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), leading to inflammatory gene transcription and junctional permeability increases. This study examined the activation of RhoA and Rho kinase induced by TNF-α in primary human pulmonary microvascular ECs and its role in regulating EC responses to TNF-α. TNF-α induced a time-dependent activation of RhoA and Rho kinase in these ECs. TNF-α 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-α-induced early and late JNK activation. Inhibition of RhoA protein expression using small-interfering RNA, however, did not prevent TNF-α-induced Rho kinase activation or JNK activation. Studies using MAPK kinase 4 (MKK4) small-interfering RNA showed that MKK4 was not required for TNF-α-induced early JNK activation and that Rho kinase modulated early JNK activation through MKK4-independent mechanisms. Rho kinase, however, modulated TNF-α-induced late JNK activation mainly through MKK4-dependent mechanisms. Activation of Rho kinase was required for JNK-dependent IL-6 secretion induced by TNF-α. Moreover, inhibition of Rho kinase prevented TNF-α-induced cytoskeletal changes and permeability increases. 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. Therefore, Rho kinase plays important roles in EC responses to TNF-α by regulating permeability increases and JNK-dependent IL-6 production during pulmonary inflammation.