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Protein Kinase C-α Signals Rho-Guanine Nucleotide Dissociation Inhibitor Phosphorylation and Rho Activation and Regulates the Endothelial Cell Barrier Function*

  • Dolly Mehta
    Correspondence
    To whom correspondence should be addressed:
    Affiliations
    From the Department of Pharmacology, The University of Illinois College of Medicine, Chicago, Illinois 60612
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  • Arshad Rahman
    Affiliations
    From the Department of Pharmacology, The University of Illinois College of Medicine, Chicago, Illinois 60612
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  • Asrar B. Malik
    Affiliations
    From the Department of Pharmacology, The University of Illinois College of Medicine, Chicago, Illinois 60612
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  • Author Footnotes
    * This work was supported by the Midwest affiliate of the American Heart Association and NHLBI, National Institutes of Health Grants HL27016, HL46350, and HL45638.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:June 22, 2001DOI:https://doi.org/10.1074/jbc.M101927200
      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.
      GEF
      guanine nucleotide exchange factor
      GAP
      GTPase-activating protein
      GDI
      guanine nucleotide dissociation inhibitor
      PKC
      protein kinase C
      HUVE cells
      human umbilical venular endothelial cells
      EBM
      endothelial growth medium
      PBS
      phosphate-buffered saline
      PMA
      phorbol 12-myristate 13-acetate
      GST
      glutathione S-transferase
      RBD
      rhotekin-Rho binding domain
      PMSF
      phenylmethylsulfonyl fluoride
      SRE
      serum response element
      ANOVA
      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 (
      • Gao B.
      • Curtis T.M.
      • Blumenstock F.A.
      • Minnear F.L.
      • Saba T.M.
      ,
      • Lampugnani M.G.
      • Resnati M.
      • Dejana E.
      • Marchisio P.C.
      ,
      • Lum H.
      • Malik A.B.
      ,
      • Qiao R.L.
      • Yan W.
      • Lum H.
      • Malik A.B.
      ). 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 (
      • Gerszten R.E.
      • Chen J.
      • Ishii M.
      • Ishii K.
      • Wang L.
      • Nanevicz T.
      • Turck C.W.
      • Vu T.K.
      • Coughlin S.R.
      ,
      • Garcia J.G.
      • Patterson C.
      • Bahler C.
      • Aschner J.
      • Hart C.M.
      • English D.
      ,
      • Lum H.
      • Andersen T.T.
      • Siflinger-Birnboim A.
      • Tiruppathi C.
      • Goligorsky M.S.
      • Fenton J.W.
      • Malik A.B.
      ). 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 (
      • Lum H.
      • Malik A.B.
      ,
      • Moy A.B.
      • Van Engelenhoven J.
      • Bodmer J.
      • Kamath J.
      • Keese C.
      • Giaever I.
      • Shasby S.
      • Shasby D.M.
      ,
      • Garcia J.G.
      • Pavalko F.M.
      • Patterson C.E.
      ,
      • Garcia J.G.
      • Davis H.W.
      • Patterson C.E.
      ,
      • Garcia J.G.
      • Verin A.D.
      • Schaphorst K.
      • Siddiqui R.
      • Patterson C.E.
      • Csortos C.
      • Natarajan V.
      ).
      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 (
      • Garcia J.G.
      • Verin A.D.
      • Schaphorst K.
      • Siddiqui R.
      • Patterson C.E.
      • Csortos C.
      • Natarajan V.
      ,
      • van Nieuw Amerongen G.P.
      • Draijer R.
      • Vermeer M.A.
      • van Hinsbergh V.W.
      ,
      • Essler M.
      • Amano M.
      • Kruse H.J.
      • Kaibuchi K.
      • Weber P.C.
      • Aepfelbacher M.
      ). The multiple functions of Rho are mediated through the tightly regulated GTP-binding/GTPase cycle (
      • Hall A.
      ,
      • Sah V.P.
      • Seasholtz T.M.
      • Sagi S.A.
      • Brown J.H.
      ,
      • Kaibuchi K.
      • Kuroda S.
      • Amano M.
      ). 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 (
      • Geyer M.
      • Wittinghofer A.
      ). 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 (
      • Nomanbhoy T.K.
      • Cerione R.
      ). Thus, GDI plays a critical role in the signaling events regulated by Rho-GTPases (
      • Olofsson B.
      ).
      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 (
      • Olofsson B.
      ,
      • Hart M.J.
      • Maru Y.
      • Leonard D.
      • Witte O.N.
      • Evans T.
      • Cerione R.A.
      ,
      • Yaku H.
      • Sasaki T.
      • Takai Y.
      ). 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 (
      • Sasaki T.
      • Takai Y.
      ,
      • Takahashi K.
      • Sasaki T.
      • Mammoto A.
      • Takaishi K.
      • Kameyama T.
      • Tsukita S.
      • Takai Y.
      ). 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 (
      • Takahashi K.
      • Sasaki T.
      • Mammoto A.
      • Takaishi K.
      • Kameyama T.
      • Tsukita S.
      • Takai Y.
      ). 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 (
      • Hirao M.
      • Sato N.
      • Kondo T.
      • Yonemura S.
      • Monden M.
      • Sasaki T.
      • Takai Y.
      • Tsukita S.
      ,
      • Kotani H.
      • Takaishi K.
      • Sasaki T.
      • Takai Y.
      ).
      Rho-GDI is a family consisting of Rho-GDI-1, Ly/D4-GDI, and Rho GDI-III. Of these, Rho-GDI is ubiquitously expressed (
      • Olofsson B.
      ,
      • Sasaki T.
      • Takai Y.
      ). 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 (
      • Nishizuka Y.
      ). A PKC-dependent pathway is important in the mechanism of thrombin-induced increase in endothelial permeability (
      • Lum H.
      • Malik A.B.
      ,
      • Vuong P.T.
      • Malik A.B.
      • Nagpala P.G.
      • Lum H.
      ,
      • Aschner J.L.
      • Lum H.
      • Fletcher P.W.
      • Malik A.B.
      ,
      • Patterson C.E.
      • Davis H.W.
      • Schaphorst K.L.
      • Garcia J.G.
      ,
      • Lynch J.J.
      • Ferro T.J.
      • Blumenstock F.A.
      • Brockenauer A.M.
      • Malik A.B.
      ). 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.

      DISCUSSION

      Rho activation plays an important role in the mechanism of increased transendothelial permeability induced by mediators such as thrombin (
      • Garcia J.G.
      • Verin A.D.
      • Schaphorst K.
      • Siddiqui R.
      • Patterson C.E.
      • Csortos C.
      • Natarajan V.
      ,
      • van Nieuw Amerongen G.P.
      • Draijer R.
      • Vermeer M.A.
      • van Hinsbergh V.W.
      ,
      • Essler M.
      • Amano M.
      • Kruse H.J.
      • Kaibuchi K.
      • Weber P.C.
      • Aepfelbacher M.
      ,
      • van Nieuw Amerongen G.P.
      • van Delft S.
      • Vermeer M.A.
      • Collard J.G.
      • van Hinsbergh V.W.
      ); however, the mechanisms of activation of Rho, thereby the loss of endothelial barrier integrity, are not elucidated (
      • van Nieuw Amerongen G.P.
      • van Delft S.
      • Vermeer M.A.
      • Collard J.G.
      • van Hinsbergh V.W.
      ). The dissociation of GDI from Rho-GDP complex is a prerequisite for the activation of Rho by Rho-GEF (
      • Olofsson B.
      ,
      • Sasaki T.
      • Takai Y.
      ,
      • Regazzi R.
      • Kikuchi A.
      • Takai Y.
      • Wollheim C.B.
      ,
      • Hoffman G.R.
      • Nassar N.
      • Cerione R.A.
      ). 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 (
      • Hall A.
      ,
      • Tapon N.
      • Hall A.
      ). 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 (
      • Hippenstiel S.
      • Kratz T.
      • Krull M.
      • Seybold J.
      • von Eichel-Streiber C.
      • Suttorp N.
      ); however, these observations were not confirmed in bovine arterial endothelial cells (
      • Verin A.D.
      • Liu F.
      • Bogatcheva N.
      • Borbiev T.
      • Hershenson M.B.
      • Wang P.
      • Garcia J.G.
      ). 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 (
      • Meacci E.
      • Donati C.
      • Cencetti F.
      • Romiti E.
      • Bruni P.
      ). 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 (
      • Lum H.
      • Malik A.B.
      ,
      • Vuong P.T.
      • Malik A.B.
      • Nagpala P.G.
      • Lum H.
      ,
      • Aschner J.L.
      • Lum H.
      • Fletcher P.W.
      • Malik A.B.
      ,
      • Patterson C.E.
      • Davis H.W.
      • Schaphorst K.L.
      • Garcia J.G.
      ,
      • Lynch J.J.
      • Ferro T.J.
      • Blumenstock F.A.
      • Brockenauer A.M.
      • Malik A.B.
      ). We, therefore, measured changes in transendothelial electrical resistance (the basis of increased paracellular endothelial permeability) (
      • Tiruppathi C.
      • Malik A.B.
      • Del Vecchio P.J.
      • Keese C.R.
      • Giaever I.
      ) 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 (
      • Regazzi R.
      • Kikuchi A.
      • Takai Y.
      • Wollheim C.B.
      ); 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 (
      • Olofsson B.
      ,
      • Hart M.J.
      • Maru Y.
      • Leonard D.
      • Witte O.N.
      • Evans T.
      • Cerione R.A.
      ). In bovine neutrophils, phosphorylation/dephosphorylation events have been implicated in the regulation of the dissociation of the Rho/Rho-GDI complex (
      • Bourmeyster N.
      • Vignais P.V.
      ). 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.

      Acknowledgments

      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.

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