Activation of Rho Kinase by TNF-α Is Required for JNK Activation in Human Pulmonary Microvascular Endothelial Cells

Abstract

TNF‐α induces complex signaling events in endothelial cells (ECs), resulting in gene transcription and increased junctional permeability. This study examined the activation of the RhoA/Rho kinase pathway induced by TNF‐α in primary human pulmonary microvascular ECs and its role in EC responses to TNF‐α. Activation of RhoA was induced by TNF‐α within five minutes. This increase in RhoA activity rapidly decreased, and a late activation of RhoA was induced three hours after TNF‐α treatment. This late but not early RhoA activation was associated with its accumulation in focal aggregates near EC edges. TNF‐α also induced Rho kinase activation that was observed within five minutes and lasted for at least three hours. Inhibition of Rho kinase prevented TNF‐α‐induced cytoskeletal changes and permeability increases. TNF‐α also induced activation of JNK that peaked at fifteen minutes and lasted for at least three hours, and inhibition of Rho kinase prevented TNF‐α‐induced early and late JNK activation. Inhibition of JNK activation, however, did not prevent TNF‐α‐induced cytoskeletal changes, suggesting that Rho kinase did not modulate cytoskeletal changes through JNK activation. These data demonstrate that RhoA/Rho kinase pathway is rapidly activated by TNF‐α and regulates several downstream responses that include JNK activation, cytoskeletal changes and increases in permeability. Supported by NIH HL070009.

RhoA/Rho Kinase Mediates Neuronal Death Through Regulating cPLA2 Activation

“…Previous studies have demonstrated that TNF-α or glutamate can activate RhoA and Rho kinase [39][40][41]. RhoA is an essential component of the excitotoxicity pathway, and is sufficient to induce the activation of such pathway [42].…”

Activation of Rho Kinase Isoforms in Lung Endothelial Cells during Inflammation

“…Previous studies have demonstrated activation of ROCK in response to TNF-␣ that lasts for hours in primary human pulmonary microvascular ECs (15). To begin to evaluate isoform-specific ROCK activation, ROCK1 or ROCK2 was immunoprecipitated from primary human pulmonary microvascular ECs, and ROCK isoform activity was evaluated by an in vitro kinase activity assay using purified MYPT-1 (714-1004) as a substrate.…”

“…ECs were treated for 4 h with 10 nM ROCK1 or ROCK2 siRNA or their corresponding control siRNA premixed with 6 g/ml lipofectin (15). The cells were then incubated with normal culture medium for 72 h before experiments.…”

“…The fluxes of dextran across ECs were evaluated using Transwell chambers (15). ECs were seeded onto Transwell inserts (6.5 mm diameter and 0.4 m pore size) coated with 4 g/ml fibronectin.…”

“…Confluent ECs were treated with either vehicle (PBS containing 0.1% BSA) or 20 ng/ml recombinant human TNF-␣ for 5 min to 24 h by adding vehicle or a stock solution directly to EC cultures (15).…”