Loss of methylation in CpG sites in the NF‐κB enhancer elements of inducible nitric oxide synthase is responsible for gene induction in human articular chondrocytes

Abstract

Objective To investigate whether the abnormal expression of inducible nitric oxide synthase (iNOS) by osteoarthritic (OA) human chondrocytes is associated with changes in the DNA methylation status in the promoter and/or enhancer elements of iNOS. Methods Expression of iNOS was quantified by quantitative reverse transcriptase–polymerase chain reaction. The DNA methylation status of the iNOS promoter and enhancer regions was determined by bisulfite sequencing or pyrosequencing. The effect of CpG methylation on iNOS promoter and enhancer activities was determined using a CpG-free luciferase vector and a CpG methyltransferase. Cotransfections with expression vectors encoding NF-κB subunits were carried out to analyze iNOS promoter and enhancer activities in response to changes in methylation status. Results The 1,000-bp iNOS promoter has only 7 CpG sites, 6 of which were highly methylated in both control and OA samples. The CpG site at −289 and the sites in the starting coding region were largely unmethylated in both groups. The NF-κB enhancer region at −5.8 kb was significantly demethylated in OA samples compared with control samples. This enhancer element was transactivated by cotransfection with the NF-κB subunit p65, alone or together with p50. Critically, methylation treatment of the iNOS enhancer element significantly decreased its activity in a reporter assay. Conclusion These findings demonstrate the association between demethylation of specific NF-κB–responsive enhancer elements and the activation of iNOS transactivation in human OA chondrocytes, consistent with the differences in methylation status observed in vivo in normal and human OA cartilage and, importantly, show association with the OA process.

Association of Reduced Type IX Collagen Gene Expression in Human Osteoarthritic Chondrocytes With Epigenetic Silencing by DNA Hypermethylation

“…This is contrary to our recent findings on the NOS2 enhancer (14) and MMP13 promoter (15). It is possible that in the absence of differential methylation, the increased expression of COL2A1 observed in OA chondrocytes could be explained by the regenerative efforts of the cells to restore the ECM as an anabolic response within a degradative environment.…”

Mitochondrial epigenetics in bone remodeling during hyperhomocysteinemia

“…Another gene which is found to be associated with catabolic activity in cartilage is the inducible nitric oxide synthase (iNOS). The association between demethylation of specific NF-kappaB-responsive enhancer elements and the activation of iNOS transactivation in human OA chondrocytes was seen which was consistent with the differences in methylation status observed in vivo in normal and human OA cartilage and, importantly, show association with the OA process [59]. The pattern of methylation (hypo-, or hyper-mehylation) have earlier been seen in rheumatoid arthritis synovial fibroblast (RASF) by Nakano et al [60].…”

Inflammation and epigenetic regulation in osteoarthritis

“…Here, Takahashi et al showed that increased demethylation of the IL8 promoter in OA chondrocytes correlated with enhanced IL8 expression and that expression was mediated by the activity of C/EBP, AP-1, and NF-κB (96). In addition, demethylation of an NF-κB-responsive enhancer was shown to increase the expression of inducible nitric oxide synthase (iNOS), a gene known to be dysregulated in OA (97). Recent analysis from methylation data of hip OA patients identified that the promoter region of a subset of inflammation-associated genes including IL1α and TNF was hypo-methylated, which further led to increased MMP13 expression in OA chondrocytes through zinc ZIP8-MTF 1 axis (88).…”