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. 2009 Apr;17(4):513-7.
doi: 10.1016/j.joca.2008.08.003. Epub 2008 Sep 30.

Methylation of the OP-1 promoter: potential role in the age-related decline in OP-1 expression in cartilage

R F Loeser  1 , H-J Im, B Richardson, Q Lu, S Chubinskaya
Affiliations

Methylation of the OP-1 promoter: potential role in the age-related decline in OP-1 expression in cartilage

R F Loeser et al. Osteoarthritis Cartilage. 2009 Apr.

Abstract

Objective: An age-related decline in chondrocyte production of osteogenic protein-1 (OP-1) (Bone Morphogenetic Protein-7) may contribute to cartilage loss in osteoarthritis. This study was designed to determine if increased methylation of the OP-1 promoter might serve as a mechanism for the age-related decline in OP-1 expression.

Methods: Human articular chondrocytes were isolated from cartilage obtained after death from tissue donors (ages 19-86 years) without a known history of arthritis. DNA was obtained from isolated chondrocytes in primary culture and analyzed for OP-1 promoter methylation by polymerase chain reaction (PCR) after bisulfite treatment. Cultured cells were treated with the DNA methyltransferase inhibitor 5-azacytidine and OP-1 production was measured in the media by enzyme-linked immunosorbent assay (ELISA). RNA was isolated to measure expression of insulin-like growth factor-1 (IGF-1), the IGF-1 receptor (IGF-1R), aggrecan, and OP-1 by real-time PCR.

Results: Methylation of the OP-1 promoter was detected in chondrocytes isolated from tissue obtained from older adults and there was a positive correlation between age and OP-1 methylation status (n=22, R(2)=0.277, P=0.014). Inhibition of methylation in cultured cells with 5-azacytidine increased chondrocyte production of OP-1 protein and increased the expression of the IGF-1, the IGF-1R, aggrecan, and OP-1 genes but not GAPDH.

Conclusion: Age-related methylation of the OP-1 promoter may contribute to a decrease in OP-1 production in cartilage and a decrease in expression of OP-1 responsive genes such as IGF-1, the IGF-1R, and aggrecan.

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Figures

Fig. 1
Fig. 1
Age-related increase in methylation of OP-1 promoter DNA. (A). Methylation specific PCR for OP-1 using DNA isolated from cultures of young (20 years-old) and old (74 years-old) donor chondrocytes. DNA was amplified using primers recognizing sequences corresponding to methylated and unmethylated regions of bisulfite treated DNA. Samples with H2O substituted for DNA were used as a control to exclude primer-dimer formation. (B). Relationship of age to OP-1 promoter methylation status. The graph shows the ratio of PCR products obtained using the methylation specific primers normalized to results with primers from the unmethylated region (n=22, R2=0.277, p=0.014).
Fig. 2
Fig. 2
Treatment of chondrocytes with 5-azacytidine (0-10μM) increases the production of OP-1. An ELISA was used to measure OP-1 levels in the media from cells of two different donors aged 72 and 80 years. OP-1 results were normalized to the DNA content measured in the cell-layer. *p<0.001 vs control
Fig. 3
Fig. 3
Effects of 5-azacytidine on chondrocyte gene expression. Primary chondrocytes isolated from donors with ages of 71-82 years were treated in culture with the indicated doses of 5-azacytidine for 120 hours. RNA was isolated and used for real-time-PCR with primers specific for IGF-1, the IGF-1 receptor (IGF-1R), OP-1, aggrecan, or GAPDH as a control. (A-D) Results using cells cultured from the ankle joint (n=6) and (B) a comparison of cells cultured from ankle (n=7) and knee cartilage (n=2). *p<0.05.
Fig. 3
Fig. 3
Effects of 5-azacytidine on chondrocyte gene expression. Primary chondrocytes isolated from donors with ages of 71-82 years were treated in culture with the indicated doses of 5-azacytidine for 120 hours. RNA was isolated and used for real-time-PCR with primers specific for IGF-1, the IGF-1 receptor (IGF-1R), OP-1, aggrecan, or GAPDH as a control. (A-D) Results using cells cultured from the ankle joint (n=6) and (B) a comparison of cells cultured from ankle (n=7) and knee cartilage (n=2). *p<0.05.
Fig. 3
Fig. 3
Effects of 5-azacytidine on chondrocyte gene expression. Primary chondrocytes isolated from donors with ages of 71-82 years were treated in culture with the indicated doses of 5-azacytidine for 120 hours. RNA was isolated and used for real-time-PCR with primers specific for IGF-1, the IGF-1 receptor (IGF-1R), OP-1, aggrecan, or GAPDH as a control. (A-D) Results using cells cultured from the ankle joint (n=6) and (B) a comparison of cells cultured from ankle (n=7) and knee cartilage (n=2). *p<0.05.
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