Expression of a calmodulin inhibitor peptide in progenitor alveolar type II cells disrupts lung development

1996 ◽  
Vol 271 (2) ◽  
pp. L245-L250 ◽  
Author(s):  
J. Wang ◽  
B. Campos ◽  
M. A. Kaetzel ◽  
J. R. Dedman
Keyword(s):  
Cell Growth ◽  
Epithelial Cells ◽  
Lung Development ◽  
Branching Morphogenesis ◽  
Lung Epithelial Cells ◽  
Alveolar Type ◽  
Type Ii ◽  
Calmodulin Inhibitor ◽  
Inhibitor Peptide ◽  
Cell Growth And Differentiation

Calmodulin (CaM) is a major intracellular Ca2+ mediator protein involved in cell growth and differentiation. To evaluate calmodulin function in lung, it was necessary to construct a gene that encodes a high-affinity calmodulin binding peptide, since chemically synthesized calmodulin inhibitors lack binding and targeting specificity. This calmodulin inhibitor peptide gene was targeted to type II epithelial cells in transgenic mice using the human surfactant protein C promoter. Neutralization of calmodulin function in progenitor type II epithelial pneumocytes alters epithelial cell growth and differentiation, which prevents branching morphogenesis of the bronchial tree. Newborn transgenic animals have undeveloped lungs. This study indicates that type II lung epithelial cells require functional CaM for proliferation and development. The targeting of specific inhibitor peptides to a single lung cell type is an approach to evaluate the role of calmodulin, the ubiquitous calcium-dependent regulator protein, in lung development and disease.

Fluid transport across cultured rat alveolar epithelial cells: a novel in vitro system

2004 ◽  
Vol 287 (1) ◽  
pp. L104-L110 ◽  
Author(s):  
Xiaohui Fang ◽  
Yuanlin Song ◽  
Rachel Zemans ◽  
Jan Hirsch ◽  
Michael A. Matthay
Keyword(s):  
Epithelial Cells ◽  
Fluid Transport ◽  
Alveolar Epithelial Cells ◽  
Lung Epithelial Cells ◽  
Morphological Evidence ◽  
Alveolar Type ◽  
Type Ii ◽  
In Vitro System ◽  
Alveolar Epithelial

Previous studies have used fluid-instilled lungs to measure net alveolar fluid transport in intact animal and human lungs. However, intact lung studies have two limitations: the contribution of different distal lung epithelial cells cannot be studied separately, and the surface area for fluid absorption can only be approximated. Therefore, we developed a method to measure net vectorial fluid transport in cultured rat alveolar type II cells using an air-liquid interface. The cells were seeded on 0.4-μm microporous inserts in a Transwell system. At 96 h, the transmembrane electrical resistance reached a peak level (1,530 ± 115 Ω·cm2) with morphological evidence of tight junctions. We measured net fluid transport by placing 150 μl of culture medium containing 0.5 μCi of 131I-albumin on the apical side of the polarized cells. Protein permeability across the cell monolayer, as measured by labeled albumin, was 1.17 ± 0.34% over 24 h. The change in concentration of 131I-albumin in the apical fluid was used to determine the net fluid transported across the monolayer over 12 and 24 h. The net basal fluid transport was 0.84 μl·cm−2·h−1. cAMP stimulation with forskolin and IBMX increased fluid transport by 96%. Amiloride inhibited both the basal and stimulated fluid transport. Ouabain inhibited basal fluid transport by 93%. The cultured cells retained alveolar type II-like features based on morphologic studies, including ultrastructural imaging. In conclusion, this novel in vitro system can be used to measure net vectorial fluid transport across cultured, polarized alveolar epithelial cells.

An enhancer region determines hSP-B gene expression in bronchiolar and ATII epithelial cells in transgenic mice

2003 ◽  
Vol 284 (3) ◽  
pp. L481-L488 ◽  
Author(s):  
Li Yang ◽  
Angela Naltner ◽  
Allison Kreiner ◽  
Dong Yan ◽  
Angelynn Cowen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Transgenic Mice ◽  
Transgenic Mouse ◽  
Lung Development ◽  
Alveolar Type ◽  
Transcriptional Level ◽  
Lacz Gene ◽  
Type Ii ◽  
Enhancer Region

Regulation of the surfactant protein B gene (SP-B) is developmentally controlled and highly tissue specific. To elucidate the SP-B gene temporal/spatial expression pattern in lung development at the transcriptional level, a transgenic mouse model line carrying the human SP-B (hSP-B) 1.5-kb 5′-flanking regulatory region and the lacZ gene was established. Expression of hSP-B 1.5-kb lacZ gene started at the onset of lung formation [embryonic day 9 (E9)] and was restricted to epithelial cells throughout prenatal and postnatal lung development. In the adult lung, hSP-B 1.5-kb lacZ gene expression was restricted to bronchiolar and alveolar type II epithelial cells. In lung explant culturing studies, the hSP-B 1.5-kb lacZ gene was highly expressed in newly formed epithelial tubules during the respiratory branching process. In a second transgenic mouse line, an enhancer region, which binds to thyroid transcription factor-1, retinoic acid receptor, signal transducers and activators of transcription 3, and nuclear receptor coactivators (SRC-1, ACTR, TIF2, and CBP/p300), was deleted from the hSP-B 1.5-kb lacZ gene. The deletion abolished hSP-B lacZ gene expression in bronchiolar epithelial cells and significantly reduced its expression level in alveolar type II epithelial cells in transgenic mice.

scholarly journals Rab38 targets to lamellar bodies and normalizes their sizes in lung alveolar type II epithelial cells

2011 ◽  
Vol 301 (4) ◽  
pp. L461-L477 ◽  
Author(s):  
Linghui Zhang ◽  
Kevin Yu ◽  
Kyle W. Robert ◽  
Kristine M. DeBolt ◽  
Nankang Hong ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Lamellar Body ◽  
Lung Epithelial Cells ◽  
Alveolar Type ◽  
Type Ii ◽  
Sprague Dawley ◽  
Lung Epithelial ◽  
Green Fluorescent

Rab38 is a rat Hermansky-Pudlak syndrome gene that plays an important role in surfactant homeostasis in alveolar type II (ATII) pneumocytes. We examined Rab38 function in regulating lamellar body (LB) morphology in ATII cells. Quantitative electron microscopy revealed that LBs in ATII cells were ∼77% larger in Rab38-null fawn-hooded hypertension (FHH) than control Sprague-Dawley (SD) rats. Rab38 protein expression was restricted in lung epithelial cells but was not found in primary endothelial cells. In SD ATII cells, Rab38 protein level gradually declined during 5 days in culture. Importantly, endogenous Rab38 was present in LB fractions purified from SD rat lungs, and transiently expressed enhanced green fluorescent protein (EGFP)-tagged Rab38 labeled only the limiting membranes of a subpopulation (∼30%) of LBs in cultured ATII cells. This selective targeting was abolished by point mutations to EGFP-Rab38 and was not shared by Rab7 and Rab4b, which also function in the ATII cells. Using confocal microscopy, we established a method for quantitative evaluation of the enlarged LB phenotype temporally preserved in cultured FHH ATII cells. A direct causal relationship was established when the enlarged LB phenotype was reserved and then rescued by transiently reexpressed EGFP-Rab38 in cultured FHH ATII cells. This rescuing effect was associated with dynamic EGFP-Rab38 targeting to and on LB limiting membranes. We conclude that Rab38 plays an indispensible role in maintaining LB morphology and surfactant homeostasis in ATII pneumocytes.

scholarly journals Cyclic mechanical strain increases reactive oxygen species production in pulmonary epithelial cells

2005 ◽  
Vol 289 (5) ◽  
pp. L834-L841 ◽  
Author(s):  
Kenneth E. Chapman ◽  
Scott E. Sinclair ◽  
Daming Zhuang ◽  
Aviv Hassid ◽  
Leena P. Desai ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Epithelial Cells ◽  
Cell Line ◽  
Primary Cultures ◽  
Reactive Oxygen ◽  
Lung Epithelial Cells ◽  
Alveolar Type ◽  
Ros Production ◽  
Oxygen Species ◽  
Type Ii

Overdistention of lung tissue during mechanical ventilation may be one of the factors that initiates ventilator-induced lung injury (VILI). We hypothesized that cyclic mechanical stretch (CMS) of the lung epithelium is involved in the early events of VILI through the production of reactive oxygen species (ROS). Cultures of an immortalized human airway epithelial cell line (16HBE), a human alveolar type II cell line (A549), and primary cultures of rat alveolar type II cells were cyclically stretched, and the production of superoxide (O2−) was measured by dihydroethidium fluorescence. CMS stimulated increased production of O2− after 2 h in each type of cell. 16HBE cells exhibited no significant stimulation of ROS before 2 h of CMS (20% strain, 30 cycles/min), and ROS production returned to control levels after 24 h. Oxidation of glutathione (GSH), a cellular antioxidant, increased with CMS as measured by a decrease in the ratio of the reduced GSH level to the oxidized GSH level. Strain levels of 10% did not increase O2− production in 16HBE cells, whereas 15, 20, and 30% significantly increased generation of O2−. Rotenone, a mitochondrial complex I inhibitor, partially abrogated the stretch-induced generation of O2− after 2 h CMS in 16HBE cells. NADPH oxidase activity was increased after 2 h of CMS, contributing to the production of O2−. Increased ROS production in lung epithelial cells in response to elevated stretch may contribute to the onset of VILI.

Expression of the tyrosine phosphatase LAR-PTP2 is developmentally regulated in lung epithelia

1994 ◽  
Vol 267 (3) ◽  
pp. L263-L270 ◽  
Author(s):  
D. Rotin ◽  
B. J. Goldstein ◽  
C. A. Fladd
Keyword(s):  
Epithelial Cells ◽  
Lung Development ◽  
Tyrosine Phosphatase ◽  
A549 Cells ◽  
Alveolar Epithelial Cells ◽  
Alveolar Type ◽  
Type Ii ◽  
Adult Type ◽  
Alveolar Epithelial

The role of tyrosine kinases in regulating cell proliferation, differentiation, and development has been well documented. In contrast, little is known about the role of protein tyrosine phosphatases (PTPs) in mammalian development. To identify PTPs that may be involved in lung development, we have isolated (by polymerase chain reaction) from rat fetal alveolar epithelial cells a cDNA fragment which was identified as the recently cloned tyrosine phosphatase LAR-PTP2. Analysis of tissue expression of LAR-PTP2 identified a approximately 7.5-kb message in the lung, which is also expressed weakly in brain, and an alternatively spliced approximately 6.0-kb message (LAR-PTP2B) expressed in brain. In the fetal lung, LAR-PTP2 was preferentially expressed in lung epithelial (but not fibroblast) cells grown briefly in primary culture, and its expression was tightly regulated during lung development, peaking at 20 days of gestational age (term = 22 days), when mature alveolar type II epithelium first appears. Accordingly, immunoblot analysis revealed high expression of endogenous LAR-PTP2 protein in alveolar epithelial cells from 21-day gestation fetuses. LAR-PTP2 was also expressed in lungs of newborn rats, but transcripts (and protein) were barely detectable in adult lungs and in the nonproliferating adult alveolar type II cells. Interestingly, expression was restored in the transformed adult type II-like A549 cells. These results suggest that LAR-PTP2 may play a role in the proliferation and/or differentiation of epithelial cells during lung development.

GM-CSF enhances lung growth and causes alveolar type II epithelial cell hyperplasia in transgenic mice

1997 ◽  
Vol 273 (4) ◽  
pp. L715-L725 ◽  
Author(s):  
Jacquelyn A. Huffman Reed ◽  
Ward R. Rice ◽  
Zsuzsanna K. Zsengellér ◽  
Susan E. Wert ◽  
Glenn Dranoff ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Transgenic Mice ◽  
Lung Epithelial Cells ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Cell Hyperplasia ◽  
Α Subunit ◽  
Gm Csf ◽  
Type Ii Cell

The human surfactant protein (SP)-C gene promoter was used to direct expression of mouse granulocyte macrophage colony-stimulating factor (GM-CSF; SP-C-GM mice) in lung epithelial cells in GM-CSF-replete (GM+/+) or GM-CSF null mutant (GM−/−) mice. Lung weight and volume were significantly increased in SP-C-GM mice compared with GM+/+ or GM−/− control mice. Immunohistochemical staining demonstrated marked type II cell hyperplasia, and immunofluorescent labeling for proliferating cell nuclear antigen was increased in type II cells of SP-C-GM mice. Abundance of type II cells per mouse lung was increased three- to fourfold in SP-C-GM mice compared with GM+/+ and GM−/− mice. GM-CSF increased bromodeoxyuridine labeling of isolated type II cells in vitro. Type II cells, alveolar macrophages, and endothelial and bronchiolar epithelial cells were stained by antibodies to the GM-CSF receptor α-subunit in both GM+/+ mice and GM-CSF gene-targeted mice that are also homozygous for the SP-C-GM transgene. High levels of GM-CSF expression in type II cells of transgenic mice increased lung size and caused type II cell hyperplasia, demonstrating an unexpected role for the molecule in the regulation of type II cell proliferation and differentiation.

scholarly journals Single-cell analysis of human lung epithelia reveals concomitant expression of the SARS-CoV-2 receptor ACE2 with multiple virus receptors and scavengers in alveolar type II cells

2020 ◽  
Author(s):  
Guangchun Han ◽  
Ansam Sinjab ◽  
Warapen Treekitkarnmongkol ◽  
Patrick Brennan ◽  
Kieko Hara ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Single Cell ◽  
Expression Patterns ◽  
Severe Pneumonia ◽  
Lung Epithelial Cells ◽  
Chronic Obstructive ◽  
Alveolar Type ◽  
Type I ◽  
Type Ii ◽  
Lung Epithelial

ABSTRACTThe novel coronavirus SARS-CoV-2 was identified as the causative agent of the ongoing pandemic COVID 19. COVID-19-associated deaths are mainly attributed to severe pneumonia and respiratory failure. Recent work demonstrated that SARS-CoV-2 binds to angiotensin converting enzyme 2 (ACE2) in the lung. To better understand ACE2 abundance and expression patterns in the lung we interrogated our in-house single-cell RNA-sequencing dataset containing 70,085 EPCAM+ lung epithelial cells from paired normal and lung adenocarcinoma tissues. Transcriptomic analysis revealed a diverse repertoire of airway lineages that included alveolar type I and II, bronchioalveolar, club/secretory, quiescent and proliferating basal, ciliated and malignant cells as well as rare populations such as ionocytes. While the fraction of lung epithelial cells expressing ACE2 was low (1.7% overall), alveolar type II (AT2, 2.2% ACE2+) cells exhibited highest levels of ACE2 expression among all cell subsets. Further analysis of the AT2 compartment (n = 27,235 cells) revealed a number of genes co-expressed with ACE2 that are important for lung pathobiology including those associated with chronic obstructive pulmonary disease (COPD; HHIP), pneumonia and infection (FGG and C4BPA) as well as malarial/bacterial (CD36) and viral (DMBT1) scavenging which, for the most part, were increased in smoker versus light or non-smoker cells. Notably, DMBT1 was highly expressed in AT2 cells relative to other lung epithelial subsets and its expression positively correlated with ACE2. We describe a population of ACE2-positive AT2 cells that co-express pathogen (including viral) receptors (e.g. DMBT1) with crucial roles in host defense thus comprising plausible phenotypic targets for treatment of COVID-19.

scholarly journals Senescence associated long non-coding RNA 1 regulates cigarette smoke-induced senescence of type II alveolar epithelial cells through sirtuin-1 signaling

2021 ◽  
Vol 49 (2) ◽  
pp. 030006052098604
Author(s):  
Dong Yuan ◽  
Yuanshun Liu ◽  
Mengyu Li ◽  
Hongbin Zhou ◽  
Liming Cao ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cigarette Smoke ◽  
Alveolar Epithelial Cell ◽  
Alveolar Epithelial Cells ◽  
Lung Epithelial Cells ◽  
Sirtuin 1 ◽  
Type Ii ◽  
Alveolar Epithelial ◽  
Non Coding Rna ◽  
Long Non Coding Rna

Objective The primary aim of our study was to explore the mechanisms through which long non-coding RNA (lncRNA)-mediated sirtuin-1 (SIRT1) signaling regulates type II alveolar epithelial cell (AECII) senescence induced by a cigarette smoke-media suspension (CSM). Methods Pharmacological SIRT1 activation was induced using SRT2104 and senescence-associated lncRNA 1 (SAL-RNA1) was overexpressed. The expression of SIRT1, FOXO3a, p53, p21, MMP-9, and TIMP-1 in different groups was detected by qRT-PCR and Western blotting; the activity of SA-β gal was detected by staining; the binding of SIRT1 to FOXO3a and p53 gene transcription promoters was detected by Chip. Results We found that CSM increased AECII senescence, while SAL-RNA1 overexpression and SIRT1 activation significantly decreased levels of AECII senescence induced by CSM. Using chromatin immunoprecipitation, we found that SIRT1 bound differentially to transcriptional complexes on the FOXO3a and p53 promoters. Conclusion Our results suggested that lncRNA-SAL1-mediated SIRT1 signaling reduces senescence of AECIIs induced by CSM. These findings suggest a new therapeutic target to limit the irreversible apoptosis of lung epithelial cells in COPD patients.

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