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FRI0352 Ubiquitin E3 Ligase F Box Component, Fbxo3 Modulates Pro-Inflammatory Gene Expressions in Rheumatoid Arthritis via Regulation of Nuclear Factor-Kappab
  1. K. Yong-Jin,
  2. S.-W. Lee,
  3. S. Jung-Sik,
  4. Y.-B. Park,
  5. S.-K. Lee,
  6. M.-C. Park
  1. Yonsei University College of Medicine, Seoul, Korea, Republic Of

Abstract

Background The TNFR-associated factor (TRAF) proteins have a critical role in inflammation, innate and adaptive immune responses, and programmed cell death by delivering signals from cell surface receptors to induce transcriptional activation of pro-inflammatory cytokine genes. The ubiquitin E3 ligase F box component, named Fbxo3 has recently emerged as a key regulatory role in the modulation of the inflammatory response by mediating the degradation of the TRAF inhibitory protein, Fbxl2.

Objectives The purposes of this study are to determine the expression of Fbxo3 and to investigate the effect of selective small-molecule Fbxo3 inhibitors, BC-1215, on pro-inflammatory gene expressions in fibroblast-like synoviocyte (FLS) in patients with rheumatoid arthritis (RA).

Methods FLS were isolated and cultured from synovial tissues obtained from patients with RA or osteoarthritis (OA) during total knee replacement surgery. The mRNA expressions of Fbxo3 in peripheral blood mononuclear cells (PBMCs) and FLS from patients with RA or OA were determined using real-time quantitative RT-PCR. RA FLS were subsequently treated with 0.5 μM, 5 μM, and 50 μM BC-1215 with or without TNF-α (10 ng/mL) stimulation. The mRNA expressions of IL-1β, IL-6, COX-2, MMP-9, CCL-2 and CCL-7 were determined by real-time quantitative RT-PCR, and FLS viability in response to BC-1215 treatment was measured by MTT assay. Electrophoretic mobility shift assay (EMSA) was performed to assess transcriptional activities of nuclear factor-kappaB (NF-κB) in RA FLS.

Results Expression of Fbxo3 mRNA in PMBCs from RA patients was significantly increased compared to those from OA patients, and this increase was more accentuated in RA FLS compared to OA FLS. BC-1215 treatment induced a dose-dependent reduction in constitutional mRNA expressions of IL-1β, IL-6, COX-2, MMP-9, CCL-2 and CCL-7 from RA FLS. The viability of RA FLS was not significant change in dose- and time- dependent manners. The transcriptional activities of NF-κB were down-regulated in RA FLS with BC-1215 treatment.

Conclusions Fbxo3 inhibitors, BC-1215 suppress various pro-inflammatory gene expressions via modulation of NF-κB. These results suggest that inhibition of Fbxo3 can be a novel therapeutic approach in human RA.

References

  1. Alvarez, S.E. et al. Sphingosine-1-phosphate is a missing cofactor for the E3 ubiquitin ligase TRAF2. Nature 465, 1084–1088 (2010).

  2. Hildebrand, J.M. et al. Roles of tumor necrosis factor receptor associated factor 3 (TRAF3) and TRAF5 in immune cell functions. Immunol. Rev. 244, 55–74 (2011).

  3. Tseng, P.H. et al. Different modes of ubiquitination of the adaptor TRAF3 selectively activate the expression of type I interferons and proinflammatory cytokines. Nat. Immunol. 11, 70–75 (2010).

Disclosure of Interest None declared

DOI 10.1136/annrheumdis-2014-eular.5985

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