Background/Purpose:

Synovial fibroblasts (SF) produce elevated levels of matrix degrading enzymes, including matrix metalloproteinase 1 (MMP1), in the joints of rheumatoid arthritis (RA) patients, leading to irreversible joint damage. The aggressive behavior of RASF has been associated with epigenetic alterations, including global DNA hypomethylation and altered histone methylation. Supplementation of SF with re-methylating agents, such as betaine, may attenuate the destructive behavior of SF. Here, we investigated the role of betaine in the regulation of MMP1 expression in SF focusing on histone dynamics at the MMP1 transcription start site.

Methods:

RASF (passages 5-8) were supplemented with 50 mM betaine for two weeks and were cultured for an additional week without betaine. The levels of HOTAIR and MMP1 mRNA were quantified by SYBR Green real time qPCR. The amount of MMP1 protein in cell culture supernatants was assessed by ELISA and normalized to the cell number. SF were transfected with HOTAIR siRNA (72h) using Lipofectamine 2000. The Roadmap Epigenomics database was used to determine regions enriched for histone 3 lysine 4 trimethylation (H3K4me3) and histone 3 lysine 27 trimethylation (H3K27me3) at the promoters of HOTAIR and MMP1 and in the region 500 bp downstream of their transcription start sites. The levels of H3, H3K4me3 and H3K27me3 were assessed using chromatin immunoprecipitation. Adhesion to cell culture plates and proliferation of SF were monitored with xCELLigence RTCA DP Instrument. Apoptosis was analyzed by flow cytometry using the Annexin V/PI assay.

Results:

The levels of HOTAIR (0.7±0.03 vs. 1 in untreated, p=0.005 n=3), MMP1 mRNA (0.54±0.27, p=0.008, n=6) and MMP1 protein (0.4±0.3, p=0.02, n=6) were significantly reduced in SF supplemented with 50 mM betaine for two weeks and were down regulated by 10%±7.8% (n=3) after an additional week without betaine. Moreover, betaine supplemented SF adhered less strongly to the cell culture plate (cell index=0.7±0.1 vs. 1 in untreated SF, n=3) and proliferated slower than untreated SF (doubling time=1.4±0.3 vs.1 in untreated SF, n=3). The number of apoptotic cells did not increase after supplementation. Silencing of HOTAIR resulted in significant up regulation of MMP1 mRNA (2.6±0.8 vs. 1 in control transfected, p<0.0001, n=8), indicating that HOTAIR is not involved in MMP1 repression in betaine supplemented SF. In contrast, the levels of activating H3K4me3 mark 74 to 220 bp downstream the transcription start site of MMP1 (0.66±0.23 vs. 1 in untreated, n=2) and at the HOTAIR promoter (-1908-2008 bp, 0.66±0.09 vs. 1 in untreated, n=2) were reduced in betaine supplemented SF, whereas the repressive H3K27me3 mark was significantly increased 74 to 220 bp downstream the transcription start site of MMP1 (2.4±0.2 vs. 1 in untreated, p=0.046, n=2). Of interest, the levels of H3 at the HOTAIR promoter diminished in supplemented SF, suggesting that betaine induced eviction of H3.

Conclusion:

We provide the first evidence that, by altering the histone dynamics at gene regulatory sites, betaine supplementation may regulate the transcription of RA-relevant genes such as MMP1, thus significantly reducing the matrix destructive behavior of RASF.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/altered-histone-3-dynamics-at-the-matrix-metalloproteinase-1-mmp1-transcription-start-site-contributes-to-mmp1-suppression-in-betaine-supplemented-synovial-fibroblasts-in-rheumatoid-arthritis/