Background 5-hydroxymethyl cytosine (5-hmC) was recently discovered as a new epigenetic mark widely distributed in all types of tissues with varying degrees of abundance. 5-hmC is formed by the oxidation of 5-mC by ten-eleven translocation (TET) family of enzymes (TET1, TET2 and TET3), which need α-ketoglutarate as a co-factor produced by isocitratedehydrogenases (IDHs). 5-hmC plays an important role in the regulation of gene expression by acting as an intermediate state of complete demethylation as well as by modulating the binding of transcription regulatory proteins. Pro-inflammatory cytokines IL-1β and TNF-α have been shown to modulate the expression of many genes in chondrocytes, resulting in the activation of catabolic mechanisms in the joint and ultimately causing the damage to articular cartilage.
Objectives To determine (a) whether pro-inflammatory cytokines IL-1β and TNF-α regulate gene expression in human chondrocytes via modulating the level of 5-hmC; (b) the global occurrence of 5-hmC in primary human chondrocytes; and (c) whether pro-inflammatory cytokines modulate the expression and activity of TET and IDH enzymes and genomic DNA hydroxymethylation in human chondrocytes.
Methods Primary human chondrocytes were isolated from the deep zone of the cartilage obtained from OA patients who underwent total joint replacement (n=8). Global 5-hmC content in the genomic DNA was quantified using a 5-hmC-specific ELISA (Epigentek, Farmingdale, NY). Total TET activity was determined by an ELISA based activity assay kit (Epigentek). Total IDH activity was determined by a colorimetric assay kit (Sigma Aldrich, St Louis, MO). Gene expression of MMP-3, TET-1, TET-2, TET-3, IDH-1 and IDH-2 was quantitated using the TaqMan assays (Applied Biosystems, Carlsbad, CA). Involvement of NF-kB in gene-regulation of TET-1 was investigated by using a small molecule inhibitor BAY 11-7082. Data were derived using Origin 6.1 software and P<0.05 was considered significant.
Results The global 5-hmC content in human chondrocytes was found to be 0.1-0.2% of the total genomic DNA. There was approximately 70% decrease (P<0.05) in the 5-hmC content upon treatment with IL-1b plus TNFa for 48 hrs. This correlated with the reduction of total TET enzyme activity after treatment with IL-1b alone or with IL-1b + TNF-a. There was a 20 fold reduction in the level of TET-1 mRNA expression while there was a 2-3 fold increase in the expression of TET-3 mRNA. The level of TET-2 mRNA expression did not change upon treatment with the cytokines. Down-regulation of TET-1 gene expression by cytokines was NF-kB dependent. Treatment of human chondrocytes with IL-1b and TNF-a also resulted in down-regulation of IDH-1 and IDH-2 mRNA expression and decrease in total IDH enzymatic activity.
Conclusions Our data demonstrate for the first time the presence of a significant amount of 5-hmC in human chondrocyte DNA. We also for the first time show that pro-inflammatory cytokines IL-1β and TNF-α modulate the 5-hmC content in the genomic DNA via modulating the gene expression and activities of TET and IDH enzymes. Taken together our novel data (a) suggests that inflammatory cytokines-induced hydroxymethylation of genomic DNA in OA chondrocytes may be involved in active demethylation of target promoters; and (b) these data identify novel targets for the development of OA therapeutics.
Acknowledgements Supported by AT 003627 and AT 005520 from NIH.
Disclosure of Interest A. Haseeb: None Declared, T. Haqqi Grant/research support from: NIH