Boosting mitochondria activity by silencing MCJ overcomes cholestasis-induced liver injury

Abstract

Background & aims: Mitochondria are the major organelles for the formation of reactive oxygen species (ROS) in the cell, and mitochondrial dysfunction has been described as a key factor in the pathogenesis of cholestatic liver disease. The methylation-controlled J-protein (MCJ) is a mitochondrial protein that interacts with and represses the function of complex I of the electron transport chain. The relevance of MCJ in the pathology of cholestasis has not yet been explored.

Methods: We studied the relationship between MCJ and cholestasis-induced liver injury in liver biopsies from patients with chronic cholestatic liver diseases, and in livers and primary hepatocytes obtained from WT and MCJ-KO mice. Bile duct ligation (BDL) was used as an animal model of cholestasis, and primary hepatocytes were treated with toxic doses of bile acids. We evaluated the effect of MCJ silencing for the treatment of cholestasis-induced liver injury.

Results: Elevated levels of MCJ were detected in the liver tissue of patients with chronic cholestatic liver disease when compared with normal liver tissue. Likewise, in mouse models, the hepatic levels of MCJ were increased. After BDL, MCJ-KO animals showed significantly decreased inflammation and apoptosis. In an in vitro model of bile-acid induced toxicity, we observed that the loss of MCJ protected mouse primary hepatocytes from bile acid-induced mitochondrial ROS overproduction and ATP depletion, enabling higher cell viability. Finally, the in vivo inhibition of the MCJ expression, following BDL, showed reduced liver injury and a mitigation of the main cholestatic characteristics.

Conclusions: We demonstrated that MCJ is involved in the progression of cholestatic liver injury, and our results identified MCJ as a potential therapeutic target to mitigate the liver injury caused by cholestasis.

Lay summary: In this study, we examine the effect of mitochondrial respiratory chain inhibition by MCJ on bile acid-induced liver toxicity. The loss of MCJ protects hepatocytes against apoptosis, mitochondrial ROS overproduction, and ATP depletion as a result of bile acid toxicity. Our results identify MCJ as a potential therapeutic target to mitigate liver injury in cholestatic liver diseases.

Keywords: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AMA-M2, antimitochondrial M2 antibody; ANA, antinuclear antibodies; APRI, AST to platelet ratio index; AST, aspartate aminotransferase; Abs, antibodies; BA, bile acid; BAX, BCL2 associated X; BCL-2, B-cell lymphoma 2; BCL-Xl, B-cell lymphoma-extra large; BDL, bile duct ligation; Bile duct ligation; CLD, cholestatic liver disease; Ccl2, C-C motif chemokine ligand 2; Ccr2, C-C motif chemokine receptor 2; Ccr5, C-C motif chemokine receptor 5; Cholestasis; Cxcl1, C-X-C motif chemokine ligand 1; Cyp7α1, cholesterol 7 alpha-hydroxylase; DCA, deoxycholic acid; ETC, electron transport chain; Ezh2, enhancer of zeste homolog 2; Fxr, farnesoid X receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GCDCA, glycochenodeoxycholic acid; HSC, hepatic stellate cells; Hif-1α, hypoxia-inducible factor 1-alpha; JNK, c-Jun N-terminal kinase; KO, knockout; LSM, liver stiffness; MAPK, mitogen-activated protein kinase; MCJ; MCJ, methylation-controlled J; MLKL, mixed-lineage kinase domain-like pseudokinase; MMP, mitochondrial membrane potential; MPO, myeloperoxidase; MPT, mitochondrial permeability transition; Mitochondria; Nrf1, nuclear respiratory factor 1; PARP, poly (ADP-ribose) polymerase; PBC, primary biliary cholangitis; PSC, primary sclerosing cholangitis; Pgc1α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; Pgc1β, peroxisome proliferator-activated receptor gamma coactivator 1-beta; ROS; ROS, reactive oxygen species; RT, room temperature; SDH2, succinate dehydrogenase; TNF, tumour necrosis factor; Tfam, transcription factor A mitochondrial; Trail, TNF-related apoptosis-inducing ligand; UDCA, ursodeoxycholic acid; Ucp2, uncoupling protein 2; VCTE, vibration-controlled transient elastography; WT, wild-type; mRNA, messenger ribonucleic acid; p-JNK, phosphor-JNK; p-MLKL, phosphor-MLKL; shRNA, small hairpin RNA; siRNA, small interfering RNA; tBIL, total bilirubin; α-SMA, alpha-smooth muscle actin.

Graphical abstract

Fig. 1

MCJ expression is increased in cholestasis. (A) Liver biopsies from individuals with histologically normal liver (healthy) (n = 18) and from patients with CLD (n = 37) where MCJ expression was determined by immunohistochemistry and quantified. (B) The same liver biopsies where MCJ expression by immunohistochemistry was analysed according to disease stage. (C) MCJ mRNA expression in liver tissue from other cohort subjects (healthy, n = 7; and patients with CLD, n=25). (D) MCJ mRNA expression in liver tissue from patients with CLD according to disease stage. (E) MCJ levels by Western blotting in WT liver extracts. (F) MCJ mRNA expression was determined by qPCR in livers from WT mice. (G) MCJ mRNA expression of hepatocytes and non-parenchymal cells (Kupffer cells and hepatic stellate cells -HSC-). Values are represented as mean ± SEM. ∗p <0.05; ∗∗p <0.01; ∗∗∗∗p <0.0001 (Student t test). BDL, bile duct ligation; CLD, cholestatic liver disease; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HSC, hepatic stellate cells; MCJ, methylation-controlled J; WT, wild-type.

Fig. 2

MCJ deficiency attenuates acute cholestasis-induced liver injury in vivo. WT (n = 5) and MCJ-KO (n = 5) mice were subjected to BDL. Liver injury was evaluated 48 hours after the procedure. (A) Serum ALT levels in WT and MCJ-KO 48 h after BDL. (B) Biliary infarcts were assessed by H&E staining, liver inflammation by F4/80 staining, and fibrogenesis by α-SMA staining. (C) Serum TNF levels in WT and MCJ-KO 48 h after BDL. (D) Systemic neutrophil activation evaluated by MPO activity. (E) Assessment of complex II activity by colourimetry with an SDH₂ activity assay kit. (F) JNK activation by Western blotting in liver extracts. (G) Apoptosis measured by caspase-3 activity. Values are represented as mean ± SEM. ∗p <0.05; ∗∗p <0.01 (Student t test). α-SMA, alpha-smooth muscle actin; BDL, bile duct ligation; CLD, cholestatic liver disease; JNK, c-Jun N-terminal kinase; KO, knockout; MCJ, methylation-controlled J; MPO, myeloperoxidase; SDH₂, succinate dehydrogenase; TNF, tumour necrosis factor; WT, wild-type.

Fig. 3

MCJ deficiency prevents chronic cholestasis-induced liver injury in vivo. WT (n = 5) and MCJ-KO (n = 4) mice were subjected to BDL. Liver injury was evaluated 14 days after the procedure. (A) Serum ALT levels in WT and MCJ-KO 14 days after BDL. (B) Biliary infarcts were assessed by H&E staining, and fibrogenesis by α-SMA staining 14 days after BDL. (C) Relative mRNA expression of inflammatory related genes in liver tissue. (D) Assessment of complex II activity by colourimetry with SDH₂ activity assay kit. (E) Apoptosis measured by caspase-3 activity. (F) Expression of proteins related to apoptosis and necroptosis by Western blotting in WT and MCJ-KO liver extracts at 14 days after BDL. Values are represented as mean ± SEM. ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001 (Student t test). α-SMA, alpha-smooth muscle actin; ALT, alanine aminotransferase; BAX, BCL2 associated X; BDL, bile duct ligation; KO, knockout; MCJ, methylation-controlled J; MLKL, mixed-lineage kinase domain-like pseudokinase; p-MLKL; phosphor-MLKL; SDH₂, succinate dehydrogenase; WT, wild-type.

Fig. 4

Lack of MCJ protects against bile acid-mediated toxicity in hepatocytes. WT and MCJ-KO hepatocytes were treated with GCDCA 100 μM. Duplicates, triplicates, or quadruplicates were used for each experimental condition. (A) Apoptosis measured by caspase-3 activity. (B) Relative mRNA expression in hepatocytes of different inflammatory and apoptosis-related genes after 1-h GCDCA treatment. (C) JNK activation by Western blotting. (D) mROS in primary WT and MCJ-KO hepatocytes determined by staining with MitoSOX reagent. (E) Total ATP levels in primary WT and MCJ-KO hepatocytes. Values are represented as mean ± SEM. ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001; ∗∗∗∗p <0.0001 (Student t test). GCDCA, glycochenodeoxycholic acid; JNK, c-Jun N-terminal kinase; KO, knockout; MCJ, methylation-controlled J; ROS, reactive oxygen species; WT, wild-type.

Fig 5

MCJ in vivo silencing counteracts cholestasis-induced liver injury. BDL was performed on WT mice, and 3 and 5 days later, siControl (n = 6) or siMcj (n = 5) was injected i.v. into these mice. (A) Serum ALT levels in siControl and siMcj-treated animals. (B) Biliary infarcts were assessed by H&E staining, inflammation by F4/80 staining, fibrogenesis by α-SMA staining, and connective tissue by Sirius Red staining. (C) Relative mRNA expression of different inflammatory genes. (D) PARP (full length [f.l.] and catalytic domain [c.d.]) by Western blotting. (E) Apoptosis measured by caspase-3 activity. (F) Assessment of complex II activity by colourimetry with an SDH₂ activity assay kit. Values are represented as mean ± SEM. ∗p <0.05; ∗∗p <0.01 (Student t test). α-SMA, alpha-smooth muscle actin; ALT, alanine aminotransferase; BDL, bile duct ligation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KO, knockout; MCJ, methylation-controlled J; PARP, poly (ADP-ribose) polymerase; SDH₂, succinate dehydrogenase; WT, wild-type.