Hypoxia and STAT3 signalling interactions regulate pro-inflammatory pathways in rheumatoid arthritis. Issue 6 (13th February 2014)

• Record Type: Journal Article
• Authors: Gao, Wei; McCormick, Jennifer; Connolly, Mary; Balogh, Emese; Veale, Douglas J; Fearon, Ursula
• Is Part Of: Annals of the rheumatic diseases. Volume 74:Issue 6(2015)
• Page Start: 1275
• Page End: 1283
• Publication Date: 2014-02-13
• Subjects: Rheumatoid Arthritis -- Cytokines -- Fibroblasts; Rheumatism -- Periodicals; 616.723005
• DOI: 10.1136/annrheumdis-2013-204105
• Languages: English
• ISSNs: 0003-4967

Abstract

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.