Hypoxia and STAT3 signalling interactions regulate pro-inflammatory pathways in rheumatoid arthritis

Ann Rheum Dis. 2015 Jun;74(6):1275-83. doi: 10.1136/annrheumdis-2013-204105. Epub 2014 Feb 13.


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.

Keywords: Cytokines; Fibroblasts; Rheumatoid Arthritis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Arthritis, Rheumatoid / genetics
  • Arthritis, Rheumatoid / immunology*
  • Arthritis, Rheumatoid / metabolism
  • Blotting, Western
  • Cells, Cultured
  • Enzyme-Linked Immunosorbent Assay
  • Female
  • Fibroblasts / immunology*
  • Fibroblasts / metabolism
  • Humans
  • Hypoxia / genetics
  • Hypoxia / immunology*
  • Hypoxia / metabolism
  • Hypoxia-Inducible Factor 1, alpha Subunit / metabolism
  • Interleukin-10 / immunology
  • Interleukin-10 / metabolism
  • Interleukin-6 / immunology
  • Interleukin-6 / metabolism
  • Interleukin-8 / immunology
  • Interleukin-8 / metabolism
  • Male
  • Matrix Metalloproteinase 3 / metabolism
  • Middle Aged
  • Receptor, Notch1 / genetics
  • Receptor, Notch1 / metabolism
  • STAT3 Transcription Factor
  • Signal Transduction
  • Synovial Membrane / immunology*
  • Synovial Membrane / metabolism


  • CXCL8 protein, human
  • HIF1A protein, human
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • IL10 protein, human
  • IL6 protein, human
  • Interleukin-6
  • Interleukin-8
  • NOTCH1 protein, human
  • Receptor, Notch1
  • STAT3 Transcription Factor
  • STAT3 protein, human
  • Interleukin-10
  • MMP3 protein, human
  • Matrix Metalloproteinase 3