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Transforming Growth Factor-1 Effects on Endothelial Monolayer Permeability Involve Focal Adhesion Kinase/Src

¹ Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland; ² Tufts—New England Medical Center, Tupper Research Institute, Division of Pulmonary, Critical Care, and Sleep, Boston, Massachusetts; and ³ Department of Cell Biology, Georgetown University Medical Center, Washington, DC

Correspondence and requests for reprints should be addressed to Regina M. Day, Ph.D., Department of Pharmacology, Bldg C, Rm 2023, 4301 Jones Bridge Rd, Bethesda, MD 20814-4799. E-mail: rday{at}usuhs.mil

Transforming growth factor (TGF)-1 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-1 increases permeability, and a number of studies have demonstrated the alteration of cell–cell contacts by TGF-1. 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-1; inhibition of both of these kinases using pp2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), ameliorates TGF-1–induced monolayer permeability. Activation of FAK/Src requires activation of the epidermal growth factor receptor downstream of the TGF- 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-1; these proteins are released from focal adhesion complexes. Rearrangement of paxillin and vinculin by TGF-1 is not blocked by the FAK/Src inhibitor, pp2, or by SB431542 inhibition of the TGF- 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- receptors, does block paxillin and vinculin rearrangement. Hic-5 protein rearrangement requires FAK/Src activity. Together, these results suggest that TGF-1–induced monolayer permeability involves focal adhesion and cytoskeletal rearrangement through both FAK/Src-dependent and -independent pathways.

Key Words: focal adhesion complex • hydrogen peroxide–inducible clone 5 • nuclear translocation • paxillin • vinculin

CLINICAL RELEVANCE Pulmonary edema is a critical factor in acute respiratory distress syndrome. Transforming growth factor (TGF)-1 is involved in this activity, and we have investigated early signal transduction of TGF-1, which is involved in alterations of the endothelial cell monolayer.