Pharmacologic transglutaminase inhibition attenuates drug-primed liver hypertrophy but not Mallory body formation

FEBS Letters 580 (2006) 2351–2357

Pharmacologic transglutaminase inhibition attenuates drug-primed liver hypertrophy but not Mallory body formation Pavel Strnada, Matthew Siegelb, Diana M. Toivolaa, Kihang Choib,1, Jon C. Kosekc, Chaitan Khoslab, M. Bishr Omarya,* a

Department of Medicine, Palo Alto VA Medical Center, 3801 Miranda Avenue, Mail Code 154J, Palo Alto, CA 94304, United States b Department of Chemistry and Chemical Engineering, Stanford University, Stanford, CA 94305, United States c Department of Pathology, Stanford University Digestive Disease Center, United States Received 15 February 2006; revised 14 March 2006; accepted 15 March 2006 Available online 29 March 2006 Edited by Barry Halliwell

Abstract Mallory bodies (MBs) are characteristic of several liver disorders, and consist primarily of keratins with transglutaminase-generated keratin crosslinks. We tested the effect of the transglutaminase-2 (TG2) inhibitor KCC009 on MB formation in a mouse model fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). KCC009 decreased DDC-induced liver enlargement without affecting MB formation or extent of liver injury. TG2 protein and activity increased after DDC feeding and localized within and outside hepatocytes. KCC009 inhibited DDC-induced hepatomegaly by affecting hepatocyte cell size rather than proliferation. Hence, TG2 is a potential mediator of injury-induced hepatomegaly via modulation of hepatocyte hypertrophy, and KCC009-mediated TG2 inhibition does not affect mouse MB formation.  2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Transglutaminase; Keratin; Mallory body

1. Introduction Transglutaminases (TGs) are a family of enzymes which catalyze a reaction between c-carboxamide groups of glutamines and e-amino groups of lysines, thereby forming e-(c-glutamyl) lysine intra- or inter-molecular crosslinks [1,2]. TG-generated crosslinks can be beneficial as occurs during blood clotting or formation of the epidermal cell-envelope, but can be pathologic since they may lead to autoimmune disorders such as celiac disease [3]. Within the TG family, TG2 (also known as tissue transglutaminase) is ubiquitously expressed and impli* Corresponding author. Fax: +1 650 852 3259. E-mail address: [email protected] (M.B. Omary). 1 Present address: Department of Chemistry, Korea University, Seoul 136-701, Republic of Korea.

Abbreviations: Ab, antibody; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; DDC, 3,5diethoxycarbonyl-1,4-dihydrocollidine; ECM, extracellular matrix; H&E, hematoxylin and eosin; IF, intermediate filament; Inh, transglutaminase inhibitor; ip, intraperitoneal; K, keratin; KCC009, dihydroisoxazole-derived transglutaminase inhibitor; MB, Mallory body; MDC, monodansylcadaverine; PCNA, proliferating cell nuclear antigen; Re, recovery; TG, transglutaminase; WGA, wheat germ agglutinin

cated in apoptosis, organization of extracellular matrix (ECM) and signal transduction [1,2]. Many proteins are known TG2 substrates, including intermediate filament (IF) proteins [4,5]. The significance of IF proteins as substrates for TG2 is that IFs are the major components of a variety of cytoplasmic inclusions that are associated with a range of diseases that reflect the tissue-specific expression of IF proteins [6]. Among the inclusions are Mallory bodies (MBs) that are found in a variety of liver disorders [7], desmin bodies in muscle [8], and neurofilament and a-internexin inclusions in brain [9]. IFs make up a large family of tissue-specific nuclear and cytoplasmic proteins. Types I–IV cytoplasmic IFs include keratins (K) type I (K9–K20) and II (K1–K8) of epithelial cells, several type III proteins (e.g., desmin), and several type IV proteins (e.g., neurofilaments and a-internexin) [10]. Epithelial cells may express four or more keratins but adult hepatocytes are unique among glandular epithelia in that they express K8/ K18 exclusively [11]. K8/K18 are the major constituents of MBs [7,11,12] and animal model studies showed that K8 is essential for MB formation [13,14]. Targeting of TG2 as a potential modulator of inclusion diseases is clinically attractive for several reasons. Transglutaminase crosslinks have been found in animal models of MB [15] and in patients with Parkinson [16] and Alzheimer [17] diseases. In addition, transglutaminase inhibition improves the phenotype and prolongs the lifespan of the Huntington mice [18] and mating of these mice with transglutaminase knockouts has a similar effect [19]. A recently described group of TG2 inhibitors [20] offer potential advantages over other existing inhibitors such as monodansylcadaverine (MDC) and cystamine due to their irreversible nature, tolerability (so far tested only in rodents), specificity and short half-life [20]. Based on these findings, we tested the hypothesis that pharmacologic inhibition of TG2 may block MB formation, using the recently described irreversible TG2-inhibitor halo-dihydroisoxazolederivative transglutaminase inhibitor (KCC009) (also termed compound-1b [20]).

2. Materials and methods 2.1. Animal experiments and TG2 activity C3H mice were obtained from Taconic Farms (Germantown, NY) and permitted ad libitum consumption of water and diet. The animal experiments protocol was approved by the institutional Animal Care Committee. For the inhibitor experiments, 2–3-months-old, age- and sex-matched mice were injected intraperitoneally (ip) with KCC009

0014-5793/$32.00  2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.03.051

2352 (20–60 mg/kg) or 110 mg/kg cystamine (Sigma) both dissolved in 60% DMSO in PBS (v/v); or 200 mg/kg MDC (Fluka) (in DMSO). Similar cystamine/MDC concentrations were previously used in rodents [21,22], while KCC009 (chemically synthesized [20]) concentration is based on its upper-range solubility in 60% DMSO. MBs were induced by feeding a powdered Formulab Diet 5008 (Deans Animal Feeds) containing 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) (Aldrich) for 3–3.5 months. Mice were recovered for 4 weeks on a standard diet (termed ‘‘primed mice’’ after recovery, Fig. 1A) then divided into three groups (8 mice/group). One group was given KCC009 alone (20 mg/kg, 3·/d), and the remaining two groups were re-fed the DDC-containing diet (6 d) either alone or with KCC009 (20 mg/kg, 3·/d; first KCC009 dose given 2 h before beginning DDC feeding) in order to induce re-appearance of MBs. Mice were sacrificed by CO2 inhalation 1–2 h after the last injection, and blood was collected by intracardiac puncture for serologic testing. Mouse livers were snap frozen in liquid nitrogen and stored ( 80 C). Thawed tissues were dounced and sonicated in 50 mM mannitol, 2 mM Tris–HCl (pH 7.2). TG activity is based on [1,4-14C]putrescine (Amersham) incorporation into N,N-dimethyl casein (Sigma) [23,24].

P. Strnad et al. / FEBS Letters 580 (2006) 2351–2357 2.2. Histological and immunofluorescence staining, and in situ TG2 activity Tissues were fixed then stained with hematoxylin and eosin. For immunofluorescence staining, freshly-frozen liver pieces were fixed with 3% paraformaldehyde (actin staining) or acetone (all other stainings). Immunofluorescence and in situ TG2-activity staining were performed as described [25,26] using the antibodies (Ab): Troma I (antiK8; [26]), anti-TG2 antibodies CUB7402 and TG100 (Labvision), and anti-fibronectin (Sigma). Cell borders were visualized using Texas red-conjugated wheat germ agglutinin (WGA) and Alexa 488-coupled phalloidin (Molecular Probes). Pictures were obtained using a TE300 microscope, a BioRad confocal and Lasersharp software (Zeiss). For cell size measurement, five random high-power-field images/sample were acquired and cell borders were manually selected and the area determined using Volocity software (Improvision). 2.3. Quantification of keratin-containing MBs and immunoblotting The percent cells with keratin-containing aggregates was estimated using mouse liver sections stained with Troma I Ab. The relative MB content was scored from 0 to 4 which corresponded to 0%, 1– 25%, 26–50%, 51–75% and 76–100% of cells with keratin-containing

Fig. 1. Impact of TG2 inhibition on re-appearance of MBs, animal weights and serology. (A) Schematic of MB induction, priming and treatment algorithm. Inh, the inhibitor KCC009. (B) Hematoxylin and eosin (H&E) (a–c) and K8 immunofluorescence (d–f) staining of livers isolated from mice treated for 6 d with KCC009 (Inh) (a,d), DDC alone (b,e), or DDC + Inh (c,f). Arrows point to MBs which were similarly noted in the DDC and DDC + Inh groups, whereas no MBs were apparent in livers of mice treated with KCC009 alone. Bar = 50 lm (bar in ‘‘c’’, a–c; bar in ‘‘f’’, d–f). (C) Serum transaminases (ALT, AST), alkaline phosphatase (ALP), bilirubin, percent weight loss ([final weight initial weight/initial weight] · 100) and corrected percent weight loss (by subtracting the final liver weight from the final animal weight) are shown. There were no significant differences in serum tests between the primed groups that received DDC or DDC + Inh, while DDC re-challenge led to a significant increase in all serum tests. Serum tests from control mice (two untreated and two vehicle-treated mice) are shown in the upper row, 8 mice were used for the remaining groups. Values are shown as means ± S.D. n.a. = not applicable. When comparing the two groups highlighted by ‘‘*’’, P = 0.02; when comparing the groups highlighted by ‘‘**’’, P = 0.07.

P. Strnad et al. / FEBS Letters 580 (2006) 2351–2357 aggregates (all sizes), respectively. At least 10 fields (20·) were evaluated/section and eight different livers were scored/group. Immunoblotting was done using total liver homogenates (in 2% SDS) and primary Abs [mouse anti-PCNA and anti-tubulin, rabbit anti-TG2 (all Labvision); mouse anti-caspase 9 (Immunotech)] as described [26].

3. Results 3.1. KCC009 is well tolerated in a mouse MB reformation model, but does not affect MB generation In preliminary experiments, we compared the efficacy of three TG2 inhibitors: cystamine, MDC and KCC009. All three led to 25% inhibition of liver TG2 activity (not shown) but MDC and cystamine appeared toxic leading to decreased animal movement and heavy breathing. In contrast, KCC009-injected animals behaved similar to non-injected controls (not shown), which led us to focus on KCC009. Presence of free KCC009 was observed in liver tissue 1 h after drug administration, as confirmed by mass spectrometry (not shown). The dosing frequency of KCC009 was optimized by testing TG2 activity in mouse liver at different intervals after a single ip injection. The inhibitory activity of KCC009 remains up to 3 h but rebounds by 8 h (not shown). Hence, we used a 3·/d ip dosing in the experiments described below. In order to test the potential role of TG2 in MB formation, we used the well-established rapid mouse MB reformation model (Fig. 1A), whereby MB are initially induced by DDC feeding, after which mice recover and most if not all MBs resolve after regular diet feeding for one month (which generates ‘‘primed mice’’) followed by re-feeding with DDC for <7 d which induces a robust MB formation [27,28]. The effect of KCC009 on MB re-formation (rather than formation) was tested during DDC re-feeding (instead of initial feeding), which allows a finite use of the drug and avoids what would otherwise be prohibitively large-scale synthesis. We administered KCC009 alone or in combination with DDC for 6 d and compared the liver phenotype to livers from DDC re-fed mice (Fig. 1A). KCC009 did not significantly alter the toxicity of the re-introduced DDC, as determined by histological and

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serologic assessment (Fig. 1B and C). However, the KCC009 + DDC led to a significantly higher weight loss when compared to administering either drug individually (Fig. 1C). This difference is related at least in part to the smaller liver enlargement seen in the DDC + Inh regimen (see below), since the corrected weight loss (excluding liver weights) difference becomes smaller (Fig. 1C). With regard to MBs, DDC re-feeding with or without simultaneous administration of KCC009 for 6 d led to re-appearance of keratin-containing aggregates (Fig. 1B; arrows in panels e and f), which were also apparent in histological staining (Fig. 1B; arrows in panels b and c). There was no significant difference in MB formation upon KCC009 administration (MB scores were 2.4 and 2.9 for mice treated with DDC alone and DDC + KCC009, respectively). 3.2. TG2 activity and distribution in response to DDC We measured liver TG2 activity and protein levels. DDC treatment caused a 4-fold increase in TG2 activity, which returned to near baseline after 4 weeks of recovery. DDC refeeding elevated the TG2 activity 2.5-fold but less so when KCC009 was included (Fig. 2A). The TG2 activity level changes paralleled changes at the protein level as determined by immunoblotting using anti-TG2 Ab (Fig. 2B). Similar changes in transglutaminase activity were previously described when mice were fed the MB-inducing agent griseofulvin [29]. Next we examined whether upregulation of TG2 after DDC re-challenge affects its distribution. A similar TG2 staining pattern was found in DDC + Inh versus Inh-alone livers although TG2 staining was brighter after DDC exposure with a prominent staining that co-localizes with the extracellular matrix protein fibronectin (Fig. 3). Staining of TG2 protein and its activity showed significant co-localization although some areas stained with anti-TG2 antibody but did not show any activity staining (Fig. 4A, a–c). A significant portion of TG2 activity did not co-localize with cytoplasmic K8 (Fig. 4A, d–f; Fig. 4B) but co-localized with fibronectin (Fig. 4B, arrows in panel a) and had intense staining just outside the keratin staining (Fig. 4B, arrowheads in panel b). In addition to extracellular TG2 staining, there was also diffuse cytoplasmic staining

Fig. 2. Upregulation of TG2 in the liver after DDC feeding. (A) TG2 activity was measured in livers (2 mice/measurement) of the indicated groups. Mice were sacrificed 1–2 h after the last KCC009 dose. TG2 activity (y-axis) represents pmol incorporated putrescine/mg protein/min. (B) Total tissue homogenates were obtained from the livers used in panel A and used for immunoblotting with anti-caspase 9 (used as loading control) and anti-TG2 Ab. Equal amounts of protein were loaded/lane, and each lane corresponds to a homogenate from an individual liver. Asterisk points to a TG2 degradation product seen in mice fed DDC for 3.5 months. The TG2 blot of ‘‘Re + Inh’’ had a similar intensity to the ‘‘Re alone’’ intensity (not shown, but analyzed on a separate gel).

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Fig. 3. Immunofluorescence staining shows co-localization of fibronectin and TG2 in livers of re-challenged animals. Livers from the indicated treatments were double-stained using antibodies to fibronectin (a–c; initial fluorochrome of the secondary antibody was green) and TG2 (d–f; initial fluorochrome was red). The merged (green + red) images are displayed in the bottom panels. Note the extensive co-localization of TG2 and fibronectin. Bar = 50 lm.

without obvious co-localization with keratin filaments (Fig. 4B). The TG2 distribution was largely identical when visualized with two independent anti-TG2 antibodies (Figs. 3 and 4A) or with a TG2 in situ activity assay (Fig. 4A). 3.3. TG2 inhibition blocks DDC-induced hepatocyte hypertrophy DDC treatment for 3.5 months led to more than a 3-fold increase in liver size, which returned to near normal after four weeks of recovery (Fig. 5A). DDC re-feeding of primed mice for 6 d caused a 75% increase in liver size whereas treatment with KCC009 had no significant effect (Fig. 5A). Interestingly, simultaneous administration of KCC009 and DDC for 6 d led to only a 57% increase in liver size, which represents 24% inhibition of the DDC-induced liver enlargement (Fig. 5A). The KCC009 effect was not related to cell proliferation since proliferating cell nuclear antigen (PCNA) levels were similar in the KCC009 +/ groups (Fig. 5B). However, the effect of KCC009 on DDC-induced hepatomegaly is related to inhibition of hepatocyte hypertrophy as determined by quantification of hepatocyte cell size (Fig. 5C and D). As such, the average difference in hepatocyte cell area of 67 lm2 (358–291 = 67 lm2) (Fig. 5D) represents a 19% inhibition in cell size enlargement, which is consistent with the 24% observed inhibition in liver weight increase (Fig. 1C).

4. Discussion The major conclusions of our study are: (i) modest inhibition of TG2 in the liver by KCC009 has no effect on MB formation, but has a significant effect on DDC-induced hepatomegaly which suggests a role for TG2 in hepatocyte hypertrophy, and (ii) the effect of KCC009 on hepatocyte hypertrophy likely represents a primarily ‘‘extracellular response’’ based on triple staining results of TG2, TG2 activity and intra/extra cellular markers (K8 and fibronectin, respectively). KCC009 acts as a pseudosubstrate that irreversibly inhibits only the active enzyme pool [20]. In general, 70–80% of TG2 is intracellular in a dormant state due to low Ca2+ and high GTP, whereas extracellular TG2 is preferentially active [30]. Therefore, we posit that the limited liver TG2 inhibition by KCC009 likely corresponds to inhibition of the catalytically-active extracellular pool. Support for extracellular TG2 protein/activity are demonstrated by co-localization of TG2, TG2 activity and fibronectin (Figs. 3 and 4). KCC009 did not have an added toxic effect when co-administered with DDC (Fig. 1C), but higher doses may be mildly hepatotoxic when given alone for 14 d ([20] and not shown). Inhibition of TG2 by KCC009 did not affect MB reformation upon DDC re-challenge in our experimental system. This raises the possibility that TG2 may be more relevant for MB

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Fig. 4. Localization of TG2 staining and activity in relation to K8 and fibronectin. (A) Livers from mice with the indicated treatments were stained to show: TG2 protein and activity (red and green, respectively; a–c), TG2 activity and K8 protein (green and blue, respectively; d–f) and an overlay (g–i). Bar = 50 lm. (B) High magnification images of livers from untreated control mice show extensive co-localization between TG2 and fibronectin immunostaining (arrows in panel a) and the presence of significant TG2 activity (arrowheads in panel b) outside the K8 staining that locates on the inner aspect of the cell perimeter keratin-enriched zone (b). Bar = 10 lm.

formation as compared with reformation, or that it may not play any role in MB formation. If so then TG2-mediated keratin crosslinking seen in MBs [15] may not be important for MB formation or may be more relevant for MB formation rather than reformation. For example, the highly stable TG2 crosslinks induced after prolonged DDC feeding may persist in recovered mice and induce a rapid TG2-independent MB re-appearance. However, TG2 induction is similar upon initial and re-challenge exposures to DDC (Fig. 2), which suggests that TG2 is likely to respond similarly in both biologic contexts. Alternatively, TG2-mediated crosslinking may not be di-

rectly involved in protein aggregate formation, or may be important depending on the inclusion type or experimental setting. Also, TG2 crosslinking effects may be counterbalanced by its role in apoptosis (e.g., promotion of cell death of inclusioncontaining cells), adhesion or cell signaling [1,2,30]. Furthermore, experiments using pharmacological and genetic ablation of TG2 may vary due to confounding factors inherent to both models. For example, neuronal inclusions became more prominent when Huntington mice were mated with transglutaminase knockouts [19]. However, cystamine treatment of Huntington mice decreased the number and size of aggregates

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Fig. 5. Effect of KCC009 on DDC-induced liver enlargement. (A) The percent liver to body weight is shown for the indicated mouse groups. Each category of re-challenged mice included livers from 8 mice, while livers from 4 mice were used in the remaining groups. (B) Equal amounts of liver homogenates (from the mice in Panel A) were immunoblotted using antibodies to tubulin (loading control) and PCNA. (C) and (D) Liver sections from the indicated groups were double-stained with phalloidin (F-actin) and wheat germ agglutinin (WGA; binds carbohydrate moieties of the cell surface). The average cell size was determined in four randomly selected mice (1–4) from each group of 8 mice. The mean cell area (±S.D.) is shown for each group. Bar = 50 lm.

and delayed the neuropathological sequela [22], albeit cystamine has additional non-TG2-related effects such as caspase inhibition [31]. In contrast, TG inhibitors, including KCC009 and MDC, have pro-apoptotic effects that enhance chemosensitivity of glioblastomas [32]. The effect of KCC009 on hepatocyte hypertrophy and the link between TG2 and hepatocyte size is a novel finding. This in vivo link may be due to intracellular or/and extracellular effects. Relevant to these findings, TG2 mediates, possibly via extracellular effects, chondrocyte hypertrophy; and cardiac TG2 over-expression induces organ enlargement [33,34]. A TG2 extracellular effect on hepatocyte hypertrophy may be supported by the established role of TG2 in crosslinking extracellular matrix proteins, which may provide mechanical extracellular stabilizing effects to promote cell growth. In the experimental system herein, further support is based on the lack of a KCC009 effect on MB formation (a predicted intracellular effect). Taken together, our findings suggest that TG2 is a mediator of hepatic hypertrophy which meshes well with the up-regulation of TG2 in multiple liver injury models and the associated observed liver enlargement. Acknowledgements: We are grateful to Evelyn Resurrection for assistance with immunofluorescence staining. This work was supported by National Institutes of Health (NIH) Grant DK52951 and the Department of Veterans Affairs (M.B.O.), NIH Grant DK063198 (C.K.), Digestive Disease Center NIH Grant DK56339 and a European Molecular Biology Organization postdoctoral fellowship (P.S.).

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