RhoGDI-1 Modulation of the Activity of Monomeric RhoGTPase RhoA Regulates Endothelial Barrier Function in Mouse Lungs

Rho family GTPases have been implicated in the regulation of endothelial permeability via their actions on actin cytoskeletal organization and integrity of interendothelial junctions. In cell culture studies, activation of RhoA disrupts interendothelial junctions and increases endothelial permeability, whereas activation of Rac1 and Cdc42 enhances endothelial barrier function by promoting the formation of restrictive junctions. The primary regulators of Rho proteins, guanine nucleotide dissociation inhibitors (GDIs), form a complex with the GDP-bound form of the Rho family of monomeric G proteins, and thus may serve as a nodal point regulating the activation state of RhoGTPases. In the present study, we addressed the in vivo role of RhoGDI-1 in regulating pulmonary microvascular permeability using RhoGDI-1−/− mice. We observed that basal endothelial permeability in lungs of RhoGDI-1−/− mice was 2-fold greater than wild-type mice. This was the result of opening of interendothelial junctions in lung microvessels which are normally sealed. The activity of RhoA (but not of Rac1 or Cdc42) was significantly increased in RhoGDI-1−/− lungs as well as in cultured endothelial cells on downregulation of RhoGDI-1 with siRNA, consistent with RhoGDI-1–mediated modulation RhoA activity. Thus, RhoGDI-1 by repressing RhoA activity regulates lung microvessel endothelial barrier function in vivo. In this regard, therapies augmenting endothelial RhoGDI-1 function may be beneficial in reestablishing the endothelial barrier and lung fluid balance in lung inflammatory diseases such as acute respiratory distress syndrome.

[1]  J. Couchman,et al.  RhoGDI: multiple functions in the regulation of Rho family GTPase activities. , 2005, The Biochemical journal.

[2]  C. Dermardirossian,et al.  GDIs: central regulatory molecules in Rho GTPase activation. , 2005, Trends in cell biology.

[3]  A. Ishizaka,et al.  Attenuation of endotoxin-induced acute lung injury by the Rho-associated kinase inhibitor, Y-27632. , 2005, American journal of respiratory cell and molecular biology.

[4]  A. Malik,et al.  Integrated control of lung fluid balance. , 2004, American journal of physiology. Lung cellular and molecular physiology.

[5]  C. Dermardirossian,et al.  Phosphorylation of RhoGDI by Pak1 mediates dissociation of Rac GTPase. , 2004, Molecular cell.

[6]  M. Broman,et al.  Cdc42 Regulates the Restoration of Endothelial Barrier Function , 2004, Circulation research.

[7]  J. Niu,et al.  G Protein &bgr;&ggr; Subunits Stimulate p114RhoGEF, a Guanine Nucleotide Exchange Factor for RhoA and Rac1: Regulation of Cell Shape and Reactive Oxygen Species Production , 2003 .

[8]  K. Wennerberg,et al.  Serine Phosphorylation Negatively Regulates RhoA in Vivo* , 2003, Journal of Biological Chemistry.

[9]  S. Crawford,et al.  Protein kinase involved in lung injury susceptibility: Evidence from enzyme isoform genetic knockout and in vivo inhibitor treatment , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[10]  G. Tamma,et al.  cAMP-induced AQP2 translocation is associated with RhoA inhibition through RhoA phosphorylation and interaction with RhoGDI , 2003, Journal of Cell Science.

[11]  A. Ridley,et al.  Rho GTPases and the regulation of endothelial permeability. , 2002, Vascular pharmacology.

[12]  V. Braga.  Cell-cell adhesion and signalling. , 2002, Current opinion in cell biology.

[13]  E. Van Obberghen-Schilling,et al.  Distinct signals via Rho GTPases and Src drive shape changes by thrombin and sphingosine-1-phosphate in endothelial cells. , 2002, Journal of cell science.

[14]  G. Truskey,et al.  Effect of Fluid Shear Stress on the Permeability of the Arterial Endothelium , 2002, Annals of Biomedical Engineering.

[15]  D. Gingras,et al.  Phosphorylation states of Cdc42 and RhoA regulate their interactions with Rho GDP dissociation inhibitor and their extraction from biological membranes. , 2002, The Biochemical journal.

[16]  A. Ridley,et al.  Rho family proteins: coordinating cell responses. , 2001, Trends in cell biology.

[17]  A. Malik,et al.  Protein Kinase C-α Signals Rho-Guanine Nucleotide Dissociation Inhibitor Phosphorylation and Rho Activation and Regulates the Endothelial Cell Barrier Function* , 2001, The Journal of Biological Chemistry.

[18]  A. Ridley,et al.  Rho and Rac but not Cdc42 regulate endothelial cell permeability. , 2001, Journal of cell science.

[19]  A. Malik,et al.  Abrogation of thrombin-induced increase in pulmonary microvascular permeability in PAR-1 knockout mice. , 2000, Physiological genomics.

[20]  A. Malik,et al.  Reversibility of increased microvessel permeability in response to VE-cadherin disassembly. , 2000, American journal of physiology. Lung cellular and molecular physiology.

[21]  John G. Collard,et al.  Activation of RhoA by Thrombin in Endothelial Hyperpermeability: Role of Rho Kinase and Protein Tyrosine Kinases , 2000, Circulation research.

[22]  A. Takeshita,et al.  Inhibition of myosin phosphatase by upregulated rho-kinase plays a key role for coronary artery spasm in a porcine model with interleukin-1beta. , 2000, Circulation.

[23]  S. Kuroda,et al.  Cell adhesion and Rho small GTPases. , 1999, Journal of cell science.

[24]  Fumio Matsumura,et al.  Phosphorylation of Myosin-Binding Subunit (Mbs) of Myosin Phosphatase by Rho-Kinase in Vivo , 1999, The Journal of cell biology.

[25]  R. Schaeffer,et al.  RhoA inactivation enhances endothelial barrier function. , 1999, American journal of physiology. Cell physiology.

[26]  S. Nishikawa,et al.  Progressive impairment of kidneys and reproductive organs in mice lacking Rho GDIα , 1999, Oncogene.

[27]  John G. Collard,et al.  Rac regulates phosphorylation of the myosin-II heavy chain, actinomyosin disassembly and cell spreading , 1999, Nature Cell Biology.

[28]  W. Kiosses,et al.  Regulation of the small GTP‐binding protein Rho by cell adhesion and the cytoskeleton , 1999, The EMBO journal.

[29]  T. Sasaki,et al.  The Rho small G protein family-Rho GDI system as a temporal and spatial determinant for cytoskeletal control. , 1998, Biochemical and biophysical research communications.

[30]  J. Camonis,et al.  RhoGDI-3 Is a New GDP Dissociation Inhibitor (GDI) , 1996, The Journal of Biological Chemistry.

[31]  Richard A. Cerione,et al.  Characterization of the Interaction between RhoGDI and Cdc42Hs Using Fluorescence Spectroscopy (*) , 1996, The Journal of Biological Chemistry.

[32]  J. Chernoff,et al.  Identification of a Mouse p21Cdc42/Rac Activated Kinase (*) , 1995, The Journal of Biological Chemistry.

[33]  R. Sheldon,et al.  Role of myosin light-chain phosphorylation in endothelial cell retraction. , 1993, The American journal of physiology.

[34]  T. Sasaki,et al.  The nucleotide exchange rates of rho and rac small GTP-binding proteins are enhanced to different extents by their regulatory protein Smg GDS. , 1993, Biochemical and Biophysical Research Communications - BBRC.

[35]  L. Staudt,et al.  Ly-GDI, a GDP-dissociation inhibitor of the RhoA GTP-binding protein, is expressed preferentially in lymphocytes. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[36]  C. Wollheim,et al.  The small GTP-binding proteins in the cytosol of insulin-secreting cells are complexed to GDP dissociation inhibitor proteins. , 1992, The Journal of biological chemistry.

[37]  K. Kaibuchi,et al.  Both stimulatory and inhibitory GDP/GTP exchange proteins, smg GDS and rho GDI, are active on multiple small GTP-binding proteins. , 1992, Biochemical and biophysical research communications.

[38]  K. Kaibuchi,et al.  Post-translational modifications of the C-terminal region of the rho protein are important for its interaction with membranes and the stimulatory and inhibitory GDP/GTP exchange proteins. , 1991, Oncogene.

[39]  K. Kaibuchi,et al.  Molecular cloning and characterization of a novel type of regulatory protein (GDI) for the rho proteins, ras p21-like small GTP-binding proteins. , 1990, Oncogene.

[40]  Y. Takai,et al.  Purification and characterization from bovine brain cytosol of a novel regulatory protein inhibiting the dissociation of GDP from and the subsequent binding of GTP to rhoB p20, a ras p21-like GTP-binding protein. , 1990, The Journal of biological chemistry.

[41]  B. Hinz.  Cell adhesion and signaling , 2008 .

[42]  A. Malik,et al.  Signaling mechanisms regulating endothelial permeability. , 2006, Physiological reviews.

[43]  J. Niu,et al.  G Protein betagamma subunits stimulate p114RhoGEF, a guanine nucleotide exchange factor for RhoA and Rac1: regulation of cell shape and reactive oxygen species production. , 2003, Circulation research.

[44]  V. V. van Hinsbergh,et al.  Cytoskeletal effects of rho-like small guanine nucleotide-binding proteins in the vascular system. , 2001, Arteriosclerosis, thrombosis, and vascular biology.