Role of ERBB3 in the interplay between epithelial cells and fibroblasts during late-stage lung development and disease

Zhang, Xin (2022): Role of ERBB3 in the interplay between epithelial cells and fibroblasts during late-stage lung development and disease. Dissertation, LMU München: Medizinische Fakultät

Abstract

Background Lung development is subdivided into 5 stages: embryonic, pseudoglandular, canalicular, saccular, and alveolar stages. The first four stages are completed in the fetal lung through airway branching. About 90% gas exchange area is formed during the last stage of alveolarization by secondary septation, which concludes the coordinated growth of septae from the alveolar walls to subdivide the distal saccules into alveoli, accompanied by microvascular maturation and underlying matrix formation and remodeling. Interplay between the mesenchy-mal-alveolar epithelial cells is key to understanding the septation process during alveolarization. The epidermal growth factor receptor family (also known as ERBBs) pathways play a crucial role in orchestrating epithelial progenitor cell differentiation and organ maturation. The current study aimed at unraveling the role of ERBB3 in lung development and disease with a specific focus on the epithelial-mesenchymal crosstalk. Methods and Results Temporal and spatial resolution of ERBB3 signaling Neonatal Balb/c mice were sacrificed on postnatal day 3, 5, 7, 10, 14, 21, or 60, and their lungs were excised and prepared into formalin fixed paraffin embedded (FFPE) lung slides and stained for phosphorylated Erbb3 (pErbb3). Data of single-cell RNA-sequencing time-series were obtained from online databases published by collaborating partners and analyzed for gene expression. Phosphorylated Erbb3 receptor level peaked on postnatal day (PND) 5 and remained at a high level until PND14, corresponding to the structural changes of increased alveolar number and enlarged air exchange area featured in postnatal alveolarization. The Erbb3 is expressed in type 1 alveolar epithelial cells (AEC1) and type 2 alveolar epithelial cells (AEC2) in the lung periphery, co-localized with other crucial components that drive lung scaffold formation, i.e., elastic fiber, and Pdgf-Rα positive fibroblasts at the septal tips in the postnatally developing lung. Epithelial cells-fibroblasts interaction and underlying pathway crosstalk An ex vivo model of lung organoids was derived from co-culture of primary mouse epithelial cells (PMECs) and murine lung fibroblasts cell line (Mlg) or primary mouse fibroblasts culture) in Matrigel to study the interaction between epithelial cells and fibroblasts. Samples were collected at day-7 or 14 for analysis of morphology and gene expression. Organoids derived from neonatal PMEC + Mlg revealed higher expression of Pdgf-Rα in fibroblasts when compared to the adult organoids and had increased organoid size. Increased fibroblasts number resulted in organoid segmentation, similar to septation. Supplemental Nrg1 treatment provoked epithelial cell proliferation by Erbb3 activation. Murine lung epithelial cell line (MLE12) cells were co-cultured with primary fibroblasts in a transwell system for 24h without direct contact. Gene expression was analyzed in both cell types with comparison to monoculture. Cell-cell interaction by soluble molecules resulted in upregulation of neuregulin (NRG1) in fibroblasts and ERBB3 in MLE12 cells, together with upregulation of GATA Binding Protein 6 (GATA6), homeodomain-only protein (HOPX), Vascular Endothelial Growth Factor A (VEGFA), ATP Binding Cassette Subfamily A Member 3 (ABCA3), and Collagen Type I Alpha 1 Chain (COL1A1). Gene expression in Platelet Derived Growth Factor Subunit A (PDGF-A), NRG1, and Aquaporin 5 (AQP5) remained unchanged. Impact of moderate hyperoxia exposure on ERBB3 signaling In vitro monoculture of both MLE12 and primary mouse fibroblasts were exposed to moderate hyperoxia (FiO2=0.4) for 48h and analyzed for mRNA and protein expression. In a subsequent experiment, supernatant from hyperoxia-exposed fibroblasts was collected to treat MLE12 cells for 12h, and gene analysis was performed in MLE12 cells. Organoids derived from neonatal PMEC and Mlg were cultured under normoxia for 7 days and exposed to hyperoxia for another 7 days, with or without supplemental NRG1 in the medium, and analyzed for morphological changes. Neonatal mice at PND 5-7 were exposed to hyperoxia for 8h and sacrificed for lung protein expression analysis. After hyperoxia exposure, ERBB3 was downregulated in MLE12 cells accompanied by decreased proliferation. Likewise, protein level of ERBB3 also decreased in neonatal mouse lungs after hyperoxia exposure. Ligand (NRG1) expression was upregulated in primary wildtype fibroblasts together with PDGF-Rα and COLLAGEN1 expression when co-cultured with MLE12 cells, whereas ACTA2 expression was downregulated. These changes were less prominent in platelet-derived growth factor receptor alpha (PDGF-Rα+/-) fibroblasts. Incubation with supernatant derived from hyperoxia-exposed fibroblast cultures did not affect ERBB3 signaling in MLE12 cells. In ex vivo organoids, hyperoxia reduced epithelial cell proliferation and organoid expansion, while NRG1 treatment partially counterbalanced the repression effect. Conclusion In summary, temporal ERBB3 receptors expression in neonatal mouse lung was most prominent during the postnatal alveolarization period (PND3-14), and local-ized at the septal tip in alveolar epithelial cells. Activation of ERBB3 signaling led to epithelial cell proliferation, indicating a crucial role of ERBB signaling in alveo-lar epithelium elongation during secondary septation. Epithelial cell-fibroblast interaction was essential in activating ERBB3 signaling. Hyperoxia exposure downregulated ERBB3 signaling in epithelial cells and repressed epithelial cell proliferation, and exogenous NRG1 treatment partially counterbalanced the re-pression.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: ERBB3, epithelial cells, Pdgf-Ra, fibroblasts, lung development, alveolarization, secondary septation, hyperoxia, organoids
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 17. November 2022
• 1. Bericht­erstatter:in: Hilgendorff, Anne