Congenital Deletion of Nedd4-2 in Lung Epithelial Cells Causes Progressive Alveolitis and Pulmonary Fibrosis in Neonatal Mice.

Dominik H W Leitz,Surafel Mulugeta,Marcus A Mall,Hiroshi Kawabe,Stefan Zimmermann,Alexander H Dalpke,Michael F Beers,Ayça Seyhan Agircan,Julia Duerr

doi:10.3390/ijms22116146

Abstract

Recent studies found that expression of NEDD4-2 is reduced in lung tissue from patients with idiopathic pulmonary fibrosis (IPF) and that the conditional deletion of Nedd4-2 in lung epithelial cells causes IPF-like disease in adult mice via multiple defects, including dysregulation of the epithelial Na+ channel (ENaC), TGFβ signaling and the biosynthesis of surfactant protein-C proprotein (proSP-C). However, knowledge of the impact of congenital deletion of Nedd4-2 on the lung phenotype remains limited. In this study, we therefore determined the effects of congenital deletion of Nedd4-2 in the lung epithelial cells of neonatal doxycycline-induced triple transgenic Nedd4-2fl/fl/CCSP-rtTA2S-M2/LC1 mice, with a focus on clinical phenotype, survival, lung morphology, inflammation markers in BAL, mucin expression, ENaC function and proSP-C trafficking. We found that the congenital deletion of Nedd4-2 caused a rapidly progressive lung disease in neonatal mice that shares key features with interstitial lung diseases in children (chILD), including hypoxemia, growth failure, sterile pneumonitis, fibrotic lung remodeling and high mortality. The congenital deletion of Nedd4-2 in lung epithelial cells caused increased expression of Muc5b and mucus plugging of distal airways, increased ENaC activity and proSP-C mistrafficking. This model of congenital deletion of Nedd4-2 may support studies of the pathogenesis and preclinical development of therapies for chILD.

Highlights

Nedd4-2 is an E3 ubiquitin-protein ligase that participates in the posttranscriptional regulation of several proteins including epithelial Na+ channel (ENaC), Smad2/3 and proSP-C, which play key roles in multiple cellular processes such as epithelial ion and fluid transport, TGFβ signaling and surfactant biogenesis that are essential for epithelial homeostasis and lung health [1,2,3,4,5,6,7,8]
Mice [9], we found that proSP-C mistrafficking alone was insufficient to drive the abnormal lung phenotype found in neonatal mice with the congenital deletion of Nedd4-2
Mice and induced in utero with doxycycline, the genetic deletion of surfactant protein C (Sftpc) in quadruple transgenic mice had no effect on survival (Figure 7d), the number of BAL macrophages (Figure 7e), neutrophils (Figure 7f), eosinophils (Figure 7g) or on structural lung disease compared to triple transgenic congenital Nedd4-2−/− mice

Summary

Introduction

Nedd is an E3 ubiquitin-protein ligase that participates in the posttranscriptional regulation of several proteins including ENaC, Smad2/3 and proSP-C, which play key roles in multiple cellular processes such as epithelial ion and fluid transport, TGFβ signaling and surfactant biogenesis that are essential for epithelial homeostasis and lung health [1,2,3,4,5,6,7,8]. Nedd4-2fl/fl /CCSP-rtTA2S -M2/LC1 mice, hereafter referred to as conditional Nedd4-2−/− mice, causes a chronic progressive, restrictive lung disease that shares key features with IPF in patients including signature lesions such as radiological and histological honeycombing and fibroblast foci [9]. These studies identified the dysregulation of (i) ENaC, leading to airway surface liquid depletion and reduced mucociliary clearance; (ii) proSP-C biogenesis and (iii) TGFβ/Smad signaling, promoting fibrotic remodeling as epithelial defects and potential mechanisms triggering IPF-like disease in adult conditional Nedd4-2−/− mice [9]. The lung phenotype of neonatal Nedd4-2fl/fl /CCSP-rtTA2S -M2/LC-1 mice, facilitating “tight” deletion of Nedd in alveolar type 2 (AT2) cells as well as club cells of the conducting airways under control of the club cells 10 kDa secretory protein (CCSP) [12] promoter, has not been studied

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