To examine the methylation profile of the nuclear factor (NF)-κB enhancer region at -5.8 kb of inducible nitric oxide synthase (iNOS) and the subsequent role in the induction of osteoarthritis (OA) via cell cycle regulation.
Percentage methylation was determined by pyrosequencing, gene expression by qRT-PCR and cell proliferation was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Transient transfections were induced to determine the effect of the NF-κB enhancer region on cell proliferation and the influence of DNA methylation.
In vitro de-methylation with 5-aza-dC showed decreased levels of DNA methylation at CpG sites localised at -5.8 kb, which correlated with higher levels of iNOS expression. In vitro methylation of the NF-κB enhancer region at -5.8 kb increased the percentage of cells at G0/G1 cell cycle phase. Loss of methylation within this region correlated with, enhanced proliferation and increased number of cells at G2/M phase. OA chondrocytes demonstrated up-regulation of the G0/G1 cell cycle progression markers Cyclin D1 and CDK6 in contrast to control cells. We demonstrate the loss of methylation that occurs at specific CpG sites localised at the -5.8 kb NF-κB enhancer region of the iNOS gene in OA chondrocytes permits the binding of this transcription factor activating the expression of iNOS. This results in subsequent altered cell cycle regulation, altered proliferative phenotype and transmission of the pathogenic phenotype to daughter cells.
This study indicates that inhibition of cell cycle progression by iNOS enhancer hypermethylation is capable of reducing pro-inflammatory responses via down-regulation of NF-κB with important therapeutic implications in OA.