Aging modulates susceptibility to mouse liver Mallory-Denk body formation

Abstract

Mallory-Denk bodies (MDBs) are hepatocyte cytoplasmic inclusions found in several liver diseases and consist primarily of the cytoskeletal proteins, keratins 8 and 18 (K8/K18). Recent evidence indicates that the extent of stress-induced protein misfolding, a K8>K18 overexpression state, and transglutaminase-2 activation promote MDB formation. In addition, the genetic background and gender play an important role in mouse MDB formation, but the effect of aging on this process is unknown. Given that oxidative stress increases with aging, the authors hypothesized that aging predisposes to MDB formation. They used an established mouse MDB model-namely, feeding non-transgenic male FVB/N mice (1, 3, and 8 months old) with 3,5 diethoxycarbonyl-1,4-dihydrocollidine for 2 months. MDB formation was assessed using immunofluorescence staining and biochemically by demonstrating keratin and ubiquitin-containing crosslinks generated by transglutaminase-2. Immunofluorescence staining showed that old mice had a significant increase in MDB formation compared with young mice. MDB formation paralleled the generation of high molecular weight ubiquitinated keratin-containing complexes and induction of p62. Old mouse livers had increased oxidative stress. In addition, 20S proteasome activity and autophagy were decreased, and endoplasmic reticulum stress was increased in older livers. Therefore, aging predisposes to experimental MDB formation, possibly by decreased activity of protein degradation machinery.

Figure 1.

Assessment for Mallory-Denk body (MDB) formation using immunofluorescence staining for K8/K18 and Ub in livers of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)–fed mice. Livers were double-labeled with Abs to K8/K18 (red) and Ub (green). MDBs are seen as yellow clumps due to colocalization of keratins and Ub (arrows). Eight-month-old mouse livers had the highest number of MDBs as compared with livers of younger mice (A–D) and had no MDBs under basal condition (E, F). Scale bars: (A–C, E) 200 µm; (D) 50 µm; (F) 20 µm.

Figure 2.

Biochemical evidence for age-related formation of Mallory-Denk bodies (MDBs) in the livers of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)–fed mouse livers. (A) Human K8 transgenic mice were fed a DDC-containing diet or a regular diet, and liver homogenates were isolated and blotted with an antibody to human K8. Crosslinked human K8-containing complexes are highlighted by the arrows. (B) Liver homogenates were isolated from 1-, 3-, and 8-month-old mice fed a DDC-containing diet. Four independent livers were analyzed from each age group. The homogenates were blotted with Abs to the indicated antigens. The immunoblots show the presence of high molecular weight K8- and Ub-containing complexes (arrows) at the top of the stacking gel (S). Relative intensity of these bands was quantified by densitometry. (C) Duplicate samples to those used in panel B were analyzed for the expression of K18, p62, TG2, HNE, and Hsp60 (used as a loading control). Arrowheads highlight HNE-modified proteins that manifest increased intensity in livers of the older mice.

Figure 3.

Immunohistochemistry for malondialdehyde (MDA)–modified proteins in the liver. Liver sections from the control and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)–treated mice were stained with an Ab to MDA-modified proteins and visualized by diaminobenzidine (DAB) staining. MDA-positive hepatocytes were increased in old mouse livers (C). Scale bars: (A–C) 100 µm.

Figure 4.

Decreased proteasome and autophagy activity upon aging in response to 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) administration. (A, B) Proteasome activity in liver homogenates was measured by detection of the fluorophore after cleavage of the substrate as described in Materials and Methods. The activity was measured in the livers of 1-, 3-, and 8-month-old mice under basal conditions (A) and after DDC feeding (B). (C, D) Autophagy activity was determined using the LC3-II/I ratio as a surrogate marker. For this, liver homogenates from DDC-fed mice with feeding starting at the indicated ages were immunoblotted with antibodies to LC3-I/II. Note that the LC3-II/I ratio showed a significant decrease in 8-month-old mouse livers. Values in panels A, B, and D represent the mean ± SD (n=4). *p<0.05 compared with 3-month-old group (B, D). **p<0.01 compared with 1-month-old group.

Figure 5.

Increased endoplasmic reticulum (ER) stress upon aging in response to 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) administration. Liver homogenates were isolated from 1-, 3-, and 8-month-old mice fed a DDC-containing diet. Four independent livers were analyzed from each age group. The homogenates were blotted with Abs to ER stress markers, GRP78 and XBP-1. Note that the expression of these proteins was increased in 8-month-old mouse livers.