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

Abstract

TNF-alpha induces complex signaling events in endothelial cells (ECs), leading to inflammatory gene transcription and junctional permeability increases. This study examined the activation of RhoA and Rho kinase induced by TNF-alpha in primary human pulmonary microvascular ECs and its role in regulating EC responses to TNF-alpha. TNF-alpha induced a time-dependent activation of RhoA and Rho kinase in these ECs. TNF-alpha also induced activation of JNK that peaked at 15 min and lasted for at least 3 h. Inhibition of Rho kinase using a specific pharmacological inhibitor, Y27632, prevented TNF-alpha-induced early and late JNK activation. Inhibition of RhoA protein expression using small-interfering RNA, however, did not prevent TNF-alpha-induced Rho kinase activation or JNK activation. Studies using MAPK kinase 4 (MKK4) small-interfering RNA showed that MKK4 was not required for TNF-alpha-induced early JNK activation and that Rho kinase modulated early JNK activation through MKK4-independent mechanisms. Rho kinase, however, modulated TNF-alpha-induced late JNK activation mainly through MKK4-dependent mechanisms. Activation of Rho kinase was required for JNK-dependent IL-6 secretion induced by TNF-alpha. Moreover, inhibition of Rho kinase prevented TNF-alpha-induced cytoskeletal changes and permeability increases. Inhibition of JNK activation, however, did not prevent TNF-alpha-induced cytoskeletal changes, suggesting that Rho kinase did not modulate cytoskeletal changes through JNK activation. Therefore, Rho kinase plays important roles in EC responses to TNF-alpha by regulating permeability increases and JNK-dependent IL-6 production during pulmonary inflammation.