The roles of negative regulators of notch signaling, itch and numb, in activation of macrophages

Abstract

Activation of macrophages triggers the release of pro-inflammatory cytokines leading to inflammation. Numb and Itch are negative regulators of Notch signaling. Although the role of Itch has been well characterized, the role of Numb in macrophages is not fully understood. In this study, murine macrophages wherein Numb was silenced, secreted significantly less TNFα, IL-6 and IL-12p70 upon activation via Toll-like receptor 4 (TLR4), despites activation of Notch signaling. Furthermore, downstream signaling pathways of TLR4, including p65 NF-κB and p38 MAPK, were affected by Numb silencing. Numb regulated Il6 and Il12p40 at transcriptional level through p65 NF-κB. In contrast, it regulated Tnfα mRNA stability at post-transcriptional level possibly through p38 MAPK. Inhibition of the Notch signaling pathway did not further reduce level of TNFα, suggesting a Notch-independent role of Numb in regulation of TNFα. In addition, Numb directly interacted with Itch in resting macrophages. Manipulation of Numb expression level influenced the level of Itch and TNFα. Proteomics data revealed a novel role of Numb that modulated inflammation-related signaling cascades downstream of TLR4 activation in macrophages possibly through Akt/p65 NF-κB/p38 MAPK axis. Moreover, Itch silencing reduced production of pro-inflammatory cytokines in macrophages possibly through p38 and JNK MAPKs. This regulation of pro-inflammatory cytokines by Itch was partially dependent on Notch signaling. Taken together, this study reported a novel role of Numb as a positive regulator of inflammatory response in macrophages and an involvement of Notch signaling in regulation of the pro-inflammatory cytokines by Itch.