Bile acid-induced Mallory body formation in drug-primed mouse liver

Abstract

Chronic cholestasis is associated with retention of bile acids and profound cytoskeletal alterations in hepatocytes including Mallory body (MB) formation. The mechanisms responsible for MB formation in cholestatic liver diseases are unclear. The aim of our study was to determine the relevance of cholestasis and bile acids for MB formation. For this purpose mice received a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-supplemented diet for 2.5 months to induce MB formation. After recovery from DDC intoxication for 4 weeks followed by disappearance of MBs, these drug-primed mice were subjected to DDC refeeding, common bile duct ligation (CBDL), and feeding of a cholic acid (CA)-supplemented diet for 7 days, respectively. Cytokeratin (CK) 8 and CK 18 expression was studied by competitive reverse transcriptase-polymerase chain reaction and Western blot analysis. Cytoskeletal alterations of hepatocytes and MB formation were monitored by immunofluorescence microscopy and immunohistochemistry using CK-, ubiquitin-, and MB-specific antibodies. Like DDC refeeding, both CBDL and CA feeding of drug-primed mice significantly increased CK 8 and CK 18 mRNA and protein levels (with excess of CK 8) and resulted in ubiquitination and abnormal phosphorylation of CKs. Furthermore, CBDL and CA feeding resulted in rapid neoformation of MBs in drug-primed mice. It is concluded that MB formation in cholestatic liver diseases may be triggered by the action of potentially toxic bile acids.

Figure 1.

Experimental design to study the role of cholestasis and bile acids in MB formation in drug-primed mice. Mice were fed a control diet or 0.1% DDC-supplemented diet for 2.5 months to induce MBs. One group of animals was sacrificed to study DDC-induced cytoskeletal alterations (including MB formation), whereas another group was sacrificed 4 weeks after discontinuation of DDC feeding (recovery) to assess the reversibility of these changes. In addition, recovered (primed) mice received control diet (Co) or were subjected to DDC refeeding (DDC), CBDL, CA feeding, and sham operation for 7 days, respectively. Five animals were studied in each group.

Figure 2.

DDC, CBDL, and CA feeding induce overexpression of CK 8 and CK 18 and MB formation in drug-primed mice. A: Competitive reverse transcriptase-polymerase chain reaction revealed a significant increase of CK 8 (open bars) and of CK 18 (filled bars) mRNA in DDC-refed (primed DDC), bile duct-ligated (primed CBDL), and CA-fed (primed CA) drug-primed mouse liver in comparison to drug-primed recovered mice (primed Co) and naive control diet-fed mice (control). Data (means + SEM) are expressed as copy numbers/200 ng of total RNA (n = 5 in each group; * and #, P < 0.05, DDC, CBDL, and CA versus controls). B: Representative Western blots for CK 8, CK 18, and β-actin in naive control diet-fed (control), drug-primed recovered (primed Co), DDC-refed (primed DDC), bile duct-ligated (primed CBDL), and CA-fed (primed CA) drug-primed mouse liver. DDC refeeding, CBDL feeding, and CA feeding significantly increased CK 8 and CK 18 protein levels, whereas β-actin levels remained unchanged. Data (means + SEM) are expressed as relative protein amounts (n = 3 in each group; * and #, P < 0.05, primed Co, DDC, CBDL, and CA versus controls). The increase of CK 8 (open bars) was more pronounced than that of CK 18 (filled bars). The corresponding Western blots are shown at top of B. C to F: Double-immunofluorescence microscopy was performed on frozen liver sections of recovered (primed/Co) (C), DDC-refed (primed/DDC) (D), bile duct-ligated (primed/CBDL) (E), and CA-fed (primed/CA) (F) drug-primed mice combining the monoclonal antibody M_M120-1 specifically recognizing MBs (yellow because of co-localization with CKs) with the polyclonal antibody 50K160 against CKs (red). Note the increased density of CK-IF network (particularly in D and F) and the increasing number and size of MBs (arrowheads) in hepatocytes after DDC refeeding, CBDL feeding, and CA feeding, whereas in the primed-recovered mouse liver (primed/Co) only small residual MBs still remain in enlarged hepatocytes with diminished or absent immunostainable IF network (arrowheads). Scale bar, 20 μm.

Figure 3.

DDC, CBDL, and CA feeding induce abnormal phosphorylation of the CK-IF network and MBs in drug-primed mice. Double-immunofluorescence microscopy was performed on frozen liver sections of recovered (primed/Co) (A), DDC-refed (primed/DDC) (B), bile duct-ligated (primed/CBDL) (C), and CA-fed (primed/CA) (D) drug-primed mice combining the monoclonal antibody 5B3 specifically recognizing an abnormally phosphorylated epitope of CK 8 (yellow because of co-localization with CK) with the polyclonal antibody 50K160 against CKs (red). Note the abnormal phosphorylation of the CK-IF network (arrows) and newly formed MBs (arrowheads) in hepatocytes after DDC refeeding, CBDL feeding, and CA feeding (B–D). Abnormally phosphorylated larger MBs are usually present in enlarged hepatocytes with diminished or missing IF network. In the primed recovered liver (primed/Co) there are only small residual MBs in still enlarged hepatocytes with diminished IF network (arrowheads) (A). Scale bar, 20 μm.

Figure 4.

DDC, CBDL, and CA feeding induce ubiquitination of neoformed MBs in drug-primed mice. Immunohistochemistry was performed on liver sections of recovered (primed/Co) (A), DDC-refed (primed/DDC) (B), bile duct-ligated (primed/CBDL) (C), and CA-fed (primed/CA) (D) drug-primed mice using an antibody against ubiquitin. DDC refeeding, CBDL feeding, and CA feeding resulted in newly formed ubiquitin-positive MBs (arrowheads) (B–D). Arrows designate pigment deposits (A, B). Original magnifications, ×20.