MLN51 and GM-CSF involvement in the proliferation of fibroblast-like synoviocytes in the pathogenesis of rheumatoid arthritis

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease of unclear etiology. This study was conducted to identify critical factors involved in the synovial hyperplasia in RA pathology. We applied cDNA microarray analysis to profile the gene expressions of RA fibroblast-like synoviocytes (FLSs) from patients with RA. We found that the MLN51 (metastatic lymph node 51) gene, identified in breast cancer, is remarkably upregulated in the hyperactive RA FLSs. However, growth-retarded RA FLSs passaged in vitro expressed small quantities of MLN51. MLN51 expression was significantly enhanced in the FLSs when the growth-retarded FLSs were treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) or synovial fluid (SF). Anti-GM-CSF neutralizing antibody blocked the MLN51 expression even though the FLSs were cultured in the presence of SF. In contrast, GM-CSF in SFs existed at a significant level in the patients with RA (n = 6), in comparison with the other inflammatory cytokines, IL-1beta and TNF-alpha. Most RA FLSs at passage 10 or more recovered from their growth retardation when cultured in the presence of SF. The SF-mediated growth recovery was markedly impaired by anti-GM-CSF antibody. Growth-retarded RA FLSs recovered their proliferative capacity after treatment with GM-CSF in a dose-dependent manner. However, MLN51 knock-down by siRNA completely blocked the GM-CSF/SF-mediated proliferation of RA FLSs. Taken together, our results imply that MLN51, induced by GM-CSF, is important in the proliferation of RA FLSs in the pathogenesis of RA.     

A role of monocyte chemoattractant protein-4 (MCP-4)/CCL13 from chondrocytes in rheumatoid arthritis

We studied the role of monocyte chemoattractant (MCP)-4/CCL13 in the pathogenesis of rheumatoid arthritis (RA). MCP-4 was highly expressed in cartilage from RA patients. Interferon-gamma significantly stimulated MCP-4/CCL13 production in human chondrocytes, and this effect was enhanced in combination with interleukin-1beta or tumor necrosis factor-alpha. MCP-4/CCL13 induces the phosphorylation of extracellular signal-regulated kinase in fibroblast-like synoviocytes and activates cell proliferation, and PD98059 completely inhibits these effects. These data suggest that interferon-gamma in combination with interleukin-1beta/tumor necrosis factor-alpha activates the production of MCP-4/CCL13 from chondrocytes in RA joints, and that secreted MCP-4/CCL13 enhances fibroblast-like synoviocyte proliferation by activating the extracellular signal-regulated kinase mitogen-activated protein kinase cascade.     

Regulation of apoptosis and cell cycle activity in rheumatoid arthritis

The regulation of proliferation and cell death is vital for homeostasis, but the mechanisms that coordinately balances these two events in rheumatoid arthritis (RA) remains largely unknown. In RA, the synovial lining increases through enhanced proliferation, migration, and/or decreased cell death. The aberrant decrease in apoptosis or increased cell cycle activity of fibroblast-like or macrophage-like synoviocytes is responsible for the synovial hyperplasia and contributes to the destruction of cartilage and bone. Recently, numerous molecules that modulate apoptosis and cell cycle have been implicated to play a role in RA. This review will describe the current understanding of the molecular mechanisms that govern apoptosis and cell cycle and their relationship to RA pathogenesis.     

Anti-arthritis effects of vitamin K(2) (menaquinone-4)--a new potential therapeutic strategy for rheumatoid arthritis

Vitamin K(2) (menaquinone-4, MK-4) has been reported to induce apoptosis in hepatocellular carcinoma, leukemia and myelodysplastic syndrome cell lines. The effects of MK-4 on the development of arthritis have never been addressed thus far. In the present study, we investigated the effect of MK-4 upon the proliferation of rheumatoid synovial cells and the development of arthritis in collagen-induced arthritis. We analyzed the effect of MK-4 on the proliferation of fibroblast-like synoviocytes using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The pro-apoptotic effect of MK-4 upon fibroblast-like synoviocytes was investigated with annexin V staining and DNA fragmentation and caspase 3/7 assays. Moreover, we analyzed the effect of MK-4 on the development of collagen-induced arthritis in female dark agouti rats. Our results indicated that MK-4 inhibited the proliferation of fibroblast-like synoviocytes and the development of collagen-induced arthritis in a dose-dependent manner. We conclude that MK-4 may represent a new agent for the treatment of rheumatoid arthritis in the setting of combination therapy with other disease-modifying antirheumatic drugs.     

Serum amyloid A-induced IL-6 production by rheumatoid synoviocytes

In this study, we investigated the role of serum amyloid A protein (SAA) in the production of interleukin-6 (IL-6) using rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). Recombinant SAA stimulation induced the production of pro-inflammatory cytokine, IL-6, from RA-FLS. The signaling events induced by SAA included the activation of the mitogen-activated protein kineases, p38 and JNK1/2 and the activation of nuclear factor-kappa B (NF-kappaB). Inhibitor studies have shown SAA-induced IL-6 production to be down-regulated by NF-kappaB inhibition and partially inhibited by p38 or JNK inhibitors. Our findings demonstrate that SAA is a significant inducer of IL-6, which is critically involved in RA pathogenesis.     

The microRNA miR-23b suppresses IL-17-associated autoimmune inflammation by targeting TAB2, TAB3 and IKK-α

Inflammatory cytokines such as interleukin-17 (IL-17) promote inflammatory autoimmune diseases. Although several microRNAs (miRNAs) have been shown to regulate autoimmune pathogenesis by affecting lymphocyte development and function, the role of miRNAs in resident cells present in inflammatory lesions remains unclear. Here we show that miR-23b is downregulated in inflammatory lesions of humans with lupus or rheumatoid arthritis, as well as in the mouse models of lupus, rheumatoid arthritis or multiple sclerosis. IL-17 downregulates miR-23b expression in human fibroblast-like synoviocytes, mouse primary kidney cells and astrocytes and is essential for the downregulation of miR-23b during autoimmune pathogenesis. In turn, miR-23b suppresses IL-17-, tumor necrosis factor α (TNF-α)- or IL-1β-induced NF-κB activation and inflammatory cytokine expression by targeting TGF-β-activated kinase 1/MAP3K7 binding protein 2 (TAB2), TAB3 and inhibitor of nuclear factor κ-B kinase subunit α (IKK-α) and, consequently, represses autoimmune inflammation. Thus, IL-17 contributes to autoimmune pathogenesis by suppressing miR-23b expression in radio-resident cells and promoting proinflammatory cytokine expression.     

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces rheumatoid arthritis synovial fibroblast proliferation through mitogen-activated protein kinases and phosphatidylinositol 3-kinase/Akt

A hallmark of rheumatoid arthritis (RA) is the pseudo-tumoral expansion of fibroblast-like synoviocytes (FLSs), and the RA FLS has therefore been proposed as a therapeutic target. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been described as a pro-apoptotic factor on RA FLSs and, therefore, suggested as a potential drug. Here we report that exposure to TRAIL-induced apoptosis in a portion (up to 30%) of RA FLSs within the first 24 h. In the cells that survived, TRAIL induced RA FLS proliferation in a dose-dependent manner, with maximal proliferation observed at 0.25 nm. This was blocked by a neutralizing anti-TRAIL antibody. RA FLSs were found to express constitutively TRAIL receptors 1 and 2 (TRAIL-R1 and TRAIL-R2) on the cell surface. TRAIL-R2 appears to be the main mediator of TRAIL-induced stimulation, as RA FLS proliferation induced by an agonistic anti-TRAIL-R2 antibody was comparable with that induced by TRAIL. TRAIL activated the mitogen-activated protein kinases ERK and p38, as well as the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway with kinetics similar to those of TNF-alpha. Moreover, TRAIL-induced RA FLS proliferation was inhibited by the protein kinase inhibitors PD98059, SB203580, and LY294002, confirming the involvement of the ERK, p38, and PI3 kinase/Akt signaling pathways. This dual functionality of TRAIL in stimulating apoptosis and proliferation has important implications for its use in the treatment of RA.     

Gene expression induced by interleukin-17 in fibroblast-like synoviocytes of patients with rheumatoid arthritis: upregulation of hyaluronan-binding protein TSG-6

Interleukin-17 (IL-17) has been characterized as a proinflammatory cytokine produced by CD4+ CD45RO+ memory T cells. Overproduction of IL-17 was detected in the synovium of patients with rheumatoid arthritis (RA) compared with patients with osteoarthritis. This study examines differentially expressed genes after the stimulation of fibroblast-like synoviocytes of RA patients by IL-17. Among these genes we identified the following: tumor necrosis factor-stimulated gene-6 (TSG-6), IL-6, IL-8, GRO-beta, and bone morphogenetic protein-6 with an expression 3.6-10.6-fold that in the unstimulated control. IL-17 augmented the expression of TSG-6, a hyaluronan-binding protein, in a time- and dose-dependent manner. IL-17 showed additive effects with IL-1beta and tumour necrosis factor-alpha on the expression of TSG-6, IL-6 and IL-8. The mitogen-activated protein kinase p38 seems to be necessary for the regulation of TSG-6 expression by IL-17, as shown by inhibition with SB203580. Our results support the hypothesis that IL-17 is important in the pathogenesis of RA, contributing to an unbalanced production of cytokines as well as participating in connective tissue remodeling.     

Treatment with recombinant interferon-beta reduces inflammation and slows cartilage destruction in the collagen-induced arthritis model of rheumatoid arthritis

We investigated the therapeutic potential and mechanism of action of IFN-beta protein for the treatment of rheumatoid arthritis (RA). Collagen-induced arthritis was induced in DBA/1 mice. At the first clinical sign of disease, mice were given daily injections of recombinant mouse IFN-beta or saline for 7 days. Disease progression was monitored by visual clinical scoring and measurement of paw swelling. Inflammation and joint destruction were assessed histologically 8 days after the onset of arthritis. Proteoglycan depletion was determined by safranin O staining. Expression of cytokines, receptor activator of NF-kappaB ligand, and c-Fos was evaluated immunohistochemically. The IL-1-induced expression of IL-6, IL-8, and granulocyte/macrophage-colony-stimulating factor (GM-CSF) was studied by ELISA in supernatant of RA and osteoarthritis fibroblast-like synoviocytes incubated with IFN-beta. We also examined the effect of IFN-beta on NF-kappaB activity. IFN-beta, at 0.25 microg/injection and higher, significantly reduced disease severity in two experiments, each using 8-10 mice per treatment group. IFN-beta-treated animals displayed significantly less cartilage and bone destruction than controls, paralleled by a decreased number of positive cells of two gene products required for osteoclastogenesis, receptor activator of NF-kappaB ligand and c-Fos. Tumor necrosis factor alpha and IL-6 expression were significantly reduced, while IL-10 production was increased after IFN-beta treatment. IFN-beta reduced expression of IL-6, IL-8, and GM-CSF in RA and osteoarthritis fibroblast-like synoviocytes, correlating with reduced NF-kappaB activity. The data support the view that IFN-beta is a potential therapy for RA that might help to diminish both joint inflammation and destruction by cytokine modulation.     

MDM4 overexpression contributes to synoviocyte proliferation in patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease with features of inflammatory cell infiltration, synovial cell invasive proliferation, and ultimately, irreversible joint destruction. It has been reported that the p53 pathway is involved in RA pathogenesis. MDM4/MDMX is a major negative regulator of p53. To determine whether MDM4 contributes to RA pathogenesis, MDM4 mRNA and protein expression were assessed in fibroblast-like synoviocytes (FLS) by real-time PCR, western blotting, and in synovial tissues by immunohistochemistry. Furthermore, MDM4 was knocked down and overexpressed by lentivirus-mediated expression, and the proliferative capacity of FLS was determined by MTS assay. We found that cultured FLS from RA and osteoarthritis (OA) patients exhibited higher levels of MDM4 mRNA and protein expression than those from trauma controls. MDM4 protein was highly expressed in the synovial lining and sublining cells from both types of arthritis. Finally, MDM4 knockdown inhibited the proliferation of RA FLS by enhancing functional p53 levels while MDM4 overexpression promoted the growth of RA FLS by inhibiting p53 effects. Taken together, our results suggest that the abundant expression of MDM4 in FLS may contribute to the hyperplasia phenotype of RA synovial tissues.     

Arthritis is associated with T-cell-induced upregulation of Toll-like receptor 3 on synovial fibroblasts

Introduction  

Toll-like receptors (TLRs) are likely to play crucial roles in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to determine the key TLRs in synovium and explore their roles in the activation of fibroblast-like synoviocytes (FLSs) mediated by T cells in arthritis.     

Apigenin-induced apoptosis is mediated by reactive oxygen species and activation of ERK1/2 in rheumatoid fibroblast-like synoviocytes

Fibroblast-like synovial cells play a crucial role in the pathophysiology of rheumatoid arthritis (RA), as these cells are involved in inflammation and joint destruction. Apigenin, a dietary plant-flavonoid, is known to have many functions in animal cells including anti-proliferative and anticancer activities, but its role in human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) has not been reported. In this study, we investigated the roles of apigenin in RA-FLSs. The survival rate decreased, and apoptotic cell death was induced by apigenin treatment in RA-FLSs. Apigenin treatment resulted in activation of the mitogen-activated protein kinase (MAPK) ERK1/2, and pretreatment with an ERK inhibitor PD98059 dramatically reduced apigenin-induced apoptosis. We found that apigenin-mediated production of a large amount of intracellular reactive oxygen species (ROS) caused activation of ERK1/2 and apoptosis; treatment with the antioxidant Tiron strongly inhibited the apigenin-induced generation of ROS, phosphorylation of ERK1/2, and apoptotic cell death. Apigenin-induced apoptotic cell death was mediated through activation of the effectors caspase-3 and caspase-7, and was blocked by pretreatment with Z-VAD-FMK (a pan-caspase inhibitor). These results showed that apigenin-induced ROS and oxidative stress-activated ERK1/2 caused apoptotic cell death in apigenin-treated RA-FLSs.     

Regulation of the JNK pathway by TGF-beta activated kinase 1 in rheumatoid arthritis synoviocytes

c-Jun N-terminal kinase (JNK) contributes to metalloproteinase (MMP) gene expression and joint destruction in inflammatory arthritis. It is phosphorylated by at least two upstream kinases, the mitogen-activated protein kinase kinases (MEK) MKK4 and MKK7, which are, in turn, phosphorylated by MEK kinases (MEKKs). However, the MEKKs that are most relevant to JNK activation in synoviocytes have not been determined. These studies were designed to assess the hierarchy of upstream MEKKs, MEKK1, MEKK2, MEKK3, and transforming growth factor-β activated kinase (TAK)1, in rheumatoid arthritis (RA). Using either small interfering RNA (siRNA) knockdown or knockout fibroblast-like synoviocytes (FLSs), MEKK1, MEKK2, or MEKK3 deficiency (either alone or in combination) had no effect on IL-1β-stimulated phospho-JNK (P-JNK) induction or MMP expression. However, TAK1 deficiency significantly decreased P-JNK, P-MKK4 and P-MKK7 induction compared with scrambled control. TAK1 knockdown did not affect p38 activation. Kinase assays showed that TAK1 siRNA significantly suppressed JNK kinase function. In addition, MKK4 and MKK7 kinase activity were significantly decreased in TAK1 deficient FLSs. Electrophoretic mobility shift assays demonstrated a significant decrease in IL-1β induced AP-1 activation due to TAK1 knockdown. Quantitative PCR showed that TAK1 deficiency significantly decreased IL-1β-induced MMP3 gene expression and IL-6 protein expression. These results show that TAK1 is a critical pathway for IL-1β-induced activation of JNK and JNK-regulated gene expression in FLSs. In contrast to other cell lineages, MEKK1, MEKK2, and MEKK3 did not contribute to JNK phosphorylation in FLSs. The data identify TAK1 as a pivotal upstream kinase and potential therapeutic target to modulate synoviocyte activation in RA.     

Synoviocyte-derived CXCL12 is displayed on endothelium and induces angiogenesis in rheumatoid arthritis

CXCL12 (stromal cell-derived factor-1) is a potent CXC chemokine that is constitutively expressed by stromal resident cells. Although it is considered a homeostatic rather than an inflammatory chemokine, CXCL12 has been immunodetected in different inflammatory diseases, but also in normal tissues, ant its potential functions and regulation in inflammation are not well known. In this study, we examined the cellular sources of CXCL12 gene expression and the mechanism and effects of its interactions with endothelial cells in rheumatoid arthritis synovium. We show that CXCL12 mRNA was not overexpressed nor induced in cultured rheumatoid synoviocytes, but it specifically accumulated in the rheumatoid hyperplastic lining layer and endothelium. CXCL12 gene expression was restricted to fibroblast-like synoviocytes, whereas endothelial cells did not express CXCL12 mRNA, but displayed the protein on heparitinase-sensitive factors. CXCL12 colocalized with the angiogenesis marker alpha(v)beta(3) integrin in rheumatoid endothelium and induced angiogenesis in s.c. Matrigel plugs in mice. The angiogenic activity of rheumatoid synovial fluid in vivo was abrogated by specific immunodepletion of CXCL12. Our results indicate that synoviocyte-derived CXCL12 accumulates and it is immobilized on heparan sulfate molecules of endothelial cells, where it can promote angiogenesis and inflammatory cell infiltration, supporting a multifaceted function for this chemokine in the pathogenesis of rheumatoid arthritis.     

α-Mangostin promotes apoptosis of human rheumatoid arthritis fibroblast-like synoviocytes by reactive oxygen species-dependent activation of ERK1/2 mitogen-activated protein kinase

α-Mangostin (α-M) is a commonly used traditional medicine with various biological and pharmacological activities. Our study aimed to explore the effects and mechanism of α-M in regulating apoptosis of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). α-M of 10 to 100 μM was used to treat RA-FLS for 24 hours, followed by measuring cell viability and apoptosis. The involvement of reactive oxygen species (ROS) and mitogen-activated protein kinases was detected. Treatment of α-M promoted apoptosis and reduced viability of RA-FLS in a dose-dependent manner. The mitochondrial membrane potential in RA-FLS was remarkably reduced by α-M treatment, accompanied by the cytochrome c accumulation in the cytosol and increased activities of caspase-3 and caspase-9. Moreover, we found that α-M treatment promoted ROS production and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. The proapoptotic activity of α-M in RA-FLS was markedly reversed by the co-induction with the ERK1/2 inhibitor LY3214996 or ROS scavenger N-acetyl-l -cysteine. In conclusion, our studies found that α-M had remarkable proapoptotic activities in RA-FLS, which is regulated by the induction of ROS accumulation and ERK1/2 phosphorylation. α-M may thus have potential therapeutic effects for rheumatoid arthritis.     

Regulation of c-Jun N-terminal kinase by MEKK-2 and mitogen-activated protein kinase kinase kinases in rheumatoid arthritis

The mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK) is a critical regulator of collagenase-1 production in rheumatoid arthritis (RA). The MAPKs are regulated by upstream kinases, including MAPK kinases (MAPKKs) and MAPK kinase kinases (MAP3Ks). The present study was designed to evaluate the expression and regulation of the JNK pathway by MAP3K in arthritis. RT-PCR studies of MAP3K gene expression in RA and osteoarthritis synovial tissue demonstrated mitogen-activated protein kinase/ERK kinase kinase (MEKK) 1, MEKK2, apoptosis-signal regulating kinase-1, TGF-beta activated kinase 1 (TAK1) gene expression while only trace amounts of MEKK3, MEKK4, and MLK3 mRNA were detected. Western blot analysis demonstrated immunoreactive MEKK2, TAK1, and trace amounts of MEKK3 but not MEKK1 or apoptosis-signal regulating kinase-1. Analysis of MAP3K mRNA in cultured fibroblast-like synoviocytes (FLS) showed that all of the MAP3Ks examined were expressed. Western blot analysis of FLS demonstrated that MEKK1, MEKK2, and TAK1 were readily detectable and were subsequently the focus of functional studies. In vitro kinase assays using MEKK2 immunoprecipitates demonstrated that IL-1 increased MEKK2-mediated phosphorylation of the key MAPKKs that activate JNK (MAPK kinase (MKK)4 and MKK7). Furthermore, MEKK2 immunoprecipitates activated c-Jun in an IL-1 dependent manner and this activity was inhibited by the selective JNK inhibitor SP600125. Of interest, MEKK1 immunoprecipitates from IL-1-stimulated FLS appeared to activate c-Jun through the JNK pathway and TAK1 activation of c-Jun was dependent on JNK, ERK, and p38. These data indicate that MEKK2 is a potent activator of the JNK pathway in FLS and that signal complexes including MEKK2, MKK4, MKK7, and/or JNK are potential therapeutic targets in RA.     

Involvement of PDCD5 in the regulation of apoptosis in fibroblast-like synoviocytes of rheumatoid arthritis

Apoptosis is reduced in the synovial tissue of patients with rheumatoid arthritis (RA), possibly due to decreased expression of pro-apoptotic genes. Programmed Cell Death 5 (PDCD5) has been recently identified as a protein that mediates apoptosis. Although PDCD5 is down-regulated in many human tumors, the role of PDCD5 in RA has not been investigated. Here we report that reduced levels of PDCD5 mRNA and protein are detected in RA synovial tissue (ST) and fibroblast-like synoviocytes (FLS) than in tissue and cells from patients with osteoarthritis (OA). We also report differences in the PDCD5 expression pattern in tissues from patients with these two types of arthritis. PDCD5 showed a scattered pattern in rheumatoid synovium compared with OA, in which the protein labeling was stronger in the synovial lining layer than in the sublining. We also observed increased expression and nuclear translocation of PDCD5 in RA patient-derived FLS undergoing apoptosis. Finally, overexpression of PDCD5 led to enhanced apoptosis and activation of caspase-3 in triptolide-treated FLS. We propose that PDCD5 may be involved in the pathogenesis of RA. These data also suggest that PDCD5 may serve as a therapeutic target to enhance sensitivity to antirheumatic drug-induced apoptosis in RA.