| Abstract: | Increased macrophage vulnerability is associated with progression of systemic lupus erythematosus. Our previous studies have shown that cystamine, an inhibitor of transglutaminase 2 (TG2), alleviated the apoptosis of hepatocyte and brain cell in lupus-prone mice NZB/W-F1. In present study, we further investigated the effects of cystamine on apoptosis-prone macrophages (APMs) in the lupus mice. Using two-dimensional gel electrophoresis (2-DE) analysis, we found that cystamine induced a differential protein expression pattern of APM as comparing to the PBS control. The protein spots presenting differential level between cystamine and PBS treatment were then identified by peptide-mass fingerprinting (PMF). After bioinformatic analysis, these identified proteins were found involved in mitochondrial apoptotic pathway, oxidative stress, and mitogen-activated protein (MAP) kinase-mediated pathway. Further investigation revealed that cystamine significantly decreased the levels of apoptotic Bax and Apaf-1 and the activity of caspase-3, and increased the levels of anti-apoptotic Bcl-2 in APM. We also found that these apoptotic mediators were up-regulated in a correlation with the progression of lupus severity in NZB/W-F1, which were little affected in BALB/c mice. We also found that the reduced serum glutathione was restored by cystamine in NZB/W-F1. Interestingly, the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in APM and the phagocytic ability was diminished in presence of cystamine. In conclusion, our findings indicate that cystamine significantly inhibited mitochondrial pathway, induced antioxidant proteins, and diminished phosphorylation of extracellular ERK1/2, which may alleviate the apoptosis and the phagocytic ability of APM. J. Cell. Biochem. 110: 660–670, 2010. ? 2010 Wiley-Liss, Inc.
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease, which is usually characterized by loss of tolerance to self-antigens and production of circulating autoantibodies to nuclear antigens, and consequent immune-mediated tissue injury of multiple organs [Munoz et al., 2005]. Apoptotic cells might provide self-antigens; therefore, persistence of apoptosis and impaired clearance of pro-apoptotic cells, causing the spillage of potential immunogenic macromolecules to the exterior, could enhance the production of auto-reactive T and B cells and then promote pathogenesis of SLE [Wu et al., 2001; Cohen, 2006]. Actually, increase of apoptosis in polymorphonuclear cells and macrophages leading to impaired clearance of pro-apoptotic cells was commonly observed in SLE patients [Ren et al., 2003]. Similarly, the apoptosis of lupus-prone macrophages (APM) in lupus-prone mice was markedly expended that it was proportional to the severity and development of lupus syndrome [Russell et al., 1985].
Recently, transglutaminase 2 (TG2) has been implicated in the pathogenesis of SLE [Szondy et al., 2003; Pitidhammabhorn et al., 2006]. TG2 is known as a multi-functional enzyme that is expressed in the majority of tissues and implicated in numerous biological functions, including remodeling of the extracellular matrix [Aeschlimann et al., 1995], stimulus-secretion coupling [Bungay et al., 1986], receptor-mediated endocytosis [Davies et al., 1980], cell differentiation [Aeschlimann et al., 1993], tumor growth [Johnson et al., 1994], and apoptosis [Fesus et al., 1987]. Recent study reported that TG2-knockout mice developed typical lupus-prone symptoms, such as autoantibodies, immune complex glomerulonephritis, and impaired engulfment of apoptotic cells by macrophages [Falasca et al., 2005]. These evidences strongly suggest that TG2 may contribute to SLE pathogenesis.
Cystamine, the FDA-approved precursor of cysteamine, is known to inhibit the transamidation activity of TG2 [Jeon et al., 2004] and to exhibit anti-oxidant activity. It is also reported that cystamine inactivates protein kinase C-epsilon, gamma-glutamylcysteine synthetase and tissue transglutaminase (tTG) by S-cysteaminylation-triggered mechanisms [Seelig and Meister, 1984; Chu et al., 2005; O'Brian and Chu, 2005]. Our previous study showed that cystamine diminished the activity of matrix metalloproteinase-9, the mRNA expression of tumor necrosis factor- and transforming growth factor-β in APM, and suppressed the production of anti-cardiolipin autoantibody in lupus-prone mice NZB/W-F1 [Hsu et al., 2007]. Additionally, cystamine also shows neuroprotective effects, prolongs cell survival and alleviates abnormal cell movements in the transgenic model of Huntington disease [Karpuj et al., 2002]. However, the effects of cystamine on lupus-induced apoptosis of APM in NZB/W-F1 are still unclear.
In this study, we used two-dimensional gel electrophoresis (2-DE) to investigate the effects of cystamine on protein expression of APM. The proteins with different expression level regulated by cystamine were further identified by peptide-mass fingerprinting (PMF) and characterized by bioinformatic analysis. The findings revealed that cystamine significantly regulated the expression of the proteins associated with mitochondrial apoptotic signaling, oxidative stress, mitogen-activated protein (MAP) kinase signaling, protein degradation, and energy metabolism, which were further evaluated by immunoblotting. Moreover, the serum reduced glutathione (GSH) and the phagocytic ability of APM in cystamine-treated mice were also determined. |
