FRI0041 Pro-inflammatory cytokines IL-1[beta] and tnf-[alpha] regulate the genomic 5-hydroxymethylcytosine levels by modulating the expression and activity of tet-1 and idhs in human oa chondrocytes

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-...

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Veröffentlicht in:Annals of the rheumatic diseases 2013-06, Vol.72, p.A381
Hauptverfasser: Haseeb, A, Haqqi, T M
Format: Artikel
Sprache:eng
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Zusammenfassung: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
ISSN:0003-4967
1468-2060
DOI:10.1136/annrheumdis-2013-eular.1169