DNA hypomethylation in rheumatoid arthritis synovial fibroblasts.
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.


CONCLUSIONS
DNA hypomethylation contributes to the chronicity of RA and could be responsible for the limitation of current therapies.
Karouzakis
Emmanuel
E
Zurich Center of Integrative Human Physiology, University Hospital, Zurich, Switzerland.
Gay
Renate E
RE
Michel
Beat A
BA
Gay
Steffen
S
Neidhart
Michel
M
D001172:Arthritis, Rheumatoid / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D001374:Azacitidine / Q000494:pharmacology; D002478:Cells, Cultured; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D015870:Gene Expression / Q000187:drug effects; D020868:Gene Silencing; D006801:Humans; D020084:Long Interspersed Nucleotide Elements / Q000187:drug effects / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D010003:Osteoarthritis / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D013583:Synovial Membrane / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology
D004791:Enzyme Inhibitors; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human; D001374:Azacitidine