Rheumatoid arthritis (RA), which affects ∼1% of the population, is a chronic autoimmune disease involving progressive destruction of the affected joints. Proinflammatory cytokines, such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor α (TNFα), are involved in its pathogenesis (1–3). Anti-TNFα therapies in particular have been shown to provide substantial benefit to patients not only through the reduction of signs and symptoms of the disease, but also by the inhibition of joint destruction (1). However, treatments with current biologics, including anti-TNFα, anti–T cell, and anti–B cell therapies, are only successful in at best 60% of the treated patients and are still unable to cure the disease. A cytokine-independent pathway appears responsible for the ongoing joint destruction mediated by synovial fibroblasts (SFs) (2). Since implantation of RASFs with human cartilage into SCID mice causes invasion into the cartilage without the support of the cells of the human immune system, it has been proposed that the activated phenotype is an “intrinsic” property of these cells (3).
SFs, more than other types of fibroblasts, acquire phenotypic characteristics commonly associated with transformed cells (4). RASFs show “spontaneous” activities associated with aggressive behavior, and they differ from the SFs of patients with osteoarthritis (OASFs) or normal SFs. For example, RASFs up-regulate protooncogenes (5), specific matrix-degrading enzymes (6), adhesion molecules (7), and cytokines (8). These observations of an intrinsically activated cellular phenotype prompted us to search for epigenetic modifications.
In somatic cells, Dnmt1 is the predominant DNA methyltransferase (9, 10). Reduction of Dnmt1 levels leads to hypomethylation, genomic instability, and tumorigenesis. Direct interaction between Dnmt1 and proliferating cell nuclear antigen (PCNA) ensures that patterns of methylation are faithfully preserved in DNA synthesis (10). Moreover, repetitive sequences such as L1, Alu, and satellite alpha repeats are silenced by methylation in normal cells and can be used as markers of global hypomethylation (11). Our group and others (12, 13) reported a reactivation of the endogenous retroviral element L1 in the RA synovial lining and at sites of invasion. These reports suggest that global genomic hypomethylation plays a role in the pathogenesis of RA, and that genes normally silenced by methylation might contribute to the activated phenotype of RASFs (12).
Here we show that DNA demethylation of normal SFs induces a cellular phenotype resembling that of activated RASFs. Genomic hypomethylation is a characteristic of RASFs and is involved in the pathogenesis of RA.
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Here we report that genomic hypomethylation developed in situ is conserved in RASFs in vitro even after >5 passages, and we confirm that the expression of L1 proteins in RASFs is associated with a partially hypomethylated L1 promoter. The degree of CG hypomethylation (78% methylation in RASFs versus 85% in OASFs) in the L1 promoter is similar to the degree of hypomethylation in tumor cells (9).
It is essential to understand more about the induction and maintenance of this global genomic hypomethylation. Proinflammatory cytokines such as TNFα, IL-1β, and IL-6 have multiple influences on the pathogenesis of RA. IL-1β (14) and IL-6 (15) can affect genomic methylation. TNFα, however, had not previously been associated with epigenetic changes. We observed that a physiologic dose of TNFα accelerated the cell cycle within 24 hours of exposure even more than IL-1β or PDGF. In OASFs, stimulation of cell proliferation was accompanied by increased DNA methylation. This was not the case in RASFs, and therefore the relative degree of DNA hypomethylation remained in RASFs treated with TNFα or IL-1β. As a consequence, it can be hypothesized that RASFs become progressively more hypomethylated during inflammation. This results in further activation of genes in RASFs.
Previous studies have shown that deficiency of Dnmt1 is associated with genomic hypomethylation (10, 16, 17). Dnmt1 interacts with PCNA at the DNA replication fork, and this system is responsible for the correct transmission of methylation marks to daughter cells. In RASFs, however, the expression of Dnmt1 appeared deficient, either in unstimulated cells or after exposure to proinflammatory cytokines. Thus, in RASFs, a relative deficiency of Dnmt1 during cell proliferation could result in the observed genomic hypomethylation.
Our work raises the question of whether the global genomic hypomethylation is accompanied, or followed by, specific promoter hypermethylation, since this is the case in various tumors (9). At least 1 example has been reported in the literature (i.e., by silencing death receptor 3 ), which could, at least in part, explain the relative resistance to apoptosis reported for RASFs in certain patients (2).
In SFs, the loss of methylation marks in daughter cells could cause an irreversible differentiation into an aggressive phenotype. Based on our observations, we hypothesized that normal SFs continuously treated with the Dnmt1 inhibitor 5-azaC will resemble RASFs. Indeed, a large number of gene transcripts (73 of 186, 39%) found to be up-regulated upon stimulation with 5-azaC and detected in cDNA microarrays were previously described to be involved in the pathogenesis of RA. It is known that DNA methylation silences genes with CpG island promoters. Therefore, from the list provided by the microarrays, we chose 3 genes that showed a >5-fold up-regulation of mRNA and the presence of CpG islands in their promoters, namely, CD10, CD29, and CD130. We confirmed that they were expressed on the surfaces of RASFs more than on the surfaces of OASFs and that their expression was increased within 2 weeks of treatment with a low dose of 5-azaC. It is well established that RASFs attach to cartilage through adhesion molecules, including CD29 and CD61 (β1 and β3 integrins) (7, 19). Invasion of RASFs into cartilage requires the availability of these 2 integrins (19, 20). CD10, a neutral endopeptidase, is highly expressed on RASFs (21) and presumably plays a critical role in the local regulation of peptide levels in the joint. IL-6 signaling involves both a specific IL-6 receptor (IL-6Rα) and a ubiquitous signal-transducing protein, CD130 (gp130), which is also used by oncostatin M. Both IL-6 and oncostatin M are involved in the pathogenesis of RA (8, 22, 23).
We also evaluated the expression of genes that have no CpG island in their promoters but that do have a CpG island in exon 1, namely, CD26, MMP-14, and TGFβRII. CD26 (dipeptidylpeptidase 4) was found to be highly expressed in RA synovial tissue (24) and in proliferating RASFs (25). The destruction of cartilage and bone in RA is in large part mediated by MMPs (20). MMP-14 has a central role because it cleaves other proMMPs and converts them into active forms. Inhibition of MMP-1 and/or MMP-14 results in a significant reduction of cartilage invasion by RASFs (26). Our results are in line with previously reported data in pancreatic cancer cells showing that 5-azaC also up-regulates the expression of MMP-14 and MMP-1 (27). Expression of TGFβRII was already maximal on RASFs, as proposed earlier (28).
Approximately one-fourth of genes (51 of 186, 27%) (see Supplementary Tables 1 and 2, available on the Arthritis & Rheumatism Web site at http://www.mrw.interscience.wiley.com/suppmat/0004-3591/suppmat/) that were up-regulated upon treatment with 5-azaC in normal SFs contained no CpG island (e.g., CD36, CD46, CTK, caspase 1, and IL-1RI). For example, CD36, abundantly expressed in RA synovial tissue, binds proinflammatory oxidized low-density lipoproteins (29) and thrombospondin 1 (30). CD46 is a C3b binding protein, which could be involved in tissue damage in RA (31), whereas CTK, a key enzyme in bone resorption, is highly expressed in RA synovial tissue, not only by osteoclasts, but also by RASFs (32). Caspase 1 (IL-1β–converting enzyme), which activates IL-1β in RA synovial tissue (33), is also up-regulated in normal SFs by 5-azaC. IL-1β stimulates both the synthesis and the activity of MMPs involved in cartilage destruction (34).
Of the gene products that were up-regulated in normal SFs upon 5-azaC treatment, a particularly high proportion (52 of 73, 71%) are involved in intercellular processes and interactions with the extracellular matrix and have been described in RA synovial tissue and/or RASFs (see Supplementary Table 1, available on the Arthritis & Rheumatism Web site at http://www.mrw.interscience.wiley.com/suppmat/0004-3591/suppmat/). These include 22 interleukins, growth factors, and their receptors, 13 extracellular matrix proteins and related enzymes, 10 matrix-degrading enzymes and their inhibitors, and 4 adhesion molecules. Most importantly, among them, cathepsins, MMPs, mannosidase α1, carbonic anhydrases, and ADAM-12 are involved in joint destruction, and lysyl oxidase has been shown to increase the crosslinking of mature collagen, an early step in cartilage destruction.
Many of the gene products also up-regulated in normal SFs upon 5-azaC treatment are involved in intracellular processes and play a role in RA (20 of 94, 21%) (see Supplementary Table 2, available on the Arthritis & Rheumatism Web site at http://www.mrw.interscience.wiley.com/suppmat/0004-3591/suppmat/). They include 4 protein kinases, 10 transcription factors, 2 proteins in the Wnt pathway, 2 proteins involved in the regulation of actin filaments and Rho signaling, and 2 regulators of apoptosis. Furthermore, transcription factors whose expression increased upon 5-azaC treatment (18 of 22, 82%) (see Supplementary Table 2, available on the Arthritis & Rheumatism Web site at http://www.mrw.interscience.wiley.com/suppmat/0004-3591/suppmat/) had CpG islands in their promoters and/or exon 1 more often than other genes revealed by the cDNA arrays. Many of them may play a role in RA, including Ets-related transcription factor, activating transcription factor 2 (ATF-2; which binds to activator protein 1), CCAAT/enhancer binding protein δ, nuclear factor of activated T cells 5, CREB/ATF, hypoxia-inducible factor 2α, and STAT-1. The sustained up-regulation of multiple signaling and transcription pathways in a hypomethylating milieu clearly could be responsible for the intrinsically activated phenotype of RASFs. The cDNA arrays identified proteins that are implicated in the normal or pathologic function of SFs, including 1 recently reported as a potential autoantigen in RA, human cartilage gp-39 (35).
CD10, CD36, and CD46 were also more up-regulated in OASFs than in RASFs upon 5-azaC treatment. The expression of all other genes tested was up-regulated to the same extent in OASFs and RASFs. Bisulfite sequencing of the CD10 CpG island, however, showed that it is hypomethylated even in normal SFs (data not shown). Therefore, the gene is regulated indirectly or by a methylation-independent mechanism. Other investigators have reported similar effects of 5-azaC on myeloid leukemia genes (36). CD46 and CD36 do not have a CpG island in their gene promoter; most likely they are regulated by indirect mechanisms. Other 5-azaC microarray studies have shown up-regulation of genes in the absence of CpG islands in their promoters (37). Thus, 5-azaC can apparently influence the expression of certain genes by different mechanisms. For example, it can affect histone modifications and up-regulate transcription factors, transcriptional repressors, and/or the expression of microRNA (38, 39). We have provided a list of transcription factors that could be candidates for future investigations (see Supplementary Table 2, available on the Arthritis & Rheumatism Web site at http://www.mrw.interscience.wiley.com/suppmat/0004-3591/suppmat/).
In summary, we report reduced 5-methylcytosine DNA in RA synovial tissue and in cultured RASFs. Specifically, the promoter of an L1 element was partially demethylated, confirming the global genomic hypomethylation in RASFs. Moreover, our observations suggested a progressive loss of methylation marks. It can be hypothesized 1) that the loss of methylation marks could be responsible for the intrinsically activated and aggressive phenotype of RASFs and 2) that tissue-specific transcription factors, which are not normally expressed in synovial tissue, are up-regulated in the disease and can be responsible for the activation of many genes involved in the pathogenesis of RA. Moreover, genomic hypomethylation could explain the increased expression of multiple receptors, adhesion molecules, and matrix-degrading enzymes, which play a role in RA and explain the enhanced response of RASFs to proinflammatory cytokines, leading all together to joint destruction. Thus, the epigenetic modifications of RASFs may be responsible, at least in part, for the fact that current therapies do not work in all patients and do not yet cure the disease.