title: IL-6 trans-signalling directly induces RANKL on fibroblast-like synovial cells and is involved in RANKL induction by TNF-alpha and IL-17.
abstract: OBJECTIVE
  We investigated the influence of cytokines on the expression of RANK ligand (RANKL) in fibroblast-like synoviocytes from RA patients (RA-FLS).
  
  
  METHODS
  RA-FLS were stimulated by IL-6, TNF-alpha, IL-17 and IL-1beta with or without soluble IL-6 receptor (sIL-6R) for 24 h. The expression of RANKL was measured by real-time PCR, western blotting and immunostaining. In proliferation assay, RA-FLS were cultured with cytokines for 3 days. RA-FLS were co-cultured with RAW cell in the presence of IL-6/sIL-6R for 3 days and then NFATc1 mRNA expression in RAW cells was examined. RA-FLS was cultured with parthenolide [PAR, signal transducer and activator of transcription (STAT) inhibitor] or PD98059 (PD, mitogen-activated protein kinase inhibitor) in the presence of IL-6/sIL-6R and then the influence of these drugs on phosphorylation of STAT3 and ERK1/2, and RANKL expression was examined.
  
  
  RESULTS
  RANKL expression was induced by IL-6/sIL-6R (but not IL-6 alone) and by IL-1beta. On the other hand, TNF-alpha and IL-17 did not induce RANKL expression, although TNF-alpha, IL-17 or IL-1beta stimulated cell growth and IL-6 production. However, in the presence of sIL-6R, TNF-alpha or IL-17 induced RANKL expression. By the co-culture of RA-FLS, NFATc1 mRNA expression was induced in RAW cells. Finally, IL-6/sIL-6R induced phosphorylation of STAT3 and ERK1/2 in RA-FLS, and was completely inhibited by PAR and PD, respectively. PAR completely inhibited IL-6/sIL-6R-induced RANKL expression, but PD did not.
  
  
  CONCLUSIONS
  IL-6/sIL-6R directly induced RANKL expression in RA-FLS and it is essential for RANKL induction by TNF-alpha and IL-17. Moreover, RANKL induction by IL-6/sIL-6R is mediated by the janus kinase/STAT signalling pathway.
authors.affiliation: Product Research Department, Chugai Pharmaceutical Co. Ltd, Komakado, Gotemba, Shizuoka, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Hashizume
authors.forename: N
authors.initials: N
authors.lastname: Hayakawa
authors.forename: M
authors.initials: M
authors.lastname: Mihara
mesh_terms: D000911:Antibodies, Monoclonal / Q000494:pharmacology; D061067:Antibodies, Monoclonal, Humanized; D001172:Arthritis, Rheumatoid / Q000378:metabolism*; D015153:Blotting, Western / Q000379:methods; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D005347:Fibroblasts; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D020381:Interleukin-17 / Q000494:pharmacology*; D053583:Interleukin-1beta / Q000494:pharmacology; D015850:Interleukin-6 / Q000378:metabolism* / Q000494:pharmacology; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D053245:RANK Ligand / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D053246:Receptor Activator of Nuclear Factor-kappa B / Q000378:metabolism; D019947:Receptors, Interleukin-6 / Q000037:antagonists & inhibitors / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D050796:STAT3 Transcription Factor / Q000378:metabolism; D012717:Sesquiterpenes / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D013268:Stimulation, Chemical; D013583:Synovial Membrane / Q000187:drug effects / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D005419:Flavonoids; D020381:Interleukin-17; D053583:Interleukin-1beta; D015850:Interleukin-6; D053245:RANK Ligand; D012333:RNA, Messenger; D053246:Receptor Activator of Nuclear Factor-kappa B; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins; D050796:STAT3 Transcription Factor; D012717:Sesquiterpenes; D014409:Tumor Necrosis Factor-alpha; C002669:parthenolide; D020929:Mitogen-Activated Protein Kinase Kinases; C502936:tocilizumab; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one