Background Angiogenesis is one of the primary events in the pathogenesis of inflammatory arthritis. Notch/HIF signalling interactions regulate angiogenesis and endothelial cell (EC) fate in Rheumatoid Arthritis (RA), however the precise mechanisms involved remain to be elucidated.
Objectives To examine the role of signal transducer and activator of transcription 3 (STAT3) in mediating hypoxia/Notch-induced interactions and downstream functional pathways in RA.
Methods Phospho-STAT3 (p-STAT3) expression in RA synovial tissue was quantified by Immunohistology/Immunofluorescence and Western Blot. Notch-1 IC, HIF1α, p-STAT3 and total-STAT3 protein levels were assessed in RA synovial fibroblast cells (RASFC) and an immortalised synoviocyte cell line (K4IM) under normoxic and hypoxic (3%) conditions by Western Blot. Gene expression of the Notch-1 receptor, its ligand DLL-4 and downstream target genes (hrt-1, hrt-2) were quantified by Real-time PCR. Migration, invasion, matrigel network formation and MMP2/9 in-gel zymography were quantified under normoxic and hypoxic (3%) conditions in the presence of STAT3 siRNA or STAT3 inhibitor (WP1066). Using RA synovial explants ex-vivo, the effect of the STAT3 inhibitor (WP1066) on IL-6, IL-8 and IL-10 expression were assessed by ELISA.
Results Nuclear expression of p-STAT3 was demonstrated in RA synovial tissue, localised to the sub-lining and lining layer regions. p-STAT3 expression was significantly higher in inflamed synovial tissue compared to normal synovial tissue. Hypoxia-induced p-STAT3, Notch-1 IC, HIF1α protein expression in RASFC and K4IM, an effect that was inhibited by the presence of STAT3 siRNA or WP1066. Similarly hypoxia-induced Notch-1 receptor, DLL-4 and hrt-1, hrt-2 gene expression were inhibited in the presence of WP1066. Functionally hypoxia-induced RASFC/K4IM invasion, matrigel network formation, migration, and pro-MMP-2/-9 activities, were inhibited in the presence of STAT3 siRNA and the STAT3 inhibitor. Finally we demonstrated in RA synovial explants ex-vivo that WP1066 significantly decreased IL-6, IL-8 expression and significantly increased anti-inflammatory cytokine IL-10 expression.
Conclusions This study demonstrates regulatory mechanisms between p-STAT3, HIF1α and Notch-1 signaling in RA. STAT3 blockade by siRNA or WP1066 inhibits pro-inflammatory pathways in RA, suggesting that STAT3 may be a potential therapeutic agent for the treatment of RA.
Disclosure of Interest W. Gao: None Declared, J. McCormick: None Declared, M. Connolly: None Declared, D. Veale Grant/research support from: Abbott, Opsona, Pfizer, Roche, Consultant for: MSD, Pfizer, Roche, Speakers bureau: Janssen, MSD, Pfizer, UCB, U. Fearon: None Declared
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