The matrin3-CAR-thrombin axis in hepatic inflammation and fibrosis

About This Grant

PROJECT SUMMARY Metabolic dysfunction-associated steatotic liver disease affects approximately 30% of U.S. adults with rising prevalence. It can progress to metabolic dysfunction-associated steatohepatitis (MASH). Although steatosis, inflammation, and coagulation contribute to the development of fibrosis in MASH, the molecular mechanisms linking them are elusive, hindering the development of therapeutic approaches to treat MASH. Matrin-3 is a DNA- and RNA-binding protein involved in different biological processes by regulating RNA processing, RNA stability, and chromatin remodeling. Matrin-3 genetic variations are associated with amyotrophic lateral sclerosis and cardiometabolic traits in humans. However, the role of matrin-3 and its downstream molecules in regulating signaling pathways and gene expression remains largely unknown in MASH. We found that matrin-3 liver- specific knockout (LKO) mice display increased hepatic steatosis compared with matrin-3 floxed mice. Integrated analysis of our transcriptomic and genomic data suggests that constitutive androstane receptor (CAR) is a main transcription factor that mediates transcriptome changes and hepatic steatosis in the liver of matrin-3 LKO mice. Importantly, we found that matrin-3 LKO mice display increased infiltration of inflammatory cells and hepatic fibrosis in a mouse model of MASH. To reveal mechanistic insights, we performed single-nucleus Multiome analysis and inferred intercellular communications. The study revealed that hepatocytes and hepatic stellate cells (HSC) have the strongest intercellular communication in the livers of LKO mice. The protease-activated receptors (PARs) mediate the topmost incoming signaling pattern in HSCs of LKO livers. Among the four PARs, PAR1, PAR3, and PAR4, except PAR2, are thrombin receptors. We identified F2, a gene that encodes prothrombin, as a new target gene of CAR through a combination of genomic and biochemical approaches. We found that CAR signaling is required to repress F2 expression through an unknown mechanism in healthy livers. In support of the potential role of thrombin in hepatic fibrosis, we found that: 1) F2 expression is induced in the hepatocyte cluster revealed by single-nucleus RNA-seq in the livers of LKO mice and patients with MASH; 2) PAR1 is the dominantly expressed thrombin receptor in the HSC cluster; and 3) thrombin activates mouse primary HSCs. Together, these preliminary data lead to our hypothesis that the matrin-3-CAR axis constrains hepatic inflammation and fibrosis in part by repressing the paracrine effects of hepatocytes on hepatic stellate cells through thrombin and its receptor PAR1 in diet-induced MASH. Three specific aims are to: 1) identify the molecular mechanisms by which matrin-3 regulates CAR signaling, 2) determine the protective role of the matrin- 3-CAR axis in hepatic inflammation and fibrosis in a preclinical model of MASH, and 3) reveal mechanistic insights into the matrin-3-CAR axis-regulated hepatic stellate cells activation and liver fibrosis. Completion of the project will identify the matrin-3-CAR-thrombin axis as a new connection linking steatosis, inflammation, and coagulation that contributes to liver fibrosis and will provide novel therapeutic targets with translational impact.