title: Cdc42 and k-Ras Control Endothelial Tubulogenesis through Apical Membrane and Cytoskeletal Polarization: Novel Stimulatory Roles for GTPase Effectors, the Small GTPases, Rac2 and Rap1b, and Inhibitory Influence of Arhgap31 and Rasa1.
abstract: A critical and understudied property of endothelial cells is their ability to form lumens and tube networks. Although considerable information has been obtained concerning these issues, including the role of Cdc42 and Rac1 and their effectors such as Pak2, Pak4, Par6b, and co-regulators such as integrins, MT1-MMP and Par3; many key questions remain that are necessary to elucidate molecular and signaling requirements for this fundamental process. In this work, we identify new small GTPase regulators of EC tubulogenesis including k-Ras, Rac2 and Rap1b that act in conjunction with Cdc42 as well as the key downstream effectors, IQGAP1, MRCKβ, beta-Pix, GIT1, and Rasip1 (which can assemble into multiprotein complexes with key regulators including α2β1 integrin and MT1-MMP). In addition, we identify the negative regulators, Arhgap31 (by inactivating Cdc42 and Rac) and Rasa1 (by inactivating k-Ras) and the positive regulator, Arhgap29 (by inactivating RhoA) which play a major functional role during the EC tubulogenic process. Human EC siRNA suppression or mouse knockout of Rasip1 leads to identical phenotypes where ECs form extensive cord networks, but cannot generate lumens or tubes. Essential roles for these molecules during EC tubulogenesis include; i) establishment of asymmetric EC cytoskeletal polarization (subapical distribution of acetylated tubulin and basal membrane distribution of F-actin); and ii) directed membrane trafficking of pinocytic vacuoles or other intracellular vesicles along acetylated tubulin tracks to the developing apical membrane surface. Cdc42 co-localizes subapically with acetylated tubulin, while Rac1 and k-Ras strongly label vacuole/ vesicle membranes which accumulate and fuse together in a polarized, perinuclear manner. We observe polarized apical membrane and subapical accumulation of key GTPases and effectors regulating EC lumen formation including Cdc42, Rac1, Rac2, k-Ras, Rap1b, activated c-Raf and Rasip1 to control EC tube network assembly. Overall, this work defines novel key regulators and their functional roles during human EC tubulogenesis.
authors.affiliation: Department of Medical Pharmacology and Physiology, University of Missouri School of Medicine, Dalton Cardiovascular Research Center, Columbia, MO, United States of America.
authors.forename: Pieter R
authors.initials: PR
authors.lastname: Norden
authors.affiliation: Department of Medical Pharmacology and Physiology, University of Missouri School of Medicine, Dalton Cardiovascular Research Center, Columbia, MO, United States of America.
authors.forename: Dae Joong
authors.initials: DJ
authors.lastname: Kim
authors.affiliation: Department of Molecular Biology, UT Southwestern Medical Center, Dallas, TX, United States of America.
authors.forename: David M
authors.initials: DM
authors.lastname: Barry
authors.affiliation: Department of Molecular Biology, UT Southwestern Medical Center, Dallas, TX, United States of America.
authors.forename: Ondine B
authors.initials: OB
authors.lastname: Cleaver
authors.affiliation: Department of Medical Pharmacology and Physiology, University of Missouri School of Medicine, Dalton Cardiovascular Research Center, Columbia, MO, United States of America.
authors.forename: George E
authors.initials: GE
authors.lastname: Davis
mesh_terms: D000818:Animals; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005260:Female; D020690:GTPase-Activating Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008297:Male; D051379:Mice; D008856:Microscopy, Fluorescence; D010703:Phorbol Esters / Q000494:pharmacology; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D015398:Signal Transduction / Q000187:drug effects; D014404:Tubulin / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020729:p120 GTPase Activating Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020744:rac GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020668:rap GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018631:ras Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000097766:RAC2 GTP-Binding Protein
chemical_list: D020690:GTPase-Activating Proteins; D010703:Phorbol Esters; D010750:Phosphoproteins; D014404:Tubulin; D020764:cdc42 GTP-Binding Protein; D020729:p120 GTPase Activating Protein; D020744:rac GTP-Binding Proteins; D020668:rap GTP-Binding Proteins; D018631:ras Proteins; D000097766:RAC2 GTP-Binding Protein; C514543:ARHGAP31 protein, human; C557248:RASA1 protein, human; C116753:RAP1B protein, human
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