Apoptosis In Rheumatoid Arthritis Synovial Fibroblasts Research Articles

Regulating apoptosis in fibroblasts

Apoptosis constitutes a highly selective way of eliminating aged and injured cells and is a key mechanism for the balanced growth and regeneration of tissues. In addition to genotoxic stress and the withdrawal of growth factors, apoptosis can be induced by death receptors. Such receptors trigger apoptosis through an approximately 90 amino acid death domain. Prominent and best-characterized members of the death domain receptor family are Fas (CD-95/Apo-1) and the p55 TNF receptor (TNFRI). Alterations in receptor-mediated apoptosis may result in changes of tissue homeostasis and have been found in a variety of malignancies as well as in the inflamed and hyperplastic synovium in rheumatoid arthritis. Accumulating evidence suggests that the activation of rheumatoid arthritis synovial fibroblasts (RASF) is associated with alterations in apoptosis, especially at sites of invasion into cartilage and bone. Specifically, it has been demonstrated that RASF are less susceptible to Fas-induced cell death than osteoarthritis synovial fibroblasts but show higher expression levels of Fas. Recent studies have shown that both cytokine-dependent and cytokine-independent mechanisms contribute to the resistance of RASF against Fas-induced apoptosis. Thus, tumor necrosis factor alpha (TNF-α) – a major inflammatory cytokine in the rheumatoid synovium – fails to induce apoptosis in RASF, but reduces the susceptibility of these cells to Fas-mediated cell death through the induction of the transcription factor NF-κB. Providing a link between altered apoptosis and cartilage destruction, we have shown that overexpression of TIMP-3 through gene transfer not only reduces the invasiveness of RASF but also modulates the apoptosis-inhibiting effects of TNF-α. RASF overexpressing TIMP-3 are sensitized strongly to Fas/CD95-mediated cell death by TNF-α, and gene transfer of TIMP-3 inhibits the TNF-α-induced activation of NF-κB. While these effects of TNF-α on apoptosis can be found in different fibroblasts, several anti-apoptotic molecules have been demonstrated to be upregulated specifically in RASF. In this context, we have investigated the role of the small ubiquitin-like modifier SUMO-1 in the regulation of apoptosis. We found that increased levels of SUMO-1 in RASF but not in osteoarthritis synovial fibroblasts were associated with a reduced susceptibility of RASF to Fas-induced apoptosis. Using small interfering (si)RNA to knock-down the expression of SUMO-1 as well as retroviral gene transfer of SUMO-1, we could establish a functional relationship between the expression of SUMO-1 and the resistance of RASF against apoptosis. Moreover, it was demonstrated by gene transfer of the nuclear SUMO-protease SENP1 that, rather than by interacting directly with the Fas-associated death domain, SUMO-1 inhibits apoptosis through recruiting pro-apoptotic molecules such as DAXX into nuclear PML bodies, where they cannot exert their pro-apoptotic effects. Taken together, there is growing evidence that in activated fibroblasts, such as in RASF, there is a close association between anti-apoptotic and destructive pathways. In addition to cytokine-dependent inflammatory mechanisms, the intrinsic upregulation of anti-apoptotic molecules contributes to the resistance of RASF against apoptosis. Post-translational modification of nuclear proteins through SUMO-1 appears to constitute an important mechanism of apoptosis regulation. Therefore, the inhibition of molecules that confer the resistance of RASF to apoptosis constitutes a most interesting therapeutic target.

Cooperation of Ras- and c-Myc-dependent pathways in regulating the growth and invasiveness of synovial fibroblasts in rheumatoid arthritis.

To study the specific contribution of MAP kinase activator c-Raf-1 and one of its downstream transcription factors, c-Myc, to the growth and invasive behavior of rheumatoid arthritis synovial fibroblasts (RASFs). RASFs were transduced with retroviral constructs expressing dominant-negative mutants of c-Raf-1 or c-Myc (DN c-Raf-1 or DN c-Myc, respectively) or with the mock vector. The expression of wild-type and mutant proteins was confirmed by Western blotting. Growth curves of RASFs were recorded, and apoptosis was measured by flow cytometry. Invasiveness of RASFs was assessed in the SCID mouse model of RA. Immunohistochemistry was used to study the effects of DN c-Raf-1 on phosphorylated c-Jun and matrix metalloproteinase 1 (MMP-1) in RASFs implanted into SCID mice. The phosphorylation of ERK and JNK in DN c-Raf-1- and mock-transduced RASFs was determined in vitro by Western blotting. The levels of MMPs in these cells were measured by quantitative polymerase chain reaction (PCR). Neither DN c-Raf-1 alone nor DN c-Myc alone significantly altered proliferation or apoptosis of RASFs, but both mutants together rapidly induced apoptosis. Inhibition of c-Raf-1 or c-Myc significantly reduced the invasiveness of RASFs in the SCID mouse model. DN c-Raf-1 decreased the phosphorylation of ERK and JNK in vitro and reduced the in vivo expression of phosphorylated c-Jun as well as the expression of disease-relevant MMPs. As determined by quantitative PCR, the inhibition was most pronounced for MMP-1 and MMP-3. The data demonstrate that Ras- and c-Myc-dependent signaling events cooperate to regulate the growth and invasiveness of RASFs. Targeting of both c-Raf-1 and c-Myc may constitute an interesting therapeutic approach in RA.

Central role of mitochondria and p53 in Fas-mediated apoptosis of rheumatoid synovial fibroblasts.

Fas-mediated apoptosis is preferentially observed in synoviocytes of patients with rheumatoid arthritis (RA) and is associated with the pathophysiological process of RA. To clarify the molecular mechanisms of Fas-mediated apoptosis of RA synoviocytes, we investigated the role of the mitochondrial pathway and tumour suppressor p53 in this process. Cultured synovial fibroblasts were prepared from RA patients. After treatment of RA synovial fibroblasts with anti-Fas monoclonal antibody, the expression levels of activated caspase-9 and -3, Bid cleavage, cytochrome c release and phosphorylation of p53 at Ser15 were assessed using immunoblot analysis. The mitochondrial membrane potential (DeltaPsim) was evaluated with a fluorescence-based detection assay. Apoptotic cells were determined by a DNA fragmentation assay in the presence or absence of caspase inhibitors. Expression of p53-regulated apoptosis-inducing protein 1 (p53AIP1) was measured by real-time PCR. RA synovial fibroblasts stably transfected with a dominant-negative (DN) p53 were prepared in order to investigate the role of p53 during Fas-induced apoptosis. Fas ligation induced Bid cleavage, loss of DeltaPsim, cytochrome c release to the cytosol and activation of caspase-9 and -3 in RA synovial fibroblasts. Treatment with a caspase-9-specific inhibitor almost completely inhibited Fas-mediated apoptosis. Moreover, p53 activation after Fas ligation was evidenced by its phosphorylation at Ser15 and up-regulation of the p53 target gene p53AIP1. Fas-mediated apoptosis was significantly suppressed by anti-sense p53 oligonucleotides and by p53DN. Our findings strongly suggest the involvement of mitochondria and p53 in Fas-mediated apoptosis of RA synovial fibroblasts.

The SCID mouse model: novel therapeutic targets - lessons from gene transfer.

The hallmark of rheumatoid arthritis (RA) is progressive destruction of the joints, preceded and accompanied by synovial hyperplasia and chronic inflammation. Spontaneous and induced animal models of RA reflect predominantly the inflammatory aspects of the disease. To reproduce the destruction of cartilage and bone mediated by an activated synovium, it was desirable to develop models that allow the dissection of cellular and molecular components derived from human tissue. The SCID mouse co-implantation model of human RA focuses on RA synovial fibroblasts (RA-SF) and their role in cartilage destruction. The model has provided the best evidence that RA-SF contribute significantly to matrix degradation, even in the absence of human lymphocytes and macrophages, since highly purified RA-SF invade the co-implanted normal human cartilage. Moreover, it became clear that they maintained their aggressive phenotype over long periods of time, particularly at sites of invasion into the co-implanted human cartilage. Targeting different signaling molecules, cytokines and matrix-degrading enzymes by soluble receptors, antagonists or negative mutants in the SCID mouse model of RA has implicated many of them in the mechanisms leading to cartilage destruction. However, since inhibition of a single molecule or pathway is not sufficient to inhibit the aggressive behavior of RA-SF it appears necessary to co-express in the synoviocytes genes for two or even more antagonists of e.g. cytokines, matrix-degrading enzymes or molecules interfering specifically with signaling pathways involved in the apoptosis of RA-SF. Based on the recent observation that the L1 (line-1) endogenous retroviral element appears responsible for the cytokine- independent activation via the MAPK p38delta, the current understanding of disease pathogenesis suggests that both the cytokine-dependent as well as the cytokine-independent pathways of joint destruction must be inhibited. Modulation of both pathways by gene transfer approaches in the SCID mouse model is a feasible method aimed at identifying novel targets for the prevention of cartilage destruction in RA.

Induction of apoptosis in rheumatoid synovial fibroblasts by celecoxib, but not by other selective cyclooxygenase 2 inhibitors.

Selective cyclooxygenase 2 (COX-2) inhibitors are now being used as antiinflammatory agents that cause fewer gastrointestinal complications, compared with other antiinflammatory drugs, in patients with rheumatoid arthritis (RA). This study was undertaken to investigate whether selective COX-2 inhibitors could induce apoptosis of RA synovial fibroblasts (RASFs). RASFs were exposed to selective COX-2 inhibitors, i.e., celecoxib, etodolac, meloxicam, nimesulide, N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methanesulfonamide, and rofecoxib, under various conditions. Cell proliferation and cell viability were assessed by incorporation of 5-bromo-2'-deoxyuridine and by the 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt assay, respectively. Apoptosis was detected by identifying DNA fragmentation. Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) was measured by the luciferase reporter gene assay with a PPAR response element-driven luciferase reporter plasmid and a PPARgamma expression plasmid. Celecoxib strongly inhibited the proliferation of RASFs, whereas other selective COX-2 inhibitors had little or no effect. In addition, celecoxib reduced the viability of RASFs by induction of apoptosis, in a concentration-dependent manner. This action was abolished by addition of caspase inhibitors. Interleukin-1beta had a weak enhancing effect on celecoxib-induced apoptosis in RASFs. In contrast, other selective COX-2 inhibitors at concentrations up to 100 microM did not induce apoptosis of RASFs. Indomethacin, a nonselective COX inhibitor, activated PPARgamma transcription, while celecoxib did not. Celecoxib suppressed the proliferation of RASFs by COX-2-independent and PPARgamma-independent induction of apoptosis. Although the mechanism involved remains unclear, celecoxib may have not only antiinflammatory activity, but also a disease-modifying effect on rheumatoid synovial proliferation.

Effects of TIMP-1 and TIMP-3 gene transfer on invasiveness, proliferation and apoptosis of rheumatoid arthritis (RA) synovial fibroblasts (RA-SF)

TIMPs play a key role in counter balancing the action of MMPs and have been associated with cell proliferation, inhibition of angiogenesis and induction of apoptosis. Here, we investigated the effects of TIMP-1 and TIMP-3 gene transfer on cartilage invasion, proliferation and apoptosis of RA-SF. RA-SF were transduced with an adenoviral vector expressing human TIMP-1 (AdTIMP-1) or TIMP-3 (AdTIMP-3). Transduction efficacy was assessed by LacZ staining of RA-SF that were transduced with an adenoviral β-galactosidase construct. Untransduced and mock transduced RA-SF were used as controls. TIMP-1 was measured by ELISA in the culture supernatants of AdTIMP-1 transduced and mock transduced cells every 10 days until 60 days after transduction. Proliferation was assessed by 3H-thymidine incorporation, and the rate of spontaneous apoptosis as well as FasL induced cell death was determined by a histon fragmentation assay. AdTIMP-1 and AdTIMP-3 transduced RA-SF and control RA-SF were co-implanted with human articular cartilage under the renal capsule of SCID mice for 60 days and their invasiveness was evaluated on paraffin sections using a semiquantitative score. Transduction efficacy was 67%, and TIMP-1 levels in the supernatants of AdTIMP-1 transduced cells were 51.5 ± 6.5 μg/ml as compared to 8.7 ± 3.4 μg/ml in the mock transduced cells. These levels of TIMP expression were maintained for at least 60 days. AdTIMP-1 and AdTIMP-3 gene transfer resulted in an inhibition of proliferation (35% and 40% vs. mock, respectively; P < 0.05). Transduction of RA-SF with AdTIMP-3 but not TIMP-1 increased spontaneous apoptosis (+24%; vs. mock, P < 0.05) as well as susceptibility to FasL-induced cell death (+23% vs. mock, P < 0.05). In the SCID mouse model, untransduced and mock transduced RA.SF deeply invaded the cartilage (scores: 2.5 ± 0.2 and 3.2 respectively). In the AdTIMP-1 and AdTIMP-3 transduced RA-SF, invasion was inhibited clearly (scores 0.9 ± 0.4 and 1.2 ± 0.2 respectively) Both AdTIMP-1 and AdTIMP-3 gene transfer inhibit proliferation of RA-SF and reduce cartilage invasion. In contrast to TIMP-1, adenoviral gene transfer with TIMP-3. has a strong pro-apoptotic effect on RA-SF and facilitates Fas mediated cell death. These results indicate that gene transfer of TIMPs may be a useful approach to inhibit joint destruction in RA.

Article omitted to mention victims of left wing revolutionary politics

<h3>Objective</h3> To analyse the expression of SIRT1 in synovial tissues and cells of patients with rheumatoid arthritis (RA) and to study the function of SIRT1 in inflammation and apoptosis in RA. <h3>Methods</h3> Levels of SIRT1 expression were analysed in synovial tissues and cells from patients with RA by real-time PCR and western blotting before and after stimulation with toll-like receptor ligands, tumour necrosis factor α (TNFα) and interleukin 1β (IL-1β). Immunohistochemistry was used to study the localisation of SIRT1. Fluorescence activated cell sorting analysis was performed to investigate the effect of SIRT1 on apoptosis. Peripheral blood monocytes and rheumatoid arthritis synovial fibroblasts (RASFs) were transfected with wild-type or enzymatically inactive SIRT1 expression vectors or with siRNA targeting SIRT1. Cytokine analysis of IL-6, IL-8 and TNFα were performed by ELISA to study the role of SIRT1 on proinflammatory mediators of RA. <h3>Results</h3> SIRT1 was found to be constitutively upregulated in synovial tissues and cells from patients with RA compared to osteoarthritis. TNFα stimulation of RASFs and monocytes resulted in further induced expression levels of SIRT1. Silencing of SIRT1 promoted apoptosis in RASFs, whereas SIRT1 overexpression protected cells from apoptosis. Inhibition of SIRT1 enzymatic activity by inhibitors, siRNA and overexpression of an enzymatically inactive form of SIRT1 reduced lipopolysaccharide-induced levels of TNFα in monocytes. Similarly, knockdown of SIRT1 resulted in a reduction of proinflammatory IL-6 and IL-8 in RASFs. <h3>Conclusion</h3> The TNFα-induced overexpression of SIRT1 in RA synovial cells contributes to chronic inflammation by promoting proinflammatory cytokine production and inhibiting apoptosis.