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United States Department of Agriculture

Agricultural Research Service

Research Project: GENOMIC AND IMMUNOLOGIC STRATEGIES TO IMPROVE MILK PRODUCTION EFFICIENCY AND CONTROL MASTITIS Title: Bacterial lipopolysaccharide increases tyrosine phosphorylation of zonula adherens proteins and opens the paracellular pathway in lung microvascular endothelia through TLR4, TRAF6, and src family kinase activation

Authors
item Gong, Ping - U OF MD SCHOOL OF MED
item Yang, Shiqi - U OF MD SCHOOL OF MED
item Mann, Dean - U OF MD SCHOOL OF MED
item Bannerman, Douglas
item Vogel, Stefanie - U OF MD SCHOOL OF MED
item Goldblum, Simeon - U OF MD SCHOOL OF MED

Submitted to: American Society of Cell Biology Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: September 24, 2007
Publication Date: December 1, 2007
Citation: Gong, P., Yang, S., Mann, D., Bannerman, D.D., Vogel, S.N., Goldblum, S.E. 2007. Bacterial lipopolysaccharide increases tyrosine phosphorylation of zonula adherens (ZA) proteins and opens the paracellular pathway in lung microvascular endothelia through TLR4, TRAF6, and src family kinase (SFK)activation. 47th Annual Meeting of the American Society of Cell Biology. p. 98, Dec. 1-5, 2007, Washington DC.

Technical Abstract: Objective: LPS is a key mediator in vascular leak syndromes associated with Gram-negative bacterial infections and opens the pulmonary vascular endothelial paracellular pathway through protein tyrosine kinase (PTK) activation. We asked which PTKs and signaling molecules mediate LPS-induced endothelial barrier disruption. Methods: In human lung microvascular endothelial cell (HMVEC-L)s, RT-PCR, Western blotting, and FACS analysis were used to detect TLR4, TRAF6, and SFK expression. Purified E. coli K235 LPS was used as a specific TLR4 agonist. Transendothelial 14C-albumin flux was used to assess paracellular permeability. Immunoprecipitation and phosphotyrosine immunoblotting were used to study ZA protein phosphorylation. siRNA was used to knockdown target genes. Results: In HMVEC-Ls, TLR4, TRAF6, c-src, fyn, yes, and lyn were expressed at both mRNA and protein levels. LPS increased src family kinase (SFK) activation and disrupted endothelial barrier integrity in a dose- and time-dependent manner. Prior broad-spectrum PTK inhibition, as well as SFK-selective inhibition with either PP2 or SU6656, protected against these LPS-induced effects. Prior knockdown of TLR4, or its downstream adapter protein, TRAF6, blocked both LPS-induced SFK activation and barrier disruption. LPS increased tyrosine phosphorylation of the ZA proteins, VE cadherin, '-catenin, and p120 catenin. PP2 and SU6656 blocked LPS-induced VE cadherin and p120ctn phosphorylation. Prior knockdown of c-src, fyn, or yes, but not lyn, diminished LPS-induced SFK activation and barrier disruption. Prior knockdown of fyn prevented VE-cadherin tyrosine phosphorylation whereas knockdown of either c-src or fyn diminished p120ctn phosphorylation. Conclusions: In HMVEC-Ls, the LPS stimulus is coupled to increased tyrosine phosphorylation of ZA proteins and opening of the paracellular pathway, through TLR4- and TRAF6-dependent SFK activation. LPS-induced SFK activation can be ascribed to c-src, fyn, and yes but not lyn.

Last Modified: 7/31/2014
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