Background/Purpose:

5-hydroxymethylcytosine (5-hmC), which is formed by the oxidation of 5-methylcytosine (5-mC), is a recently discovered epigenetic mark and is highest in brain and in embryonic stem cells. There have been reports supporting the notion that generation of 5-hmC may be a mechanism of DNA demethylation and thus important in the regulation of gene expression. In the present study we investigated the global and locus-specific 5-hmC content in the promoter region of MMP-3 gene and its expression in human OA chondrocytes. We also investigated the effect of IL-1β and TNF-α on the level of 5-hmC, expression and activity of the enzymes responsible for hydroxylation of 5-mC to generate 5-hmC and MMP-3 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) and were treated with IL-1β and TNF-α for 48 hr. Global 5-hmC content in genomic DNA was quantified using a 5-hmC-specific ELISA (Epigentek, Farmingdale, NY). Total TET methylcytosine dioxygenase (Tet) activity was determined by an ELISA based activity assay kit (Epigentek). Tet-1, Tet-2 and Tet-3 and MMP-3 mRNA levels were quantitated by using the TaqMan assays (Applied Biosystems, Carlsbad, CA). Locus specific 5-mC and 5-hmC content in the MMP-3 promoter was examined by using EpiMark 5-mC and 5-hmC analysis kit (NEB, Ipswich, MA), which uses methylation sensitive HpaII and glucosylation sensitive enzyme MspI following the treatment of genomic DNA with T4-β-glucosyl transferase, and PCR using specific primers. Data were derived using Origin 6.1 software and P<0.05 was considered significant.

Results:

The global content of 5-hmC in human chondrocytes was found to be 0.1-0.2% of the total genome. There was approximately 70% decrease (P<0.05) in the 5-hmC content upon treatment with IL-1β in combination with TNF-α for 48 hrs. This correlated with the reduction of Tet enzyme activity in chondrocytes after treatment with IL-1β and IL-1β + TNF-α. There was a significant (upto 20 fold) reduction in the level of Tet-1 mRNA expression while expression of Tet-3 mRNA was increased slightly (2-3 fold). The level of Tet-2 mRNA expression did not change upon treatment with the cytokines. MMP-3 promoter contained a high percentage (~70%) of 5-hmC at HpaII locus in deep zone chondrocytes and these chondrocytes showed a dramatic (40 fold) increase in the expression level of MMP-3 upon treatment with IL-1β and TNF-α.

Conclusion:

Our results 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 the 5-hmC content of the genomic DNA can be modulated by treatment with proinflammatory cytokines IL-1β and TNF-α in a short span of 48 hrs. The changes in 5-hmC levels correlated with the Tet-1 gene expression level and Tet enzyme activity in chondrocytes stimulated with IL-1β + TNF-α. Our data also demonstrate that the expression of MMP-3 was significantly (P <0.005) increased in chondrocytes with high content of 5-hmC in its promoter region. Taken together our novel data identify an important role for DNA hydroxymethylation in cartilage and may be important in understanding the mechanism and pathogenesis of OA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/il-1%ce%b2-and-tnf-%ce%b1-regulate-the-global-and-locus-specific-hydroxymethylation-of-genomic-dna-by-modulating-the-expression-and-activity-of-tet-1-in-human-oa-chondrocytes/