title: Hypoxia and STAT3 signalling interactions regulate pro-inflammatory pathways in rheumatoid arthritis.
abstract: OBJECTIVE
  To examine the effect of hypoxia on Signal Transducer and Activator of Transcription 3 (STAT3)-induced pro-inflammatory pathways in rheumatoid arthritis (RA).
  
  
  METHODS
  Detection of phospho-STAT3 was assessed in RA synovial tissue and fibroblasts (RASFC) by immunohistology/immunofluorescence. Primary RASFCs and a normal synoviocyte cell line (K4IM) were cultured under hypoxic and normoxic conditions±Stat3-siRNA, HIF-siRNA or WP1066 (JAK2-inhibitor). HIF1α, p-STAT3, p-STAT1 and Notch-1IC protein expression were analysed by western blot. Functional mechanisms were quantified by invasion chamber, matrigel and migration assays. IL-6, IL-8, IL-10 and matrixmetalloproteinases (MMP)-3 were quantified by ELISA. Notch-1 receptor, its DLL-4 ligand and downstream target genes (hrt-1, hrt-2) were quantified by real-time PCR. The effect of WP1066 on spontaneous secretion of pro/anti-inflammatory cytokines and Notch signalling was examined in RA synovial explants ex vivo.
  
  
  RESULTS
  p-STAT3 was increased in RA synovium compared with control (p<0.05). Hypoxia induced p-STAT3, p-STAT1 and HIF1α expression, an effect blocked by Stat3-siRNA and WP1066. Hypoxia-induced cell invasion, migration and cytokine production were inhibited by Stat3-siRNA (p<0.05) and WP1066 (p<0.05). While HIF1α siRNA inhibited hypoxia-induced p-STAT3 detection, Stat3-siRNA also inhibited hypoxia-induced HIF1α. Furthermore, hypoxia-induced Notch-1IC, DLL4, hrt-1 and -2 expression were significantly inhibited by WP1066 (p<0.05). Finally, in RA synovial explant cultures ex vivo, WP1066 decreased spontaneous secretion of IL-6, IL-8 and MMP3 (p<0.05), Notch-1 mRNA (p<0.05) and induced IL-10 (p<0.05).
  
  
  CONCLUSIONS
  This is the first study to provide evidence of a functional link between HIF1α, STAT3 and Notch-1 signalling in the regulation of pro-inflammatory mechanisms in RA, and further supports a role for STAT blockade in the treatment of RA.
authors: Translational Research Group, Dublin Academic Medical Centre, St Vincent's University Hospital, Dublin, Ireland.
authors: Wei
authors: W
authors: Gao
authors: Translational Research Group, Dublin Academic Medical Centre, St Vincent's University Hospital, Dublin, Ireland.
authors: Jennifer
authors: J
authors: McCormick
authors: Translational Research Group, Dublin Academic Medical Centre, St Vincent's University Hospital, Dublin, Ireland.
authors: Mary
authors: M
authors: Connolly
authors: Translational Research Group, Dublin Academic Medical Centre, St Vincent's University Hospital, Dublin, Ireland.
authors: Emese
authors: E
authors: Balogh
authors: Translational Research Group, Dublin Academic Medical Centre, St Vincent's University Hospital, Dublin, Ireland.
authors: Douglas J
authors: DJ
authors: Veale
authors: Translational Research Group, Dublin Academic Medical Centre, St Vincent's University Hospital, Dublin, Ireland.
authors: Ursula
authors: U
authors: Fearon
mesh_terms: D000328:Adult; D000368:Aged; D001172:Arthritis, Rheumatoid / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005347:Fibroblasts / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D000860:Hypoxia / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D016753:Interleukin-10 / Q000276:immunology / Q000378:metabolism; D015850:Interleukin-6 / Q000276:immunology / Q000378:metabolism; D016209:Interleukin-8 / Q000276:immunology / Q000378:metabolism; D008297:Male; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism; D008875:Middle Aged; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism; D050796:STAT3 Transcription Factor; D015398:Signal Transduction; D013583:Synovial Membrane / Q000276:immunology* / Q000378:metabolism
chemical_list: C508606:CXCL8 protein, human; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C508609:IL10 protein, human; C508600:IL6 protein, human; D015850:Interleukin-6; D016209:Interleukin-8; C497761:NOTCH1 protein, human; D051881:Receptor, Notch1; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D016753:Interleukin-10; C511768:MMP3 protein, human; D019278:Matrix Metalloproteinase 3
keywords: Cytokines; Fibroblasts; Rheumatoid Arthritis