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. 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. 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. 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.

中文翻译：

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GCN5L1通过调节小鼠肺泡上皮细胞的层状体生物发生和运输来调节肺表面活性剂的产生

肺泡内空气-液体表面的肺表面活性物质是气体交换所必需的蛋白质-脂质混合物。表面活性剂脂质和蛋白质在从肺泡 II 型 (AT2) 细胞分泌之前合成并储存在层状体 (LB) 中。调节这些过程的分子和细胞机制尚不完全清楚。我们之前确定了氨基酸合成 5 like 1 (GCN5L1) 的一般控制和溶酶体相关细胞器复合物 1 亚基 1 (BLOS1) 的生物发生在斑马鱼表面活性剂系统发育中的重要作用。在这里，我们探讨了 GCN5L1 在肺表面活性物质调节中的作用。GCN5L1 敲除细胞系是使用 CRISPR/Cas9 系统生成的。通过MTT测定分析细胞活力。通过 ELISA 测量释放的表面活性蛋白。基于耦合酶反应测量释放的表面活性剂脂质。基因过表达是通过慢病毒介导的。通过RNA测序（RNA-seq）和定量逆转录（qRT）-聚合酶链反应（PCR）检测RNA水平。通过蛋白质印迹法检测蛋白质水平。通过免疫荧光分析细胞定位。通过透射电子显微镜 (TEM)、Lysotracker 染色和 BODIPY 磷脂酰胆碱标记分析层状体的形态。敲除 MLE-12 中的 GCN5L1 显着减少表面活性​​蛋白和脂质的释放。我们检测到突变细胞中一些表面活性剂相关基因的下调和 ROS-Erk-Foxo1-Cebpα 轴的失调。调节轴的活性或重建 GCN5L1 的线粒体表达可以部分恢复这些表面活性剂相关基因的表达。我们进一步表明，MLE-12 细胞含有许多类 LB 细胞器，这些细胞器富含脂质且对多种 LB 标记物呈阳性。这些细胞器尺寸较小，并且在没有 GCN5L1 的情况下积累，表明生物发生和运输缺陷。在突变细胞中检测到三磷酸腺苷结合盒转运蛋白 A3 (ABCA3) 阳性细胞器中积累的内源表面活性蛋白 (SP)-B 或外源表达的 SP-B/SP-C。GCN5L1 定位于线粒体和 LB。线粒体 GCN5L1 表达的重建挽救了细胞器形态，但未能恢复运输缺陷和表面活性剂释放，表明与不同亚细胞定位相关的特定作用。总之，我们的研究确定 GCN5L1 是一种新的肺表面活性剂调节剂，在含表面活性剂 LB 的生物发生和定位/运输中发挥作用。