Induction of cross-linking and silencing of Sp1 by transglutaminase during liver injury in ASH and NASH via different ER stress pathways

Alcoholic steatohepatitis (ASH) and non-alcoholic steatohepatitis (NASH) share many histological similarities, but the molecular mechanisms responsible for hepatic apoptosis remain unclear. We previously reported that transglutaminase 2 (TG2), a protein cross-linking enzyme, is induced in the nucleus of ethanol-treated hepatocytes, and cross-links and inactivates a general transcription factor Sp1, which eventually leads to reduced expression of c-Met and caspase-independent hepatic apoptosis [Tatsukawa et al., Gastroenterology 2009;136:1783-1795]. In this study, we investigated if a similar change might be observed also in NASH and if yes how TG2 and cross-linked Sp1 (CLSp1) would be induced in NASH and ASH. We obtained elevated nuclear TG2 and CLSp1 formation in NASH patients, as well as in HepG2 cells treated with free fatty acids (FFAs). Biochemical analyses on this culture model revealed that both ethanol and FFAs provoked fat accumulation, endoplasmic reticulum (ER) stress, increased nuclear factor-κB (NFκB) and nuclear TG2, but the synergistic effect was not obvious between FFA and ethanol. Salubrinal, a selective inhibitor against dephosphorylation of eukaryotic initiation factor-2α in ER stress-induced pancreatic ER kinase (PERK) signal pathway, inhibited NFκB activation, nuclear TG2 expression and apoptosis only induced by FFAs, but not those induced by ethanol, while retinoid antagonist blocks ethanol induction of NFκB and TG2. These results suggest that FFA and ethanol may increase ER stress and lead to nuclear NFκB activation and TG2 induction through respectively distinctive pathways, leading to TG2-mediated apoptosis via cross-linking and inactivation of Sp1 and reduction in c-Met.