title: The role of Act1, a NF-κB-activating protein, in IL-6 and IL-8 levels induced by IL-17 stimulation in SW982 cells.
abstract: BACKGROUND
  Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation in the synovial membrane of affected joints. It has been shown that several kinds of cytokine were increased in synovial fluid, while the underlying mechanism remains poorly understood.
  
  
  OBJECTIVE
  NF-κB activator 1 (Act1) is a recently identified protein binding to the IκB kinase complex. Our study aimed to investigate the expression of Act1 induced by cytokine IL-17 stimulation in SW982 cells.
  
  
  METHODS
  The human synovial sarcoma cell line SW982 and primary cultured RA fibroblast-like synovial cells were used. RT-PCR and Western blot assays were selected to investigate the genetic and protein expression of Act1. Additionally, four independent Act1 small interfering RNA (siRNA) oligonucleotides were designed and obtained according to the GenBank cDNA, the sequence of Act1 (Traf3ip2). Finally, enzyme-linked immunosorbent assay (ELISA) double antibody sandwich was used to assay supernatant IL-6 and IL-8 concentrations.
  
  
  RESULTS
  The Act1 mRNA expression level increased significantly after stimulation with IL-17 (5-100 ng/ml) in SW982 cells. Additionally, the level of Act1 mRNA expression correlated positively with the concentration of IL-17 (p < 0.01). IL-17 induced IL-6 and IL-8 in SW982 cells was in a concentration- and time-dependent way. Furthermore, ELISA assay revealed that IL-17 (20 ng/ml) significantly increased IL-6 (1927.4 ± 288.77 versus 786.5 ± 172.42 ng/ml, p < 0.01) and IL-8 levels (984.8 ± 95.09 ng/ml versus 307.1 ± 90.83 ng/ml, p < 0.01) compared with control group after stimulation for 24 h. However, transfection of Traf3ip2 siRNA markedly decreased IL-6 (995.9 ± 115.30 ng/ml versus 1816.1 ± 273.27 ng/ml, p < 0.01) and IL-8 levels (575.6 ± 65.96 ng/ml versus 929.4 ± 124.39 ng/ml, p < 0.01) compared to transfection negative control. These findings suggested that IL-6 and IL-8 level induced by IL-17 in SW982 cells could be reversed by down-regulation of Act1 expression level with Traf3ip2 siRNA.
  
  
  CONCLUSIONS
  Our results suggested that Act1 might play a key role in the pathophysiology and the treatment of RA.
authors.affiliation: Department of Rheumatology, Qilu Hospital of Shandong University , Jinan , China.
authors.forename: Fang
authors.initials: F
authors.lastname: Lv
authors.forename: Li-Jun
authors.initials: LJ
authors.lastname: Song
authors.forename: Xiu-Hua
authors.initials: XH
authors.lastname: Wang
authors.forename: Feng
authors.initials: F
authors.lastname: Qiu
authors.forename: Xing-Fu
authors.initials: XF
authors.lastname: Li
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D001172:Arthritis, Rheumatoid / Q000276:immunology / Q000378:metabolism; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D006801:Humans; D020381:Interleukin-17 / Q000494:pharmacology*; D015850:Interleukin-6 / Q000378:metabolism*; D016209:Interleukin-8 / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013583:Synovial Membrane / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C508606:CXCL8 protein, human; C508600:IL6 protein, human; D020381:Interleukin-17; D015850:Interleukin-6; D016209:Interleukin-8; D012333:RNA, Messenger; C414202:TRAF3IP2 protein, human; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins