Molecular and functional characterization of alveolar epithelial type I (AT1) cells has been challenging due to difficulty in isolating sufficient numbers of viable cells. Here we performed ...single-cell RNA-sequencing (scRNA-seq) of tdTomato
cells from lungs of AT1 cell-specific Aqp5-Cre-IRES-DsRed (ACID);R26tdTomato reporter mice. Following enzymatic digestion, CD31
CD45
E-cadherin
tdTomato
cells were subjected to fluorescence-activated cell sorting (FACS) followed by scRNA-seq. Cell identity was confirmed by immunofluorescence using cell type-specific antibodies. After quality control, 92 cells were analyzed. Most cells expressed 'conventional' AT1 cell markers (
,
,
,
), with heterogeneous expression within this population. The remaining cells expressed AT2, club, basal or ciliated cell markers. Integration with public datasets identified three robust AT1 cell- and lung-enriched genes,
,
and
, that were conserved across species. GPRC5A co-localized with HOPX and was not expressed in AT2 or airway cells in mouse, rat and human lung. GPRC5A co-localized with AQP5 but not pro-SPC or CC10 in mouse lung epithelial cell cytospins. We enriched mouse AT1 cells to perform molecular phenotyping using scRNA-seq. Further characterization of putative AT1 cell-enriched genes revealed GPRC5A as a conserved AT1 cell surface marker that may be useful for AT1 cell isolation.
The molecular signals that control decisions regarding progenitor/stem cell proliferation versus differentiation are not fully understood. Differentiation of motile cilia from progenitor/stem cells ...may offer a simple tractable model to investigate this process. Wnt and Notch represent two key signaling pathways in progenitor/stem cell behavior in a number of tissues. Adenomatous Polyposis Coli, Apc is a negative regulator of the Wnt pathway and a well known multifunctional protein. Using the cre-LoxP system we inactivated the Apc locus via Foxj1-cre, which is expressed in cells committed to ciliated cell lineage. We then characterized the consequent phenotype in two select tissues that bear motile cilia, the lung and the testis. In the lung, Apc deletion induced β-catenin accumulation and Jag1 expression in ciliated cells and by lateral induction, triggered Notch signaling in adjacent Clara cells. In the bronchiolar epithelium, absence of Apc blocked the differentiation of a subpopulation of cells committed to the ciliogenesis program. In the human pulmonary adenocarcinoma cells, Apc over-expression inhibited Jag1 expression and promoted motile ciliogenic gene expression program including Foxj1, revealing the potential mechanism. In the testis, Apc inactivation induced β-catenin accumulation in the spermatogonia, but silenced Notch signaling and depleted spermatogonial stem cells, associated with reduced proliferation, resulting in male infertility. In sum, the present comparative analysis reveals the tissue-dependent consequences of Apc inactivation on proliferation and differentiation of ciliated cell progenitors by coordinating Wnt and Notch signaling.
Abstract We investigated trafficking of polystyrene nanoparticles (PNP; 20 and 100 nm; carboxylate, sulfate, or aldehyde-sulfate modified negatively charged and amidine-modified positively charged) ...across rat alveolar epithelial cell monolayers (RAECM). Apical-to-basolateral fluxes of nanoparticles were estimated as functions of apical PNP concentration (PNP) and temperature. Uptake of nanoparticles into RAECM was determined using confocal microscopy. Fluxes increased as charge density became less negative/more positive, with positively charged PNPs trafficking 20–40 times faster than highly negatively charged PNP of comparable size. Trafficking rates decreased with increasing PNP diameter. PNP fluxes tended to level off at high apical PNP. Fluxes at 4°C were significantly lower than those at 37°C. Confocal microscopy revealed nanoparticles localized to cell cytoplasm, whereas cell junctions and nuclei appeared free of PNP. These data indicate that (1) trafficking of PNP across RAECM is strongly influenced by charge density, size, and temperature, (2) PNP translocate primarily transcellularly, and (3) PNP translocation requires cellular energy.
Abstract Studies of polystyrene nanoparticle (PNP) trafficking across mouse alveolar epithelial cell monolayers (MAECM) show apical-to-basolateral flux of 20 and 120 nm amidine-modified PNP is ~ 65 ...times faster than that of 20 and 100 nm carboxylate-modified PNP, respectively. Calcium chelation with EGTA has little effect on amidine-modified PNP flux, but increases carboxylate-modified PNP flux ~ 50-fold. PNP flux is unaffected by methyl-β-cyclodextrin, while ~ 70% decrease in amidine- (but not carboxylate-) modified PNP flux occurs across chlorpromazine- or dynasore-treated MAECM. Confocal microscopy reveals intracellular amidine- and carboxylate-modified PNP and association of amidine- (but not carboxylate-) modified PNP with clathrin heavy chain. These data indicate (1) amidine-modified PNP translocate across MAECM primarily via clathrin-mediated endocytosis and (2) physicochemical properties (e.g., surface charge) determine PNP interactions with mouse alveolar epithelium. Uptake/trafficking of nanoparticles into/across epithelial barriers is dependent on both nanoparticle physicochemical properties and (based on comparison with our prior results) specific epithelial cell type. From the Clinical Editor In this study of polystyrene nanoparticle trafficking across mouse alveolar epithelial cell monolayers, the authors determined that uptake/trafficking of nanoparticles into/across epithelial barriers is dependent on both nanoparticle physicochemical properties and the specific type of epithelial cells.
Epithelial-mesenchymal transition (EMT), a process whereby fully differentiated epithelial cells undergo transition to a mesenchymal phenotype giving rise to fibroblasts and myofibroblasts, is ...increasingly recognized as playing an important role in repair and scar formation following epithelial injury. The extent to which this process contributes to fibrosis following injury in the lung is a subject of active investigation. Recently, it was demonstrated that transforming growth factor (TGF)-beta induces EMT in alveolar epithelial cells (AEC) in vitro and in vivo, and epithelial and mesenchymal markers have been colocalized to hyperplastic type II (AT2) cells in lung tissue from patients with idiopathic pulmonary fibrosis (IPF), suggesting that AEC may exhibit extreme plasticity and serve as a source of fibroblasts and/or myofibroblasts in lung fibrosis. In this review, we describe the characteristic features of EMT and its mechanistic underpinnings. We further describe the contribution of EMT to fibrosis in adult tissues following injury, focusing especially on the critical role of TGF-beta and its downstream mediators in this process. Finally, we highlight recent descriptions of EMT in the lung and the potential implications of this process for the treatment of fibrotic lung disease. Treatment for fibrosis of the lung in diseases such as IPF has heretofore focused largely on amelioration of potential inciting processes such as inflammation. It is hoped that this review will stimulate further consideration of the cellular mechanisms of fibrogenesis in the lung and especially the role of the epithelium in this process, potentially leading to innovative avenues of investigation and treatment.
Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease of unknown aetiology. It has a very poor prognosis and no effective treatment. There are two major barriers to the development of ...novel treatments in IPF: an incomplete understanding of its pathogenesis and the fact that current models of the disease are poorly predictive of therapeutic response. Recent studies suggest an important role for the alveolar epithelium in the pathogenesis of IPF. However, practical limitations associated with isolation and culture of primary alveolar epithelial cells have hampered progress towards further elucidating their role in the pathogenesis of the disease or developing disease models that accurately reflect the epithelial contribution. The practical limitations of primary alveolar epithelial cell culture can be divided into technical, logistical and regulatory hurdles that need to be overcome to ensure rapid progress towards improved treatment for patients with IPF. To develop a strategy to facilitate alveolar epithelial cell harvest, retrieval and sharing between IPF research groups and to determine how these cells contribute to IPF, a workshop was organised to discuss the central issues surrounding epithelial cells in IPF (ECIPF). The central themes discussed in the workshop have been compiled as the proceedings of the ECIPF.
Claudins are a family of integral tight junction proteins that regulate paracellular permeability in polarized epithelia. Overexpression or reduction of claudins can both promote and limit cancer ...progression, revealing complex dichotomous roles for claudins depending on cellular context. In contrast, recent studies demonstrating tumor formation in claudin knockout mouse models indicate a role for several claudin family members in suppressing tumor initiation. For example, intestine-specific claudin-7 knockout mice spontaneously develop atypical hyperplasia and intestinal adenomas, while claudin-18 knockout mice develop carcinomas in the lung and stomach. Claudin-4, -11, and -15 knockout mice show increased cell proliferation and/or hyperplasia in urothelium, Sertoli cells, and small intestinal crypts, respectively, possibly a precursor to cancer development. Pathways implicated in both cell proliferation and tumorigenesis include Yap/Taz and insulin-like growth factor-1 receptor (IGF-1R)/Akt pathways, among others. Consistent with the tumor suppressive role of claudins shown in mice, in humans, claudin-low breast cancer has been described as a distinct entity with a poor prognosis, and claudin-18-Rho GTPase activating protein 26 (CLDN18-ARHGAP26) fusion protein as a driver gene aberration in diffuse-type gastric cancer due to effects on RhoA. Paradoxically, claudins have also garnered interest as targets for therapy, as they are sometimes aberrantly expressed in cancer cells, which may or may not promote cancer progression. For example, a chimeric monoclonal antibody which targets cells expressing claudin-18.2 through antibody-dependent cell-mediated cytotoxicity has shown promise in multiple phase II studies. In this review, we focus on new findings supporting a tumor suppressive role for claudins during cancer initiation.
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