Notch1 Signaling Contributed To TLR4-Triggered NF-kB Activation In Macrophages

Background: Macrophages substantially shape the development, progression, and complications of inflammation-driven diseases. Although numerous researches support a critical role for Notch signaling in most inflammatory diseases, there is limited data on the role of Notch signaling in TLR4-induced macrophage activation and the interaction of Notch signaling with other signaling pathways(e.g., the NF-kB pathway during inflammation) in macrophages. Methods and Results: In this study, we confirmed that stimulation with TLR4 ligand LPS up-regulates Notch1 expression in RAW264.7 monocyte/macrophage-like cell line. LPS also induced the expression of Notch target genes Notch1 and Hes1 mRNA in macrophages, suggesting that TLR4 signaling enhances Notch pathway activation. The upregulation of Notch1, NICD, and Hes1 protein by LPS treatment was inhibited by the Notch1 inhibitor of DAPT. The increase of TNF-a, IL-6, and IL-1b induced by LPS was inhibited by DAPT while jagged1, the Notch1 ligand, rescued them. Furthermore, the suppression of Notch signaling by DAPT up-regulated CYLD expression but down-regulated TRAF6, IKKa/bphosphorylation, and subsequently phosphorylation and degradation of IκB-α, indicating the inhibition of TLR4-triggered NF-kB activation by DAPT. Interestingly, DAPT showed no inhibitory effect on the increase of MyD88 expression induced by LPS in our study. Conclusions: Our study shows that the stimulation of macrophages via the TLR4 signaling cascade triggers the activation of Notch1 signaling, which regulates the expression patterns of genes involved in pro-inflammatory responses through by activating NF-kB. It may be dependent on the CYLD-TRAF6-IKK pathway. The Notch1 signaling may be considered as a potential therapeutic target against infectious and inflammatory driven diseases.