Abstract
In osteoarthritis (OA), articular chondrocytes undergo a phenotypic change and acquire a gene expression repertoire that is characterized by the aberrant expression of numerous catabolic genes including matrix metalloproteinases 3, 9 and 13, ADAMTS-4 and interleukin-1beta (IL1B = gene, IL-1b=protein). Previous studies (Arthritis Rheum 52;3110-24) have shown that epigenetic DNA demethylation at specific CpG sites in the relevant promoters accounts for the aberrant expression and that inflammatory cytokines (TNF-alpha, oncostatin M, IL-1b) can cause both aberrant expression and loss of DNA methylation, at least in vitro (Arthritis Rheum. 2009, 60,3303-3313). If the mechanisms of DNA de-methylation were understood, they might provide a new molecular target for therapeutic intervention. We hypothesize that nuclear translocation of the transcription factor NF-kB is involved in de-methylation because 1) we and others have demonstrated that cytokine-induced expression of IL1B in healthy chondrocytes requires NF-kB and 2) DNA de-methylation during B cell maturation was crucially dependent on the rel/NF-kB family (Nat Genet. 1996, 13,435-441). The aims of the study were to determine whether the NF-kB inhibitor BAY 11-7082 (BAY) could prevent the cytokine-induced loss of DNA de-methylation and thereby show that NF-kB is required for DNA de-methylation.
METHODS
Healthy chondrocytes were isolated from the articular cartilage of six femoral heads, obtained with ethical permission after operations following neck of femur fractures. Chondrocytes were cultured for 5 weeks in 4 separate groups: without treatment (control culture); with 2.5ng/ml IL-1b and 2.5ng/ml oncostatin M (IL-1b+OSM); with 1.0mM BAY alone; and IL-1b+OSM+BAY. Total RNA and genomic DNA were extracted from each sample. Gene expression of IL1B was determined by SybrGreen-based qRT-PCR. The % DNA methylation at a specific CpG site in the IL1B promoter (which had previously been identified as a crucial CpG site) was quantified after bisulfite modification with a pyrosequencer (Biotage). The data for IL1B expression and % DNA methylation were analyzed in Microsoft Excel using Wilcoxon's signed rank test. P values < 0.05 were considered significant.
RESULTS
Although there was considerable variation between samples, expression of IL1B was increased by > 1000 fold in the IL-1b+OSM group compared with control culture, confirming previous results. When BAY was present together with IL-1b+OSM, the increase in IL1B expression was reduced from ∼1000-fold to ∼200-300-fold (P< 0.01). In addition, the % DNA methylation had changed. At the -299 CpG site of IL1B promoter the % methylation was 57% in control culture and 60% in the BAY alone group. IL-1b+OSM caused a decrease to 37% (P<0.01 compared with all other groups), whereas presence of BAY prevented this loss, since the % methylation was 58% in IL-1b+OSM+BAY group.
DISCUSSION
The novel findings of this study are that when nuclear translocation of NF-kB is inhibited by BAY, the IL-1b induced increase of IL1B expression was ameliorated and the loss of DNA methylation in the IL1B promoter was prevented. The data confirm our hypothesis that NF-kB is required for the DNA de-methylation initiated by IL-1b+OSM.
