Oxidative stress, Nrf2 and keratin upregulation 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, 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.

INTRODUCTION

Erythropoietic protoporphyria (EPP) is an inherited disorder caused by mutations in the ferrochelatase (Fch) gene (1, 2). More than 40 molecular defects have been described in Fch gene in EPP patients (3). Mitochondrial ferrochelatase catalyzes the insertion of ferrous iron into protoporphyrin IX (PP-IX), thereby regulating heme biosynthesis (1). Reduced ferrochelatase activity in EPP causes excessive accumulation of PP-IX in RBCs, skin and liver (4). The disease is characterized by cutaneous photosensitivity as PP-IX becomes phototoxic upon light exposure (4). Approximately 20% of patients exhibit hepatic manifestations, and 5–10% progress to end-stage liver disease (4). Genetic background has been suggested as a key determinant in the variable clinical symptoms in EPP (3).

The Fech^m1Pas mutant Balb/c mice (fch/fch) were previously reported by others (5). These mice harbor a point mutation in the ferrochelatase gene (resulting in 95% enzymatic activity loss) and suffer from phototoxicity, hemolytic anemia and severe hepatic dysfunction (5). They have elevated levels of serum transaminases, bilirubin and hyperlipidemia (6). Fech^m1Pas mice develop biliary and parenchymal hepatic injury as evidenced by the presence of hepatocyte ballooning, acidophil bodies, necrosis and Mallory-Denk bodies (MDBs) (7). MDBs are markers of hepatocellular injury and are seen after feeding mice for 12 or more weeks with the porphyrinogenic compounds, griseofulvin or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) (8). Interestingly, MDB formation has not been described in EPP patients and the characterization and mechanism of MDB formation in the fch/fch mice has not been addressed.

MDBs are cytoplasmic hyaline inclusions that associate with various liver disorders, including alcoholic and nonalcoholic steatohepatitis (8). In context of hepatitis C virus infection, MDB presence associates with poor prognosis (9). Biochemically, MDBs are composed primarily of the intermediate filament proteins keratins 8 and 18 (K8/K18), ubiquitin (Ub) and the ubiquitin-binding protein p62 (8). The major molecular processes involved in MDB formation include induction of K8/K18 expression with an increase in K8-to-K18 ratio, transglutaminase-2 (TG2) induction, resulting in transamidation and keratin crosslinking, and increased p62 levels (8). TG2 induction concomitant with an increase in K8-to-K18 ratio leaves excess K8 that is unbound to its obligate binding partner K18, which is significant because K8 is a superior TG2 substrate (compared with K18) and TG2-null mice do not form DDC-induced MDBs (10).

Oxidative stress strongly associates with DDC-induced MDB formation in FVB/N mice (male>female) due to differences in DDC metabolite formation (11). Similarly, C3H mice form far fewer MDBs, as compared with C57BL mice, due to their ability to maintain levels and subcellular distribution of the protective enzymes glyceraldehyde-3-phosphate-dehydrogenase and nucleoside-diphosphate-kinase under oxidative stress (12). Additionally, S-adenosyl methionine prevents the formation of MDBs in DDC-primed mice by, in part, normalizing an otherwise compromised proteasomal activity (13). Expression-profile analysis of fch/fch mouse livers showed major alterations in several oxidative stress-response genes including glutathione-S-transferase, superoxide dismutase, and nuclear factor E2 derived 2-like-2 (Nrf2) (6). Further, exogenous treatment of HepG2 cells with protoporphyrin increases intracellular H₂O₂, thereby contributing to oxidative stress (14).

We hypothesized that fch mutation creates a molecular milieu that mimics experimental drug-induced MDBs. Upon testing this hypothesis we delineated the genetic susceptibility of Fech^m1Pas mice to spontaneous and drug-induced MDB formation using various biochemical and molecular approaches. Our results support the importance of oxidative stress and Nrf2 in both spontaneous and DDC-induced MDB formation in Fech^m1Pas mice.

MATERIALS AND METHODS

Antibodies used

K8 (Troma-I) (Developmental-Studies-Hybridoma-Bank); Glutathione-S-transferase-M-1 (GSTM1), Ub and Nrf2 (Santa Cruz Biotechnology); mouse/human K8 and K18 (antibody-8592); mouse/human K18 (antibody-4668); p62 (Sigma-Aldrich); TG2, β-tubulin, hK8 (TS1) and hK18 (DC10) (Thermo Scientific).

Animal experiments

All animal studies were approved by the Animal Use and Care Committee (University of Michigan). Fech^m1Pas mice (Balb/c background; Jackson Laboratories) were screened using a PCR-based protocol. 13 and 20-week old mice were used to study spontaneous MDB formation. MDB induction in 20-week-old mice was done by feeding with LabDiet 5001 (PMI Nutrition International) containing 0.1% DDC (Sigma-Aldrich) (3-weeks). Age-matched controls were fed a standard mouse diet. FVB mice were also fed 0.1% DDC (2–10d) to examine oxidative stress and proteasome inhibition. Mice were euthanized by CO₂ inhalation, and intracardiac blood was collected for measurement of alkaline phosphatase (ALP), alanine aminotransaminase (ALT) and bile acids (BA) [using the Vetscan-vs2 instrument (Abaxis)]. Livers were harvested and apportioned for: hematoxylin-eosin (HE) staining, biochemical analysis and immunofluorescence staining.

RNA extraction and quantitative real time PCR

Total RNA was extracted utilizing Qiagen RNeasy kits. TaqMan Reverse Transcriptase kit (Applied Biosystems) was used to translate RNA into cDNA which was subjected to real time PCR (MyiQ real time PCR detection system, Biorad) and amplified by Brilliant SYBR green master mix utilizing gene specific primers (Supplemental Table 1).

Total lysates, high salt extracts (HSE) preparation and immunoblotting

For total lysates livers were homogenized in SDS-containing sample buffer. HSE were prepared as described (15) to obtain keratin-enriched fractions. Equal protein amounts were loaded (4–20% gradient SDS-polyacrylamide gels) and stained by Coomassie blue or transferred to polyvinylidene-difluoride membranes and immunoblotted using relevant antibodies.

Histological and immunofluorescence studies

HE-stained liver sections were assessed for MDBs. For immunofluorescence studies, liver sections were fixed in acetone, air-dried and stained as described (16). MDBs were counted using 40X lens, and 7 fields were examined per liver specimen.

Mitochondria isolation, protein oxidation assay and ATP content

Liver mitochondria were isolated as described (17). Mitochondrial lysates were analyzed for oxidized proteins using an OxyBlot Protein Oxidation Detection Kit (Millipore). DNP signals were quantified using ImageJ software and expressed as relative intensity values (mean±SEM). Mitochondrial ATP content was measured using ATP determination kit (Invitrogen).

Nuclear extracts and 20S proteasome activity

Nuclear extracts were prepared using nuclear and cytoplasmic extraction reagent kit (Pierce Biotechnology). Extracts were immunoblotted with anti-Nrf2 antibody. Liver 20S proteasomal activity was determined using 20S proteasome assay kit (Cayman Chemical).

Cell culture and transfections

HepG2 hepatoblastoma cells were transfected with control or Nrf2 siRNA (Santa Cruz Biotechnology) using Lipofectamine RNAiMax (Invitrogen) (48h) then used to prepare RNA extracts and protein lysates.

Statistical Analysis

One way ANOVA with Dunnett’s post test or unpaired t-test was used for statistical analysis. Differences were considered significant at P<0.05.

RESULTS

Fech^m1Pas mice exhibit spontaneous MDB formation

The presence of MDBs was previously reported in a mouse model of EPP (7), although the underlying mechanisms are unknown. We carried out detailed biochemical, serologic and histological analyses of the livers of these animals. Initial assessment of 20-week old fch/fch mice and their wild-type (wt/wt) and heterozygous (wt/fch) controls indicated no body weight differences, while the percent liver-to-body weight ratio was 1.7–1.8-fold higher in fch/fch mice relative to wt/wt and wt/fch mice (Table S2). Serological analysis of the 20-week old fch/fch mice revealed increased ALP, ALT and BA levels (Fig. 1A), in agreement with previously reported hepatobiliary injury in fch/fch mice (6, 18).

Fig. 1. Serum and biochemical analysis of livers of 20-week old mice.

(A) Serum was collected from 20-week old wt/wt, wt/fch and fch/fch mice and used to measure ALT (U/L), ALP (U/L) and BA (μmol/L) levels. N=3/group and *p<0.05 compared to wt/wt. (B) K8 and K18 mRNA levels in indicated genotypes were quantified using real time PCR. The ratio of K8 to K18 mRNA in fch/fch is 1.94. *p<0.05 compared to wt/wt. (C) Top panel (a) Coomassie stain for HSEs showing increased K8 and K18 expression in fch/fch mice and (b) Immunoblots for K8, ubiquitin and p62 of the HSEs (prepared using 100 mg of liver tissue, equal fractions were loaded). (c) Total liver lysates were used to examine TG2 levels (β-tubulin as a loading control). Each lane corresponds to a separate liver fraction. (D) Quantification of K8/K18 protein bands (from Panel Ca) by densitometric scanning using ImageJ software.

Given that an essential component of drug-induced MDB formation is an upregulation of keratins with a K8>K18 ratio and formation of high molecular weight K8-containing crosslinked species, we examined if the same features occur in the genetic fch model. As shown in Fig. 1B, K8 and K18 mRNA levels were increased in fch/fch mice with a K8-to-K18 ratio of 1.94. Biochemical analysis of fch/fch livers revealed increased K8/K18 expression (per mg of liver tissue) as shown by Coomassie staining of the HSE in Fig. 1C. Immunoblotting showed the presence of K8-ubiquitin high-molecular-weight complexes and increased expression of p62 and TG2 in fch/fch mice (Fig. 1C). These data provide biochemical support for spontaneous MDB formation in 20-week old fch/fch mice. Densitometric analysis showed ~2-fold increase in K8/K18 protein in fch/fch mice (Fig. 1D).

Susceptibility of younger Fech^m1Pas mice (13-week old) to MDB formation was also examined. Similar to the 20-week old mice, 13-week old fch/fch mice showed a 1.8–1.9 higher percent liver/body weight (Table S2), and had elevated levels of ALP, ALT and BA (Fig. S1A). 13-week old mice showed K8 and K18 mRNA induction, with a K8:K18 ratio of 1.25 (Fig. S1B) that is less than the 1.94 ratio seen in the 20-week old fch/fch mice (Fig. 1B). K8, K18 and TG2 protein levels were increased in 13-week old fch/fch mice (Fig. S1C). These mice exhibited K8 dimer formation in the absence of K8 high molecular weight complexes.

MDB formation in Fech^m1Pas mice is age-dependent and gender-independent

The above data suggest that young mice begin to show some of the initiating events of MDB formation (increased keratins/TG2) but have not yet progressed to mature MDBs that can be visualized histologically, since HE staining revealed the presence of MDBs in 20-week (Fig. 2A, panels c,d), but not in 13-week-old, fch/fch mice (Fig. S2, panels c,d)). This was further confirmed biochemically by the limited formation of high-molecular-weight K8-containing complexes and lack of induction of p62, another key constituent of MDBs (Fig. 2B). Immunofluorescence staining revealed prominent keratin and ubiquitin co-localized deposits in 20-week old (Fig. 3, panels c,d) but not in 13-week old fch/fch livers (Fig. S3), supporting our histological and biochemical findings. Keratin and ubiquitin staining for 20-week old livers is shown in Fig. S4. Taken together, these data indicate that MDB formation in Fech^m1Pas mice occurs in an age-dependent manner.

Fig. 2. fch/fch mice exhibit age dependent and gender independent formation of MDBs.

(A) HE staining of livers from 20-week old animals. Panel c represents the image taken using a 40X objective, whereas panel d is 100X image of 20-week old fch/fch livers. Black arrows (panels c,d) highlight the MDBs in the 20-week old fch/fch mice (scale bar = 50 μm). (B) K8 crosslinking was visualized by immunoblotting HSEs from 13 and 20-week old fch/fch mice. (C) HSEs from 20-week old male and female mice were immunoblotted for K8. For panel B, each lane corresponds to separate liver fractions.

Fig. 3. Analysis of MDB formation using immunofluorescence staining.

Liver sections from 20-week old mice of the indicated genotypes were double stained using K8/K18 (red) and Ub (green) antibodies. Nuclei are shown in blue. MDBs (yellow dots due to co-localization of two epitopes) were noted using 40X lens in the fch/fch livers and are highlighted by white arrows (panel c). Panel d represents the higher magnification of the boxed area in panel c. Scale bar=20 μm.

Given that male FVB/N mice are more susceptible to drug-induced MDB formation (11), potential gender differences in spontaneous MDB formation were analyzed. Notably, Fech^m1Pas mice (Balb/c background) did not show a gender-based (M>F) predilection to MDB formation which was confirmed using biochemical (Fig. 2C) and histological (not shown) means.

Fech^m1Pas mice have increased susceptibility to drug-induced MDBs

The possibility that the underlying genetic predisposition of spontaneous MDBs renders Fech^m1Pas mice more susceptible to drug-induced MDBs was tested using a modified, 3-week DDC feeding protocol, which is significantly shorter than the standard 12–16 weeks (8). Short-term DDC feeding markedly induced K8/K18 mRNA in all three genotypes relative to their non-DDC-fed controls (Fig. 4A). Biochemically, wt/fch mice respond by a dramatic upregulation of K8 and K18 (compare Fig. 4A, lanes 2+3 with 5+6), which parallels increased K8 crosslinking (Fig. 4B). There was a significant increase in MDB numbers in wt/fch mice (13.2±0.4) after DDC feeding, while MDB numbers was 16.7±1.9 in control fch/fch mice and 19.2±2.4 in DDC-fed fch/fch mice. There was also an increase in TG2 levels in DDC-fed mice (Fig. 4B). Histological analysis confirmed the increase in DDC-induced MDBs in wt/fch and fch/fch mice (Fig. 5). These data indicate that the Fech^m1Pas mice have a markedly enhanced propensity to form drug-induced MDBs.

Fig. 4. Short-term DDC feeding of fch/fch mice induces prominent MDB formation.

20-week old wt/wt, wt/fch and fch/fch mice were DDC-fed for 3 weeks. (A) RNA (extracted from the control and DDC livers) was subjected to real time PCR to examine K8 and K18 mRNA levels. N=3, *p<0.05 vs. wt/wt. (B) HSEs from the control and DDC livers were (a) stained by Coomassie blue or (b) immunoblotted using antibodies to K8 or TG2. Each lane corresponds to a separate liver fraction.

Fig. 5. HE staining of liver sections from control and DDC-fed fch/fch and wt/fch mice.

HE staining was performed to examine the MDBs in DDC-fed livers. MDBs are marked using black arrows (scale bar=50 μm). Panels a–c are 40X images and d–f are the 100X images.

Mitochondrial oxidative stress is altered in Fech^m1Pas mice

Prior expression-profile analysis of livers from Fech^m1Pas mice revealed alterations in biological pathway genes involving heme metabolism, inflammation and oxidative stress (6). Since mitochondria play an important role in reactive oxygen species generation, we examined several mitochondrial oxidative stress markers. fch/fch livers had increased protein oxidized by-products relative to wt/wt and wt/fch livers (Fig. 6A). Additionally, glutathione-S-transferase-mu-1 [GSTM1, induced during oxidative stress (19)] was markedly enhanced under basal conditions in the fch/fch livers (Fig. 6B, compare lanes 9+10 with 1+2 or 5+6).

Fig. 6. Comparison of oxidative stress markers in livers of 20-week old fch/fch versus controls.

(A) Protein oxidation was measured as described in Methods. (B) Lysates of livers from the indicated genotypes (DDC-fed or normal chow) were blotted with GSTM1 antibody. For panels A and B, each of the lanes corresponds to liver preparations from independent mice. (C) Mitochondrial ATP content was measured in the livers isolated from control and DDC-fed mice. (D) 20S proteasomal activity was measured in liver homogenates from control and DDC-treated mice. N=3, *p<0.05 compared vs wt/wt.

Oxidative damage to mitochondria can lead to injury through ATP. Notably, there was a significant decrease in ATP content in fch/fch livers relative to wt/wt and wt/fch livers, and DDC feeding led to further reduction of ATP, particularly in wt/fch and fch/fch livers (Fig. 6C). Given that increased oxidative stress has been shown to decrease proteasomal activity, which is associated with MDB formation (13, 20), we tested and observed a 2-fold inhibition of the 20S proteasomal activity in the fch/fch mice and also in the DDC fed wt/wt, wt/fch and fch/fch mice (Fig. 6D). These results suggest that oxidative stress in fch/fch mice decreases proteasomal activity.

Fech^m1Pas mice show increased expression of Nrf2

Nuclear factor E2 derived 2-like-2 (Nrf2) is a redox sensitive transcription factor expressed in the liver (21). Nrf2 regulates multiple antioxidant pathways (21) and its mRNA is up-regulated based on un-validated microarray studies in Fech^m1Pas mice (6). Therefore, mRNA levels and nuclear protein expression of Nrf2 in control and DDC-fed wt/wt, wt/fch and fch/fch livers were analyzed. There was increased Nrf2 liver mRNA expression (~3 fold) in fch/fch relative to wt/wt mice (Fig. 7A). Short-term DDC feeding induced liver Nrf2 mRNA levels in all mice. Consistent with the mRNA findings, control and DDC-fed fch/fch mice had significant induction in nuclear Nrf2 protein (Fig. 7B). These results indicate Nrf2 up-regulation in control and DDC-fed fch/fch mice, likely in response to oxidative-stress. To further examine the sequence of events, FVB mice were fed DDC for 2–10d. Analysis of the DDC-fed livers showed an increase in Nrf2 mRNA and protein expression after 5d of DDC feeding (Fig. S5A,B). However, there was no inhibition in proteasome activity in 2d and 5d DDC fed livers (Fig. S5C), suggesting that there is another mechanism involved in increasing Nrf2 expression. Further, we examined the mitochondrial oxidized proteins, and Oxyblot analysis (Fig. S5D) showed an increase in oxidized protein by-products in 5d DDC-fed mice suggesting that oxidative stress is upstream of proteasome inhibition. Nrf2 protein levels were greatly stabilized in M132-treated HepG2 cells, suggesting that proteasome inhibition also help stabilize Nrf2 protein (Fig. S5E).

Fig. 7. K8 is a target gene for Nrf2.

(A) RNA was isolated from the livers of control and DDC-fed mice was quantified for Nrf2 mRNA. (B) Immunoblot analysis (using anti-Nrf2 antibody) of nuclear extracts from control and DDC-fed livers (Coomassie stain is included as a loading control). Representative blot of 3 independent experiments is shown, and quantification was done form the 3 experimetns (*p<0.05 vs wt/wt). (C) HepG2 cells were transfected with control and Nrf2 siRNA. Real time PCR was utilized to examine K8 and K18 mRNA levels. (D) Total lysates were immunoblotted using K8, K18 and Nrf2 antibodies. β-actin served as a loading control. For all the experiments, N=3, *p<0.05.

Nrf2 regulates K8 but not K18 expression

Nrf2 was shown previously to regulate keratin-16 (K16) gene expression in human keratinocytes (22). Therefore, we examined the effect of Nrf2 on K8 and K18 expression in HepG2 cells using siRNA knockdown. Nrf2 siRNA but not control siRNA significantly inhibited K8 but not K18 mRNA and protein expression (Fig. 7C,D). These findings suggest that the K8 gene is a target for Nrf2.

DISCUSSION

Relevance of MDBs to EPP in humans and mouse models of EPP

Porphyrias are inherited metabolic disorders that occur due to defects in the heme biosynthetic pathway, and are divided into acute and non-acute porphyrias (23). EPP is a non-acute porphyria caused by mutation in the Fch gene. Two mouse models of EPP have been described to date. The first model was generated by an Fch exon deletion which led to embryo-lethality of the homozygous mice, while heterozygous mice developed mild protoporphyria without liver disease (24). The second model (Fech^m1Pas) was generated by inducing a point mutation in the Fch gene (5), which has been a valuable animal model for EPP since it mimics the most severe hepatic forms of the human disease.

The presence of MDBs has been described in the Fech^m1Pas mouse strain and also in the griseofulvin-induced mouse model of EPP (7, 25). However, MDBs have not been reported in EPP patients. It should be noted that MDBs are visualized in clinical contexts using histological approaches (e.g., HE staining), which lack sensitivity for detecting small and early MDBs (16). It is possible that some patients with EPP may form MDBs if sensitive methods are employed for their detection, and if indeed found then the genetic background of an individual may be a contributing factor. For example, Hispanics appear to be more predisposed to MDB formation (9, 26), and clear differences in MDB susceptibility are also noted in different mouse strains (12, 16). Other potential factors that may contribute to the apparent absence of MDBs in EPP patients (versus the animal models) may be related to differences in porphyrin metabolites across species. In this context, the contribution of porphyria per se to MDB formation is unclear. Certainly, porphyrin accumulation leads to oxidative stress (14) though other factors are likely to be important. The range of accumulation of protoporphyrin-IX (PP-IX) in DDC-fed male (~550 nmol/g protein) and female (~1900 nmol/g protein) mice (11) is similar to the porphyria in the fch/fch mice [1000–5000 nmol/g protein (6, 27)]. By comparison, patients with EPP have PP-IX levels in the range of 3000–8000nmol/g tissue (1), though the genetic modifiers that result in severe human liver disease in the 5–10% of affected EPP patients are not known.

fch/fch mice manifest spontaneous MDB accumulation and enhanced susceptibility to drug-induced MDB formation

Our findings herein characterized the formation of spontaneous and DDC-induced MDBs in fch/fch mice and showed that MDBs formation in these mice is age dependent but gender independent. The lack of gender-dependency in this model, in contrast to the FVB/N drug-induced model may be due to genetic mouse strain differences (Balb/c versus FVB/N) or may be related to the increased porphyria that might overcome genetic gender-related influences.

The enhanced susceptibility to drug-induced MDB formation, even in the heterozygous wt/fch mice, renders these mice very useful for studying MDBs given that MDBs can be induced in these mice within 3 weeks as compared with the standard 12–16 week DDC regimen. Hence, these mice are even more susceptible to MDB formation when compared to another genetic model, which is mice that overexpress K8 (which develop MDBs within 6 weeks of DDC exposure (28). The spontaneous formation of MDBs in fch/fch mice is also reminiscent of their formation in the K8-overexpressor mice and in the K18-null mice upon aging (both have a K8>K18 ratio) (28, 29).

Oxidative stress, proteasome function and Nrf2 in MDB formation in fch/fch mice

Oxidative stress plays a major role in several protein aggregation disorders (30, 31). It is also involved in MDB formation in FVB/N and K8-overexpressing mice in response to DDC feeding (11). Our findings demonstrate a role for oxidative-stress in MDB formation in fch/fch mice, and support previously-reported alterations in oxidative-stress genes in fch/fch mice and in EPP patients utilizing microarray analysis (3, 6). In addition, griseofulvin-induced EPP in mice results in ultra-structural changes in liver mitochondria, accumulation of large amounts of protoporphyrin and low-energy coupling in mitochondria which suggest blunting of mitochondrial function (32). Moreover, treatment of HepG2 cells with protoporphyrin in the absence of light increases intracellular H₂O₂ levels which supports the role of protoporphyrin in causing oxidative stress (14). Our data are consistent with the presence of mitochondrial oxidative stress in the fch/fch livers as demonstrated by decreased mitochondrial ATP and increased liver oxidized proteins (Fig. 6, Fig. S5), possibly due to accumulation of protoporphyrin deposits.

Time course of DDC feeding to FVB animals demonstrated that oxidative stress is upstream of proteasome inhibition and correlates with the increase Nrf2 expression which becomes further magnified upon proteasome inhibition. Therefore, the observed 50% decrease in proteasome function in fch/fch mice under basal conditions are likely intimately linked to the increased oxidative milieu in the livers of these animals. Reduction in mitochondrial ATP content impairs proteasomal activity which in turn leads to mitochondrial dysfunction in various diseased states (20). Furthermore, proteasome inhibition in vivo accelerates MDB formation from 6 weeks to 2 weeks in K8-overexpressing mice (33), and a decrease in proteasome activity became normalized upon S-adenosylmethionine administration to DDC-fed mice in a short-term re-challenge model (13).

Nrf2 is a redox-sensitive transcription factor that protects cells from oxidative stress (21). It is sequestered in the cytoplasm by Keap-1 (Kelch-like ECH associating protein 1) under non-stress conditions and is targeted for ubiquitination and proteasomal degradation (21). During stress, Nrf2 escapes Keap-1 mediated repression and translocates to the nucleus to up-regulate various genes such as glutathione-S-transferases and heme oxygenase-1 (21). Notably, the phytochemical sulforaphane, which activates Nrf2, up-regulates epidermal keratins K16 and K17 (34) and Nrf2 up-regulates K16 in human keratinocytes (22). Furthermore, autophagy deficiency and upregulation of p62 are known to stabilize Nrf2 resulting in activation of Nrf2 target genes (35). Our findings showed a decrease of K8, but not K18, mRNA and protein upon Nrf2 knockdown in HepG2 cells, and also showed Nrf2 nuclear accumulation in fch/fch mice under basal conditions and in fch/fch, wt/fch and wt/wt livers after exposure to DDC (Fig. 7). Furthermore, sequence analysis of the 5′-proximal promoter region of K8 and K18 revealed the presence of Nrf2 consensus binding sequence (not shown). This was consistent with the study showing the Nrf2 binding sites on K16 promoter region (22). Based on the findings herein, and the established importance of K8 in MDB formation (8) and of PPIX in oxidative stress (14), we propose a model (Fig. 8) whereby accumulation of PPIX leads to increased mitochondrial oxidative stress which inhibits the Keap-1-mediated Nrf2 degradation thereby resulting in translocation of Nrf2 to nucleus and upregulation of K8 and other target genes.

Fig. 8. Schematic of the crosstalk between oxidative stress and MDB formation in the context of Fech mutation.

A defect in ferrochelatase leads to accumulation of protoporphyrin-IX (PP-IX) in liver mitochondria, which results in increased mitochondrial oxidative stress and reduced ATP content. The increase in oxidative stress leads to proteasome inhibition, decreased Nrf2 degradation, with subsequent nuclear translocation of Nrf2. Nuclear Nrf2 then binds to the antioxidant response element (ARE) of the K8 gene and increases K8 mRNA, among additional transcriptional regulatory events. The increase in K8, TG2 and p62 promotes MDB formation.

Supplementary Material

Supp Fig S1. Fig. S1: Serum and biochemical analysis of livers of 13-week old mice.

(A) Serum from 13-week old mice of the indicated genotypes were used to measure ALT, ALP and BA levels (N=3, *p<0.05 compared to wt/wt). (B) RNA isolated from livers of 13-week old mice was used to quantify K8 and K18 mRNA levels. The ratio of K8 to K18 mRNA is 1.25 in fch/fch mice. *p<0.05 compared to wt/wt. (C) Top panel (a) Coomassie stain for the HSEs (b) Immunoblot analysis of HSEs for K8 and ubiquitin. Ub crosslinks that appear at the top of the gel are highlighted by brackets. (c) total lysates were used to examine TG2 levels (β-tubulin is shown as a loading control).

Supp Fig S2. Fig. S2: HE staining of 13-week old liver sections.

Liver sections from 13-week old mice were HE stained to examine the MDBs. No MDBs were noted in the 13-week old fch/fch mice. Scale bar = 50 μm

Supp Fig S3. Fig. S3: Immunofluorescence staining of 13-week old mouse liver sections.

Liver sections from 13-week old mice were double-stained with K8/K18 (red) and ubiquitin (green) antibodies. No MDBs (yellow dots) were noted in the 13-week old fch/fch mice. Scale bar = 20 μm

Supp Fig S4. Fig. S4: Immunofluorescence staining of 20-week old mouse liver sections.

Keratins (red), ubiquitin (green) and nuclei (blue) staining of liver sections from 20-week old mice. Scale bar = 20 μm

Supp Fig S5. Fig. S5: Oxidative stress is upstream of proteasomal inhibition.

FVB animals were fed 0.1% DDC for 2, 5 and 10 days. (A) Quantification of Nrf2 mRNA expression in control and DDC-treated livers. N=3, *p<0.05. (B) Immunoblot analysis of the nuclear extracts from control and DDC-treated livers using anti-Nrf2 antibody. Coomassie stain of the nuclear extracts serve as loading control. (C) 20S proteasomal activity was measured in the liver lysates. N=3, *p < 0.05. (D) Mitochondrial protein oxidation was measured and relative intensity of bands was quantified using ImageJ software. (E) Nrf2 levels are increased in MG132-treated HepG2 cells.

Abbreviations

EPP

erythropoietic protoporphyria

Fch

ferrochelatase; fch/fch

PPIX

protoporphyrin IX

RBCs

red blood cells; Fech^m1Pas mutant mice

DDC

3,5-diethoxycarbonyl-1,4-dihydrocollidine

K18

keratin 18

K8

keratin 8; ALP

Ub

ubiquitin

TG2

transglutaminase-2

Nrf2

nuclear factor E2 derived 2-like 2

GSTM1

glutathione S-transferase M1; alkaline phosphatase

ALT

alanine aminotransferase

BA

bile acids

HE

hematoxylin-eosin

HSE

high salt extracts

Keap-1

Kelch-like ECH associating protein 1