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


CONCLUSIONS
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.
Loeser
R F
RF
Department of Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA. rloeser@wfubmc.edu
Im
H-J
HJ
Richardson
B
B
Lu
Q
Q
Chubinskaya
S
S
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D053674:Aggrecans / Q000096:biosynthesis / Q000235:genetics; D000375:Aging / Q000378:metabolism*; D001374:Azacitidine / Q000494:pharmacology; D055419:Bone Morphogenetic Protein 7 / Q000096:biosynthesis / Q000235:genetics*; D002358:Cartilage, Articular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis / Q000235:genetics; D008875:Middle Aged; D011401:Promoter Regions, Genetic; D017526:Receptor, IGF Type 1 / Q000096:biosynthesis / Q000235:genetics; D055815:Young Adult
D053674:Aggrecans; C525819:BMP7 protein, human; D055419:Bone Morphogenetic Protein 7; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1; D001374:Azacitidine