title: Rasip1-Mediated Rho GTPase Signaling Regulates Blood Vessel Tubulogenesis via Nonmuscle Myosin II.
abstract: BACKGROUND
  Vascular tubulogenesis is essential to cardiovascular development. Within initial vascular cords of endothelial cells, apical membranes are established and become cleared of cell-cell junctions, thereby allowing continuous central lumens to open. Rasip1 (Ras-interacting protein 1) is required for apical junction clearance, as well as for regulation of Rho GTPase (enzyme that hydrolyzes GTP) activity. However, it remains unknown how activities of different Rho GTPases are coordinated by Rasip1 to direct tubulogenesis.
  
  
  OBJECTIVE
  The aim of this study is to determine the mechanisms downstream of Rasip1 that drive vascular tubulogenesis.
  
  
  RESULTS
  Using conditional mouse mutant models and pharmacological approaches, we dissect GTPase pathways downstream of Rasip1. We show that clearance of endothelial cell apical junctions during vascular tubulogenesis depends on Rasip1, as well as the GTPase Cdc42 (cell division control protein 42 homolog) and the kinase Pak4 (serine/threonine-protein kinase 4). Genetic deletion of Rasip1 or Cdc42, or inhibition of Pak4, all blocks endothelial cell tubulogenesis. By contrast, inactivation of RhoA (Ras homologue gene family member A) signaling leads to vessel overexpansion, implicating actomyosin contractility in control of lumen diameter. Interestingly, blocking activity of NMII (nonmuscle myosin II) either before, or after, lumen morphogenesis results in dramatically different tubulogenesis phenotypes, suggesting time-dependent roles.
  
  
  CONCLUSIONS
  Rasip1 controls different pools of GTPases, which in turn regulate different pools of NMII to coordinate junction clearance (remodeling) and actomyosin contractility during vascular tubulogenesis. Rasip1 promotes activity of Cdc42 to activate Pak4, which in turn activates NMII, clearing apical junctions. Once lumens open, Rasip1 suppresses actomyosin contractility via inhibition of RhoA by Arhgap29, allowing controlled expansion of vessel lumens during embryonic growth. These findings elucidate the stepwise processes regulated by Rasip1 through downstream Rho GTPases and NMII.
authors.affiliation: From the Department of Molecular Biology and Center for Regenerative Science and Medicine (D.M.B., Y.K., K.X., D.B.A., O.C.) and Department of Pharmacology (C.W., M.H.C.), University of Texas Southwestern Medical Center, Dallas; Department of Medical Pharmacology and Physiology, University of Missouri, Columbia (P.R.N., G.E.D.); Department of Biology, University of North Carolina, Chapel Hill (L.A.W.); and Division of Experimental Hematology and Cancer Biology, Children's Hospital Research Foundation, University of Cincinnati, OH (Y.Z.).
authors.forename: David M
authors.initials: DM
authors.lastname: Barry
authors.affiliation: From the Department of Molecular Biology and Center for Regenerative Science and Medicine (D.M.B., Y.K., K.X., D.B.A., O.C.) and Department of Pharmacology (C.W., M.H.C.), University of Texas Southwestern Medical Center, Dallas; Department of Medical Pharmacology and Physiology, University of Missouri, Columbia (P.R.N., G.E.D.); Department of Biology, University of North Carolina, Chapel Hill (L.A.W.); and Division of Experimental Hematology and Cancer Biology, Children's Hospital Research Foundation, University of Cincinnati, OH (Y.Z.).
authors.forename: Yeon
authors.initials: Y
authors.lastname: Koo
authors.affiliation: From the Department of Molecular Biology and Center for Regenerative Science and Medicine (D.M.B., Y.K., K.X., D.B.A., O.C.) and Department of Pharmacology (C.W., M.H.C.), University of Texas Southwestern Medical Center, Dallas; Department of Medical Pharmacology and Physiology, University of Missouri, Columbia (P.R.N., G.E.D.); Department of Biology, University of North Carolina, Chapel Hill (L.A.W.); and Division of Experimental Hematology and Cancer Biology, Children's Hospital Research Foundation, University of Cincinnati, OH (Y.Z.).
authors.forename: Pieter R
authors.initials: PR
authors.lastname: Norden
authors.affiliation: From the Department of Molecular Biology and Center for Regenerative Science and Medicine (D.M.B., Y.K., K.X., D.B.A., O.C.) and Department of Pharmacology (C.W., M.H.C.), University of Texas Southwestern Medical Center, Dallas; Department of Medical Pharmacology and Physiology, University of Missouri, Columbia (P.R.N., G.E.D.); Department of Biology, University of North Carolina, Chapel Hill (L.A.W.); and Division of Experimental Hematology and Cancer Biology, Children's Hospital Research Foundation, University of Cincinnati, OH (Y.Z.).
authors.forename: Lyndsay A
authors.initials: LA
authors.lastname: Wylie
authors.affiliation: From the Department of Molecular Biology and Center for Regenerative Science and Medicine (D.M.B., Y.K., K.X., D.B.A., O.C.) and Department of Pharmacology (C.W., M.H.C.), University of Texas Southwestern Medical Center, Dallas; Department of Medical Pharmacology and Physiology, University of Missouri, Columbia (P.R.N., G.E.D.); Department of Biology, University of North Carolina, Chapel Hill (L.A.W.); and Division of Experimental Hematology and Cancer Biology, Children's Hospital Research Foundation, University of Cincinnati, OH (Y.Z.).
authors.forename: Ke
authors.initials: K
authors.lastname: Xu
authors.affiliation: From the Department of Molecular Biology and Center for Regenerative Science and Medicine (D.M.B., Y.K., K.X., D.B.A., O.C.) and Department of Pharmacology (C.W., M.H.C.), University of Texas Southwestern Medical Center, Dallas; Department of Medical Pharmacology and Physiology, University of Missouri, Columbia (P.R.N., G.E.D.); Department of Biology, University of North Carolina, Chapel Hill (L.A.W.); and Division of Experimental Hematology and Cancer Biology, Children's Hospital Research Foundation, University of Cincinnati, OH (Y.Z.).
authors.forename: Chonlarat
authors.initials: C
authors.lastname: Wichaidit
authors.affiliation: From the Department of Molecular Biology and Center for Regenerative Science and Medicine (D.M.B., Y.K., K.X., D.B.A., O.C.) and Department of Pharmacology (C.W., M.H.C.), University of Texas Southwestern Medical Center, Dallas; Department of Medical Pharmacology and Physiology, University of Missouri, Columbia (P.R.N., G.E.D.); Department of Biology, University of North Carolina, Chapel Hill (L.A.W.); and Division of Experimental Hematology and Cancer Biology, Children's Hospital Research Foundation, University of Cincinnati, OH (Y.Z.).
authors.forename: D Berfin
authors.initials: DB
authors.lastname: Azizoglu
authors.affiliation: From the Department of Molecular Biology and Center for Regenerative Science and Medicine (D.M.B., Y.K., K.X., D.B.A., O.C.) and Department of Pharmacology (C.W., M.H.C.), University of Texas Southwestern Medical Center, Dallas; Department of Medical Pharmacology and Physiology, University of Missouri, Columbia (P.R.N., G.E.D.); Department of Biology, University of North Carolina, Chapel Hill (L.A.W.); and Division of Experimental Hematology and Cancer Biology, Children's Hospital Research Foundation, University of Cincinnati, OH (Y.Z.).
authors.forename: Yi
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: From the Department of Molecular Biology and Center for Regenerative Science and Medicine (D.M.B., Y.K., K.X., D.B.A., O.C.) and Department of Pharmacology (C.W., M.H.C.), University of Texas Southwestern Medical Center, Dallas; Department of Medical Pharmacology and Physiology, University of Missouri, Columbia (P.R.N., G.E.D.); Department of Biology, University of North Carolina, Chapel Hill (L.A.W.); and Division of Experimental Hematology and Cancer Biology, Children's Hospital Research Foundation, University of Cincinnati, OH (Y.Z.).
authors.forename: Melanie H
authors.initials: MH
authors.lastname: Cobb
authors.affiliation: From the Department of Molecular Biology and Center for Regenerative Science and Medicine (D.M.B., Y.K., K.X., D.B.A., O.C.) and Department of Pharmacology (C.W., M.H.C.), University of Texas Southwestern Medical Center, Dallas; Department of Medical Pharmacology and Physiology, University of Missouri, Columbia (P.R.N., G.E.D.); Department of Biology, University of North Carolina, Chapel Hill (L.A.W.); and Division of Experimental Hematology and Cancer Biology, Children's Hospital Research Foundation, University of Cincinnati, OH (Y.Z.).
authors.forename: George E
authors.initials: GE
authors.lastname: Davis
authors.affiliation: From the Department of Molecular Biology and Center for Regenerative Science and Medicine (D.M.B., Y.K., K.X., D.B.A., O.C.) and Department of Pharmacology (C.W., M.H.C.), University of Texas Southwestern Medical Center, Dallas; Department of Medical Pharmacology and Physiology, University of Missouri, Columbia (P.R.N., G.E.D.); Department of Biology, University of North Carolina, Chapel Hill (L.A.W.); and Division of Experimental Hematology and Cancer Biology, Children's Hospital Research Foundation, University of Cincinnati, OH (Y.Z.). ondine.cleaver@utsouthwestern.edu.
authors.forename: Ondine
authors.initials: O
authors.lastname: Cleaver
mesh_terms: D000818:Animals; D001808:Blood Vessels / Q000196:embryology* / Q000378:metabolism*; D002352:Carrier Proteins / Q000502:physiology*; D047108:Embryonic Development / Q000502:physiology; D005260:Female; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D024462:Myosin Type II / Q000378:metabolism*; D011247:Pregnancy; D015398:Signal Transduction / Q000502:physiology*; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C541054:Rasip1 protein, mouse; D024462:Myosin Type II; D020741:rho GTP-Binding Proteins
keywords: Ras homologue gene family member A GTP-binding protein; Ras interacting protein 1; actin cytoskeleton; endothelial cells; morphogenesis; myosin type II; tubulogenesis