ACR Meeting Abstracts

ACR Meeting Abstracts

Abstract Number: 932

Hypoxia and Signal Transducer and Activator Of Transcription 3 Signalling Interactions Regulate Pro-Inflammatory Pathways In Rheumatoid Arthritis

Wei Gao1, Jennifer McCormick1, Mary Connolly2, Emese Balogh2, Douglas J. Veale1 and Ursula Fearon1, 1Dublin Academic Medical Centre, Translational Rheumatology Research Group, Dublin, Ireland, 2Rheumatology, Translation Research Group, Dublin Academic Medical Centre, St. Vincent's University Hospital, Dublin, Ireland

Meeting: 2013 ACR/ARHP Annual Meeting

Keywords: ,

Session Information

Title: Cytokines, Mediators, Cell-cell Adhesion, Cell Trafficking and Angiogenesis I

Session Type: Abstract Submissions (ACR)

Background/Purpose: Signal Transducer and Activator of Transcription 3 (STAT3), a critical transcription activator in angiogenesis, plays a crucial role in the pathogenesis of Rheumatoid Arthritis (RA). Hypoxia and Notch signalling regulate angiogenesis, cell proliferation, invasion and survival. This study was to examine the effect of hypoxia on STAT3-induced pro-inflammatory pathways in RA.

Methods: Expression of phospho-STAT3 in synovial tissue and RA synovial fibroblasts (RASFC) was assessed by immunohistology/immunofluorescence. Primary RASFC and synoviocyte cell lines (K4IM) were cultured under 3% hypoxia and normoxia conditions±Stat3-siRNA, HIF-siRNA or WP1066 (potent STAT inhibitor). HIF1α, p-STAT3 and Notch-1IC (intracellular domain) protein expression were analyzed by Western blot. Functional mechanisms were quantified by invasion chamber, matrigel and wound repair assays. IL-6, IL-8, IL-10 and 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. Finally the effect of WP1066 on spontaneous secretion of pro/anti-inflammatory cytokines was examined in RA synovial explants ex-vivo.

Results: Increased p–STAT3 expression was demonstrated in RA synovium compared to healthy control (p<0.05) and 3% hypoxia induced nuclear translocation of p-STAT3 in RASFC. Hypoxia induced p-STAT3 and HIF1α expression, an effect blocked by Stat3-siRNA and WP1066. Hypoxia-induced cell invasion, migration and cytokine production were inhibited by Stat3-siRNA (all p<0.05) and WP1066 (all p<0.05). While HIF1α siRNA inhibited hypoxia-induced p-STAT3 expression, Stat3-siRNA also inhibited hypoxia-induced HIF1α. Furthermore hypoxia-induced Notch-1IC, DLL4, hrt-1 and -2 expression were significantly inhibited by STAT3 blockade (p<0.05). Finally, in RA synovial explant cultures ex-vivo, STAT3 blockade decreased spontaneous secretion of IL-6, IL-8 and MMP3 (p<0.05), and induced IL-10 (p<0.05).

Conclusion: 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.

Disclosure:

W. Gao,
None;

J. McCormick,
None;

M. Connolly,
None;

E. Balogh,
None;

D. J. Veale,

AbbVie, ,

2,

Pfizer Inc,

2,

MSD,

2,

Roche Pharmaceuticals,

2,

Pfizer Inc,

5,

Roche Pharmaceuticals,

5,

Abbott Laboratories,

8,

Pfizer Inc,

8,

MSD,

8,

Roche Pharmaceuticals,

8;

U. Fearon,
None.

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