Objectives Changes in DNA methylation and histone marks have been associated with diseases such as cancer, rheumatoid arthritis (RA) and systemic lupus erythematosus. Previously, we performed methylation immunoprecipitation (MeDIP) in combination with human promoter arrays and identified TBX5 as a differentially methylated gene in RA synovial fibroblasts (SF). Now, we want to further examine its role in the pathogenesis of RA and identify specific TBX5 gene targets.
Methods Synovial tissues from RA and OA patients were stained with antibodies for TBX5, a fibroblast marker specific for collagen prolyl 4 hydroxylases, CD3+ on T cells, CD68+ on macrophages and analysed by immunohistochemistry. mRNA and DNA were prepared from synovial tissues, CD3+ T cells and CD14+ monocytes of RA patients. Intracellular FACS staining was used to quantify the levels of TBX5 in T cells and monocytes. The expression and DNA methylation status of TBX5 were determined by quantitative SYBR PCR and bisulfite sequencing. Transient transfections with TBX5 expression vector were performed in OASF and RASF. The transfected SF cells were analysed with quantitative SYBR PCR for TBX5 gene targets.
Results Immunohistochemistry of synovial tissues confirmed that TBX5 is expressed in RA (n=6), but rarely in OA (n=6). The expression of TBX5 was nuclear and mostly localised in the synovial lining of RA tissues. Double labelling of RA tissues with a fibroblast marker showed that the sublining fibroblasts were labelled with TBX5 antibodies. Next, we examined the CD3+ T cells and CD68+ macrophages of synovium and found that the CD3+ T cells and CD68+ macrophages were expressing variable levels of TBX5. TBX5 transcripts were significantly more expressed in RA than OA tissues (RA dCt: 15.5±0.4 n=10; OA dCt: 18.1±0.9 n=5, p<0.04). The TBX5 promoter was significantly more methylated in OA than in RA tissues as shown by bisulfite sequencing. In addition, CD3+ T cells and CD14+ monocytes were expressing significantly higher protein levels of TBX5 than the healthy controls as shown by FACS staining.Transient overexpression of TBX5 in RASF and OASF (n=3 each) showed a 1.5 fold increase of TNFR1 and TNFR2 mRNA as shown recently also in cancer cell lines.
Conclusions DNA methylation is responsible for the overexpression of TBX5 in RA synovial tissue in vivo. Consequently, TBX5 appears to be a novel regulator of TNFα receptor expression in RA and thereby associated with the chronically activated phenotype of RASF.
Disclosure of Interest None Declared
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