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 Fechm1Pas 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, that 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 ATP content, and Nrf2 (redox sensitive transcription factor) up-regulation. Nrf2 knockdown in HepG2 cells down-regulated K8, but not K18. Conclusions 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.

Lamin aggregation is an early sensor of porphyria-induced liver injury

“…1A). To determine if the lamin aggregation is drug-specific or if it can be similarly observed in a genetic model of spontaneous MDB formation that is also associated with porphyria (Singla et al, 2012), we isolated the nuclear fractions from the Fech m1Pas mice [which harbor a mutation in the ferrochelatase (fch) gene] (Tutois et al, 1991). We found prominent formation of lamin high MW complexes in homozygous (fch/fch) mice as compared to wild-type (wt/wt) and heterozygous (wt/fch) mice (Fig.…”

“…Given that MDB formation in fch/fch mice occurs progressively as the mice age (Singla et al, 2012), we examined the time course of lamin aggregation relative to keratin aggregation. Notably, fch/fch (2/2) livers accumulates lamin-containing high MW complexes as early as 1 month of age, at a time when keratin aggregates are not detectable (Fig.…”

“…Several lines of evidence support the increase in oxidative load in the presence of porphyria. For example, the porphyria that is induced by DDC feeding or in the context of Fch mutation is associated with enhanced protein oxidation as measured biochemically (Hanada et al, 2010;Singla et al, 2012), altered bioenergetics with decreased liver ATP levels (Singla et al, 2012), and increased levels of hepatocyte reactive oxygen species (ROS) (Snider et al, 2011). In addition, PPIX increases intracellular H 2 O 2 levels as noted in cultured HepG2 cells (Koningsberger et al, 1995).…”

“…Age and gender matched control mice were fed a standard mouse diet. Fech m1Pas mice (Balb/c background) were also obtained from Jackson Laboratories and were described previously (Singla et al, 2012). Mice were euthanized by CO 2 inhalation and livers were harvested and snap-frozen.…”

Cellular and Gene Therapy for Erythropoietic Porphyrias

“…8 PPIX is highly cytotoxic and induces tissue damage triggered by light exposure through free radical formation. 9 EPP usually presents in early childhood, but in rare cases, symptoms manifest in adulthood. 10 The main clinical manifestation is acute skin photosensitivity after sun exposure, inducing pruritus, erythema, swelling and pain.…”