RhoC maintains vascular homeostasis by regulating VEGF-induced signaling in endothelial cells

ABSTRACT Vasculogenesis and angiogenesis are controlled by vascular endothelial growth factor A (VEGF-A). Dysregulation of these physiological processes contributes to the pathologies of heart disease, cancer and stroke. Rho GTPase proteins play an integral role in VEGF-mediated formation and maintenance of blood vessels. The regulatory functions of RhoA and RhoB in vasculogenesis and angiogenesis are well defined, whereas the purpose of RhoC remains poorly understood. Here, we describe how RhoC promotes vascular homeostasis by modulating endothelial cell migration, proliferation and permeability. RhoC stimulates proliferation of human umbilical vein endothelial cells (HUVECs) by stabilizing nuclear β-catenin, which promotes transcription of cyclin D1 and subsequently drives cell cycle progression. RhoC negatively regulates endothelial cell migration through MAPKs and downstream MLC2 signaling, and decreases vascular permeability through downregulation of the phospholipase Cγ (PLCγ)–Ca2+–eNOS cascade in HUVECs. Using a VEGF-inducible zebrafish (Danio rerio) model, we observed significantly less vascular permeability in RhoC morpholino (MO)-injected zebrafish than control MO-injected zebrafish. Taken together, our findings suggest that RhoC is a key regulator of vascular homeostasis in endothelial cells. Highlighted Article: RhoC maintains vascular homeostasis in endothelial cells yet is dispensable for vascular development. Inhibition of RhoC represents an attractive therapeutic approach to prevent cancer metastasis.

[1]  Y. Hozumi,et al.  RhoC Upregulation Is Correlated with Reduced E-cadherin in Human Breast Cancer Specimens After Chemotherapy and in Human Breast Cancer MCF-7 Cells , 2014, Hormones and Cancer.

[2]  Wen-feng Gou,et al.  The role of RhoC in epithelial-to-mesenchymal transition of ovarian carcinoma cells , 2014, BMC Cancer.

[3]  C. Vettel,et al.  The activation of RhoC in vascular endothelial cells is required for the S1P receptor type 2-induced inhibition of angiogenesis. , 2013, Cellular signalling.

[4]  C. Der,et al.  The RhoGEF TEM4 Regulates Endothelial Cell Migration by Suppressing Actomyosin Contractility , 2013, PloS one.

[5]  G. Blatch,et al.  Knockdown of Hop downregulates RhoC expression, and decreases pseudopodia formation and migration in cancer cell lines. , 2013, Cancer letters.

[6]  Luke H. Hoeppner,et al.  Revealing the role of phospholipase Cβ3 in the regulation of VEGF-induced vascular permeability. , 2012, Blood.

[7]  S. Kutschera,et al.  VEGFR2 induces c-Src signaling and vascular permeability in vivo via the adaptor protein TSAd , 2012, The Journal of experimental medicine.

[8]  A. Eichmann,et al.  VEGF signaling inside vascular endothelial cells and beyond. , 2012, Current opinion in cell biology.

[9]  C. Addison,et al.  RhoB controls endothelial cell morphogenesis in part via negative regulation of RhoA , 2012, Vascular cell.

[10]  E. Keshet,et al.  Vascular endothelial growth factor and vascular homeostasis. , 2011, Proceedings of the American Thoracic Society.

[11]  P. Andreassen,et al.  RhoA GTPase Is Dispensable for Actomyosin Regulation but Is Essential for Mitosis in Primary Mouse Embryonic Fibroblasts* , 2011, The Journal of Biological Chemistry.

[12]  P. Savagner.  The epithelial-mesenchymal transition (EMT) phenomenon. , 2010, Annals of oncology : official journal of the European Society for Medical Oncology.

[13]  J. Liao,et al.  RhoA/ROCK signaling is essential for multiple aspects of VEGF‐mediated angiogenesis , 2010, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[14]  Andrew M. Petzold,et al.  SCORE imaging: specimen in a corrected optical rotational enclosure. , 2010, Zebrafish.

[15]  Michael C. Ostrowski,et al.  Erk1 and Erk2 Regulate Endothelial Cell Proliferation and Migration during Mouse Embryonic Angiogenesis , 2009, PloS one.

[16]  T. Thompson,et al.  Caveolin-1 regulates VEGF-stimulated angiogenic activities in prostate cancer and endothelial cells , 2009, Cancer biology & therapy.

[17]  A. Pathak.  Model system takes us a step closer to efficacious imaging biomarkers of angiogenesis in head and neck cancer , 2009, Cancer biology & therapy.

[18]  E. Dejana,et al.  VE-Cadherin-Mediated Cell-Cell Interaction Suppresses Sprouting via Signaling to MLC2 Phosphorylation , 2009, Current Biology.

[19]  Wei Wang,et al.  RhoC is essential for angiogenesis induced by hepatocellular carcinoma cells via regulation of endothelial cell organization , 2008, Cancer science.

[20]  C. Cooper,et al.  Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell diapedesis , 2008, Clinical & Experimental Metastasis.

[21]  P. Oettgen,et al.  Reciprocal regulation of RhoA and RhoC characterizes the EMT and identifies RhoC as a prognostic marker of colon carcinoma , 2006, Oncogene.

[22]  M. Glotzer,et al.  Cytokinesis: welcome to the Rho zone. , 2005, Trends in cell biology.

[23]  T. Mak,et al.  RhoC is dispensable for embryogenesis and tumor initiation but essential for metastasis. , 2005, Genes & development.

[24]  C. Wernstedt,et al.  VEGF receptor‐2 Y951 signaling and a role for the adapter molecule TSAd in tumor angiogenesis , 2005, The EMBO journal.

[25]  A. Meijer,et al.  Genomic annotation and expression analysis of the zebrafish Rho small GTPase family during development and bacterial infection. , 2005, Genomics.

[26]  Xiao-pei Gao,et al.  Angiopoietin-1 Opposes VEGF-Induced Increase in Endothelial Permeability by Inhibiting TRPC1-Dependent Ca2+ Influx , 2005, Circulation research.

[27]  A. Ridley,et al.  Why three Rho proteins? RhoA, RhoB, RhoC, and cell motility. , 2004, Experimental cell research.

[28]  E. Piek,et al.  TGF-beta induces proangiogenic and antiangiogenic factors via parallel but distinct Smad pathways. , 2004, Kidney international.

[29]  G. Prendergast,et al.  RhoB controls Akt trafficking and stage-specific survival of endothelial cells during vascular development. , 2003, Genes & development.

[30]  Anne J. Ridley,et al.  ROCKs: multifunctional kinases in cell behaviour , 2003, Nature Reviews Molecular Cell Biology.

[31]  V. V. van Hinsbergh,et al.  Involvement of RhoA/Rho Kinase Signaling in VEGF-Induced Endothelial Cell Migration and Angiogenesis In Vitro , 2003, Arteriosclerosis, thrombosis, and vascular biology.

[32]  D. Mukhopadhyay,et al.  KDR Stimulates Endothelial Cell Migration through Heterotrimeric G Protein Gq/11-mediated Activation of a Small GTPase RhoA* , 2002, The Journal of Biological Chemistry.

[33]  E. Van Obberghen,et al.  Regulation of Vascular Endothelial Growth Factor Expression by Advanced Glycation End Products* , 2001, The Journal of Biological Chemistry.

[34]  G. Prendergast,et al.  RhoB Is Dispensable for Mouse Development, but It Modifies Susceptibility to Tumor Formation as Well as Cell Adhesion and Growth Factor Signaling in Transformed Cells , 2001, Molecular and Cellular Biology.

[35]  D. Mukhopadhyay,et al.  Tyrosine Residues 951 and 1059 of Vascular Endothelial Growth Factor Receptor-2 (KDR) Are Essential for Vascular Permeability Factor/Vascular Endothelial Growth Factor-induced Endothelium Migration and Proliferation, Respectively* , 2001, The Journal of Biological Chemistry.

[36]  M. Shibuya,et al.  A single autophosphorylation site on KDR/Flk‐1 is essential for VEGF‐A‐dependent activation of PLC‐γ and DNA synthesis in vascular endothelial cells , 2001, The EMBO journal.

[37]  N. Voelkel,et al.  Hypoxia-induced pulmonary vascular remodeling: a model for what human disease? , 2000, The Journal of clinical investigation.

[38]  S. Merajver,et al.  RhoC GTPase overexpression modulates induction of angiogenic factors in breast cells. , 2000, Neoplasia.

[39]  A. Hall,et al.  Rho GTPases and their effector proteins. , 2000, The Biochemical journal.

[40]  Jacques Landry,et al.  Vascular Endothelial Growth Factor (VEGF)-driven Actin-based Motility Is Mediated by VEGFR2 and Requires Concerted Activation of Stress-activated Protein Kinase 2 (SAPK2/p38) and Geldanamycin-sensitive Phosphorylation of Focal Adhesion Kinase* , 2000, The Journal of Biological Chemistry.

[41]  P. Carmeliet.  Mechanisms of angiogenesis and arteriogenesis , 2000, Nature Medicine.

[42]  R. Busse,et al.  Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation , 1999, Nature.

[43]  D. Johnson,et al.  Cyclins and cell cycle checkpoints. , 1999, Annual review of pharmacology and toxicology.

[44]  H. Granger,et al.  Role of phospholipase C, protein kinase C, and calcium in VEGF-induced venular hyperpermeability. , 1999, The American journal of physiology.

[45]  A. Ridley,et al.  The GTP-binding protein Rho. , 1997, The international journal of biochemistry & cell biology.

[46]  Shuh Narumiya,et al.  Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension , 1997, Nature.

[47]  L. Van Aelst,et al.  Rho GTPases and signaling networks. , 1997, Genes & development.

[48]  David A. Cheresh,et al.  Regulation of Cell Motility by Mitogen-activated Protein Kinase , 1997, The Journal of cell biology.

[49]  W. Risau,et al.  Mechanisms of angiogenesis , 1997, Nature.

[50]  X. Q. Chen,et al.  The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton , 1996, Molecular and cellular biology.

[51]  S. Izumo,et al.  Molecular aspects of signal transduction of shear stress in the endothelial cell. , 1994, Journal of hypertension.

[52]  S. Narumiya,et al.  Inhibition of lysophosphatidate- and thrombin-induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein Rho , 1994, The Journal of cell biology.

[53]  T. Sasaki,et al.  rac p21 is involved in insulin-induced membrane ruffling and rho p21 is involved in hepatocyte growth factor- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced membrane ruffling in KB cells , 1994, Molecular and cellular biology.

[54]  Anne J. Ridley,et al.  The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors , 1992, Cell.

[55]  T. Sasaki,et al.  Involvement of rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction. , 1992, The Journal of biological chemistry.

[56]  A. Hall,et al.  Microinjection of recombinant p21rho induces rapid changes in cell morphology , 1990, The Journal of cell biology.

[57]  S. Narumiya,et al.  ADP-ribosylation of the rho/rac proteins induces growth inhibition, neurite outgrowth and acetylcholine esterase in cultured PC-12 cells. , 1990, Biochemical and biophysical research communications.

[58]  H. Dvorak,et al.  Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. , 1983, Science.

[59]  B. Thisse,et al.  High-resolution in situ hybridization to whole-mount zebrafish embryos , 2007, Nature Protocols.

[60]  T. Sasaki,et al.  Involvement of Rho p21 small GTP-binding protein and its regulator in the HGF-induced cell motility. , 1994, Oncogene.

[61]  H. Granger,et al.  Molecular and cellular basis of myocardial angiogenesis. , 1994, Cellular & molecular biology research.

[62]  H. Dvorak,et al.  Tumor-secreted vascular permeability factor increases cytosolic Ca2+ and von Willebrand factor release in human endothelial cells. , 1991, The American journal of pathology.