W F1 mice

International Immunopharmacology (2008) 8, 589–596

w w w. e l s e v i e r. c o m / l o c a t e / i n t i m p

Treatment with cystamine reduces apoptosis in liver from NZB/W F1 mice Bor-Show Tzang a,⁎, Szu-Yi Chiang b , Wen-Xian Lai a , Chun-Chou Tsai c , Jen-Huang Wu a , Tsai-Ching Hsu c,⁎ a

Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan, ROC Department of Health, Executive Yuan, Hua-Lien Hospital, Hua-Lien, Taiwan, ROC c Institute of Immunology, Chung Shan Medical University, Taichung, Taiwan, ROC b

Received 14 November 2007; received in revised form 17 December 2007; accepted 13 January 2008

KEYWORDS Systemic lupus erythematosus (SLE); Liver; Apoptosis; Cystamine; NF-κB

Abstract Increased population with hepatic diseases and apoptosis were found in patients with SLE and implicated in the pathogenesis of SLE. Since cystamine has been demonstrated to be beneficial to NZB/W F1 mice in our previous report, this study intends to investigate the effects of cystamine in liver from NZB/W F1 mice. Decreased apoptosis was detected in liver from NZB/W F1 mice given cystamine as compared to those given PBS by TUNEL and caspase-3 activity assay. Fasdependent apoptotic proteins including Fas, cleaved caspase-8 and tBid were reduced in liver from NZB/W F1 mice given cystamine as compared to those given PBS. Additionally, the mitochondria-dependent apoptotic proteins including cytochrome c and Apaf-1 were reduced in liver from NZB/W F1 mice given cystamine as compared to those given PBS. Moreover, increased BCL-2 protein was observed in liver from both mice. Notably, increased NF-κB protein was detected in liver from NZB/W F1 mice given cystamine as compared to those given PBS. These experimental results suggest the effect of cystamine in reducing apoptosis in liver from NZB/W F1 mice through Fas-dependent and mitochondrial-dependent pathways. The phosphorylation of NF-κB (p65) could be a possible mechanism involving anti-apoptotic effects of cystamine in liver from NZB/W F1 mice. © 2008 Elsevier B.V. All rights reserved.

1. Introduction

⁎ Corresponding authors. Hsu is to be contacted at fax: +886 4 23248172. Tzang, fax: +886 4 23248195. E-mail addresses: [email protected] (B.-S. Tzang), [email protected] (T.-C. Hsu). 1567-5769/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2008.01.010

Systemic lupus erythematosus (SLE) is known as an autoimmune disorder with unknown etiology [1] that impacts various organs including skin, joints, cardiovascular system, nervous system, kidneys and liver [2–7]. However, the underlying pathogenic mechanisms of associated tissues or organs in SLE are still obscure. Recent reports indicated that

590 increased population with liver disease was found in patients with SLE [8–10]. A previous study of 19 patients with SLE indicated that 11 patients showed liver abnormality including fatty change, portal tract fibrosis, cellular infiltration, or even cirrhosis [8]. Another study of patients with SLE indicated that 124 of 206 patients tested had at least one abnormal result, and 43 met strict criteria for the existence of liver disease [9]. Although the occurrence of liver disease is not routinely screened, the incidence of hepatic abnormality in patients with SLE was reported as varying from 12% to 55% [10]. Increased Fas and FasL have been implicated in the pathogenesis of SLE [11,12] since the precise role of Fas and FasL in the pathogenesis of SLE is still unclear. Recently, Fas and FasL, the apoptosis-associated molecules, have been implicated in liver damage in patients with SLE by causing liver dysfunction and apoptosis [6,7]. Fas (CD95/Apo-1) is a TNF-receptor alike type 1 membrane protein with a molecular weight of 45–48 kDa [13] and known to be associated with apoptosis in various cells [14]. Moreover, growing evidences and consensus exist that impaired functions of

B.-S. Tzang et al. macrophages in clearance for dying cells are contributed to the accumulation of apoptotic cells in tissues of SLE patients [15–17]. Therefore, these findings indicated that persistence of apoptosis and impaired clearance of apoptotic cells in liver is intimate with the pathogenesis of SLE. Cystamine is known to be an inhibitor of transglutaminase 2 (TG2) by interfering TG2 activity [18,19]. Cystamine is also reported to inhibit caspase-3 activity and indicated in preventing apoptosis [20,21]. Additionally, cystamine has been demonstrated as playing important roles in neuroprotection [22] and prolonged survival and decreased abnormal movements in a transgenic model of Huntington disease [23]. Notably, our recent experimental results demonstrated the beneficial effects of cystamine in reducing MMP-9 activity, TNF-α and TGF-β mRNA expression in macrophage from NZB/W F1 mice and generation of anti-cardiolipin autoantibody level [24]. However, rare study of cystamine in liver apoptosis in SLE is investigated. In this study, we identified the protective effects of cystamine in liver from NZB/W F1 mice against apoptosis.

Figure 1 Detection of apoptosis. (A) Activity of caspase-3 was measured in 20 μg liver lysates from BALB/c or NZB/W F1 mice that were given PBS or cystamine at the age of 188-day or 202-day, respectively. The UV-induced apoptotic U937 lysate comes along with the kit was used as positive control. (B) TUNEL assay of liver sections from NZB/W F1 mice given PBS or cystamine. FITC-labeled terminal deoxy-transferase was bound to nicked end of DNA as arrows indicated. Three independent experiments were performed and ⁎ indicates significant difference.

Treatment with cystamine reduces apoptosis in liver from NZB/W F1 mice

2. Materials and methods 2.1. Materials Sodium chloride (NaCl), potassium chloride (KCl), sodium phosphate dibasic dodecahydrate (NA2HPO4–12 H2O), potassium phosphate monobasic (KH2PO4), paraformaldehyde, cystamine, 2-mercaptoethanol, sodium dodecyl sulfate (SDS), glycerol, Tris–HCl were purchased from Sigma (St. Louis, USA). OCT compound was from Tissue-Tek (Miles Inc., Elkhart, IN, USA). TUNEL assay kit was from Roche (Applied Science, Inc., USA). Caspase-3 ELISA kit was from BD Pharmingen (San Diego, CA, USA). Nitrocellulose membrane was from Amersham Biosciences Piscataway (NJ, USA). Antibodies against Fas, caspase-8, cytochrome c, Apaf-1, BCL-2, tBid, AKT-p, Erk1/2-p, cJUN-p, p38-p and NF-κB (p65-p) and actin were from Upstates (Charlottesville, Virginia, USA). Pierce's Supersignal West Dura HRP Detection Kit was from Pierce Biotechnology Inc. (Rockford, IL).

2.2. Mice and liver samples Female NZB/W F1 mice were purchased from National Taiwan University, Laboratory Animal Center, Taiwan and housed under supervision of the Institutional Animal Care and Use Committee at Chung Shan Medical University, Taichung, Taiwan. NZB/W F1 mice is a well-known lupus mice strain and the clinical symptoms will appear at the age of 8 to 12 weeks and will reach the peak at the age of 24 to 28 weeks [25–27]. Disease activity was determined by monitoring the proteinuria biweekly with Albustix test strips from the age of 14 weeks for ten weeks as described previously [24] and scored according to the manufacturer's scoring system (Bayer Diagnostics, Hong Kong). Mice of 188-day-old were divided into two groups (8 mice/group) given PBS or cystamine for experiments. For cystamine (Sigma, St. Louis, MO, USA) treatment, NZB/W F1 mice were injected intraperitoneally (ip) with 100 μl of cystamine (10 mM) for 14 days that was recognized as suitable for analysis. [23,24] Liver samples of mice were obtained next to CO2 sacrifice. Mice were sacrificed by CO2 asphyxiation and rinsed in 70% ethanol solution. The abdomen of mouse was then incised and the liver was dissected and stored at −80 °C until use.

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as the representative results. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using 12.5% acrylamide gel, was performed as previously described [28]. Protein samples were denatured for 5 min in boiling water with sample buffer (0.0625 M Tris–HCl buffer, pH 6.8, containing 2.3% SDS, 5% 2-mercaptoethanol, and 10% glycerol). Samples applied to the gel were run at 100–150 V for 1.5 h and electrophoretically transferred to nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA). The membrane was then soaked in PBS with 5% nonfat dry milk for 30 min at room temperature to saturate irrelevant protein binding sites. Antibodies against Fas, caspase-8, cytochrome c, Apaf-1, BCL-2, tBid, AKT-p, Erk1/2-p, c-JUN-p, p38-p and NF-κB (p65-p) and actin (Upstates, Charlottesville, Virginia, USA) were diluted in PBS with 2.5% BSA and incubated for 1.5 h with gentle agitation at room temperature. The membranes were washed twice with PBS-Tween for 1 h and secondary antibody conjugated with horseradish peroxidase (HRP) was added. Pierce's Supersignal West Dura HRP Detection Kit (Pierce Biotechnology Inc., Rockford, IL) was used to detect antigen–antibody complexes. The blots were also scanned and quantified by densitometry (Appraise, Beckman-Coulter, Brea, California, USA).

2.6. Statistical analysis The paired t-test was used to analyze for statistical significance. A P value b 0.05 was considered significant. 3. Results 3.1. Cystamine reduces apoptosis in liver from NZB/W F1 mice Liver dysfunction could induce apoptosis that is reported in patients with SLE and considered as an important factor for self-antigens exposure [6,7]. To clarify the anti-apoptotic effect of cystamine in liver in SLE, liver samples from NZB/W F1 mice were detected with TUNEL and caspase-3 activity assay. Higher increase of caspase-3 activity was detected in liver from NZB/W F1 mice at the age of 188day before receiving the cystamine. Notably, significant reduction of

2.3. TUNEL assay Liver tissues obtained as described above were embedded into OCT compound (Tissue-Tek, Miles Inc., Elkhart, IN, USA) and snap frozen in liquid nitrogen. The frozen tissue blocks were sectioned at 5 μm and fixed in 4% paraformaldehyde (Sigma, Saint Louis Mo, USA) in 0.1 M PBS, pH 7.4 for 20 min at RT. After washing for 30 min with 0.1 M PBS, the tissue sections were incubated with 3% H2O2 in methanol for 10 min at RT. TUNEL reaction mixture was freshly prepared according to the manufacture's instruction (Roche Applied Science, Inc., USA) and a total volume of 100 μl terminal deoxytransferase reaction mixture was incubated with the tissue sections for 1 h at RT in the dark. The tissue sections were then rinsed with 0.1 M PBS and observed with a fluorescence microscope.

2.4. Caspase-3 activity assay A caspase-3 ELISA kit (BD Pharmingen, San Diego, CA, USA) was used for in vitro determination of caspase-3 enzymatic activity in 20 μg liver lysates derived from NZB/W F1 mice given PBS or cystamine according to the manufacturer's instruction.

2.5. Immunoblotting The liver samples from the 8 mice of each group were analyzed for immunoblotting and similar results were observed in the 8 mice of the same group. Three random selected mice from each group were used

Figure 2 Expression of Fas protein in liver from NZB/W F1 mice. (A) Liver lysates obtained from 3 different NZB/W F1 mice given PBS or cystamine were probed with anti-Fas antibody. (B) Densitometric analysis is shown in the lower panel and the P value is 0.0167. Similar results were obtained in three independent experiments and ⁎ indicates significant difference.

592 caspase-3 activity (P= 0.004) was also detected in liver from NZB/W F1 mice given cystamine as compared to those given PBS (Fig. 1A). No significant variation of caspase-3 activity was observed in livers from BALB/c mice at the age of 188-day or 202-day that received cystamine or not. Additionally, Fig. 1B illustrates the fluorescence results of nicked-DNA labeled by FITC-labeled terminal deoxy-transferase (TdT) in liver from NZB/W F1 mice treated with PBS or cystamine. However, apparent nicked-DNA was detected in liver from NZB/W F1 mice given PBS, whereas significant reduction of nicked-DNA was observed in those given cystamine.

B.-S. Tzang et al. 3.2. Cystamine reduces the expression of Fas-dependent apoptotic proteins in liver from NZB/W F1 mice

Since Fas engagement is known to induce hepatic cell apoptosis [29], here we investigate the effect of cystamine on Fas-dependent apoptosis in liver from NZB/W F1 mice. The expressions of Fas, caspase-8 and tBid proteins were examined by immunoblot. As shown in Fig. 2, significant reduction of Fas protein was detected in liver from NZB/W F1 mice treated with cystamine as compared to those treated

Figure 3 Expression of caspase-8 protein in liver from NZB/W F1 mice. (A) Liver lysates obtained from 3 different NZB/W F1 given with PBS or cystamine were probed with anti-caspase-8 antibody. (B) Densitometric analyses are shown below and the P values are 0.003, 0.042 and 0.037, respectively. Similar results were obtained in three independent experiments and ⁎ indicates significant difference.

Treatment with cystamine reduces apoptosis in liver from NZB/W F1 mice

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with PBS (Fig. 2). The expression of caspase-8, a downstream molecule of Fas protein, was also examined. Fig. 3 shows the immunoblot results of procaspase-8 and its two cleaved forms with a molecular weight at 40 kDa and 23 kDa, respectively. Notably, significantly increased expression of procaspase-8 was detected in liver from NZB/W F1 mice treated with cystamine as compared to those treated with PBS (Fig. 3). The two cleaved forms of caspase-8 at molecular weight of 40 kDa and 23 kDa were found to be decreased significantly in liver from NZB/W F1 mice treated with cystamine (Fig. 3). Additionally, significantly reduced tBid, the downstream molecular of caspase-8, was detected in liver from NZB/W F1 mice treated with cystamine as compared to those treated with PBS (Fig. 4). 3.3. Cystamine reduces the expression of mitochondriadependent apoptotic proteins in liver from NZB/W F1 mice To further clarify whether cystamine influences other apoptotic pathway, we also examine the molecules involving mitochondriadependent apoptotic pathway. Fig. 5 illustrates the representative immunoblot results of mitochondria-dependent apoptotic proteins. Notably, cytochrome c protein was reduced significantly in liver from NZB/W F1 mice as compared to those treated with PBS (Fig. 5A). Additionally, Apaf-1 protein, a downstream molecule of cytochrome c, was also examined and the expression of Apaf-1 protein was significantly decreased in liver from NZB/W F1 mice as compared to those treated with PBS (Fig. 5B). Since BCL-2 is reported to play an important role in inhibiting cytochrome c expression [30], herein we further examined the influence of cystamine on BCL-2 protein expression. Intriguingly, the expression of BCL-2 protein was significantly increased in liver from NZB/W F1 mice treated with cystamine as compared to those treated with PBS (Fig. 6). 3.4. The NF-κB signaling pathway participates in cystamine mediated anti-apoptosis in liver from NZB/W F1 mice To clarify the possible signaling pathway involved in the antiapoptosis in liver from NZB/W F1 mice treated with cystamine,

Figure 5 Expression of mitochondrial-dependent apoptotic proteins in liver from NZB/W F1 mice. Liver lysates obtained from 3 different NZB/W F1 mice given PBS or cystamine were probed with (A) anti-cytochrome c and (B) anti-Apaf-1 antibodies. Densitometric analysis is shown in the lower panel and the P values are 0.039 and 0.001, respectively. Similar results were obtained in three independent experiments and ⁎ indicates significant difference.

various signaling molecules including, AKT-p, Erk1/2-p, c-JUN-p, p38-p and NF-κB (p65-p), were examined. Notably, the expression of NF-κB (p65-p) protein was significantly increased in liver from NZB/W F1 mice treated with cystamine as compared to those treated with PBS (Fig. 7). However, no significant variations of AKT-p, Erk1/2-p, cJUN-p and p38-p were observed (data not shown).

4. Discussion Figure 4 Expression of tBid protein in liver from NZB/W F1 mice. (A) Liver lysates obtained from 3 different NZB/W F1 mice given PBS or cystamine were probed with anti-Fas antibody. (B) Densitometric analysis is shown in the lower panel and the P value is 0.035. Similar results were obtained in three independent experiments and ⁎ indicates significant difference.

Apoptosis induced by liver dysfunction is associated with the pathogenesis of SLE [12]; however, the precise mechanism involved and the possible treatments are still unclear. This study indicated the anti-apoptotic function of cystamine in liver from NZB/W F1 mice by inhibiting Fas- and mitochondria-dependent cell death pathways. Moreover, the phosphorylation of NF-κB

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Figure 6 Expression of Bcl-2 protein in liver from NZB/W F1 mice. (A) Liver lysates obtained from 3 different NZB/W F1 mice given PBS or cystamine were probed with anti-Bcl-2 antibody. (B) Densitometric analysis is shown in the lower panel and the P value is 0.003. Similar results were obtained in three independent experiments and ⁎ indicates significant difference.

(p65) protein could be a possible mechanism involving antiapoptotic effects of cystamine in liver from NZB/W F1 mice. Liver is the largest gland of the human body with various functions that maintain the homeostasis. Indeed, dysfunction of the liver in SLE is more common than previously recognized and known as a primary disorder associated with the pathogenesis of SLE [10]. Apoptosis is known to occur by two major pathways including death-receptor and organellebased intrinsic pathway [31]. However, the mechanism of apoptosis in liver is complicated and appears to be usually triggered through activation of death receptors including Fas [32]. Recently, serum soluble Fas (sFas) [33] and Fas ligand (FasL) on T cells [34] have been associated with liver injury and apoptosis in mice. Growing studies have also implicated that up-regulated expression of Fas and FasL contributed to the pathogenesis of SLE [12,35] including liver injury by causing hepatic dysfunction, apoptosis and fibrosis [6,7,36,37]. Moreover, delivery of Fas siRNA reduces liver damage and improves the survival of a septic C3H/HeN male mice model [38]. In this study, beneficial effects of cystamine are observed in NZB/W F1 mice. Elevated Fasand mitochondria-dependent apoptosis in liver from NZB/W F1 mice were detected. However, significant reductions of Fas, cleaved caspase-8, tBid, cytochrome c and Apaf-1 were observed in liver from NZB/W F1 mice treated with cystamine. These results indicated the complicated apoptotic machinery in liver from NZB/W F1 mice and suggest the multiple anti-apoptosis effects of cystamine in liver from NZB/W F1 mice by inhibiting of Fas- and mitochondriadependent pathways. Previous studies have demonstrated the involvement of NF-κB in anti-apoptosis in various cell types including human

B.-S. Tzang et al. synovial fibroblasts, squamous cell, gastric epithelium cells, mouse B cells and human leukemic cells [39–44]. Moreover, a recent study reported the protective effect of NF-κB by preventing TNF-induced apoptosis in beta-cells of autoimmune type 1 diabetes mice [45]. In this study, increased phosphorylation of NF-κB (p65) was detected in liver from NZB/W F1 mice treated with cystamine and provide an explanation of cystamine in its anti-apoptotic effects in liver from NZB/W F1 mice. Since the role of NF-κB in liver from SLE is still unclear, further investigations are merited to clarify the underlying mechanisms. Although cystamine is known as an inhibitor of transglutaminase (TG) [19], it has been recently demonstrated to inhibit caspase-3 through TG-independent pathway [20]. Cystamine is known to inhibit thiol-dependent enzymes activity or proteins interaction by forming a mixed disulfide [18,19,22]. Recently, cystamine is demonstrated to prolong survival and decrease abnormal movements in a transgenic model of Huntington disease [23]. Additionally, the dimer form of cystamine, cysteamine, is now under Phase I clinical trail in patients with Huntington's disease [46]. In our recent study, beneficial effects of cystamine are demonstrated in macrophage from NZB/W F1 mice by reducing MMP-9 activity, TNF-α and TGF-β mRNA expression and generation of anti-cardiolipin autoantibody [24]. Up to now, only rare study directly indicated the hepatic apoptosis in patients with SLE [7] and no related report was found in studying the anti-apoptotic effect of drugs in treatment with SLE. In the current study, we firstly investigated the effect of cystamine on anti-hepatic apoptosis in NZB/W F1 mice. We demonstrated the possible machineries of cystamine against apoptosis in liver from NZB/W F1 mice including reduction of Fas-dependent apoptosis, mitochondria-dependent apoptosis and phosphorylation of NF-κB and suggested the potentials of cystamine in treatment of SLE.

Figure 7 Expression of phosphorylated p65 protein in liver from NZB/W F1 mice. (A) Liver lysates obtained from 3 different NZB/W F1 mice given PBS or cystamine were probed with antiphosphorylated p65 antibody (NF-kB). (B) Quantified results were shown in the lower panel and the P value is 0.042. Similar results were obtained in three independent experiments and ⁎ indicates significant difference.

Treatment with cystamine reduces apoptosis in liver from NZB/W F1 mice

Acknowledgements This study was supported by the grants DOH96-TD-9627 and NSC96-2320-B-040-025-MY3 from the National Science Council and Department of Health, Taiwan, Republic of China.

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