TNF-alpha increases tyrosine phosphorylation of vascular endothelial cadherin and opens the paracellular pathway through fyn activation in human lung endothelia.
Tumor necrosis factor (TNF)-alpha is a key mediator of sepsis-associated multiorgan failure, including the acute respiratory distress syndrome. We examined the role of protein tyrosine phosphorylation in TNF-alpha-induced pulmonary vascular permeability. Postconfluent human lung microvascular and pulmonary artery endothelial cell (EC) monolayers exposed to human recombinant TNF-alpha displayed a dose- and time-dependent increase in transendothelial [(14)C]albumin flux in the absence of EC injury. TNF-alpha also increased tyrosine phosphorylation of EC proteins, and several substrates were identified as the zonula adherens proteins vascular endothelial (VE)-cadherin, and beta-catenin, gamma-catenin, and p120 catenin (p120(ctn)). Prior protein tyrosine kinase (PTK) inhibition protected against the TNF-alpha effect. TNF-alpha activated multiple PTKs, including src family PTKs. Prior PTK inhibition with the src-selective agents PP1 and PP2 each protected against approximately 60% of the TNF-alpha-induced increment in [(14)C]albumin flux. PP2 also blocked TNF-alpha-induced tyrosine phosphorylation of VE-cadherin, gamma-catenin, and p120(ctn). To identify which src family kinase(s) was required for TNF-alpha-induced vascular permeability, small interfering RNA (siRNA) targeting each of the three src family PTKs expressed in human EC, c-src, fyn, and yes, were introduced into the barrier function assay. Only fyn siRNA protected against the TNF-alpha effect, whereas the c-src and yes siRNAs did not. These combined data suggest that TNF-alpha regulates the pulmonary vascular endothelial paracellular pathway, in part, through fyn activation.
Angelini
Daniel J
DJ
Division of Infectious Disease and Pulmonary, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Hyun
Sang-Won
SW
Grigoryev
Dmitry N
DN
Garg
Pallavi
P
Gong
Ping
P
Singh
Ishwar S
IS
Passaniti
Antonino
A
Hasday
Jeffery D
JD
Goldblum
Simeon E
SE
D000818:Animals; D015820:Cadherins / Q000187:drug effects / Q000378:metabolism*; D002460:Cell Line; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D008168:Lung; D051379:Mice; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn / Q000187:drug effects / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology; D020545:Respiratory Mucosa / Q000187:drug effects / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D015820:Cadherins; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D019000:Phosphotyrosine; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn