Volume 132, Issue 4, Pages 1515-1526 (April 2007) Transglutaminase 2 Regulates Mallory Body Inclusion Formation and Injury-Associated Liver Enlargement  Pavel Strnad, Masaru Harada, Matthew Siegel, Robert A. Terkeltaub, Robert M. Graham, Chaitan Khosla, M. Bishr Omary  Gastroenterology  Volume 132, Issue 4, Pages 1515-1526 (April 2007) DOI: 10.1053/j.gastro.2007.02.020 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 TG2−/− mice exhibit pronounced jaundice and an ameliorated liver hypertrophy response after DDC treatment. (A) The livers of TG2+/+ mice show significantly higher TG activities than TG2−/− livers before (27 vs 115 pmol putrescine/min/mg) and after 3.5 months of DDC treatment (70 vs 189 pmol putrescine/min/mg). Four mice were analyzed per genotype and treatment condition. (B) Liver enzymes and the ratio of liver weight to total body weight are analyzed in TG2+/+ and TG2−/− mice before and after 3.5 months of DDC feeding. After DDC treatment, TG2+/+ mice display a higher ratio of liver/total body weight than their TG2−/− counterparts. The nontreated TG2−/− mice have slightly elevated ALT and lower total bilirubin without obvious histological signs of damage. DDC ingestion leads to marked jaundice and ductal damage with an increase in total bilirubin (Total Bili) and alkaline phosphatase (Alk Phos). n.d., none detected; ns, not significant. *Liver weight, body weight, and the ratio of liver/body weight were determined from 7 pairs of nontreated and 11 pairs of DDC-fed mice. There is also a trend for smaller liver/body weights in the non-DDC fed TG2−/− mice as they aged (not shown). (C) Three and one-half months of DDC treatment leads to more prominent hepatomegaly in TG2+/+ versus TG2−/− mice. L, liver. (D) DDC ingestion for 3.5 months increases liver TG2 protein levels in TG2+/+ mice, whereas no TG2 is detected in TG2−/− mice. Tubulin is used as a loading control. Gastroenterology 2007 132, 1515-1526DOI: (10.1053/j.gastro.2007.02.020) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 TG2−/− mice are protected from DDC-induced MB formation and from the accumulation of p62 and ubiquitinated proteins. (A) Morphometric analysis was carried out using hematoxylin and eosin–stained liver sections of TG2+/+ and TG2−/− DDC-fed (3.5 months) mice. The most prominent findings in the TG2+/+ mice are the development of MBs, hepatocyte ballooning, and the increased number of hepatocytes lacking eosin staining (termed empty cells). DDC-fed TG2−/− mice have a more pronounced ductal proliferation and pigment deposition within hepatocytes. ns, not significant. (B) Hematoxylin and eosin staining of TG2+/+ (a, c) and TG2−/− mouse livers (b, d), before (a, b) and after 3.5 months of DDC feeding (c, d). Arrows highlight the MBs in the DDC-fed TG2+/+ mice. (C) Immunofluorescence double-staining depicting the localization of K8/K18 (red) and ubiquitin (green) in TG2+/+ (a, c) and TG2−/− (b, d) mice before (a, b) and after 3.5 months of DDC ingestion (c, d). Arrows highlight the MBs that appear as yellow dots because of the red and green costaining. (D) Total mouse liver homogenates of nontreated and 3.5 months DDC-fed TG2+/+ and TG2−/− mice were analyzed by blotting using antibodies to the indicated proteins. Three independent livers (1 to 3) were used per condition. The tubulin blot is included as a loading control. Scale bars: 100 μm (B), 20 μm (C). Gastroenterology 2007 132, 1515-1526DOI: (10.1053/j.gastro.2007.02.020) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 K8/K18 mRNA and protein levels are differentially altered after DDC treatment. (A) Quantitative real-time PCR shows the up-regulation of K8 (a) and K18 mRNA (b) in mouse livers after 3.5 months of DDC feeding. The K8 and K18 mRNA levels in the non-DDC fed TG2+/+ mice are arbitrarily set as 1, and all other levels represent a ratio. Four mice were analyzed per genotype and treatment condition. (B) Immunoblot analysis of total mouse liver lysates before and after 3.5 months of DDC ingestion. After DDC treatment, TG2+/+ livers have decreased K8/K18 protein, whereas DDC feeding increases the detectable K8/K18 in the TG2−/− mice. Three independent livers (1 to 3) were used/condition. (C) Livers of TG2+/+ mice (3 livers, each, from mice fed DDC or a normal diet for 6 weeks) were homogenized in SDS-containing buffer. Tissue homogenates were analyzed by blotting using antibodies to the indicated proteins. Tubulin was used as a loading control. (D) The insoluble extracts of TG2+/+ and TG2−/− mouse livers (+/− 3.5 months of DDC mouse feeding, 2 mice/condition) were isolated as described in the Methods section. The extracts were stained with Coomassie blue or were analyzed by blotting using antibodies to ubiquitin or to the lysine-glutamine isopeptide links. Note that the abundant K8 and K18 proteins can be readily seen after Coomassie staining (arrowheads in upper panel). Gastroenterology 2007 132, 1515-1526DOI: (10.1053/j.gastro.2007.02.020) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 MB formation is paralleled by increased levels of high Mr proteins. (A) Immunoblotting of total liver homogenates from DDC-fed TG2+/+ and TG2−/− mice reveals elevated p62, ubiquitinated proteins, and an accumulation of high Mr proteins that are recognized by antibodies to K8 and ubiquitin but not to K18 (or p62 or tubulin; the stacking/resolving part of the corresponding gels is not shown). The histologic presence (+) or absence (−) of MBs for each of the independent 22 analyzed livers is shown. S, stacking gel; R, resolving gel. (B) Liver homogenates from nontreated, 6 weeks, and 3.5-month-DDC-treated TG2+/+ mice were analyzed by immunoblotting. Six weeks of DDC-treatment, which is not sufficient to induce MB formation (displayed as MBs + or −), results in the appearance of an 180 kDa intermediate size K8-containing species (arrow) but no accumulation of p62 or ubiquitinated proteins. In contrast, 3.5 months of DDC ingestion leads to MB formation with increased p62/ubiquitinated proteins/high Mr species. TG2 expression increases gradually during DDC treatment. Three independent livers (1 to 3) were analyzed per condition. (C) Mice were fed DDC for 3.5 months to induce MBs and were allowed to recover for 1 month (Rec.) on a standard diet that led to MB resolution. Liver homogenates were subsequently prepared (2 livers/condition) followed by blotting with antibodies to K8 or tubulin. S, position of the stacking gel. (D) The high Mr band located at the top of the stacking gel was isolated from liver homogenates of DDC-fed TG2+/+ mice and then analyzed by mass spectrometry as described in the Methods section. Peptide scores >35 are considered significant. % coverage, % amino acid sequence identified. Gastroenterology 2007 132, 1515-1526DOI: (10.1053/j.gastro.2007.02.020) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 K8 is preferentially transamidated both in vitro and in vivo. (A) Recombinant human K8 and K18 were incubated separately or together (2 hours) with TG2 followed by Coomassie staining or immunoblotting with the indicated antibodies. Lanes 1, 2, 4, and 6 show the input proteins without added substrate or enzyme. TG2 in lane 1 was mixed immediately with sample buffer without incubation. Arrows highlight the high Mr species, which either do not enter the stacking gel or remain at the border between the stacking and resolving gels. (B, C) Total liver homogenates from nontransgenic littermates (control) and from mice that overexpress mouse K8 (B) or human (h) K18 (C) were isolated after feeding the mice DDC for 6 weeks (B) or for 3.5 months (C). The homogenates were analyzed by blotting using antibodies to the indicated antigens. Only the stacking gel portion is shown for the K8 blot while the remaining blots (tubulin, K18, and Hsp60) show only the resolving part of the gel that includes the monomeric proteins because the stacking portion of these blots are blank (ie, without cross-linked species, not shown). Arrows highlight the K8-containing high Mr species. Hsp60 was used as a loading control. Gastroenterology 2007 132, 1515-1526DOI: (10.1053/j.gastro.2007.02.020) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 TG2 is critical for MB formation through transamidation of K8. MB-inducing exposures promote several early and late changes, with the late changes ultimately leading to MB formation. Essential among these late changes is the transamidation of K8, which is promoted in situations of increased K8-greater-than-K18 ratio coupled with induction of TG2, as noted after DDC and griseofulvin treatment or genetic manipulation such as the overexpression of K8. Gastroenterology 2007 132, 1515-1526DOI: (10.1053/j.gastro.2007.02.020) Copyright © 2007 AGA Institute Terms and Conditions