title: Epac1 and PDZ-GEF cooperate in Rap1 mediated endothelial junction control.
abstract: Epac1 and its effector Rap1 are important mediators of cAMP induced tightening of endothelial junctions and consequential increased barrier function. We have investigated the involvement of Rap1 signalling in basal, unstimulated, barrier function of a confluent monolayer of HUVEC using real time Electric Cell-substrate Impedance Sensing. Depletion of Rap1, but not Epac1, results in a strong decrease in barrier function. This decrease is also observed when cells are depleted of the cAMP independent Rap exchange factors PDZ-GEF1 and 2, showing that PDZ-GEFs are responsible for Rap1 activity in control of basal barrier function. Monolayers of cells depleted of PDZ-GEF or Rap1 show an irregular, zipper-like organization of VE-cadherin and live imaging of VE-cadherin-GFP reveals enhanced junction motility upon depletion of PDZ-GEF or Rap1. Importantly, activation of Epac1 increases the formation of cortical actin bundles at the cell-cell junctions, inhibits junction motility and restores barrier function of PDZ-GEFs depleted, but not Rap1 depleted cells. We conclude that PDZ-GEF activates Rap1 under resting conditions to stabilize cell-cell junctions and maintain basal integrity. Activation of Rap1 by cAMP/Epac1 induces junctional actin to further tighten cell-cell contacts.
authors.affiliation: Molecular Cancer Research, Centre of Biomedical Genetics and Cancer Genomics Centre, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Willem-Jan
authors.initials: WJ
authors.lastname: Pannekoek
authors.forename: Jantine J G
authors.initials: JJ
authors.lastname: van Dijk
authors.forename: On Ying A
authors.initials: OY
authors.lastname: Chan
authors.forename: Stephan
authors.initials: S
authors.lastname: Huveneers
authors.forename: Jelena R
authors.initials: JR
authors.lastname: Linnemann
authors.forename: Emma
authors.initials: E
authors.lastname: Spanjaard
authors.forename: Patricia M
authors.initials: PM
authors.lastname: Brouwer
authors.forename: Anne Jan
authors.initials: AJ
authors.lastname: van der Meer
authors.forename: Fried J T
authors.initials: FJ
authors.lastname: Zwartkruis
authors.forename: Holger
authors.initials: H
authors.lastname: Rehmann
authors.forename: Johan
authors.initials: J
authors.lastname: de Rooij
authors.forename: Johannes L
authors.initials: JL
authors.lastname: Bos
mesh_terms: D036002:ADP Ribose Transferases / Q000494:pharmacology; D000199:Actins / Q000378:metabolism; D022005:Adherens Junctions / Q000378:metabolism*; D015703:Antigens, CD / Q000378:metabolism; D001905:Botulinum Toxins / Q000494:pharmacology; D015820:Cadherins / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017097:Electric Impedance; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology; D055785:Gene Knockdown Techniques; D020662:Guanine Nucleotide Exchange Factors / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D000089804:Shelterin Complex; D034501:Telomere-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D059008:Time-Lapse Imaging; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D000099152:Cadherin 5
chemical_list: D036002:ADP Ribose Transferases; D000199:Actins; D015703:Antigens, CD; D001905:Botulinum Toxins; D015820:Cadherins; D000242:Cyclic AMP; D020662:Guanine Nucleotide Exchange Factors; D009419:Nerve Tissue Proteins; D000089804:Shelterin Complex; D034501:Telomere-Binding Proteins; D020741:rho GTP-Binding Proteins; D000099152:Cadherin 5; C473217:8-(4-chloro-phenylthio)-2'-O-methyladenosine-3'-5'-cyclic monophosphate; C403525:RAPGEF2 protein, human; C116122:RAPGEF3 protein, human; C440809:TERF2IP protein, human; C063007:exoenzyme C3, Clostridium botulinum