Myeloid Notch1 deficiency activates the 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/Rho-associated protein kinase (ROCK) pathway promotes leukocyte accumulation in tissue injury. However, it remains unknown whether Notch signaling regulates ras homolog gene family member A/ROCK–mediated immune responses in liver ischemia and reperfusion (IR) injury. 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 showed aggravated hepatocellular damage, with increased serum alanine aminotransferase levels, hepatocellular apoptosis, macrophage/neutrophil trafficking, and proinflammatory mediators compared to Notch1-proficient controls. Unlike in the 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 phosphatase and tensin homolog (PTEN) activation in ischemic livers. Disruption of JSAP1 in myeloid-specific Notch1 knockout livers improved hepatocellular function and reduced JNK, ROCK1, PTEN, and toll-like receptor 4 activation. Moreover, ROCK1 knockdown inhibited PTEN and promoted Akt, leading to depressed toll-like receptor 4. In parallel in vitro studies, transfection of lentivirus-expressing Notch1 intracellular domain promoted Hes1 and inhibited JSAP1 in lipopolysaccharide-stimulated bone marrow–derived macrophages. Hes1 deletion enhanced JSAP1/JNK activation, whereas clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9–mediated JSAP1 knockout diminished ROCK1/PTEN and toll-like receptor 4 signaling. Conclusion: Myeloid Notch1 deficiency activates the ras homolog gene family member A/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 IR injury in transplant recipients. (Hepatology 2018;67:1041–1055).