Differential effect of MLC kinase in TNF-alpha-induced endothelial cell apoptosis and barrier dysfunction

Tumor necrosis factor (TNF)-alpha is released in acute inflammatory lung sy ndromes linked to the extensive vascular dysfunction associated with increa sed permeability and endothelial cell apoptosis. TNF-alpha induced signific ant decreases in transcellular electrical resistance across pulmonary endot helial cell monolayers, reflecting vascular barrier dysfunction (beginning at 4 h and persisting for 48 h). TNF-alpha also triggered endothelial cell apoptosis beginning at 4 h, which was attenuated by the caspase inhibitor Z -Val-Ala-Asp-fluoromethylketone. Exploring the involvement of the actomyosi n cytoskeleton in these important endothelial cell responses, we determined that TNF-alpha significantly increased myosin light chain (MLC) phosphoryl ation, with prominent stress fiber and paracellular gap formation, which pa ralleled the onset of decreases in transcellular electrical resistance and enhanced apoptosis. Reductions in MLC phosphorylation by the inhibition of either MLC kinase (ML-7, cholera toxin) or Rho kinase (Y-27632) dramaticall y attenuated TNF-alpha -induced stress fiber formation, indexes of apoptosi s, and caspase-8 activity but not TNF-alpha -induced barrier dysfunction. T hese studies indicate a central role for the endothelial cell cytoskeleton in TNF-alpha -mediated apoptosis, whereas TNF-alpha -induced vascular perme ability appears to evolve independently of contractile tension generation.