Myeloid Notch1 Deficiency Activates RhoA/ROCK Pathway and Aggravates Hepatocellular Damage In Mouse Ischemic Livers

Abstract

Notch signaling plays an emerging role in the regulation of immune cell development and function during inflammatory response. Activation of the ras homolog gene family, member A (RhoA)/Rho-associated protein kinase (ROCK) pathway promotes leukocyte accumulation in tissue injury. However, it remains unknown whether Notch signaling regulates RhoA/ROCK-mediated immune responses in liver ischemia and reperfusion injury (IRI). This study investigated intracellular signaling pathways regulated by Notch receptors in the IR-stressed liver and in vitro. In a mouse model of IR-induced liver inflammatory injury, we found that mice with myeloid specific Notch1 knockout (Notch1^M-KO) showed aggravated hepatocellular damage, with increased serum ALT levels, hepatocellular apoptosis, macrophage/neutrophil trafficking, and pro-inflammatory mediators compared to the Notch1-proficient (Notch1^FL/FL) controls. Unlike in the Notch1^FL/FL controls, myeloid Notch1 ablation diminished hairy and enhancer of split-1 (Hes1) and augmented c-Jun N-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), JNK, ROCK1, and PTEN activation in ischemic livers. Disruption of JSAP1 in Notch1^M-KO livers improved hepatocellular function and reduced JNK, ROCK1, PTEN, and TLR4 activation. Moreover, ROCK1 knockdown inhibited PTEN and promoted Akt, leading to depressed TLR4. In parallel in vitro studies, transfection of lentivirus-expressing Notch1 intracellular domain (NICD) promoted Hes1 and inhibited JSAP1 in LPS-stimulated bone marrow-derived macrophages (BMMs). Hes1 deletion enhanced JSAP1/JNK activation whereas CRISPR/Cas9-mediated JSAP1 knockout diminished ROCK1/PTEN and TLR4 signaling. Conclusions: Myeloid Notch1 deficiency activates the RhoA/ROCK pathway and exacerbates hepatocellular injury by inhibiting transcriptional repressor Hes1 and inducing scaffold protein JSAP1 in IR-triggered liver inflammation. Our findings underscore the crucial role of the Notch-Hes1 axis as a novel regulator of innate immunity-mediated inflammation and imply the therapeutic potential for the management of organ IRI in transplant recipients. This article is protected by copyright. All rights reserved.

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