Keratin mutation predisposes to mouse liver fibrosis and unmasks differential effects of the carbon tetrachloride and thioacetamide models

Abstract

Background & aims: Keratins 8 and 18 (K8/K18) are important hepatoprotective proteins. Animals expressing K8/K18 mutants show a marked susceptibility to acute/subacute liver injury. K8/K18 variants predispose to human end-stage liver disease and associate with fibrosis progression during chronic hepatitis C infection. We sought direct evidence for a keratin mutation-related predisposition to liver fibrosis using transgenic mouse models because the relationship between keratin mutations and cirrhosis is based primarily on human association studies.

Methods: Mouse hepatofibrosis was induced by carbon tetrachloride (CCl(4)) or thioacetamide. Nontransgenic mice, or mice that over express either human Arg89-to-Cys (R89C mice) or wild-type K18 (WT mice) were used. The extent of fibrosis was evaluated by quantitative real-time reverse-transcription polymerase chain reaction of fibrosis-related genes, liver hydroxyproline measurement, and Picro-Sirius red staining and collagen immunofluorescence staining.

Results: Compared with control animals, CCl(4) led to similar liver fibrosis but increased injury in K18 R89C mice. In contrast, thioacetamide caused more severe liver injury and fibrosis in K18 R89C as compared with WT and nontransgenic mice and resulted in increased messenger RNA levels of collagen, tissue inhibitor of metalloproteinase 1, matrix metalloproteinase 2, and matrix metalloproteinase 13. Analysis in nontransgenic mice showed that thioacetamide and CCl(4) have dramatically different molecular expression responses involving cytoskeletal and chaperone proteins.

Conclusions: Over expression of K18 R89C predisposes transgenic mice to thioacetamide- but not CCl(4)-induced liver fibrosis. Differences in the keratin mutation-associated fibrosis response among the 2 models raise the hypothesis that keratin variants may preferentially predispose to fibrosis in unique human liver diseases. Findings herein highlight distinct differences in the 2 widely used fibrosis models.

Figure 1. K18 R89C mice develop more pronounced liver injury after CCl₄ and TAA treatment

Nontransgenic mice (FVB; a,d,g) and mice overexpressing human K18 (K18 WT; b,e,h) or K18 R89C (c,f,i) were treated with TAA- (d–f) or CCl₄ (g–i) for 6 and 8 weeks, respectively. Note that both TAA- and CCl₄-treated K18 R89C mice develop more pronounced liver injury as evidenced by an increased inflammatory reaction after H&E staining (arrows in f,i) which is quantified in Table 1. Scale bar=100 μm.

Figure 2. K18 R89C mice develop increased liver fibrosis after TAA but not CCl₄ treatment

The extent of liver fibrosis in control mice (a–c) and mice given TAA for 6 weeks (d–f) or CCl₄ for 8 weeks (g–i) was evaluated using PSR staining. Nontransgenic mice (FVB; a,d,g), and mice overexpressing human K18 WT (b,e,h) or K18 R89C (c,f,i) were analyzed. Note that TAA-treated K18 R89C mice exhibit more pronounced fibrosis than K18 WT and nontransgenic mice (arrows in d–f), whereas no difference is noted in the three strains treated with CCl₄ (arrows in g–i). Scale bar=200 μm.

Figure 3. K18 R89C livers harbor increased collagen mRNA and protein, and hydroxyproline levels, after TAA but not CCl₄ treatment as compared with nontransgenic and K18 WT livers

(A) Double immunofluorescence staining using antibodies to K8 (green) and collagen (red) highlights the extent of collagen deposition in nontransgenic mice (FVB; a,d,g), mice overexpressing K18 (K18 WT; b,e,h) or K18 R89C (c,f,i) under basal conditions (a–c) or after six weeks of TAA (d–f) or eight weeks of CCl₄ administration (g–i). Note the increased collagen staining in livers of TAA-treated (arrows, d–f), but not CCl₄-treated (arrows, g–i) K18 R89C versus K18 WT and nontransgenic mice. Scale bar=200 μm. (B) 6 weeks of TAA treatment (left panel) causes a higher hepatic collagen mRNA expression in K18 R89C versus K18 WT and nontransgenic livers as determined by quantitative real time RT-PCR. In contrast, similar collagen mRNA levels are observed in all three strains after eight weeks of CCl₄ administration (right panel). The expression in nontransgenic mice was arbitrarily set as 1. Four mice were analyzed/strain/treatment condition. (C) Hydroxyproline is measured as described in “Materials and Methods” using livers from control (untreated), TAA-treated or CCl₄-treated nontransgenic (FVB), K18 WT and K18 R89C mice. The number of independent livers used for each condition is shown below the x-axis. All three CCl₄-treated strains of mice and the TAA-treated K18 R89C mice have significantly elevated levels of hydroxyproline content (p <0.05, not shown) when compared with their counterparts in the untreated control groups.

Figure 4. TAA but not CCl₄ treatment increases keratin expression and phosphorylation

(A) Liver K8 (upper panel) and K18 (lower panel) expression levels in nontransgenic mice increase after TAA but not CCl₄ treatment as determined by quantitative real time RT-PCR. Keratin expression in untreated mice (Control) was arbitrarily set as 1, and four mice were analyzed/strain/treatment condition. (B) Immunoblot analysis of nontransgenic mouse total liver lysates before (Control) and after TAA and CCl₄ administration. Four individual livers/genotype were analyzed. Note that TAA leads to a significant increase in total K8/K18 protein levels and K8 phospho-S431, whereas CCl₄ administration shows less prominent changes. (C) Livers from two nontransgenic mice/treatment group, from control or TAA/CCl₄-treated mice, were homogenized to isolate the high-salt insoluble keratin fraction followed by analysis by SDS-PAGE then Coomassie staining. Equal fractions were loaded in each lane and normalization was done by using equal weights of liver pieces. (D) Immunofluorescence double-staining was carried out on livers similar to those used in panels A–C, using antibodies to K8 phospho-S431 (green) and K18 phospho-S33 (red). Yellow color indicates co-localization of the two epitopes. Scale bar=100 μm.

Figure 5. TAA and CCl₄ are distinct fibrosis models that lead to unique molecular alterations

(A) β-actin and α-tubulin mRNA levels were compared in livers from four nontransgenic mice/treatment group (control or TAA/CCl₄-treated mice) using quantitative real time RT-PCR. The levels observed in untreated mice (Control) were arbitrarily set as 1. (B) Liver pieces from the same mice analyzed in panel A were homogenized and the homogenates were blotted using antibodies to the indicated cytoskeletal and chaperone proteins. Note that TAA induces prominent overexpression of Hsp27, which is not seen in control or CCl₄-treated livers. Both models result in decreased actin and tubulin levels, but the change is much more prominent after CCl₄ administration. The decrease in actin and tubulin protein levels is related at least in part to actin (arrow) and tubulin (not shown) degradation. (C) (a–c): Liver sections from nontransgenic mice similar to those used in Panels A,B were double-stained using antibodies to K8/K18 (red) and Hsp27 (green). Scale bar=200 μm. (d–f): A higher magnification of a liver from animals given TAA (similar to that shown in image b) is displayed to highlight each individual staining and the merged image. Scale bar=50 μm.

Figure 6. Summary of findings from the CCl₄ and TAA fibrosis models in the context of normal and mutant K18

Both CCl₄ and TAA induce fibrosis in nontransgenic and K18 WT-overexpressing mice, but the K18 R89C mutation leads to an accelerated fibrosis in the TAA but not the CCl₄ model. Hepatomegaly (liver-to-body weight size) is also more prominent in the K18 R89C mice and in the TAA model in general. In nontransgenic mice, the CCl₄ and TAA models evoke very different molecular responses as manifested by markedly different effects on cytoskeletal proteins and chaperones.