An SFTPC BRICHOS mutant links epithelial ER stress and spontaneous lung fibrosis
Jeremy Katzen, … , Robin R. Deterding, Michael F. Beers
Jeremy Katzen, … , Robin R. Deterding, Michael F. Beers
Published February 5, 2019
Citation Information: JCI Insight. 2019;4(6):e126125. https://doi.org/10.1172/jci.insight.126125.
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Research Article Pulmonology

An SFTPC BRICHOS mutant links epithelial ER stress and spontaneous lung fibrosis

Abstract

Alveolar type 2 (AT2) cell endoplasmic reticulum (ER) stress is a prominent feature in adult and pediatric interstitial lung disease (ILD and ChILD), but in vivo models linking AT2 cell ER stress to ILD have been elusive. Based on a clinical ChILD case, we identified a critical cysteine residue in the surfactant protein C gene (SFTPC) BRICHOS domain whose mutation induced ER stress in vitro. To model this in vivo, we generated a knockin mouse model expressing a cysteine-to-glycine substitution at codon 121 (C121G) in the Sftpc gene. SftpcC121G expression during fetal development resulted in a toxic gain-of-function causing fatal postnatal respiratory failure from disrupted lung morphogenesis. Induced SftpcC121G expression in adult mice resulted in an ER-retained pro-protein causing AT2 cell ER stress. SftpcC121G AT2 cells were a source of cytokines expressed in concert with development of polycellular alveolitis. These cytokines were subsequently found in a high-dimensional proteomic screen of bronchoalveolar lavage fluid from ChILD patients with the same class of SFTPC mutations. Following alveolitis resolution, SftpcC121G mice developed spontaneous pulmonary fibrosis and restrictive lung impairment. This model provides proof of concept linking AT2 cell ER stress to fibrotic lung disease coupled with translationally relevant biomarkers.

Authors

Jeremy Katzen, Brandie D. Wagner, Alessandro Venosa, Meghan Kopp, Yaniv Tomer, Scott J. Russo, Alvis C. Headen, Maria C. Basil, James M. Stark, Surafel Mulugeta, Robin R. Deterding, Michael F. Beers

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Figure 1

In vitro modeling of a clinical SFTPCC121Y mutation identifies mistrafficked pro-protein and epithelial ER stress with mutagenesis of codon 121 in SFTPC.

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In vitro modeling of a clinical SFTPCC121Y mutation identifies mistraffi...
(A) Chest imaging of a 3-month-old patient with heterozygous SFTPCC121Y mutation shows bilateral diffuse hazy opacity on chest radiograph (left) and diffuse ground glass opacities on chest CT (right). (B) H&E staining of patient lung biopsy demonstrates diffuse abnormality in lobular architecture, with mild enlargement of airspaces and mild to moderate interstitial widening, airspaces filled by foamy macrophages, scattered neutrophils, and eosinophilic material suggestive of proteinosis (original magnification, ×4 left and ×20 right). (C) HEK293 cells were transiently transfected with either EGFP/SP-CWT or EGFP/SP-CC121 mutants as indicated, and cell lysates harvested at 48 hours were subjected to Western blotting for EGFP. An SP-C primary translation product (black arrow) was detected in all transfections; however, no processing intermediates (black bracket) were observed with either EGFP/SP-CC121 mutation. Representative of 3 independent experiments. (D) HEK293 cells transiently cotransfected with either EGFP/SP-CWT or EGFP/SP-CC121G and DsRed/ABCA3 and subjected to immunfluorescence microscopy, demonstrating colocalization of EGFP/SP-CWT with DsRed/ABCA3, but a reticular expression pattern of EGFP/SP-CC121G without DsRed/ABCA3 colocalization (original magnification, ×60). (E) Cell lysates of HEK293 cells 48 hours after transfection with either EGFP/SP-CWT or EGFP/SP-CC121 mutants as indicated were subjected to Western analysis for BiP (top). Representative Western blot of n = 3. Quantitative densitometry (bottom) normalized to β-actin showing increases in BiP with the cysteine mutants compared to EGFP/SP-CWT. n = 3. *P < 0.05 vs. EGFP/SP-CWT by 1-way ANOVA with Dunnett’s multiple comparison test.