OC01: INFLAMMATORY CYTOKINES CAUSE LOSS OF DNA METHYLATION TOGETHER WITH INDUCTION OF ABNORMAL GENE EXPRESSION IN HEALTHY HUMAN ARTICULAR CHONDROCYTES

Abstract

Epigenetic DNA de-methylation at specific CpG promoter sites is associated with abnormal synthesis of matrix-degrading enzymes in human osteoarthritis (Arthritis Rheum 52:3110–24), but the mechanisms that trigger or cause loss of DNA methylation are not known. Since inflammatory cytokines are known to induce abnormal gene expression in cultured chondrocytes, we wanted to know whether this induction also involved loss of DNA methylation. If so, the abnormal gene expression would be permanent and transmitted to daughter cells rather than a simple up-regulation. To test this hypothesis, we selected IL-1b as the abnormally expressed gene. Healthy chondrocytes, harvested from human femoral head cartilage following a fracture, were divided into five groups: non-culture; control culture; culture with the de-methylating agent 5-aza-deoxycyti-dine (5-aza-dC); culture with the inflammatory cytokine IL-1b; or with TNF-a/oncostatin M. Total RNA and genomic DNA were extracted at confluency, relative mRNA expression of IL-1b was quantified by Syb-rGreen-based real-time PCR, and a method for quantifying the percent of cells with DNA methylation at a specific CpG site was developed (Epigenetics 2: 86–95).

The methylation status of 16 CpG sites in the promoter of IL-1b was determined by the bisulfite modification method. The two CpG sites important for the epigenetic regulation of IL-1b were at -247bp and -290bp, the latter was selected to quantify DNA methylation. 5-aza-dC halved DNA methylation, which resulted in 4–8 fold increases in IL-1b expression; showing that DNA de-methylation per se increases gene expression. However, far greater effects were seen with the inflammatory cytokines. IL-1b increased its own expression 50–100 fold, whereas TNF-a/OSM increased IL-1b expression 500–1000 fold. DNA methylation varied inversely, IL-1b reducing methylation to ~15% and TNF-a/OSM abolishing DNA methylation almost completely.

This is the first demonstration that inflammatory cytokines have the capacity to cause loss of DNA methylation. We also confirmed previous work that IL-1b induces its own expression in healthy chondrocytes, thus setting up a dangerous positive feed-back mechanism. If true in vivo, both the auto-induction and the heritable expression of IL-1b by a growing number of chondrocytes could explain the unrelenting progression of osteoarthritis.