Project description:Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Persistent NAFLD can progress to nonalcoholic steatohepatitis (NASH) with inflammation and fibrosis, which predisposes patients to cirrhosis and hepatocellular carcinoma. CCAAT/enhancer-binding protein alpha (CEBPA) is a transcription factor that modulates glycolipid homeostasis, cell differentiation and tumor progression. MTL-CEBPA, a first-in-human small activating RNA therapeutic, has been used to overexpress CEBPA and treat hepatocellular carcinoma by targeting myeloid cells in clinical trials. However, whether and how hepatocyte-specific CEBPA modulates NASH are unknown. Here we found that CEBPA expression was inhibited in the livers of high-fat, high-cholesterol, and high-fructose diet (HFCFD)-fed mice, NASH patients and palmitic/oleic acid-treated primary hepatocytes. Liver-specific Cebpa knockout mice (Cebpa-dLiv) and liver-specific tamoxifen-inducible ERT2-Cre mice (Cebpa-dLiv-ERT2) were then generated. Both Cebpa-dLiv and Cebpa-dLiv-ERT2 mice (treated with tamoxifen) developed enhanced NASH and fibrosis compared to control mice. Global transcriptome analyses demonstrated hepatic secreted phosphoprotein 1 (Spp1) encoding the fibrosis-promoting factor SPP1, was markedly induced by hepatocyte CEBPA knockout in HFCFD-fed mice. Consistently, hepatic SPP1 was upregulated by NASH and negatively correlated with CEBPA expression in humans. Hepatocyte loss of CEBPA enhanced hepatic SPP1 expression and serum SPP1 levels both at the early and late stage of NASH in mice. CEBPA-knockout primary hepatocytes released more SPP1 to the culture medium than wild-type mouse hepatocytes, resulting in enhanced fibrogenesis of hepatic stellate cells, a phenotype that was rescued by the Spp1 shRNA. Hepatocyte-directed AAV8-deliverey of Spp1 shRNA rescued the fibrosis in HFCFD-fed Cebpa-dLiv mice. Mechanistically, CEBPA overexpression reduced, while CEBPA knockout enhanced, SPP1 expression in primary hepatocytes in vitro. The Spp1 promoter had functional CEBPA response elements, while CEBPA modulated histone acetylation to restrict SPP1 expression in a novel feedback loop. Furthermore, overexpression of CEBPA in hepatocytes with AAV8-TBG-CEBPA, inhibited hepatic SPP1 expression and reduced fibrosis in HFCFD-fed wild-type mice. This study demonstrates a novel hepatocyte CEBPA-SPP1 axis involved in modulating NASH fibrosis that supports hepatocyte CEBPA as an anti-NASH target.

Project description:Background: Senescent hepatocytes accumulate in parallel with fibrosis progression during NASH. The mechanisms that enable progressive expansion of nonreplicating cell populations and the significance of that process in determining NASH outcomes are unclear. Many types of senescing cells upregulate the THBD-PAR-1 signaling axis to remain viable. Vorapaxar, an FDA-approved PAR-1 inhibitor, blocks the activity of that pathway. We used vorapaxar to determine if and how THBD-PAR1 signaling promotes fibrosis progression in NASH. Methods: We evaluated the THBD-PAR1 pathway in liver biopsies from NAFLD patient cohorts with a spectrum of liver fibrosis. Chow fed mice were treated with viral vectors to over-express p16 specifically in hepatocytes and induce replicative senescence. Effects on the THBD-PAR-1 axis and regenerative capacity were assessed; the transcriptome of p16 over-expressing hepatocytes was characterized and we examined how conditioned medium from senescent but viable (dubbed ‘undead’) hepatocytes reprograms hepatic stellate cells. A genetically obese mouse model of NASH with little liver fibrosis, and a diet-induced mouse model of NASH with advanced fibrosis were treated with vorapaxar to determine effects on hepatocyte senescence and liver damage. Results: Inducing senescence up-regulates the THBD-PAR1 signaling axis in hepatocytes and induces their expression of fibrogenic factors, including hedgehog ligands. Hepatocyte THBD-PAR1 signaling increases in NAFLD and supports sustained hepatocyte senescence that limits effective liver regeneration and promotes maladaptive repair. Inhibiting PAR-1 signaling with vorapaxar interrupts this process, reduces the burden of ‘undead’ senescent cells, and safely improves NASH and fibrosis despite ongoing lipotoxic stress Conclusion: The THBD-PAR1 signaling axis is a novel therapeutic target for NASH because blocking this pathway prevents accumulation of senescing but viable hepatocytes that generate factors that promote maladaptive liver repair.

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Hepatocyte CCAAT/enhancer-binding protein alpha restricts liver fibrosis by inhibiting secreted phosphoprotein 1 expression (II)

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