Abstract
In rheumatoid arthritis (RA), synovial fibroblasts (RA-SFs) accumulate in affected joints, where they play roles in inflammation and joint destruction. RA-SFs exhibit tumor-like proliferation and are resistant to apoptosis. Although RA-SF activation is well described, negative regulators of RA-SF activation are unknown. We previously reported that histone deacetylase (HDAC) inhibitors facilitate apoptosis in RA-SFs. Here we found that RA-SFs treated with the HDAC inhibitor Trichostatin A (TSA) exhibited an upregulation of the immediate early response gene X-1 (IEX-1). IEX-1 has roles in apoptosis sensitivity, cell-cycle progression, and proliferation, and is reported to be involved in immune responses, inflammation, and tumorigenesis, and to have anti-arthritic properties. To investigate IEX-1’s role in RA-SFs, we used in vitro-cultured synovial fibroblasts from RA and osteoarthritis (OA) patients. We confirmed that TSA upregulated the IEX-1 protein and mRNA expressions in RA-SFs by western blotting and quantitative RT-PCR. Inhibiting HDAC1, 2, and 3 (but not 6 or 8) also upregulated IEX-1. The IEX-1 mRNA levels were higher in RA-SFs than in OA-SFs, and were further upregulated in RA-SFs by the pro-inflammatory cytokines TNFα and IL-1β. The staining of surgical specimens showed that IEX-1 was present in the pannus from affected RA joints. Si-RNA-mediated IEX-1 knockdown upregulated the lipopolysaccharide (LPS)-induced expression of TNFα and various chemokine mRNAs, indicating that IEX-1 downregulates TNFα and chemokines. Furthermore, apoptosis analysis showed that IEX-1 knockdown protected RA-SFs from apoptosis induced by TSA or by an anti-Fas mAb, indicating that IEX-1 is pro-apoptotic in RA-SFs. Collectively, our results showed that IEX-1 is induced by TNFα and IL-1β in RA-SFs, in which it suppresses TNFα and chemokine production and induces apoptosis; thus, IEX-1 negatively regulates RA-SF activation. Further investigation of IEX1’s functions in RA-SFs may lead to new therapeutic approaches for RA.
Highlights
Rheumatoid arthritis (RA) is a systemic inflammatory disease that progressively destroys the joints [1]
We previously reported that inhibiting histone deacetylase (HDAC) induces apoptosis in rheumatoid arthritis synovial fibroblasts (RA-SF)
immediate early response gene X-1 (IEX-1) knockdown did not augmented cell survival in the presence of the anti-Fas mAb alone or in combination with trichostatin A (TSA) (Fig 8). These results indicated that IEX-1 has no effects of apoptosis induced in the presence of the anti-Fas mAb and HDAC inhibitors in OA-SFs, suggesting that the roles for IEX-1 in apoptosis are different between RA-SF and OA-SF
Summary
Rheumatoid arthritis (RA) is a systemic inflammatory disease that progressively destroys the joints [1]. RA-SFs are actively involved in persistent inflammation and joint destruction [2,3,4]. RA-SFs are characterized by increased cell survival and destruction of surrounding tissue, and play a pro-inflammatory role in immune responses. RA-SFs have tumor-like proliferative properties and are resistant to apoptosis. This resistance to apoptosis may be related to somatic p53 mutations, the activation of the NF-κB pathway in RA-SFs, and the elevated expression of such anti-apoptotic molecules as Bcl-2, Fasassociated death domain-like interleukin-1β-converting enzyme-inhibitory protein (FLIP), and sentrin-1/small ubiquitin-like modifier (SUMO-1) [5]. We previously showed that histone deacetylase (HDAC) inhibitors facilitate apoptosis in RA-SFs in the presence of an anti-Fas mAb [6]. We used a differential display technique to search for genes that were up- or downregulated in RA-SFs by the HDAC inhibitor trichostatin A (TSA), and found that the immediate early response gene X-1 (IEX-1) was upregulated in TSA-treated RA-SFs (data not shown)
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