Submitted to: Journal of Biological Chemistry
Publication Type: Peer reviewed journal
Publication Acceptance Date: 3/31/2009
Publication Date: 5/15/2008
Citation: Gong, P., Angelini, D.J., Yang, S., Xia, G., Cross, A.S., Mann, D., Bannerman, D.D., Vogel, S.N., Goldblum, S.E. 2008. Toll-like receptor 4 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. Journal of Biological Chemistry. 283(19):13437-13449. Interpretive Summary: Toll-like receptor 4 (TLR-4) plays a critical role in the innate immune response to Gram-negative bacteria, which are responsible for approximately 40% of the clinical cases of mastitis in dairy cattle and a leading cause of septic shock in humans. In both cattle and humans, a major complication of these infections is the development of vascular dysfunction and accompanying acute respiratory distress. The current manuscript describes studies that mechanistically link TLR-4 to bacterial endotoxin-induced alterations in vascular endothelial permeability. The findings from this study identify cell signal transduction molecules that can be targeted with novel therapeutics (e.g., small interfering RNA, protein tyrosine kinase inhibitors, etc.) to ameliorate the complications that accompany these infections.
Technical Abstract: 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 kinase (PTK)s and their substrate(s) required for LPS-induced protein tyrosine phosphorylation and opening of the paracellular pathway in human lung microvascular endothelial cell (HMVEC-L)s. 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 (ZA) proteins, VE cadherin, gamma-catenin and p120ctn. Two src family PTK (SFK)-selective inhibitors, PP2 and SU6656, blocked LPS-induced increments in tyrosine phosphorylation of VE cadherin and p120ctn and paracellular permeability. In HMVEC-Ls, c-src, yes, fyn, and lyn were expressed at both mRNA and protein levels. Selective siRNA-induced knockdown of c-src, fyn or yes diminished LPS-induced src Y416 phosphorylation, tyrosine phosphorylation of VE-cadherin and p120ctn, and barrier disruption whereas knockdown of lyn had no such effects. For VE-cadherin phosphorylation, knockdown of either c-src or fyn provided total protection whereas yes knockdown was only partially protective. For p120ctn phosphorylation, knockdown of fyn, c-src, or yes each provided comparable but partial protection. Toll-like receptor (TLR)4 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 c-src, fyn and yes, and both increases tyrosine phosphorylation of ZA proteins and opens the endothelial paracellular pathway through SFK activation.