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1.
Stem Cells ; 40(7): 691-703, 2022 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-35429397

RESUMO

Lung maturation is not limited to proper structural development but also includes differentiation and functionality of various highly specialized alveolar cell types. Alveolar type 1 (AT1s) cells occupy nearly 95% of the alveolar surface and are critical for establishing efficient gas exchange in the mature lung. AT1 cells arise from progenitors specified during the embryonic stage as well as alveolar epithelial progenitors expressing surfactant protein C (Sftpcpos cells) during postnatal and adult stages. Previously, we found that Wnt5a, a non-canonical Wnt ligand, is required for differentiation of AT1 cells during the saccular phase of lung development. To further investigate the role of Wnt5a in AT1 cell differentiation, we generated and characterized a conditional Wnt5a gain-of-function mouse model. Neonatal Wnt5a gain-of-function disrupted alveologenesis through inhibition of cell proliferation. In this setting Wnt5a downregulated ß-catenin-dependent canonical Wnt signaling, repressed AT2 (anti-AT2) and promoted AT1 (pro-AT1) lineage-specific gene expression. In addition, we identified 2 subpopulations of Sftpchigh and Sftpclow alveolar epithelial cells. In Sftpclow cells, Wnt5a exhibits pro-AT1 and anti-AT2 effects, concurrent with inhibition of canonical Wnt signaling. Interestingly, in the Sftpchigh subpopulation, although increasing AT1 lineage-specific gene expression, Wnt5a gain-of-function did not change AT2 gene expression, nor inhibit canonical Wnt signaling. Using primary epithelial cells isolated from human fetal lungs, we demonstrate that this property of Wnt5a is evolutionarily conserved. Wnt5a therefore serves as a selective regulator that ensures proper AT1/AT2 balance in the developing lung.


Assuntos
Células Epiteliais Alveolares , Via de Sinalização Wnt , Células Epiteliais Alveolares/metabolismo , Animais , Diferenciação Celular/genética , Células Epiteliais/metabolismo , Expressão Gênica , Humanos , Recém-Nascido , Camundongos , Via de Sinalização Wnt/genética , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo
2.
PLoS Genet ; 16(9): e1009023, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32925947

RESUMO

Lung cancer is the leading cause of cancer-related death and lung adenocarcinoma is its most common subtype. Although genetic alterations have been identified as drivers in subsets of lung adenocarcinoma, they do not fully explain tumor development. Epigenetic alterations have been implicated in the pathogenesis of tumors. To identify epigenetic alterations driving lung adenocarcinoma, we used an improved version of the Tracing Enhancer Networks using Epigenetic Traits method (TENET 2.0) in primary normal lung and lung adenocarcinoma cells. We found over 32,000 enhancers that appear differentially activated between normal lung and lung adenocarcinoma. Among the identified transcriptional regulators inactivated in lung adenocarcinoma vs. normal lung, NKX2-1 was linked to a large number of silenced enhancers. Among the activated transcriptional regulators identified, CENPA, FOXM1, and MYBL2 were linked to numerous cancer-specific enhancers. High expression of CENPA, FOXM1, and MYBL2 is particularly observed in a subgroup of lung adenocarcinomas and is associated with poor patient survival. Notably, CENPA, FOXM1, and MYBL2 are also key regulators of cancer-specific enhancers in breast adenocarcinoma of the basal subtype, but they are associated with distinct sets of activated enhancers. We identified individual lung adenocarcinoma enhancers linked to CENPA, FOXM1, or MYBL2 that were associated with poor patient survival. Knockdown experiments of FOXM1 and MYBL2 suggest that these factors regulate genes involved in controlling cell cycle progression and cell division. For example, we found that expression of TK1, a potential target gene of a MYBL2-linked enhancer, is associated with poor patient survival. Identification and characterization of key transcriptional regulators and associated enhancers in lung adenocarcinoma provides important insights into the deregulation of lung adenocarcinoma epigenomes, highlighting novel potential targets for clinical intervention.


Assuntos
Adenocarcinoma de Pulmão/genética , Epigênese Genética/genética , Elementos Reguladores de Transcrição/genética , Adenocarcinoma/genética , Adulto , Idoso , Proteínas de Ciclo Celular/genética , Epigenômica , Proteína Forkhead Box M1/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes Homeobox , Humanos , Pulmão/metabolismo , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Sequências Reguladoras de Ácido Nucleico/genética
3.
Am J Respir Crit Care Med ; 201(2): 198-211, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31738079

RESUMO

Rationale: 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.Objectives: To investigate a causal role for ER stress in the pathogenesis of pulmonary fibrosis (PF) and therapeutic potential of ER stress inhibition in PF.Methods: The role of ER stress in AEC dysfunction and fibrosis was studied in mice with tamoxifen (Tmx)-inducible deletion of ER chaperone Grp78, a key regulator of ER homeostasis, in alveolar type II (AT2) cells, progenitors of distal lung epithelium, and in IPF lung slice cultures.Measurements and Main Results:Grp78 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 Grp78 knockout (KO) mice. Grp78 KO AT2 cells showed evidence of ER stress, apoptosis, senescence, impaired progenitor capacity, and activation of TGF-ß (transforming growth factor-ß)/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 Grp78 KO mouse and IPF lung slice cultures.Conclusions: 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.


Assuntos
Células Epiteliais Alveolares/metabolismo , Estresse do Retículo Endoplasmático/genética , Proteínas de Choque Térmico/genética , Fibrose Pulmonar/genética , Células-Tronco/metabolismo , Fatores Etários , Células Epiteliais Alveolares/patologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Antioxidantes/farmacologia , Apoptose/genética , Senescência Celular/genética , Dasatinibe/farmacologia , Chaperona BiP do Retículo Endoplasmático , Técnicas de Inativação de Genes , Proteínas de Choque Térmico/metabolismo , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/efeitos dos fármacos , Glicoproteínas de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Inibidores de Proteínas Quinases/farmacologia , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Quercetina/farmacologia , Quinolinas/farmacologia , Proteínas Smad/metabolismo , Ácido Tauroquenodesoxicólico/farmacologia , Fator de Transcrição CHOP/efeitos dos fármacos , Fator de Transcrição CHOP/metabolismo , Fator de Crescimento Transformador beta/metabolismo
4.
Am J Physiol Lung Cell Mol Physiol ; 319(1): L173-L184, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32432919

RESUMO

The alveolar epithelium is comprised of two cell types, alveolar epithelial type 1 (AT1) and type 2 (AT2) cells, the latter being capable of self-renewal and transdifferentiation into AT1 cells for normal maintenance and restoration of epithelial integrity following injury. MicroRNAs (miRNAs) are critical regulators of several biological processes, including cell differentiation; however, their role in establishment/maintenance of cellular identity in adult alveolar epithelium is not well understood. To investigate this question, we performed genome-wide analysis of sequential changes in miRNA and gene expression profiles using a well-established model in which human AT2 (hAT2) cells transdifferentiate into AT1-like cells over time in culture that recapitulates many aspects of transdifferentiation in vivo. We defined three phases of miRNA expression during the transdifferentiation process as "early," "late," and "consistently" changed, which were further subclassified as up- or downregulated. miRNAs with altered expression at all time points during transdifferentiation were the largest subgroup, suggesting the need for consistent regulation of signaling pathways to mediate this process. Target prediction analysis and integration with previously published gene expression data identified glucocorticoid signaling as the top pathway regulated by miRNAs. Serum/glucocorticoid-regulated kinase 1 (SGK1) emerged as a central regulatory factor, whose downregulation correlated temporally with gain of hsa-miR-424 and hsa-miR-503 expression. Functional validation demonstrated specific targeting of these miRNAs to the 3'-untranslated region of SGK1. These data demonstrate the time-related contribution of miRNAs to the alveolar transdifferentiation process and suggest that inhibition of glucocorticoid signaling is necessary to achieve the AT1-like cell phenotype.


Assuntos
Diferenciação Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Genoma Humano , MicroRNAs/metabolismo , Alvéolos Pulmonares/metabolismo , Transcriptoma/genética , Sequência de Bases , Diferenciação Celular/genética , Linhagem Celular , Transdiferenciação Celular/genética , Regulação da Expressão Gênica , Glucocorticoides/metabolismo , Humanos , Proteínas Imediatamente Precoces/metabolismo , MicroRNAs/genética , Proteínas Serina-Treonina Quinases/metabolismo
5.
Cell Mol Life Sci ; 76(23): 4663-4672, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31332482

RESUMO

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.


Assuntos
Claudinas/genética , Neoplasias/etiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Anticorpos Monoclonais/uso terapêutico , Carcinogênese , Claudinas/metabolismo , Camundongos , Camundongos Knockout , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Fatores de Transcrição/metabolismo
6.
Hum Mol Genet ; 26(15): 3014-3027, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28854564

RESUMO

Smoking-associated DNA hypomethylation has been observed in blood cells and linked to lung cancer risk. However, its cause and mechanistic relationship to lung cancer remain unclear. We studied the association between tobacco smoking and epigenome-wide methylation in non-tumor lung (NTL) tissue from 237 lung cancer cases in the Environment And Genetics in Lung cancer Etiology study, using the Infinium HumanMethylation450 BeadChip. We identified seven smoking-associated hypomethylated CpGs (P < 1.0 × 10-7), which were replicated in NTL data from The Cancer Genome Atlas. Five of these loci were previously reported as hypomethylated in smokers' blood, suggesting that blood-based biomarkers can reflect changes in the target tissue for these loci. Four CpGs border sequences carrying aryl hydrocarbon receptor binding sites and enhancer-specific histone modifications in primary alveolar epithelium and A549 lung adenocarcinoma cells. A549 cell exposure to cigarette smoke condensate increased these enhancer marks significantly and stimulated expression of predicted target xenobiotic response-related genes AHRR (P = 1.13 × 10-62) and CYP1B1 (P < 2.49 × 10-61). Expression of both genes was linked to smoking-related transversion mutations in lung tumors. Thus, smoking-associated hypomethylation may be a consequence of enhancer activation, revealing environmentally-induced regulatory elements implicated in lung carcinogenesis.


Assuntos
Ilhas de CpG/genética , Neoplasias Pulmonares/genética , Fumar/efeitos adversos , Células A549/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores Tumorais/sangue , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Metilação de DNA/genética , Elementos Facilitadores Genéticos/genética , Epigênese Genética/genética , Epigenômica/métodos , Estudo de Associação Genômica Ampla , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fumar/genética , Nicotiana
7.
Int J Cancer ; 143(12): 3169-3180, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30325015

RESUMO

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.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Claudinas/fisiologia , Neoplasias Pulmonares/metabolismo , Transdução de Sinais/fisiologia , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Animais , Western Blotting , Proliferação de Células , Claudinas/genética , Metilação de DNA , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Knockout , Invasividade Neoplásica , Metástase Neoplásica , Fosforilação , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-yes/metabolismo , Receptor IGF Tipo 1/metabolismo , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional
8.
Am J Respir Cell Mol Biol ; 56(3): 310-321, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27749084

RESUMO

Diseases involving the distal lung alveolar epithelium include chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and lung adenocarcinoma. Accurate labeling of specific cell types is critical for determining the contribution of each to the pathogenesis of these diseases. The distal lung alveolar epithelium is composed of two cell types, alveolar epithelial type 1 (AT1) and type 2 (AT2) cells. Although cell type-specific markers, most prominently surfactant protein C, have allowed detailed lineage tracing studies of AT2 cell differentiation and the cells' roles in disease, studies of AT1 cells have been hampered by a lack of genes with expression unique to AT1 cells. In this study, we performed genome-wide expression profiling of multiple rat organs together with purified rat AT2, AT1, and in vitro differentiated AT1-like cells, resulting in the identification of 54 candidate AT1 cell markers. Cross-referencing with genes up-regulated in human in vitro differentiated AT1-like cells narrowed the potential list to 18 candidate genes. Testing the top four candidate genes at RNA and protein levels revealed GRAM domain 2 (GRAMD2), a protein of unknown function, as highly specific to AT1 cells. RNA sequencing (RNAseq) confirmed that GRAMD2 is transcriptionally silent in human AT2 cells. Immunofluorescence verified that GRAMD2 expression is restricted to the plasma membrane of AT1 cells and is not expressed in bronchial epithelial cells, whereas reverse transcription-polymerase chain reaction confirmed that it is not expressed in endothelial cells. Using GRAMD2 as a new AT1 cell-specific gene will enhance AT1 cell isolation, the investigation of alveolar epithelial cell differentiation potential, and the contribution of AT1 cells to distal lung diseases.


Assuntos
Células Epiteliais Alveolares/metabolismo , Perfilação da Expressão Gênica , Especificidade de Órgãos/genética , Animais , Biomarcadores/metabolismo , Canais Epiteliais de Sódio/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Reprodutibilidade dos Testes , Especificidade da Espécie
9.
J Biol Chem ; 291(12): 6569-82, 2016 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-26833564

RESUMO

Maintenance of stem/progenitor cell-progeny relationships is required for tissue homeostasis during normal turnover and repair. Wnt signaling is implicated in both maintenance and differentiation of adult stem/progenitor cells, yet how this pathway serves these dichotomous roles remains enigmatic. We previously proposed a model suggesting that specific interaction of ß-catenin with either of the homologous Kat3 co-activators, p300 or CREB-binding protein, differentially regulates maintenance versus differentiation of embryonic stem cells. Limited knowledge of endogenous mechanisms driving differential ß-catenin/co-activator interactions and their role in adult somatic stem/progenitor cell maintenance versus differentiation led us to explore this process in defined models of adult progenitor cell differentiation. We focused primarily on alveolar epithelial type II (AT2) cells, progenitors of distal lung epithelium, and identified a novel axis whereby WNT5a/protein kinase C (PKC) signaling regulates specific ß-catenin/co-activator interactions to promote adult progenitor cell differentiation. p300/ß-catenin but not CBP/ß-catenin interaction increases as AT2 cells differentiate to a type I (AT1) cell-like phenotype. Additionally, p300 transcriptionally activates AT1 cell-specific gene Aqp-5. IQ-1, a specific inhibitor of p300/ß-catenin interaction, prevents differentiation of not only primary AT2 cells, but also tracheal epithelial cells, and C2C12 myoblasts. p300 phosphorylation at Ser-89 enhances p300/ß-catenin interaction, concurrent with alveolar epithelial cell differentiation. WNT5a, a traditionally non-canonical WNT ligand regulates Ser-89 phosphorylation and p300/ß-catenin interactions in a PKC-dependent manner, likely involving PKCζ. These studies identify a novel intersection of canonical and non-canonical Wnt signaling in adult progenitor cell differentiation that has important implications for targeting ß-catenin to modulate adult progenitor cell behavior in disease.


Assuntos
Células-Tronco Adultas/fisiologia , Diferenciação Celular , Proteína p300 Associada a E1A/fisiologia , Proteína Quinase C/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/fisiologia , Células Epiteliais Alveolares/fisiologia , Animais , Aquaporina 5/genética , Aquaporina 5/metabolismo , Linhagem Celular , Impedância Elétrica , Expressão Gênica , Camundongos , Camundongos Knockout , Fosforilação , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Ratos , Via de Sinalização Wnt , Proteína Wnt-5a
10.
Am J Physiol Lung Cell Mol Physiol ; 312(1): L131-L142, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-27864284

RESUMO

Previous studies have demonstrated resistance to naphthalene-induced injury in proximal airways of mice with lung epithelial-specific deletion of the tumor-suppressor gene Pten, attributed to increased proliferation of airway progenitors. We tested effects of Pten loss following bleomycin injury, a model typically used to study distal lung epithelial injury, in conditional PtenSFTPC-cre knockout mice. Pten-deficient airway epithelium exhibited marked hyperplasia, particularly in small bronchioles and at bronchoalveolar duct junctions, with reduced E-cadherin and ß-catenin expression between cells toward the luminal aspect of the hyperplastic epithelium. Bronchiolar epithelial and alveolar epithelial type II (AT2) cells in PtenSFTPC-cre mice showed decreased expression of epithelial markers and increased expression of mesenchymal markers, suggesting at least partial epithelial-mesenchymal transition at baseline. Surprisingly, and in contrast to previous studies, mutant mice were exquisitely sensitive to bleomycin, manifesting rapid weight loss, respiratory distress, increased early mortality (by day 5), and reduced dynamic lung compliance. This was accompanied by sloughing of the hyperplastic airway epithelium with occlusion of small bronchioles by cellular debris, without evidence of increased parenchymal lung injury. Increased airway epithelial cell apoptosis due to loss of antioxidant defenses, reflected by decreased expression of superoxide dismutase 3, in combination with deficient intercellular adhesion, likely predisposed to airway sloughing in knockout mice. These findings demonstrate an important role for Pten in maintenance of airway epithelial phenotype integrity and indicate that responses to Pten deletion in respiratory epithelium following acute lung injury are highly context-dependent and region-specific.


Assuntos
Células Epiteliais/metabolismo , Especificidade de Órgãos , PTEN Fosfo-Hidrolase/metabolismo , Mucosa Respiratória/metabolismo , Animais , Apoptose , Biomarcadores/metabolismo , Bleomicina , Caderinas/metabolismo , Complacência (Medida de Distensibilidade) , Regulação da Expressão Gênica , Hiperplasia , Marcação In Situ das Extremidades Cortadas , Inflamação/patologia , Integrases/metabolismo , Junções Intercelulares/metabolismo , Pulmão/patologia , Pulmão/fisiopatologia , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Lesão Pulmonar/fisiopatologia , Mesoderma/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , PTEN Fosfo-Hidrolase/deficiência , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coloração e Rotulagem , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
11.
Am J Respir Cell Mol Biol ; 55(1): 135-49, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26816051

RESUMO

Bronchopulmonary dysplasia (BPD), a chronic lung disease of prematurity, has been linked to endoplasmic reticulum (ER) stress. To investigate a causal role for ER stress in BPD pathogenesis, we generated conditional knockout (KO) mice (cGrp78(f/f)) with lung epithelial cell-specific KO of Grp78, a gene encoding the ER chaperone 78-kD glucose-regulated protein (GRP78), a master regulator of ER homeostasis and the unfolded protein response (UPR). Lung epithelial-specific Grp78 KO disrupted lung morphogenesis, causing developmental arrest, increased alveolar epithelial type II cell apoptosis, and decreased surfactant protein and type I cell marker expression in perinatal lungs. cGrp78(f/f) pups died immediately after birth, likely owing to respiratory distress. Importantly, Grp78 KO triggered UPR activation with marked induction of the proapoptotic transcription factor CCAAT/enhancer-binding proteins (C/EBP) homologous protein (CHOP). Increased expression of genes involved in oxidative stress and cell death and decreased expression of genes encoding antioxidant enzymes suggest a role for oxidative stress in alveolar epithelial cell (AEC) apoptosis. Increased Smad3 phosphorylation and expression of transforming growth factor-ß/Smad3 targets Cdkn1a (encoding p21) and Gadd45a suggest that interactions among the apoptotic arm of the UPR, oxidative stress, and transforming growth factor-ß/Smad signaling pathways contribute to Grp78 KO-induced AEC apoptosis and developmental arrest. Chemical chaperone Tauroursodeoxycholic acid reduced UPR activation and apoptosis in cGrp78(f/f) lungs cultured ex vivo, confirming a role for ER stress in observed AEC abnormalities. These results demonstrate a key role for GRP78 in AEC survival and gene expression during lung development through modulation of ER stress, and suggest the UPR as a potential therapeutic target in BPD.


Assuntos
Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/metabolismo , Homeostase , Células Epiteliais Alveolares/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ácido Tauroquenodesoxicólico/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos
12.
Am J Physiol Lung Cell Mol Physiol ; 310(2): L114-20, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26545903

RESUMO

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.


Assuntos
Células Epiteliais Alveolares/citologia , Antígenos de Diferenciação/metabolismo , Transdiferenciação Celular/fisiologia , Células Epiteliais/citologia , Alvéolos Pulmonares/citologia , Envelhecimento , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Células Epiteliais/metabolismo , Camundongos , Ratos
13.
PLoS Genet ; 9(6): e1003513, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23818859

RESUMO

Elucidation of the epigenetic basis for cell-type specific gene regulation is key to gaining a full understanding of how the distinct phenotypes of differentiated cells are achieved and maintained. Here we examined how epigenetic changes are integrated with transcriptional activation to determine cell phenotype during differentiation. We performed epigenomic profiling in conjunction with transcriptomic profiling using in vitro differentiation of human primary alveolar epithelial cells (AEC). This model recapitulates an in vivo process in which AEC transition from one differentiated cell type to another during regeneration following lung injury. Interrogation of histone marks over time revealed enrichment of specific transcription factor binding motifs within regions of changing chromatin structure. Cross-referencing of these motifs with pathways showing transcriptional changes revealed known regulatory pathways of distal alveolar differentiation, such as the WNT and transforming growth factor beta (TGFB) pathways, and putative novel regulators of adult AEC differentiation including hepatocyte nuclear factor 4 alpha (HNF4A), and the retinoid X receptor (RXR) signaling pathways. Inhibition of the RXR pathway confirmed its functional relevance for alveolar differentiation. Our incorporation of epigenetic data allowed specific identification of transcription factors that are potential direct upstream regulators of the differentiation process, demonstrating the power of this approach. Integration of epigenomic data with transcriptomic profiling has broad application for the identification of regulatory pathways in other models of differentiation.


Assuntos
Diferenciação Celular/genética , Linhagem da Célula/genética , Células Epiteliais , Perfilação da Expressão Gênica , Neoplasias Pulmonares/genética , Adulto , Animais , Epigenômica/métodos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Cultura Primária de Células , Ratos , Transdução de Sinais/genética , Ativação Transcricional/genética
14.
Am J Respir Cell Mol Biol ; 51(2): 210-22, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24588076

RESUMO

Claudin proteins are major constituents of epithelial and endothelial tight junctions (TJs) that regulate paracellular permeability to ions and solutes. Claudin 18, a member of the large claudin family, is highly expressed in lung alveolar epithelium. To elucidate the role of claudin 18 in alveolar epithelial barrier function, we generated claudin 18 knockout (C18 KO) mice. C18 KO mice exhibited increased solute permeability and alveolar fluid clearance (AFC) compared with wild-type control mice. Increased AFC in C18 KO mice was associated with increased ß-adrenergic receptor signaling together with activation of cystic fibrosis transmembrane conductance regulator, higher epithelial sodium channel, and Na-K-ATPase (Na pump) activity and increased Na-K-ATPase ß1 subunit expression. Consistent with in vivo findings, C18 KO alveolar epithelial cell (AEC) monolayers exhibited lower transepithelial electrical resistance and increased solute and ion permeability with unchanged ion selectivity. Claudin 3 and claudin 4 expression was markedly increased in C18 KO mice, whereas claudin 5 expression was unchanged and occludin significantly decreased. Microarray analysis revealed changes in cytoskeleton-associated gene expression in C18 KO mice, consistent with observed F-actin cytoskeletal rearrangement in AEC monolayers. These findings demonstrate a crucial nonredundant role for claudin 18 in the regulation of alveolar epithelial TJ composition and permeability properties. Increased AFC in C18 KO mice identifies a role for claudin 18 in alveolar fluid homeostasis beyond its direct contributions to barrier properties that may, at least in part, compensate for increased permeability.


Assuntos
Claudinas/metabolismo , Células Epiteliais/metabolismo , Alvéolos Pulmonares/metabolismo , Junções Íntimas/metabolismo , Animais , Células Cultivadas , Claudina-3/metabolismo , Claudina-4/metabolismo , Claudina-5/metabolismo , Claudinas/deficiência , Claudinas/genética , Citoesqueleto/metabolismo , Modelos Animais de Doenças , Impedância Elétrica , Genótipo , Homeostase , Humanos , Transporte de Íons , Camundongos , Camundongos Knockout , Ocludina/metabolismo , Permeabilidade , Fenótipo , Alvéolos Pulmonares/fisiopatologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Lesão Pulmonar Induzida por Ventilação Mecânica/genética , Lesão Pulmonar Induzida por Ventilação Mecânica/metabolismo , Lesão Pulmonar Induzida por Ventilação Mecânica/fisiopatologia
15.
Am J Physiol Lung Cell Mol Physiol ; 307(6): L449-59, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25106429

RESUMO

The molecular mechanisms for lung cell repair are largely unknown. Previous studies identified tripartite motif protein 72 (TRIM72) from striated muscle and linked its function to tissue repair. In this study, we characterized TRIM72 expression in lung tissues and investigated the role of TRIM72 in repair of alveolar epithelial cells. In vivo injury of lung cells was introduced by high tidal volume ventilation, and repair-defective cells were labeled with postinjury administration of propidium iodide. Primary alveolar epithelial cells were isolated and membrane wounding and repair were labeled separately. Our results show that absence of TRIM72 increases susceptibility to deformation-induced lung injury whereas TRIM72 overexpression is protective. In vitro cell wounding assay revealed that TRIM72 protects alveolar epithelial cells through promoting repair rather than increasing resistance to injury. The repair function of TRIM72 in lung cells is further linked to caveolin 1. These data suggest an essential role for TRIM72 in repair of alveolar epithelial cells under plasma membrane stress failure.


Assuntos
Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Membrana Celular/patologia , Células Epiteliais , Alvéolos Pulmonares , Cicatrização , Animais , Proteínas de Transporte/genética , Caveolina 1/genética , Caveolina 1/metabolismo , Membrana Celular/genética , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Proteínas de Membrana , Camundongos , Camundongos Knockout , Alvéolos Pulmonares/lesões , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia
16.
J Biol Chem ; 287(10): 7026-38, 2012 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-22241478

RESUMO

Interactions between transforming growth factor-ß (TGF-ß) and Wnt are crucial to many biological processes, although specific targets, rationale for divergent outcomes (differentiation versus block of epithelial proliferation versus epithelial-mesenchymal transition (EMT)) and precise mechanisms in many cases remain unknown. We investigated ß-catenin-dependent and transforming growth factor-ß1 (TGF-ß1) interactions in pulmonary alveolar epithelial cells (AEC) in the context of EMT and pulmonary fibrosis. We previously demonstrated that ICG-001, a small molecule specific inhibitor of the ß-catenin/CBP (but not ß-catenin/p300) interaction, ameliorates and reverses pulmonary fibrosis and inhibits TGF-ß1-mediated α-smooth muscle actin (α-SMA) and collagen induction in AEC. We now demonstrate that TGF-ß1 induces LEF/TCF TOPFLASH reporter activation and nuclear ß-catenin accumulation, while LiCl augments TGF-ß-induced α-SMA expression, further confirming co-operation between ß-catenin- and TGF-ß-dependent signaling pathways. Inhibition and knockdown of Smad3, knockdown of ß-catenin and overexpression of ICAT abrogated effects of TGF-ß1 on α-SMA transcription/expression, indicating a requirement for ß-catenin in these Smad3-dependent effects. Following TGF-ß treatment, co-immunoprecipitation demonstrated direct interaction between endogenous Smad3 and ß-catenin, while chromatin immunoprecipitation (ChIP)-re-ChIP identified spatial and temporal regulation of α-SMA via complex formation among Smad3, ß-catenin, and CBP. ICG-001 inhibited α-SMA expression/transcription in response to TGF-ß as well as α-SMA promoter occupancy by ß-catenin and CBP, demonstrating a previously unknown requisite TGF-ß1/ß-catenin/CBP-mediated pro-EMT signaling pathway. Clinical relevance was shown by ß-catenin/Smad3 co-localization and CBP expression in AEC of IPF patients. These findings suggest a new therapeutic approach to pulmonary fibrosis by specifically uncoupling CBP/catenin-dependent signaling downstream of TGF-ß.


Assuntos
Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Fibrose Pulmonar/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , beta Catenina/metabolismo , Actinas/biossíntese , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteína de Ligação a CREB , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Fibrose Pulmonar/genética , Pirimidinonas/farmacologia , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/genética , beta Catenina/genética
17.
J Pathol ; 226(4): 633-44, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21984393

RESUMO

Evidence suggests epithelial-mesenchymal transition (EMT) as one potential source of fibroblasts in idiopathic pulmonary fibrosis. To assess the contribution of alveolar epithelial cell (AEC) EMT to fibroblast accumulation in vivo following lung injury and the influence of extracellular matrix on AEC phenotype in vitro, Nkx2.1-Cre;mT/mG mice were generated in which AECs permanently express green fluorescent protein (GFP). On days 17-21 following intratracheal bleomycin administration, ~4% of GFP-positive epithelial-derived cells expressed vimentin or α-smooth muscle actin (α-SMA). Primary AECs from Nkx2.1-Cre;mT/mG mice cultured on laminin-5 or fibronectin maintained an epithelial phenotype. In contrast, on type I collagen, cells of epithelial origin displayed nuclear localization of Smad3, acquired spindle-shaped morphology, expressed α-SMA and phospho-Smad3, consistent with activation of the transforming growth factor-ß (TGFß) signalling pathway and EMT. α-SMA induction and Smad3 nuclear localization were blocked by the TGFß type I receptor (TßRI, otherwise known as Alk5) inhibitor SB431542, while AEC derived from Nkx2.1-Cre;Alk5(flox/KO) mice did not undergo EMT on collagen, consistent with a requirement for signalling via Alk5 in collagen-induced EMT. Inability of a pan-specific TGFß neutralizing antibody to inhibit effects of collagen together with absence of active TGFß in culture supernatants is consistent with TGFß ligand-independent activation of Smad signalling. These results support the notion that AECs can acquire a mesenchymal phenotype following injury in vivo and implicate type I collagen as a key regulator of EMT in AECs through signalling via Alk5, likely in a TGFß ligand-independent manner.


Assuntos
Células Epiteliais Alveolares/patologia , Colágeno Tipo I/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Fibrose Pulmonar/patologia , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Actinas/metabolismo , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Angiotensina II/metabolismo , Animais , Antibióticos Antineoplásicos/toxicidade , Benzamidas/farmacologia , Bleomicina/toxicidade , Células Cultivadas , Dioxóis/farmacologia , Modelos Animais de Doenças , Feminino , Ligantes , Masculino , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Transdução de Sinais , Vimentina/metabolismo
18.
Proc Natl Acad Sci U S A ; 107(32): 14309-14, 2010 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-20660310

RESUMO

Idiopathic pulmonary fibrosis (IPF)/usual interstitial pneumonia is a ravaging condition of progressive lung scarring and destruction. Anti-inflammatory therapies including corticosteroids have limited efficacy in this ultimately fatal disorder. An important unmet need is to identify new agents that interact with key molecular pathways involved in the pathogenesis of pulmonary fibrosis to prevent progression or reverse fibrosis in these patients. Because aberrant activation of the Wnt/beta-catenin signaling cascade occurs in lungs of patients with IPF, we have targeted this pathway for intervention in pulmonary fibrosis using ICG-001, a small molecule that specifically inhibits T-cell factor/beta-catenin transcription in a cyclic AMP response-element binding protein binding protein (CBP)-dependent fashion. ICG-001 selectively blocks the beta-catenin/CBP interaction without interfering with the beta-catenin/p300 interaction. We report here that ICG-001 (5 mg/kg per day) significantly inhibits beta-catenin signaling and attenuates bleomycin-induced lung fibrosis in mice, while concurrently preserving the epithelium. Administration of ICG-001 concurrent with bleomycin prevents fibrosis, and late administration is able to reverse established fibrosis and significantly improve survival. Because no effective treatment for IPF exists, selective inhibition of Wnt/beta-catenin-dependent transcription suggests a potential unique therapeutic approach for pulmonary fibrosis.


Assuntos
Proteína de Ligação a CREB/metabolismo , Fibrose Pulmonar/prevenção & controle , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Bleomicina/administração & dosagem , Bleomicina/toxicidade , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Camundongos , Fibrose Pulmonar/induzido quimicamente , Pirimidinonas/administração & dosagem , Pirimidinonas/farmacologia , Pirimidinonas/uso terapêutico , Transcrição Gênica/efeitos dos fármacos
19.
bioRxiv ; 2023 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-37398026

RESUMO

Lymphangioleiomyomatosis (LAM) is a debilitating, progressive lung disease with few therapeutic options, largely due to a paucity of mechanistic knowledge of disease pathogenesis. Lymphatic endothelial cells (LECs) are known to envelope and invade clusters of LAM-cells, comprising of smooth muscle α-actin and/or HMB-45 positive "smooth muscle-like cells" however the role of LECs in LAM pathogenesis is still unknown. To address this critical knowledge gap, we investigated wether LECs interact with LAM-cells to augment their metastatic behaviour of LAM-cells. We performed in situ spatialomics and identified a core of transcriptomically related cells within the LAM nodules. Pathway analysis highlights wound and pulmonary healing, VEGF signaling, extracellular matrix/actin cytoskeletal regulating and the HOTAIR regulatory pathway enriched in the LAM Core cells. We developed an organoid co-culture model combining primary LAM-cells with LECs and applied this to evaluate invasion, migration, and the impact of Sorafenib, a multi-kinase inhibitor. LAM-LEC organoids had significantly higher extracellular matrix invasion, decreased solidity and a greater perimeter, reflecting increased invasion compared to non-LAM control smooth muscle cells. Sorafenib significantly inhibited this invasion in both LAM spheroids and LAM-LEC organoids compared to their respective controls. We identified TGFß1ι1, a molecular adapter coordinating protein-protein interactions at the focal adhesion complex and known to regulate VEGF, TGFß and Wnt signalling, as a Sorafenib-regulated kinase in LAM-cells. In conclusion we have developed a novel 3D co-culture LAM model and have demonstrated the effectiveness of Sorafenib to inhibit LAM-cell invasion, identifying new avenues for therapeutic intervention.

20.
bioRxiv ; 2023 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-37905051

RESUMO

Alveolar epithelial regeneration is critical for normal lung function and becomes dysregulated in disease. While alveolar type 2 (AT2) and club cells are known distal lung epithelial progenitors, determining if alveolar epithelial type 1 (AT1) cells also contribute to alveolar regeneration has been hampered by lack of highly specific mouse models labeling AT1 cells. To address this, the Gramd2 CreERT2 transgenic strain was generated and crossed to Rosa mTmG mice. Extensive cellular characterization, including distal lung immunofluorescence and cytospin staining, confirmed that GRAMD2 + AT1 cells are highly enriched for green fluorescent protein (GFP). Interestingly, Gramd2 CreERT2 GFP + cells were able to form organoids in organoid co-culture with Mlg fibroblasts. Temporal scRNAseq revealed that Gramd2 + AT1 cells transition through numerous intermediate lung epithelial cell states including basal, secretory and AT2 cell in organoids while acquiring proliferative capacity. Our results indicate that Gramd2 + AT1 cells are highly plastic suggesting they may contribute to alveolar regeneration.

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