Background/Purpose

In the pathogenesis of Rheumatoid arthritis (RA) RA fibroblast-like synoviocytes (RA FLS) exhibit a unique aggressive phenotype that contributes to the cytokine milieu and joint destruction. We previously revealed that persistent exposure of pro-inflammatory cytokines contribute to passive DNA demethylation through decreased expression of DNMT in FLS. We here assessed the involvement of a novel active DNA demethylation enzyme, Ten-eleven-translocation (TET), in cytokine-mediated activation of RA-FLS. 

Methods

FLS were obtained from patients with RA and osteoarthritis (OA) synovium at total joint replacement and studied in the 4^th through 6^th passage. The study was approved by the ethical committee of the university and informed consents were obtained by each patient. cDNA, Nuclear extracts and genomic DNA were purified from control or stimulated FLS. Gene expression was determined by qPCR and protein expression by Western blot and immunostaining. 5-hmC was determined by dot blot. Secretion of cytokines and MMPs was measured by Cytometric Bead Array and latex agglutination method. Cell migration was assessed using a wound healing scratch assay.

Results

TET3 and its product, 5-hydroxymethylcytosine (5-hmC), are detected in the intimal lining layer of synovium in patients with active RA, while TET1 and TET2 were observed marginally there. TET3 was characteristically expressed in both cytoplasm and nuclear of FLS, whereas TET1 and TET2 were not detected in nucleus. Although unstimulated RA and OA FLS expressed similar amounts of TET3 mRNA (n=6 each; n.s.), stimulation with TNFα (10 ng/ml) and IL-1β (1 ng/ml) for 2 hrs significantly increased TET3 mRNA expression in FLS (>3-fold and >4-fold respectively). Western blot analysis showed expression levels of TET3 protein in nucleus in RA FLS were significantly increased by stimulation with TNFα within 48 hrs and were maintained for 96 hrs. Dot blot analysis showed that 5-hmC was also increased by TNFα when FLS were cultured continuously for 96 hrs with TNFα. TET3-knockdown of FLS with siRNA not only inhibited TNF-induced expression of key migratory genes, including CCL2 and ICAM-1, but also reduced TNF-induced FLS-migration completely.

Conclusion

We initially report that a novel DNA methylation enzyme TET3 is characteristically induced in RA FLS through TNF/IL-1 stimulation. Taken together with our previous report, persistent exposure to pro-inflammatory cytokines in the synovium not only decreases DNMT expression but also increases TET3 expression, resulting in promotion of DNA demethylation. In addition to demonstrating a critical role for TET3 in FLS in cytokine milieu, our study suggests that targeting the TET3-5-hmC pathway may be a therapeutic strategy for preventing FLS from TNF-mediated imprinting in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-role-of-tet3-mediated-dna-demethylation-by-pro-inflammatory-cytokines-in-rheumatoid-arthritis/