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• W2049967046 abstract "The Rho-GDP guanine nucleotide dissociation inhibitor (GDI) complexes with the GDP-bound form of Rho and inhibits its activation. We investigated the role of protein kinase C (PKC) isozymes in the mechanism of Rho activation and in signaling the loss of endothelial barrier function. Thrombin and phorbol 12-myristate 13-acetate induced rapid phosphorylation of GDI and the activation of Rho-A in human umbilical venular endothelial cells. Inhibition of PKC by chelerythrine chloride abrogated the thrombin-induced GDI phosphorylation and Rho activation. Depletion of PKC prevented the thrombin-induced GDI phosphorylation and Rho activation, thereby indicating that these events occurred downstream of phorbol ester-sensitive PKC isozyme activation. The depletion of PKC or inhibition of Rho by C3 toxin also prevented the thrombin-induced decrease in transendothelial electrical resistance (a measure of increased transendothelial permeability), thus indicating that PKC-induced barrier dysfunction was mediated through Rho-dependent pathway. Using inhibitors and dominant-negative mutants, we found that Rho activation was regulated by PKC-α. Moreover, the stimulation of human umbilical venular endothelial cells with thrombin induced rapid association of PKC-α with Rho. Activated PKC-α but not PKC-ε induced marked phosphorylation of GDI in vitro. Taken together, these results indicate that PKC-α is critical in regulating GDI phosphorylation, Rho activation, and in signaling Rho-dependent endothelial barrier dysfunction. The Rho-GDP guanine nucleotide dissociation inhibitor (GDI) complexes with the GDP-bound form of Rho and inhibits its activation. We investigated the role of protein kinase C (PKC) isozymes in the mechanism of Rho activation and in signaling the loss of endothelial barrier function. Thrombin and phorbol 12-myristate 13-acetate induced rapid phosphorylation of GDI and the activation of Rho-A in human umbilical venular endothelial cells. Inhibition of PKC by chelerythrine chloride abrogated the thrombin-induced GDI phosphorylation and Rho activation. Depletion of PKC prevented the thrombin-induced GDI phosphorylation and Rho activation, thereby indicating that these events occurred downstream of phorbol ester-sensitive PKC isozyme activation. The depletion of PKC or inhibition of Rho by C3 toxin also prevented the thrombin-induced decrease in transendothelial electrical resistance (a measure of increased transendothelial permeability), thus indicating that PKC-induced barrier dysfunction was mediated through Rho-dependent pathway. Using inhibitors and dominant-negative mutants, we found that Rho activation was regulated by PKC-α. Moreover, the stimulation of human umbilical venular endothelial cells with thrombin induced rapid association of PKC-α with Rho. Activated PKC-α but not PKC-ε induced marked phosphorylation of GDI in vitro. Taken together, these results indicate that PKC-α is critical in regulating GDI phosphorylation, Rho activation, and in signaling Rho-dependent endothelial barrier dysfunction. guanine nucleotide exchange factor GTPase-activating protein guanine nucleotide dissociation inhibitor protein kinase C human umbilical venular endothelial cells endothelial growth medium phosphate-buffered saline phorbol 12-myristate 13-acetate glutathione S-transferase rhotekin-Rho binding domain phenylmethylsulfonyl fluoride serum response element analysis of variance The vascular endothelium consisting of the monolayer of endothelial cells and the extracellular matrix represents the major barrier for the exchange of liquid and solutes across the vessel wall (1Gao B. Curtis T.M. Blumenstock F.A. Minnear F.L. Saba T.M. J. Cell Sci. 2000; 113: 247-257Crossref PubMed Google Scholar, 2Lampugnani M.G. Resnati M. Dejana E. Marchisio P.C. J. Cell Biol. 1991; 112: 479-490Crossref PubMed Scopus (241) Google Scholar, 3Lum H. Malik A.B. Am. J. Physiol. 1994; 267: L223-L241Crossref PubMed Google Scholar, 4Qiao R.L. Yan W. Lum H. Malik A.B. Am. J. Physiol. 1995; 269: C110-C117Crossref PubMed Google Scholar). Thrombin by binding to the endothelial cell surface protease-activated receptor-1 induces a repertoire of signaling events that result in the development of minute gaps among cells, thereby mediating increased vascular permeability, a hallmark of tissue inflammation (5Gerszten R.E. Chen J. Ishii M. Ishii K. Wang L. Nanevicz T. Turck C.W. Vu T.K. Coughlin S.R. Nature. 1994; 368: 648-651Crossref PubMed Scopus (183) Google Scholar, 6Garcia J.G. Patterson C. Bahler C. Aschner J. Hart C.M. English D. J. Cell. Physiol. 1993; 156: 541-549Crossref PubMed Scopus (120) Google Scholar, 7Lum H. Andersen T.T. Siflinger-Birnboim A. Tiruppathi C. Goligorsky M.S. Fenton J.W. Malik A.B. J. Cell Biol. 1993; 120: 1491-1499Crossref PubMed Scopus (70) Google Scholar). Loss of endothelial barrier function primarily occurs as a result of cell shape change via actinomyosin driven contraction activated by myosin light chain phosphorylation and actin polymerization (3Lum H. Malik A.B. Am. J. Physiol. 1994; 267: L223-L241Crossref PubMed Google Scholar, 8Moy A.B. Van Engelenhoven J. Bodmer J. Kamath J. Keese C. Giaever I. Shasby S. Shasby D.M. J. Clin. Invest. 1996; 97: 1020-1027Crossref PubMed Scopus (172) Google Scholar, 9Garcia J.G. Pavalko F.M. Patterson C.E. Blood Coagul. Fibrinolysis. 1995; 6: 609-626Crossref PubMed Scopus (97) Google Scholar, 10Garcia J.G. Davis H.W. Patterson C.E. J. Cell. Physiol. 1995; 163: 510-522Crossref PubMed Scopus (488) Google Scholar, 11Garcia J.G. Verin A.D. Schaphorst K. Siddiqui R. Patterson C.E. Csortos C. Natarajan V. Am. J. Physiol. 1999; 276: L989-L998PubMed Google Scholar). Studies have shown an important role of the small GTPase, Rho, in the regulation of cytoskeletal dynamics, actin stress fiber formation, and myosin light chain-phosphorylation, and thus by inference, in the control of endothelial barrier function (11Garcia J.G. Verin A.D. Schaphorst K. Siddiqui R. Patterson C.E. Csortos C. Natarajan V. Am. J. Physiol. 1999; 276: L989-L998PubMed Google Scholar, 12van Nieuw Amerongen G.P. Draijer R. Vermeer M.A. van Hinsbergh V.W. Circ. Res. 1998; 83: 1115-1123Crossref PubMed Google Scholar, 13Essler M. Amano M. Kruse H.J. Kaibuchi K. Weber P.C. Aepfelbacher M. J. Biol. Chem. 1998; 273: 21867-21874Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). The multiple functions of Rho are mediated through the tightly regulated GTP-binding/GTPase cycle (14Hall A. Science. 1998; 280: 2074-2075Crossref PubMed Scopus (166) Google Scholar, 15Sah V.P. Seasholtz T.M. Sagi S.A. Brown J.H. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 459-489Crossref PubMed Scopus (298) Google Scholar, 16Kaibuchi K. Kuroda S. Amano M. Annu. Rev. Biochem. 1999; 68: 459-486Crossref PubMed Scopus (891) Google Scholar). Three different classes of proteins are required for this regulation: (i) guanine nucleotide exchange factors (GEFs),1 which stimulate the GTP-GDP exchange reaction; (ii) GTPase-activating proteins (GAPs), which stimulate the GTP-hydrolytic reaction; and (iii) guanine nucleotide dissociation inhibitors (GDIs), which by binding to Rho block the dissociation of GDP from Rho GTPases (17Geyer M. Wittinghofer A. Curr. Opin. Struct. Biol. 1997; 7: 786-792Crossref PubMed Scopus (143) Google Scholar). Furthermore, GDI is also capable of inhibiting GTP hydrolysis by Rho family GTPases as well as stimulating the release of Rho-GTPases from cellular membranes, thereby shutting off the Rho cycle (18Nomanbhoy T.K. Cerione R. J. Biol. Chem. 1996; 271: 10004-10009Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Thus, GDI plays a critical role in the signaling events regulated by Rho-GTPases (19Olofsson B. Cell. Signal. 1999; 11: 545-554Crossref PubMed Scopus (412) Google Scholar). The GDP-bound form of Rho complexed with GDI is not activated by Rho-GEFs, suggesting that Rho activation critically depends upon upstream factors mediating the dissociation of GDI from Rho (19Olofsson B. Cell. Signal. 1999; 11: 545-554Crossref PubMed Scopus (412) Google Scholar, 20Hart M.J. Maru Y. Leonard D. Witte O.N. Evans T. Cerione R.A. Science. 1992; 258: 812-815Crossref PubMed Scopus (122) Google Scholar, 21Yaku H. Sasaki T. Takai Y. Biochem. Biophys. Res. Commun. 1994; 198: 811-817Crossref PubMed Scopus (74) Google Scholar). The mechanisms activating the dissociation of Rho-GDI from the Rho-GDP complex remain to be determined. It has been suggested that the dissociation of Rho-GDI might be facilitated by members of ezrin/radixin/moesin family of proteins (22Sasaki T. Takai Y. Biochem. Biophys. Res. Commun. 1998; 245: 641-645Crossref PubMed Scopus (167) Google Scholar, 23Takahashi K. Sasaki T. Mammoto A. Takaishi K. Kameyama T. Tsukita S. Takai Y. J. Biol. Chem. 1997; 272: 23371-23375Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). However, Rho-GDI was found to interact only with the N-terminal fragment of radixin but not the full-length radixin, indicating the need of upstream effectors that are required to induce the unfolding of radixin (23Takahashi K. Sasaki T. Mammoto A. Takaishi K. Kameyama T. Tsukita S. Takai Y. J. Biol. Chem. 1997; 272: 23371-23375Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). Furthermore, several studies indicate that the translocation and activation of ezrin/radixin/moesin proteins to the membrane are critically dependent on Rho, thereby indicating the intervention of other molecules that activate dissociation of GDI from Rho (24Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita S. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (512) Google Scholar, 25Kotani H. Takaishi K. Sasaki T. Takai Y. Oncogene. 1997; 14: 1705-1713Crossref PubMed Scopus (71) Google Scholar). Rho-GDI is a family consisting of Rho-GDI-1, Ly/D4-GDI, and Rho GDI-III. Of these, Rho-GDI is ubiquitously expressed (19Olofsson B. Cell. Signal. 1999; 11: 545-554Crossref PubMed Scopus (412) Google Scholar, 22Sasaki T. Takai Y. Biochem. Biophys. Res. Commun. 1998; 245: 641-645Crossref PubMed Scopus (167) Google Scholar). The structure of Rho-GDI-1 indicates that it contains sequences for phosphorylation by serine-threonine kinases, raising the possibility that Rho-GDI is regulated by signaling mechanisms that induce its phosphorylation. Protein kinase C (PKC) isozymes are serine-threonine kinases that induce phosphorylation of multiple proteins, which in turn regulate intracellular signaling (26Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4232) Google Scholar). A PKC-dependent pathway is important in the mechanism of thrombin-induced increase in endothelial permeability (3Lum H. Malik A.B. Am. J. Physiol. 1994; 267: L223-L241Crossref PubMed Google Scholar, 27Vuong P.T. Malik A.B. Nagpala P.G. Lum H. J. Cell. Physiol. 1998; 175: 379-387Crossref PubMed Scopus (40) Google Scholar, 28Aschner J.L. Lum H. Fletcher P.W. Malik A.B. J. Cell. Physiol. 1997; 173: 387-396Crossref PubMed Scopus (44) Google Scholar, 29Patterson C.E. Davis H.W. Schaphorst K.L. Garcia J.G. Microvasc. Res. 1994; 48: 212-235Crossref PubMed Scopus (77) Google Scholar, 30Lynch J.J. Ferro T.J. Blumenstock F.A. Brockenauer A.M. Malik A.B. J. Clin. Invest. 1990; 85: 1991-1998Crossref PubMed Scopus (238) Google Scholar). Because of the possibility that PKC may activate Rho by mediating Rho-GDI phosphorylation, we investigated the role of PKC in the mechanism of thrombin-induced Rho activation and in signaling the loss of endothelial barrier function in human umbilical venular endothelial (HUVE) cells. The present findings suggest the existence of a novel pathway by which thrombin can stimulate Rho activation. This pathway involves PKC-α-mediated phosphorylation of GDI, which may stimulate GDI dissociation, thereby resulting in Rho activation and increased endothelial permeability. Human α-thrombin was obtained from Enzyme Research Laboratories (South Bend, IN). HUVE cells and endothelial growth medium (EBM-2) were obtained from Clonetics (San Diego, CA). Phosphate-buffered saline (PBS) and trypsin were obtained from Life Technologies, Inc. Anti-Rho-A, anti-Rho-GDI, and anti-PKC-α, -ε, -δ, and -ζ polyclonal antibodies were obtained from Santa Cruz Biotechnology (San Diego, CA). Purified GST-Rho-GDI was purchased from Cytoskeleton, Inc. (Denver, CO). HUVE cells were cultured in a T-75 flask coated with 0.1% gelatin in EBM-2 medium supplemented with 10% fetal bovine serum. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air until they formed a confluent monolayer. Cells from each of the primary flasks were detached with 0.05% trypsin, 0.02% EDTA and resuspended in fresh culture medium and passaged as described below. In all experiments, unless otherwise indicated, a confluent monolayer of HUVE cells was washed twice with serum-free medium and incubated in serum-free medium for 3–4 h before treatment with the drug. In all experiments, cells between passages 4 and 8 were used. The time course of endothelial cell retraction, a measure of increased endothelial permeability, was measured according to the procedures described previously (31Tiruppathi C. Malik A.B. Del Vecchio P.J. Keese C.R. Giaever I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7919-7923Crossref PubMed Scopus (379) Google Scholar). HUVE cells grown to confluence on a gelatin-coated small gold electrode (4.9 × 10−4cm2) were pretreated with 500 nm phorbol 12-myristate 13-acetate (PMA) overnight or with 10 μg/ml of C3 transferase for 16 h in EBM-10% fetal bovine serum medium. After serum deprivation, cells were stimulated with thrombin to measure change in electrical resistance of the endothelial monolayer. The small electrode and larger counter electrode were connected to a phase-sensitive lock-in amplifier. A constant current of 1 μA was supplied by a 1-V 4000-Hz alternating current connected serially to a 1-megohm resistor between the small electrode and the larger counter electrode. The voltage between the small and large electrode was monitored by a lock-in amplifier, stored, and processed on a computer. Data are presented as change in resistive (in phase) portion of impedance normalized to its initial value at time zero. pGEX-2T containing rhotekin-Rho binding domain was provided by Dr. M. A. Schwartz (Scripps Research Institute, La Jolla, CA). Bacterial-expressed GST-rhotekin-Rho binding domain (RBD) protein was purified from isopropyl-1-thio-β-d-galactopyranozide (1 mm)-induced DH5α cells previously transformed with the appropriate plasmid as described previously (32Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1369) Google Scholar). Confluent HUVE cells grown in 100-mm dishes were stimulated for the indicated times with 50 nm thrombin or 100 nm PMA. Cells were then quickly washed with ice-cold Tris-buffered saline and lysed in lysis buffer (50 mm Tris, pH 7.4, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mm NaCl, 10 mmMgCl2, 10 μg/ml each of aprotinin and leupeptin, and 1 mm phenylmethylsulfonyl fluoride (PMSF)). 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-RBD beads (15 μg) at 4 °C for 1 h. The beads were washed 3 times with wash buffer (50 mm Tris, pH 7.4, 1% Triton X-100, 150 mm NaCl, 10 mm MgCl2, 10 μg/ml each of aprotinin and leupeptin, and 0.1 mm PMSF), and bound Rho was eluted by boiling each sample in Laemmli sample buffer. Eluted samples from beads and total cell lysate were then electrophoresed on 12.5% SDS-polyacrylamide gel electrophoresis gels, transferred to nitrocellulose, blocked with 5% nonfat milk, and analyzed by Western blotting using a polyclonal anti-Rho-A antibody. The amount of RBD-bound Rho was normalized to the total amount of Rho in cell lysates for quantitation of Rho activity in different samples using scanning densitometry. A serum-starved confluent monolayer of HUVE cells was labeled with 300 μCi/ml 32P for 4 h in phosphate-free medium, after which they were stimulated with 50 nm thrombin or 100 nm PMA at indicated times. Cells were quickly rinsed twice with ice-cold PBS and then lysed for 20 min on ice with 300 μl of radioimmune precipitation buffer (10 mm Tris, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 0.5% Nonidet P-40, 1% Triton-X, 1 mmsodium orthovanadate, 1 mm PMSF, and 1 μg/ml each of leupeptin, pepstatin A, and aprotinin). After clearing the lysate by centrifuging at 4 °C at 14,000 × g for 10 min, the lysate was incubated with anti-rabbit polyclonal Rho-GDI antibody for 1 h followed by the addition of protein A-agarose beads overnight at 4 °C. The beads were collected by centrifugation, washed 4 times with radioimmune precipitation buffer, electrophoresed on 4–15% gradient SDS-polyacrylamide gels, and transferred to nitrocellulose for visualization of GDI phosphorylation by autoradiography and for Western blotting with Rho-GDI antibody to verify equal protein loading. Specificity of the Rho-GDI antibody was confirmed by using peptide immunogen as a negative control. Rho has been shown to be required primarily for agonist-induced SRE reporter gene activity (33Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1207) Google Scholar). Therefore, we determined using the SRE reporter gene activity, the role of PKC isozymes in the mechanism of Rho activation. The pSRE-luciferase plasmid was kindly provided by Dr. T. Kozasa (University of Illinois, Chicago, IL). C3 transferase, produced by Clostridium botulinum that specifically ADP-ribosylates and inhibits Rho protein (34Paterson H.F. Self A.J. Garrett M.D. Just I. Aktories K. Hall A. J. Cell Biol. 1990; 111: 1001-1007Crossref PubMed Scopus (571) Google Scholar), was purified from an Escherichia colipGEX-2r-bcr recombinant vector expression system as described previously (35Morii N. Narumiya S. Methods Enzymol. 1995; 256: 196-206Crossref PubMed Scopus (55) Google Scholar). The expression vector pcDNA3-containing tagged dominant-negative form of PKC-α and PKC-δ isozymes were provided by Dr. I. B. Weinstein (Columbia University, New York, NY). The dominant-negative PKC-α, PKC-ε, and PKC-δ mutants lacking a functional catalytic domain were generated by a substitution of lysine 368, 437, or 376 for arginine, respectively (36Soh J.W. Lee E.H. Prywes R. Weinstein I.B. Mol. Cell. Biol. 1999; 19: 1313-1324Crossref PubMed Scopus (249) Google Scholar). Transfections were performed with DEAE-dextran method (37Rahman A. Anwar K.N. Malik A.B. Am. J. Physiol. 2000; 279: C906-C914Crossref PubMed Google Scholar). 5 μg of DNA were mixed with 50 μg/ml DEAE-dextran in serum-free EBM medium, and the mixture was added onto 70–80% confluent cells. pTKRLUC plasmid (0.125 μg) (Promega Corp., Madison, WI) containing Renilla luciferase gene driven by the constitutively active thymidine kinase promoter was added to normalize the transfection efficiencies. After 1 h, cells were incubated for 4 min with 10% dimethyl sulfoxide (Me2SO) in serum-free medium, washed twice with EBM-containing 10% fetal bovine serum, and grown to confluence. Cell extracts were prepared and assayed for luciferase activity using the Dual Luciferase Reporter Assay System (Promega, Madison, WI). SRE-luciferase activity was expressed as the ratio of firefly andRenilla luciferase activity. Cell viability (>95%) after transfection was confirmed using trypan blue (Sigma) exclusion assay. Phosphorylation of Rho-GDI in vitro was performed using immunocomplexes of PKC-α or PKC-ε obtained after immunoprecipitation of cell lysate with respective PKC antibodies as described previously (38Jain N. Zhang T. Kee W.H. Li W. Cao X. J. Biol. Chem. 1999; 274: 24392-24400Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Confluent cells grown in 100-mm dishes were stimulated for 1 min with 50 nm thrombin, washed quickly with ice-cold PBS, and lysed in radioimmune precipitation buffer containing 50 mm Tris, pH 7.4, 150 mm NaCl, 0.25 mm EDTA, pH 8.0, 1% deoxycholic acid, 1% Triton-X, 5 mm NaF, 1 mmsodium orthovanadate, 1 mm PMSF, and 5 μg/ml each of leupeptin and aprotinin, and 1 μg/ml pepstatin A. The lysate was scraped and cleared by centrifugation at 4 °C at 14,000 ×g for 10 min. Cell lysate containing an equal amount of protein was then incubated with anti-rabbit polyclonal PKC-α or PKC-ε antibody for 1 h followed by an addition of protein A-agarose beads overnight at 4 °C. Beads from each sample were collected by centrifugation, washed twice with ice-cold lysate buffer, then washed 3 times with PBS, and once with PKC kinase assay buffer (25 mm Tris, pH 7.4, 5 mm MgCl2, 0.5 mm EGTA, 1 mm dithiothreitol, and 20 μg of phosphatidylserine/reaction). The purified GST-Rho-GDI fusion proteins (3 μg) were incubated with immunocomplexes of PKC dissolved in kinase assay buffer for 10 min at 30 °C followed by an addition of cold ATP (20 μm) and 10 μCi of [γ-32P]ATP, after which the mixture was incubated for an additional 30 min. The reaction was stopped by the addition of Laemmli sample buffer, and each sample was electrophoresed on 10% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and exposed to x-ray films. The blots were then subjected to Western blotting with anti-PKC-α or PKC-ε antibodies to verify the equal amount of the protein in each reaction. Comparisons among experimental groups were made by ANOVA or Kruskal-Wallis one-way analysis of variance using SigmaStat software. Differences in mean values were considered significant at p < 0.05. We used GST-rhotekin fusion protein, which specifically binds to activated Rho, to quantitate Rho activation (32Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1369) Google Scholar, 39van Nieuw Amerongen G.P. van Delft S. Vermeer M.A. Collard J.G. van Hinsbergh V.W. Circ. Res. 2000; 87: 335-340Crossref PubMed Google Scholar). Thrombin induced a 3–4-fold increase in Rho activity in a time-dependent manner with a maximum response occurring at 1 min followed by a decline at 10 min (Fig. 1, A and B). We also used PMA, a direct activator of PKC, to determine the role of PKC activation in mediating Rho activation. As shown in Fig. 1,C and D, PMA induced a 2–3-fold increase in Rho activity in a time-dependent manner with maximum activation at 10 min. To investigate whether PKC acts upstream of Rho activation, we used chelerythrine chloride, a specific (but not isozyme-selective) PKC inhibitor belonging to a new class of PKC inhibitors that interfere with the phosphate acceptor site and non-competitively inhibit the ATP binding site (40Herbert J.M. Augereau J.M. Gleye J. Maffrand J.P. Biochem. Biophys. Res. Commun. 1990; 172: 993-999Crossref PubMed Scopus (1196) Google Scholar, 41Laudanna C. Mochly-Rosen D. Liron T. Constantin G. Butcher E.C. J. Biol. Chem. 1998; 273: 30306-30315Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Chelerythrine pretreatment of HUVE cells prevented thrombin-induced Rho activation, indicating that PKC is an upstream regulator of Rho activation (Fig.2). To determine whether PKC isozymes involved in thrombin-induced Rho activation were phorbol-sensitive, we studied the effects of PKC depletion by PMA on thrombin-induced Rho activation. HUVE cells were pretreated without or with 500 nm PMA overnight, after which they were stimulated with thrombin to measure Rho activation. Fig. 3 A shows that overnight pretreatment of HUVE cells with PMA prevented Rho activation in response to thrombin. Western blot analysis of the cell lysate from these samples showed that exposure of HUVE cells to phorbol esters resulted in the depletion of PKC-α, PKC-ε, and PKC-δ with PKC-α and PKC-ε being most sensitive to phorbol esters, whereas residual levels of PKC-δ remained detectable. In contrast phorbol ester treatment had no effect on PKC-ζ (Fig. 3 B). Thus, these results indicate that thrombin-induced Rho activation in HUVE cells is regulated by phorbol-sensitive PKC isozymes but not by the atypical PKC isozymes. Using LY379196 and rottlerin, which inhibit PKC-β and PKC-δ isozymes, respectively (42Jirousek M.R. Gillig J.R. Gonzalez C.M. Heath W.F. McDonald J.H. Neel D.A. Rito C.J. Singh U. Stramm L.E. Melikian-Badalian A. Baevsky M. Ballas L.M. Hall S.E. Winneroski L.L. Faul M.M. J. Med. Chem. 1996; 39: 2664-2671Crossref PubMed Scopus (326) Google Scholar, 43Gschwendt M. Muller H.J. Kielbassa K. Zang R. Kittstein W. Rincke G. Marks F. Biochem. Biophys. Res. Commun. 1994; 199: 93-98Crossref PubMed Scopus (762) Google Scholar, 44Pongracz J. Webb P. Wang K. Deacon E. Lunn O.J. Lord J.M. J. Biol. Chem. 1999; 274: 37329-37334Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), we ruled out the involvement of PKC-β or PKC-δ isoforms in regulating thrombin-induced Rho activation (data not shown). As these data and the results in Fig. 2 pointed to the phorbol ester-sensitive PKC isozymes, such as PKC-α, as being responsible for thrombin-induced Rho activation, we studied the specific role of PKC-α in the mechanism of thrombin-induced Rho activation. Using the SRE reporter gene activity, we determined the role of PKC isozymes in the mechanism of Rho activation. We used the dominant-negative mutant of PKC-α to address its role in thrombin-induced Rho activation. The dominant-negative mutants of PKC-δ and PKC-ε were also included in these experiments. In addition, we studied the effect of C3 transferase, which inhibits Rho activation (34Paterson H.F. Self A.J. Garrett M.D. Just I. Aktories K. Hall A. J. Cell Biol. 1990; 111: 1001-1007Crossref PubMed Scopus (571) Google Scholar), on thrombin-induced SRE activation. HUVE cells were cotransfected with SRE-luciferase reporter gene construct together without or with C3 transferase and assayed for thrombin-induced SRE luciferase activity. Thrombin increased SRE reporter gene activity by 4-fold, whereas it failed to increase SRE activation in HUVE cells that were cotransfected with C3 transferase. These results indicate that thrombin-induced SRE reporter gene activation is mediated by Rho (Fig.4 A). The results of thrombin-induced SRE reporter gene activity in cells cotransfected with SRE-luciferase reporter without or with dominant-negative mutants of PKC-α, PKC-δ, or PKC-ε are shown in Fig. 4 B. Thrombin-induced SRE reporter gene activity was completely prevented in cells transfected with the dominant-negative mutant of PKC-α. In contrast, dominant-negative PKC-δ or PKC-ε had no significant effect on thrombin-induced SRE activation. Thus, these results, which show that PKC-α is critical in stimulating Rho activation, are in accord with the above findings, which were obtained using pharmacological inhibitors. To determine whether thrombin-induced regulation of Rho by PKC-α occurs as a result of their physical interaction, lysates from cells stimulated without or with thrombin (1-min challenge period) were incubated with GST-rhotekin fusion protein to pull down activated Rho, after which they were subjected to Western blotting with anti-Rho, PKC-α, or PKC-ε antibody. Fig. 5shows that thrombin induced the rapid association of PKC-α and Rho, whereas under similar conditions, Rho and PKC-ε did not associate. The GDP-bound form of Rho complexed with GDI is not activated by Rho-GEFs (19Olofsson B. Cell. Signal. 1999; 11: 545-554Crossref PubMed Scopus (412) Google Scholar), suggesting that Rho activation critically depends on upstream factors that activate the dissociation of GDI. Since Rho-GDI contains sequence for phosphorylation, we addressed the possibility that the activation of HUVE cells results in the phosphorylation of GDI. Fig. 6 shows the autoradiograph of GDI phosphorylation in 32P-labeled HUVE cells in response to thrombin or PMA stimulation. Thrombin as well as PMA induced the rapid phosphorylation of GDI that returned to near basal level at 5 min after challenge (Fig. 6). Since thrombin-induced Rho activation was blocked by either PKC depletion of cells or by the inhibition of PKC using chelerythrine, we determined the role of PKC in mediating the phosphorylation of GDI in response to thrombin. Fig.7 shows the role of PKC in mediating thrombin-induced GDI phosphorylation. We found that depletion of PKC by overnight pretreatment with PMA abrogated thrombin-induced phosphorylation of Rho-GDI. Similarly, the inhibition of PKC by chelerythrine pretreatment prevented the phosphorylation of GDI in response to thrombin stimulation of endothelial cells. Thus, these results demonstrate that phorbol-sensitive PKC isozymes are involved in regulating the phosphorylation of GDI in response to thrombin. As Rho activation was regulated by PKC-α, we next performed an immunocomplex protein kinase assay to determine whether PKC-α can directly phosphorylate GDI in vitro. HUVE cells were stimulated without or with thrombin, and the lysates were immunoprecipitated with anti-PKC-α antibody. These immunocomplexes were used for kinase assay using GST-GDI as a substrate. In parallel, control kinase reactions were performed using immunocomplexes obtained from cells immunoprecipitated using anti-PKC-ε antibody. As shown in Fig.8, GST-GDI was slightly phosphorylated by PKC-α in unstimulated cells, but GDI phosphorylation increased markedly in the kinase reaction in cells treated with thrombin. In contrast, PKC-ε failed to induce the phosphorylation of GST-GDI in kinase reaction regardless of whether the immunocomplex was obtained from stimulated or unstimulated cells. The amounts of PKC-α or PKC-ε in each reaction were equivalent (Fig. 8). Thus, these results indicate that activated PKC-α is directly capable of phosphorylating Rho-GDI in vitro. The biochemical events by which PKC activation regulates thrombin-induced barrier dysfunction are incompletely understood (3Lum H. Malik A.B. Am. J. Physiol. 1994; 267: L223-L241Crossref PubMed Google Scholar, 27Vuong P.T. Malik A.B. Nagpala P.G. Lum H. J. Cell. Physiol. 1998; 175: 379-387Crossref PubMed Scopus (40) Google Scholar, 28Aschner J.L. Lum H. Fletcher P.W. Malik A.B. J. Cell. Physiol. 1997; 173: 387-396Crossref PubMed Scopus (44) Google Scholar, 29Patterson C.E. Davis H.W. Schaphorst K.L. Garcia J.G. Microvasc. Res. 1994; 48: 212-235Crossref PubMed Scopus (77) Google Scholar, 30Lynch J.J. Ferro T.J. Blumenstock F.A. Brockenauer A.M. Malik A.B. J. Clin. Invest. 1990; 85: 1991-1998Crossref PubMed Scopus (238) Google Scholar). Because the results of this study directly implicate PKC as the upstream regulator of Rho, we measured changes in transendothelial electrical resistance (the basis of increased paracellular endothelial permeability) in PKC-depleted cells or in cells treated with C3 transferase to block Rho activation. As shown in Fig. 9, thrombin caused a significant decrease in resistance in untreated cells, whereas the decreases in resistance in response to thrombin were significantly reduced in cells, which were depleted of phorbol ester-sensitive PKC isozymes, as well as in cells treated with C3 transferase. Rho activation plays an important role in the mechanism of increased transendothelial permeability induced by mediators such as thrombin (11Garcia J.G. Verin A.D. Schaphorst K. Siddiqui R. Patterson C.E. Csortos C. Natarajan V. Am. J. Physiol. 1999; 276: L989-L998PubMed Google Scholar, 12van Nieuw Amerongen G.P. Draijer R. Vermeer M.A. van Hinsbergh V.W. Circ. Res. 1998; 83: 1115-1123Crossref PubMed Google Scholar, 13Essler M. Amano M. Kruse H.J. Kaibuchi K. Weber P.C. Aepfelbacher M. J. Biol. Chem. 1998; 273: 21867-21874Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 39van Nieuw Amerongen G.P. van Delft S. Vermeer M.A. Collard J.G. van Hinsbergh V.W. Circ. Res. 2000; 87: 335-340Crossref PubMed Google Scholar); however, the mechanisms of activation of Rho, thereby the loss of endothelial barrier integrity, are not elucidated (39van Nieuw Amerongen G.P. van Delft S. Vermeer M.A. Collard J.G. van Hinsbergh V.W. Circ. Res. 2000; 87: 335-340Crossref PubMed Google Scholar). The dissociation of GDI from Rho-GDP complex is a prerequisite for the activation of Rho by Rho-GEF (19Olofsson B. Cell. Signal. 1999; 11: 545-554Crossref PubMed Scopus (412) Google Scholar, 22Sasaki T. Takai Y. Biochem. Biophys. Res. Commun. 1998; 245: 641-645Crossref PubMed Scopus (167) Google Scholar, 45Regazzi R. Kikuchi A. Takai Y. Wollheim C.B. J. Biol. Chem. 1992; 267: 17512-17519Abstract Full Text PDF PubMed Google Scholar, 46Hoffman G.R. Nassar N. Cerione R.A. Cell. 2000; 100: 345-356Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar). As GDI may play a critical role in mediating thrombin-induced Rho activation and thus in signaling increased endothelial permeability, we addressed in this study the basis of Rho activation and its contribution in mediating the loss of endothelial barrier function induced by thrombin. The present results provide several lines of evidence that Rho-GDI phosphorylation and Rho activation are regulated by a PKC-dependent pathway in endothelial cells. We showed that thrombin as well as the direct activation of PKC by PMA induced the phosphorylation of Rho-GDI and that the Rho-GDI phosphorylation occurred concurrently with the thrombin-induced activation of Rho. Furthermore, the inhibition of PKC by chelerythrine (a specific but not isozyme-selective inhibitor of PKC) abrogated not only thrombin-induced Rho activation but also Rho-GDI phosphorylation. We also showed that the phosphorylation of GDI and Rho activation is regulated by phorbol ester-sensitive isozymes as the depletion of these isozymes by exposing HUVE cells to phorbol esters in the standard manner prevented thrombin-induced GDI phosphorylation and Rho activation. Because the PKC isozymes, -α, -β, -δ, and -ε, expressed in endothelial cells are all phorbol ester-sensitive, we used both pharmacological and genetic approaches to further identify the specific PKC isozyme regulating GDI phosphorylation and Rho activation. We found that the treatment of HUVE cells with LY379196 or rottlerin, which inhibits PKC-β or PKC-δ isozymes, respectively, failed to prevent Rho activation in response to thrombin in endothelial cells. Using dominant-negative mutant constructs, we showed that dominant-negative PKC-δ failed to prevent thrombin-induced SRE reporter gene activity that is regulated by Rho. Furthermore, we found that Rho-mediated SRE reporter gene activity in response to thrombin was completely prevented in endothelial cells transfected with the dominant-negative mutant of PKC-α, whereas PKC-ε had no significant effect on thrombin-induced SRE activation. Thus, these data demonstrate that PKC-α is the major kinase regulating Rho activation in endothelial cells. As the above findings indicate the critical role of PKC-α activation in the regulation of Rho activation, we usedin vitro kinase assay to test the possibility that PKC-α can directly phosphorylate GDI. Results of the in vitrokinase assay using PKC-α and PKC-ε immunoprecipitates from unstimulated and stimulated cells indicated that only PKC-α from activated cells was capable of inducing phosphorylation of GST-GDI. Thus, these findings indicate that the activation of PKC-α is required for phosphorylation of Rho-GDI, although the possibility cannot be ruled out that PKC may also activate another protein kinase controlling the phosphorylation state of GDI in HUVE cells. We also found in the pull-down assay that stimulation of endothelial cells with thrombin leads to a rapid association of PKC-α with the activated Rho, although it failed to associate with PKC-ε. The association of PKC-α with Rho after activation, although not in resting cells, indicates that the protein complex formation is probably important in targeting and regulating Rho function (14Hall A. Science. 1998; 280: 2074-2075Crossref PubMed Scopus (166) Google Scholar, 47Tapon N. Hall A. Curr. Opin. Cell Biol. 1997; 9: 86-92Crossref PubMed Scopus (698) Google Scholar). However, our results do not distinguish between the possibility that association of PKC-α with Rho can occur directly or whether it is mediated by intermediate factors. There is little information regarding the role of different PKC isoforms in regulating the activation of Rho. Studies in endothelial and epithelial cells have implicated Rho in PMA-induced recruitment of PKC-α to the cell membrane (48Hippenstiel S. Kratz T. Krull M. Seybold J. von Eichel-Streiber C. Suttorp N. Biochem. Biophys. Res. Commun. 1998; 245: 830-834Crossref PubMed Scopus (65) Google Scholar); however, these observations were not confirmed in bovine arterial endothelial cells (49Verin A.D. Liu F. Bogatcheva N. Borbiev T. Hershenson M.B. Wang P. Garcia J.G. Am. J. Physiol. 2000; 279: L360-L370PubMed Google Scholar). A permissive role of PKC-α but not PKC-δ in sphingosine 1-phosphate-induced Rho-A translocation from cytosol to membrane (an indirect measure of Rho activation) was also recently reported in C2C12 myoblasts (50Meacci E. Donati C. Cencetti F. Romiti E. Bruni P. FEBS Lett. 2000; 482: 97-101Crossref PubMed Scopus (36) Google Scholar). Thus, on the basis of using multiple approaches our results provide unequivocal evidence that PKC-α is a key upstream regulator of Rho activation. Several studies have implicated a critical role of PKC-dependent pathway in regulating thrombin-induced increase in endothelial permeability (3Lum H. Malik A.B. Am. J. Physiol. 1994; 267: L223-L241Crossref PubMed Google Scholar, 27Vuong P.T. Malik A.B. Nagpala P.G. Lum H. J. Cell. Physiol. 1998; 175: 379-387Crossref PubMed Scopus (40) Google Scholar, 28Aschner J.L. Lum H. Fletcher P.W. Malik A.B. J. Cell. Physiol. 1997; 173: 387-396Crossref PubMed Scopus (44) Google Scholar, 29Patterson C.E. Davis H.W. Schaphorst K.L. Garcia J.G. Microvasc. Res. 1994; 48: 212-235Crossref PubMed Scopus (77) Google Scholar, 30Lynch J.J. Ferro T.J. Blumenstock F.A. Brockenauer A.M. Malik A.B. J. Clin. Invest. 1990; 85: 1991-1998Crossref PubMed Scopus (238) Google Scholar). We, therefore, measured changes in transendothelial electrical resistance (the basis of increased paracellular endothelial permeability) (31Tiruppathi C. Malik A.B. Del Vecchio P.J. Keese C.R. Giaever I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7919-7923Crossref PubMed Scopus (379) Google Scholar) using cells depleted of PKC by PMA treatment or cells treated with C3 transferase to inhibit Rho. We used these cells to address the possibility that PKC-induced barrier dysfunction can be explained by Rho activation. The results showed that thrombin caused a decrease in transendothelial electrical resistance, whereas depletion of PKC or inhibition of Rho reduced the response. Thus, these observations indicate that PKC induces the permeability increase activated by thrombin via the Rho-mediated pathway. What are the implications of PKC-α-induced GDI phosphorylation in the mechanism of Rho activation? It has been shown that the cytoplasmic pool of Rho-GTPase is complexed with GDI proteins (45Regazzi R. Kikuchi A. Takai Y. Wollheim C.B. J. Biol. Chem. 1992; 267: 17512-17519Abstract Full Text PDF PubMed Google Scholar); thus, GDI can influence both the cellular localization and cycling of Rho proteins between GDP- and GTP-bound states. The dissociation of GDI from the Rho protein is a prerequisite for membrane association and its activation by Rho-GEFs (19Olofsson B. Cell. Signal. 1999; 11: 545-554Crossref PubMed Scopus (412) Google Scholar, 20Hart M.J. Maru Y. Leonard D. Witte O.N. Evans T. Cerione R.A. Science. 1992; 258: 812-815Crossref PubMed Scopus (122) Google Scholar). In bovine neutrophils, phosphorylation/dephosphorylation events have been implicated in the regulation of the dissociation of the Rho/Rho-GDI complex (51Bourmeyster N. Vignais P.V. Biochem. Biophys. Res. Commun. 1996; 218: 54-60Crossref PubMed Scopus (28) Google Scholar). Based on our results of thrombin-induced phosphorylation of GDI and Rho activation through a PKC-dependent pathway, we hypothesize that phosphorylation/dephosphorylation of GDI may play a role in the mechanism of PKC-α-induced activation of Rho. Thus, the results of this study describe a novel pathway of GDI phosphorylation and Rho activation regulated by PKC-α and in signaling PKC-induced loss of endothelial barrier function. We thank Dr. Martin Schwartz for providing rhotekin-Rho binding domain construct and Dr. Tohru Kozasa for providing SRE-luciferase construct. We thank Dr. I. B. Weinstein for providing PKC-α, PKC-δ, and PKC-ε isozyme constructs. We also thank Arash Jalali for expert technical assistance and Mike Holinstat and Dr. K. Anwar for help in purifying SRE construct and luciferase activity measurements." @default.
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• W2049967046 title "Protein Kinase C-α Signals Rho-Guanine Nucleotide Dissociation Inhibitor Phosphorylation and Rho Activation and Regulates the Endothelial Cell Barrier Function" @default.
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