Modulation of HSP27 alters hypoxia-induced endothelial permeability and related signaling pathways.
This manuscript describes how the permeability of pulmonary artery microvascular endothelial cell (RPMEC) monolayer is elevated by hypoxia and the role played by HSP27 phosphorylation. p38 MAP kinase activation leading to HSP27 phosphorylation was previously shown by our laboratory to alter the actin cytoskeleton and tethering properties of RPMEC. This effect was independent of hypoxia-induced contractility which was ROCK-dependent rather than HSP27-dependent. Results described here show that increased HSP27 phosphorylation not only does not underlie hypoxia-induced permeability, but may actually augment the endothelial barrier. Hypoxia causes gap formation between RPMEC and increases MLC2 phosphorylation. The phosphorylation of MYPT1, which inhibits MLC2 phosphatase, is also increased in hypoxia. In addition, FAK phosphorylation, which alters focal adhesion signaling, is increased in hypoxia. Overexpressing phosphomimicking HSP27 (pmHSP27), which induces significant actin stress fiber formation, surprisingly renders RPMEC resistant to hypoxia- or TGFbeta-induced permeability. siRNA against pmHSP27 reverses the increased actin stress fiber formation in pmHSP27-overexpressing cells, and disrupting actin stress fibers in pmHSP27-overexpressing RPMEC renders them more susceptible to hypoxia. Finally, hypoxia-induced gap formation, as well as phosphorylation of MLC2, MYPT1 and FAK are almost abolished by overexpressing pmHSP27 in RPMEC. These effects of pmHSP27 overexpression might represent decreased cytoskeletal plasticity and increased tethering which counteracts permeability-inducing contractility. Thus hypoxia activates two pathways one leading to contractility and increased permeability, the other leading to actin stress fibers, stronger adhesion, and reduced permeability. Altering HSP27 phosphorylation, which tips the balance towards decreased permeability, might be targeted in managing endothelial barrier dysfunction.
Tufts Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Tiegang
T
Liu
Oscar E
OE
Guevara
Rod R
RR
Warburton
Nicholas S
NS
Hill
Matthias
M
Gaestel
Usamah S
US
Kayyali
D000818:Animals; D002199:Capillary Permeability; D024722:Cardiac Myosins / Q000378:metabolism; D015687:Cell Hypoxia; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000201:enzymology / Q000378:metabolism*; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism; D017629:Gap Junctions / Q000201:enzymology / Q000378:metabolism*; D055551:HSP27 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009154:Mutation; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation; D054645:Protein Phosphatase 1 / Q000378:metabolism; D034622:RNA Interference; D051381:Rats; D015398:Signal Transduction; D022502:Stress Fibers / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D054460:rho-Associated Kinases / Q000378:metabolism
D055551:HSP27 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; C486114:Hspb1 protein, rat; D018832:Molecular Chaperones; D018994:Myosin Light Chains; D016212:Transforming Growth Factor beta; C107184:myosin light chain 2; D051417:Focal Adhesion Kinase 1; C496052:Ptk2 protein, rat; D054460:rho-Associated Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C499850:Ppp1r12a protein, rat; D054645:Protein Phosphatase 1; D024722:Cardiac Myosins