Apoptosis Of Rheumatoid Arthritis Synovial Cells Research Articles

NK4 inhibits the proliferation and induces apoptosis of human rheumatoid arthritis synovial cells.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by proliferation and insufficient apoptosis of synovial cells. NK4 is a hepatocyte growth factor antagonist and is implicated in cell proliferation, viability, and apoptosis of many tumour cells. This study aimed to investigate the role of NK4 in the regulation of human RA synovial cell proliferation and apoptosis. Fibroblast-like synoviocytes (FLSs) isolated from RA patients and MH7A synovial cells were subjected to MTT, flow cytometry, and Western blot analysis. We found that NK4 suppressed cell proliferation through cell cycle arrest at the G0/G1 phase and induced apoptosis in RA synovial cells. Furthermore, NK4 altered the expression of cell cycle and apoptosis-related proteins such as cyclin D1, cyclin B1, PCNA, p21, p53, Bcl-2, Bax, cleaved caspase-9, and cleaved caspase-3. Additionally, NK4 reduced the phosphorylation level of NF-κB p65 and upregulated the expression of sirt1, but did not change the levels of p38 and p-p38 in RA-FLS and MH7A cells. In conclusion, NK4 inhibits the proliferation and induces apoptosis of human RA synovial cells. NK4 is a promising therapeutic target for RA. We demonstrated that NK4 inhibited cell proliferation by inducing apoptosis and arresting cell cycle in RA-FLS and MH7A cells. The apoptotic effects of NK4 may be mediated in part by decreasing Bcl-2 protein level, increasing Bax and caspase 3 protein levels, and inhibiting NF-κB signalling in RA-FLS and MH7A cells. These findings reveal potential mechanism underlying the role of NK4 in RA synovial cells and suggest that NK4 is a promising agent for RA treatment.

Involvement of thioredoxin reductase 1 in the regulation of redox balance and viability of rheumatoid synovial cells

Rheumatoid arthritis (RA), a chronic and systemic disease of unknown etiology, is characterized by hyperplasia of synovial cells, which ultimately lead to the destruction of cartilage and bone. To elucidate the molecular mechanisms that lead to RA, we analyzed synovial cells established from patients with RA by oligonucleotide microarrays. Gene expression profiles clearly suggested that oxidative stress is enhanced in RA synovial cells, which was confirmed by measuring cellular levels of reactive oxygen species. One of the highly up-regulated proteins in RA synovial cells was thioredoxin reductase 1 (TRXR1), a protein that plays an important role in antioxidant defense system. Subsequent analysis demonstrated that TRXR1 suppresses hydrogen peroxide and inhibits apoptosis of RA synovial cells. Thus, our results reveal a novel pathophysiologic function of RA synovial cells as a generator of oxidative stress, and a self-defense mechanism against self-generated oxidative stress.

Resistance to endoplasmic reticulum stress is an acquired cellular characteristic of rheumatoid synovial cells

Synoviolin is an endoplasmic reticulum (ER)-resident E3 ubiquitin ligase which plays a critical role in ER-associated degradation (ERAD). We found that Synoviolin is a novel causative factor for rheumatoid arthritis (RA), which is especially up-regulated in proliferating synovial cells in the disease. We attempted to examine the role of Synoviolin in ER stress-induced apoptosis and proliferation of synovial cells. RA synovial cells (RSCs) were refractory to ER stress-induced apoptosis compared with HEK293 or HeLa cells. RSCs were also more resistant to the apoptosis than synovial cells from osteoarthritis patients, significantly. Down-regulation of Synoviolin by siRNA increased the susceptibility to ER stress-induced apoptosis in RSCs. Knock-down of Synoviolin by siRNA did not only induce apoptosis of RSCs but also inhibited their proliferation in vitro. These data suggest that RSCs are extraordinarily refractory to ER stress-induced apoptosis, and we termed this special property 'hyper-ERAD'. Since Synoviolin is overexpressed in RSCs, and is known to play a critical role in the ERAD system as E3 ubiquitin ligase, hyper-ERAD is likely to present in these cells. Subsequently, the hyper-ERAD may cause synovial hyperplasia through its anti-apoptotic effect in RA. Further analyses are necessary to address this point, however, resistance to ER stress-induced apoptosis, or hyper-ERAD is a noteworthy new cellular characteristic of RSCs.

Hypermethylated promoter region of DR3, the death receptor 3 gene, in rheumatoid arthritis synovial cells

To examine the promoter activity and protein expression of the death receptor 3 gene DR3, a member of the apoptosis-inducing Fas gene family, with particular reference to the methylation status of its promoter region in rheumatoid arthritis (RA). Genomic DNA was prepared from peripheral blood mononuclear cells obtained from healthy individuals and from patients with RA and synovial cells obtained from patients with RA and osteoarthritis. The methylation status of the DR3 promoter was analyzed by bisulfite genomic sequencing and methylation-specific polymerase chain reaction techniques. Gene promoter activity and protein expression were examined using the luciferase reporter and Western blotting techniques. The promoter region of the DR3 gene contained many CpG motifs, including one CpG island that was specifically hypermethylated in synovial cells from patients with RA. Promoter assays showed that the promoter CpG island was essential for the transactivation of the DR3 gene and that forced hypermethylation of the CpG island with the bacterial methylase Sss I in vitro resulted in inhibition of the DR3 gene expression. Furthermore, the expression of DR-3 protein was down-modulated in association with methylation of the promoter CpG island in RA synovial cells. The CpG island in the DR3 gene promoter was specifically methylated to down-modulate the expression of DR-3 protein in rheumatoid synovial cells, which may provide resistance to apoptosis in RA synovial cells.

TRAIL-R2 (DR5) mediates apoptosis of synovial fibroblasts in rheumatoid arthritis.

TRAIL has been proposed as an anti-inflammatory cytokine in animal models of rheumatoid arthritis (RA). Using two agonistic mAbs specific for TRAIL-R1 (DR4) and TRAIL-R2 (DR5), we examined the expression and function of these death receptors in RA synovial fibroblast cells. The synovial tissues and primary synovial fibroblast cells isolated from patients with RA, but not those isolated from patients with osteoarthritis, selectively expressed high levels of cell surface DR5 and were highly susceptible to anti-DR5 Ab (TRA-8)-mediated apoptosis. In contrast, RA synoviocytes did not show increased expression of TRAIL-R1 (DR4), nor was there any difference in expression of Fas between RA and osteoarthritis synovial cells. In vitro TRA-8 induced apoptosis of RA synovial cells and inhibited production of matrix metalloproteinases induced by pro-inflammatory cytokines. In vivo TRA-8 effectively inhibited hypercellularity of a SV40-transformed RA synovial cell line and completely prevented bone erosion and cartilage destruction induced by these cells. These results indicate that increased DR5 expression and susceptibility to DR5-mediated apoptosis are characteristic of the proliferating synovial cells in RA. As highly proliferative transformed-appearing RA synovial cells play a crucial role in bone erosion and cartilage destruction in RA, the specific targeting of DR5 on RA synovial cells with an agonistic anti-DR5 Ab may be a potential therapy for RA.

Synergistic induction of apoptosis of rheumatoid arthritis synovial cells by H2O2 and N-acetyl-leucyl-leucyl-norleucinal

The effects of proteolysis inhibitors on hydrogen peroxide (H2O2)-induced apoptosis were examined in cultured human synovial cells of rheumatoid arthritis (RA) patients. RA synovial cells were resistant to apoptosis induced by H2O2. In the presence of 100 μM N-acetyl-leucyl-leucylnorleucinal (ALLN, known as calpain inhibitor 1 and also a proteasome inhibitor), but not N-acetyl-leucyl-leucylmethioninal (ALLM), apoptotic cell death was elicited by 400 μM H2O2 at a concentration that alone never induced cell death. ALLN induced the expression of tumor suppressor p53 protein and p21WAF-1 protein, probably through inhibition of proteasome. H2O2 further potentiated ALLN-induced p53 expression. H2O2 appeared to activate c-Jun N-terminal kinase (JNK) as well as extracellular signal-regulated kinase (ERK) and AKT. After administration of H2O2 and p53 induction by ALLN, we found that either one alone is insufficient to induce apoptosis of RA synovial cells but their combination synergistically does so. These results suggest that induction of p53 by ALLN may be potentially important for triggering H2O2-induced apoptosis processes in RA synovial cells.

Modulation by proinflammatory cytokines of Fas/Fas ligand-mediated apoptotic cell death of synovial cells in patients with rheumatoid arthritis (RA).

Synovial cell hyperplasia is a characteristic of patients with RA. Excessive proliferation of RA synovial cells is, in part, responsible for the synovial cell hyperplasia. In addition, synovial cell death that would reduce synovial cell number may be defective, leading to the hyperplasia. Thus, the defective control of cell death as well as cell proliferation may be of central importance in the pathogenesis of RA. In this study we analysed effects of proinflammatory cytokines on Fas/Fas ligand (FasL)-induced synovial cell apoptosis, and evaluated apoptosis-associated protein expression in the synovial cells in patients with RA. RA synovial cells expressed Fas antigen and lymphocytes infiltrating into RA synovium expressed FasL. Apoptotic synovial cells were detected within the sublining layer of RA synovium. Anti-Fas MoAb induced apoptosis of RA synovial cells in vitro, and proinflammatory cytokines tumour necrosis factor-alpha (TNF-alpha) and IL-1beta, but not IL-6 or IL-8, inhibited the anti-Fas-induced apoptosis accompanying up-regulation of Bcl-2 protein expression and reduced expression of CPP32 and ICH-1L. Immunohistochemical study revealed that CPP32 and ICH-1L were expressed weakly in the RA synovial lining cells compared with osteoarthritis (OA) synovial lining cells. Thus, we found that although RA synovial cells could die via apoptosis through Fas/FasL pathway, apoptosis of synovial cells was inhibited by proinflammatory cytokines present within the synovium. Inhibition of apoptosis by the proinflammatory cytokines may contribute outgrowth of synovial cells that leads to pannus formation and the destruction of joints in patients with RA.

Cell cycle implications in the pathogenesis of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by hyperplasia of the synovial lining cells, angiogenesis, and infiltration of mononuclear cells resulting in pannus formation, cartilage erosion and ultimately joint destruction. Synovial tissue (ST) fibroblast hyperplasia is reminiscent of tumor-like proliferation and is a major cause of cartilage destruction in the RA joint. The RA joint is replete with cytokines and growth factors which exert a synergistic mitogenic effect on ST fibroblasts. As a result, RA ST fibroblasts exhibit elevated gene expression of proto- oncogenes, such as c-Myc, c-Ras, and c-Jun and apoptosis inhibitors such as Bcl-2. At the same time, RA ST fibroblasts contain mutations in tumor suppressor genes such as p53. The altered rates of proliferation and apoptosis of RA synovial cells result in the hyperplasia of synovial tissue and in concert with the chronic inflammatory environment ultimately lead to the destruction of the RA joint.

Selective activation of the JNK/AP-1 pathway in Fas-mediated apoptosis of rheumatoid arthritis synoviocytes.

To evaluate the Fas-dependent signaling pathway, we examined the involvement of protein tyrosine phosphorylation and the DNA binding activity of AP-1 in rheumatoid arthritis (RA) cultured synovial cells. The number of dead cells was counted after treatment with anti-Fas antibody in the presence of protein tyrosine kinase or phosphatase inhibitor. Protein tyrosine phosphorylation in synoviocytes after Fas ligation was examined by immunoblot and immunoprecipitation analyses. The DNA binding activity of AP-1 was examined by electrophoretic mobility shift assay. Treatment with the protein tyrosine phosphatase inhibitor, orthovanadate, significantly enhanced the apoptosis of RA synoviocytes after Fas ligation. Ligation of the Fas molecule on RA synoviocytes induced a rapid tyrosine phosphorylation of JNK (c-Jun amino-terminal kinase) and formation of the AP-1 transcription factor. Our results strongly suggest that the JNK/AP-1 signaling pathway is activated during the process of Fas-mediated apoptosis of RA synovial cells.

Fas-Mediated Stimulation Induces IL-8 Secretion by Rheumatoid Arthritis Synoviocytes Independently of CPP32-Mediated Apoptosis

In this study, we investigated the IL-1β converting enzyme (ICE) family cysteine proteases responsible for the Fas-mediated apoptosis of rheumatoid arthritis (RA) synoviocytes and their involvement in proinflammatory cytokine production. CPP32 inhibitor, but not ICE inhibitor, was capable of inhibiting the Fas-mediated apoptosis of RA synovial cells. CPP32, but not ICE, was activated in response to anti-Fas stimulation. IL-8, but not IL-1β, was secreted from the anti-Fas-stimulated RA synoviocytes even in the presence of CPP32 inhibitor. These results demonstrated that CPP32, but not ICE, is the predominant cysteine protease that mediates the Fas-mediated apoptosis of RA synovial cells. We also demonstrated that anti-Fas stimulation of RA synoviocytes leads to IL-8 secretion independently of the CPP32-mediated apoptosis, which would accelerate inflammation.

Apoptosis and functional Fas antigen in rheumatoid arthritis synoviocytes.

To determine whether apoptosis occurs in rheumatoid arthritis (RA) synoviocytes, and if this phenomenon is dependent on the Fas/Apo-1 pathway. Apoptotic change in vivo was examined in RA synovial cells by several standard methods. The ability of cells to undergo Fas-induced apoptosis was determined in vitro. Typical apoptotic change was demonstrated in RA synovial cells by each method. Anti-Fas antibody induced apoptotic synovial cell death in vitro. This is the first reported study to demonstrate apoptosis in RA synovial cells. The findings indicate that rheumatoid synoviocytes undergo Fas-mediated apoptosis.