Thus, deletion of NHEJ proteins caused a similar phenotype

pathophysiological point of view, the ability of IL-4 to counteract IL-1b-dependent RANTES induction is 1022150-57-7 highly relevant. We also analyzed the effects of IL-4 on other soluble factors strongly involved in cartilage matrix break-down, namely ECM degrading enzymes: MMP-13, ADAMTS-4,-5 and tissue inhibitors TIMP-1 and -3. The latter were chosen from among the four TIMPs because TIMP-1 is the most expressed tissue inhibitor in both normal and OA cartilage, and TIMP-3 has the unique property of being able to counteract both MMPs and ADAMTS. IL-1b-stimulated gene expression of MMP-13 and ADAMTS-4 was strongly inhibited by IL-4, whereas neither IL-1b nor IL-4 had any effect on ADAMTS-5, TIMP-1 or 3 gene expression. The combined action of IL-4 on proteases active on both the aggrecan and the collagen 2 components of the extracellular matrix is particularly interesting in light of recent findings in OA pathogenesis. A first, potentially reversible step in ECM remodeling, is proteoglycan depletion mediated by the ADAMTS enzymes. This results in the activation of the chondrocyte discoidin domain receptor through interaction with denuded collagen type II. DDR-2 activation then triggers the production of MMP-13, the true noreturn point in OA pathogenesis, being able to sustain the production of collagen 2 neoepitopes, which then positively feed back into ECM remodeling. In OA cartilage, MMP-13 and ADAMTS-4,-5 are therefore the foremost degrading enzymes of collagen II and aggrecan, IL-4 Expression and Effects in Human Osteoarthritic Chondrocytes respectively, and a growing body of evidence underlines their contribution to OA disease. It is worth noting that ADAMTS-4 is selectively over-expressed in human OA cartilage, with a positive correlation with the degree of cartilage destruction, whereas PubMed ID: ADAMTS-5 is constitutively expressed in both healthy and OA cartilage. The high IL-1b-inducibility of ADAMTS-4 has been recently detailed at the transcriptional level with very recent findings regarding its post-transcriptional regulation. In this study, we evaluated ADAMTS-4 protein by western blotting in lysates obtained from the high-density cultures together with their extracellular matrix, since it is known that ADAMTS have the unique ability to bind to ECM via their TS domains. Prominent bands at 75 and 65 KD were observed, without any differences across the various conditions. The lack of ADAMTS-4 protein induction upon IL-1b stimulation was previously reported by Pratta and colleagues who hypothesized that IL-1b may act through the activation of a constitutively produced protein rather than by increased protein production. Proteolytic activation of the zymogen results in C-terminal truncation, release of the enzyme from the ECM and alteration of its activity profile. Shorter activated forms are mainly 75 kDa, 60 kDa and 50 kDa in pig tissue, but slight variances in these molecular masses have been reported by different research groups. Therefore, most of the ADAMTS-4 protein in our cultures corresponded to the activated form. Time course experiments previously showed that the maximum mRNA level for ADAMTS4 expression is at 24 hours, whereas for MMP13 it is at 12 hours. In our 24-hour experimental design IL-1b stimulation as the best compromise between cytokines and ECM enzyme expression, we saw modulation in MMP13 at both mRNA and protein level, whereas ADAMTS-4 was only modulated at mRNA level. We can speculate that in our experimental setting, chondr

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