TAK1 mediates the collagen-II-dependent induction of the COX-2 gene and PGE2 release in primary human chondrocytes.

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.

Matrilin-3 activates the expression of osteoarthritis-associated genes in primary human chondrocytes.

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-1beta, TNFalpha, 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-1beta. 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.

Discoidin domain receptor 2 mediates the collagen II-dependent release of interleukin-6 in primary human chondrocytes.

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.

A critical role for collagen II in cartilage matrix degradation: collagen II induces pro-inflammatory cytokines and MMPs in primary human chondrocytes.

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-1beta. 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 IkappaB concentration were monitored by immunoblot analysis to detect NFkappaB 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-1beta, IL-6, and IL-8. At the same time, inhibition of p38 and IkappaB degradation revealed that collagen II-dependent gene induction also involves MAPK p38 and NFkappaB 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.

RNAi in primary human chondrocytes: efficiencies, kinetics, and non-specific effects of siRNA-mediated gene suppression.

RNAi-mediated gene silencing is a recent, powerful tool to investigate gene function. Controlling for experimental factors such as transfection efficiencies, siRNA concentration, gene suppression levels, gene suppression kinetics, or non-specific effects is key to robust results. In this methods paper, we compare the efficiencies of different transfection reagents in primary human chondrocytes (PHCs). We investigated TAK1 gene suppression efficiencies and kinetics on the mRNA and protein level depending on the siRNA concentration used. Furthermore, we evaluated PKR, IL-6, and TNF-alpha induction, as well as IkappaB degradation and NFkappaB activation as control parameters of non-specific siRNA effects. PKR and IL-6 proved to be appropriate markers of cellular inflammatory responses resulting from siRNA transfection. In addition, we compared different siRNAs (silencing, non-silencing, classic 21-mer, and 25-mer stealth siRNA) with respect to their capacity to induce cellular inflammatory responses. We found the occurrence of cellular responses in PHCs to be a function of the specific siRNA sequence in use. Hence, it is essential to analyze and to compare gene silencing siRNAs and control siRNAs with respect to their off-target effects prior to any functional gene validation.

TAK1 downregulation reduces IL-1beta induced expression of MMP13, MMP1 and TNF-alpha.

The paper provides evidence that transforming growth factor-beta activated kinase 1 (TAK1, MEKK7), a downstream mediator of IL-1beta signal transduction, plays an important role in the regulation of catabolic events and inflammatory processes in the context of degenerative joint diseases. We investigated the expression of TAK1 in human articular chondrocytes and in the murine growth plate by cDNA array, quantitative RT-PCR and immunohistochemistry, respectively. The human chondrosarcoma cell line SW1353 was stimulated with the proinflammatory cytokine IL-1beta. The subsequent expression of proteolytic enzymes and proinflammatory cytokines was quantified. TAK1 specific siRNA was used to study the influence of TAK1 downregulation on the expression of MMP-13, MMP1 and TNF-alpha. As a result we demonstrated the expression of TAK1 in normal and osteoarthritic human articular cartilage. Expression of TAK1 in the hypertrophic zone of the growth plate gave us a first evidence for a catabolic function of TAK1 concerning cartilage metabolism. By gene suppression with RNAi technology we could show that TAK1 downregulation leads to a 60-70% reduced release of TNF-alpha, a 40-50% reduced release of MMP13, and a 20-30% reduction of MMP1 release. As TNF-alpha is a main player in inflammatory processes, and MMP13 is one of the major proteases involved in cartilage degradation, our results suggests that TAK1 has an important regulatory role in the context of degenerative joint diseases and thus is an attractive drug target in attempts to reduce inflammation and suppress structural changes in OA induced by IL-1beta.