Decreased levels of transforming growth factor beta (TGFbeta) have been related to the failure of cartilage repair in experimental models of osteoarthritis. This study aimed to examine this aspect of osteoarthritis in human cartilage.
Cartilage samples were obtained from 11 patients with hip osteoarthritis and 11 patients with femoral neck fracture who were undergoing total hip replacement. Gene expression of the three TGFbeta isoforms, collagen type II (COL2A1) and aggrecan (AGC1) was analysed by reverse transcription quantitative PCR and immunohistochemistry.
Expression of the three TGFbeta isoforms was increased in osteoarthritis cartilage. The upregulation was more marked for the TGFbeta3 isoform (2.3-fold) than for TGFbeta1 (1.6-fold) or TGFbeta2 (1.7-fold). The messenger RNA levels of TGFbeta1 and TGFbeta2 were strongly correlated in osteoarthritis cartilage (r(s) = 0.83, p = 0.002), but levels of TGFbeta3 were uncorrelated with any of the two other TGFbeta isoforms. Immunohistochemistry showed an extension of immunoreactivity for the three TGFbeta isoforms to more chondrocytes and to deeper cartilage layers in the more severe osteoarthritis lesions. No correlation of TGFbeta isoforms with COL2A1 or AGC1 expression levels was found.
The three isoforms of TGFbeta were differentially upregulated in late osteoarthritis in relation to an increased percentage of TGFbeta-positive chondrocytes. These results indicate that cartilage damage progresses in spite of the TGFbeta stimulus for cartilage anabolism and that other causes of the failure to cope with the increased cartilage catabolism of osteoarthritis should be investigated.
"As TGF-β production is significantly increased in OA [24,25], we examined whether TGF-β could interfere with the translocation of NFAT3 and prevent its action. As expected (Figure 7A, B), ionomycin significantly triggered the translocation of NFAT3 (47%, P ≤0.001), and TGF-β triggered that of SMAD3 (52%, P ≤0.0001) in OA chondrocytes. "
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) down-regulate their target genes. The intronic miR-140, present in the WW domain containing E3 ubiquitin protein ligase 2 (WWP2) gene, decreases the expression of genes that play detrimental roles in osteoarthritis (OA). As the expression level of miR-140 is significantly decreased in human OA chondrocytes, we investigated its regulation in those cells.
Gene expression in human chondrocytes was determined by quantitative polymerase chain reaction (qPCR) and gene silencing was done in OA chondrocytes by transient transfection with specific small interfering RNAs (siRNAs). Binding sites of the miR-140 regulatory sequence (rsmiR-140) were identified by mutagenesis and chromatin immunoprecipitation (ChIP) in OA chondrocytes. The effects of translocation on OA chondrocytes were determined by immunocytochemistry and qPCR.
In contrast to miR-140, the expression of WWP2 was similar in both normal and OA cells, suggesting that miR-140 has an additional level of regulation. rsmiR-140 showed activity and predicted binding sites for nuclear matrix transcription factor 4 (NMP4), myc-associated zinc (MAZ), nuclear factor of activated T-cells (NFAT), and mothers against decapentaplegic homolog 3 (SMAD3). Silencing NFAT3 (P <=0.01) and SMAD3 (P <=0.05) differentially regulated miR-140 independently of WWP2. Silencing NFAT5 decreased both miR-140 and WWP2 (P <=0.003 and P <=0.05, respectively). NFAT3 activation increased and transforming growth factor-beta (TGF-beta) decreased rsmiR-140 activity. Mutagenesis of rsmiR-140 and ChIP assays identified binding sites at which NFAT3 (activator) and SMAD3 (repressor) directly regulated miR-140. TGF-beta interfered with NFAT3 translocation, and subsequently with miR-140 expression.
This is the first study to provide evidence of a regulatory mechanism of miR-140 independent of WWP2, and new and differential roles for NFAT3 and SMAD3 in the OA process in the regulation of miR-140 transcription. Such knowledge could advance therapeutic strategies targeting OA.
[Show abstract][Hide abstract] ABSTRACT: We have demonstrated previously that a 12-day course of FK506 permits the induction of tolerance to fully MHC-mismatched renal transplants in miniature swine. In the present study, we examined the mechanism of this tolerance by assessing the possibility that the survival of one-haplotype mismatched third-party kidneys might be prolonged via linked suppression. Ten SLA(d/d) miniature swine received fully MHC-mismatched renal allografts from SLA(c/c) donors with 12 days of FK506. Six animals received second SLA(c/c) kidneys without immunosuppression to confirm tolerance. Regulatory mechanisms were assessed by mixed lymphocyte reaction (MLR) and cell-mediated lympholysis coculture assays and ELISA for regulatory cytokines. Linked suppression was investigated by transplanting SLA(a/c) or SLA(a/d) allografts into long-term tolerant recipients without immunosuppression. All recipients showed donor-specific unresponsiveness in standard cell-mediated lympholysis and MLR assays. Tolerant cells prestimulated with donor Ag and then cocultured with naive recipient MHC-matched cells inhibited antidonor responses, confirming the presence of regulatory cells. ELISA and MLR assays showed that TGF-beta2 was involved in mediating the suppression in vitro. SLA(a/d) renal allografts transplanted into tolerant recipients were rejected by postoperative day 8 (median, 7 days; range, 6-8). In contrast, SLA(a/c) allografts showed markedly prolonged survival (median, 52 days; range, 28-78; p = 0.0246), suggesting linked suppression. Animals not challenged with a second donor-matched graft did not manifest linked suppression consistent with in vitro data showing that re-exposure to tolerated Ags is important for generation of regulatory cells. To our knowledge, these data represent the first evidence of linked suppression across fully MHC-mismatched barriers in a large animal model.
The Journal of Immunology 10/2008; 181(6):4027-36. DOI:10.4049/jimmunol.181.6.4027 · 4.92 Impact Factor
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