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Published on: October 16, 2016
DNA hypomethylation in rheumatoid arthritis synovial fibroblasts
Emmanuel Karouzakis1, Renate E Gay, Beat A Michel
1Zurich Center of Integrative Human Physiology, University Hospital, Zurich, Switzerland.
Objective:
Rheumatoid arthritis synovial fibroblasts (RASFs) are phenotypically activated and aggressive. We undertook this study to investigate whether the intrinsic activation of RASFs is due to global genomic hypomethylation, an epigenetic modification.
Methods:
Global genomic hypomethylation was assessed by immunohistochemistry, flow cytometry, and L1 promoter bisulfite sequencing. The levels of Dnmt1 were determined in synovial tissue and cultured SFs by Western blotting before and after treatment with cytokines and growth factors. Normal SFs were treated for 3 months with a nontoxic dose of the DNA hypomethylation drug 5-azacytidine (5-azaC), and changes in gene expression were revealed using complementary DNA arrays. The phenotypic changes were confirmed by flow cytometry.
Results:
In situ and in vitro, RASF DNA had fewer 5-methylcytosine and methylated CG sites upstream of an L1 open-reading frame than did DNA of osteoarthritis SFs, and proliferating RASFs were deficient in Dnmt1. Using 5-azaC, we reproduced the activated phenotype of RASFs in normal SFs. One hundred eighty-six genes were up-regulated>2-fold by hypomethylation, with enhanced protein expression. These included growth factors and receptors, extracellular matrix proteins, adhesion molecules, and matrix-degrading enzymes. The hypomethylating milieu induced irreversible phenotypic changes in normal SFs, which resembled those of the activated phenotype of RASFs.
Conclusion:
DNA hypomethylation contributes to the chronicity of RA and could be responsible for the limitation of current therapies.
Insights
Global genomic hypomethylation drives rheumatoid arthritis synovial fibroblast activation. This epigenetic change contributes to rheumatoid arthritis (RA) chronicity and may limit current treatment efficacy.
Area of Science:
- Epigenetics
- Molecular Biology
- Rheumatology
Background:
- Rheumatoid arthritis synovial fibroblasts (RASFs) exhibit an activated and aggressive phenotype.
- The underlying mechanisms of RASF activation are not fully understood.
- Epigenetic modifications, such as DNA methylation, play a crucial role in cellular function.
Purpose of the Study:
- To investigate if global genomic hypomethylation is responsible for the intrinsic activation of RASFs.
- To explore the role of DNA methylation in the pathogenesis of rheumatoid arthritis.
Main Methods:
- Global genomic hypomethylation assessed via immunohistochemistry, flow cytometry, and L1 promoter bisulfite sequencing.
- Dnmt1 levels determined in synovial tissue and cultured SFs using Western blotting.
- Normal SFs treated with 5-azacytidine (5-azaC) to induce hypomethylation, with gene expression analyzed by cDNA arrays.
Main Results:
- RASF DNA showed reduced 5-methylcytosine and methylated CG sites compared to osteoarthritis SFs.
- Proliferating RASFs demonstrated a deficiency in Dnmt1.
- Treatment of normal SFs with 5-azaC replicated the activated RASF phenotype, up-regulating 186 genes and inducing irreversible phenotypic changes.
Conclusions:
- DNA hypomethylation is a significant contributor to the development and chronicity of rheumatoid arthritis.
- This epigenetic alteration may explain the limited effectiveness of current RA therapies.
- Targeting DNA hypomethylation could offer novel therapeutic strategies for RA.
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