Small GTP-binding protein Rho-mediated signaling promotes proliferation of rheumatoid synovial fibroblasts

Rho is a major small GTP-binding protein that is involved in the regulation of various cell functions, including proliferation and cell migration, through activation of multiple signaling molecules in various types of cells. We studied its roles in synovial fibroblasts (SFs) in patients with rheumatoid arthritis (RA) and clarified its relevance to RA synovitis, with the following results. 1)We found that the thrombin receptor was overexpressed on RA synovial fibroblasts (RA SFs) and that thrombin induced a marked proliferation and progression of the cell cycle to the S phase in these cells. 2)We also found that thrombin efficiently activated Rho. 3)Rho activation and proliferation and the progression of the cell cycle to the S phase were completely blocked by p115RGS (an N-terminal regulator of the G-protein signaling domain of p115RhoGEF) and by the C-terminal fragments of Gα13 (an inhibitor of the interaction of receptors with G13). 4)Thrombin induced the secretion of IL-6 by RA SFs, but this action was blocked by p115RGS or Gα13. Our findings show that the actions of thrombin on the proliferation of RA SFs, cell-cycle progression to the S phase, and IL-6 secretion were mainly mediated by the G13 and RhoGEF pathways. These results suggest that p115RGS and Gα13 could be potent inhibitors of such functions. A rational design of future therapeutic strategies for RA synovitis could perhaps include the exploitation of the Rho pathway to directly reduce the growth of synovial cells.     

Mcl-1 is essential for the survival of synovial fibroblasts in rheumatoid arthritis

Mcl-1 is a Bcl-2-family, antiapoptotic molecule that is critical for the survival of T and B lymphocytes and macrophages; however, its role in nonhemopoietic cells remains to be fully elucidated. The current study focuses on the role of Mcl-1 in rheumatoid arthritis (RA). Mcl-1 was strongly expressed in the synovial lining and was increased in the sublining fibroblasts of patients with RA, compared with control synovial tissue. The expression of Mcl-1 in sublining fibroblasts correlated with the degree of inflammation and TNF-alpha, and IL-1beta treatment of cultured synovial fibroblasts resulted in the increased expression of Mcl-1 at the mRNA and protein levels. Mcl-1 was critical for the survival of RA synovial fibroblasts, because the forced reduction of Mcl-1 using a Mcl-1 antisense-expressing adenoviral vector induced apoptotic cell death, which was mediated through Bax, Bak, and Bim. These observations document a critical role for Mcl-1 in protecting against apoptosis in RA and suggest that Mc1-1 is a potential therapeutic target in this disease.     

Endothelial protein C receptor-associated invasiveness of rheumatoid synovial fibroblasts is likely driven by group V secretory phospholipase A2

Introduction

Rheumatoid synovial fibroblasts (RASFs) mediate joint inflammation and destruction in rheumatoid arthritis (RA). Endothelial protein C receptor (EPCR) is a specific receptor for the natural anticoagulant activated protein C (APC). It mediates the cytoprotective properties of APC and is expressed in rheumatoid synovial tissue. A recent report shows that group V secretory phospholipase A2 (sPLA₂V) prevents APC from binding to EPCR in endothelium and inhibits EPCR/APC function. The aim of this study was to investigate the expression and function of EPCR on RASFs.

Methods

Human synovial fibroblasts (SFs) were isolated from RA or osteoarthritis (OA) synovial tissues and treated with control, EPCR, or sPLA₂V small interfering RNA (siRNA); recombinant human APC, tumor necrosis factor-alpha (TNF-α), or sPLA₂V. RASF viability and migration/invasion were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and collagen gel migration/invasion assays, respectively, and cartilage degradation by 1,9-dimethylmethylene blue (DMMB) assay in the presence of human OA articular cartilage explants. The expression or activation of cytokines, EPCR, cadherin-11, mitogen-activated protein (MAP) kinases, and nuclear factor-kappa-B (NF-κB) or both were detected by enzyme-linked immunosorbent assay, Western blotting, or immunostaining.

Results

EPCR was expressed by both OASFs and RASFs but was markedly increased in RASFs. When EPCR was suppressed by siRNA or blocking antibody cell viability, cell invasion and cartilage degradation were reduced by more than 30%. Inflammatory mediators interleukin-1-beta (IL-1β), cadherin-11, and NF-κB were significantly reduced by EPCR suppression under control or TNF-α-stimulated conditions. The expression or activation (or both) of MAP kinases ERK, p38, and JNK were also markedly decreased in cells transfected with EPCR siRNA. Further analysis revealed that sPLA₂V co-localized with EPCR on RASFs. Suppression of sPLA₂V reduced cell viability and cartilage degradation and increased APC binding to RASFs. Conversely, recombinant sPLA₂V increased cartilage degradation, blocked APC binding to RASFs, and could not rescue the effects induced by EPCR suppression.

Conclusions

Our results demonstrate that EPCR is overexpressed by RASFs and mediates the aggressive behavior of RASFs. This function of EPCR is contrary to its cytoprotective role in other settings and is likely driven by sPLA₂V.     

Susceptibility of rheumatoid arthritis synovial fibroblasts to FasL- and TRAIL-induced apoptosis is cell cycle-dependent

Introduction  

The rheumatoid arthritis (RA) synovium is characterised by the presence of an aggressive population of activated synovial fibroblasts (RASFs) that are prominently involved in the destruction of articular cartilage and bone. Accumulating evidence suggests that RASFs are relatively resistant to Fas-ligand (FasL)-induced apoptosis, but the data concerning tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) have been conflicting. Here, we hypothesise that the susceptibility of RASFs to receptor-mediated apoptosis depends on the proliferation status of these cells and therefore analysed the cell cycle dependency of FasL- and TRAIL-induced programmed cell death of RASFs in vitro.     

Adiponectin stimulates IL-8 production by rheumatoid synovial fibroblasts

The adipokines are linked not only to metabolic regulation, but also to immune responses. Adiponectin, but not leptin or resistin induced interleukin-8 production from rheumatoid synovial fibroblasts (RSF). The culture supernatant of RSF treated with adiponectin induced chemotaxis, although adiponectin itself had no such effect. Addition of antibody against adiponectin, and inhibition of adiponectin receptor gene decreased adiponectin-induced IL-8 production. Nuclear translocation of nuclear factor-kappa B was increased by adiponectin. The induction of interleukin-8 was inhibited by mitogen-activated protein kinase inhibitors. These findings suggest that adiponectin contributes to the pathogenesis of rheumatoid arthritis.     

Apoptosis is not the major death mechanism induced by celecoxib on rheumatoid arthritis synovial fibroblasts

Synovial hyperplasia in rheumatoid arthritis (RA) has been associated with apoptosis deficiency of RA fibroblast-like synoviocytes (FLSs). Celecoxib is a non-steroidal anti-inflammatory drug that has been demonstrated to induce apoptosis in some cellular systems. We have therefore examined the dose- and time-dependent effects of celecoxib on RA FLS viability. Treatment of RA FLSs with celecoxib for 24 hours reduced their viability in a dose-dependent manner. Analysis of celecoxib-treated RA FLSs for their content of apoptotic and necrotic cells by Annexin V staining and TO-PRO-3 uptake displayed only few apoptotic cells. Caspase 3, a key mediator of apoptosis, was not activated in celecoxib-treated RA FLSs, and the presence of specific caspase 3 or pan-caspase inhibitors did not affect celecoxib-induced cell death. Moreover, we could not detect other signs of apoptosis, such as cleavage of poly(ADP-ribose) polymerase, caspase 8 or 9, or DNA fragmentation. We therefore conclude that apoptosis is not the major death pathway in celecoxib-treated RA FLSs.     

Interleukin-18 induces serum amyloid A (SAA) protein production from rheumatoid synovial fibroblasts

Interleukin-18 (IL-18) is a novel proinflammatory cytokine that was recently found in synovial fluids and synovial tissues from patients with rheumatoid arthritis (RA). To investigate the role of IL-18 in rheumatoid synovitis, the levels of IL-18 and serum amyloid A (SAA) were measured in synovial fluids from 24 patients with rheumatoid arthritis (RA) and 13 patients with osteoarthritis (OA). The levels of IL-18 and SAA in the synovial fluids were elevated in RA patients. In contrast, the levels of IL-18 in synovial fluids from OA patients were significantly lower compared to those of RA patients. SAA was not detected in synovial fluids from OA patients. The expression of SAA mRNA in rheumatoid synovial cells was also examined. SAA4 mRNA, which was constitutively expressed by rheumatoid synovial cells, was not affected by IL-18 stimulation. Although acute phase SAA (A-SAA, SAA1 + 2) mRNA was not detected in unstimulated synovial cells, its expression was induced by IL-18 stimulation. By immunoblot, we demonstrated that IL-18 induced the SAA protein synthesis from rheumatoid synovial cells in a dose-dependent manner. These results indicate a novel role for IL-18 in rheumatoid inflammation through the synovial SAA production.     

Migratory potential of rheumatoid arthritis synovial fibroblasts: Additional perspectives

Cell migration is a central part of physiological and pathophysiological processes including wound healing, immune defense, matrix remodeling and organ homeostasis. Different cell types have migratory potential including cells of the immune system and cells required in wound healing and tissue repair. These cells migrate locally through the tissue to the site of damage. The fibroblast is a central cell type of wound healing. In rheumatoid arthritis (RA), activated synovial fibroblasts (SFs) have the ability to invade joint cartilage, actively contributing to joint destruction in RA. Recently, RASFs have been shown to be able to migrate to non-affected areas and joints through the blood stream and to invade distant cartilage. RASFs most likely use similar mechanisms comparable to lymphocytes and tumor cells for long-distance and vascular trans-migration. Future experiments will address the goal to keep the transformed-appearing fibroblasts in the affected joints using therapeutical strategies that inhibit the pathophysiological changes of transformed-appearing RASFs but do not interfere with the physiological processes of ‘normal’ fibroblasts.     

Inhibition of protein geranylgeranylation induces apoptosis in synovial fibroblasts

Statins, competitive inhibitors of hydroxymethylglutaryl-CoA reductase, have recently been shown to have a therapeutic effect in rheumatoid arthritis (RA). In RA, synovial fibroblasts in the synovial lining, are believed to be particularly important in the pathogenesis of disease because they recruit leukocytes into the synovium and secrete angiogenesis-promoting molecules and proteases that degrade extracellular matrix. In this study, we show a marked reduction in RA synovial fibroblast survival through the induction of apoptosis when the cells were cultured with statins. Simvastatin was more effective in RA synovial fibroblasts than atorvastatin, and both statins were more potent on tumor necrosis factor-α-induced cells. In contrast, in osteoarthritis synovial fibroblasts, neither the statin nor the activation state of the cell contributed to the efficacy of apoptosis induction. Viability of statin-treated cells could be rescued by geranylgeraniol but not by farnesol, suggesting a requirement for a geranylgeranylated protein for synovial fibroblast survival. Phase partitioning experiments confirmed that in the presence of statin, geranylgeranylated proteins are redistributed to the cytoplasm. siRNA experiments demonstrated a role for Rac1 in synovial fibroblast survival. Western blotting showed that the activated phosphorylated form of Akt, a protein previously implicated in RA synovial fibroblast survival, was decreased by about 75%. The results presented in this study lend further support to the importance of elevated pAkt levels to RA synovial fibroblast survival and suggest that statins might have a beneficial role in reducing the aberrant pAkt levels in patients with RA. The results may also partly explain the therapeutic effect of atorvastatin in patients with RA.     

Fibroblast biology: Role of synovial fibroblasts in the pathogenesis of rheumatoid arthritis

There is growing evidence that activated synovial fibroblasts, as part of a complex cellular network, play an important role in the pathogenesis of rheumatoid arthritis. In recent years, significant progress has been made in elucidating the specific features of these fibroblasts. It has been understood that although macrophage and lymphocyte secreted factors contribute to their activation, rheumatoid arthritis synovial fibroblasts (RA-SFs) do not merely respond to stimulation by pro-inflammatory cytokines, but show a complex pattern of molecular changes also maintained in the absence of external stimulation. This pattern of activation is characterized by alterations in the expression of regulatory genes and signaling cascades, as well as changes in pathways leading to apoptosis. These together result in the upregulation of adhesion molecules that mediate the attachment of RA-SFs to the extracellular matrix and in the overexpression of matrix degrading enzymes that mediate the progressive destruction of the joints. In addition, activated RA-SFs exert specific effects on other cell types such as macrophages and lymphocytes. While the initiating step in the activation of RA-SFs remains elusive, several key pathways of RA-SF activation have been identified. However, there is so far no single, specific marker for this phenotype of RA-SF. It appears that activated RA-SFs are characterized by a set of specific properties which together lead to their aggressive behavior.     

Fibroblast biology. Role of synovial fibroblasts in the pathogenesis of rheumatoid arthritis

There is growing evidence that activated synovial fibroblasts, as part of a complex cellular network, play an important role in the pathogenesis of rheumatoid arthritis. In recent years, significant progress has been made in elucidating the specific features of these fibroblasts. It has been understood that although macrophage and lymphocyte secreted factors contribute to their activation, rheumatoid arthritis synovial fibroblasts (RA-SFs) do not merely respond to stimulation by pro-inflammatory cytokines, but show a complex pattern of molecular changes also maintained in the absence of external stimulation. This pattern of activation is characterized by alterations in the expression of regulatory genes and signaling cascades, as well as changes in pathways leading to apoptosis. These together result in the upregulation of adhesion molecules that mediate the attachment of RA-SFs to the extracellular matrix and in the overexpression of matrix degrading enzymes that mediate the progressive destruction of the joints. In addition, activated RA-SFs exert specific effects on other cell types such as macrophages and lymphocytes. While the initiating step in the activation of RA-SFs remains elusive, several key pathways of RA-SF activation have been identified. However, there is so far no single, specific marker for this phenotype of RA-SF. It appears that activated RA-SFs are characterized by a set of specific properties which together lead to their aggressive behavior.     

Slit3 inhibits Robo3-induced invasion of synovial fibroblasts in rheumatoid arthritis

Introduction  

The repellent factor family of Slit molecules has been described to have repulsive function in the developing nervous system on growing axons expressing the Robo receptors. However, until today no data are available on whether these repellent factors are involved in the regulation of synovial fibroblast (SF) activity in rheumatoid arthritis (RA).     

Origin and evolution of Kinesin-like calmodulin-binding protein

Kinesin-like calmodulin-binding protein (KCBP), a member of the Kinesin-14 family, is a C-terminal microtubule motor with three unique domains including a myosin tail homology region 4 (MyTH4), a talin-like domain, and a calmodulin-binding domain (CBD). The MyTH4 and talin-like domains (found in some myosins) are not found in other reported kinesins. A calmodulin-binding kinesin called kinesin-C (SpKinC) isolated from sea urchin (Strongylocentrotus purpuratus) is the only reported kinesin with a CBD. Analysis of the completed genomes of Homo sapiens, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and a red alga (Cyanidioschyzon merolae 10D) did not reveal the presence of a KCBP. This prompted us to look at the origin of KCBP and its relationship to SpKinC. To address this, we isolated KCBP from a gymnosperm, Picea abies, and a green alga, Stichococcus bacillaris. In addition, database searches resulted in identification of KCBP in another green alga, Chlamydomonas reinhardtii, and several flowering plants. Gene tree analysis revealed that the motor domain of KCBPs belongs to a clade within the Kinesin-14 (C-terminal motors) family. Only land plants and green algae have a kinesin with the MyTH4 and talin-like domains of KCBP. Further, our analysis indicates that KCBP is highly conserved in green algae and land plants. SpKinC from sea urchin, which has the motor domain similar to KCBP and contains a CBD, lacks the MyTH4 and talin-like regions. Our analysis indicates that the KCBPs, SpKinC, and a subset of the kinesin-like proteins are all more closely related to one another than they are to any other kinesins, but that either KCBP gained the MyTH4 and talin-like domains or SpKinC lost them.     

Constitutive upregulation of the transforming growth factor-beta pathway in rheumatoid arthritis synovial fibroblasts

Genome-wide gene expression was comparatively investigated in early-passage rheumatoid arthritis (RA) and osteoarthritis (OA) synovial fibroblasts (SFBs; n = 6 each) using oligonucleotide microarrays; mRNA/protein data were validated by quantitative PCR (qPCR) and western blotting and immunohistochemistry, respectively. Gene set enrichment analysis (GSEA) of the microarray data suggested constitutive upregulation of components of the transforming growth factor (TGF)-beta pathway in RA SFBs, with 2 hits in the top 30 regulated pathways. The growth factor TGF-beta1, its receptor TGFBR1, the TGF-beta binding proteins LTBP1/2, the TGF-beta-releasing thrombospondin 1 (THBS1), the negative effector SkiL, and the smad-associated molecule SARA were upregulated in RA SFBs compared to OA SFBs, whereas TGF-beta2 was downregulated. Upregulation of TGF-beta1 and THBS1 mRNA (both positively correlated with clinical markers of disease activity/severity) and downregulation of TGF-beta2 mRNA in RA SFBs were confirmed by qPCR. TGFBR1 mRNA (only numerically upregulated in RA SFBs) and SkiL mRNA were not differentially expressed. At the protein level, TGF-beta1 showed a slightly higher expression, and the signal-transducing TGFBR1 and the TGF-beta-activating THBS1 a significantly higher expression in RA SFBs than in OA SFBs. Consistent with the upregulated TGF-beta pathway in RA SFBs, stimulation with TGF-beta1 resulted in a significantly enhanced expression of matrix-metalloproteinase (MMP)-11 mRNA and protein in RA SFBs, but not in OA SFBs. In conclusion, RA SFBs show broad, constitutive alterations of the TGF-beta pathway. The abundance of TGF-beta, in conjunction with an augmented mRNA and/or protein expression of TGF-beta-releasing THBS1 and TGFBR1, suggests a pathogenetic role of TGF-beta-induced effects on SFBs in RA, for example, the augmentation of MMP-mediated matrix degradation/remodeling.     

Annexin-1 mediates TNF-alpha-stimulated matrix metalloproteinase secretion from rheumatoid arthritis synovial fibroblasts

Annexins are intracellular molecules implicated in the down-regulation of inflammation. Recently, annexin-1 has also been identified as a secreted molecule, suggesting it may have more complex effects on inflammation than previously appreciated. We studied the role of annexin-1 in mediating MMP-1 secretion from rheumatoid arthritis (RA) synovial fibroblasts (SF) stimulated with TNF-alpha. TNF-alpha induced a biphasic secretion of annexin-1 from RA SF. Early (< or = 60 min), cycloheximide-independent secretion from preformed intracellular pools was followed by late (24 h) cycloheximide-inhibitable secretion requiring new protein synthesis. Exogenous annexin-1 N-terminal peptide Ac2-26 stimulated MMP-1 secretion in a dose- (EC(50) approximately 25 microM) and time- (8-24 h) dependent manner; full-length annexin-1 had a similar effect. Down-regulation of annexin-1 using small interfering RNA resulted in decreased secretion of both annexin-1 and MMP-1, confirming that annexin-1 mediates TNF-alpha-stimulated MMP-1 secretion. Erk, Jnk, and NF-kappaB have been implicated in MMP-1 secretion. Erk, Jnk, and NF-kappaB inhibitors had no effect on annexin-1 secretion stimulated by TNF-alpha but inhibited MMP-1 secretion in response to Ac2-26, indicating that these molecules signal downstream of annexin-1. Annexin-1 stimulation of MMP-1 secretion was inhibited by both a formyl peptide receptor antagonist and pertussis toxin, suggesting that secreted annexin-1 acts via formyl peptide family receptors, most likely FPLR-1. In contrast to its commonly appreciated anti-inflammatory roles, our data indicate that annexin-1 is secreted by RA SF in response to TNF-alpha and acts in an autacoid manner to engage FPRL-1, activate Erk, Jnk, and NF-kappaB, and stimulate MMP-1 secretion.