H2O2-induced filamin redistribution in endothelial cells is modulated by the cyclic AMP-dependent protein kinase pathway.
Hypoxia/reoxygenation injury in vitro causes endothelial cell cytoskeletal rearrangement that is related to increased monolayer permeability. Nonmuscle filamin (ABP-280) promotes orthogonal branching of F-actin and links microfilaments to membrane glycoproteins. Human umbilical vein endothelial cell monolayers are exposed to H2O2 (100 microM) for 1-60 min, with or without modulators of cAMP-dependent second-messenger pathways, and evaluated for changes in filamin distribution, cAMP levels, and the formation of gaps at interendothelial junctions. Filamin translocates from the membrane-cytoskeletal interface to the cytosol within 1 min of exposure to H2O2. This is associated with a decrease in endothelial cell cAMP levels from 83 pmoles/mg protein to 15 pmoles/mg protein. Intercellular gaps form 15 min after H2O2 treatment and progressively increase in number and diameter through 60 min. Both filamin redistribution and actin redistribution are associated with decreased phosphorylation of filamin and are prevented by activation of the cAMP-dependent protein kinase pathway. A synthetic peptide corresponding to filamin's C-terminal, cAMP-dependent, protein kinase phosphorylation site effectively induces filamin translocation and intercellular gap formation, which suggests that decreased phosphorylation of filamin at this site causes filamin redistribution and destabilization of junctions. These data indicate that H2O2-induced filamin redistribution and interendothelial cell gap formation result from inhibition of the cAMP-dependent protein kinase pathway.
Hastie
L E
LE
Microvascular Research Laboratory, Boston University, Massachusetts 02215, USA.
Patton
W F
WF
Hechtman
H B
HB
Shepro
D
D
D000262:Adenylyl Cyclases / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D003285:Contractile Proteins / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D064448:Filamins; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007365:Intercellular Junctions / Q000187:drug effects / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation
D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D010446:Peptide Fragments; D005576:Colforsin; D006861:Hydrogen Peroxide; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases