physiol. 2004; 287: L1303-L1313Crossref PubMed Scopus (133) Google Scholar, 19Ahmmed G.U. Mehta D. Vogel S. Holinstat M. Paria B.C. Tiruppathi C. Malik A.B. J. Biol. Chem. 2004; 279: 20941-20949Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 20Brough G.H. Wu S. Cioffi D. Moore T.M. Li M. Dean N. Stevens T. FASEB J. 2001; 15: 1727-1738Crossref PubMed Scopus (151) Google Scholar, 21Cioffi D.L. Wu S. Alexeyev M. Goodman S.R. Zhu M.X. Stevens T. Circ. Res. 2005; 97: 1164-1172Crossref PubMed Scopus (86) Google Scholar, 22Moore T. Brough G. Kelly J. Babal P. Li M. Stevens T. Chest. 1998; 114 (Suppl. 1): 36S-38SAbstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 23Moore T.M. Brough G.H. Babal P. Kelly J.J. Li M. Stevens T. Am. J. Physiol. 1998; 275: L574-L582Crossref PubMed Google Scholar, 24Moore T.M. Norwood N.R. Creighton J.R. Babal P. Brough G.H. Shasby D.M. Stevens T. Am. J. Physiol. 2000; 279: L691-L698Crossref PubMed Google Scholar, 25Pocock T.M. Foster R.R. Bates D.O. Am. J. Physiol. 2004; 286: H1015-H1026Crossref PubMed Scopus (102) Google Scholar). Because at the mRNA level TRPC1 and TRPC6 are more abundantly expressed in human endothelial cells than other cell types (11Tiruppathi C. Minshall R.D. Paria B.C. Vogel S.M. Malik A.B. Vascul. Pharmacol. 2002; 39: 173-185Crossref PubMed Scopus (247) Google Scholar, 18Paria B.C. Vogel S.M. Ahmmed G.U. Alamgir S. Shroff J. Malik A.B. Tiruppathi C. Am. J. Physiol. 2004; 287: L1303-L1313Crossref PubMed Scopus (133) Google Scholar, 23Moore T.M. Brough G.H. Babal P. Kelly J.J. Li M. Stevens T. Am. J. Physiol. 1998; 275: L574-L582Crossref PubMed Google Scholar, 26Paria B.C. Malik A.B. Kwiatek A.M. Rahman A. May M.J. Ghosh S. Tiruppathi C. J. Biol. Chem. 2003; 278: 37195-37203Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), in the present study we addressed the roles of TRPC1 and TRPC6 in regulating RhoA activation. We demonstrate here the essential involvement of TRPC6 in mediating RhoA activity and endothelial cell contraction downstream of activation of PKCα in response to thrombin. These results for the first time establish a causal link between the Gαq-mediated increase in cytosolic Ca2+ via TRPC6 and activation of RhoA, which in turn mediates endothelial cell contraction and the increase in endothelial permeability.EXPERIMENTAL PROCEDURESMaterials—Human α-thrombin was obtained from Enzyme Research Laboratories (South Bend, IN). Human pulmonary arterial endothelial cells (HPAEC) and endothelial growth medium 2 were obtained from Clonetics (San Diego, CA). Fura 2-AM and Alexa-phalloidin, were purchased from Molecular Probes (Eugene, OR). U73122, OAG, and thapsigargin were obtained from Calbiochem (La Jolla, CA). Trypsin was purchased from Invitrogen. Electrodes for endothelial monolayer electrical resistance measurements were from Applied Biophysics (Troy, NY). Constitutively active Gαq (GαqQ209L) and RGS2 (HA-tagged) mutants were obtained from UMR cDNA Resource Center (Rolla, MO). Transfection reagents for siRNA (Nucleofector HCAEC kit) and the electroporation system were from Amaxa (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 obtained from Dharmacon (Lafayette, CO). Anti-RhoA, anti-TRPC1, anti-TRPC6, anti-Gαq, anti-actin, and anti-PKCα antibodies and siRNA transfection reagent were purchased from Santa Cruz Biotechnology (San Diego, CA), whereas phospho-PKCα antibody was purchased from Upstate (Lake Placid, NY). Rho activity was determined using GST-rhotekin-Rho binding domain beads from Cytoskeleton (Denver, CO). Anti-phospho-MLC antibody was a gift from Dr. Jerold Turner (University of Chicago).Endothelial Cell Culture—HPAEC were cultured in T-75 flasks coated with 0.1% gelatin in endothelial growth medium 2 supplemented with 10% fetal bovine serum. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air until confluent. Cells from each primary flask were detached with 0.025% trypsin/EDTA and plated on either 60-mm dishes for the Rho pulldown assay or coverslips for calcium and confocal imaging studies. In all experiments, HPAEC between passages 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 were trypsinized, mixed with 2.8 μg of siRNA or 3.0 μg of cDNA along with 100 μl of nucleofector solution. Cells were rapidly electroporated by the Amaxa nucleofector device using the manufacturer’s recommended program (S-05) dedicated for human coronary arterial endothelial cells. The cells were removed, mixed in endothelial growth medium 2, and plated on either 60-mm dishes. HPAE cells plated on coverslips or gold-plated 10-well electrodes were transfected with 100 nm siRNA using 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 a 7 or 12.5% polyacrylamide gel, and transferred to nitrocellulose membrane for Western blotting with the indicated antibodies (17Mehta D. Ahmmed G.U. Paria B.C. Holinstat M. Voyno-Yasenetskaya T. Tiruppathi C. Minshall R.D. Malik A.B. J. Biol. Chem. 2003; 278: 33492-33500Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 19Ahmmed G.U. Mehta D. Vogel S. Holinstat M. Paria B.C. Tiruppathi C. Malik A.B. J. Biol. Chem. 2004; 279: 20941-20949Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar).Cytosolic Ca2+ Measurements—An increase in intracellular Ca2+ was measured using the Ca2+-sensitive fluorescent dye Fura 2-AM as described (17Mehta D. Ahmmed G.U. Paria B.C. Holinstat M. Voyno-Yasenetskaya T. Tiruppathi C. Minshall R.D. Malik A.B. J. Biol. Chem. 2003; 278: 33492-33500Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 19Ahmmed G.U. Mehta D. Vogel S. Holinstat M. Paria B.C. Tiruppathi C. Malik A.B. J. Biol. Chem. 2004; 279: 20941-20949Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Briefly, cells grown on 25-mm coverslips were incubated with 3 μm Fura 2-AM for 15 min at 37 °C. Cells were washed three to four times with Hank’s balanced salt solution and imaged using an Attofluor Ratio Vision digital fluorescence microscopy system (Atto Instruments, Inc., Rockville, MD) equipped with a Zeiss Axiovert S100 inverted microscope and F-Fluor ×40 1.3-numerical aperture oil immersion objective. Regions of interests in individual cells were marked and excited at 334 and 380 nm with emission at 520 nm. The 340/380 nm excitation ratio, which increases as a function 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 activated RhoA as described (4Mehta D. Rahman A. Malik A.B. J. Biol. Chem. 2001; 276: 22614-22620Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 17Mehta D. Ahmmed G.U. Paria B.C. Holinstat M. Voyno-Yasenetskaya T. Tiruppathi C. Minshall R.D. Malik A.B. J. Biol. Chem. 2003; 278: 33492-33500Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). HPAE cell monolayers were stimulated for the indicated times with 50 nm thrombin, 100 μm OAG, or 2 μm thapsigargin. Cells were quickly washed with ice-cold Tris-buffered saline, and lysed with 200 μl of lysis buffer (50 mm Tris, pH 7.5, 10 mm MgCl2, 0.5 m NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 10 μg/ml each of aprotinin and leupeptin). Cell lysates were clarified by centrifugation at 14,000 × g at 4 °C for 2 min and equal volumes of cell lysates were incubated with GST-Rho binding domain beads (15 μg) at 4 °C for 2 h. The beads were washed three times with wash buffer (25 mm Tris, pH 7.5, 30 mm MgCl2, and 40 mm NaCl), and bound RhoA was eluted by boiling each sample in Laemmli sample buffer. Eluted samples from the beads and total cell lysate were then electrophoresed on 12.5% SDS-polyacrylamide gels and Western blotted with rabbit polyclonal anti-RhoA antibody.PKCα Translocation—HPAEC monolayer stimulated with 50 nm thrombin for the indicated times were quickly washed with ice-cold phosphate-buffered saline and cells were scrapped using Tris buffer (pH 7.5 containing in mm; 10 Tris, 1 MgCl2, 5 EDTA, 10 EGTA, 1 Na3VO4) and a mixture of protease inhibitors. Cell lysates were sonicated for 10 s and an aliquot of the lysates was saved for determination of total PKCα. Lysates were then centrifuged at 100,000 × g for 1 h at 4 °C to separate cytosolic fraction. The pellets were suspended in the above lysis buffer plus 1% Triton X-100, sonicated, and incubated for 30 min at 4 °C followed by centrifugation at 14,000 × g at 4 °C to separate membrane fractions as described (27Javaid K. Rahman A. Anwar K.N. Frey R.S. Minshall R.D. Malik A.B. Circ. Res. 2003; 92: 1089-1097Crossref PubMed Scopus (118) Google Scholar). Total cell lysates, cytosol, and membrane fractions were immunoblotted with PKCα antibody to determine PKCα translocation in the membrane fraction following thrombin stimulation.PKCα Activity—We used GST-GDI-1 fusion protein as a substrate to assess PKCα activity (4Mehta D. Rahman A. Malik A.B. J. Biol. Chem. 2001; 276: 22614-22620Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Briefly, HPAEC monolayer stimulated with 50 nm thrombin for 5 min was quickly washed with phosphate-buffered saline and lysed using immunoprecipitation assay buffer (1% Triton X-100, 150 mm NaCl, 10 mm Tris, 1 mm EDTA, 1 mm EGTA, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 0.5% Nonidet P-40, and 2 μg/ml each of pepstatin A, leupeptin, and aprotinin). Cell lysates were cleared by centrifugation at 4 °C at 14,000 × g for 10 min and immunoprecipitated with anti-rabbit polyclonal PKCα antibody. PKCα immunocomplexes were used to phosphorylate GST-GDI-1 fusion protein.Immunofluorescence—Cells were stimulated with 50 nm thrombin for the indicated times, rinsed quickly with ice-cold Hank’s balanced salt solution, and fixed with 2% paraformaldehyde. Cells were permeabilized for 3 min with 0.1% Triton X-100 in Hank’s balanced salt solution followed by incubation for 40 min with 1% ovalbumin. Cells were then incubated with anti-PKCα antibody followed by incubation with Alexa-labeled secondary antibody for another 1 h. Cells were then washed three times with Hank’s balanced salt solution and mounted with anti-fade media. For determining actin stress fiber formation, cells were rinsed and incubated with Alexa-labeled phalloidin to label actin stress fibers. Cells were viewed using a 63× 1.2 NA objective and appropriate filters using a Zeiss LSM510 confocal microscope.MLC Phosphorylation—HPAEC monolayer was stimulated with thrombin for the indicated times. Endothelial cells were scraped off and mixed with Laemmli sample buffer and Western blotted with antibodies for phosphorylated-MLC or pan-MLC antibodies to determine MLC phosphorylation.Transendothelial Resistance Measurement—The time course of endothelial cell retraction in real time, a measure of increased endothelial permeability, was measured according to the procedure described previously (4Mehta D. Rahman A. Malik A.B. J. Biol. Chem. 2001; 276: 22614-22620Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar).Statistical Analysis—Two-tailed Student’s t test and one-way analysis of variance with the Bonferroni post hoc test were used for statistical comparisons. Differences were considered significant at p < 0.05.RESULTSActivation of Gαq-PLC Pathway Induces RhoA Activity—Stimulation of PAR-1 receptor by thrombin increases intracellular Ca2+ by the Gαq-PLC pathway (11Tiruppathi C. Minshall R.D. Paria B.C. Vogel S.M. Malik A.B. Vascul. Pharmacol. 2002; 39: 173-185Crossref PubMed Scopus (247) Google Scholar, 14Ahmmed G.U. Malik A.B. Pflugers Arch. 2005; 451: 131-142Crossref PubMed Scopus (83) Google Scholar). In the present experiments, we sought to determine the contribution of Gαq and the PLC-mediated increase in intracellular Ca2+ in thrombin-induced RhoA activation. We transduced the constitutively active Gαq mutant in endothelial cells and determined RhoA activity using rhotekin-bound fusion proteins. HPAEC transducing the active mutant of Gαq showed a 3.8 ± 0.4-fold increase in RhoA activity (Fig. 1A)(p < 0.05). To corroborate these findings, we inhibited Gαq function using RGS2, which predominantly increases the intrinsic rate of Gαq to hydrolyze GTP to GDP, thereby inhibiting Gαq function (28Heximer 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. Steinberg T.H. Wyss J.M. Mecham R.P. Blumer K.J. J. Clin. Investig. 2003; 111: 445-452Crossref PubMed Scopus (156) Google Scholar, 29Heximer S.P. Srinivasa S.P. Bernstein L.S. Bernard J.L. Linder M.E. Hepler J.R. Blumer K.J. J. Biol. Chem. 1999; 274: 34253-34259Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 30Heximer S.P. Watson N. Linder M.E. Blumer K.J. Hepler J.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14389-14393Crossref PubMed Scopus (311) Google Scholar). We observed that expression of RGS2 inhibited RhoA activation in response to thrombin (Fig. 1, B and G)(p < 0.05). Next, we pretreated HPAEC with U73122, an inhibitor of PLC (31Smith R.J. Sam L.M. Justen J.M. Bundy G.L. Bala G.A. Bleasdale J.E. J. Pharmacol. Exp. Ther. 1990; 253: 688-697PubMed Google Scholar), to assess the requirement of PLC activity in thrombin-induced activation of RhoA. We observed that U73122 prevented RhoA activation in response to thrombin (Fig. 1, C and G)(p < 0.05). To determine whether the thrombin-activated increase in intracellular Ca2+ and RhoA activity is causally related, intracellular Ca2+ was chelated with the membrane permeant Ca2+ chelator BAPTA-2AM. We observed that thrombin failed to induce RhoA activation in BAPTA-pretreated cells (Fig. 1, D and G) (p < 0.05). Simultaneous measurement of intracellular Ca2+ using Fura 2-AM confirmed that these interventions significantly suppressed thrombin-induced intracellular Ca2+ rise (Fig. 1E). In other studies, we depleted extracellular Ca2+ to 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 significantly reduced in the absence of extracellular Ca2+ (p < 0.05). These findings indicated that the increase in intracellular Ca2+ induced by the Gαq-PLC pathway mediated Ca2+ entry contributed to RhoA activation.Receptor-operated Ca2+ Entry Induces RhoA Activation—Gαq upon activation with thrombin stimulates PLC, which in turn activates Ca2+ entry through SOC and ROC channels (11Tiruppathi C. Minshall R.D. Paria B.C. Vogel S.M. Malik A.B. Vascul. Pharmacol. 2002; 39: 173-185Crossref PubMed Scopus (247) Google Scholar, 14Ahmmed G.U. Malik A.B. Pflugers Arch. 2005; 451: 131-142Crossref PubMed Scopus (83) Google Scholar). Thus, we determined the role of Ca2+ entry mediated by SOC and ROC channels in inducing RhoA activation in response to thrombin. We used thapsigargin because it activates SOC channels independently of ligand-receptor-G protein-coupled receptors (12Nilius B. Droogmans G. Physiol. Rev. 2001; 81: 1415-1459Crossref PubMed Scopus (753) Google Scholar). Thapsigargin increased intracellular Ca2+ (Fig. 2A); however, it failed to induce RhoA activation (Fig. 2, B and C), indicating SOC channels are not sufficient to activate RhoA. To determine the role of ROC channels, we used OAG, a membrane-permeable analogue of DAG, known to activate ROC channels (13Yao X. Garland C.J. Circ. Res. 2005; 97: 853-863Crossref PubMed Scopus (228) Google Scholar, 14Ahmmed G.U. Malik A.B. Pflugers Arch. 2005; 451: 131-142Crossref PubMed Scopus (83) Google Scholar). OAG in a dose-dependent manner induced sustained Ca2+ entry in endothelial cells (Fig. 3A). We also observed that OAG significantly increased RhoA activity, a response sustained up to 20 min (Fig. 3, B and C). OAG failed to induce Ca2+ entry (Fig. 3A) as well as RhoA activation (Fig. 3D) in the absence of extracellular Ca2+, indicating that OAG increases the intracellular Ca2+ concentration and RhoA activity by activating ROC channels and does not require ER store depletion. To address the possibility that OAG effects on Ca2+ entry are the result of PKCα activation by OAG, we overexpressed the dominant negative (dn) mutant of PKCα by infecting endothelia

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