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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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 5

Loss of Emc3 rescues I73T-associated alveolar simplification and mitochondrial dysfunction in adult mice.

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Loss of Emc3 rescues I73T-associated alveolar simplification and mitocho...
(A) AT2-specific Emc3 deletion was induced by administration of tamoxifen chow to control (WT/CreERT), SftpcI73T heterozygous (I73T/CreERT), and I73T/CreERT;Emc3Δ/Δ adult mice (6–8 weeks of age). Lung tissues were obtained 14 days later. Representative H&E-stained lung tissue is shown. Scale bars: 200 μm. (B) ImageJ was used to quantify the H&E staining results in A. Both defects of volume density of alveolar septa (VVsep) and mean linear intercept of the airspaces (Lm) in I73T/CreERT lungs were rescued by deletion of Emc3. Mean ± SEM; **P < 0.01, ***P < 0.001 using 1-way ANOVA multiple comparisons test, n = 6. (C) Lung sections from adult mice treated as in A were stained for proSP-C (green), LAMP1 (red) and DAPI (blue). Emc3 deletion restored the intracellular distribution of SP-C(I73T). Scale bar: 20 μm. (D) Oxygen consumption rate (OCR) and extracellular acidification rates (ECAR) of AT2 cells isolated from adult mice treated as in A were tested with the Seahorse XF96 analyzer using 1.2 × 105 cells for each sample. OCR was measured under basal conditions, followed by addition of oligomycin (Oligo; 2 μM), FCCP (2 μM), and antimycin A (Ant A; 2.5 μM), as indicated. ECAR was measured under basal conditions followed by addition of glucose (10 nM), oligomycin (Oligo; 2 μM), and 2-DG (50 mM), as indicated. Emc3 deletion restored maximal respiration and glycolytic capacity of AT2 cells expressing SP-C(I73T). Mean ± SEM; **P < 0.01, ****P < 0.0001 using 2-way ANOVA multiple comparisons test, n = 6 (WT/CreERT), n = 3 (I73T/CreERT), n = 5 (I73T/CreERT;Emc3Δ/Δ).