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 8

Rescue of I73T-iAT2 cells by EMC3 shRNA and VCP inhibition.

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Rescue of I73T-iAT2 cells by EMC3 shRNA and VCP inhibition. (A) I73T/tdT...
(A) I73T/tdT and WT/tdT iAT2 cells were transfected with control shRNA (shControl) or EMC3 shRNA (shEMC3). Fourteen days after transfection, cells were fixed and paraffin sections were stained for proSP-C (green), EMC4 (red), and DAPI (blue). EMC4 staining was used to identify sites of Emc3 inhibition. Scale bars: 20 μm. (B) Western blots were performed on lysates of iAT2 cells in A. In I73T/tdT iAT2 cells, shEMC3 transfection reduced EMC3 and TOM20 expression. (C) WT/tdT and I73T/tdT iAT2 cells were treated with DMSO (vehicle) or 0.25 μM CB5083 for 14 days and stained for proSP-C (green), proSP-B (red), LAMP1 (white), and DAPI (blue). Scale bars: 20 μm. (D) Western blots on lysates of iAT2 cells treated with DMSO or CB5083 as in C. (E) Quantification of Western blots in D. Total proSP-C (n = 3), EMC3 (n = 5), and TOM20 (n = 4) were normalized to that of actin. Mean ± SEM; *P < 0.05, **P < 0.01, ****P < 0.0001 using 2way ANOVA multiple comparisons test. (F) I73T/tdT iAT2 cells were treated with CB5083 as in C and stained for proSP-C (green), TOM20 (red), and DAPI (blue). Scale bars: 20 μm. (G) Oxygen consumption rate (OCR) of WT/tdT or I73T/tdT iAT2 cells treated with vehicle (DMSO) or CB5083 were tested with the Seahorse XF96 analyzer using 2 × 104 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. Emc3 deletion restored basal respiration, proton leak, and maximal respiration of I73T/tdT iAT2 cells. Mean ± SEM; **P < 0.01, ***P < 0.001, ****P < 0.0001 using 1-way ANOVA multiple comparisons test, n = 5 (WT/tdT+DMSO), n = 6 (WT/tdT+CB5083), n = 5 (I73T/tdT+DMSO), n = 6 (I73T/tdT+CB5083).