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

PLoS One. 2016 Oct 13;11(10):e0164350. doi: 10.1371/journal.pone.0164350. eCollection 2016.


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.

MeSH terms

  • Apoptosis
  • Apoptosis Regulatory Proteins / genetics*
  • Apoptosis Regulatory Proteins / metabolism*
  • Arthritis, Rheumatoid / genetics
  • Arthritis, Rheumatoid / metabolism
  • Arthritis, Rheumatoid / pathology*
  • Cells, Cultured
  • Chemokines / genetics
  • Chemokines / metabolism
  • Fibroblasts / cytology*
  • Fibroblasts / drug effects
  • Fibroblasts / metabolism
  • Humans
  • Hydroxamic Acids / pharmacology
  • Membrane Proteins / genetics*
  • Membrane Proteins / metabolism*
  • Osteoarthritis / genetics
  • Osteoarthritis / metabolism
  • Osteoarthritis / pathology
  • Synovial Membrane / cytology*
  • Synovial Membrane / drug effects
  • Synovial Membrane / metabolism
  • Tumor Necrosis Factor-alpha / genetics
  • Tumor Necrosis Factor-alpha / metabolism
  • Up-Regulation


  • Apoptosis Regulatory Proteins
  • Chemokines
  • Hydroxamic Acids
  • IER3 protein, human
  • Membrane Proteins
  • TNF protein, human
  • Tumor Necrosis Factor-alpha
  • trichostatin A

Grant support

This work was supported by a Grant-in-Aid for scientific research (16590981, 21591265) from the Japanese Ministry of Education, Culture, Sports, Science and Technology ( The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.