Research ArticleCell biologyPulmonology Open Access | 10.1172/JCI173861

Xiaofang Tang,1,2 Wei Wei,1 Yuqing Sun,1 Timothy E. Weaver,2 Ernesto S. Nakayasu,3 Geremy Clair,3 John M. Snowball,2 Cheng-Lun Na,2 Karen S. Apsley,2 Emily P. Martin,2 Darrell N. Kotton,4 Konstantinos-Dionysios Alysandratos,4 Jiuzhou Huo,5 Jeffery D. Molkentin,5 William A. Gower,6 Xinhua Lin,1 and Jeffrey A. Whitsett2

1State Key Laboratory of Genetic Engineering, Greater Bay Area Institute of Precision Medicine (Guangzhou), School of Life Sciences, Shanghai Key Laboratory of Lung Inflammation and Injury, Zhongshan Hospital, Fudan University, Shanghai, China .

2Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.

4Department of Medicine, The Pulmonary Center, Center for Regenerative Medicine, Boston University School of Medicine, Boston, Massachusetts, USA.

5Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

6Division of Pediatric Pulmonology and Program for Rare and Interstitial Lung Disease, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

Address correspondence to: Jeffrey A. Whitsett, Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229, USA. Phone: 513.803.2790; Email: jeffrey.whitsett@cchmc.org. Or to: Xiaofang Tang, Greater Bay Area Institute of Precision Medicine (Guangzhou), Fudan University, 2nd Nanjiang Rd, Nansha District, Guangzhou, 511458, China. Phone: 86.020.66333080; Email: tangxiaofang@ipm-gba.org.cn.

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1State Key Laboratory of Genetic Engineering, Greater Bay Area Institute of Precision Medicine (Guangzhou), School of Life Sciences, Shanghai Key Laboratory of Lung Inflammation and Injury, Zhongshan Hospital, Fudan University, Shanghai, China .

2Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.

4Department of Medicine, The Pulmonary Center, Center for Regenerative Medicine, Boston University School of Medicine, Boston, Massachusetts, USA.

5Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

6Division of Pediatric Pulmonology and Program for Rare and Interstitial Lung Disease, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

Address correspondence to: Jeffrey A. Whitsett, Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229, USA. Phone: 513.803.2790; Email: jeffrey.whitsett@cchmc.org. Or to: Xiaofang Tang, Greater Bay Area Institute of Precision Medicine (Guangzhou), Fudan University, 2nd Nanjiang Rd, Nansha District, Guangzhou, 511458, China. Phone: 86.020.66333080; Email: tangxiaofang@ipm-gba.org.cn.

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1State Key Laboratory of Genetic Engineering, Greater Bay Area Institute of Precision Medicine (Guangzhou), School of Life Sciences, Shanghai Key Laboratory of Lung Inflammation and Injury, Zhongshan Hospital, Fudan University, Shanghai, China .

2Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.

4Department of Medicine, The Pulmonary Center, Center for Regenerative Medicine, Boston University School of Medicine, Boston, Massachusetts, USA.

5Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

6Division of Pediatric Pulmonology and Program for Rare and Interstitial Lung Disease, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

Address correspondence to: Jeffrey A. Whitsett, Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229, USA. Phone: 513.803.2790; Email: jeffrey.whitsett@cchmc.org. Or to: Xiaofang Tang, Greater Bay Area Institute of Precision Medicine (Guangzhou), Fudan University, 2nd Nanjiang Rd, Nansha District, Guangzhou, 511458, China. Phone: 86.020.66333080; Email: tangxiaofang@ipm-gba.org.cn.

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1State Key Laboratory of Genetic Engineering, Greater Bay Area Institute of Precision Medicine (Guangzhou), School of Life Sciences, Shanghai Key Laboratory of Lung Inflammation and Injury, Zhongshan Hospital, Fudan University, Shanghai, China .

2Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.

4Department of Medicine, The Pulmonary Center, Center for Regenerative Medicine, Boston University School of Medicine, Boston, Massachusetts, USA.

5Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

6Division of Pediatric Pulmonology and Program for Rare and Interstitial Lung Disease, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

Address correspondence to: Jeffrey A. Whitsett, Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229, USA. Phone: 513.803.2790; Email: jeffrey.whitsett@cchmc.org. Or to: Xiaofang Tang, Greater Bay Area Institute of Precision Medicine (Guangzhou), Fudan University, 2nd Nanjiang Rd, Nansha District, Guangzhou, 511458, China. Phone: 86.020.66333080; Email: tangxiaofang@ipm-gba.org.cn.

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1State Key Laboratory of Genetic Engineering, Greater Bay Area Institute of Precision Medicine (Guangzhou), School of Life Sciences, Shanghai Key Laboratory of Lung Inflammation and Injury, Zhongshan Hospital, Fudan University, Shanghai, China .

2Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.

4Department of Medicine, The Pulmonary Center, Center for Regenerative Medicine, Boston University School of Medicine, Boston, Massachusetts, USA.

5Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

6Division of Pediatric Pulmonology and Program for Rare and Interstitial Lung Disease, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

Address correspondence to: Jeffrey A. Whitsett, Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229, USA. Phone: 513.803.2790; Email: jeffrey.whitsett@cchmc.org. Or to: Xiaofang Tang, Greater Bay Area Institute of Precision Medicine (Guangzhou), Fudan University, 2nd Nanjiang Rd, Nansha District, Guangzhou, 511458, China. Phone: 86.020.66333080; Email: tangxiaofang@ipm-gba.org.cn.

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1State Key Laboratory of Genetic Engineering, Greater Bay Area Institute of Precision Medicine (Guangzhou), School of Life Sciences, Shanghai Key Laboratory of Lung Inflammation and Injury, Zhongshan Hospital, Fudan University, Shanghai, China .

2Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.

4Department of Medicine, The Pulmonary Center, Center for Regenerative Medicine, Boston University School of Medicine, Boston, Massachusetts, USA.

5Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

6Division of Pediatric Pulmonology and Program for Rare and Interstitial Lung Disease, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

Address correspondence to: Jeffrey A. Whitsett, Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229, USA. Phone: 513.803.2790; Email: jeffrey.whitsett@cchmc.org. Or to: Xiaofang Tang, Greater Bay Area Institute of Precision Medicine (Guangzhou), Fudan University, 2nd Nanjiang Rd, Nansha District, Guangzhou, 511458, China. Phone: 86.020.66333080; Email: tangxiaofang@ipm-gba.org.cn.

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1State Key Laboratory of Genetic Engineering, Greater Bay Area Institute of Precision Medicine (Guangzhou), School of Life Sciences, Shanghai Key Laboratory of Lung Inflammation and Injury, Zhongshan Hospital, Fudan University, Shanghai, China .

2Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.

4Department of Medicine, The Pulmonary Center, Center for Regenerative Medicine, Boston University School of Medicine, Boston, Massachusetts, USA.

5Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

6Division of Pediatric Pulmonology and Program for Rare and Interstitial Lung Disease, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

Address correspondence to: Jeffrey A. Whitsett, Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229, USA. Phone: 513.803.2790; Email: jeffrey.whitsett@cchmc.org. Or to: Xiaofang Tang, Greater Bay Area Institute of Precision Medicine (Guangzhou), Fudan University, 2nd Nanjiang Rd, Nansha District, Guangzhou, 511458, China. Phone: 86.020.66333080; Email: tangxiaofang@ipm-gba.org.cn.

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1State Key Laboratory of Genetic Engineering, Greater Bay Area Institute of Precision Medicine (Guangzhou), School of Life Sciences, Shanghai Key Laboratory of Lung Inflammation and Injury, Zhongshan Hospital, Fudan University, Shanghai, China .

2Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.

4Department of Medicine, The Pulmonary Center, Center for Regenerative Medicine, Boston University School of Medicine, Boston, Massachusetts, USA.

5Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

6Division of Pediatric Pulmonology and Program for Rare and Interstitial Lung Disease, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

Address correspondence to: Jeffrey A. Whitsett, Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229, USA. Phone: 513.803.2790; Email: jeffrey.whitsett@cchmc.org. Or to: Xiaofang Tang, Greater Bay Area Institute of Precision Medicine (Guangzhou), Fudan University, 2nd Nanjiang Rd, Nansha District, Guangzhou, 511458, China. Phone: 86.020.66333080; Email: tangxiaofang@ipm-gba.org.cn.

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1State Key Laboratory of Genetic Engineering, Greater Bay Area Institute of Precision Medicine (Guangzhou), School of Life Sciences, Shanghai Key Laboratory of Lung Inflammation and Injury, Zhongshan Hospital, Fudan University, Shanghai, China .

2Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.

4Department of Medicine, The Pulmonary Center, Center for Regenerative Medicine, Boston University School of Medicine, Boston, Massachusetts, USA.

5Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

6Division of Pediatric Pulmonology and Program for Rare and Interstitial Lung Disease, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

Address correspondence to: Jeffrey A. Whitsett, Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229, USA. Phone: 513.803.2790; Email: jeffrey.whitsett@cchmc.org. Or to: Xiaofang Tang, Greater Bay Area Institute of Precision Medicine (Guangzhou), Fudan University, 2nd Nanjiang Rd, Nansha District, Guangzhou, 511458, China. Phone: 86.020.66333080; Email: tangxiaofang@ipm-gba.org.cn.

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1State Key Laboratory of Genetic Engineering, Greater Bay Area Institute of Precision Medicine (Guangzhou), School of Life Sciences, Shanghai Key Laboratory of Lung Inflammation and Injury, Zhongshan Hospital, Fudan University, Shanghai, China .

2Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.

4Department of Medicine, The Pulmonary Center, Center for Regenerative Medicine, Boston University School of Medicine, Boston, Massachusetts, USA.

5Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

6Division of Pediatric Pulmonology and Program for Rare and Interstitial Lung Disease, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

Address correspondence to: Jeffrey A. Whitsett, Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229, USA. Phone: 513.803.2790; Email: jeffrey.whitsett@cchmc.org. Or to: Xiaofang Tang, Greater Bay Area Institute of Precision Medicine (Guangzhou), Fudan University, 2nd Nanjiang Rd, Nansha District, Guangzhou, 511458, China. Phone: 86.020.66333080; Email: tangxiaofang@ipm-gba.org.cn.

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1State Key Laboratory of Genetic Engineering, Greater Bay Area Institute of Precision Medicine (Guangzhou), School of Life Sciences, Shanghai Key Laboratory of Lung Inflammation and Injury, Zhongshan Hospital, Fudan University, Shanghai, China .

2Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.

4Department of Medicine, The Pulmonary Center, Center for Regenerative Medicine, Boston University School of Medicine, Boston, Massachusetts, USA.

5Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

6Division of Pediatric Pulmonology and Program for Rare and Interstitial Lung Disease, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

Address correspondence to: Jeffrey A. Whitsett, Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229, USA. Phone: 513.803.2790; Email: jeffrey.whitsett@cchmc.org. Or to: Xiaofang Tang, Greater Bay Area Institute of Precision Medicine (Guangzhou), Fudan University, 2nd Nanjiang Rd, Nansha District, Guangzhou, 511458, China. Phone: 86.020.66333080; Email: tangxiaofang@ipm-gba.org.cn.

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1State Key Laboratory of Genetic Engineering, Greater Bay Area Institute of Precision Medicine (Guangzhou), School of Life Sciences, Shanghai Key Laboratory of Lung Inflammation and Injury, Zhongshan Hospital, Fudan University, Shanghai, China .

2Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.

4Department of Medicine, The Pulmonary Center, Center for Regenerative Medicine, Boston University School of Medicine, Boston, Massachusetts, USA.

5Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

6Division of Pediatric Pulmonology and Program for Rare and Interstitial Lung Disease, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

Address correspondence to: Jeffrey A. Whitsett, Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229, USA. Phone: 513.803.2790; Email: jeffrey.whitsett@cchmc.org. Or to: Xiaofang Tang, Greater Bay Area Institute of Precision Medicine (Guangzhou), Fudan University, 2nd Nanjiang Rd, Nansha District, Guangzhou, 511458, China. Phone: 86.020.66333080; Email: tangxiaofang@ipm-gba.org.cn.

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1State Key Laboratory of Genetic Engineering, Greater Bay Area Institute of Precision Medicine (Guangzhou), School of Life Sciences, Shanghai Key Laboratory of Lung Inflammation and Injury, Zhongshan Hospital, Fudan University, Shanghai, China .

2Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.

4Department of Medicine, The Pulmonary Center, Center for Regenerative Medicine, Boston University School of Medicine, Boston, Massachusetts, USA.

5Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

6Division of Pediatric Pulmonology and Program for Rare and Interstitial Lung Disease, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

Address correspondence to: Jeffrey A. Whitsett, Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229, USA. Phone: 513.803.2790; Email: jeffrey.whitsett@cchmc.org. Or to: Xiaofang Tang, Greater Bay Area Institute of Precision Medicine (Guangzhou), Fudan University, 2nd Nanjiang Rd, Nansha District, Guangzhou, 511458, China. Phone: 86.020.66333080; Email: tangxiaofang@ipm-gba.org.cn.

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1State Key Laboratory of Genetic Engineering, Greater Bay Area Institute of Precision Medicine (Guangzhou), School of Life Sciences, Shanghai Key Laboratory of Lung Inflammation and Injury, Zhongshan Hospital, Fudan University, Shanghai, China .

2Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.

4Department of Medicine, The Pulmonary Center, Center for Regenerative Medicine, Boston University School of Medicine, Boston, Massachusetts, USA.

5Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

6Division of Pediatric Pulmonology and Program for Rare and Interstitial Lung Disease, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

Address correspondence to: Jeffrey A. Whitsett, Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229, USA. Phone: 513.803.2790; Email: jeffrey.whitsett@cchmc.org. Or to: Xiaofang Tang, Greater Bay Area Institute of Precision Medicine (Guangzhou), Fudan University, 2nd Nanjiang Rd, Nansha District, Guangzhou, 511458, China. Phone: 86.020.66333080; Email: tangxiaofang@ipm-gba.org.cn.

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1State Key Laboratory of Genetic Engineering, Greater Bay Area Institute of Precision Medicine (Guangzhou), School of Life Sciences, Shanghai Key Laboratory of Lung Inflammation and Injury, Zhongshan Hospital, Fudan University, Shanghai, China .

2Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.

4Department of Medicine, The Pulmonary Center, Center for Regenerative Medicine, Boston University School of Medicine, Boston, Massachusetts, USA.

5Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

6Division of Pediatric Pulmonology and Program for Rare and Interstitial Lung Disease, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

Address correspondence to: Jeffrey A. Whitsett, Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229, USA. Phone: 513.803.2790; Email: jeffrey.whitsett@cchmc.org. Or to: Xiaofang Tang, Greater Bay Area Institute of Precision Medicine (Guangzhou), Fudan University, 2nd Nanjiang Rd, Nansha District, Guangzhou, 511458, China. Phone: 86.020.66333080; Email: tangxiaofang@ipm-gba.org.cn.

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1State Key Laboratory of Genetic Engineering, Greater Bay Area Institute of Precision Medicine (Guangzhou), School of Life Sciences, Shanghai Key Laboratory of Lung Inflammation and Injury, Zhongshan Hospital, Fudan University, Shanghai, China .

2Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.

4Department of Medicine, The Pulmonary Center, Center for Regenerative Medicine, Boston University School of Medicine, Boston, Massachusetts, USA.

5Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

6Division of Pediatric Pulmonology and Program for Rare and Interstitial Lung Disease, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

Address correspondence to: Jeffrey A. Whitsett, Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229, USA. Phone: 513.803.2790; Email: jeffrey.whitsett@cchmc.org. Or to: Xiaofang Tang, Greater Bay Area Institute of Precision Medicine (Guangzhou), Fudan University, 2nd Nanjiang Rd, Nansha District, Guangzhou, 511458, China. Phone: 86.020.66333080; Email: tangxiaofang@ipm-gba.org.cn.

Find articles by Lin, X. in: JCI | PubMed | Google Scholar

1State Key Laboratory of Genetic Engineering, Greater Bay Area Institute of Precision Medicine (Guangzhou), School of Life Sciences, Shanghai Key Laboratory of Lung Inflammation and Injury, Zhongshan Hospital, Fudan University, Shanghai, China .

2Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.

4Department of Medicine, The Pulmonary Center, Center for Regenerative Medicine, Boston University School of Medicine, Boston, Massachusetts, USA.

5Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.

6Division of Pediatric Pulmonology and Program for Rare and Interstitial Lung Disease, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

Address correspondence to: Jeffrey A. Whitsett, Perinatal Institute, Divisions of Neonatology, Perinatal and Pulmonary Biology, Cincinnati, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229, USA. Phone: 513.803.2790; Email: jeffrey.whitsett@cchmc.org. Or to: Xiaofang Tang, Greater Bay Area Institute of Precision Medicine (Guangzhou), Fudan University, 2nd Nanjiang Rd, Nansha District, Guangzhou, 511458, China. Phone: 86.020.66333080; Email: tangxiaofang@ipm-gba.org.cn.

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Published October 15, 2024 - More info

Published in Volume 134, Issue 23 on December 2, 2024
J Clin Invest. 2024;134(23):e173861. https://doi.org/10.1172/JCI173861.
© 2024 Tang et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published October 15, 2024 - Version history
Received: July 11, 2023; Accepted: October 8, 2024 View PDF 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.

Introduction

Pulmonary surfactant lipids and proteins, synthesized and secreted by alveolar type 2 epithelial (AT2) cells, are required for the adaptation to air breathing during evolution (1). The gene encoding surfactant protein C, Sftpc, emerged in lungfish as a gene critical for the water-to-land transition (2). Surfactant proteins B and C (SP-B and SP-C) are synthesized as larger proproteins that are proteolytically processed during their transit from the endoplasmic reticulum (ER) to specialized storage organelles termed lamellar bodies (LBs). The fully processed mature SP-B and SP-C interact with surfactant lipids to form a lipid protein complex required for surfactant function and homeostasis (3).

While expression of SP-B is essential to perinatal respiration and postnatal survival (4), SP-C is dispensable for postnatal lung function but is required for homeostatic responses to lung injury (5). Monoallelic variants in the SFTPC gene are associated with diffuse parenchymal lung disease in children and adults (6, 7). The most common disease-associated SFTPC mutation, a missense isoleucine-to-threonine substitution at position 73 (I73T), causes autosomal dominantly inherited interstitial lung disease (ILD) (8, 9) with phenotypic variability ranging from mild respiratory symptoms to respiratory failure in infancy and childhood to idiopathic pulmonary fibrosis-like (IPF-like) disease in adults (10, 11).

Findings from human and mouse studies demonstrated abnormal trafficking and processing of SP-C(I73T) proprotein, causing AT2 cell toxicity, inhibition of macroautophagy, and impairment of cellular proteostasis and mitophagy (12–15). Current mouse and cell culture models expressing SP-C(I73T) model the effects of acute and high levels of expression, perhaps consistent with pathology seen in clinical exacerbations. To assess the natural history of SFTPCI73T-related disease, we inserted the I73T mutation into the mouse Sftpc locus. Subsequent morphological, biochemical, and proteomic analysis indicated that the knock-in mouse model phenocopied the mistrafficking and misprocessing of SP-C(I73T) and multiple aspects of AT2 cell dysfunction.

EMC3 is a key subunit of the ER membrane complex (EMC), which plays essential roles in the routing and processing of proteins through the ER (16–19). EMC3 is required for surfactant homeostasis at birth, regulating the stability of the surfactant phospholipid transporter ABCA3 and the routing of surfactant proteins SP-B and SP-C in vivo (20). In the current study, we identified the role of EMC3 in the pathogenesis of alveolar abnormalities caused by SP-C(I73T). Postnatal deletion of Emc3 in AT2 cells altered the mistrafficking of SP-C(I73T), rescued mitochondrial function, and restored alveolar morphology. We identified valosin containing protein (VCP) as an interacting partner with EMC3. Inhibition of EMC3 or VCP restored alveolar morphology in vivo and restored homeostasis in patient-specific induced AT2 (iAT2) cells derived from SFTPCI73T patient-specific induced pluripotent stem cells (iPSCs), supporting the concept that EMC3, VCP, and other components of the EMC complex provide a framework for development of new therapies for SFTPCI73T-associated ILD and other disorders caused by similar mistrafficking of variant proteins.

Results

Generation of a constitutive SftpcI73T knock-in mouse model. To produce a mouse model constitutively expressing SP-C(I73T), we inserted a mouse cDNA construct encoding the 218 T>C mutation into the mouse Sftpc locus in frame with exon 1, resulting in an amino acid substitution at position 73 (I73T) of the mouse proSP-C (Figure 1A). The floxed neomycin (neo) resistance cassette was excised by breeding mice to an Ella-Cre transgenic line, resulting in germline deletion of the neomycin cassette (Figure 1A). In all of the following experiments, we used the neomycin-free SftpcI73T knock-in allele. As early as postnatal day 7, SftpcI73T/I73T mice (referred to as I73T/I73T) had simplified alveoli (shown as focal airspace enlargement) in the peripheral lung (Figure 1, B and C and Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/JCI173861DS1). Subtle airspace enlargement was observed in SftpcWT/I73T (referred to as WT/I73T) lungs when compared with SftpcWT/WT controls (referred to as WT/WT) (Supplemental Figure 1, A and B). No obvious fibrosis was detected in I73T/I73T lungs as measured by hydroxyproline assay and quantitative reverse transcription PCR (qRT-PCR) on extracellular matrix genes Acta2, Col1a1, and Col3a1 (Supplemental Figure 1, C and D). AT2 cells from I73T/I73T mice expressed Sftpc transcripts at approximately 20% of the levels in control AT2 cells (Figure 1D). Compared with the intracellular punctate distribution of WT SP-C proprotein (proSP-C(WT)) in AT2 cells, SP-C(I73T) proprotein accumulated (Supplemental Figure 2) with dense staining detected in proximity to cell surfaces (Figure 1E). In AT2 cell lysates, whole lung homogenates, and BALF, intracellular and secreted proSP-C(I73T) was detected at higher molecular weights and was not processed efficiently into the mature SP-C peptide, with misprocessed peptides accumulating as multiple intermediates (Figure 1, F–H). BiP (Figure 1, F and G), eIF2-α phosphorylation, ATF4, CHOP, and Xbp1 splicing (Supplemental Figure 3) were detected at similar levels among different genotypes, indicating that proSP-C(I73T) was not subject to ER-associated degradation (ERAD) in vivo. Autophagy, which manifested in some patients with SFTPCI73T and a mouse model expressing acute and high levels of SP-C(I73T) (12–15), was not altered in the I73T/I73T AT2 cells, as reflected by unchanged protein levels of P62 and LC3B (Supplemental Figure 3A). With no alterations in autophagy and pulmonary fibrosis — likely due to low expression levels of SP-C(I73T) — findings in the SftpcI73T knock-in mouse model are consistent with previous studies demonstrating abnormal trafficking and processing of the mutant protein, and the knock-in mouse model is suitable for study of the primary effects of SP-C(I73T) expression in vivo.

Figure 1

Generation of SftpcI73T knock-in mouse model. (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.

AT2-specific deletion of Emc3 rescued alveolar simplification in SftpcI73T neonates. We previously demonstrated that loss of EMC3 in embryonic lung epithelial cells caused accumulation of proSP-C in the multivesicular bodies (MVB) and reduced its processing to the mature SP-C peptides (mSP-C) (20). Since EMC3 and other EMC subunits, including EMC4, are regulated simultaneously at protein levels (20), lacking suitable EMC3 antibody for immunostaining on lung sections, we examined the subcellular distribution of EMC4 in AT2 cells in our SftpcI73T knock-in mouse model (Supplemental Figure 4). Distinct patterns of EMC4 staining were detected in AT2 cells from I73T/I73T mutant and WT/WT control mice, with dense staining near cell surfaces in I73T/I73T AT2 cells, contrasting with cytoplasmic puncta seen in controls, suggesting that EMC is involved in the regulation of SP-C(I73T) proteostasis. To delete Emc3 in postnatal AT2 cells, we used a tamoxifen-inducible AT2-specific Cre allele, Sftpc-CreERT. In contrast to the indispensable role of EMC3 in perinatal surfactant metabolism (20), deletion of Emc3 in postnatal AT2 cells did not alter lung morphology or subcellular localization of surfactant proteins SP-B and SP-C (Supplemental Figure 5). Compared with the normal lung structure in SftpcWT/CreERT (referred to as WT/CreERT) mice, SftpcI73T/CreERT (referred to as I73T/CreERT) mice demonstrated alveolar simplification on postnatal day 21 (P21) (Figure 2A). When Emc3 deletion was induced in SftpcI73T/CreERT;Emc3fl/fl (referred to as I73T/CreERT;Emc3Δ/Δ) neonatal mice, lung morphology was remarkably restored (Figure 2, A and B) on P21. Alveolar protection was not due to increased mature SP-C production, since no major changes in mSP-C(I73T) levels were detected (Figure 2, C and D). Emc3 deletion failed to ameliorate the increased protein levels of proSP-C(I73T) or proSP-B in SP-C(I73T)–expressing AT2 cells (Figure 2E), suggesting the concept that restoration of alveolar structure was not caused by changes in SP-C(I73T) protein levels. Unlike embryonic Emc3 deletion, ABCA3 stability was not altered by deletion of Emc3 (Figure 2, F–H), demonstrating a dispensable role of EMC3 in maintaining postnatal ABCA3. Deletion of Emc3 decreased the dense periplasma membrane staining of proSP-C(I73T) (Figure 2F), thus, altered transport of proSP-C(I73T) may contribute to the alveolar simplification defect associated with the mutation.

Figure 2

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