Background Accumulating evidence implicates hypoxia in the pathogenesis of rheumatoid arthritis (RA). The effect of hypoxia on the expression of Slug, a transcriptional repressor that impairs apoptosis of RA fibroblast-like synoviocytes (FLS), remains unknown.
Objectives The aim of this study is to investigate the role of hypoxia in the expression of Slug in RA FLS and to delineate the signaling pathway involved in the process.
Methods After RA FLS were exposed to hypoxia, expression of HIF1-a and phosphorylation of ERK, JNK, and STAT3 were analyzed by Western blot. The experiment was repeated with RA FLS pretreated with WP1066, an inhibitor of the JAK/STAT pathway. RT-PCR was performed to measure HIF-1a mRNA and Slug mRNA expressions in RA FLS under hypoxia. RA FLS was transfected with HIF-1a cDNA to investigate the effect of overexpression of HIF-a on Slug expression. Immunohistochemistry was used to assess the presence HIF-1a and Slug in RA synovial tissue. Microarray analysis was performed to investigate the change in Slug gene expression when FLS were exposed to hypoxia. Finally, the effect of TNF-α on Slug and HIF-1α expressions under normoxic conditions was assessed in RA FLS by Western blot.
Results Hypoxia induced the expression of HIF-1a and phosphorylation of STAT3, but not JNK and ERK was increased in RA FLS. Pre-treatment of RA FLS with WP1066 before exposure to hypoxia inhibited the expression of p-STAT and HIF-1a. Hypoxic conditions induced Slug mRNA expression from RA FLS, and similarly, overexpression of HIF-1a in RA FLS reproduced enhanced Slug expression. Microarray analysis revealed a significantly up-regulated Slug expression. Immunohistochemical staining showed that HIF-1a and Slug were co-localized in the lining area of RA synovium. Stimulation of FLS with TNF-α alone without hypoxia resulted in increased expression of Slug HIF-1a and Slug.
Conclusions Our study demonstrates that HIF-1a expression from RA FLS induced by hypoxia or TNF-a alone is mediated through JAK/STAT3 signaling pathway, which ultimately leads to the expression of Slug. Hypoxia-driven pathway of Slug expression may become a novel treatment target in RA.
Disclosure of Interest None declared
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