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AB0047 Decoy receptor 3 increases the expression of il-12b in rheumatoid synovial fibroblasts
  1. K. Fukuda1,
  2. Y. Miura1,2,
  3. T. Maeda1,
  4. S. Hayashi1,
  5. M. Kurosaka1
  1. 1Department Of Orthopaedic Surgery, KOBE UNIVERSITY GRADUATE SCHOOL OF MEDICINE, KOBE, JAPAN
  2. 2Department of Rehabilitation Science, Kobe University Graduate School of Health Sciences, Kobe, Japan

Abstract

Background Decoy receptor 3 (DcR3) is a secreted decoy tumor necrosis factor receptor (TNFR) and competitively binds and inhibits the TNF family including Fas-ligand (FasL), LIGHT, and TL1A. DcR3 is overexpressed in tumor cells and might benefit tumors by helping them to avoid cytotoxic and regulatory effects of the ligands. We previously reported that DcR3 overexpressed in rheumatoid synovial fibroblasts (RA-FLS) stimulated by TNF-alpha protects the cells from Fas-induced apoptosis [1]. Meanwhile, recent studies suggested that DcR3 directly induces osteoclast formation from monocytes, and that DcR3 triggers enhanced adhesion of monocytes via reverse signaling. We recently reported that DcR3 induces VLA-4 expression in THP-1 macrophages to inhibit cycloheximide-induced apoptosis [2], and that DcR3 binds to TL1A expressed on RA-FLS resulting in the negative regulation of cell proliferation induced by inflammatory cytokines [3]. Therefore, DcR3 may regulate gene expressions in RA-FLS by binding to TL1A on RA-FLS as a ligand.

Objectives In the present study, we studied the genes expression profiles in RA-FLS regulated by DcR3 to identify key molecules in DcR3-TL1A signaling.

Methods Microarray assay. Four individual lines of primary cultured RA-FLS were incubated with either 1.0 μg/ml recombinant human DcR3-Fc protein or control IgG1 for 12 hours. Gene expressions were detected by microarray assay.

Real-time polymerase chain reaction (real-time PCR). RA-FLS was stimulated with 10, 100 or 1000 ng/ml DcR3-Fc or 1000ng/ml control IgG1 for 12 hours. The relative expression levels of IL-12B mRNA were quantified by real-time PCR.

Western blotting. RA-FLS was stimulated with 1000 ng/ml DcR3-Fc or control IgG1 for 24 hours. The expression of IL-12B p40 protein in RA-FLS was investigated by western blotting.

Results Microarray data analysis revealed that DcR3 up-regulates and down-regulates gene expression in RA-FLS. Among the most significantly regulated 100 genes by DcR3, 45 genes were up-regulated and 55 genes were down-regulated. The profile indicated that shared p40 subunit (IL-12B) of IL-12 and IL-23 was up-regulated by DcR3-Fc. Real-time PCR revealed that IL-12B mRNA in RA-FLS was significantly increased in a dose dependent manner when stimulated with DcR3-Fc. Western blotting confirmed that IL-12B p40 protein in RA-FLS was increased when stimulated with DcR3-Fc.

Conclusions IL-12 consisting of IL-12A (p35) and IL-12B induces Th1 immune responses. Meanwhile, IL-23 consisting of IL-23A (p19) and IL-12B is involved in the inflammatory pathway via IL-17. In this study, we revealed that DcR3 increased the expression of IL-12B in RA-FLS. Combined with our previous findings, DcR3 may up-regulate the expression of IL-12B in RA-FLS by binding to membrane-bound TL1A as a ligand to affect the pathogenesis of RA.

  1. Hayashi S. et al., Arthritis and Rheum, 56,1067-1074,2007

  2. Tateishi K. et al., Biochem Biophys Res Commun. 389,593-598,2009

  3. Takahashi M. et al., Int J Mol Med., 28,423-427,2011

Disclosure of Interest None Declared

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