Notch‐1 and Notch‐3 Mediate Hypoxia‐Induced Activation of Synovial Fibroblasts in Rheumatoid Arthritis

Objective To investigate the molecular mechanism of hypoxia‐induced rheumatoid arthritis synovial fibroblast (RASF) activation via Notch‐1 and Notch‐3 signaling, and to evaluate its potential as a therapeutic target. Methods Expression of Notch‐1 intracellular domain (N1ICD), N3ICD, and hypoxia‐inducible factor 1α (HIF‐1α) was assessed by immunhistology in synovial tissue from patients with RA. RASFs were cultured under hypoxic conditions and normoxic conditions with or without small interfering RNAs (siRNAs), and N1ICD and N3ICD were overexpressed under normoxic conditions. Rats with collagen‐induced arthritis (CIA) were administered LY411575 (inhibitor of N1ICD and N3ICD) for 15 days and 28 days, and its therapeutic efficacy was assessed by histologic and radiologic evaluation of the rat synovial tissue, and by analysis of inflammatory cytokine production in the serum of rats. Results N1ICD, N3ICD, and HIF‐1α were expressed abundantly in the synovial tissue of RA patients. HIF‐1α was shown to directly regulate the expression of Notch‐1 and Notch‐3 genes under hypoxic conditions. Moreover, hypoxia‐induced N1ICD and N3ICD expression in RASFs was blocked by HIF‐1α siRNA. Notch‐1 siRNA and Notch‐3 siRNA inhibited hypoxia‐induced RASF invasion and angiogenesis in vitro, whereas overexpression of N1ICD and N3ICD promoted these processes. In addition, Notch‐1 was shown to regulate RASF migration and epithelial–mesenchymal transition under hypoxic conditions, whereas Notch‐3 was shown to regulate the processes of anti‐apoptosis and autophagy. Furthermore, in vivo studies in rats with CIA showed that the N1ICD and N3ICD inhibitor LY411575 had a therapeutic effect in terms of ameliorating the symptoms and severity of the disease. Conclusion This study identified a functional link between HIF‐1α, Notch‐1, and Notch‐3 signaling in regulating activation of RASFs and the processes involved in the pathogenesis of RA.