NOTCH3 signaling is essential for NF-\u03baB activation in TLR-activated macrophages

Susana López-López,Isabella Screpanti,Eva María Monsalve,José Javier García-Ramírez,Francisco Ruiz-Marcos,Julia González-Gómez,Jorge Laborda,Victoriano Baladrón,María José M Díaz-Guerra,María Pía Felli,Natalia Hernández De León,María Jesús García-León,María José Romero De Ávila,María Luisa Nueda

doi:10.1038/s41598-020-71810-4

Abstract

Macrophage activation by Toll receptors is an essential event in the development of the response against pathogens. NOTCH signaling pathway is involved in the control of macrophage activation and the inflammatory processes. In this work, we have characterized NOTCH signaling in macrophages activated by Toll-like receptor (TLR) triggering and determined that DLL1 and DLL4 are the main ligands responsible for NOTCH signaling. We have identified ADAM10 as the main protease implicated in NOTCH processing and activation. We have also observed that furin, which processes NOTCH receptors, is induced by TLR signaling in a NOTCH-dependent manner. NOTCH3 is the only NOTCH receptor expressed in resting macrophages. Its expression increased rapidly in the first hours after TLR4 activation, followed by a gradual decrease, which was coincident with an elevation of the expression of the other NOTCH receptors. All NOTCH1, 2 and 3 contribute to the increased NOTCH signaling detected in activated macrophages. We also observed a crosstalk between NOTCH3 and NOTCH1 during macrophage activation. Finally, our results highlight the relevance of NOTCH3 in the activation of NF-κB, increasing p65 phosphorylation by p38 MAP kinase. Our data identify, for the first time, NOTCH3 as a relevant player in the control of inflammation.

Highlights

Macrophage activation by Toll receptors is an essential event in the development of the response against pathogens
OP9 stromal cells expressing the human NOTCH ligands DLL1, DLL4 or JAGGED were co-cultured with control or activated Raw 264.7 macrophages to analyze their effects on the activation of a NOTCH-dependent luciferase reporter gene
Our results suggest that NOTCH3 is the most relevant NOTCH receptor soon after Toll-like receptor (TLR) activation, but other NOTCH receptors, mostly NOTCH1 and NOTCH2, appear to be more relevant to control the expression of NOTCH target genes at later times

Summary

Introduction

Macrophage activation by Toll receptors is an essential event in the development of the response against pathogens. The canonical NOTCH activation pathway depends on a proteolytic cascade induced by ligand binding that triggers a first cleavage just outside the membrane mediated by ADAM metalloproteases, followed by a second cleavage, just inside the inner leaflet of the cell membrane, by a presenilin-γ-secretase complex. These proteolytic cleavages release the intracellular domain of the NOTCH receptor (NICD), which translocates into the nucleus and promotes the displacement of a repressor complex, favoring the activation of its target g enes[10]

Methods

Results

Conclusion