The Interplay of TGF-?1 and Cholesterol Orchestrating Hepatocyte Cell Fate, EMT, and Signals for HSC Activation

BACKGROUND & AIMS: Transforming growth factor -ss1 (TGFss1) plays important roles in chronic liver diseases, including metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD involves various biological processes including dysfunctional cholesterol metabolism and contributes to progression to metabolic dysfunction-associated steatohepatitis and hepatocellular carcinoma. However, the reciprocal regulation of TGF-ss1 signaling and cholesterol metabolism in MASLD is yet unknown. METHODS: Changes in transcription of genes associated with cholesterol metabolism were assessed by RNA sequencing of murine hepatocyte cell line (alpha mouse liver 12/AML12) and mouse primary hepatocytes treated with TGF-ss1. Functional assays were performed on AML12 cells (untreated, TGF-ss1 treated, or subjected to cholesterol enrichment [CE] or cholesterol depletion [CD]), and on mice injected with adenovirus-associated virus 8-control/TGF-ss1. RESULTS: TGF-ss1 inhibited messenger RNA expression of several cholesterol metabolism regulatory genes, including rate-limiting enzymes of cholesterol biosynthesis in AML12 cells, mouse primary hepatocytes, and adenovirus-associated virus-TGF- ss1-treated mice. Total cholesterol levels and lipid droplet accumulation in AML12 cells and liver tissue also were reduced upon TGF-ss1 treatment. Smad2/3 phosphorylation after 2 hours of TGFss1 treatment persisted after CE or CD and was mildly increased after CD, whereas TGF-ss1-mediated AKT phosphorylation (30 min) was inhibited by CE. Furthermore, CE protected AML12 cells from several effects mediated by 72 hours of incubation with TGF-ss1, including epithelial-mesenchymal transition, actin polymerization, and apoptosis. CD mimicked the outcome of long-term TGF-ss1 administration, an effect that was blocked by an inhibitor of the type I TGF-ss receptor. In addition, the supernatant of CE- or CDtreated AML12 cells inhibited or promoted, respectively, the activation of LX -2 hepatic stellate cells. CONCLUSIONS: TGF-ss1 inhibits cholesterol metabolism whereas cholesterol attenuates TGF-ss1 downstream effects in hepatocytes. (Cell Mol Gastroenterol Hepatol 2024