Transglutaminase‐mediated cytokeratin modifications implicated in bile‐acid‐induced hepatocyte death

Transglutaminases (TGs) are crosslinking enzymes that catalyze the formation of isopeptide bonds between glutamine and lysine residues. They consist of eight isozymes: TG1-TG7 and factor XIIIa. Our previous studies have shown that TG1 and TG2 facilitate hepatic apoptosis, contributing to liver fibrosis, and that their crosslinking substrates-cytokeratin 18 (K18) and cytokeratin 8 (K8)-are also targeted by TGs in a bile-duct-ligation-induced mouse model of liver fibrosis. However, the precise mechanisms by which TGs and keratins contribute to hepatocyte damage remain unclear. This study investigates the molecular mechanisms underlying TG1- and TG2-mediated cell death in hepatocytes exposed to bile acids. HepG2 cells and primary hepatocytes were treated with glycochenodeoxycholic acid (GCDCA), a toxic bile salt elevated in cholestasis. GCDCA-reduced cell viability and induced apoptosis in a dose-dependent manner. Knockdown of K18/K8 or TG1/TG2 by siRNA significantly attenuated GCDCA-induced apoptosis, indicating their contributory roles in hepatocyte injury. GCDCA-treated cells showed increased levels of proteins crosslinked by TG1 and TG2. In vivo analysis using cholestatic model mice also showed elevated high-molecular-weight protein complexes involving K18/K8, suggesting early-stage Mallory body formation, as observed in chronic liver injury. Mass spectrometry identified cytoskeletal proteins, such as vimentin and periplakin, and regulatory proteins, such as ATP synthase subunit β and PI3K adapter protein, as K18-crosslinked partners. These results suggest that TG1/TG2-mediated aggregation of K18 sequesters essential structural and survival proteins, promoting hepatocyte apoptosis. Targeting these pathological interactions may provide a novel therapeutic strategy to mitigate liver fibrosis and improve hepatocyte survival.