RhoA/ROCK Signaling Triggers Innate Immune Responses in Ischemia-Stressed Liver Through Activation of c-Jun N-Terminal Kinase Pathway.

Abstract

Background: The RhoA/Rho kinase (ROCK) signaling plays an important role in innate immune cell activation and the development of inflammatory diseases. However, little is known on the functional role of RhoA/ROCK signaling in the activation of innate immune responses during organ ischemia-reperfusion injury (IRI). This study was designed to explore potential molecular mechanisms of Rho/ROCK signaling in innate immune responses triggered by IR stress in a mouse liver. Methods: Using a partial hepatic warm ischemia model (90 min), C57BL/6 wide-type (WT) mice (n=6/gr) were pretreated with ROCK inhibitor H1152 (3 mg/kg, i.p.) or vehicle at 1h prior to ischemia, and then sacrificed at 6 h of reperfusion. In parallel in vitro study, bone marrow-derived WT macrophages (BMMs) were transfected with JNK1 siRNA/nonspecific siRNA (100 nM) or treated with H1152 (10μM), and then incubated with LPS (100 ng/ml). Results: Hepatic IR increased ROCK1 and c-Jun N-terminal kinase (JNK) expression and enhanced PTEN activity, whereas anti-ROCK (H1152) treatment decreased JNK and PTEN/TLR4 activation yet increased PI3K/Akt expression. Unlike in vehicle-treated WT controls, H1152 preconditioning improved hepatic function, reduced hepatocellular damage, and depressed macrophage/neutrophil trafficking, pro-inflammatory cytokine programs, and hepatocellular necrosis/apoptosis, while increasing anti-apoptotic functions in ischemic liver lobes. LPS-stimulated BMMs in vitro revealed increased expression of ROCK1, JNK, PTEN, and TLR4. However, knockdown of JNK with siRNA readily suppressed PTEN/TLR4, leading to diminished NF-κB activation and decreased expression of proinflammatory mediators. Consistent with our in vivo findings, pretreatment of LPS-stimulated BMMs with H1152 decreased JNK expression and upregulated Akt phosphorylation. Conclusion: This study demonstrates that IR-triggered RhoA/ROCK signaling induced JNK activation, leading to enhanced PTEN/TLR4 signaling. Inhibition of RhoA/ROCK protected livers from IR-mediated inflammation. Knockdown of JNK depressed PTEN yet promoted PI3K/Akt, which in turn negatively regulated TLR4 activation. By documenting the critical role of RhoA/ROCK-JNK signaling cascade in the mechanism of innate immune-driven local inflammation responses, our findings provide the rationale for a novel therapeutic strategy against IRI in organ transplant recipients.