The lung microvasculature is essential for gas exchange and commonly considered homogeneous. We show that VEGFA from the epithelium is required for a distinct endothelial cell (EC) population in the ...mouse lung. Vegfa is predominantly expressed by alveolar type 1 (AT1) cells and locally required to specify a subset of ECs. Single-cell RNA sequencing (scRNA-seq) reveals that ∼15% of lung ECs are transcriptionally distinct—marked by Carbonic anhydrase 4 (Car4)—and arise from bulk ECs, as suggested by trajectory analysis. Car4 ECs have extensive cellular projections and are separated from AT1 cells by a limited basement membrane without intervening pericytes. Car4 ECs are specifically lost upon epithelial Vegfa deletion; without Car4 ECs, the alveolar space is aberrantly enlarged despite the normal appearance of myofibroblasts. Lung Car4 ECs and retina tip ECs have common and distinct features. These findings support a signaling role of AT1 cells and shed light on alveologenesis.
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•AT1 epithelial cells express VEGFA, which promotes local alveolar angiogenesis•scRNA-seq identifies a molecularly distinct lung EC population, labeled by CAR4•CAR4 ECs display extended morphology and are lost upon epithelial Vegfa deletion•CAR4 ECs contribute to alveolar morphogenesis independent of myofibroblasts
Using imaging and single-cell RNA-seq, Vila Ellis et al. identify a lung endothelial cell (EC) population with a unique transcriptome, location, morphology, and function in lung development. These ECs are involved in alveolar morphogenesis independent of myofibroblasts.
Alveolar epithelial cell (AEC) injury and dysregulated repair are implicated in the pathogenesis of pulmonary fibrosis. Endoplasmic reticulum (ER) stress in AEC has been observed in idiopathic ...pulmonary fibrosis (IPF), a disease of aging.
To investigate a causal role for ER stress in the pathogenesis of pulmonary fibrosis (PF) and therapeutic potential of ER stress inhibition in PF.
The role of ER stress in AEC dysfunction and fibrosis was studied in mice with tamoxifen (Tmx)-inducible deletion of ER chaperone
, a key regulator of ER homeostasis, in alveolar type II (AT2) cells, progenitors of distal lung epithelium, and in IPF lung slice cultures.
deletion caused weight loss, mortality, lung inflammation, and spatially heterogeneous fibrosis characterized by fibroblastic foci, hyperplastic AT2 cells, and increased susceptibility of old and male mice, all features of IPF. Fibrosis was more persistent in more severely injured
knockout (KO) mice.
KO AT2 cells showed evidence of ER stress, apoptosis, senescence, impaired progenitor capacity, and activation of TGF-β (transforming growth factor-β)/SMAD signaling. Glucose-regulated protein 78 is reduced in AT2 cells from old mice and patients with IPF, and ER stress inhibitor tauroursodeoxycholic acid ameliorates ER stress and fibrosis in
KO mouse and IPF lung slice cultures.
These results support a causal role for ER stress and resulting epithelial dysfunction in PF and suggest ER stress as a potential mechanism linking aging to IPF. Modulation of ER stress and chaperone function may offer a promising therapeutic approach for pulmonary fibrosis.
Lesions and neurologic disability in inflammatory CNS diseases such as multiple sclerosis (MS) result from the translocation of leukocytes and humoral factors from the vasculature, first across the ...endothelial blood-brain barrier (BBB) and then across the astrocytic glia limitans (GL). Factors secreted by reactive astrocytes open the BBB by disrupting endothelial tight junctions (TJs), but the mechanisms that control access across the GL are unknown. Here, we report that in inflammatory lesions, a second barrier composed of reactive astrocyte TJs of claudin 1 (CLDN1), CLDN4, and junctional adhesion molecule A (JAM-A) subunits is induced at the GL. In a human coculture model, CLDN4-deficient astrocytes were unable to control lymphocyte segregation. In models of CNS inflammation and MS, mice with astrocyte-specific Cldn4 deletion displayed exacerbated leukocyte and humoral infiltration, neuropathology, motor disability, and mortality. These findings identify a second inducible barrier to CNS entry at the GL. This barrier may be therapeutically targetable in inflammatory CNS disease.
The extraordinarily thin alveolar type 1 (AT1) cell constitutes nearly the entire gas exchange surface and allows passive diffusion of oxygen into the blood stream. Despite such an essential role, ...the transcriptional network controlling AT1 cells remains unclear. Using cell-specific knockout mouse models, genomic profiling, and 3D imaging, we found that NK homeobox 2-1 (Nkx2-1) is expressed in AT1 cells and is required for the development and maintenance of AT1 cells. Without Nkx2-1, developing AT1 cells lose 3 defining features— molecular markers, expansive morphology, and cellular quiescence— leading to alveolar simplification and lethality. NKX2-1 is also cellautonomously required for the same 3 defining features in mature AT1 cells. Intriguingly, Nkx2-1 mutant AT1 cells activate gastrointestinal (GI) genes and form dense microvilli-like structures apically. Single-cell RNA-seq supports a linear transformation of Nkx2-1 mutant AT1 cells toward a GI fate. Whole lung ChIP-seq shows NKX2-1 binding to 68% of genes that are down-regulated upon Nkx2-1 deletion, including 93% of known AT1 genes, but near-background binding to up-regulated genes. Our results place NKX2-1 at the top of the AT1 cell transcriptional hierarchy and demonstrate remarkable plasticity of an otherwise terminally differentiated cell type.
Claudins, the integral tight junction (TJ) proteins that regulate paracellular permeability and cell polarity, are frequently dysregulated in cancer; however, their role in neoplastic progression is ...unclear. Here, we demonstrated that knockout of Cldn18, a claudin family member highly expressed in lung alveolar epithelium, leads to lung enlargement, parenchymal expansion, increased abundance and proliferation of known distal lung progenitors, the alveolar epithelial type II (AT2) cells, activation of Yes-associated protein (YAP), increased organ size, and tumorigenesis in mice. Inhibition of YAP decreased proliferation and colony-forming efficiency (CFE) of Cldn18-/- AT2 cells and prevented increased lung size, while CLDN18 overexpression decreased YAP nuclear localization, cell proliferation, CFE, and YAP transcriptional activity. CLDN18 and YAP interacted and colocalized at cell-cell contacts, while loss of CLDN18 decreased YAP interaction with Hippo kinases p-LATS1/2. Additionally, Cldn18-/- mice had increased propensity to develop lung adenocarcinomas (LuAd) with age, and human LuAd showed stage-dependent reduction of CLDN18.1. These results establish CLDN18 as a regulator of YAP activity that serves to restrict organ size, progenitor cell proliferation, and tumorigenesis, and suggest a mechanism whereby TJ disruption may promote progenitor proliferation to enhance repair following injury.
Activation of an apical Ca ²⁺-activated Cl ⁻ channel (CaCC) triggers the secretion of saliva. It was previously demonstrated that CaCC-mediated Cl ⁻ current and Cl ⁻ efflux are absent in the acinar ...cells of systemic Tmem16A (Tmem16A Cl ⁻ channel) null mice, but salivation was not assessed in fully developed glands because Tmem16A null mice die within a few days after birth. To test the role of Tmem16A in adult salivary glands, we generated conditional knockout mice lacking Tmem16A in acinar cells ( Tmem16A ⁻/⁻). Ca ²⁺-dependent salivation was abolished in Tmem16A ⁻/⁻ mice, demonstrating that Tmem16A is obligatory for Ca ²⁺-mediated fluid secretion. However, the amount of saliva secreted by Tmem16A ⁻/⁻ mice in response to the β-adrenergic receptor agonist isoproterenol (IPR) was comparable to that seen in controls, indicating that Tmem16A does not significantly contribute to cAMP-induced secretion. Furthermore, IPR-stimulated secretion was unaffected in mice lacking Cftr (Cftr F⁵⁰⁸/F⁵⁰⁸) or ClC-2 (Clcn2 ⁻/⁻) Cl ⁻ channels. The time course for activation of IPR-stimulated fluid secretion closely correlated with that of the IPR-induced cell volume increase, suggesting that acinar swelling may activate a volume-sensitive Cl ⁻ channel. Indeed, Cl ⁻ channel blockers abolished fluid secretion, indicating that Cl ⁻ channel activity is critical for IPR-stimulated secretion. These data suggest that β-adrenergic–induced, cAMP-dependent fluid secretion involves a volume-regulated anion channel. In summary, our results using acinar-specific Tmem16A ⁻/⁻ mice identify Tmem16A as the Cl ⁻ channel essential for muscarinic, Ca ²⁺-dependent fluid secretion in adult mouse salivary glands.
Significance There are no effective treatments for dry mouth, a common problem that affects millions of people in the United States. Salivary gland hyposecretion is caused by multiple diseases, including Sjáñögren's syndrome, as well as by radiation therapy and medications. The resulting dry mouth is often associated with dysphagia, reduced taste sensation, and opportunistic infections. The mammalian fluid secretion model predicts that Ca ²⁺-activated Tmem16A and/or cAMP-dependent Cftr Cl ⁻ channels are critical in this process. We demonstrate that activation of the Tmem16A channel is required for muscarinic, Ca ²⁺-dependent salivation, but that β-adrenergic receptor-mediated salivation is independent of Tmem16A, Cftr, and ClC-2 Cl ⁻ channels. A better understanding of the β-adrenergic stimulated secretion mechanism may prove important in the development of therapeutic targets for dry mouth.
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.
Claudins are a family of transmembrane proteins integral to the structure and function of tight junctions (TJ). Disruption of TJ and alterations in claudin expression are important features of ...invasive and metastatic cancer cells. Expression of CLDN18.1, the lung‐specific isoform of CLDN18, is markedly decreased in lung adenocarcinoma (LuAd). Furthermore, we recently observed that aged Cldn18
−/− mice have increased propensity to develop LuAd. We now demonstrate that CLDN18.1 expression correlates inversely with promoter methylation and with LuAd patient mortality. In addition, when restored in LuAd cells that have lost expression, CLDN18.1 markedly attenuates malignant properties including xenograft tumor growth in vivo as well as cell proliferation, migration, invasion and anchorage‐independent colony formation in vitro. Based on high throughput analyses of Cldn18
−/− murine lung alveolar epithelial type II cells, as well as CLDN18.1‐repleted human LuAd cells, we hypothesized and subsequently confirmed by Western analysis that CLDN18.1 inhibits insulin‐like growth factor‐1 receptor (IGF‐1R) and AKT phosphorylation. Consistent with recent data in Cldn18
−/− knockout mice, expression of CLDN18.1 in human LuAd cells also decreased expression of transcriptional co‐activator with PDZ‐binding motif (TAZ) and Yes‐associated protein (YAP) and their target genes, contributing to its tumor suppressor activity. Moreover, analysis of LuAd cells in which YAP and/or TAZ are silenced with siRNA suggests that inhibition of TAZ, and possibly YAP, is also involved in CLDN18.1‐mediated AKT inactivation. Taken together, these data indicate a tumor suppressor role for CLDN18.1 in LuAd mediated by a regulatory network that encompasses YAP/TAZ, IGF‐1R and AKT signaling.
What's new?
Mice deficient in expression of the lung‐specific claudin, CLDN18.1, develop lung cancer but insight into the precise role of this tight junction protein in lung cancer suppression remains limited. Here the authors show that CLDN18.1 expression in human lung adenocarcinomas is inversely correlated with promoter methylation and patient mortality. Restoration of CLDN18.1 expression in human lung cancer cells suppresses cell proliferation and impairs the protein kinase B/AKT signaling pathway, implicating CLDN18.1 as a new prognostic marker and therapeutic target in lung adenocarcinoma patients.
Lung adenocarcinoma (LUAD) is the most prevalent subtype of lung cancer and presents clinically with a high degree of biological heterogeneity and distinct clinical outcomes. The current paradigm of ...LUAD etiology posits alveolar epithelial type II (AT2) cells as the primary cell of origin, while the role of AT1 cells in LUAD oncogenesis remains unknown. Here, we examine oncogenic transformation in mouse Gram-domain containing 2 (Gramd2)+ AT1 cells via oncogenic KRASG12D. Activation of KRASG12D in AT1 cells induces multifocal LUAD, primarily of papillary histology. Furthermore, KRT8+ intermediate cell states were observed in both AT2- and AT1-derived LUAD, but SCGB3A2+, another intermediate cell marker, was primarily associated with AT1 cells, suggesting different mechanisms of tumor evolution. Collectively, our study reveals that Gramd2+ AT1 cells can serve as a cell of origin for LUAD and suggests that distinct subtypes of LUAD based on cell of origin be considered in the development of therapeutics.
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•AT1 cells can give rise to KRAS-driven LUAD using transgenic mouse models•Alveolar cell of origin influences histologic presentation of LUAD•KRT8+ intermediate cell state marker is associated with both subtypes of LUAD•KRT8+ cells were SCGB3A2+ only in AT1-derived LUAD
Yang et al. show that alveolar epithelial type I (AT1) cells can serve as a cell of origin for lung adenocarcinoma (LUAD). In addition, cell of origin can be associated with known properties of LUAD, including histologic subtypes and transcriptomic signatures, suggesting different mechanisms in tumor evolution.
Epigenetic regulation of differentiation-related genes is poorly understood. We previously reported that transcription factors GATA6 and Sp1 interact with and activate the rat proximal 358-bp ...promoter/enhancer (p358P/E) of lung alveolar epithelial type I (AT1) cell-specific gene aquaporin-5 (Aqp5). In this study, we found that histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) increased AQP5 expression and Sp1-mediated transcription of p358P/E. HDAC3 overexpression inhibited Sp1-mediated Aqp5 activation, while HDAC3 knockdown augmented AQP5 protein expression. Knockdown of GATA6 or transcriptional co-activator/histone acetyltransferase p300 decreased AQP5 expression, while p300 overexpression enhanced p358P/E activation by GATA6 and Sp1. GATA6 overexpression, SAHA treatment or HDAC3 knockdown increased histone H3 (H3) but not histone H4 (H4) acetylation within the homologous p358P/E region of mouse Aqp5. HDAC3 binds to Sp1 and HDAC3 knockdown increased interaction of GATA6/Sp1, GATA6/p300 and Sp1/p300. These results indicate that GATA6 and HDAC3 control Aqp5 transcription via modulation of H3 acetylation/deacetylation, respectively, through competition for binding to Sp1, and suggest that p300 modulates acetylation and/or interacts with GATA6/Sp1 to regulate Aqp5 transcription. Cooperative interactions among transcription factors and histone modifications regulate Aqp5 expression during alveolar epithelial cell transdifferentiation, suggesting that HDAC inhibitors may enhance repair by promoting acquisition of AT1 cell phenotype.
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