Sites of collagenase cleavage and denaturation of type II collagen in aging and osteoarthritic articular cartilage and their relationship to the distribution of matrix metalloproteinase 1 and matrix metalloproteinase 13.
(65/1192)
OBJECTIVE: To determine the sites of cleavage and denaturation of type II collagen (CII) by collagenase(s) in healthy and osteoarthritic (OA) human articular cartilage and their relationship to the distribution of matrix metalloproteinase 1 (MMP-1) and MMP-13. METHODS: Single (per subject) full-depth specimens from femoral condylar cartilage were isolated from articulating surfaces at autopsy from 8 subjects without arthritis and during arthroplasty from 10 patients with OA. Fixed frozen sections of cartilage were examined by immunoperoxidase localization, using antibodies to the collagenase-generated cleavage site in CII, to an intrachain epitope recognized only in denatured CII, and to MMP-1 and MMP-13 (proenzyme, activated enzyme, or enzyme/inhibitor complex). RESULTS: Staining for collagen cleavage, denaturation, and both MMPs was weak to moderate and was frequently observed in pericellular sites in cartilage from younger, nonarthritic subjects. In specimens from older subjects, this staining was often more widespread and of greater intensity. Similar staining was usually, but not always, seen for all antibodies. In OA cartilage, staining was often stronger and more intense than that in normal cartilage from older subjects, and the distribution of staining was often similar for the different antibodies. Pericellular staining in the deep zone was frequently more pronounced in arthritic cartilage and extended to territorial and sometimes interterritorial sites. In very degenerate specimens, staining was distributed throughout most of the cartilage matrix. CONCLUSION: These observations provide evidence for the presence of limited cleavage and denaturation of CII restricted to mainly pericellular and superficial sites in cartilage from younger, healthy subjects, where MMP-1 and MMP-13 are also selectively localized. Collagen degradation is more extensive and often more pronounced in cartilage from older, nonarthritic subjects. Characteristic changes in early OA are similar to those seen with aging in cartilage from older, healthy subjects, with collagen damage and collagenases concentrated closer to the articular surface. There was usually a close correspondence between the cleavage and denaturation of CII and the sites at which these collagenases were detected, suggesting that both MMPs are involved in the physiology and pathology. There was no evidence that the damage to CII is ordinarily initiated in sites other than at and near the articular surface and around chondrocytes. (+info)
The TATA-containing core promoter of the type II collagen gene (COL2A1) is the target of interferon-gamma-mediated inhibition in human chondrocytes: requirement for Stat1 alpha, Jak1 and Jak2.
(66/1192)
Interferon-gamma (IFN-gamma) inhibits the synthesis of the cartilage-specific extracellular matrix protein type II collagen, and suppresses the expression of the type II collagen gene ( COL2A1 ) at the transcriptional level. To further examine this mechanism, the responses of COL2A1 regulatory sequences to IFN-gamma and the role of components of the Janus kinase/signal transducer and activators of transcription (JAK/STAT) pathway were examined in the immortalized human chondrocyte cell line, C-28/I2. IFN-gamma inhibited the mRNA levels of COL2A1 and aggrecan, but not Sox9, L-Sox5 and Sox6, all of which were expressed by these cells as markers of the differentiated phenotype. IFN-gamma suppressed the expression of luciferase reporter constructs containing sequences of the COL2A1 promoter spanning -6368 to +125 bp in the absence and presence of the intronic enhancer and stimulated activity of the gamma-interferon-activated site (GAS) luciferase reporter vector, associated with induction of Stat1 alpha-binding activity in nuclear extracts. These responses to IFN-gamma were blocked by overexpression of the JAK inhibitor, JAK-binding protein (JAB), or reversed by dominant-negative Stat1 alpha Y701F containing a mutation at Tyr-701, the JAK phosphorylation site. IFN-gamma had no effect on COL2A1 promoter expression in Jak1 (U4A)-, Jak2 (gamma 2A)- and Stat1 alpha (U3A)-deficient cell lines. In the U3A cell line, the response to IFN-gamma was rescued by overexpression of Stat1 alpha, but not by either Stat1 alpha Y701F or Stat1 beta. Functional analysis using deletion constructs showed that the IFN-gamma response was retained in the COL2A1 core promoter region spanning -45 to +11 bp, containing the TATA-box and GC-rich sequences but no Stat1-binding elements. Inhibition of COL2A1 promoter activity by IFN-gamma persisted in the presence of multiple deletions within the -45/+11 bp region. Our results indicate that repression of COL2A1 gene transcription by IFN-gamma requires Jak1, Jak2 and Stat1 alpha and suggest that this response involves indirect interaction of activated Stat1 alpha with the general transcriptional machinery that drives constitutive COL2A1 expression. (+info)
Epitope-specific recognition of type II collagen by rheumatoid arthritis antibodies is shared with recognition by antibodies that are arthritogenic in collagen-induced arthritis in the mouse.
(67/1192)
OBJECTIVE: To analyze the fine specificity of IgG autoantibodies in sera from rheumatoid arthritis (RA) patients for type II collagen (CII) epitopes that are arthritogenic in collagen-induced arthritis (CIA), a relevant murine model of RA. METHODS: For enzyme-linked immunosorbent assay (ELISA) analysis of conformation-dependent autoantibody binding, recombinant chimeric collagens that harbor the respective CII epitopes as an insertion within the frame of a constant type X collagen triple helix were constructed. In addition, synthetic peptides mimicking the native collagen structures were applied for the first time in the ELISA assessment of humoral CII autoimmunity. RESULTS: The pathogenicity of IgG responses to certain CII determinants in CIA was demonstrated by arthritis development in BALB/c mice upon the combined transfer of 2 mouse monoclonal antibodies specific for precisely mapped conformational CII epitopes (amino acid residues 359-369 [C1(III)] and 551-564 [J1]), whereas antibodies to another epitope (F4) were not arthritogenic. To test whether human autoimmune responses are similarly directed to these conserved CII determinants, serum IgG was analyzed. The prevalence of sera with increased IgG binding to the C1(III) epitope was significantly higher in RA compared with sera from healthy donors or from patients with other rheumatic conditions, e.g., osteoarthritis (OA), systemic lupus erythematosus (SLE), or relapsing polychondritis (RP), whereas levels of antibodies specific for the nonarthritogenic F4 epitope were associated with OA rather than RA. CONCLUSION: Autoimmunity to CII, although detectable in different rheumatic conditions, differs in fine specificity between distinct disease entities. In RA, in contrast to degenerative joint disease, RP, and SLE, autoantibody responses are directed to an evolutionary conserved CII structure that is also targeted by pathogenic autoimmune responses in murine models of arthritis. (+info)
Reduced susceptibility to collagen-induced arthritis in DBA/1J mice expressing the TSG-6 transgene.
(68/1192)
OBJECTIVE: Expression of TSG-6 (tumor necrosis factor-stimulated gene 6) is induced by proinflammatory cytokines. This study was undertaken to examine the effects of local expression of TSG-6 in arthritic joints of TSG-6 transgenic mice, in the collagen-induced arthritis (CIA) model. METHODS: We generated transgenic mice that harbored the TSG-6 gene under the control of the T cell-specific lck promoter. Arthritis was induced by immunization with bovine type II collagen (CII), and its progression was monitored based on the incidence of arthritis, the arthritis index, and footpad swelling. Anti-CII antibodies and cytokine production were determined by enzyme-linked immunosorbent assay. Gene expression arrays were used to compare gene expression profiles of transgenic and control mice at various stages of CIA. RESULTS: TSG-6 was expressed in limbs of transgenic mice after immunization with CII, while its expression in nontransgenic animals was insignificant. The incidence of CIA was reduced in TSG-6 transgenic animals, its onset delayed, and all parameters of clinical arthritis significantly reduced. However, the immune response against CII was not significantly inhibited in TSG-6 transgenic mice. CONCLUSION: TSG-6 expression has been demonstrated in patients with rheumatoid and other forms of arthritis. Our data show that local expression of TSG-6 at sites of inflammation results in potent inhibition of inflammation and joint destruction in a model of autoimmune arthritis in mice. Therefore, it is likely that TSG-6 plays a similar modulatory role in human rheumatoid arthritis and related diseases and may have potential for the treatment of autoimmune arthritis in humans. (+info)
Skeletal abnormalities and ultrastructural changes of cartilage in transgenic mice expressing a collagen II gene (COL2A1) with a Cys for Arg-alpha1-519 substitution.
(69/1192)
OBJECTIVE: To examine the mechanism by which the Arg-->Cys 519 mutation causes the clinical phenotype employing transgenic mice that express the mutated human COL2A1. METHODS: A DNA construct under the control of a COL2A1 specific promoter was prepared from genomic DNA isolated from fibroblasts from the proband with primary generalized osteoarthritis (OA) associated with a mild chondrodysplasia. Transgenic mice were obtained by injection of the constructs into pro-nuclei of fertilized eggs from the FVB/N inbred mouse strain. Transgenic mice harboring two alleles of the mutated human COL2A1 were examined for morphological abnormalities and for alterations of their skeletal development. Ultrastructural examination was performed to identify changes in the organization and density of collagen II fibrils in articular cartilage of the transgenic mice. RESULTS: Transgenic mice harboring two alleles of the mutated human collagen gene were smaller than their normal littermates, had a cleft palate, and disorganized growth plate. Electron microscopy of articular cartilage showed a decreased density of collagen II fibrils and revealed chondrocytes with dilated Golgi cysternae. CONCLUSIONS: Expression of a COL2A1 with an Arg-->Cys 519 substitution in transgenic mice causes retardation of skeletal development and ultrastructural alterations in articular cartilage with a profound reduction of the density of the collagen II fibrils in the tissue. These alterations may be responsible for the phenotype of precocious generalized OA and chondrodysplasia displayed by patients harboring this COL2A1 mutation. (+info)
Amelioration of collagen-induced arthritis by blockade of inducible costimulator-B7 homologous protein costimulation.
(70/1192)
B7 homologous protein (B7h)/B7-related protein 1 (B7RP-1) is a new member of the B7 family of costimulatory molecules that specifically interacts with inducible costimulator (ICOS) expressed on activated T cells. Collagen type II (CII)-induced arthritis (CIA) is an experimental model of arthritis that has been used to dissect the pathogenesis of human rheumatoid arthritis. In this study, we have investigated the effect of neutralizing anti-B7h mAb on the development and disease progression of CIA. Administration of anti-B7h mAb significantly ameliorated the disease as assessed by clinical arthritis score and histology in the joints, and a beneficial effect was also obtained by a delayed treatment after the onset of disease. Expression of ICOS and B7h was observed in the inflamed synovial tissue as well as in the draining lymph nodes (LNs) and expansion of ICOS(+) T cells in the LN was reduced by the anti-B7h mAb treatment. Expression of mRNA for proinflammatory cytokines such as TNF-alpha, IL-1beta, and IL-6 in the joints was inhibited by the treatment. Proliferative responses and production of IFN-gamma and IL-10 upon restimulation with CII in vitro were significantly inhibited in LN cells from the anti-B7h mAb-treated mice. Serum anti-CII IgG1, IgG2a, and IgG2b levels were also reduced. Our present results showed a beneficial effect of the B7h blockade on CIA through anti-inflammatory actions and inhibition of both Th1- and Th2-mediated immune responses, suggesting that the ICOS-B7h interaction plays an important role in the pathogenesis of CIA and thus the blockade of this pathway may be beneficial for the treatment of human rheumatoid arthritis. (+info)
Effects of overexpression of membrane-bound transferrin-like protein (MTf) on chondrogenic differentiation in Vitro.
(71/1192)
Membrane-bound transferrin-like protein (MTf) is expressed in parallel with the expression of cartilage-characteristic genes during differentiation of chondrocytes, and the MTf level is much higher in cartilage than in other tissues. To investigate the role of MTf in cartilage, we examined the effects of growth factors on MTf expression in mouse prechondrogenic ATDC5 cells and the effect of MTf overexpression on differentiation of ATDC5 and mouse pluripotent mesenchymal C3H10T1/2 cells. In ATDC5 cultures, bone morphogenetic protein-2 and transforming growth factor-beta as well as insulin induced MTf mRNA expression when these peptides induced chondrogenic differentiation. Forced expression of rabbit MTf in ATDC5 cells induced aggrecan, type II collagen, matrilin-1, type X collagen mRNAs, and cell-shape changes from fibroblastic cells to spherical chondrocytes. Accordingly, the synthesis and accumulation of proteoglycans were higher in MTf-expressing cultures than in control cultures. These effects of MTf overexpression correlated with the MTf protein level on the cell surface and decreased in the presence of anti-MTf antibody. However, the aggrecan mRNA level in the ATDC5 cells overexpressing MTf was lower than that in wild type ATDC5 cells exposed to 10 microg/ml insulin. MTf overexpression in C3H10T1/2 cells also induced aggrecan and/or type II collagen mRNA but not the spherical phenotype. These findings suggest that the expression of MTf on the cell surface facilitates the differentiation of prechondrogenic cells, although MTf overexpression alone seems to be insufficient to commit pluripotent mesenchymal cells to the chondrocyte lineage. (+info)
A new long form of c-Maf cooperates with Sox9 to activate the type II collagen gene.
(72/1192)
A new long form of the c-Maf transcription factor (Lc-Maf) was identified and shown to interact specifically with SOX9 in a yeast two-hybrid cDNA library screening. Lc-Maf encodes an extra 10 amino acids at the carboxyl terminus of c-Maf and contains a different 3'-untranslated region compared with c-Maf. The interaction between SOX9 and Lc-Maf was further confirmed by co-immunoprecipitation and glutathione S-transferase pull-down assays, which mapped the interacting domain of SOX9 to the high mobility group box DNA binding domain and that of Lc-Maf to the basic leucine zipper motif. In situ hybridizations showed that Lc-Maf RNA was coexpressed with Sox9 and Col2a1 RNA in areas of precartilaginous mesenchymal condensations during mouse embryo development. A DNA binding site of Lc-Maf was identified at the 5'-end of a 48-bp Col2a1 enhancer element near the high mobility group binding site of SOX9. Lc-Maf and SOX9 synergistically activated a luciferase reporter plasmid containing a Col2a1 enhancer and increased the transcription of the endogenous Col2a1 gene. In summary, Lc-Maf is the first transcription factor shown to interact with Sox9, to be coexpressed with Sox9 during an early step of chondrogenesis and to cooperate with Sox9 in activating a downstream target gene of Sox9. (+info)