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.
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, gamma-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 Tyr(416) 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.
Gong
Ping
P
Department of Pathology, University of Maryland, Baltimore, Maryland 21201, USA.
Angelini
Daniel J
DJ
Yang
Shiqi
S
Xia
Guanjun
G
Cross
Alan S
AS
Mann
Dean
D
Bannerman
Douglas D
DD
Vogel
Stefanie N
SN
Goldblum
Simeon E
SE
D022005:Adherens Junctions / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008168:Lung / Q000098:blood supply / Q000187:drug effects / Q000378:metabolism*; D008833:Microcirculation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D012710:Serum Albumin, Bovine / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013379:Substrate Specificity; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
D008070:Lipopolysaccharides; D034741:RNA, Small Interfering; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D019000:Phosphotyrosine; D012710:Serum Albumin, Bovine; D019061:src-Family Kinases