Background A healthy alveolar epithelium is critical to the gas exchange function of the lungs. As the major cell type of alveolar epithelium, alveolar type 2 (AT2) cells play a critical role in maintaining pulmonary homeostasis by serving as alveolar progenitors during lung injury, inflammation, and repair. Dysregulation of AT2 cells may lead to the development of acute and chronic lung diseases and cancer. The lack of clinically relevant AT2 cell models hampers our ability to understand pulmonary diseases. Here, we sought to establish reversibly immortalized mouse pulmonary alveolar type 2 cells (imPAC2) and investigate their potential in forming alveolar organoids to model pulmonary diseases. Methods Primary mouse pulmonary alveolar cells (mPACs) were isolated and immortalized with a retroviral expression of SV40 Large T antigen (LTA). Cell proliferation and survival was assessed by crystal violet staining and WST-1 assays. Marker gene expression was assessed by qPCR, Western blotting, and/or immunostaining. Alveolar organoids were generated by using matrigel. Ad-TGF-β1 was used to transiently express TGF-β1. Stable silencing β-catenin or overexpression of mutant KRAS and TP53 was accomplished by using retroviral vectors. Subcutaneous cell implantations were carried out in athymic nude mice. The retrieved tissue masses were subjected to H & E histologic evaluation. Results We immortalized primary mPACs with SV40 LTA to yield the imPACs that were non-tumorigenic and maintained long-term proliferative activity that was reversible by FLP-mediated removal of SV40 LTA. The EpCAM+ AT2-enriched subpopulation (i.e., imPAC2) was sorted out from the imPACs, and was shown to express AT2 markers and form alveolar organoids. Functionally, silencing β-catenin decreased the expression of AT2 markers in imPAC2 cells, while TGF-β1 induced fibrosis-like response by regulating the expression of epithelial-mesenchymal transition markers in the imPAC2 cells. Lastly, concurrent expression of oncogenic KRAS and mutant TP53 rendered the imPAC2 cells a tumor-like phenotype and activated lung cancer-associated pathways. Collectively, our results suggest that the imPAC2 cells may faithfully represent AT2 populations that can be further explored to model pulmonary diseases.
Background Alveolar type II epithelial cells (AECIIs), a crucial part of the alveolar epithelium, can secrete surfactant-associated proteins and act as progenitor cells of the alveoli. The number of AECIIs in lung tissues is closely related to the pathogenesis and pathological process of numerous lung diseases. Brahma-related geng1 (Brg1), a catalytic subunit of ATPase, is a core component of the mammalian switch/sucrose non-fermentable (SWI/SNF) ATP-dependent chromatin-remodeling complex, which achieves chromosome recombination and further regulates gene expression in an ATP-dependent manner. Brgl plays a pivotal role in regulating cell differentiation, proliferation, and apoptosis. Unfortunately, almost no research exists on the relationship between Brg1 and AECIIs. Therefore, we aimed to investigate the effect of Brg1 on the quantity of AECIIs as well as the possible mechanisms. Methods C57BL/6 mice with the Brg1 gene specifically knocked out in AECII epithelial cells (Brg1fl/fl mice) were constructed to analyze the effect of brg1 gene expression on the number of AECIIs in vivo. The number of ACEIIs was detected and compared in the Brg1fl/fl group and wild-type (WT) group using immunohistochemistry, flow cytometry, and immunofluorescence. The Brg1 gene in immortalized mouse pulmonary alveolar type II (ImpacII) cells was knocked down using lentiviral vectors. The migration and invasion of ImpacII were observed using cell scratch assay and transwell migration assay. The proliferation of ImpacII was monitored using the cell clone assay, CCK-8 cell proliferation assay, and cell cycle assay. The proliferation-related proteins including Ki67, p-JAK1/2/JAK1/2, p-STAT6/STAT6, p-PI3K/PI3K, and p-AKT/AKT were detected using Western blot and immunofluorescence in ImpacII cells. To explore the specific molecular mechanism of Brg1 regulating ImpacII proliferation, the binding sequences of Brg1 in ImpacII cells were sought using chromatin immunoprecipitation-sequence (CHIP-seq) and further confirmed by chromatin immunoprecipitation-qPCR (CHIP-qPCR). The interactive relationship between JAK1/2 and PI3K was verified by co-immunoprecipitation (Co-IP) assay. Results Knocking out brg1 facilitated the proliferation of AECIIs in vivo. Knocking down brg1 induced the proliferation in association with the migration and invasion of ImpacII in vitro. Mechanistically, knocking down brg1 activated the JAK1/2-PI3K/AKT signaling pathway and induced the expression of proliferation-related protein Ki67. Furthermore, CHIP-seq and CHIP-qPCR results showed that Brg1 could bind to the JAK1/2 promoter region and regulate the activity of the JAK1/2-PI3K/AKT signaling pathway. Co-IP confirmed that JAK1/2 interacted with PI3K. Conclusion Knocking out Brg1 promoted the proliferation, migration, and invasion of AECIIs via the JAK1/2-PI3K/AKT signaling pathway. This represents a potential therapeutic target and a novel prognostic indicator in various pulmonary diseases.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.