Mutation of PXR phosphorylation motif at Ser347 disrupts lipid and bile acid homeostasis in diet-induced metabolic dysfunction–associated steatohepatitis in mice

The pregnane X receptor (PXR), a ligand-activated transcription factor, regulates the expression of genes involved in endobiotic and xenobiotic metabolism, inflammation, and fibrosis. Disruption of PXR functions can affect processes critical to metabolic dysfunction-associated steatohepatitis (MASH) progression. Although ligand-dependent PXR functions are well studied, its regulation by post-translational modification, particularly phosphorylation, remains unclear. PXR has a conserved phosphorylation motif within its ligand binding domain (Ser347 in mice; Ser350 in humans). In vitro studies showed that this site mutation impairs human PXR transcriptional activity; however, the mechanism remains elusive. To investigate this phosphorylation site role in MASH development, wild-type and PXR Ser347Ala knock-in mutation (PXR-KI) mice were fed either a high-fat diet or a control chow diet for 16 weeks. On control chow diet, PXR-KI mice exhibited decreased expression of alternative bile acid (BA) synthesis genes compared with wild-type mice. On a high-fat diet, PXR-KI mice manifested more severe hepatic steatosis, revealed by elevated serum total cholesterol, and increased expression of genes involved in lipid metabolism. In addition, changes in BA metabolism and transporter genes suggested a cholestatic pattern in this group of mice. BA profiling showed higher levels of conjugated, hydrophilic, primary BA in the serum and liver, and increased unconjugated BA in the intestine. The data suggest that PXR Ser347 phosphorylation motif is essential for regulating PXR functions to maintain endobiotic metabolism and alleviate hepatotoxicity during MASH progression. SIGNIFICANT STATEMENT: The ligand-independent role of pregnane X receptor (PXR) is unclear. In phosphodeficient PXR knock-in mice, loss of Ser347 phosphorylation worsened hepatic steatosis and altered bile acid homeostasis under high-fat diet feeding, uncovering a novel role and therapeutic potential of PXR phosphorylation in fatty liver diseases.