Epigenetics and Rheumatoid Arthritis (RA)

Epigenetics: The study of heritable changes in function of genes that occur without a change in the sequence of nuclear DNA. There are three epigenetic mechanisms: DNA methylation, histone modifications (e.g. acetylation) and microRNAs. DNA methylation is accomplished by the addition of a methyl group to a cytosine in the context of a cytosine/guanidine (CG) pair. The enzyme DNA methyltransferase (DNMT) catalyses the reaction. Three DNMTS are important: DNMT1 (for maintanceof methylation in somatic cells) , DMNT3A and DMNT3B (for de novo methylation during embryogenesis). The surface of nucleosomes (composed of an octamer of the four core Histones, H3, H4, H2A and H2B, around which 147 base pairs of DNA are wrapped) is studded with a multiplicity of modifications: e.g. acetylation, methylation, ubiquitination, phosphorylation. Together with DNA methylation, histone modifications have the potential to influence gene expression.

MicroRNAs are a class of post-transcriptional regulators. They are short ~22 nucleotide RNA sequences that bind to complementary sequences in the 3 UTR of multiple target mRNAs, usually resulting in their silencing. MicroRNAs target ~60% of all genes, are abundantly presen in all human cells and are able to repress hundreds of targets each.

Global DNA hypomethylation in RA synovial fibroblasts

Synovial fibroblasts in RA are the effector cells of cartilage and bone destruction. They have an intrinsic aggressive behaviour that occurs even in absence of cells of the immune sytem (e.g. T-, B-cells or macrophages). The expression of endogenous retroviruses, like Line-1 mRNA and proteins, which are normaly silenced by DNA methylation, was the first evidence that DNA methylation was dysregulated in RA synovial fibroblasts (1-3). Later, it has been shown that DNMT1 is deficient in proliferating cells, leading to a progressive demethylation (4). DNMT1 is deficient in spite of a normal transcription, suggesting that it is degraded in proteosomes. The global DNA hypomethylation may explain the increased expression of many genes that are related to the pathogenesis of RA: e.g., matrix-degrading enzymes, chemokines and cytokine receptors. For example, the promoter of the chemokine CXCL12 has been found to be demethylation in RA synovial fibroblasts (5).

Cartilage and bone destruction DNMT1 PCNA 5-methylcytosine (DNA) Endogenous retroviruses Gene expression Matrixdegrading enzymes, cytokine recerptors,

Increased recycling of polyamines and global DNA hypomethylation

S-Adenosyl methionin (SAM) is the methyl donor during DNA methylation. SAM is produced by the essential amino acid methionine. Putrescine is processed into spermidine and spermine. This pathway requires decarboxy-SAM (dSAM) that is produced from SAM by S-adenosyl methionine decarboxylase (AMD). In addition, spermidine and spermine can be reconverted into putrescine through the action of spermidine/spermine N1-acetyltransferase (SSAT1). Increased spermine expression indirectly stimulates the expression of SSAT1 through activation of the transcription factor polyamine-modulating factor 1-binding protein (PMFBP1). The intermediaries (diacetylpolyamines) can be secreted through a transporter system (SLC3A2). In turn, increased putrescine stabilizes AMD. In RA synovial fibroblasts, AMD, SSAT1, PMFBP1 and SLC3A2 are up-regulated (6). Increased diacetylpolamines in the medium also indicates that the recycling pathway of polyamines is intrinsicaly activated. As a consequence, SAM is excessively consumed in RA synovial fibroblasts, leading to a progressive DNA demethylation.
Methionine AMD dSAM Putrescine Spermidine Spermine PMFBP1 SSAT1 SAM Methyl DNMT1 DNA methylation

SLC3A2

Histone modifications and microRNAs in RA synovial fibroblasts

DNA methylation is only one of the several mechanisms of epigenetic regulation implicated in RA that also involve histone modifications (7) and microRNAs (8). Together these epigenetic changes might cause stable activation of RA synovial fibroblsts and alter the inflammatory response, thereby promoting the development of a chronic disease.

References
1. Neidhart M, Rethage J, Kuchen S, Knzler P, Crowl RM, Billingham ME, Gay RE, Gay S. Retrotransposable L1 elements expressed in rheumatoid arthritis synovial tissue: association with genomic DNA hypomethylation and influence on gene expression. Arthritis Rheum. 2000 Dec;43(12):2634-47. 2. Kuchen S, Seemayer CA, Rethage J, von Knoch R, Kuenzler P, Beat AM, Gay RE, Gay S, Neidhart M. The L1 retroelementrelated p40 protein induces p38delta MAP kinase. Autoimmunity. 2004 Feb;37(1):57-65. 3. Neidhart M, Karouzakis E, Schumann GG, Gay RE, Gay S. Trex-1 deficiency in rheumatoid arthritis synovial fibroblasts. Arthritis Rheum. 2010 Sep;62(9):2673-9. 4. Karouzakis E, Gay RE, Michel BA, Gay S, Neidhart M. DNA hypomethylation in rheumatoid arthritis synovial fibroblasts. Arthritis Rheum. 2009 Dec;60(12):3613-22. 5. Karouzakis E, Rengel Y, Jngel A, Kolling C, Gay RE, Michel BA, Tak PP, Gay S, Neidhart M, Ospelt C. DNA methylation regulates the expression of CXCL12 in rheumatoid arthritis synovial fibroblasts. Genes Immun. 2011 Dec;12(8):643-52. 6. Karouzakis E, Gay RE, Gay S, Neidhart M. Increased recycling of polyamines is associated with global DNA hypomethylation in rheumatoid arthritis synovial fibroblasts. Arthritis Rheum. 2012, in press. 7. Trenkmann M, Brock M, Gay RE, Kolling C, Speich R, Michel BA, Gay S, Huber LC. Expression and function of EZH2 in synovial fibroblasts: epigenetic repression of the Wnt inhibitor SFRP1 in rheumatoid arthritis. Ann Rheum Dis. 2011 Aug;70(8):1482-8. 8. Niederer F, Trenkmann M, Ospelt C, Karouzakis E, Neidhart M, Stanczyk J, Kolling C, Gay RE, Detmar M, Gay S, Jngel A, Kyburz D. Downregulation of microRNA-34a* in rheumatoid arthritis synovial fibroblasts promotes apoptosis resistance. Arthritis Rheum. 2012, in press.