A metalloproteinase from human rheumatoid synovial fibroblasts that digests connective tissue matrix components. Purification and characterization

Human rheumatoid synovial cells in culture stimulated with the conditioned culture medium of rabbit macrophages secrete three distinct latent metalloproteinases. One of them, a proteinase that digests proteoglycan and other connective tissue matrix components, was purified as two active forms after activation with 4-aminophenylmercuric acetate. The two forms were homogeneous on sodium dodecyl sulfate-gel electrophoresis with Mr = 45,000 and Mr = 28,000, whereas the latent precursor was estimated to have Mr = 51,000 by gel permeation chromatography. Both active enzymes had optimal activity at pH 7.5-7.8 and were inhibited by EDTA and 1,10-phenanthroline but not by inhibitors for cysteine, serine, or aspartic proteinases. Removal of Ca2+ from the enzyme solution resulted in a complete loss of activity that could be fully restored by the addition of 1 mM Ca2+. The activity of the apoenzyme was restored by the addition of 0.5 mM Zn2+, 5 mM Co2+, or 5 mM Mn2+ in the presence of Ca2+ but not by each metal ion alone. The identical digestion patterns of reduced, carboxymethylated protein substrates indicated that both active forms of the enzyme have the same substrate specificity. The enzyme degraded cartilage proteoglycans, type I gelatin, type IV collagen, laminin, and fibronectin, and removed the NH2-terminal propeptides from chick type I procollagen. This enzyme may play a role in the normal turnover of the connective tissue matrix as well as in the joint destruction of chronic synovitis.     

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.     

Chemokine secretion of rheumatoid arthritis synovial fibroblasts stimulated by Toll-like receptor 2 ligands

To analyze the role of Toll-like receptors (TLR) in the pathogenesis of rheumatoid arthritis, we have assessed the effects of stimulation of cultured synovial fibroblasts by the TLR-2 ligand bacterial peptidoglycan. By using high density oligonucleotide microarray analysis we identified 74 genes that were up-regulated >2.5-fold. Fourteen CC and CXC chemokine genes were among the genes with the highest up-regulation. Quantitative real-time PCR analysis confirmed up-regulation of granulocyte chemotactic protein (GCP)-2, RANTES, monocyte chemoattractant protein (MCP)-2, IL-8, growth-related oncogene-2, and to a lesser extent, macrophage-inflammatory protein 1alpha, MCP-1, EXODUS, and CXCL-16. GCP-2, RANTES, and MCP-2 were detected in culture supernatants of synovial fibroblasts stimulated with peptidoglycan. Chemokine secretion induced by stimulation of rheumatoid arthritis synovial fibroblasts via TLR-2 was functionally relevant as demonstrated by chemotaxis assays. GCP-2 and MCP-2 expression, which have not been reported previously in rheumatoid arthritis, was demonstrated in synovial tissue sections of patients diagnosed with rheumatoid arthritis but not in those with osteoarthritis. Correspondingly, synovial fluid levels were significantly higher in patients diagnosed with rheumatoid arthritis as compared with osteoarthritis. Thus, we present evidence for an induction of chemokine secretion by activation of synovial fibroblasts via TLR-2, possibly contributing to the formation of inflammatory infiltrates characteristically found in rheumatoid arthritis joints.     

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.     

Fucosyltransferase 1 mediates angiogenesis,cell adhesion and rheumatoid arthritis synovial tissue fibroblast proliferation

Introduction

We previously reported that sialyl Lewis^y, synthesized by fucosyltransferases, is involved in angiogenesis. Fucosyltransferase 1 (fut1) is an α(1,2)-fucosyltransferase responsible for synthesis of the H blood group and Lewis^y antigens. However, the angiogenic involvement of fut 1 in the pathogenesis of rheumatoid arthritis synovial tissue (RA ST) has not been clearly defined.

Methods

Assay of α(1,2)-linked fucosylated proteins in RA was performed by enzyme-linked lectin assay. Fut1 expression was determined in RA ST samples by immunohistological staining. We performed angiogenic Matrigel assays using a co-culture system of human dermal microvascular endothelial cells (HMVECs) and fut1 small interfering RNA (siRNA) transfected RA synovial fibroblasts. To determine if fut1 played a role in leukocyte retention and cell proliferation in the RA synovium, myeloid THP-1 cell adhesion assays and fut1 siRNA transfected RA synovial fibroblast proliferation assays were performed.

Results

Total α(1,2)-linked fucosylated proteins in RA ST were significantly higher compared to normal (NL) ST. Fut1 expression on RA ST lining cells positively correlated with ST inflammation. HMVECs from a co-culture system with fut1 siRNA transfected RA synovial fibroblasts exhibited decreased endothelial cell tube formation compared to control siRNA transfected RA synovial fibroblasts. Fut1 siRNA also inhibited myeloid THP-1 adhesion to RA synovial fibroblasts and RA synovial fibroblast proliferation.

Conclusions

These data show that α(1,2)-linked fucosylated proteins are upregulated in RA ST compared to NL ST. We also show that fut1 in RA synovial fibroblasts is important in angiogenesis, leukocyte-synovial fibroblast adhesion, and synovial fibroblast proliferation, all key processes in the pathogenesis of RA.     

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.     

Cross-talk between IL-1 and IL-6 signaling pathways in rheumatoid arthritis synovial fibroblasts.

The balance between pro- and anti-inflammatory cytokines plays an important role in determining the severity of inflammation in rheumatoid arthritis (RA). Antagonism between opposing cytokines at the level of signal transduction plays an important role in many other systems. We have begun to explore the possible contribution of signal transduction cross-talk to cytokine balance in RA by examining the effects of IL-1, a proinflammatory cytokine, on the signaling and action of IL-6, a pleiotropic cytokine that has both pro- and anti-inflammatory actions, in RA synovial fibroblasts. Pretreatment with IL-1 suppressed Janus kinase-STAT signaling by IL-6, modified patterns of gene activation, and blocked IL-6 induction of tissue inhibitor of metalloproteases 1 expression. These results suggest that proinflammatory cytokines may contribute to pathogenesis by modulating or blocking signal transduction by pleiotropic or anti-inflammatory cytokines. The mechanism of inhibition did not require de novo gene activation and did not depend upon tyrosine phosphatase activity, but, instead, was dependent on the p38 stress kinase. These results identify a molecular basis for IL-1 and IL-6 cross-talk in RA synoviocytes and suggest that, in addition to levels of cytokine expression, modulation of signal transduction also plays a role in regulating cytokine balance in RA.     

Degradation of type IX collagen by matrix metalloproteinase 3 (stromelysin) from human rheumatoid synovial cells

The degradation of type IX collagen, a minor collagen in cartilage, was examined by treatment with three different types of matrix metalloproteinases (MMPs) purified from the culture medium of rheumatoid synovial cells. Neither MMP-1 (collagenase) nor MMP-2 (so-called 'gelatinase') could digest type IX collagen, but MMP-3 (stromelysin) readily degraded it into smaller fragments. This suggests that MMP-3 may be responsible for the pathological degradation and/or normal turnover of type IX collagen.     

Hypermethylation of EBF3 and IRX1 genes in synovial fibroblasts of patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disease of unknown origin, which exhibits a complex heterogeneity in its pathophysiological background, resulting in differential responses to a range of therapies and poor long-term prognosis. RA synovial fibroblasts (RASFs) are key player cells in RA pathogenesis. Identification of DNA methylation biomarkers is a field that provides potential for improving the process of diagnosis and prognosis of various human diseases. We utilized a genome-wide technique, methylated DNA isolation assay (MeDIA), in combination with a high resolution CpG microarray for discovery of novel hypermethylated genes in RASFs. Thirteen genes (APEX1, EBF3, EGR2, EN1, IRX1, IRX6, KIF12, LHX2, MIPOL1, SGTA, SIN3A, TOLLIP, and ZHX2) with three consecutive hypermethylated probes were isolated as candidate genes through two CpG microarrays. Pyrosequencing assay was performed to validate the methylation status of TGF-β signaling components, EBF3 and IRX1 genes in RASFs and osteoarthritis (OA) SFs. Hypermethylation at CpG sites in the EBF3 and IRX1 genes was observed with a high methylation index (MI) in RASFs (52.5% and 41.4%, respectively), while a lower MI was observ ed in OASFs and h ealthy SFs (13.2% for EBF3 and 4.3% for IRX1). In addition, RT-PCR analysis showed a remarkable decrease in their mRNA expression in the RA group, compared with the OA or healthy control, and their reduction levels correlated with MI. The current findings suggest that methylation-associated down-regulation of EBF3 and IRX1 genes may play an important role in a pathogenic effect of TGF-β on RASFs. However, further clinical validation with large numbers of patients is needed in order to confirm our findings.     

Matrix metalloproteinase 2 from human rheumatoid synovial fibroblasts. Purification and activation of the precursor and enzymic properties

Human rheumatoid synovial cells in culture secrete at least three related metalloproteinases that digest extracellular matrix macromolecules. One of them, termed matrix metalloproteinase 2 (MMP-2), has been purified as an inactive zymogen (proMMP-2). The final product is homogeneous on SDS/PAGE with Mr = 72,000 under reducing conditions. The NH2-terminal sequence of proMMP-2 is Ala-Pro-Ser-Pro-Ile-Ile-Lys-Phe-Pro-Gly-Asp-Val-Ala-Pro-Lys-Thr, which is identical to that of the so-called '72-kDa type IV collagenase/gelatinase'. The zymogen can be rapidly activated by 4-aminophenylmercuric acetate to an active form of MMP-2 with Mr = 67,000, and the new NH2-terminal generated is Tyr-Asn-Phe-Phe-Pro-Arg-Lys-Pro-Lys-Trp-Asp-Lys-Asn-Gln-Ile. However, following 4-aminophenylmercuric acetate activation, MMP-2 is gradually inactivated by autolysis. Nine endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, neutrophil elastase, cathepsin G, matrix metalloproteinase 3, and thermolysin) were tested for their abilities to activate proMMP-2, but none had this ability. This contrasts with the proteolytic activation of proMMP-1 (procollagenase) and proMMP-3 (prostromelysin). The optimal activity of MMP-2 against azocoll is around pH 8.5, but about 50% of activity is retained at pH 6.5. Enzymic activity is inhibited by EDTA, 1,10-phenanthroline or tissue inhibitor of metalloproteinases, but not by inhibitors of serine, cysteine or aspartic proteinases. MMP-2 digests gelatin, fibronectin, laminin, and collagen type V, and to a lesser extent type IV collagen, cartilage proteoglycan and elastin. Comparative studies on digestion of collagen types IV and V by MMP-2 and MMP-3 (stromelysin) indicate that MMP-3 degrades type IV collagen more readily than MMP-2, while MMP-2 digests type V collagen effectively. Biosynthetic studies of MMPs using cultured human rheumatoid synovial fibroblasts indicated that the production of both proMMP-1 and proMMP-3 is negligible but it is greatly enhanced by the treatment with rabbit-macrophage-conditioned medium, whereas the synthesis of proMMP-2 is constitutively expressed by these cells and is not significantly affected by the treatment. This suggests that the physiological and/or pathological role of MMP-2 and its site of action may be different from those of MMP-1 and MMP-3.     

Evidence that human rheumatoid synovial matrix metalloproteinase 3 is an endogenous activator of procollagenase

The treatment of crude culture medium from human rheumatoid synovial cells with 4-aminophenylmercuric acetate (APMA) or trypsin results in the activation of procollagenase. This process was shown to be dependent on the presence of matrix metalloproteinase 3 (MMP-3). MMP-3 can directly activate procollagenase without changing the apparent molecular weight of procollagenase. This activity was accelerated in the presence of APMA. We propose that MMP-3 plays an important role in connective tissue destruction through the activation of procollagenase in addition to its direct action on components of the extracellular matrix.     

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.