Pathophysiological Mechanisms in Osteoarthritis Lead to Novel Therapeutic Strategies

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Objective. Expression of matrix metalloproteinase 9 (MMP-9) is up-regulated in osteoarthritis (OA) and usually presents as multiple bands when synovial fluid (SF) from OA patients is analyzed by zymography. Among these bands is an ϳ125-130-kd band for high molecular weight (HMW) gelatinase, which has not been characterized. This study was undertaken to characterize the HMW MMP activity in OA SF.Methods. MMP activity in OA SF was determined by gelatin zymography. Recombinant MMPs were used to identify MMP activity on the zymogram. Western immunoblotting, immunoprecipitation, and immunodepletion analyses were performed using antibodies specific for human MMP-9 and human neutrophil gelatinase-associated lipocalin (NGAL). Human cartilage matrix degradation was determined by dimethylmethylene blue assay.Results. Zymographic analysis showed that the HMW gelatinase in OA SF comigrated with a purified NGAL-MMP-9 complex. Results of Western immunoblotting showed that the HMW gelatinase was also recognized by antibodies specific for human NGAL or human MMP-9. These same antibodies also immunoprecipitated the HMW gelatinase activity from OA SF. The NGAL-MMP-9 complex was reconstituted in vitro in gelatinase buffer. In the presence of NGAL, MMP-9 activity was stabilized; in the absence of NGAL, rapid loss of MMP-9 activity occurred. MMP-9-mediated release of cartilage matrix proteoglycans was significantly higher in the presence of NGAL (P < 0.05).Conclusion. Our findings demonstrate that the HMW gelatinase activity in OA SF represents a complex of NGAL and MMP-9. The ability of NGAL to protect MMP-9 activity is relevant to cartilage matrix degradation in OA and may represent an important mechanism by which NGAL may contribute to the loss of cartilage matrix proteins in OA.The developed world's aging population has experienced a dramatic increase in the incidence of joint dysfunction and osteoarthritis (OA), leading to a compromised quality of life. Aging, biomechanical stress, and oxidative stress, in addition to genetic factors and trauma, all appear to be associated with degenerative and progressive cartilage and bone changes in OA, particularly in the weight-bearing joints. Although OA is not considered an inflammatory disease by traditional standards, elevated levels of proinflammatory cytokines, such as interleukin-1␤ (IL-1␤) and tumor necrosis factor ␣, have been observed in OA synovial fluid (SF), supporting the notion that there is an inflammatory component associated with the pathogenesis of OA (for review, see refs. 1-3).The only cell type present in articular cartilage is the chondrocyte, which is embedded within an extracellular matrix (ECM) primarily composed of type II collagen and aggrecan. Chondrocytes arise from a mesenchymal progenitor cell and are responsible for the biosynthesis of ECM proteins and their transport into the space occupied by the ECM. Chondrocytes play an important role in cartilage homeostasis by maintaining the proper balance between anabolic pathways, which Supported in part by the NIH...

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Neutrophil gelatinase–associated lipocalin is expressed in osteoarthritis and forms a complex with matrix metalloproteinase 9

Gupta¹,

Shukla²,

Cowland³

et al. 2007

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“…In OA, the excessive degradation is primarily due to the action of proteases released by chondrocytes, synovial cells, and macrophages (4). Numerous serine proteases and metalloproteases are overexpressed by these cells during the disease process, but specific metalloproteases, including the collagenase matrix metalloproteinase 13 (MMP-13) and the aggrecanase ADAMTS-5, are thought to be especially important for initiating and promoting cartilage matrix degradation in OA (5,6).…”

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confidence: 99%

Characterization of metalloprotease cleavage products of human articular cartilage

Zhen¹,

Brittain²,

Laska³

et al. 2008

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Objective. To identify, characterize, and compare proteolysis peptide products generated by metalloprotease digests of human articular cartilage.Methods. Human articular cartilage was digested by the addition of exogenous metalloproteases, including matrix metalloproteinases 2, 3, 8, 9, 12, and 13 and aggrecanases ADAMTS-4 and ADAMTS-5. Proteolyzed peptide products were identified by proteomics methods using mass spectrometry.Results. Complete sequences of the peptides proteolyzed from human articular cartilage, including Nand C-termini and hydroxylated posttranslational modifications, were determined. A wide variety of peptides, originating from types I, II, and III collagen, biglycan, prolargin, fibromodulin, fibronectin, decorin, cartilage oligomeric matrix protein, cartilage intermediate-layer protein, megakaryocyte-stimulating factor, mimecan, aggrecan, and lumican, was analyzed following metalloprotease digestion. Release of peptides varied as a function of time, enzyme specificity, and abundance. Specific type II collagen peptide biomarkers, including those containing the three-quarter-length fragment cleavage site and those containing the domains for helical peptide of type II collagen and C-telopeptide of type II collagen, were observed after release by selected proteases.Conclusion. The use of intact cartilage instead of purified protein substrates in the assay allowed for the identification of novel potential substrates and cleavage sites for individual enzymes under more physiologically relevant conditions. Characterization of these cartilage matrix peptides may help in the development of pharmacodynamic biomarkers of cartilage degradation, and also may contribute to an understanding of the bioactive peptides important in chondrocyte signaling.Osteoarthritis (OA) and rheumatoid arthritis (RA) are typified by biologic changes in the articular cartilage that lead to cartilage degradation and joint space narrowing (1,2). Articular cartilage is composed of 2 primary matrix proteins, type II collagen and aggrecan, as well as a number of other matrix proteins that provide structural functions such as rigidity, flexibility, and compression damping. In addition, the cartilage matrix influences chondrocyte function through integrin receptor signaling, initiated via ligation of either intact matrix molecules or peptide fragments resulting from proteolytic activity (3).In arthritis, an imbalance of chondrocyte matrix synthesis and degradation leads to a net loss of matrix, and eventually to joint impairment. In OA, the excessive degradation is primarily due to the action of proteases released by chondrocytes, synovial cells, and macrophages (4). Numerous serine proteases and metalloproteases are overexpressed by these cells during the disease process, but specific metalloproteases, including the collagenase matrix metalloproteinase 13 (MMP-13) and the aggrecanase ADAMTS-5, are thought to be especially important for initiating and promoting cartilage matrix degradation in OA (5,6). MMP-13 cleaves type II collage...

“…During osteoarthritis (OA), IL-1␤ is found in large amounts in synovial fluids and may actively participate in cartilage breakdown (12). It induces the activation of protein kinase cascades involving the MAPKs and/or the NF-B pathway.…”

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confidence: 99%

Interleukin‐1β impairment of transforming growth factor β1 signaling by DOWN‐REGULATION of transforming growth factor β receptor type II and up‐regulation of Smad7 in human articular chondrocytes

Baugé

Legendre

Leclercq

et al. 2007

Objective. Extracellular matrix deposition is tightly controlled by a network of regulatory cytokines. Among them, interleukin-1␤ (IL-1␤) and transforming growth factor ␤1 (TGF␤1) have been shown to play antagonistic roles in tissue homeostasis. The purpose of this study was to determine the influence of IL-1␤ on TGF␤ receptor type II (TGF␤RII) regulation and TGF␤1 responsiveness in human articular chondrocytes.Methods. TGF␤1-induced gene expression was analyzed through plasminogen activator inhibitor 1 and p3TP-Lux induction. Receptor-activated Smad (RSmad) phosphorylation, TGF␤ receptors, and Smad expression were determined by Western blotting and real-time reverse transcription-polymerase chain reaction techniques. Signaling pathways were investigated using specific inhibitors, messenger RNA (mRNA) silencing, and expression vectors.Results. IL-1␤ down-regulated TGF␤RII expression at both the protein and mRNA levels and led to inhibition of the TGF␤1-induced gene expression and Smad2/3 phosphorylation. Moreover, IL-1␤ strongly stimulated the expression of inhibitory Smad7.TGF␤RII overexpression abolished the loss of TGF␤1 responsiveness induced by IL-1␤. The decrease in TGF␤RII required de novo protein synthesis and involved both the NF-B and JNK pathways.Conclusion. We demonstrate that IL-1␤ impairs TGF␤1 signaling through down-regulation of TGF␤RII, which is mediated by the p65/NF-B and activator protein 1/JNK pathways, and secondarily through the up-regulation of Smad7. These findings show that there is cross-talk in the signaling of 2 regulatory cytokines involved in inflammation.