Oxidative stress, Nrf2 and keratin up-regulation associate with Mallory-Denk body formation in mouse erythropoietic protoporphyria

Abstract

Mallory-Denk bodies (MDBs) are hepatocyte inclusions commonly seen in steatohepatitis. They are induced in mice by feeding 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) for 12 weeks, which also causes porphyrin accumulation. Erythropoietic protoporphyria (EPP) is caused by mutations in ferrochelatase (fch), and a fraction of EPP patients develop liver disease that is phenocopied in Fech(m1Pas) mutant (fch/fch) mice, which have an inactivating fch mutation. fch/fch mice develop spontaneous MDBs, but the molecular factors involved in their formation and whether they relate to DDC-induced MDBs are unknown. We tested the hypothesis that fch mutation creates a molecular milieu that mimics experimental drug-induced MDBs. In 13- and 20-week-old fch/fch mice, serum alkaline phosphatase, alanine aminotransferase, and bile acids were increased. The 13-week-old fch/fch mice did not develop histologically evident MDBs but manifested biochemical alterations required for MDB formation, including increased transglutaminase-2 and keratin overexpression, with a greater keratin 8 (K8)-to-keratin 18 (K18) ratio, which are critical for drug-induced MDB formation. In 20-week-old fch/fch mice, spontaneous MDBs were readily detected histologically and biochemically. Short-term (3-week) DDC feeding markedly induced MDB formation in 20-week-old fch/fch mice. Under basal conditions, old fch/fch mice had significant alterations in mitochondrial oxidative-stress markers, including increased protein oxidation, decreased proteasomal activity, reduced adenosine triphosphate content, and Nrf2 (redox sensitive transcription factor) up-regulation. Nrf2 knockdown in HepG2 cells down-regulated K8, but not K18.

Conclusion: Fch/fch mice develop age-associated spontaneous MDBs, with a marked propensity for rapid MDB formation upon exposure to DDC, and therefore provide a genetic model for MDB formation. Inclusion formation in the fch/fch mice involves oxidative stress which, together with Nrf2-mediated increase in K8, promotes MDB formation.