EMC3 regulates trafficking and pulmonary toxicity of the SFTPCI73T mutation associated with interstitial lung disease
Xiaofang Tang, … , Xinhua Lin, Jeffrey A. Whitsett
Xiaofang Tang, … , Xinhua Lin, Jeffrey A. Whitsett
Published October 15, 2024
Citation Information: J Clin Invest. 2024;134(23):e173861. https://doi.org/10.1172/JCI173861.
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Research Article Cell biology Pulmonology

EMC3 regulates trafficking and pulmonary toxicity of the SFTPCI73T mutation associated with interstitial lung disease

Abstract

The most common mutation in surfactant protein C gene (SFTPC), SFTPCI73T, causes interstitial lung disease with few therapeutic options. We previously demonstrated that EMC3, an important component of the multiprotein endoplasmic reticulum membrane complex (EMC), is required for surfactant homeostasis in alveolar type 2 epithelial (AT2) cells at birth. In the present study, we investigated the role of EMC3 in the control of SFTPCI73T metabolism and its associated alveolar dysfunction. Using a knock-in mouse model phenocopying the I73T mutation, we demonstrated that conditional deletion of Emc3 in AT2 cells rescued alveolar remodeling/simplification defects in neonatal and adult mice. Proteomic analysis revealed that Emc3 depletion reversed the disruption of vesicle trafficking pathways and rescued the mitochondrial dysfunction associated with I73T mutation. Affinity purification-mass spectrometry analysis identified potential EMC3 interacting proteins in lung AT2 cells, including valosin containing protein (VCP) and its interactors. Treatment of SftpcI73T knock-in mice and SFTPCI73T-expressing iAT2 cells derived from SFTPCI73T patient-specific iPSCs with the VCP inhibitor CB5083 restored alveolar structure and SFTPCI73T trafficking, respectively. Taken together, the present work identifies the EMC complex and VCP in the metabolism of the disease-associated SFTPCI73T mutant, providing therapeutical targets for SFTPCI73T-associated interstitial lung disease.

Authors

Xiaofang Tang, Wei Wei, Yuqing Sun, Timothy E. Weaver, Ernesto S. Nakayasu, Geremy Clair, John M. Snowball, Cheng-Lun Na, Karen S. Apsley, Emily P. Martin, Darrell N. Kotton, Konstantinos-Dionysios Alysandratos, Jiuzhou Huo, Jeffery D. Molkentin, William A. Gower, Xinhua Lin, Jeffrey A. Whitsett

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Figure 1

Generation of SftpcI73T knock-in mouse model.

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Generation of SftpcI73T knock-in mouse model. (A) Design of the SftpcI73...
(A) Design of the SftpcI73T knock-in allele. Founder mice carrying the knock-in allele were crossed to EIIA-Cre deleter mice to excise the neomycin resistance cassette. (B) Representative H&E-stained sections from 6–8 week-old WT/WT and I73T/I73T mice. Scale bars: 200 μm. (C) Quantitative morphometry of B using ImageJ expressed as the volume density of alveolar septa (VVsep), mean linear intercept of the airspaces (Lm), the mean transsectional wall length (Lmw), and the surface area density of the air spaces (SVair). I73T/I73T lungs showed significantly reduced volume density of alveolar septa and increased mean linear intercept of the airspaces. Mean ± SEM; **P < 0.01 using student t test, n = 5. (D) Reduced Sftpc mRNA in isolated AT2 cells from 8-week-old I73T/I73T mice compared with WT/WT mice. Levels of the Sftpc transcript were normalized to that of 18S by qPCR. Mean ± SEM; **P < 0.01 using unpaired, 2-tailed Student’s t test, n = 4/group. (E) 8-week-old I73T/I73T and WT/WT lung sections were stained for SP-C proprotein (proSP-C, green) and DAPI (blue). WT proSP-C is detected in a punctate pattern while proSP-C(I73T) is detected as a dense staining stripe near the cell surface. Red signal is autofluorescence of erythrocytes. Scale bars: 20 μm. (F and G) Western blot of lysates of isolated AT2 cells (F) or whole lung homogenates (G) from 6–8 week-old WT/WT and I73T/I73T mice. Processing of SP-C(I73T) proprotein into mature peptide (mSP-C) is decreased and processing intermediates are increased. (H) Western blot of bronchoalveolar lavage fluid (BALF) detected secreted proteins from 6–8 week-old WT/WT and I73T/I73T mice. Decreased mature SP-C and increased proSP-C(I73T) processing intermediates were present in BALF of I73T/I73T mice. Lysozyme was used as a loading control.