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FGF signaling plays an essential role in lung development, homeostasis, and regeneration. We employed mouse 3D cell culture models and imaging to study the role of FGF ligands and the interplay of FGF signaling with epithelial growth factor (EGF) and WNT signaling pathways in lung epithelial morphogenesis and differentiation. In non-adherent conditions, FGF signaling promoted formation of lungospheres from lung epithelial stem/progenitor cells (LSPCs). Ultrastructural and immunohistochemical analyses showed that LSPCs produced more differentiated lung cell progeny. In a 3D extracellular matrix, FGF2, FGF7, FGF9, and FGF10 promoted lung organoid formation. FGF9 showed reduced capacity to promote lung organoid formation, suggesting that FGF9 has a reduced ability to sustain LSPC survival and/or initial divisions. FGF7 and FGF10 produced bigger organoids and induced organoid branching with higher frequency than FGF2 or FGF9. Higher FGF concentration and/or the use of FGF2 with increased stability and affinity to FGF receptors both increased lung organoid and lungosphere formation efficiency, respectively, suggesting that the level of FGF signaling is a crucial driver of LSPC survival and differentiation, and also lung epithelial morphogenesis. EGF signaling played a supportive but non-essential role in FGF-induced lung organoid formation. Analysis of tissue architecture and cell type composition confirmed that the lung organoids contained alveolar-like regions with cells expressing alveolar type I and type II cell markers, as well as airway-like structures with club cells and ciliated cells. FGF ligands showed differences in promoting distinct lung epithelial cell types. FGF9 was a potent inducer of more proximal cell types, including ciliated and basal cells. FGF7 and FGF10 directed the differentiation toward distal lung lineages. WNT signaling enhanced the efficiency of lung organoid formation, but in the absence of FGF10 signaling, the organoids displayed limited branching and less differentiated phenotype. In summary, we present lung 3D cell culture models as useful tools to study the role and interplay of signaling pathways in postnatal lung development and homeostasis, and we reveal distinct roles for FGF ligands in regulation of mouse lung morphogenesis and differentiation .

Palmitoylation is a reversible post-translational lipid modification of proteins, catalyzed by the Zinc finger DHHC domain-containing (ZDHHC) family of palmitoyltransferases. Palmitoylation plays a pivotal role in regulating localization, stability, trafficking, and interactions, thereby contributing to a wide range of cellular processes. Dysregulation of palmitoylation has been implicated in numerous pathological conditions, including metabolic disorders, muscular diseases, mitochondrial disorders, cancer, and neurodegeneration. In this review, we summarize recent advances in understanding S-palmitoylation, emphasizing its critical roles in protein regulation, cellular and physiological processes, and its implications in both health and disease. Additionally, we highlight emerging therapeutic opportunities and novel strategies in therapeutic applications targeting this lipid modification.

Fibroblast growth factor 2 (FGF2) plays important roles in tissue development and repair. Using heparan sulfates (HS)/heparin as a cofactor, FGF2 binds to FGF receptor (FGFR) and induces downstream signaling pathways, such as ERK pathway, that regulate cellular behavior. In most cell lines, FGF2 signaling displays biphasic dose-response profile, reaching maximal response to intermediate concentrations, but weak response to high levels of FGF2. Recent reports demonstrated that the biphasic cellular response results from competition between binding of FGF2 to HS and FGFR that impinge upon ERK signaling dynamics. However, the role of HS/heparin in FGF signaling has been controversial. Several studies suggested that heparin is not required for FGF-FGFR complex formation and that the main role of heparin is to protect FGF from degradation. In this study, we investigated the relationship between FGF2 stability, heparin dependence and ERK signaling dynamics using FGF2 variants with increased thermal stability (FGF2-STABs). FGF2-STABs showed higher efficiency in induction of FGFR-mediated proliferation, lower affinity to heparin and were less dependent on heparin than wild-type FGF2 (FGF2-wt) for induction of FGFR-mediated mitogenic response. Interestingly, in primary mammary fibroblasts, FGF2-wt displayed a sigmoidal dose-response profile, while FGF2-STABs showed a biphasic response. Moreover, at low concentrations, FGF2-STABs induced ERK signaling more potently and displayed a faster dynamics of full ERK activation and higher amplitudes of ERK signaling than FGF2-wt. Our results suggest that FGF2 stability and heparin dependence are important factors in FGF-FGFR signaling complex assembly and ERK signaling dynamics.

Extracellular matrix (ECM) is an essential component of the tissue microenvironment, actively shaping cellular behavior. In vitro culture systems are often poor in ECM constituents, thus not allowing for naturally occurring cell–ECM interactions. This study reports on a straightforward and efficient method for the generation of ECM scaffolds from lung tissue and its subsequent in vitro application using primary lung cells. Mouse lung tissue was subjected to decellularization with 0.2% sodium dodecyl sulfate, hypotonic solutions, and DNase. Resultant ECM scaffolds were devoid of cells and DNA, whereas lung ECM architecture of alveolar region and blood and airway networks were preserved. Scaffolds were predominantly composed of core ECM and ECM-associated proteins such as collagens I-IV, nephronectin, heparan sulfate proteoglycan core protein, and lysyl oxidase homolog 1, among others. When homogenized and applied as coating substrate, ECM supported the attachment of lung fibroblasts (LFs) in a dose-dependent manner. After ECM characterization and biocompatibility tests, a novel in vitro platform for three-dimensional (3D) matrix repopulation that permits live imaging of cell–ECM interactions was established. Using this system, LFs colonized the ECM scaffolds, displaying a close-to-native morphology in intimate interaction with the ECM fibers, and showed nuclear translocation of the mechanosensor yes-associated protein (YAP), when compared with cells cultured in two dimensions. In conclusion, we developed a 3D-like culture system, by combining an efficient decellularization method with a live-imaging culture platform, to replicate in vitro native lung cell–ECM crosstalk. This is a valuable system that can be easily applied to other organs for ECM-related drug screening, disease modeling, and basic mechanistic studies.

Progressive pulmonary fibrosis results from a dysfunctional tissue repair response and is characterized by fibroblast proliferation, activation, and invasion and extracellular matrix accumulation. Lung fibroblast heterogeneity is well recognized. With single-cell RNA sequencing, fibroblast subtypes have been reported by recent studies. However, the roles of fibroblast subtypes in effector functions in lung fibrosis are not well understood. In this study, we incorporated the recently published single-cell RNA-sequencing datasets on murine lung samples of fibrosis models and human lung samples of fibrotic diseases and analyzed fibroblast gene signatures. We identified and confirmed the novel fibroblast subtypes we reported recently across all samples of both mouse models and human lung fibrotic diseases, including idiopathic pulmonary fibrosis, systemic sclerosis-associated interstitial lung disease, and coronavirus disease (COVID-19). Furthermore, we identified specific cell surface proteins for each fibroblast subtype through differential gene expression analysis, which enabled us to isolate primary cells representing distinct fibroblast subtypes by flow cytometry sorting. We compared matrix production, including fibronectin, collagen, and hyaluronan, after profibrotic factor stimulation and assessed the invasive capacity of each fibroblast subtype. Our results suggest that in addition to myofibroblasts, lipofibroblasts and Ebf1 (Ebf transcription factor 1 ) fibroblasts are two important fibroblast subtypes that contribute to matrix deposition and also have enhanced invasive, proliferative, and contraction phenotypes. The histological locations of fibroblast subtypes are identified in healthy and fibrotic lungs by these cell surface proteins. This study provides new insights to inform approaches to targeting lung fibroblast subtypes to promote the development of therapeutics for lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an aging-associated interstitial lung disease resulting from repeated epithelial injury and inadequate epithelial repair. Alveolar type II cells (AEC2s) are progenitor cells that maintain epithelial homeostasis and repair the lung after injury. In the current study, we assessed lipid metabolism in AEC2s from human lungs of patients with IPF and healthy donors, as well as AEC2s from bleomycin-injured young and old mice. Through single-cell RNA sequencing, we observed that lipid metabolism-related genes were downregulated in IPF AEC2s and bleomycin-injured mouse AEC2s. Aging aggravated this decrease and hindered recovery of lipid metabolism gene expression in AEC2s after bleomycin injury. Pathway analyses revealed downregulation of genes related to lipid biosynthesis and fatty acid β-oxidation in AEC2s from IPF lungs and bleomycin-injured, old mouse lungs compared with the respective controls. We confirmed decreased cellular lipid content in AEC2s from IPF lungs and bleomycin-injured, old mouse lungs using immunofluorescence staining and flow cytometry. Futhermore, we show that lipid metabolism was associated with AEC2 progenitor function. Lipid supplementation and PPARγ (peroxisome proliferator activated receptor γ) activation promoted progenitor renewal capacity of both human and mouse AEC2s in three-dimensional organoid cultures. Lipid supplementation also increased AEC2 proliferation and expression of in AEC2s. In summary, we identified a lipid metabolism deficiency in AEC2s from lungs of patients with IPF and bleomycin-injured old mice. Restoration of lipid metabolism homeostasis in AEC2s might promote AEC2 progenitor function and offer new opportunities for therapeutic approaches to IPF.

Alveolar type II (AT2) progenitor cell exhaustion and impaired regenerative capacity are key pathogenic hallmarks in idiopathic pulmonary fibrosis (IPF). Nicotinamide adenine dinucleotide (NAD ) functions as a central regulator of cellular energy metabolism. We have reported that downregulation of NAD -dependent sirtuin signaling contributes to the impaired progenitor function of IPF AT2s. In this study, we identified that a key NAD biosynthesis enzyme, nicotinamide phosphoribosyltransferase (NAMPT), is significantly downregulated in IPF AT2s. NAMPT deficiency impairs AT2 renewal and enhances lung fibrosis through downregulation of SIRT7 and SOD2, which results in increased oxidative stress, mitochondrial dysfunction, induction of pathological transitional gene expression and impaired regenerative capacity to generate alveolar type I (AT1) cell required for gas exchange. Mice with deletion of Nampt in AT2s showed severely impaired AT2 renewal and increased susceptibility to bleomycin lung injury and spontaneous fibrois. Activation of NAMPT by small molecule activators promoted AT2 renewal, restored homeostasis, and reversed lung fibrosis. NAMPT activation could be a therapeutic strategy for restoring AT2 progenitor function and halting or reversing progressive pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal lung disease marked by alveolar type 2 (AT2) stem cell dysfunction and excessive matrix deposition, with no effective treatments. Recent advances have recognized that AT2 cells act as stem cells, in addition to their role in the production of pulmonary surfactants in the distal alveolar space. We and others have reported a failure of AT2 regeneration and a loss of AT2 cells in IPF. We recently further reported that there is a defect in lipid metabolism in IPF AT2 cells and we discovered a selective loss of lysophosphatidylcholine acyltransferase 1 (LPCAT1) in AT2 cells from IPF, as well as in AT2 cells from bleomycin-injured mice. Pharmacological and genetic experiments confirm that LPCAT1 is required for AT2 cell renewal in 3D organoid assays. AT2 cell-specific deletion resulted in reduced AT2 renewal, spontaneous lung fibrosis, and heightened susceptibility to bleomycin-induced fibrosis in mice in vivo. Expression-based high-content drug screening with an LPCAT1 knock-in cell line identified several drug families that upregulated LPCAT1 expression. We further confirmed that anti-malarial artesunate and PLA2 inhibitor ONO-RS-082 increased LPCAT1 mRNA expression, promoted AT2 renewal, and attenuated bleomycin-induced lung fibrosis in mice in vivo. Our findings establish LPCAT1 as a critical regulator of AT2 renewal and lipid metabolism in IPF, suggesting that reactivation of LPCAT1 could offer a novel therapeutic strategy for restoring alveolar progenitor function and mitigating lung fibrosis.

FGF signaling plays an essential role in lung development, homeostasis, and regeneration. Several FGF ligands were detected in the developing lungs, however, their roles have not been fully elucidated. We employed mouse 3D cell culture models and imaging to ex vivo study of a) the role of FGF ligands in lung epithelial morphogenesis and b) the interplay of FGF signaling with epithelial growth factor (EGF) and WNT signaling pathways. In non-adherent conditions, FGF signaling promoted formation of lungospheres from lung epithelial stem/progenitor cells (LSPCs). Based on their architecture, we defined three distinct phenotypes of lungospheres. Ultrastructural and immunohistochemical analyses showed that LSPCs produced more differentiated lung cell progeny. In 3D extracellular matrix, FGF2, FGF7, FGF9, and FGF10 promoted lung organoid formation with similar efficiency. However, FGF9 showed reduced capacity to promote lung organoid formation, suggesting that FGF9 has a reduced ability to sustain LSPCs survival and/or initial divisions. Analysis of lung organoid phenotypes revealed that FGF7 and FGF10 produce bigger organoids and induce organoid branching with higher frequency than FGF2 and FGF9. Higher FGF concentration and/or the use of FGF2 with increased stability and affinity to FGF receptors both increased lung organoid and lungosphere formation efficiency, respectively, suggesting that the level of FGF signaling is a crucial driver of LSPC survival and differentiation, and also lung epithelial morphogenesis. EGF signaling played a supportive but nonessential role in FGF-induced lung organoid formation. Moreover, analysis of tissue architecture and cell type composition confirmed that the lung organoids contained alveolar-like regions with cells expressing alveolar type I and type II cell markers, as well as airway-like structures with club cells and ciliated cells. WNT signaling enhanced the efficiency of lung organoid formation, but in the absence of FGF10 signaling, the organoids displayed limited branching and less differentiated phenotype. In summary, we present lung 3D cell culture models as useful tools to study the role and interplay of signaling pathways in lung development and we reveal roles for FGF ligands in regulation of mouse lung morphogenesis ex vivo.