title: Transforming growth factor-beta1 effects on endothelial monolayer permeability involve focal adhesion kinase/Src.
abstract: Transforming growth factor (TGF)-beta1 activity has been shown to increase vascular endothelial barrier permeability, which is believed to precede several pathologic conditions, including pulmonary edema and vessel inflammation. In endothelial monolayers, TGF-beta1 increases permeability, and a number of studies have demonstrated the alteration of cell-cell contacts by TGF-beta1. We hypothesized that focal adhesion complexes also likely contribute to alterations in endothelial permeability. We examined early signal transduction events associated with rapid changes in monolayer permeability and the focal adhesion complex of bovine pulmonary artery endothelial cells. Western blotting revealed rapid tyrosine phosphorylation of focal adhesion kinase (FAK) and Src kinase in response to TGF-beta1; inhibition of both of these kinases using pp2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), ameliorates TGF-beta1-induced monolayer permeability. Activation of FAK/Src requires activation of the epidermal growth factor receptor downstream of the TGF-beta receptors, and is blocked by the epidermal growth factor receptor inhibitor AG1478. Immunohistochemistry showed that actin and the focal adhesion proteins paxillin, vinculin, and hydrogen peroxide-inducible clone-5 (Hic-5) are rearranged in response to TGF-beta1; these proteins are released from focal adhesion complexes. Rearrangement of paxillin and vinculin by TGF-beta1 is not blocked by the FAK/Src inhibitor, pp2, or by SB431542 inhibition of the TGF-beta type I receptor, anaplastic lymphoma kinase 5; however, pp1 (4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), which inhibits both type I and type II TGF-beta receptors, does block paxillin and vinculin rearrangement. Hic-5 protein rearrangement requires FAK/Src activity. Together, these results suggest that TGF-beta1-induced monolayer permeability involves focal adhesion and cytoskeletal rearrangement through both FAK/Src-dependent and -independent pathways.
authors.affiliation: Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA.
authors.forename: Young H
authors.initials: YH
authors.lastname: Lee
authors.forename: Usamah S
authors.initials: US
authors.lastname: Kayyali
authors.forename: Anne Marie
authors.initials: AM
authors.lastname: Sousa
authors.forename: Thomas
authors.initials: T
authors.lastname: Rajan
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Lechleider
authors.forename: Regina M
authors.initials: RM
authors.lastname: Day
mesh_terms: D000077548:Anaplastic Lymphoma Kinase; D000818:Animals; D002417:Cattle; D002463:Cell Membrane Permeability / Q000187:drug effects*; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D066246:ErbB Receptors / Q000235:genetics; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D022001:Focal Adhesions / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D051419:Paxillin / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases; D015533:Transcriptional Activation / Q000187:drug effects; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D016596:Vinculin / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D051419:Paxillin; D053773:Transforming Growth Factor beta1; D016596:Vinculin; D019000:Phosphotyrosine; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D019950:Mitogen-Activated Protein Kinase 1