GCN5L1 regulates pulmonary surfactant production by modulating lamellar body biogenesis and trafficking in mouse alveolar epithelial cells
Abstract Background The pulmonary surfactant that lines the air–liquid surface within alveoli is a protein–lipid mixture essential for gas exchange. Surfactant lipids and proteins are synthesized and stored in the lamellar body (LB) before being secreted from alveolar type II (AT2) cells. The molecu...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-11-01
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| Series: | Cellular & Molecular Biology Letters |
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| Online Access: | https://doi.org/10.1186/s11658-023-00506-0 |
| _version_ | 1797630082179661824 |
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| author | Wenqin Xu Xiaocui Ma Qing Wang Jingjing Ye Nengqian Wang Zhenzhen Ye Tianbing Chen |
| author_facet | Wenqin Xu Xiaocui Ma Qing Wang Jingjing Ye Nengqian Wang Zhenzhen Ye Tianbing Chen |
| author_sort | Wenqin Xu |
| collection | DOAJ |
| description | Abstract Background The pulmonary surfactant that lines the air–liquid surface within alveoli is a protein–lipid mixture essential for gas exchange. Surfactant lipids and proteins are synthesized and stored in the lamellar body (LB) before being secreted from alveolar type II (AT2) cells. The molecular and cellular mechanisms that regulate these processes are incompletely understood. We previously identified an essential role of general control of amino acid synthesis 5 like 1 (GCN5L1) and the biogenesis of lysosome-related organelle complex 1 subunit 1 (BLOS1) in surfactant system development in zebrafish. Here, we explored the role of GCN5L1 in pulmonary surfactant regulation. Method GCN5L1 knockout cell lines were generated with the CRISPR/Cas9 system. Cell viability was analyzed by MTT assay. Released surfactant proteins were measured by ELISA. Released surfactant lipids were measured based on coupled enzymatic reactions. Gene overexpression was mediated through lentivirus. The RNA levels were detected through RNA-sequencing (RNA-seq) and quantitative reverse transcription (qRT)- polymerase chain reaction (PCR). The protein levels were detected through western blotting. The cellular localization was analyzed by immunofluorescence. Morphology of the lamellar body was analyzed through transmission electron microscopy (TEM), Lysotracker staining, and BODIPY phosphatidylcholine labeling. Results Knocking out GCN5L1 in MLE-12 significantly decreased the release of surfactant proteins and lipids. We detected the downregulation of some surfactant-related genes and misregulation of the ROS–Erk–Foxo1–Cebpα axis in mutant cells. Modulating the activity of the axis or reconstructing the mitochondrial expression of GCN5L1 could partially restore the expression of these surfactant-related genes. We further showed that MLE-12 cells contained many LB-like organelles that were lipid enriched and positive for multiple LB markers. These organelles were smaller in size and accumulated in the absence of GCN5L1, indicating both biogenesis and trafficking defects. Accumulated endogenous surfactant protein (SP)-B or exogenously expressed SP-B/SP-C in adenosine triphosphate-binding cassette transporterA3 (ABCA3)-positive organelles was detected in mutant cells. GCN5L1 localized to the mitochondria and LBs. Reconstruction of mitochondrial GCN5L1 expression rescued the organelle morphology but failed to restore the trafficking defect and surfactant release, indicating specific roles associated with different subcellular localizations. Conclusions In summary, our study identified GCN5L1 as a new regulator of pulmonary surfactant that plays a role in the biogenesis and positioning/trafficking of surfactant-containing LBs. |
| first_indexed | 2024-03-11T11:01:53Z |
| format | Article |
| id | doaj.art-17b9b54f6f0842e4a045d707840a1135 |
| institution | Directory Open Access Journal |
| issn | 1689-1392 |
| language | English |
| last_indexed | 2024-03-11T11:01:53Z |
| publishDate | 2023-11-01 |
| publisher | BMC |
| record_format | Article |
| series | Cellular & Molecular Biology Letters |
| spelling | doaj.art-17b9b54f6f0842e4a045d707840a11352023-11-12T12:24:09ZengBMCCellular & Molecular Biology Letters1689-13922023-11-0128112110.1186/s11658-023-00506-0GCN5L1 regulates pulmonary surfactant production by modulating lamellar body biogenesis and trafficking in mouse alveolar epithelial cellsWenqin Xu0Xiaocui Ma1Qing Wang2Jingjing Ye3Nengqian Wang4Zhenzhen Ye5Tianbing Chen6Central Laboratory, Yijishan Hospital of Wannan Medical CollegeHenan Clinical Research Center of Childhood Diseases, Children’s Hospital Affiliated to Zhengzhou UniversityCentral Laboratory, Yijishan Hospital of Wannan Medical CollegeCentral Laboratory, Yijishan Hospital of Wannan Medical CollegeDepartment of Pediatrics, Yijishan Hospital of Wannan Medical CollegeDepartment of Pediatrics, Yijishan Hospital of Wannan Medical CollegeCentral Laboratory, Yijishan Hospital of Wannan Medical CollegeAbstract Background The pulmonary surfactant that lines the air–liquid surface within alveoli is a protein–lipid mixture essential for gas exchange. Surfactant lipids and proteins are synthesized and stored in the lamellar body (LB) before being secreted from alveolar type II (AT2) cells. The molecular and cellular mechanisms that regulate these processes are incompletely understood. We previously identified an essential role of general control of amino acid synthesis 5 like 1 (GCN5L1) and the biogenesis of lysosome-related organelle complex 1 subunit 1 (BLOS1) in surfactant system development in zebrafish. Here, we explored the role of GCN5L1 in pulmonary surfactant regulation. Method GCN5L1 knockout cell lines were generated with the CRISPR/Cas9 system. Cell viability was analyzed by MTT assay. Released surfactant proteins were measured by ELISA. Released surfactant lipids were measured based on coupled enzymatic reactions. Gene overexpression was mediated through lentivirus. The RNA levels were detected through RNA-sequencing (RNA-seq) and quantitative reverse transcription (qRT)- polymerase chain reaction (PCR). The protein levels were detected through western blotting. The cellular localization was analyzed by immunofluorescence. Morphology of the lamellar body was analyzed through transmission electron microscopy (TEM), Lysotracker staining, and BODIPY phosphatidylcholine labeling. Results Knocking out GCN5L1 in MLE-12 significantly decreased the release of surfactant proteins and lipids. We detected the downregulation of some surfactant-related genes and misregulation of the ROS–Erk–Foxo1–Cebpα axis in mutant cells. Modulating the activity of the axis or reconstructing the mitochondrial expression of GCN5L1 could partially restore the expression of these surfactant-related genes. We further showed that MLE-12 cells contained many LB-like organelles that were lipid enriched and positive for multiple LB markers. These organelles were smaller in size and accumulated in the absence of GCN5L1, indicating both biogenesis and trafficking defects. Accumulated endogenous surfactant protein (SP)-B or exogenously expressed SP-B/SP-C in adenosine triphosphate-binding cassette transporterA3 (ABCA3)-positive organelles was detected in mutant cells. GCN5L1 localized to the mitochondria and LBs. Reconstruction of mitochondrial GCN5L1 expression rescued the organelle morphology but failed to restore the trafficking defect and surfactant release, indicating specific roles associated with different subcellular localizations. Conclusions In summary, our study identified GCN5L1 as a new regulator of pulmonary surfactant that plays a role in the biogenesis and positioning/trafficking of surfactant-containing LBs.https://doi.org/10.1186/s11658-023-00506-0Pulmonary surfactantType II alveolar epithelial cellLamellar bodyTraffickingGCN5L1 |
| spellingShingle | Wenqin Xu Xiaocui Ma Qing Wang Jingjing Ye Nengqian Wang Zhenzhen Ye Tianbing Chen GCN5L1 regulates pulmonary surfactant production by modulating lamellar body biogenesis and trafficking in mouse alveolar epithelial cells Cellular & Molecular Biology Letters Pulmonary surfactant Type II alveolar epithelial cell Lamellar body Trafficking GCN5L1 |
| title | GCN5L1 regulates pulmonary surfactant production by modulating lamellar body biogenesis and trafficking in mouse alveolar epithelial cells |
| title_full | GCN5L1 regulates pulmonary surfactant production by modulating lamellar body biogenesis and trafficking in mouse alveolar epithelial cells |
| title_fullStr | GCN5L1 regulates pulmonary surfactant production by modulating lamellar body biogenesis and trafficking in mouse alveolar epithelial cells |
| title_full_unstemmed | GCN5L1 regulates pulmonary surfactant production by modulating lamellar body biogenesis and trafficking in mouse alveolar epithelial cells |
| title_short | GCN5L1 regulates pulmonary surfactant production by modulating lamellar body biogenesis and trafficking in mouse alveolar epithelial cells |
| title_sort | gcn5l1 regulates pulmonary surfactant production by modulating lamellar body biogenesis and trafficking in mouse alveolar epithelial cells |
| topic | Pulmonary surfactant Type II alveolar epithelial cell Lamellar body Trafficking GCN5L1 |
| url | https://doi.org/10.1186/s11658-023-00506-0 |
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