Vitamin D nuclear receptor deficiency promotes cholestatic liver injury by disruption of biliary epithelial cell junctions in mice

Abstract

Alterations in apical junctional complexes (AJCs) have been reported in genetic or acquired biliary diseases. The vitamin D nuclear receptor (VDR), predominantly expressed in biliary epithelial cells in the liver, has been shown to regulate AJCs. The aim of our study was thus to investigate the role of VDR in the maintenance of bile duct integrity in mice challenged with biliary-type liver injury. Vdr(-/-) mice subjected to bile duct ligation (BDL) displayed increased liver damage compared to wildtype BDL mice. Adaptation to cholestasis, ascertained by expression of genes involved in bile acid metabolism and tissue repair, was limited in Vdr(-/-) BDL mice. Furthermore, evaluation of Vdr(-/-) BDL mouse liver tissue sections indicated altered E-cadherin staining associated with increased bile duct rupture. Total liver protein analysis revealed that a truncated form of E-cadherin was present in higher amounts in Vdr(-/-) mice subjected to BDL compared to wildtype BDL mice. Truncated E-cadherin was also associated with loss of cell adhesion in biliary epithelial cells silenced for VDR. In these cells, E-cadherin cleavage occurred together with calpain 1 activation and was prevented by the silencing of calpain 1. Furthermore, VDR deficiency led to the activation of the epidermal growth factor receptor (EGFR) pathway, while EGFR activation by EGF induced both calpain 1 activation and E-cadherin cleavage in these cells. Finally, truncation of E-cadherin was blunted when EGFR signaling was inhibited in VDR-silenced cells.

Conclusion: Biliary-type liver injury is exacerbated in Vdr(-/-) mice by limited adaptive response and increased bile duct rupture. These results indicate that loss of VDR restricts the adaptation to cholestasis and diminishes bile duct integrity in the setting of biliary-type liver injury.

Fig 1

Liver injury in Vdr^−/− mice. (A) Liver samples were collected from Sham or BDL, wildtype (WT), and Vdr^−/− mice 3 days post-BDL and stained by HPS. Liver was morphologically normal in WT and Vdr^−/− sham mice (upper panels). After BDL the number and extent of bile infarcts (arrows) were higher in Vdr^−/− (right lower panel) than in WT (left lower panel) mice. Scale bar = 50 μm. (B) Blood samples were collected from Sham or BDL, WT, and Vdr^−/− mice 3 days post-BDL. Panel shows plasma concentrations of aspartate aminotransferase (left histogram), and alanine aminotransferase (right histogram). Data represent means ± SEM of 5-6 animals. *P < 0.05. (C) Blood samples were collected from Sham or BDL, WT, and Vdr^−/− mice 3 days post-BDL. Panel shows plasma concentrations of bile acids. Data represent means ± SEM of 5-6 animals. *P < 0.05.

Fig 2

Adaptive changes in bile acid metabolism and transport in Vdr^−/− mice after BDL. Liver samples of Sham or BDL, wildtype (WT), and Vdr^−/− mice were analyzed 3 days post-BDL by qPCR for mRNA expression of Bsep (A), Mdr2 (B), Mrp3 (C), and Cyp3a11 (D). Data were normalized to Gapdh and represent means ± SEM of 5-6 animals. *P < 0.05. (E) Tetrahydroxylated bile acids were analyzed by mass spectrometry in liver samples of Sham or BDL, WT, and Vdr^−/− mice 3 days post-BDL. Data represent means ± SEM of 5-6 animals and are expressed as arbitrary units. Total tetrahydroxylated bile acids represent no more than 4% of the total bile acid pool in the setting of BDL. *P < 0.05.

Fig 3

Fibrogenic and ductular response in Vdr^−/− mice after BDL. Liver samples were collected from Sham or BDL, wildtype (WT), and Vdr^−/− mice 3 days post-BDL. (A) Collagen deposits evaluated by Sirius red staining were increased 3 days post-BDL but were not different between genotypes. Scale bar = 50 μm. mRNA expression of Tgf-β (B), α-Sma (C), and Col1a1 (D) were analyzed by qPCR. Data were normalized to Gapdh and represent means ± SEM of 5-6 animals. *P < 0.05. (E) Compared with WT mice, Vdr^−/− mice displayed little ductular reaction 3 days post-BDL (arrowheads). Scale bar = 25 μm. mRNA expression of Ck19 (F) was examined by qPCR. Data were normalized to Gapdh and represent means ± SEM of 5-6 animals. *P < 0.05.

Fig 4

E-cadherin expression in Vdr^−/− mice. (A) Representative immunostaining of E-cadherin in the liver of Sham or BDL, wildtype (WT), and Vdr^−/− mice 3 days post-BDL. E-cadherin staining was interrupted (arrows) more often in BDL Vdr^−/− (lower right panel) than in WT mice (lower left panel), indicative of higher incidence of bile duct rupture (33.0 ± 0.04% and 14.8 ± 0.08% of bile ducts, respectively). Scale bar = 10 μm. (B) Liver samples were collected from Sham or BDL, WT, and Vdr^−/− mice 3 days post-BDL and were assessed for E-cadherin expression by immunoblot. β-Actin was used as an internal control. (C) Whole liver protein extracts were immunoprecipitated with an antibody directed against E-cadherin. After gel migration, the 100-kDa protein signal was extracted and submitted to mass spectrometry analysis. The full E-cadherin protein sequence is shown, with dark boxes representing the peptides identified by mass spectrometry. The underlined sequence corresponds to the calpain cleavage site consensus sequence.

Fig 5

E-cadherin cleavage in biliary epithelial cells with diminished VDR expression. Biliary epithelial cells were transfected either with scramble or VDR siRNA for 3 days and were subjected to (A) detection of E-cadherin, Calpain 1, Calpain 2, VDR, and β-actin expression by immunoblot. Histograms represent the densitometric analysis of relative E-cadherin 100 kDa and activated Calpain 1 expression. Data represent means ± SEM of at least six independent experiments. *P < 0.05. (B) Morphologic analysis and E-cadherin (green) localization by immunocytofluorescence. Nuclei were stained with DAPI (blue). Scale bar = 25 μm. (C) Biliary epithelial cells were transfected either with scramble, VDR siRNA, Calpain 1 siRNA, or a combination of VDR and Calpain 1 siRNA for 3 days. Total protein extracts were then subjected to detection of E-cadherin, Calpain 1, Calpain 2, VDR, and β-actin expression by immunoblot. Histogram represent the densitometric analysis of relative E-cadherin 100 kDa expression. Data represent means ± SEM of six independent experiments.

Fig 6

E-cadherin cleavage in biliary epithelial cells with activated EGFR-signaling pathway. (A) Biliary epithelial cells were incubated with EGF (50 ng/mL) for various periods of time. Whole cell protein extracts were then submitted to pEGFR Y1068, EGFR, pERK, ERK, Calpain 1, Calpain 2, E-cadherin, and β-actin immunoblot. Representative gels of five different experiments are shown. (B) Morphologic analysis of biliary epithelial cells and E-cadherin localization by immunocytofluorescence 48 hours after EGF treatment. Histogram represents the results of a blinded analysis estimating the number of cells with intracellular E-cadherin staining. Scale bar = 25 μm. (C) Biliary epithelial cells were transfected either with scramble or EGFR siRNA before being incubated with EGF for 24 hours. Total protein extracts were then submitted to pEGFR Y1068, EGFR, pERK, ERK, E-cadherin, and β-actin immunoblot. (D) E-cadherin localization in biliary epithelial cells is evidenced by immunocytofluorescence 2 days after EGF treatment, in the presence or absence of the EGFR inhibitor, AG1478. Scale bar = 25 μm. (E) Biliary epithelial cells were transfected either with scramble or Calpain 1 siRNA before being incubated with EGF for 24 hours. Total protein extracts were then submitted to E-cadherin, Calpain 1, Calpain 2, and β-actin immunoblot. Representative gels of five different experiments are shown.

Fig 7

EGFR-signaling in biliary epithelial cells with diminished VDR expression. (A) Biliary epithelial cells were transfected either with scramble or VDR siRNA for 3 days. Total protein extracts were then submitted to pEGFR Y1068, EGFR, pERK, ERK, E-cadherin, VDR, and β-actin immunoblot. Histograms represent the densitometric analysis of the pEGFR to EGFR ratio and of the pERK to ERK ratio. Data represent means ± SEM of six independent experiments. *P < 0.05. (B) Biliary epithelial cells were transfected either with scramble, VDR siRNA, EGFR siRNA, or a combination of VDR and EGFR siRNA for 3 days. Total protein extracts were then subjected to detection of E-cadherin, Calpain 1, EGFR, VDR, and β-actin by immunoblot. Histogram represents the densitometric analysis of relative E-cadherin 100 kDa expression. Data represent means ± SEM of three independent experiments.