TGF-β controls alveolar type 1 epithelial cell plasticity and alveolar matrisome gene transcription in mice
Danielle A. Callaway, Ian J. Penkala, Su Zhou, Jonathan J. Knowlton, Fabian Cardenas-Diaz, Apoorva Babu, Michael P. Morley, Mariana Lopes, Benjamin A. Garcia, Edward E. Morrisey
Danielle A. Callaway, Ian J. Penkala, Su Zhou, Jonathan J. Knowlton, Fabian Cardenas-Diaz, Apoorva Babu, Michael P. Morley, Mariana Lopes, Benjamin A. Garcia, Edward E. Morrisey
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Research Article Pulmonology

TGF-β controls alveolar type 1 epithelial cell plasticity and alveolar matrisome gene transcription in mice

Abstract

Premature birth disrupts normal lung development and places infants at risk for bronchopulmonary dysplasia (BPD), a disease disrupting lung health throughout the life of an individual and that is increasing in incidence. The TGF-β superfamily has been implicated in BPD pathogenesis, however, what cell lineage it impacts remains unclear. We show that TGFbr2 is critical for alveolar epithelial (AT1) cell fate maintenance and function. Loss of TGFbr2 in AT1 cells during late lung development leads to AT1-AT2 cell reprogramming and altered pulmonary architecture, which persists into adulthood. Restriction of fetal lung stretch and associated AT1 cell spreading through a model of oligohydramnios enhances AT1-AT2 reprogramming. Transcriptomic and proteomic analyses reveal the necessity of TGFbr2 expression in AT1 cells for extracellular matrix production. Moreover, TGF-β signaling regulates integrin transcription to alter AT1 cell morphology, which further impacts ECM expression through changes in mechanotransduction. These data reveal the cell intrinsic necessity of TGF-β signaling in maintaining AT1 cell fate and reveal this cell lineage as a major orchestrator of the alveolar matrisome.

Authors

Danielle A. Callaway, Ian J. Penkala, Su Zhou, Jonathan J. Knowlton, Fabian Cardenas-Diaz, Apoorva Babu, Michael P. Morley, Mariana Lopes, Benjamin A. Garcia, Edward E. Morrisey

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Figure 7

TGF-β–mediated integrin binding regulates AT1 cell size, morphology, and ECM expression.

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TGF-β–mediated integrin binding regulates AT1 cell size, morphology, and...
(A) To obtain AT1 cells for culture, P5–P10 pup lungs were obtained after lineage labeling with tamoxifen at P0. Whole lung cell suspensions were obtained using a dispase, DNase, and collagenase digestion buffer after which the epithelial cell population was enriched by depleting the CD45+ and CD31+ population. Remaining cells were fluorescently sorted with FACS to obtain a CD326+ and YFP+ suspension. Cells were plated onto fibronectin-coated plates with or without TGF-β1 ligand (7.5 ng/mL) or TGF-β inhibitor SB431542 (10μM) and evaluated at days 2, 4, and 6. (B) ICC of RAGE+ (AGER+) cells treated with TGF-β ligand or inhibitor in culture at days 2, 4, and 6 with a zoomed image of cells at day 6 appearing at the bottom. Scale bars: 100 μm. (C) Quantification of mean AGER+ cell area depicted in B by 1-way ANOVA with Holms Šidák’s test for multiple comparisons (n = 135–231). (D) Quantification of mean cell roundness at day 6 depicted in B by 1-way ANOVA with Holms Šidák’s test for multiple comparisons (n = 148–167). (E) Quantification of qPCR RNA transcript expression levels (FC compared with GAPDH, normalized to controls) of the AT2 marker Sftpb, fibronectin-binding integrins (F) Itga5 and (G) Itgb1, and (H) basement membrane constituents including the collagen IV subtypes Col4a1, Col4a3, and Col4a4 and laminin-332 constituents Lama3 and Lamb3 (n = 3 per group, 1-way ANOVA with Tukey’s multiple comparisons). (I) Representative schematic of findings indicating that TGF-β regulates integrin expression to guide ECM binding and cellular spread, which affects cell identity and matrisome expression and impacts lung development. Schematics in A and I were created in BioRender. Results are representative of 3 experiments.