Dataset Information

Proliferative regulation of alveolar epithelial type 2 progenitor cells by human Scnn1d gene.

ABSTRACT: Lung epithelial sodium channel (ENaC) encoded by Scnn1 genes is essential for maintaining transepithelial salt and fluid homeostasis in the airway and the lung. Compared to ?, ?, and ? subunits, the role of respiratory ?-ENaC has not been studied in vivo due to the lack of animal models. Methods: We characterized full-length human ?802-ENaC expressed in both Xenopus oocytes and humanized transgenic mice. AT2 proliferation and differentiation in 3D organoids were analysed with FACS and a confocal microscope. Both two-electrode voltage clamp and Ussing chamber systems were applied to digitize ?802-ENaC channel activity. Immunoblotting was utilized to analyse ?802-ENaC protein. Transcripts of individual ENaC subunits in human lung tissues were quantitated with qPCR. Results: The results indicate that ?802-ENaC functions as an amiloride-inhibitable Na+ channel. Inhibitory peptide ?-13 distinguishes ?802- from ?-type ENaC channels. Modified proteolysis of ?-ENaC by plasmin and aprotinin did not alter the inhibition of amiloride and ?-13 peptide. Expression of ?802-ENaC at the apical membrane of respiratory epithelium was detected with biophysical features similar to those of heterologously expressed channels in oocytes. ?802-ENaC regulated alveologenesis through facilitating the proliferation of alveolar type 2 epithelial cells. Conclusion: The humanized mouse line conditionally expressing human ?802-ENaC is a novel model for studying the expression and function of this protein in vivo .

SUBMITTER: Zhao R

PROVIDER: S-EPMC6857051 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Proliferative regulation of alveolar epithelial type 2 progenitor cells by human Scnn1d gene.

Zhao Runzhen R Ali Gibran G Chang Jianjun J Komatsu Satoshi S Tsukasaki Yoshikazu Y Nie Hong-Guang HG Chang Yongchang Y Zhang Mo M Liu Yang Y Jain Krishan K Jung Bock-Gie BG Samten Buka B Jiang Dianhua D Liang Jiurong J Ikebe Mitsuo M Matthay Michael A MA Ji Hong-Long HL

Theranostics 20191018 26

Lung epithelial sodium channel (ENaC) encoded by Scnn1 genes is essential for maintaining transepithelial salt and fluid homeostasis in the airway and the lung. Compared to α, β, and γ subunits, the role of respiratory δ-ENaC has not been studied in vivo due to the lack of animal models. Methods: We characterized full-length human δ802-ENaC expressed in both Xenopus oocytes and humanized transgenic mice. AT2 proliferation and differentiation in 3D organoids ...[more]

PMID: 31754387

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Project description:Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by type-II alveolar epithelial cell (AECII) injury and fibroblast hyperproliferation. Severe AECII endoplasmic reticulum (ER) stress is thought to underlie IPF, but is yet incompletely understood. We studied the regulation of C/EBP homologous protein (CHOP), a proapoptotic ER-stress-related transcription factor (TF) in AECII-like cells. Interestingly, single or combined overexpression of the active ER stress transducers activating transcription factor-4 (Atf4) and activating transcription factor-6 (p50Atf6) or spliced x-box-binding protein-1 (sXbp1) in MLE12 cells did not result in a substantial Chop induction, as compared to the ER stress inducer thapsigargin. Employing reporter gene assays of distinct CHOP promoter fragments, we could identify that, next to the conventional amino acid (AARE) and ER stress response elements (ERSE) within the CHOP promoter, activator protein-1 (AP-1) and c-Ets-1 TF binding sites are necessary for CHOP induction. Serial deletion and mutation analyses revealed that both AP-1 and c-Ets-1 motifs act in concert to induce CHOP expression. In agreement, CHOP promoter activity was greatly enhanced upon combined versus single overexpression of AP-1 and c-Ets-1. Moreover, combined overexpression of AP-1 and c-Ets-1 in MLE12 cells alone in the absence of any other ER stress inducer was sufficient to induce Chop protein expression. Further, AP-1 and c-Ets-1 were upregulated in AECII under ER stress conditions and in human IPF. Finally, Chop overexpression in vitro resulted in AECII apoptosis, lung fibroblast proliferation, and collagen-I production. We propose that CHOP activation by AP-1 and c-Ets-1 plays a key role in AECII maladaptive ER stress responses and consecutive fibrosis, offering new therapeutic prospects in IPF. KEY MESSAGES: Overexpression of active ER stress sensors Atf4, Atf6, and Xbp1 does not induce Chop. AP-1 and c-Ets-1 TFs are necessary for induction of the ER stress factor Chop. AP-1 and c-Ets-1 alone induce Chop expression in the absence of any ER stress inducers. AP-1 and c-Ets-1 are induced in AECII under ER stress conditions and in human IPF. Chop expression alone triggers AECII apoptosis and consecutive profibrotic responses.

Dataset Information

Proliferative regulation of alveolar epithelial type 2 progenitor cells by human Scnn1d gene.

Publications

Proliferative regulation of alveolar epithelial type 2 progenitor cells by human <i>Scnn1d</i> gene.

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