Submitted on November 28, 2006
Revised on June 19, 2007
TGF-
1 Effects on Endothelial Monolayer Permeability Involve FAK/Src
Young H Lee1, Usamah S Kayyali2, Anne Marie Sousa2, Thomas Rajan2, Robert J Lechleider3, and Regina M Day1*
1 Department of Pharmacology, The Uniformed Services University of the Health Sciences, Bethesda, MD, USA,
2 Division of Pulmonary, Critical Care and Sleep, Tupper Research Institute, Tufts-New England Medical Center, Boston, MA, USA,
3 Department of Cell Biology, Georgetown University Medical Center, Washington, DC, USA
* To whom correspondence should be addressed. E-mail: rday{at}usuhs.mil.
Transforming growth factor beta 1 (TGF-1
) activity has been shown to increase vascular endothelial barrier permeability, which is believed to precede several pathological 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 (PAEC). 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 ameliorates TGF-1 induced monolayer permeability. Activation of FAK/Src requires activation of the epidermal growth factor receptor (EGFR) downstream of the TGF- receptors, and is blocked by the EGFR inhibitor AG1478. Immunohistochemistry showed that actin and the focal adhesion proteins paxillin, vinculin and Hic-5 are rearranged in response to TGF-1; these proteins are released from focal adhesion complexes. Rearrangement of vinculin by TGF-1 is not blocked by the FAK/Src inhibitor pp2 or by SB431542 inhibition of the TGF- type I receptor Alk5; however, pp1, 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.
