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 2

Deletion of Emc3 rescued I73T-associated alveolar simplification in the neonatal mice.

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Deletion of Emc3 rescued I73T-associated alveolar simplification in the ...
(A) AT2-specific deletion of Emc3 was induced by injection of tamoxifen to neonatal mice on P6, 7 and 8. Lung tissue was analyzed on P21. Representative H&E-stained lung sections are 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 < 0.05, **P < 0.01, ***P < 0.001 using 1-way ANOVA multiple comparisons test, n = 6 (WT/CreERT), n = 5 (I73T/CreERT), n = 6 (I73T/CreERT;Emc3Δ/Δ). (C) Whole lung homogenates from mice treated as in A were prepared on P21 for Western blotting. Mature SP-B (mSP-B) levels were similar among 3 groups. Reduced levels of mSP-C were detected in I73T/CreERT and I73T/CreERT; Emc3Δ/Δ lungs. (D) Lung sections were prepared from P21 mice treated as in A and stained for mSP-B (green), mSP-C (red) and DAPI (blue). While mSP-B was unchanged, mSP-C was decreased in I73T/CreERT and I73T/CreERT; Emc3Δ/Δ lungs. Scale bars: 20 μm. (E) Western blot of AT2 cell lysates prepared from P21 mice treated as in A. (F) Lung sections were prepared from P21 mice treated as in A and stained for proSP-C (green), ABCA3 (red), LAMP1 (white), and DAPI (blue). Deletion of Emc3 did not change ABCA3 staining or its colocalization with LAMP1-positive vesicles, while it decreased staining of proSP-C(I73T) near the plasma membrane and caused diffuse intracellular staining. Scale bars: 20 μm. (G) Western blot of AT2 cell lysates prepared from P21 mice treated as in A. (H) Quantification of Western blots in G. ABCA3 protein levels were normalized to that of Actin.