Session Title: Rheumatoid Arthritis - Human Etiology and Pathogenisis
Session Type: Abstract Submissions (ACR)
Previously we reported that methyl supplementation can reverse the global hypomethylation of rheumatoid arthritis synovial fibroblasts (RASF) and attenuate their aggressive behaviour. Now we analysed the molecular changes occurring in matrix metalloproteinases (MMPs) and transcription factors after treatment with methyl supplementation.
RASF (n=6 patients) were cultured in DMEM/F12 + 10% FCS as a control and in medium supplemented with 10-fold excess amount of folic acid (70 µM), vitamin B12 (6 µM) and L-methionine (1,1 mM) (high supplementation – HS) for 10 days. After the treatment, cells were counted to determine doubling time, their RNA isolated and supernatants were tested using MMPs ELISA assays. The gene expression of MMPs and transcription factors was evaluated by real time-PCR and gene expression array (QIAGEN RT2 Profiler PCR Human Transcription Factors Array), respectively. The untreated and treated RASF were implanted together with normal human cartilage into SCID mice. The cartilage sample slides were scored by three independent investigators.
After 10 days of culturing in HS medium, the expression of MMP1 gene was significantly down regulated (p = 0.034) while changes in the mRNA level of MMP3 were not significant (p = 0.56). There was also a significant reduction in the level of MMP1 protein in RASF grown in HS medium (p = 0.027) and no significant reduction of MMP3 protein level (p = 0.49). The effect of HS on the state of DNA methylation and gene expression is dependent on cell proliferation. Indeed, MMP1 expression strongly correlated with the cell population doubling time – r = 0.9455, p = 0.0044. The expression of 6 out of 84 analyzed genes was significantly changed by methyl supplementation. ELK1, HDAC1, HSF1 were up regulated while JUN, NFATC3 and SMAD9 were down regulated (p < 0.05). Most interestingly, HS treated RASF showed reduced invasion and perichondrocytic degradation in the SCID mouse invasion model in vivo(invasion score: control 2.25±0.27; HS 1.26±0.26, p<0.03, n=5; perichondrocytic score: control 1.91±0.18; HS 1.17±0.23, p<0.01, n=5).
Methyl supplementation reduced the expression of MMP1, significantly altered expression pattern of 6 transcription factors and inhibited the invasive properties of RASF in vivo. Therefore, this supplementation might be used as a novel therapeutic approach to inhibit joint destruction in rheumatoid arthritis.
IAR, IMI-BTCure,Novartis Stiftung,
R. E. Gay,
Masterswitch-FP7 and her institution,
IAR and his institution,
B. A. Michel,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/methyl-supplementation-of-rheumatoid-arthritis-synovial-fibroblasts-regulates-the-expression-of-transcription-factors-and-matrix-metalloproteinases/