Involvement of Notch signaling in interleukin-4-stimulated human macrophages

Abstract

Macrophages respond to various stimuli, resulting in distinct effector phenotypes that range from LPS-stimulated proinflammatory to IL-4-activated pro-healing phenotype. The regulatory mechanism of this polarization is not fully understood. Notch signaling pathway is a conserved signaling pathway involved in polarization of macrophages, especially proinflammatory macrophages. However, the role of Notch signaling in IL-4 stimulated macrophages (M(IL-4)) is not well defined. PPARgamma is a signature nuclear receptor in M(IL-4) that function mainly in regulating lipid metabolism. There are many reports that revealed the interaction of Notch signaling and PPARgamma in various cells but not in macrophages. In this study, the interaction between Notch signaling and PPARgamma in M(IL-4) using THP-1 cell line and primary human monocytes derived macrophages was investigated. M(IL-4) activated Notch signaling that required the activity of g-secretase to cleave Notch1 receptor. Notch1 intracellular domain (NIC1) overexpressing THP-1 increased PPARgamma expression in the presence or absence of IL-4. Furthermore, g-secretase inhibitor pretreated M(IL-4) decreased PPARgamma level. In contrast, overexpression of dominant negative mastermind-like (DNMAML), a dominant negative for Notch signaling, in M(IL-4) had no effect on PPARgamma level. NIC1 overexpression increased PPARgamma stability by delayed proteasome degradation but not the mRNA transcription or stability. RNAseq was performed to obtain global insight of Notch signaling in M(IL-4). NIC1 overexpressing M(IL-4) showed enrichment of proinflammatory gene sets consistent with previous reports. More importantly, some gene sets of pro-healing macrophages were enriched as well. Network analysis revealed the links between Notch and PPARgamma through NEDD4L, an E3 ubiquitin ligase and SGK1, which has 45-55% of catalytic domain similar to AKT. Deletion of NEDD4L in the presence of NIC1 overexpression in M(IL-4) reduced PPARG mRNA expression. This reduction of PPARG mRNA was accompanied by decreasing AKT phosphorylation. These results indicated that Notch signaling required NEDD4L for optimal expression of PPARG mRNA. Activation of Notch signaling increased lipid metabolism in M(IL-4) by increasing lipid accumulation via CD36 uptake mechanism. Collectively, this study provides evidences linking Notch signaling and PPARgamma via protein stabilization and NEDD4L mediated PPARG regulation and its effect on lipid uptake. The results obtained in this study indicated that Notch signaling operates in both proinflammatory and pro-healing macrophages.