scholarly journals A mammalian Wnt5a–Ror2–Vangl2 axis controls the cytoskeleton and confers cellular properties required for alveologenesis

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Kuan Zhang ◽  
Erica Yao ◽  
Chuwen Lin ◽  
Yu-Ting Chou ◽  
Julia Wong ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Planar Cell Polarity ◽  
Current Model ◽  
Alveolar Epithelial Cells ◽  
Alveolar Type ◽  
Type I ◽  
Alveolar Epithelial ◽  
Pcp Signaling ◽  
And Migration ◽  
Alveolar Formation

Alveolar formation increases the surface area for gas-exchange and is key to the physiological function of the lung. Alveolar epithelial cells, myofibroblasts and endothelial cells undergo coordinated morphogenesis to generate epithelial folds (secondary septa) to form alveoli. A mechanistic understanding of alveologenesis remains incomplete. We found that the planar cell polarity (PCP) pathway is required in alveolar epithelial cells and myofibroblasts for alveologenesis in mammals. Our studies uncovered a Wnt5a–Ror2–Vangl2 cascade that endows cellular properties and novel mechanisms of alveologenesis. This includes PDGF secretion from alveolar type I and type II cells, cell shape changes of type I cells and migration of myofibroblasts. All these cellular properties are conferred by changes in the cytoskeleton and represent a new facet of PCP function. These results extend our current model of PCP signaling from polarizing a field of epithelial cells to conferring new properties at subcellular levels to regulate collective cell behavior.

scholarly journals Transient receptor vanilloid 4 (TRPV4) channels are essential for alveolar epithelial cell function

2019 ◽  
Author(s):  
Jonas Weber ◽  
Yu-Kai Chao ◽  
Martina Kannler ◽  
Gabriela Krasteva-Christ ◽  
Suhasini Rajan ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Barrier Function ◽  
Alveolar Epithelial Cells ◽  
Transient Receptor Potential Vanilloid ◽  
Alveolar Type ◽  
Type I ◽  
Edema Formation ◽  
Genetic Ablation ◽  
Alveolar Epithelial ◽  
Transient Receptor

AbstractIschemia-reperfusion(IR)-induced edema formation can be mimicked ex-vivo in isolated perfused mouse lungs (IPL). Here we show enhanced edema formation in transient receptor potential vanilloid 4 (TRPV4)-deficient (TRPV4-/-) IPL compared to wild-type (WT) controls in response to IR, indicating a protective role of TRPV4 to maintain the alveolar epithelial barrier. By immunohistochemistry, mRNA profiling or electrophysiological analysis we detected TRPV4 in bronchial epithelium, alveolar type I (ATI) and alveolar type II (ATII) cells. Genetic ablation of TRPV4 resulted in reduced expression of aquaporin-5 (AQP-5) channels in ATI as well as decreased production of pro surfactant protein C (pSP-C) in ATII cells. Migration of TRPV4-deficient ATI cells was reduced and cell barrier function was impaired. Moreover, adult TRPV4−/− lungs developed emphysema-like changes and altered lung parameters compared to WT lungs. Therefore, our data highlight novel essential functions of TRPV4 channels in alveolar epithelial cells and in the protection from edema formation.eLife digestTransient receptor potential vanilloid 4 (TRPV4) is a non-selective Ca2+ permeable cation channel expressed in lung endothelium where increased channel activity has been shown to compromise endothelial barrier function. In other tissues however, the channel maintains physiological cell barriers, e.g. in skin, the urogenital tract and the corneal epithelium. In tracheal epithelial cells TRPV4 channels regulate ciliar beat frequency and in alveolar epithelial cells TRPV4 activation by 4α-phorbol esters produced blebs and breaks in lung septa by unknown molecular mechanisms. To understand the channels role in lung function Weber et al. employed ex-vivo isolated perfused mouse lungs (IPL) to mimic ischemia-reperfusion-induced edema as one of the most common and significant causes of morbidity and mortality after lung transplantation in human patients. TRPV4-deficient (TRPV4−/−) IPL developed enhanced edema formation compared to wild-type (WT) controls in response to ischemia and reperfusion, indicating a protective role of TRPV4 to maintain the alveolar epithelial barrier. TRPV4 was detected in bronchial epithelium, alveolar type I (ATI) and alveolar type II (ATII) cells by immunohistochemistry or mRNA profiling. Genetic ablation of TRPV4 resulted in reduced expression and plasma membrane insertion of water conducting aquaporin-5 (AQP-5) channels in ATI cells compared to WT mice. Analysis of isolated primary TRPV4−/− ATII cells revealed a reduced expression of pro surfactant protein-C (pSP-C) a precursor of a protein important for decreasing surface tension and for alveolar fluid homeostasis. Moreover, the TRPV4 activator GSK1016790A induced increases in current densities only in WT but not in TRPV4−/− ATII cells. On a molecular level ablation of TRPV4 induced less Ca2+-mediated nuclear translocation of nuclear factor of activated T-cells (NFAT) to the nucleus, which may be responsible for reduced expression of the identified proteins. Although the ability of TRPV4−/− ATII to differentiate to ATI cells was unchanged, migration of TRPV4-deficient ATI cells was reduced and cell barrier function was impaired. Moreover, TRPV4−/− lungs of adult mice developed significantly larger mean chord lengths and altered lung function compared to WT lungs. The findings of Weber et al. highlights novel essential functions of TRPV4 channels in alveolar epithelial cells and in the protection from edema formation.

scholarly journals Acquisition of cellular properties during alveolar formation requires differential activity and distribution of mitochondria

2021 ◽  
Author(s):  
Kuan Zhang ◽  
Erica Yao ◽  
Julia Wong ◽  
Paul J. Wolters ◽  
Pao-Tien Chuang
Keyword(s):  
Epithelial Cells ◽  
Mitochondrial Function ◽  
Shape Change ◽  
Alveolar Epithelial Cells ◽  
Mitochondrial Activity ◽  
Cellular Functions ◽  
Alveolar Epithelial ◽  
Cell Shape Change ◽  
And Migration ◽  
Alveolar Formation

AbstractAlveolar formation requires coordinated movement and interaction between alveolar epithelial cells, mesenchymal myofibroblasts and endothelial cells/pericytes to produce secondary septa. These processes rely on the acquisition of distinct cellular properties to enable ligand secretion for cell-cell signaling and initiate morphogenesis through cell migration and cell shape change. In this study, we showed that mitochondrial activity and distribution play a key role in bestowing cellular functions on both alveolar epithelial cells and mesenchymal myofibroblasts for generating secondary septa to form alveoli. These results suggest that mitochondrial function is tightly regulated to empower cellular machineries in a spatially specific manner. Indeed, such regulation via mitochondria is required for secretion of platelet-derived growth factor from alveolar epithelial cells to influence myofibroblast proliferation and migration. Moreover, mitochondrial function enables myofibroblast migration during alveolar formation. Together, these findings yield novel mechanistic insights into how mitochondria regulate pivotal steps of alveologenesis. They highlight selective utilization of energy and diverse energy demands in different cellular processes during development. Our work serves as a paradigm for studying how mitochondria control tissue patterning.

IL-23 amplifies the epithelial-mesenchymal transition of mechanically conditioned alveolar epithelial cells in RA-ILD through mTOR/S6 signaling

2021 ◽  
Author(s):  
Chujie Zhang ◽  
Shaohua Wang ◽  
Jessica Lau ◽  
Anja C. Roden ◽  
Eric L. Matteson ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Lung Fibrosis ◽  
Epithelial Mesenchymal Transition ◽  
Alveolar Epithelial Cells ◽  
Alveolar Type ◽  
Type I ◽  
Sequencing Analysis ◽  
Mesenchymal Transition ◽  
Caveolin 1 ◽  
Alveolar Epithelial

Epithelial-mesenchymal transition (EMT) creates an environment facilitating fibrosis following alveolar epithelial cell injury. IL-23 has important roles in chronic autoimmune conditions like rheumatoid arthritis (RA), but its role in the interstitial lung disease that affects RA patients is unclear. This study aimed to determine the pro-fibrogenic role of IL-23 on somatic alveolar type I (ATI) epithelial cells. Primary ATI cells were isolated from rats and cultured on plastic dishes for 1-3 weeks. After prolonged culture (≥14 days) on rigid culture dishes, primary ATI cells gradually acquired a mesenchymal phenotype, identified by decreased expression of caveolin-1, and reorganization of F-actin cytoskeleton, indicating the initiation of EMT by matrix stiffness. To determine how IL-23 promotes EMT in vitro, transitioning ATI cells, cultured on a stiff substrate for ≥14 days were stimulated with IL-23. The EMT phenotype was significantly enhanced by IL-23 which upregulated α-SMA, collagen I/III protein, and decreased caveolin-1. Furthermore, IL-23 significantly promoted cell invasion as well as apoptotic resistance on transitioning ATI cells. Mechanistically, IL-23 induced EMT was mTOR/S6 signaling dependent and reversible by rapamycin. Transcriptional sequencing analysis of human lung fibrosis biopsy tissue revealed key roles for IL-23 in RA-ILD. This result was further validated by significantly upregulated IL-23 expression at the mRNA level in RA-ILD lung sections. Notably, transitioning ATI epithelial cells were abundantly detected in RA-ILD tissue. Taken together, these data support a role for IL-23 in the pathogenesis of RA lung fibrosis by promoting EMT in alveolar epithelial cells through mTOR/S6 signaling.

Differential expression of Na-K-ATPase isoforms in rat alveolar epithelial cells

1997 ◽  
Vol 273 (1) ◽  
pp. L246-L255 ◽  
Author(s):  
K. M. Ridge ◽  
D. H. Rutschman ◽  
P. Factor ◽  
A. I. Katz ◽  
A. M. Bertorello ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Alveolar Epithelial Cells ◽  
Cell Phenotype ◽  
Alveolar Type ◽  
Type I ◽  
Binding Studies ◽  
Alveolar Epithelial ◽  
Functional Classes ◽  
Atpase Subunits ◽  
Ouabain Inhibition

Lung Na-K-ATPase has been shown to contribute to vectorial Na+ transport and edema clearance. The alpha 1- and beta 1-Na-K-ATPase subunits have been localized to alveolar type II (ATII) cells, and the alpha 2-Na-K-ATPase has been reported in rat lung homogenates. Expression of Na-K-ATPase alpha 1-, alpha 2-, and beta 1-subunits was investigated in rat ATII cells cultured for 7 days, a period during which they lose their phenotypic markers and differentiate to an alveolar type I (ATI)-like cell phenotype. Differentiation of ATII cells to an ATI-like phenotype resulted in a decrease of alpha 1- and an increase of alpha 2-mRNA and protein abundance without changes in the beta 1-subunit. Thus ATI-like cells exhibited a mixture of alpha 1- and alpha 2-isoforms. Nuclear run-on analysis suggests that these changes were transcriptionally regulated. The existence of the distinct functional classes of Na-K-ATPase in ATII and ATI-like cells was confirmed by ouabain inhibition of Na-K-ATPase activity. Ouabain inhibition of ATII cells was consistent with expression of the alpha 1-isozyme [50% inhibitory concentration (IC50) = 4 x 10(-5) M], whereas, in ATI-like cells, it was consistent with the presence of both alpha 1- and alpha 2-isozymes (IC50 = 9.0 x 10(-5) and 1.5 x 10(-7) M, respectively); [3H]ouabain binding studies corroborated these findings. Our results indicate that, during ATII cell cytodifferentiation with time in culture, there is a shift in isoform composition that may reflect physiological functions of alveolar epithelial cells.

scholarly journals Therapeutic Potential of Cytosolic PLA2 Isoform–Specific Inhibitor Arachidonyl Trifluromethyl Ketone in Cigarette Smoke Condensate–Induced Pathological Conditions in Alveolar Type I and II Epithelial Cells

2018 ◽  
Vol 4 (Supplement 2) ◽  
pp. 221s-221s
Author(s):  
S. Kumar ◽  
S.K. Sharma ◽  
B. Medhi ◽  
K.L. Khanduja
Keyword(s):  
Epithelial Cells ◽  
Cigarette Smoke ◽  
Therapeutic Potential ◽  
Specific Inhibitor ◽  
Alveolar Epithelial Cells ◽  
Alveolar Type ◽  
Type I ◽  
Continuous Exposure ◽  
Alveolar Epithelial ◽  
Cytosolic Pla2

Background: Cigarette smoking is responsible for various lung pathologies including chronic lung inflammation, emphysema, chronic obstructive pulmonary disease (COPD), cancer, and annually causes almost 10 million deaths globally. During smoke exposure, most affected cells are the alveolar epithelial cells where as a repair mechanism, activation of cytosolic phospholipase A2 enzymes takes place. High free radicals and cPLA2 activity due to continuous exposure of smoke exposure leads to elevated levels of secondary metabolites and various pathophysiologic conditions such as chronic inflammation, oxidative stress and cancer. To reduce the burden of chronic inflammation as well as oxidative stress, and higher levels of secondary metabolites whose role is well defined in progression of cancer, we checked the therapeutic potential of cPLA2 inhibitor arachidonyl trifluromethyl ketone (ATK) by pharmacologically targeting the most expressible cPLA2 during continuous exposure of cigarette smoke. Aim: To check the therapeutic potential of cytosolic PLA2 isoform specific inhibitor arachidonyl trifluromethyl ketone in cigarette smoke condensate–induced pathologic conditions in alveolar type I and II epithelial cells. Methods: Effect of cPLA2 inhibitor on CSC-induced cPLA2 activity were checked using colorimetric assay, cell viability using MTT assay, FDA uptake assay using fluorescence microscopy, ROS levels and apoptosis markers through flow cytometry, and ERK levels using ELISA, in both type of alveolar epithelial cells. Results: ATK significantly mimicked CSC-induced cPLA2 activity, free radicals, primary apoptosis, ratio of apoptotic/apoptotic proteins and levels of ERK whereas protected cells from loss of cell viability and membrane integrity. Conclusion: Current observations revealed cPLA2s as a potential therapeutic target and their inhibitor ATK as a potential therapeutic agent in Cigarette smoke induced pathological conditions in alveolar type I and II epithelial cells.

scholarly journals Combinations of differentiation markers distinguish subpopulations of alveolar epithelial cells in adult lung

2016 ◽  
Vol 310 (2) ◽  
pp. L114-L120 ◽  
Author(s):  
Janice M. Liebler ◽  
Crystal N. Marconett ◽  
Nicholas Juul ◽  
Hongjun Wang ◽  
Yixin Liu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Alveolar Epithelial Cells ◽  
Normal Adult ◽  
Alveolar Type ◽  
Type I ◽  
Cell Marker ◽  
Aquaporin 5 ◽  
Differentiation Markers ◽  
Adult Lung ◽  
Alveolar Epithelial

Distal lung epithelium is maintained by proliferation of alveolar type II (AT2) cells and, for some daughter AT2 cells, transdifferentiation into alveolar type I (AT1) cells. We investigated if subpopulations of alveolar epithelial cells (AEC) exist that represent various stages in transdifferentiation from AT2 to AT1 cell phenotypes in normal adult lung and if they can be identified using combinations of cell-specific markers. Immunofluorescence microscopy showed that, in distal rat and mouse lungs, ∼20–30% of NKX2.1+ (or thyroid transcription factor 1+) cells did not colocalize with pro-surfactant protein C (pro-SP-C), a highly specific AT2 cell marker. In distal rat lung, NKX2.1+ cells coexpressed either pro-SP-C or the AT1 cell marker homeodomain only protein x (HOPX). Not all HOPX+ cells colocalize with the AT1 cell marker aquaporin 5 (AQP5), and some AQP5+ cells were NKX2.1+. HOPX was expressed earlier than AQP5 during transdifferentiation in rat AEC primary culture, with robust expression of both by day 7. We speculate that NKX2.1 and pro-SP-C colocalize in AT2 cells, NKX2.1 and HOPX or AQP5 colocalize in intermediate or transitional cells, and HOPX and AQP5 are expressed without NKX2.1 in AT1 cells. These findings suggest marked heterogeneity among cells previously identified as exclusively AT1 or AT2 cells, implying the presence of subpopulations of intermediate or transitional AEC in normal adult lung.

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.

Disparate cytokine regulation of ICAM-1 in rat alveolar epithelial cells and pulmonary endothelial cells in vitro

1995 ◽  
Vol 269 (1) ◽  
pp. L127-L135 ◽  
Author(s):  
W. W. Barton ◽  
S. Wilcoxen ◽  
P. J. Christensen ◽  
R. Paine
Keyword(s):  
Endothelial Cells ◽  
Epithelial Cells ◽  
Inflammatory Cytokines ◽  
Alveolar Epithelial Cells ◽  
Normal Lung ◽  
Type I ◽  
Type Ii Cells ◽  
Type Ii ◽  
Alveolar Epithelial

Intercellular adhesion molecule-1 (ICAM-1) is expressed at high levels on type I alveolar epithelial cells in the normal lung and is induced in vitro as type II cells spread in primary culture. In contrast, in most nonhematopoetic cells ICAM-1 expression is induced in response to inflammatory cytokines. We have formed the hypothesis that the signals that control ICAM-1 expression in alveolar epithelial cells are fundamentally different from those controlling expression in most other cells. To test this hypothesis, we have investigated the influence of inflammatory cytokines on ICAM-1 expression in isolated type II cells that have spread in culture and compared this response to that of rat pulmonary artery endothelial cells (RPAEC). ICAM-1 protein, determined both by a cell-based enzyme-linked immunosorbent assay and by Western blot analysis, and mRNA were minimally expressed in unstimulated RPAEC but were significantly induced in a time- and dose-dependent manner by treatment with tumor necrosis factor-alpha, interleukin-1 beta, or interferon-gamma. In contrast, these cytokines did not influence the constitutive high level ICAM-1 protein expression in alveolar epithelial cells and only minimally affected steady-state mRNA levels. ICAM-1 mRNA half-life, measured in the presence of actinomycin D, was relatively long at 7 h in alveolar epithelial cells and 4 h in RPAEC. The striking lack of response of ICAM-1 expression by alveolar epithelial cells to inflammatory cytokines is in contrast to virtually all other epithelial cells studied to date and supports the hypothesis that ICAM-1 expression by these cells is a function of cellular differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretion of cytokines by rat alveolar epithelial cells: possible regulatory role for SP-A

1994 ◽  
Vol 266 (2) ◽  
pp. L148-L155 ◽  
Author(s):  
H. Blau ◽  
S. Riklis ◽  
V. Kravtsov ◽  
M. Kalina
Keyword(s):  
Epithelial Cells ◽  
Alveolar Epithelial Cells ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Alveolar Type Ii Cells ◽  
Long Term Culture ◽  
Alveolar Epithelial ◽  
Gm Csf

Cultured alveolar type II cells and pulmonary epithelial (PE) cells in long-term culture were found to secrete colony-stimulating factors (CSF) into the medium in similar fashion to alveolar macrophages. CSF activity was determined by using the in vitro assay for myeloid progenitor cells [colony-forming units in culture (CFU-C)]. Both lipopolisaccharide (LPS) and interleukin-1 alpha (IL-1 alpha) were found to upregulate the secretion 6.5- to 8-fold from alveolar type II cells and macrophages. However, no stimulatory effect of these factors was observed in PE cells that release CSF into the medium constitutively, possibly due to the conditions of long-term culture. The CSF activity was partially neutralized (70% inhibition) by antibodies against murine granulocyte/macrophage (GM)-CSF and IL-3, thus indicating the presence of both GM-CSF and IL-3-like factors in the CSF. However, the presence of other cytokines in the CSF is highly probable. Surfactant-associated protein A (SP-A), which is known to play a central role in surfactant homeostasis and function, was also found to upregulate secretion of CSF (at concentrations of 0.1-5 micrograms/ml) from alveolar type II cells and macrophages. Control cells such as rat peritoneal macrophages, alveolar fibroblasts, and 3T3/NIH cell line could not be elicited by SP-A to release CSF. The results are discussed in relation to the possible participation of the alveolar epithelial cells in various intercellular signaling networks. Our studies suggest that alveolar type II cells and SP-A may play an important regulatory role in the modulation of immune and inflammatory effector cells within the alveolar space.

Murine Alveolar Epithelial Cells and Their Lentivirus-mediated Immortalisation

2018 ◽  
Vol 46 (2) ◽  
pp. 73-89
Author(s):  
Sandra Sapich ◽  
Marius Hittinger ◽  
Remi Hendrix-Jastrzebski ◽  
Urska Repnik ◽  
Gareth Griffiths ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Lines ◽  
Experimental Testing ◽  
Barrier Properties ◽  
Alveolar Epithelial Cells ◽  
Mouse Strains ◽  
Alveolar Type ◽  
Inhalation Toxicity ◽  
Alveolar Epithelial

In this study, we describe the isolation and immortalisation of primary murine alveolar epithelial cells (mAEpC), as well as their epithelial differentiation and barrier properties when grown on Transwell® inserts. Like human alveolar epithelial cells (hAEpC), mAEpC transdifferentiate in vitro from an alveolar type II (ATII) phenotype to an ATI-like phenotype and exhibit features of the air–blood barrier, such as the establishment of a thin monolayer with functional tight junctions (TJs). This is demonstrated by the expression of TJ proteins (ZO-1 and occludin) and the development of high transepithelial electrical resistance (TEER), peaking at 1800ω•cm2. Transport across the air–blood barrier, for general toxicity assessments or preclinical drug development, is typically studied in mice. The aim of this work was the generation of novel immortalised murine lung cell lines, to help meet Three Rs requirements in experimental testing and research. To achieve this goal, we lentivirally transduced mAEpC of two different mouse strains with a library of 33 proliferation-promoting genes. With this immortalisation approach, we obtained two murine alveolar epithelial lentivirus-immortalised (mAELVi) cell lines. Both showed similar TJ protein localisation, but exhibited less prominent barrier properties (TEERmax ~250Ω•cm2) when compared to their primary counterparts. While mAEpC demonstrated their suitability for use in the assessment of paracellular transport rates, mAELVi cells could potentially replace mice for the prediction of acute inhalation toxicity during early ADMET studies.

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