Pulmonary endothelial cell barrier enhancement by sphingosine 1-phosphate: roles for cortactin and myosin light chain kinase.
We recently reported the critical importance of Rac GTPase-dependent cortical actin rearrangement in the augmentation of pulmonary endothelial cell (EC) barrier function by sphingosine 1-phosphate (S1P). We now describe functional roles for the actin-binding proteins cortactin and EC myosin light chain kinase (MLCK) in mediating this response. Antisense down-regulation of cortactin protein expression significantly inhibits S1P-induced barrier enhancement in cultured human pulmonary artery EC as measured by transendothelial electrical resistance (TER). Immunofluorescence studies reveal rapid, Rac-dependent translocation of cortactin to the expanded cortical actin band following S1P challenge, where colocalization with EC MLCK occurs within 5 min. Adenoviral overexpression of a Rac dominant negative mutant attenuates TER elevation by S1P. S1P also induces a rapid increase in cortactin tyrosine phosphorylation (within 30 s) critical to subsequent barrier enhancement, since EC transfected with a tyrosine-deficient mutant cortactin exhibit a blunted TER response. Direct binding of EC MLCK to the cortactin Src homology 3 domain appears essential to S1P barrier regulation, since cortactin blocking peptide inhibits both S1P-induced MLC phosphorylation and peak S1P-induced TER values. These data support novel roles for the cytoskeletal proteins cortactin and EC MLCK in mediating lung vascular barrier augmentation evoked by S1P.
Dudek
Steven M
SM
Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Jacobson
Jeffrey R
JR
Chiang
Eddie T
ET
Birukov
Konstantin G
KG
Wang
Peiyi
P
Zhan
Xi
X
Garcia
Joe G N
JG
D000199:Actins / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D051356:Cortactin; D003599:Cytoskeleton / Q000378:metabolism; D015536:Down-Regulation; D042783:Endothelial Cells / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D005799:Genes, Dominant; D005822:Genetic Vectors; D006801:Humans; D008168:Lung / Q000166:cytology*; D008246:Lysophospholipids / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism / Q000502:physiology*; D008856:Microscopy, Fluorescence; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains
D000199:Actins; C495868:CTTN protein, human; D051356:Cortactin; D008246:Lysophospholipids; D008840:Microfilament Proteins; D016376:Oligonucleotides, Antisense; D010455:Peptides; C060506:sphingosine 1-phosphate; D014443:Tyrosine; D009219:Myosin-Light-Chain Kinase; D020558:GTP Phosphohydrolases; D013110:Sphingosine