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J. Biol. Chem., Vol. 283, Issue 19, 13437-13449, May 9, 2008

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TLR4 Signaling Is Coupled to SRC Family Kinase Activation, Tyrosine Phosphorylation of Zonula Adherens Proteins, and Opening of the Paracellular Pathway in Human Lung Microvascular Endothelia^*

Ping Gong¹, Daniel J. Angelini, Shiqi Yang^¶, Guanjun Xia^||, Alan S. Cross^**, Dean Mann, Douglas D. Bannerman, Stefanie N. Vogel, and Simeon E. Goldblum^¶2

From the Mucosal Biology Research Center, Department of Pathology, ^¶Department of Medicine, Department of Microbiology and Immunology, ^||Department of Pharmaceutical Sciences, and ^**Center for Vaccine Development, University of Maryland, Baltimore, Maryland 21201 and the Bovine Functional Genomics Laboratory, United States Department of Agriculture, Beltsville, Maryland 20705

Bacterial lipopolysaccharide (LPS) is a key mediator in the vascular leak syndromes associated with Gram-negative bacterial infections. LPS opens the paracellular pathway in pulmonary vascular endothelia through protein tyrosine phosphorylation. We now have identified the protein-tyrosine kinases (PTKs) and their substrates required for LPS-induced protein tyrosine phosphorylation and opening of the paracellular pathway in human lung microvascular endothelial cells (HMVEC-Ls). LPS disrupted barrier integrity in a dose- and time-dependent manner, and prior broad spectrum PTK inhibition was protective. LPS increased tyrosine phosphorylation of zonula adherens proteins, VE-cadherin, -catenin, and p120^ctn. Two SRC family PTK (SFK)-selective inhibitors, PP2 and SU6656, blocked LPS-induced increments in tyrosine phosphorylation of VE-cadherin and p120^ctn and paracellular permeability. In HMVEC-Ls, c-SRC, YES, FYN, and LYN were expressed at both mRNA and protein levels. Selective small interfering RNA-induced knockdown of c-SRC, FYN, or YES diminished LPS-induced SRC Tyr⁴¹⁶ phosphorylation, tyrosine phosphorylation of VE-cadherin and p120^ctn, and barrier disruption, whereas knockdown of LYN did not. For VE-cadherin phosphorylation, knockdown of either c-SRC or FYN provided total protection, whereas YES knockdown was only partially protective. For p120^ctn phosphorylation, knockdown of FYN, c-SRC, or YES each provided comparable but partial protection. Toll-like receptor 4 (TLR4) was expressed both on the surface and intracellular compartment of HMVEC-Ls. Prior knockdown of TLR4 blocked both LPS-induced SFK activation and barrier disruption. These data indicate that LPS recognition by TLR4 activates the SFKs, c-SRC, FYN, and YES, which, in turn, contribute to tyrosine phosphorylation of zonula adherens proteins to open the endothelial paracellular pathway.

Received for publication, September 24, 2007 , and in revised form, February 6, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants HL-70155 and HL-84223 (to S. E. G.) and AI-18797 (to S. N. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of American Heart Association (Mid-Atlantic Affiliate) Postdoctoral Fellowship 0725389U.

² To whom correspondence should be addressed: Mucosal Biology Research Center, University of Maryland School of Medicine, 20 Penn St., HSF II-Rm. 351, Baltimore, MD 21201. Tel.: 410-706-5504; Fax: 410-706-5508; E-mail: sgoldblu{at}mbrc.umaryland.edu.

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