SMAD3 is up-regulated in human osteoarthritic cartilage independent of promoter dna ‎methylation

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. ‎