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European Journal of Pharmacology 579 (2008) 382 – 389 www.elsevier.com/locate/ejphar
Transglutaminase inhibitor cystamine alleviates the abnormality in liver from NZB/W F1 mice Tsai-Ching Hsu a , Chih-Yang Huang b,c,d , Szu-Yi Chiang e , Wen-Xian Lai f , Chang-Hai Tsai d , Bor-Show Tzang f,⁎ a Institutes of Immunology, Chung Shan Medical University, Taichung, Taiwan, ROC Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan, ROC c Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan, ROC d Department of Healthcare Administration, Asia University, Taichung, Taiwan, ROC e Department of Health, Executive Yuan, Hua-Lien Hospital, Hua-Lien 97061, Taiwan, ROC Institutes of Biochemistry and Biotechnology, Chung Shan Medical University, No. 110, Section 1, Chien Kuo N. Road, Taichung 40203, Taiwan, ROC b
f
Received 25 May 2007; received in revised form 21 October 2007; accepted 22 October 2007 Available online 30 October 2007
Abstract Increased hepatic abnormality has been observed in patients with systemic lupus erythematosus (SLE) and contributes to the elevated apoptosis that results in severe disease activity. Since cystamine has been demonstrated to be beneficial for NZB/W F1 mice, this study investigates the effects of cystamine on various inflammatory and stress-related proteins in liver from NZB/W F1 mice. Nephelometric analyses and immunoblots were conducted to detect aspartate aminotransferase (AST), alanine aminotransferase (ALT), C-reactive protein (CRP), p53, p21, Gadd45, heat shock protein 70 (HSP70) and cyclooxygenase-2 (COX-2). AST and ALT were reduced in NZB/W F1 mice that were given cystamine and CRP, p53, p21, Gadd45, HSP70 and COX-2 proteins in the liver were reduced in NZB/W F1 mice that were treated with cystamine. Moreover, cystamine has no obvious effect on BALB/c mice. These findings suggest that cystamine reduces the inflammation in liver of NZB/W F1 mice and provide a clue in treatment of SLE with liver abnormality. © 2007 Elsevier B.V. All rights reserved. Keywords: Systematic lupus erythematosus (SLE); Liver; Cystamine; NZB/W F1 mice
1. Introduction Systematic lupus erythematosus (SLE) is known as a systemic autoimmune disorder that affects various organs including liver (Hahn, 1993). Although the liver is not regarded as a major target organ for damage in patients with SLE, hepatic disease is more common in SLE than is usually reported. Indeed, evidence has revealed the involvement of liver abnormality in the pathogenesis of SLE (Abraham et al., 2004; Lu et al., 2006). Therefore, various inflammatory and stress proteins related to the liver have been involved in the pathogenesis of SLE, including C-reactive protein (CRP), aspartate aminotransferase (AST), alanine aminotransferase ⁎ Corresponding author. Tel.: +886 4 24730022x11680; fax: +886 4 23248195. E-mail address: [email protected] (B.-S. Tzang). 0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.10.059
(ALT) (Zein et al., 1979; Herlong, 1994), and heat shock proteins (HSPs) (Dhillon et al., 1993). CRP is known as a prototypic marker of inflammation in various diseases (Kushner et al., 2006) and associated with pathogenesis in SLE (Zein et al., 1979). An elevated level of CRP has been detected in serum from patients with SLE and is associated with such disease activity as vascular stiffness in women and cognitive deficits (Selzer et al., 2001; Shucard et al., 2007), as well as a previous study indicated that abnormal AST and ALT point to a hepatocyte disorder (Herlong, 1994). Additionally, elevated heat shock proteins, including HSP70, have been detected in patients with SLE and have been demonstrated to play a role in the pathogenesis of SLE which results in the generation of autoantibodies (Dhillon et al., 1993; Conroy et al., 1996; Ripley et al., 2001). Homeostasis between cell proliferation and apoptosis is required for the development and maintenance of organisms.
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During the modulation of apoptosis, various proteins including p53 can alter the balance toward either cell survival or apoptosis (Kanzler and Galle, 2000). A recent report indicated that elevated p53 level is associated with disease activity in patients with SLE (Miret et al., 2003) and suggested the importance in the pathogenesis of SLE by autoantibody production (Kuhn et al., 1999; Herkel et al., 2001). However, little is known about the roles of p53 and its downstream molecules in liver from SLE. Cystamine can interfere with transglutaminase 2 (TG2) (Folk, 1980; Lorand and Conrad, 1984) and prevent apoptosis by inhibiting caspases-3 activity (Lesort et al., 2003; Ientile et al., 2003). Recently, cystamine has been demonstrated to be beneficial in both mouse models of Huntington's disease and SLE (Karpuj et al., 2002; Hsu et al., 2007). This study describes the decline in the abnormality in liver of NZB/W F1 mice by
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treatment with cystamine and provides clues concerning the treatment of SLE with hepatic abnormality. 2. Materials and methods 2.1. Mice and liver sample Female BALB/c and NZB/W F1 mice, a well-described lupus-prone mice strain (Denman et al., 1967; Holborow and Denman, 1967; Russell and Denman, 1969), were purchased from animal center, National Taiwan University, Taiwan and housed under supervision of the Institutional Animal Care and Use Committee at Chung Shan Medical University. 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 (Hsu et al., 2007) and scored according
Fig. 1. Cystamine alleviates AST, ALT, and CRP protein expression in NZB/W F1 mice. Sera was obtained from BALB/c and NZB/W F1 treated with PBS or cystamine and the level of (A) AST and (B) ALT was measured by nephelometric meter as described above and the P values are 0.198, 0.012, 0.042 and 0.029, respectively. The expression of CRP protein in (C), (D) liver and (E) sera from BALB/c and NZB/W F1 was detected by immunoblotting. Densitometric analyses are shown in the lower panel and the P values are 0.259, 0.034 and 0.008, respectively. Similar results were obtained in three independent experiments and ⁎ indicates significant difference.
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to the manufacturer's scoring system (Bayer Diagnostics, Hong Kong). Mice of 188-day-old were divided into four groups (8 mice/group) including BALB/c and NZB/W F1 mice given PBS or cystamine for experiments. For cystamine (Sigma, Saint Louis Mo, USA) treatment, BALB/c and NZB/W F1 mice were injected intraperitoneally (i.p.) with 100 μl of 10 mM phosphate buffer saline (PBS) or 100 μl of 10 mM cystamine daily for 14 days (Karpuj et al., 2002; Hsu et al., 2007). Liver samples of mice were obtained after 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. 2.2. Detection of AST and ALT The mice were sacrificed as described above and the heart sera were collected by direct puncture with a sterilized 1 ml syringe. The serum value of AST and ALT was detected by a
nephelometric system according to the manufacturer's instruction (Cobas Mira, Roche, Swiss). 2.3. Immunoblotting Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using 12.5% acrylamide gel, was performed as previously described (Laemmli, 1970). The liver samples were homogenized sufficiently with B25 High-shear Dispersing Emulsifiers Homogenizing machine (BRT CO Shanghai, China) and centrifuged at 12,000 rpm in 4 °C for 30 min. Supernatants were isolated and 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 of 100–150 V for 90 min 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 CRP, p53, p21WAF1/CIP1, Gadd45, HSP70, COX-2, TG2 and actin (Upstates, Charlottesville, Virginia, USA; Chemicon International, Temecula, CA, USA) were diluted in PBS with 2.5% BSA and incubated for 90 min with gentle agitation at room temperature. The membranes were washed twice with PBS– Tween for 60 min 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 scanned and quantified by densitometry (Appraise, Beckman-Coulter, Brea, California, USA). 2.4. Statistical analysis All the statistical analyses were performed using SPSS 10.0 software (SPSS Inc, Chicago, IL). Three independent experiments were repeated. Statistical analyses were performed using the Student's t test and one-way ANOVA. P b 0.05 was considered statistically significant. 3. Results 3.1. Decreased expression of AST, ALT and CRP in NZB/W F1 mice treated with cystamine
Fig. 2. Expression of p53 protein. (A) Liver lysates obtained from three different BALB/c mice treated with PBS or cystamine were probed with anti-p53 antibody as shown in the upper panel. Densitometric analysis is shown in the lower panel and the P value is 0.631. (B) Liver lysates obtained from three different NZB/W F1 mice treated with PBS or cystamine were probed with antip53 antibody as shown in the upper panel. Densitometric analysis is shown in the lower panel and the P value is 0.0007. Similar results were obtained in three independent experiments and ⁎ indicates significant difference.
Since hepatic abnormality has been associated with various inflammatory or stress-related proteins including AST, ALT and CRP that may contribute to inflammation and apoptosis, this study investigates the influence of cystamine on these proteins in sera or liver lysates from NZB/W F1 mice. Fig. 1A and B reveals the serum level of AST and ALT in BALB/c or NZB/W F1 mice treated with PBS or cystamine. Sera from BALB/c mice that had been treated with PBS or cystamine exhibit no significant variation of either AST or ALT. However, sera from NZB/W F1 mice that had been given cystamine exhibited significantly lower AST and ALT levels than those treated with PBS (P = 0.028 and 0.038, respectively). Moreover, the expression of CRP protein was measured in liver from mice given PBS or
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p21WAF1/CIP1 , was examined by immunoblotting. Fig. 3 illustrates the expression of p21WAF1/CIP1 protein and no significant difference in p21WAF1/CIP1 protein level was detected between the liver from BALB/c mice treated with PBS or cystamine (Fig. 3A). Notably, the expression of the p21WAF1/CIP1 protein was significantly lower in the liver from NZB/W F1 mice (P = 0.003) (Fig. 3B). Gadd45, another downstream molecule of p53, was also examined by immunoblotting (Fig. 4). The expression of Gadd45 protein was significantly lower in liver from NZB/W F1 mice than in those that had been given PBS (P = 0.0008) (Fig. 4A). The Gadd45 protein expression levels in the liver from BALB/c mice given PBS did not detectably differ from those that had been given or cystamine (Fig. 4B). 3.3. Decreased expression of HSP70 protein in NZB/W F1 mice treated with cystamine This study also examines the effect of cystamine on HSP70 expression in liver from NZB/W F1 mice. Fig. 5 shows the immunoblotting results of HSP70 in liver from BALB/c and
Fig. 3. Expression of p21WAF1/CIP1 protein. (A) Liver lysates obtained from three different BALB/c mice treated with PBS or cystamine were probed with antip21 antibody as shown in the upper panel. Densitometric analysis is shown in the lower panel and the P value is 0.241. (B) Liver lysates obtained from three different NZB/W F1 mice treated with PBS or cystamine were probed with antip21 antibody as shown in the upper panel. 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.
cystamine. CRP in liver from NZB/W F1 mice that had been given cystamine was significantly lower than in those treated with PBS (P = 0.024) (Fig. 1D). No significant variation in CRP expression was observed in the BALB/c mice that had been given PBS or cystamine (Fig. 1C). The CRP level in sera from NZB/W F1 mice was significantly reduced, the result of which is consistent with those described above (Fig. 1E). 3.2. Decreased expression of p53, p21WAF1/CIP1 , and Gadd45 protein in NZB/W F1 mice treated with cystamine Immunoblotting of p53 was performed (Fig. 2) to clarify the effect of cystamine on p53 expression in liver from NZB/W F1 mice (Fig. 2). Fig. 2A shows no variation of p53 expression between liver from BALB/c mice that had been treated with PBS or cystamine (P = 0.631). However, in liver from NZB/W F1 mice upon treatment with cystamine had significantly less p53 protein than those that had been given PBS (P = 0.0007) (Fig. 2B). Moreover, the downstream molecule of p53,
Fig. 4. Expression of Gadd45 protein. (A) Liver lysates obtained from three different BALB/c mice treated with PBS or cystamine were probed with antiGadd45 antibody as shown in the upper panel. Densitometric analysis is shown in the lower panel and the P value is 0.357. (B) Liver lysates obtained from three different NZB/W F1 mice treated with PBS or cystamine were probed with antiGadd45 antibody as shown in the upper panel. Densitometric analysis is shown in the lower panel and the P value is 0.0006. Similar results were obtained in three independent experiments and ⁎ indicates significant difference.
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4. Discussion A consensus is forming around increasing evidence that impairment of functions of macrophages in the clearance of dying cells may result in apoptotic-cell accumulation in tissues of SLE patients (Szondy et al., 2003; Grossmayer et al., 2005; Gaipl et al., 2006). Therefore, the persistence of apoptosis and the impaired clearance of apoptotic cells in liver are associated with the pathogenesis of SLE. Although hepatic abnormality is not regarded as a major damage in patients with SLE, liver disease is more common than is usually reported (Mukai et al., 2000; Abraham et al., 2004; Lu et al., 2006). An increasing number of studies indicate that hepatic abnormality in SLE may contribute to apoptosis and promote disease activity. However, the precise mechanism of, and optical treatment for, SLE remain uncertain. This study demonstrates for the first time the beneficial effects of cystamine in SLE mice by reducing the inflammatory and stress responses in liver. The results revealed that sera from NZB/W F1 mice that had been given cystamine had lower levels of AST and ALT than those of mice that had been given PBS. Additionally, significantly reduced expressions of CRP, p53, p21WAF1/CIP1, Gadd45, HSP70, and COX-2 proteins were measured in liver from NZB/W F1 mice. AST and ALT were recognized as inflammatory proteins and released into the blood circulation when hepatic damage occurs in patients with SLE (Zein et al., 1979; Herlong, 1994). Therefore, reduced expression of AST and ALT helps to reduce hepatic inflammation in SLE. According to our experimental
Fig. 5. Expression of HSP70 protein. (A) Liver lysates obtained from three different BALB/c mice treated with PBS or cystamine were probed with antiHSP70 antibody as shown in the upper panel. Densitometric analysis is shown in the lower panel and the P value is 0.279. (B) Liver lysates obtained from three different NZB/W F1 mice treated with PBS or cystamine were probed with antiHSP70 antibody as shown in the upper panel. Densitometric analysis is shown in the lower panel and the P value is 0.0004. Similar results were obtained in three independent experiments and ⁎ indicates significant difference.
NZB/W F1 mice. No significant difference existed between HSP70 protein expressions in liver from BALB/c treated with PBS and that treated with cystamine (P = 0.279) (Fig. 5A). Notably, the expression of HSP70 in liver from NZB/W F1 mice treated with cystamine was significantly lower than in those treated with PBS (P = 0.004) (Fig. 5B). 3.4. Decreased expression of cyclooxygenase-2 (COX-2) protein in liver from NZB/W F1 mice treated with cystamine The immunoblotting of COX-2 protein was performed to investigate the effect of cystamine on COX-2 expression in liver from NZB/W F1 mice (Fig. 6). No detectable expression of COX-2 protein was measured in liver from BALB/c mice that had been treated with PBS or cystamine (Fig. 6A). However, the expression of COX-2 protein was reduced significantly in NZB/W F1 mice treated with cystamine (P = 0.0425) (Fig. 6B).
Fig. 6. Expression of COX-2 protein. (A) Liver lysates obtained from three different BALB/c mice treated with PBS or cystamine were probed with antiCOX-2 antibody as shown in the upper panel. (B) Liver lysates obtained from three different NZB/W F1 mice treated with PBS or cystamine were probed with anti-COX-2 antibody as shown in the upper panel. Densitometric analysis is shown in the lower panel and the P value is 0.0425. Similar results were obtained in three independent experiments and ⁎ indicates significant difference.
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results, reduced ALT and AST were detected in NZB/W F1 mice that had been treated with cystamine suggesting that cystamine alleviates the hepatic inflammation in SLE. Additionally, in the experiments, a reduced CRP level is detected in serum and liver from NZB/W F1 mice that had been treated with cystamine. Since cystamine is known to interfere with and inhibit other thiol-dependent enzymes, it indicates the diverse targets of cystamine. However, these experimental results suggest systemic anti-inflammatory activity of cystamine by reducing CRP expression in live from NZB/W F1 mice. Transglutaminase 2 (TG2) is known as a multi-functional enzyme and implicated in causing apoptosis. As demonstrated in numerous studies, TG2 activity can be inhibited by cystamine via forming a mixed disulfide (Lorand and Conrad, 1984; Jeitner et al., 2005). Since p53 oncoprotein has recently been identified as a substrate of TG2 and elevated TG2 is associated with increased p53 expression and apoptosis (Mishra and Murphy, 2006), this study examines the expression of TG2 in liver from BALB/c and NZB/W F1 mice that were treated with PBS or cystamine, to clarify the effects of cystamine on p53. As expected, TG2 expression in liver from mice treated with PBS did not differ from that of those treated with cystamine as indicated in our previous work (Hsu et al., 2007). Notably, decreased expression of p53 was detected in liver from NZB/W F1 mice that had been treated with cystamine (Fig. 2B) but not BALB/c mice. These results indicate that cystamine would not reduce protein expression of TG2, but did reduce p53 level by inhibiting the serine/threonine kinase activity of TG2. However, the precise mechanism by which cystamine reduces p53 in NZB/W F1 mice warrants further investigation. Since p53 is known to play important roles in balancing apoptosis and survival, its downstream molecules, including p21WAF1/CIP1 and Gadd45 need to be investigated. P53 is known as a tumor suppressor gene and is important to play a role in the pathogenesis and the disease activity of SLE. An elevated level of p53 was detected in patients with SLE contributing to the regulation of the apoptosis of autoreactive lymphocyte (Miret et al., 2003) and the generation of anti-p53 autoantibody (Kuhn et al., 1999; Herkel et al., 2001). The experimental results herein indicate the reduced expression of p53 protein in liver from NZB/W F1 mice that had been treated with cystamine, suggesting the reduced effect of cystamine on p53 expression in liver from NZB/W F1 mice. Additionally, Gadd45 is known to be a stress gene that will be up-regulated during acute inflammation (Zhang et al., 2005). These results herein reveal reduced expression of gadd45 in liver from NZB/W F1 mice that had been treated with cystamine, and reduced expression of p21WAF1/CIP1 in liver from NZB/W F1 mice that had been given cystamine. Since p21WAF1/CIP1 and Gadd45 are two well-known downstream molecules of p53, the levels of these two proteins are supposed to decrease when p53 is down-regulated. Additionally, no variation of p21WAF1/CIP1 and Gadd45 was observed in BABL/c mice, the levels of which may therefore be independent of the decreased expression of p53 and associated with the impaired functions in SLE (Mok and Lau, 2003). Additionally, complicated and multi-factorial interaction among various genetic and environmental factors is
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probably involved in SLE. T cells from patients with SLE have been known to be activated in vivo and to cause autoreactive B cells to exhibit various abnormal expressions, involving signaling molecules and defective signal transduction pathways (Mok and Lau, 2003; Nagy et al., 2005; Hsu et al., 2007). These studies may account for the comparative sensitivity of NZB/W F1 mice to cystamine. However, the precise effects of cystamine on p53 and its downstream molecules in SLE need further clarification. HSPs, especially HSP70, are expressed in both prokaryotic and eukaryotic cells and known to play protective roles in various processes including environmental stress, normal physiological processes and infection (Kiang and Tsokos, 1998). Recently, HSP70 has been implicated in modulating the cellular immune response. Various studies describe the presence of HSP70-peptide complex on the surface of cells in both cultured leukemic K562 and human carcinomatous renal cells, causing a cytotoxic T lymphocyte response (Roigas et al., 1998; Moseley, 2000; Botzler et al., 1996). These data indicate that HSP70 may be involved in modulating both the hazard signals and the antigen carrier. Additionally, HSP70 has been associated with the pathogenesis of SLE, although the precise underlying mechanism is unclear. A recent study demonstrated that HSP70 is deposited along the dermo-epidermal junction in patients with SLE and serves as a transporter or chaperone for autoantigen presentation (Villalobos-Hurtado et al., 2003). Therefore, the reduced expression of HSP70 in liver from NZB/W F1 mice that had been treated with cystamine may alleviate the presentation of autoantigen and provide clues on the treatment of SLE. Cyclooxygenase type 2 (COX-2) is known as a proinflammation gene and plays important roles in inflammation, carcinogenesis, hemodynamics, and renal function (Fung and Kirschenbaum, 1999; Weinberg, 2000). The COX-2– prostanoid pathway has been demonstrated to increase hypertension in animal models and induce the formation of ascites in patients with cirrhosis (Chan and Rodger, 1997; Tilley et al., 2001). The aberrant expression of COX-2 has been well associated with hepatocarcinogenesis. Additionally, accumulating data indicate that increased COX-2 is associated with various liver diseases including inflammatory and malignant bile duct cells (Abiru et al., 2002; Wu et al., 2002; Yu et al., 2006). However, the reduced expression of COX-2 was detected in liver from NZB/W F1 mice that are treated with cystamine in our experimental results, suggesting that cystamine reduces liver inflammation in SLE. A growing body of evidence demonstrates the beneficial effects of cystamine not only in neuro-protection (Karpuj et al., 2002; Jeitner et al., 2005) but also in inhibiting apoptosis (Lesort et al., 2003; Ientile et al., 2003). Additionally, recent studies have indicated the beneficial effects of cystamine in SLE by reducing MMP-9 activity, TNF-a and TGF-b mRNA expression as well as the decline in the level of anti-cardiolipin autoantibody (Hsu et al., 2007). Notably, this study demonstrated the alleviation of stress and inflammatory responses in liver from NZB/W F1 mice, including reduced expressions of CRP, P53, P21, Gadd45, HSP70 and COX-2 protein. However,
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the precise reactions of cystamine on these molecules need further investigations. Altogether, these experimental findings suggest that cystamine mitigates stress and inflammatory responses in liver from NZB/W F1 mice and that cystamine has therapeutic potential in cases of SLE with liver abnormality. Acknowledgements This study was supported by grants NSC 95-2314-B-040010, DOH96-TD-9627 and NSC96-2320-B-040-025-MY3 from the National Science Council and Department of Health, Taiwan, Republic of China. Ted Knoy is appreciated for his editorial assistance. References Abiru, S., Nakao, K., Ichikawa, T., Migita, K., Shigeno, M., Sakamoto, M., Ishikawa, H., Hamasaki, K., Nakata, K., Eguchi, K., 2002. Aspirin and NS398 inhibit hepatocyte growth factor-induced invasiveness of human hepatoma cells. Hepatology 35, 1117–1124. Abraham, S., Begum, S., Isenberg, D., 2004. Hepatic manifestations of autoimmune rheumatic diseases. Ann. Rheum. Dis. 63, 123–129. Botzler, C., Kolb, H.J., Issels, R.D., Multhoff, G., 1996. Noncytotoxic alkyllysophospholipid treatment increases sensitivity of leukemic K562 cells to lysis by natural killer (NK) cells. Int. J. Cancer 65, 633–638. Chan, C.C., Rodger, I.W., 1997. Selective cyclooxygenase-2 inhibitors as potential therapeutic agents for inflammatory diseases. Adv. Exp. Med. Biol. 407, 157–161. Conroy, S.E., Tucker, L., Latchman, D.S., Isenberg, D.A., 1996. Incidence of anti Hsp 90 and 70 antibodies in children with SLE, juvenile dermatomyositis and juvenile chronic arthritis. Clin. Exp. Rheumatol. 14, 99–104. Denman, A.M., Denman, E.J., Holborow, E.J., 1967. Suppression of Coombspositive haemolytic anaemia in NZB mice by antilymphocyte globulin. Lancet 1 (7499), 1084–1086 (May 20). Dhillon, V.B., McCallum, S., Norton, P., Twomey, B.M., Erkeller-Yuksel, F., Lydyard, P., Isenberq, D.A., Latchman, D.S., 1993. Differential heat shock protein overexpression and its clinical relevance in systemic lupus erythematosus. Ann. Rheum. Dis. 52, 436–442. Folk, J.E., 1980. Transglutaminases. Annu. Rev. Biochem. Allied Res. 49, 517–531. Fung, H.B., Kirschenbaum, H.L., 1999. Selective cyclooxygenase-2 inhibitors for the treatment of arthritis. Clin. Ther. 21, 1131–1157. Gaipl, U.S., Kuhn, A., Sheriff, A., Munoz, L.E., Franz, S., Voll, R.E., Kalden, J.R., Herrmann, M., 2006. Clearance of apoptotic cells in human SLE. Curr. Dir. Autoimmun. 9, 173–187. Grossmayer, G.E., Munoz, L.E., Gaipl, U.S., Franz, S., Sheriff, A., Voll, R.E., Kalden, J.R., Herrmann, M., 2005. Removal of dying cells and systemic lupus erythematosus. Mod. Rheumatol. 15, 383–390. Hahn, B.H., 1993. An overview of the pathogenesis of systemic lupus erythematosus. In: Wallace, D.J., Hahn, B.H. (Eds.), Dubois' lupus erythematosus. Williams and Wilkins, Philadelphia, pp. 69–76. Herkel, J., Mimran, A., Erez, N., Kam, N., Lohse, A.W., Marker-Hermann, E., Rotter, V., Cohen, I.R., 2001. Autoimmunity to the p53 protein is a feature of systemic lupus erythematosus (SLE) related to anti-DNA antibodies. J. Autoimmun. 17, 63–69. Herlong, H.F., 1994. Approach to the patient with abnormal liver enzymes. Hosp. Pract. (Off. Ed.) 29, 32–38. Holborow, E.J., Denman, A.M., 1967. Autoimmune disease in inbred mice. Clin. Exp. Immunol. 2, 761–767 (Dec, Suppl). Hsu, T.C., Chiang, S.Y., Huang, C.Y., Yang, C.W., Huang, C.N., Tzang, B.S., 2007. Beneficial effects of treatment with transglutaminases inhibitor cystamine on macrophage response in NZB/W F1 mice. Exp. Biol. Med. (Maywood) 232, 195–203. Ientile, R., Campisi, A., Raciti, G., Caccamo, D., Curro, M., Cannavo, G., Li, Volti G., Macaione, S., Vanella, A., 2003. Cystamine inhibits transglutami-
nase and caspase-3 cleavage in glutamate-exposed astroglial cells. J. Neurosci. Res. 74, 52–59. Jeitner, T.M., Delikatny, E.J., Ahlqvist, J., Capper, H., Cooper, A.J., 2005. Mechanism for the inhibition of transglutaminase 2 by cystamine. Biochem. Pharmacol. 69, 961–970. Kanzler, S., Galle, P.R., 2000. Apoptosis and the liver. Semin. Cancer Biol. 10, 173–184. Karpuj, M.V., Becher, M.W., Springer, J.E., Chabas, D., Youssef, S., Pedotti, R., Mitchell, D., Steinman, L., 2002. Prolonged survival and decreased abnormal movements in transgenic model of Huntington disease, with administration of the transglutaminase inhibitor cystamine. Nat. Med. 8, 143–149. Kiang, J.G., Tsokos, G.C., 1998. Heat shock protein 70 kDa: molecular biology, biochemistry, and physiology. Pharmacol. Ther. 80, 183–201. Kuhn, H.M., Kromminga, A., Flammann, H.T., Frey, M., Layer, P., Arndt, R., 1999. p53 autoantibodies in patients with autoimmune diseases: a quantitative approach. Autoimmunity 31, 229–235. Kushner, I., Rzewnicki, D., Samols, D., 2006. What does minor elevation of Creactive protein signify? Am. J. Med. 119 (166), e17–e28. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–684. Lesort, M., Lee, M., Tucholski, J., Johnson, G.V., 2003. Cystamine inhibits caspase activity. Implications for the treatment of polyglutamine disorders. J. Biol. Chem. 278, 3825–3830. Lorand, L., Conrad, S.M., 1984. Transglutaminases. Mol. Cell. Biochem. 58, 9–35. Lu, M.C., Li, K.J., Hsieh, S.C., Wu, C.H., Yu, C.L., 2006. Lupus-related advanced liver involvement as the initial presentation of systemic lupus erythematosus. J. Microbiol. Immunol. Infect. 39, 471–475. Miret, C., Molina, R., Filella, X., Garcia-Carrasco, M., Claver, G., Ingelmo, M., Ballesta, A., Font, J., 2003. Relationship of p53 with other oncogenes, cytokines and systemic lupus erythematosus activity. Tumour Biol. 24, 185–188. Mishra, S., Murphy, L.J., 2006. The p53 oncoprotein is a substrate for tissue transglutaminase kinase activity. Biochem. Biophys. Res. Commun. 339, 726–730. Mok, C.C., Lau, C.S., 2003. Pathogenesis of systemic lupus erythematosus. J. Clin. Pathol. 56, 481–490. Moseley, P., 2000. Stress proteins and the immune response. Immunopharmacology 48, 299–302. Mukai, M., Bohgaki, T., Notoya, A., Kohno, M., Tateno, M., Kobayashi, S., 2000. Liver dysfunction due to apoptosis in a patient with systemic lupus erythematosus. Lupus 9, 74–77. Nagy, G., Koncz, A., Perl, A., 2005. T- and B-cell abnormalities in systemic lupus erythematosus. Crit. Rev. Immunol. 25, 123–140. Ripley, B.J., Isenberg, D.A., Latchman, D.S., 2001. Elevated levels of the 90 kDa heat shock protein (hsp90) in SLE correlate with levels of IL-6 and autoantibodies to hsp90. J. Autoimmun. 17, 341–346. Roigas, J., Wallen, E.S., Loening, S.A., Moseley, P.L., 1998. Heat shock protein (HSP72) surface expression enhances the lysis of a human renal cell carcinoma by IL-2 stimulated NK cells. Adv. Exp. Med. Biol. 451, 225–229. Russell, A.S., Denman, A.M., 1969. Normal induction and maintenance of tolerance in (NZBxNZW) F1 mice. Clin. Exp. Immunol. 5 (3), 265–272 (Sep). Selzer, F., Sutton-Tyrrell, K., Fitzgerald, S., Tracy, R., Kuller, L., Manzi, S., 2001. Vascular stiffness in women with systemic lupus erythematosus. Hypertension 37, 1075–1082. Shucard, J.L., Gaines, J.J., Ambrus Jr., J., Shucard, D.W., 2007. C-reactive protein and cognitive deficits in systemic lupus erythematosus. Cogn. Behav. Neurol. 20, 31–37. Szondy, Z., Sarang, Z., Molnar, P., Nemeth, T., Piacentini, M., Mastroberardino, P.G., Falasca, L., Aeschlimann, D., Kovacs, J., Kiss, I., Szegezdi, E., Lakos, G., Rajnavolgyi, E., Birckbichler, P.J., Melino, G., Fesus, L., 2003. Transglutaminase 2−/− mice reveal a phagocytosis-associated crosstalk between macrophages and apoptotic cells. Proc. Natl. Acad. Sci. U. S. A. 100, 7812–7817. Tilley, S.L., Coffman, T.M., Koller, B.H., 2001. Mixed messages: modulation of inflammation and immune responses by prostaglandins and thromboxanes. J. Clin. Invest. 108, 15–23.
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nutritional steatohepatitis: role as pro-inflammatory mediator. Hepatology 43, 826–836. Zein, N., Ganuza, C., Kushner, I., 1979. Significance of serum C-reactive protein elevation in patients with systemic lupus erythematosus. Arthritis Rheum. 22, 7–12. Zhang, N., Ahsan, M.H., Zhu, L., Sambucetti, L.C., Purchio, A.F., West, D.B., 2005. NF-kappaB and not the MAPK signaling pathway regulates GADD45beta expression during acute inflammation. J. Biol. Chem. 280, 21400–21408.
European Journal of Pharmacology 579 (2008) 382 – 389 www.elsevier.com/locate/ejphar
Transglutaminase inhibitor cystamine alleviates the abnormality in liver from NZB/W F1 mice Tsai-Ching Hsu a , Chih-Yang Huang b,c,d , Szu-Yi Chiang e , Wen-Xian Lai f , Chang-Hai Tsai d , Bor-Show Tzang f,⁎ a Institutes of Immunology, Chung Shan Medical University, Taichung, Taiwan, ROC Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan, ROC c Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan, ROC d Department of Healthcare Administration, Asia University, Taichung, Taiwan, ROC e Department of Health, Executive Yuan, Hua-Lien Hospital, Hua-Lien 97061, Taiwan, ROC Institutes of Biochemistry and Biotechnology, Chung Shan Medical University, No. 110, Section 1, Chien Kuo N. Road, Taichung 40203, Taiwan, ROC b
f
Received 25 May 2007; received in revised form 21 October 2007; accepted 22 October 2007 Available online 30 October 2007
Abstract Increased hepatic abnormality has been observed in patients with systemic lupus erythematosus (SLE) and contributes to the elevated apoptosis that results in severe disease activity. Since cystamine has been demonstrated to be beneficial for NZB/W F1 mice, this study investigates the effects of cystamine on various inflammatory and stress-related proteins in liver from NZB/W F1 mice. Nephelometric analyses and immunoblots were conducted to detect aspartate aminotransferase (AST), alanine aminotransferase (ALT), C-reactive protein (CRP), p53, p21, Gadd45, heat shock protein 70 (HSP70) and cyclooxygenase-2 (COX-2). AST and ALT were reduced in NZB/W F1 mice that were given cystamine and CRP, p53, p21, Gadd45, HSP70 and COX-2 proteins in the liver were reduced in NZB/W F1 mice that were treated with cystamine. Moreover, cystamine has no obvious effect on BALB/c mice. These findings suggest that cystamine reduces the inflammation in liver of NZB/W F1 mice and provide a clue in treatment of SLE with liver abnormality. © 2007 Elsevier B.V. All rights reserved. Keywords: Systematic lupus erythematosus (SLE); Liver; Cystamine; NZB/W F1 mice
1. Introduction Systematic lupus erythematosus (SLE) is known as a systemic autoimmune disorder that affects various organs including liver (Hahn, 1993). Although the liver is not regarded as a major target organ for damage in patients with SLE, hepatic disease is more common in SLE than is usually reported. Indeed, evidence has revealed the involvement of liver abnormality in the pathogenesis of SLE (Abraham et al., 2004; Lu et al., 2006). Therefore, various inflammatory and stress proteins related to the liver have been involved in the pathogenesis of SLE, including C-reactive protein (CRP), aspartate aminotransferase (AST), alanine aminotransferase ⁎ Corresponding author. Tel.: +886 4 24730022x11680; fax: +886 4 23248195. E-mail address: [email protected] (B.-S. Tzang). 0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.10.059
(ALT) (Zein et al., 1979; Herlong, 1994), and heat shock proteins (HSPs) (Dhillon et al., 1993). CRP is known as a prototypic marker of inflammation in various diseases (Kushner et al., 2006) and associated with pathogenesis in SLE (Zein et al., 1979). An elevated level of CRP has been detected in serum from patients with SLE and is associated with such disease activity as vascular stiffness in women and cognitive deficits (Selzer et al., 2001; Shucard et al., 2007), as well as a previous study indicated that abnormal AST and ALT point to a hepatocyte disorder (Herlong, 1994). Additionally, elevated heat shock proteins, including HSP70, have been detected in patients with SLE and have been demonstrated to play a role in the pathogenesis of SLE which results in the generation of autoantibodies (Dhillon et al., 1993; Conroy et al., 1996; Ripley et al., 2001). Homeostasis between cell proliferation and apoptosis is required for the development and maintenance of organisms.
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During the modulation of apoptosis, various proteins including p53 can alter the balance toward either cell survival or apoptosis (Kanzler and Galle, 2000). A recent report indicated that elevated p53 level is associated with disease activity in patients with SLE (Miret et al., 2003) and suggested the importance in the pathogenesis of SLE by autoantibody production (Kuhn et al., 1999; Herkel et al., 2001). However, little is known about the roles of p53 and its downstream molecules in liver from SLE. Cystamine can interfere with transglutaminase 2 (TG2) (Folk, 1980; Lorand and Conrad, 1984) and prevent apoptosis by inhibiting caspases-3 activity (Lesort et al., 2003; Ientile et al., 2003). Recently, cystamine has been demonstrated to be beneficial in both mouse models of Huntington's disease and SLE (Karpuj et al., 2002; Hsu et al., 2007). This study describes the decline in the abnormality in liver of NZB/W F1 mice by
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treatment with cystamine and provides clues concerning the treatment of SLE with hepatic abnormality. 2. Materials and methods 2.1. Mice and liver sample Female BALB/c and NZB/W F1 mice, a well-described lupus-prone mice strain (Denman et al., 1967; Holborow and Denman, 1967; Russell and Denman, 1969), were purchased from animal center, National Taiwan University, Taiwan and housed under supervision of the Institutional Animal Care and Use Committee at Chung Shan Medical University. 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 (Hsu et al., 2007) and scored according
Fig. 1. Cystamine alleviates AST, ALT, and CRP protein expression in NZB/W F1 mice. Sera was obtained from BALB/c and NZB/W F1 treated with PBS or cystamine and the level of (A) AST and (B) ALT was measured by nephelometric meter as described above and the P values are 0.198, 0.012, 0.042 and 0.029, respectively. The expression of CRP protein in (C), (D) liver and (E) sera from BALB/c and NZB/W F1 was detected by immunoblotting. Densitometric analyses are shown in the lower panel and the P values are 0.259, 0.034 and 0.008, respectively. Similar results were obtained in three independent experiments and ⁎ indicates significant difference.
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to the manufacturer's scoring system (Bayer Diagnostics, Hong Kong). Mice of 188-day-old were divided into four groups (8 mice/group) including BALB/c and NZB/W F1 mice given PBS or cystamine for experiments. For cystamine (Sigma, Saint Louis Mo, USA) treatment, BALB/c and NZB/W F1 mice were injected intraperitoneally (i.p.) with 100 μl of 10 mM phosphate buffer saline (PBS) or 100 μl of 10 mM cystamine daily for 14 days (Karpuj et al., 2002; Hsu et al., 2007). Liver samples of mice were obtained after 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. 2.2. Detection of AST and ALT The mice were sacrificed as described above and the heart sera were collected by direct puncture with a sterilized 1 ml syringe. The serum value of AST and ALT was detected by a
nephelometric system according to the manufacturer's instruction (Cobas Mira, Roche, Swiss). 2.3. Immunoblotting Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using 12.5% acrylamide gel, was performed as previously described (Laemmli, 1970). The liver samples were homogenized sufficiently with B25 High-shear Dispersing Emulsifiers Homogenizing machine (BRT CO Shanghai, China) and centrifuged at 12,000 rpm in 4 °C for 30 min. Supernatants were isolated and 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 of 100–150 V for 90 min 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 CRP, p53, p21WAF1/CIP1, Gadd45, HSP70, COX-2, TG2 and actin (Upstates, Charlottesville, Virginia, USA; Chemicon International, Temecula, CA, USA) were diluted in PBS with 2.5% BSA and incubated for 90 min with gentle agitation at room temperature. The membranes were washed twice with PBS– Tween for 60 min 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 scanned and quantified by densitometry (Appraise, Beckman-Coulter, Brea, California, USA). 2.4. Statistical analysis All the statistical analyses were performed using SPSS 10.0 software (SPSS Inc, Chicago, IL). Three independent experiments were repeated. Statistical analyses were performed using the Student's t test and one-way ANOVA. P b 0.05 was considered statistically significant. 3. Results 3.1. Decreased expression of AST, ALT and CRP in NZB/W F1 mice treated with cystamine
Fig. 2. Expression of p53 protein. (A) Liver lysates obtained from three different BALB/c mice treated with PBS or cystamine were probed with anti-p53 antibody as shown in the upper panel. Densitometric analysis is shown in the lower panel and the P value is 0.631. (B) Liver lysates obtained from three different NZB/W F1 mice treated with PBS or cystamine were probed with antip53 antibody as shown in the upper panel. Densitometric analysis is shown in the lower panel and the P value is 0.0007. Similar results were obtained in three independent experiments and ⁎ indicates significant difference.
Since hepatic abnormality has been associated with various inflammatory or stress-related proteins including AST, ALT and CRP that may contribute to inflammation and apoptosis, this study investigates the influence of cystamine on these proteins in sera or liver lysates from NZB/W F1 mice. Fig. 1A and B reveals the serum level of AST and ALT in BALB/c or NZB/W F1 mice treated with PBS or cystamine. Sera from BALB/c mice that had been treated with PBS or cystamine exhibit no significant variation of either AST or ALT. However, sera from NZB/W F1 mice that had been given cystamine exhibited significantly lower AST and ALT levels than those treated with PBS (P = 0.028 and 0.038, respectively). Moreover, the expression of CRP protein was measured in liver from mice given PBS or
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p21WAF1/CIP1 , was examined by immunoblotting. Fig. 3 illustrates the expression of p21WAF1/CIP1 protein and no significant difference in p21WAF1/CIP1 protein level was detected between the liver from BALB/c mice treated with PBS or cystamine (Fig. 3A). Notably, the expression of the p21WAF1/CIP1 protein was significantly lower in the liver from NZB/W F1 mice (P = 0.003) (Fig. 3B). Gadd45, another downstream molecule of p53, was also examined by immunoblotting (Fig. 4). The expression of Gadd45 protein was significantly lower in liver from NZB/W F1 mice than in those that had been given PBS (P = 0.0008) (Fig. 4A). The Gadd45 protein expression levels in the liver from BALB/c mice given PBS did not detectably differ from those that had been given or cystamine (Fig. 4B). 3.3. Decreased expression of HSP70 protein in NZB/W F1 mice treated with cystamine This study also examines the effect of cystamine on HSP70 expression in liver from NZB/W F1 mice. Fig. 5 shows the immunoblotting results of HSP70 in liver from BALB/c and
Fig. 3. Expression of p21WAF1/CIP1 protein. (A) Liver lysates obtained from three different BALB/c mice treated with PBS or cystamine were probed with antip21 antibody as shown in the upper panel. Densitometric analysis is shown in the lower panel and the P value is 0.241. (B) Liver lysates obtained from three different NZB/W F1 mice treated with PBS or cystamine were probed with antip21 antibody as shown in the upper panel. 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.
cystamine. CRP in liver from NZB/W F1 mice that had been given cystamine was significantly lower than in those treated with PBS (P = 0.024) (Fig. 1D). No significant variation in CRP expression was observed in the BALB/c mice that had been given PBS or cystamine (Fig. 1C). The CRP level in sera from NZB/W F1 mice was significantly reduced, the result of which is consistent with those described above (Fig. 1E). 3.2. Decreased expression of p53, p21WAF1/CIP1 , and Gadd45 protein in NZB/W F1 mice treated with cystamine Immunoblotting of p53 was performed (Fig. 2) to clarify the effect of cystamine on p53 expression in liver from NZB/W F1 mice (Fig. 2). Fig. 2A shows no variation of p53 expression between liver from BALB/c mice that had been treated with PBS or cystamine (P = 0.631). However, in liver from NZB/W F1 mice upon treatment with cystamine had significantly less p53 protein than those that had been given PBS (P = 0.0007) (Fig. 2B). Moreover, the downstream molecule of p53,
Fig. 4. Expression of Gadd45 protein. (A) Liver lysates obtained from three different BALB/c mice treated with PBS or cystamine were probed with antiGadd45 antibody as shown in the upper panel. Densitometric analysis is shown in the lower panel and the P value is 0.357. (B) Liver lysates obtained from three different NZB/W F1 mice treated with PBS or cystamine were probed with antiGadd45 antibody as shown in the upper panel. Densitometric analysis is shown in the lower panel and the P value is 0.0006. Similar results were obtained in three independent experiments and ⁎ indicates significant difference.
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4. Discussion A consensus is forming around increasing evidence that impairment of functions of macrophages in the clearance of dying cells may result in apoptotic-cell accumulation in tissues of SLE patients (Szondy et al., 2003; Grossmayer et al., 2005; Gaipl et al., 2006). Therefore, the persistence of apoptosis and the impaired clearance of apoptotic cells in liver are associated with the pathogenesis of SLE. Although hepatic abnormality is not regarded as a major damage in patients with SLE, liver disease is more common than is usually reported (Mukai et al., 2000; Abraham et al., 2004; Lu et al., 2006). An increasing number of studies indicate that hepatic abnormality in SLE may contribute to apoptosis and promote disease activity. However, the precise mechanism of, and optical treatment for, SLE remain uncertain. This study demonstrates for the first time the beneficial effects of cystamine in SLE mice by reducing the inflammatory and stress responses in liver. The results revealed that sera from NZB/W F1 mice that had been given cystamine had lower levels of AST and ALT than those of mice that had been given PBS. Additionally, significantly reduced expressions of CRP, p53, p21WAF1/CIP1, Gadd45, HSP70, and COX-2 proteins were measured in liver from NZB/W F1 mice. AST and ALT were recognized as inflammatory proteins and released into the blood circulation when hepatic damage occurs in patients with SLE (Zein et al., 1979; Herlong, 1994). Therefore, reduced expression of AST and ALT helps to reduce hepatic inflammation in SLE. According to our experimental
Fig. 5. Expression of HSP70 protein. (A) Liver lysates obtained from three different BALB/c mice treated with PBS or cystamine were probed with antiHSP70 antibody as shown in the upper panel. Densitometric analysis is shown in the lower panel and the P value is 0.279. (B) Liver lysates obtained from three different NZB/W F1 mice treated with PBS or cystamine were probed with antiHSP70 antibody as shown in the upper panel. Densitometric analysis is shown in the lower panel and the P value is 0.0004. Similar results were obtained in three independent experiments and ⁎ indicates significant difference.
NZB/W F1 mice. No significant difference existed between HSP70 protein expressions in liver from BALB/c treated with PBS and that treated with cystamine (P = 0.279) (Fig. 5A). Notably, the expression of HSP70 in liver from NZB/W F1 mice treated with cystamine was significantly lower than in those treated with PBS (P = 0.004) (Fig. 5B). 3.4. Decreased expression of cyclooxygenase-2 (COX-2) protein in liver from NZB/W F1 mice treated with cystamine The immunoblotting of COX-2 protein was performed to investigate the effect of cystamine on COX-2 expression in liver from NZB/W F1 mice (Fig. 6). No detectable expression of COX-2 protein was measured in liver from BALB/c mice that had been treated with PBS or cystamine (Fig. 6A). However, the expression of COX-2 protein was reduced significantly in NZB/W F1 mice treated with cystamine (P = 0.0425) (Fig. 6B).
Fig. 6. Expression of COX-2 protein. (A) Liver lysates obtained from three different BALB/c mice treated with PBS or cystamine were probed with antiCOX-2 antibody as shown in the upper panel. (B) Liver lysates obtained from three different NZB/W F1 mice treated with PBS or cystamine were probed with anti-COX-2 antibody as shown in the upper panel. Densitometric analysis is shown in the lower panel and the P value is 0.0425. Similar results were obtained in three independent experiments and ⁎ indicates significant difference.
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results, reduced ALT and AST were detected in NZB/W F1 mice that had been treated with cystamine suggesting that cystamine alleviates the hepatic inflammation in SLE. Additionally, in the experiments, a reduced CRP level is detected in serum and liver from NZB/W F1 mice that had been treated with cystamine. Since cystamine is known to interfere with and inhibit other thiol-dependent enzymes, it indicates the diverse targets of cystamine. However, these experimental results suggest systemic anti-inflammatory activity of cystamine by reducing CRP expression in live from NZB/W F1 mice. Transglutaminase 2 (TG2) is known as a multi-functional enzyme and implicated in causing apoptosis. As demonstrated in numerous studies, TG2 activity can be inhibited by cystamine via forming a mixed disulfide (Lorand and Conrad, 1984; Jeitner et al., 2005). Since p53 oncoprotein has recently been identified as a substrate of TG2 and elevated TG2 is associated with increased p53 expression and apoptosis (Mishra and Murphy, 2006), this study examines the expression of TG2 in liver from BALB/c and NZB/W F1 mice that were treated with PBS or cystamine, to clarify the effects of cystamine on p53. As expected, TG2 expression in liver from mice treated with PBS did not differ from that of those treated with cystamine as indicated in our previous work (Hsu et al., 2007). Notably, decreased expression of p53 was detected in liver from NZB/W F1 mice that had been treated with cystamine (Fig. 2B) but not BALB/c mice. These results indicate that cystamine would not reduce protein expression of TG2, but did reduce p53 level by inhibiting the serine/threonine kinase activity of TG2. However, the precise mechanism by which cystamine reduces p53 in NZB/W F1 mice warrants further investigation. Since p53 is known to play important roles in balancing apoptosis and survival, its downstream molecules, including p21WAF1/CIP1 and Gadd45 need to be investigated. P53 is known as a tumor suppressor gene and is important to play a role in the pathogenesis and the disease activity of SLE. An elevated level of p53 was detected in patients with SLE contributing to the regulation of the apoptosis of autoreactive lymphocyte (Miret et al., 2003) and the generation of anti-p53 autoantibody (Kuhn et al., 1999; Herkel et al., 2001). The experimental results herein indicate the reduced expression of p53 protein in liver from NZB/W F1 mice that had been treated with cystamine, suggesting the reduced effect of cystamine on p53 expression in liver from NZB/W F1 mice. Additionally, Gadd45 is known to be a stress gene that will be up-regulated during acute inflammation (Zhang et al., 2005). These results herein reveal reduced expression of gadd45 in liver from NZB/W F1 mice that had been treated with cystamine, and reduced expression of p21WAF1/CIP1 in liver from NZB/W F1 mice that had been given cystamine. Since p21WAF1/CIP1 and Gadd45 are two well-known downstream molecules of p53, the levels of these two proteins are supposed to decrease when p53 is down-regulated. Additionally, no variation of p21WAF1/CIP1 and Gadd45 was observed in BABL/c mice, the levels of which may therefore be independent of the decreased expression of p53 and associated with the impaired functions in SLE (Mok and Lau, 2003). Additionally, complicated and multi-factorial interaction among various genetic and environmental factors is
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probably involved in SLE. T cells from patients with SLE have been known to be activated in vivo and to cause autoreactive B cells to exhibit various abnormal expressions, involving signaling molecules and defective signal transduction pathways (Mok and Lau, 2003; Nagy et al., 2005; Hsu et al., 2007). These studies may account for the comparative sensitivity of NZB/W F1 mice to cystamine. However, the precise effects of cystamine on p53 and its downstream molecules in SLE need further clarification. HSPs, especially HSP70, are expressed in both prokaryotic and eukaryotic cells and known to play protective roles in various processes including environmental stress, normal physiological processes and infection (Kiang and Tsokos, 1998). Recently, HSP70 has been implicated in modulating the cellular immune response. Various studies describe the presence of HSP70-peptide complex on the surface of cells in both cultured leukemic K562 and human carcinomatous renal cells, causing a cytotoxic T lymphocyte response (Roigas et al., 1998; Moseley, 2000; Botzler et al., 1996). These data indicate that HSP70 may be involved in modulating both the hazard signals and the antigen carrier. Additionally, HSP70 has been associated with the pathogenesis of SLE, although the precise underlying mechanism is unclear. A recent study demonstrated that HSP70 is deposited along the dermo-epidermal junction in patients with SLE and serves as a transporter or chaperone for autoantigen presentation (Villalobos-Hurtado et al., 2003). Therefore, the reduced expression of HSP70 in liver from NZB/W F1 mice that had been treated with cystamine may alleviate the presentation of autoantigen and provide clues on the treatment of SLE. Cyclooxygenase type 2 (COX-2) is known as a proinflammation gene and plays important roles in inflammation, carcinogenesis, hemodynamics, and renal function (Fung and Kirschenbaum, 1999; Weinberg, 2000). The COX-2– prostanoid pathway has been demonstrated to increase hypertension in animal models and induce the formation of ascites in patients with cirrhosis (Chan and Rodger, 1997; Tilley et al., 2001). The aberrant expression of COX-2 has been well associated with hepatocarcinogenesis. Additionally, accumulating data indicate that increased COX-2 is associated with various liver diseases including inflammatory and malignant bile duct cells (Abiru et al., 2002; Wu et al., 2002; Yu et al., 2006). However, the reduced expression of COX-2 was detected in liver from NZB/W F1 mice that are treated with cystamine in our experimental results, suggesting that cystamine reduces liver inflammation in SLE. A growing body of evidence demonstrates the beneficial effects of cystamine not only in neuro-protection (Karpuj et al., 2002; Jeitner et al., 2005) but also in inhibiting apoptosis (Lesort et al., 2003; Ientile et al., 2003). Additionally, recent studies have indicated the beneficial effects of cystamine in SLE by reducing MMP-9 activity, TNF-a and TGF-b mRNA expression as well as the decline in the level of anti-cardiolipin autoantibody (Hsu et al., 2007). Notably, this study demonstrated the alleviation of stress and inflammatory responses in liver from NZB/W F1 mice, including reduced expressions of CRP, P53, P21, Gadd45, HSP70 and COX-2 protein. However,
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