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.
"Moreover, MG132 proteasome inhibitor increased TAK1 levels in SOCS1-overexpressing chondrocytes. These findings suggested that SOCS1 provides a novel negative-feedback mechanism through the degradation of TAK1, which is involved in IL-1β signaling . "
[Show abstract][Hide abstract] ABSTRACT: Although IL-1beta is believed to be crucial in the pathogenesis of osteoarthritis (OA), the IL-1beta blockade brings no therapeutic benefit in human OA and results in OA aggravation in several animal models. We explored the role of a cytokine signaling 1 (SOCS1) suppressor as a regulatory modulator of IL-1beta signaling in chondrocytes.
Cartilage samples were obtained from patients with knee OA and those without OA who underwent surgery for femur neck fracture. SOCS1 expression in cartilage was assessed by immunohistochemistry. IL-1beta-induced SOCS1 expression in chondrocytes was analyzed by quantitative polymerase chain reaction and immunoblot. The effect of SOCS1 on IL-1beta signaling pathways and the synthesis of matrix metalloproteinases (MMPs) and aggrecanase-1 was investigated in SOCS1 over-expressing or knockdown chondrocytes.
SOCS1 expression was significantly increased in OA cartilage, especially in areas of severe damage (P <0.01). IL-1beta stimulated SOCS1 mRNA expression in a dose-dependent pattern (P <0.01). The IL-1beta induced production of MMP-1, MMP-3, MMP-13, and ADAMTS-4 (aggrecanase-1, a disintegrin and metalloproteinase with thrombospondin motifs 4) was affected by SOCS1 over-expression or knockdown in both SW1353 cells and primary human articular chondrocytes (all P <0.05). The inhibitory effects of SOCS1 were mediated by blocking p38, c-Jun N-terminal kinase (JNK) and nuclear factor kappaB (NF-kappaB) activation, and by downregulating transforming growth factor-beta activated kinase 1 (TAK1) expression.
Our results show that SOCS1 is induced by IL1-beta in OA chondrocytes and suppresses the IL-1beta induced synthesis of matrix degrading enzymes by inhibiting IL-1beta signaling at multiple levels. It suggests that the IL-1beta-inducible SOCS1 acts as a negative regulator of the IL-1beta response in OA cartilage.
"OA is a multifactorial disease characterized by progressive erosion of articular cartilage, proteoglycan (PG) degradation, disruption of the collagen network, and subsequent chondrocytes apoptosis and death (Das and Farooqi, 2008). Several studies have demonstrated that enzymatic cleavage by matrix metalloproteinases (MMPs) together with cytokines such as interleukin-1 (IL-1) and tumor necrosis factor ␣ (TNF␣) play critical roles in the initiation and progression of articular cartilage destruction (Klatt et al., 2006; Pujol et al., 2008). Chondrocytes apoptosis has also been identified as a critical reason for cell loss in aging OA cartilage and is now considered as an important factor contributing to the breakdown of extracellular matrix in joint diseases (Johnson et al., 2008). "
"The functional consequences of activating the TAK1-JNK-AP1 pathway can be evaluated by determining expression of key AP-1-driven genes implicated in RA. The AP-1 consensus sequence is located at -70 base-pairs in the promoter region of the gene encoding MMP3, making it a useful biomarker for TAK1 in cells such as osteocytes  and chondrocytes . siRNA studies showed that TAK1 inhibition significantly decreased MMP3 gene expression in cultured FLSs. "
[Show abstract][Hide abstract] ABSTRACT: c-Jun N-terminal kinase (JNK) contributes to metalloproteinase (MMP) gene expression and joint destruction in inflammatory arthritis. It is phosphorylated by at least two upstream kinases, the mitogen-activated protein kinase kinases (MEK) MKK4 and MKK7, which are, in turn, phosphorylated by MEK kinases (MEKKs). However, the MEKKs that are most relevant to JNK activation in synoviocytes have not been determined. These studies were designed to assess the hierarchy of upstream MEKKs, MEKK1, MEKK2, MEKK3, and transforming growth factor-beta activated kinase (TAK)1, in rheumatoid arthritis (RA). Using either small interfering RNA (siRNA) knockdown or knockout fibroblast-like synoviocytes (FLSs), MEKK1, MEKK2, or MEKK3 deficiency (either alone or in combination) had no effect on IL-1beta-stimulated phospho-JNK (P-JNK) induction or MMP expression. However, TAK1 deficiency significantly decreased P-JNK, P-MKK4 and P-MKK7 induction compared with scrambled control. TAK1 knockdown did not affect p38 activation. Kinase assays showed that TAK1 siRNA significantly suppressed JNK kinase function. In addition, MKK4 and MKK7 kinase activity were significantly decreased in TAK1 deficient FLSs. Electrophoretic mobility shift assays demonstrated a significant decrease in IL-1beta induced AP-1 activation due to TAK1 knockdown. Quantitative PCR showed that TAK1 deficiency significantly decreased IL-1beta-induced MMP3 gene expression and IL-6 protein expression. These results show that TAK1 is a critical pathway for IL-1beta-induced activation of JNK and JNK-regulated gene expression in FLSs. In contrast to other cell lineages, MEKK1, MEKK2, and MEKK3 did not contribute to JNK phosphorylation in FLSs. The data identify TAK1 as a pivotal upstream kinase and potential therapeutic target to modulate synoviocyte activation in RA.
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