TNF‐α‐induced activation of RhoA/Rho kinase pathway in human pulmonary microvascular endothelial cells

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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.