Loss of β-catenin reveals a role for glutathione in regulating oxidative stress during cholestatic liver disease

Abstract

Background: Cholestasis is an intractable liver disorder that results from impaired bile flow. We have previously shown that the Wnt/β-catenin signaling pathway regulates the progression of cholestatic liver disease through multiple mechanisms, including bile acid metabolism and hepatocyte proliferation. To further explore the impact of these functions during intrahepatic cholestasis, we exposed mice to a xenobiotic that causes selective biliary injury.

Methods: α-naphthylisothiocyanate (ANIT) was administered to liver-specific knockout (KO) of β-catenin and wild-type mice in the diet. Mice were killed at 6 or 14 days to assess the severity of cholestatic liver disease, measure the expression of target genes, and perform biochemical analyses.

Results: We found that the presence of β-catenin was protective against ANIT, as KO mice had a significantly lower survival rate than wild-type mice. Although serum markers of liver damage and total bile acid levels were similar between KO and wild-type mice, the KO had minor histological abnormalities, such as sinusoidal dilatation, concentric fibrosis around ducts, and decreased inflammation. Notably, both total glutathione levels and expression of glutathione-S-transferases, which catalyze the conjugation of ANIT to glutathione, were significantly decreased in KO after ANIT. Nuclear factor erythroid-derived 2-like 2, a master regulator of the antioxidant response, was activated in KO after ANIT as well as in a subset of patients with primary sclerosing cholangitis lacking activated β-catenin. Despite the activation of nuclear factor erythroid-derived 2-like 2, KO livers had increased lipid peroxidation and cell death, which likely contributed to mortality.

Conclusions: Loss of β-catenin leads to increased cellular injury and cell death during cholestasis through failure to neutralize oxidative stress, which may contribute to the pathology of this disease.

FIGURE 1

KO mice have decreased survival after ANIT diet but lack overt indicators of morbidity. (A) Kaplan-Meier survival curve shows significantly decreased survival in KO mice after exposure to ANIT diet. (B) There is a significant decrease in LW/BW ratio in KO mice after 6 days and 2 weeks of ANIT compared to WT. (C) WT mice have more bile infarcts (expressed as percent area of the image) after 6 days of ANIT compared to KO; however, by 14 days, these infarcts had resolved. KO had few to no infarcts at all time points. (D) Sinusoidal diameter was significantly increased in KO mice after 6 days of ANIT compared to WT mice. (E) H&E stains of liver sections show sinusoidal dilatation in KO livers 6 days after ANIT (magnification ×200). (F) Serum markers of hepatic and biliary injury were essentially unchanged between WT and KO at both time points after ANIT, although some parameters, such as ALT, AST, and bilirubin, were increased in KO over time. Data in B, C, and E represent mean±SD. *p<0.05, **p<0.01 by two-way ANOVA (multiple comparisons). Scale bars:100 μm. Data in A was analyzed by log-rank Mantel-Cox test and found to be significant with a p-value of 0.0005. Data in B was quantified from n≥8 200x fields from n=3 WT and KO at baseline and from 25 to 30 ×200 fields from at least n=4 WT and KO mice at both ANIT time points. Data in D were quantified from 12 to 15 sinusoids per field from 2 randomly selected liver sections per mouse (n=3 WT and KO mice at baseline and n=4 WT and KO mice at both ANIT time points). Inset images were taken from the central vein region for consistency. Abbreviations: ANIT, α-naphthylisothiocyanate; KO, knockout; LW/BW, liver weight to body weight; WT, wild type.

FIGURE 2

Increased mortality in KO is not a result of increased fibrosis, inflammation, or ductular response. (A) Sirius red staining shows the early development of sclerosing cholangitis 6 days after ANIT; quantification also shows more fibrosis in KO than in WT at this time point. However, by 2 weeks of ANIT, both WT and KO have equivalent fibrosis. (B) CD45 IHC demonstrates that while inflammation increases after ANIT, it is equivalent between WT and KO after 6 days of ANIT. CD45-positive cells are significantly decreased in KO compared to controls after 2 weeks of ANIT. (C) A6 IHC shows that ductular reaction is equivalent between WT and KO at both time points after ANIT. Notably, however, KO ducts are dilated at both time points and contain elongated ductular cells. Graphs in A–C represent mean±SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA (multiple comparisons). All magnifications are ×200. Scale bars:100 μm. Data were quantified from n≥8 ×200 fields from n=3 WT and KO at baseline and from 25 to 30 ×200 fields from at least n=4 WT and KO mice at both ANIT time points. Abbreviations: ANIT, α-naphthylisothiocyanate; IHC, immunohistochemistry; KO, knockout; WT, wild type.

FIGURE 3

KO has decreased bile acid export but equivalent total bile acid levels after ANIT. (A) Total bile acid levels are equivalent in WT and KO at both time points after ANIT. (B) Nr0b2 (Shp), Cyp7a1, and Cyp27 levels are not significantly different between WT and KO after 6 days of ANIT, as assessed by quantitative PCR. (C) The expression of detoxification enzyme Cyp2b10 is increased after 6 days of ANIT in both WT and KO but expressed equivalently in both; however, Cyp3a11, another detoxification enzyme, is significantly decreased in KO after ANIT. (D) WB shows that CYP3A11 protein expression is also decreased in KO mice after ANIT. (E) There is no change in the expression of uptake transporters Slc10a1 (Ntcp) and Slco1b2 (Oatp4) after ANIT in either WT or KO. (F) Apical transporter Abcc11 (Bsep) is unchanged in WT and KO after ANIT; however, Abcc2 (Mrp2) is significantly decreased in KO compared to WT both before and after ANIT treatment. (G) WB shows that MRP2 protein expression in KO is approximately half that of WT after 6 days of ANIT. (H) Basolateral transporter Abcc3 (Mrp3) is decreased in KO both before and after 6 days of ANIT, while Abcc4 (Mrp4) is equivalently expressed in both groups before and after treatment. Data represent mean±SD. *p<0.05, **p<0.01, ***p<0.001 by two-way ANOVA (multiple comparisons). For A–C, E, F, and H, each point on the graphs represents a biological replicate that is the average of duplicate technical replicates. Data in D and G represent individual data points from WB quantification. Abbreviations: ANIT, α-naphthylisothiocyanate; KO, knockout; Mrp, multidrug-resistance–associated protein; NQO1, NAD(P)H quinone dehydrogenase 1; WB, western blot; WT, wild type.

FIGURE 4

Expression of enzymes that facilitate GSH conjugation to xenobiotics are decreased in KO both before and after ANIT. Quantitative PCR for a panel of GSTs shows that 4 of 6 (GSTm1, GSTm2, GSTm6, GSTα3) are repressed in KO at baseline. GSTm1, GSTm2, GSTm3, and GSTm6 are also suppressed in KO after 6 days of ANIT, compared to WT. Data represent mean±SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA (multiple comparisons). Each point on the graphs represents a biological replicate that is the average of duplicate technical replicates. Abbreviations: ANIT, α-naphthylisothiocyanate; GST, glutathione-S-transferase; KO, knockout; WT, wild type.

FIGURE 5

Nrf2 is activated in ANIT-injured KO mice. (A) Total glutathione levels increase in WT after ANIT but remain the same in KO, leading to a significant difference between WT and KO. (B) β-catenin protein expression is decreased, and GS expression is absent in KO mice before and after ANIT. The trace amounts of β-catenin likely represent the population of nonparenchymal cells in the liver that are still β-catenin-positive. (C) IHC shows that KO livers have increased cytoplasmic and nuclear localization of Nrf2 in the periportal region after ANIT compared to WT. Magnification ×200. (D) Nrf2 target NQO1 is increased in KO compared to WT after 2 weeks of ANIT. For D, the same samples were run on 2 different WB and were standardized to either GAPDH (left) or total protein (right). Data in A represent mean±SD from duplicate technical replicates, averaged for each biological replicate. *p<0.05, **p<0.01 by two-way ANOVA (multiple comparisons). Scale bars:100 μm. Data in B and D represent individual data points from WB quantification. Abbreviations: ANIT, α-naphthylisothiocyanate; GS, glutamine synthetase; GSH, glutathione; IHC, immunohistochemistry; KO, knockout; Nrf2, nuclear factor erythroid-derived 2-like 2; NQO1, NAD(P)H quinone dehydrogenase 1; WB, western blot; WT, wild type.

FIGURE 6

Loss of β-catenin correlates with increased nuclear factor erythroid-derived 2-like 2 activation in explanted livers from patients with end-stage PSC. (A) Representative images of explanted livers from patients with PSC classified as either β-catenin inactive (absent or membranous only) or active (cytoplasmic and/or nuclear). Magnification ×200. (B) A contingency table classifying the data based on the status of β-catenin (inactive or active) shows that the majority of β-catenin inactive samples have moderate or strong levels of NQO1 staining. p=0.63628 by Fisher exact test. Scale bars:100 μm. Abbreviation: NQO1, NAD(P)H quinone dehydrogenase 1.

FIGURE 7

Nuclear factor erythroid-derived 2-like 2 activation in KO livers coincides with decreased NF-κB activation and increased oxidative stress. (A) Immunofluorescence for p65, a subunit of NF-κB, shows nuclear translocation in cholangiocytes and hepatocytes of WT but not KO after 2 weeks of ANIT. Arrowheads point to ducts with nuclear p65. In KO livers, p65 is only detected in the cytoplasm of cholangiocytes. (B) IHC shows more 4HNE, a product of lipid peroxidation, in KO livers than in WT after 6 days of ANIT. Staining is localized mainly around the vessels (central vein and periportal). For A, magnification ×400; for B, magnification ×200. Scale bars:100 μm. Abbreviations: ANIT, α-naphthylisothiocyanate; 4HNE, 4-hydroxynonenal; IHC, immunohistochemistry; KO, knockout; WT, wild type.

FIGURE 8

Loss of β-catenin in liver increases cell death and inhibits proliferation after ANIT. (A) Lipid peroxidation is insignificantly increased in KO compared to WT after 6 days of ANIT treatment. (B) KO have significantly more cleaved caspase 3–positive hepatocytes than WT at both 6 days and 14 days after ANIT. (C) WB shows that RIP3 is equivalent in WT and KO after 6 days of ANIT; however, protein expression decreases in WT but not in KO after 14 days of ANIT. (D) TUNEL staining shows levels of cell death in both WT and KO after 6 days of ANIT. By 2 weeks there is significantly more cell death in KO livers than in WT. (E) PCNA IHC shows that proliferation is significantly blunted in KO livers at both time points after ANIT diet, while WT livers show a robust regenerative response at 6 days that has returned to near-normal levels by 2 weeks of ANIT. Data in A–E represent mean±SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA (multiple comparisons). All magnifications are ×200. Scale bars:100 μm. For A, assay was performed in duplicate technical replicates and averaged for each biological replicate. For C, data represents individual data points from WB quantification. For D, data were quantified from n≥5 ×200 fields from n=3 WT and KO at baseline, and from n≥5 ×200 fields from n=4 WT and KO mice at both ANIT time points. For B and E, data were quantified from n≥3 ×200 fields from n=3 WT and KO at baseline, from n≥4 ×200 fields from n=3 WT and KO mice at 6 days ANIT, and from n≥4 ×200 fields from n=4 WT and n=2 KO mice at 14 days ANIT. Abbreviations: ANIT, α-naphthylisothiocyanate; IHC, immunohistochemistry; KO, knockout; MDA, malondialdehyde; NQO1, NAD(P)H quinone dehydrogenase 1; PCNA, proliferating cell nuclear antigen; RIP3, receptor-interacting kinase 3; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; WB, western blot; WT, wild type.