Abstract
Purpose: We previously reported that SMAD3 was associated with the total burden of radiographic osteoarthritis (OA). SMAD3 is a mediator of TGF-β signalling pathway that is known to be involved in the cartilage maintenance and repair. SMAD3 knocked-out mice are deficient for Collagen and Aggrecan, and develop OA-like features in the joints. The aim of the present study was to investigate the SMAD3 gene expression in osteoarthritic and healthy human cartilage and to examine whether the gene expression is regulated by the promoter DNA methylation. Methods: Osteoarthritic cartilage samples were collected from patients who underwent total hip/knee joint replacement surgery due to primary OA. Healthy cartilage samples were obtained from patients with hip fracture without any evidence of hip OA. DNA and RNA were extracted from the cartilage samples using Qiagen’s AllPrep DNA/RNA Mini Kit. Quantitative PCR experiment was done using ABI-7500 real time PCR system to measure RNA expression after cDNA synthesis by ThermoScript cDNA synthesis kit. DNA Methylation was assayed by Sequonom’s EpiTYPER after DNA bisulphate conversion using Qiagen’s EpiTect Bisulfite Kit. Mann-Whitney test was utilized to examine the association between OA cases and controls for SMAD3 expression and its promoter DNA methylation levels. Spearman's rank correlation analysis was performed to examine the association between the promoter methylation and gene expression. A P-value less than 0.05 was considered as significant. Results: A total of 49 OA patients (38 hip OA and 11 knee OA) and 51 controls were included in the study. Mean age was 64.4 in OA patients and 78.7 in controls. Four CpG sites, located ∼450bp upstream of the first SMAD3 exon, were assayed, and we found no difference in methylation between OA cases and controls after adjusting for age. The expression experiment was performed for 38 patients with OA (32 hips and 6 knees) and 28 healthy controls, and we found that the SMAD3 gene was expressed in OA cartilage on average 1.8 times higher than controls (p=0.0005). Similar results were obtained when we looked at hip and knee OA separately (p=0.01). We found no association between SMAD3 expression and the methylation at the promoter region of the gene. Conclusions: Our study demonstrated that SMAD3 is up-regulated in OA. This up-regulation, however, cannot be explained by the changes in the promoter DNA methylation. Given that TGF-β/SMAD3 pathway may play a protective role in the cartilage, the up-regulation of SMAD3 is more likely due to the consequence of OA, thus reflecting repairing activity stimulated by the cartilage lesion in OA.
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