Expression and Functions of Immediate Early Response Gene X-1 (IEX-1) in Rheumatoid Arthritis Synovial Fibroblasts

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 OASFs, and were further upregulated in RA-SFs by the pro-inflammatory cytokines TNF alpha and IL- 1 beta. 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 TNFa and various chemokine mRNAs, indicating that IEX- 1 downregulates TNF alpha 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 TNFa and IL- 1 beta in RA-SFs, in which it suppresses TNFa 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.