We report a process that results in the acceleration of matrix degradation in human articular cartilage, a phenomenon commonly observed in osteoarthritis (OA). The study was conducted by (1) examining the potential of collagen II in modulating the gene expression profile of primary human chondrocytes (PHCs), and (2) investigating the involvement of pro-inflammatory signaling cascades. We first tested the collagen II-dependent induction of pro-inflammatory cytokines and matrix metalloproteinases (MMPs) in PHCs. PHCs were incubated with or without monomeric (i.e., nonfibrillar) collagen II. Cells were then analyzed by RT-PCR for the expression of MMP1, MMP3, MMP13, MMP14, and IL-1b. ELISA was used to quantify IL-6 and IL-8 release. To examine the influence of collagen II signaling, specifically the role of MAPK p38, a p38-inhibitor was added prior to collagen treatment. Changes in IkB concentration were monitored by immunoblot analysis to detect NFkB signaling. Results indicated that incubation of PHCs with collagen II did produce a dose-dependent induction of MMP1, MMP3, MMP13, MMP14, as well as cytokines IL-1b, IL-6, and IL-8. At the same time, inhibition of p38 and IkB degradation revealed that collagen II-dependent gene induction also involves MAPK p38 and NFkB signaling. Thus, we provide evidence for a collagen II-dependent feed-forward mechanism whereby collagen II induces first MMPs and pro-inflammatory cytokines and then release of collagen II fragments from mature collagen II fibers. This, in turn, induces more pro-inflammatory cytokines and MMPs, and the process is repeated, which results in the acceleration and perpetuation of cartilage matrix degradation. Keywords: collagen; chondrocytes; osteoarthritis; matrix metalloproteinases; Osteoarthritis (OA) is a chronic disease of synovial joints. Although the etiology and pathogenesis of OA are poorly understood, we do know that degradation of the extracellular matrix (ECM) in articular cartilage is a major cause of joint destruction. Cartilage degradation is mediated by matrix metalloproteinases (MMPs), principally MMP1, MMP3, and MMP13.1-4 MMP1 (collagenase-1) and MMP13 (collagenase-3) are able to cleave mature collagen fibers and initiate further collagen degradation. MMP3 (stromelysin-1) cleaves a variety of ECM components, including proteoglycans, collagens, and procollagens.5 Chondrocytes respond to various stimuli, such as pro-inflammatory cytokines or mechanical load, by their induction of MMPs. Collagen II may fulfill a similar function in that both collagen II and a synthetic 29-mer collagen peptide have recently been shown to induce MMP2, MMP3, MMP9, and MMP13 in primary bovine articular chondrocytes. 6 According to other studies, collagen II can induce MMP13 in the immortalized human chondrocyte cell line C-28/I2, which transiently expresses discoidin domain receptor-2 (DDR-2) cDNA. 4 Thus far, however, the induction of MMPs by collagen II in PHCs has not been investigated. The induction of collagenase-1 or MMP14 (membranetype MMP1, MT...

Edited by Laszlo NagyKeywords: Matrilin Collagen II Osteoarthritis A disintegrin and MMP with thrombospondin motif Interleukin TNF a b s t r a c t Here, we tested the matrilin-3-dependent induction of osteoarthritis-associated genes in primary human chondrocytes. Matrilin stimulation leads to the induction of MMP1, MMP3, MMP13, COX-2, iNOS, IL-1b, TNFa, IL-6 and IL-8. Furthermore, we show the participation of ADAMTS4 and ADAMTS5 in the in vitro degradation of matrilin-3. We provide evidence for a matrilin-3-dependent feed-forward mechanism of matrix degradation, whereby proteolytically-released matrilin-3 induces pro-inflammatory cytokines as well as ADAMTS4 and -5 indirectly via IL-1b. ADAMTS4 and ADAMTS5, in turn, cleave matrilin-3 and may release more matrilin-3 from the matrix, which could lead to further release of pro-inflammatory cytokines and proteases in cartilage.

We deciphered constituent parts of a signal transduction cascade that is initiated by collagen II and results in the release of various pro-inflammatory cytokines, including interleukin-6 (IL-6), in primary human chondrocytes. This cascade represents a feed-forward mechanism whereby cartilage matrix degradation is exacerbated by the mutually inducing effect of released collagen II fragments and pro-inflammatory cytokines. We previously proposed discoidin domain receptor 2 as a central mediator in this event. Since this cascade plays a prominent role in the pathogenesis of osteoarthritis, our study further investigates the hypothesis that discoidin domain receptor 2 is a candidate receptor for collagen II, and that transcription factor NFkappaB, lipid kinase PI3K, and the MAP kinases are constituent parts of this very signal transduction cascade. To accomplish this, we selectively knocked down the molecules of interest in primary human chondrocytes, induced the specified cascade by incubating primary human chondrocytes with collagen II, and observed the outcome, specifically the changes in interleukin-6 release. Knockdown was performed by siRNA-mediated gene silencing in the case of discoidin domain receptor 2 (DDR2) or by using specific inhibitors for the remainder of the molecules. Results indicated that discoidin domain receptor 2 mediates the collagen II-dependent release of interleukin-6 in primary human chondrocytes and that MAP kinases p38, JNK and ERK, as well as transcription factor NFkappaB, are integral components of intracellular collagen II signalling. Given the detrimental role of these molecules in osteoarthritis, our findings provide new targets for more specific therapeutics, which may have fewer side effects than those currently applied.

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Based on recombinant expression of different matrilin-3 domains in both monomeric and oligomeric form, this work demonstrated that the VWA1 domain of matrilin-3 is primarily responsible for the induction of IL-6 release and that the oligomerization of the VWA1 domain markedly promotes its activity.

We investigated the role of transforming growth factor-beta activated kinase 1 (TAK1) in collagen II signaling in primary human chondrocytes (PHCs). We asked whether TAK1 acts as a modulator of collagen II signaling with respect to collagen-II-dependent induction of cyclooxigenase-2 (COX-2) in PHCs and release of PGE2 from PHCs. Therefore, PHCs were incubated with collagen II, and cells were then analyzed by RT-PCR for the expression of COX-2. ELISA was used to quantify PGE2 release. To examine the influence of TAK1 on these events, TAK1 gene silencing was performed by RNAi in PHCs prior to collagen II treatment. Results indicated that COX-2 gene expression and PGE2 release are specific outcomes of collagen II signaling and that both depend on TAK1 mediation. These findings are promising in that therapeutic inhibition of TAK1 might be used to reduce pain and relieve inflammatory symptoms that are common in osteoarthritis.