The infantile myofibromatosis NOTCH3 L1519P mutation leads to hyperactivated ligand-independent Notch signaling and increased PDGFRB expression.

Dan Wu,Thibaud Lebouvier,Shaobo Jin,Daniel V Oliveira,Christer Betsholtz,Michael Vanlandewijck,Francesca Del Gaudio,Sailan Wang,Jian Zhao,Helena Karlström,Urban Lendahl

doi:10.1242/dmm.046300

Abstract

ABSTRACTInfantile myofibromatosis (IMF) is a benign tumor form characterized by the development of nonmetastatic tumors in skin, bone, muscle and sometimes viscera. Autosomal-dominant forms of IMF are caused by mutations in the PDGFRB gene, but a family carrying a L1519P mutation in the NOTCH3 gene has also recently been identified. In this study, we address the molecular consequences of the NOTCH3L1519P mutation and the relationship between Notch and PDGFRB signaling in IMF. The NOTCH3L1519P receptor generates enhanced downstream signaling in a ligand-independent manner. Despite the enhanced signaling, the NOTCH3L1519P receptor is absent from the cell surface and instead accumulates in the endoplasmic reticulum. Furthermore, the localization of the NOTCH3L1519P receptor in the bipartite, heterodimeric state is altered, combined with avid secretion of the mutated extracellular domain from the cell. Chloroquine treatment strongly reduces the amount of secreted NOTCH3L1519P extracellular domain and decreases signaling. Finally, NOTCH3L1519P upregulates PDGFRB expression in fibroblasts, supporting a functional link between Notch and PDGF dysregulation in IMF. Collectively, our data define a NOTCH3–PDGFRB axis in IMF, in which an IMF-mutated NOTCH3 receptor elevates PDGFRB expression. The functional characterization of a ligand-independent gain-of-function NOTCH3 mutation is important for Notch therapy considerations for IMF, including strategies aimed at altering lysosome function.

Highlights

Infantile myofibromatosis (IMF, MIM 228550) patients suffer from nonmetastatic tumors that develop in skin, bone, muscle and viscera (Chung and Enzinger, 1981)
NOTCH3L1519P exhibits an imbalance between the full-length and transmembrane intracellular domain (TMIC) forms To explore the molecular consequences of the NOTCH3L1519P mutation, we engineered cell lines in which the wild-type or L1519P forms of NOTCH3 were introduced into human embryonic kidney (HEK) 293T cells from which we had first ablated the NOTCH1, NOTCH2 and NOTCH3 genes by CRISPR/Cas9 (HEK 293T ΔN1-3 cell line), to rid the cells of endogenous Notch signaling and eliminate the potential risk of antibody cross-reactivity in the cellular localization assays described below (Fig. S1A)
The wild-type or L1519P forms of NOTCH3 were stably integrated into the AAVS1 locus in the HEK 293T ΔN1-3 cell line, which is tetracycline regulated, allowing the levels of NOTCH3 receptor expression to be regulated by doxycycline stimulation (Fig. S1B)

Summary

Introduction

Infantile myofibromatosis (IMF, MIM 228550) patients suffer from nonmetastatic tumors that develop in skin, bone, muscle and viscera (Chung and Enzinger, 1981). For the autosomaldominant forms, two mutations in the PDGFRB gene (MIM 173410) have been identified (Cheung et al, 2013; Martignetti et al, 2013). Elevated expression of PDGF ligands and receptors has been observed in pediatric fibromatoses and myofibromatosis (Gibson et al, 2007), further supporting the notion that elevated PDGF signaling can cause IMF. PDGF signaling is paramount for the recruitment of pericytes during blood vessel formation, and complete knockout of either the Pdgfb or Pdgfrb gene in mice leads to perinatal death (Leveen et al, 1994; Soriano, 1994)

Methods

Results

Conclusion