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1.
Cells ; 9(2)2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-32046118

RESUMO

WNT5a is a mainly "non-canonical" WNT ligand whose dysregulation is observed in lung diseases such as idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD) and asthma. Germline deletion of Wnt5a disrupts embryonic lung development. However, the temporal-specific function of WNT5a remains unknown. In this study, we generated a conditional loss-of-function mouse model (Wnt5aCAG) and examined the specific role of Wnt5a during the saccular and alveolar phases of lung development. The lack of Wnt5a in the saccular phase blocked distal airway expansion and attenuated differentiation of endothelial and alveolar epithelial type I (AT1) cells and myofibroblasts. Postnatal Wnt5a inactivation disrupted alveologenesis, producing a phenotype resembling human bronchopulmonary dysplasia (BPD). Mutant lungs showed hypoalveolization, but endothelial and epithelial differentiation was unaffected. The major impact of Wnt5a inactivation on alveologenesis was on myofibroblast differentiation and migration, with reduced expression of key regulatory genes. These findings were validated in vitro using isolated lung fibroblasts. Conditional inactivation of the WNT5a receptors Ror1 and Ror2 in alveolar myofibroblasts recapitulated the Wnt5aCAG phenotype, demonstrating that myofibroblast defects are the major cause of arrested alveologenesis in Wnt5aCAG lungs. Finally, we show that WNT5a is reduced in human BPD lung samples, indicating the clinical relevance and potential role for WNT5a in pathogenesis of BPD.

2.
Am J Respir Crit Care Med ; 201(2): 198-211, 2020 Jan 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.

3.
Stem Cell Res Ther ; 10(1): 366, 2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31791401

RESUMO

BACKGROUND: The airways of mammalian lung are lined with highly specialized cell types that are the target of airborne toxicants and injury. Several epithelial cell types and bone marrow-derived mesenchymal stem cells have been identified to serve as stem cells during injury repair. However, the contributions of endogenous mesenchymal cells to recruitment, expansion or differentiation of stem cells, and repair and reestablishment of the normal composition of airway epithelium following injury have not been addressed. METHODS: The role of mouse pulmonary mesenchymal cells was investigated by lineage tracing using Dermo1-Cre; ROSAmTmG mice. In experimental models of lung injury by lipopolysaccharide and naphthalene, GFP-labeled Dermo1+ mesenchymal cells were traced during injury repair. In vitro lung explant culture treated with or without lipopolysaccharide was also used to verify in vivo data. RESULTS: During injury repair, a subgroup of GFP-labeled Dermo1+ mesenchymal cells were found to contribute to normal repair of the airway epithelium and differentiated into Club cells, ciliated cells, and goblet cells. In Club cell-specific naphthalene injury model, the process of Dermo1+ stem cell regenerating epithelial cells was dissected. The Dermo1+ stem cells was migrated into the airway epithelium layer sooner after injury, and sequentially differentiated transitionally to epithelial stem cells, such as neuroendocrine cells, and finally to newly differentiated Club cells, ciliated cells, and goblet cells in injury repair. CONCLUSION: In this study, a population of Dermo1+ mesenchymal stem cell was identified to serve as stem cells in airway epithelial cell regeneration during injury repair. The Dermo1+ mesenchymal stem cell differentiated into epithelial stem cells before reestablishing various epithelial cells. These findings have implications for understanding the regulation of lung repair and the potential for usage of mesenchymal stem cells in therapeutic strategies for lung diseases.

4.
Development ; 146(15)2019 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-31331942

RESUMO

Postnatal alveolar formation is the most important and the least understood phase of lung development. Alveolar pathologies are prominent in neonatal and adult lung diseases. The mechanisms of alveologenesis remain largely unknown. We inactivated Pdgfra postnatally in secondary crest myofibroblasts (SCMF), a subpopulation of lung mesenchymal cells. Lack of Pdgfra arrested alveologenesis akin to bronchopulmonary dysplasia (BPD), a neonatal chronic lung disease. The transcriptome of mutant SCMF revealed 1808 altered genes encoding transcription factors, signaling and extracellular matrix molecules. Elastin mRNA was reduced, and its distribution was abnormal. Absence of Pdgfra disrupted expression of elastogenic genes, including members of the Lox, Fbn and Fbln families. Expression of EGF family members increased when Tgfb1 was repressed in mouse. Similar, but not identical, results were found in human BPD lung samples. In vitro, blocking PDGF signaling decreased elastogenic gene expression associated with increased Egf and decreased Tgfb family mRNAs. The effect was reversible by inhibiting EGF or activating TGFß signaling. These observations demonstrate the previously unappreciated postnatal role of PDGFA/PDGFRα in controlling elastogenic gene expression via a secondary tier of signaling networks composed of EGF and TGFß.

5.
Sci Rep ; 7(1): 3473, 2017 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-28615712

RESUMO

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.


Assuntos
Células Epiteliais Alveolares/metabolismo , Aquaporina 5/genética , Fator de Transcrição GATA6/metabolismo , Regulação da Expressão Gênica , Histonas/metabolismo , Fator de Transcrição Sp1/metabolismo , Acetilação , Animais , Linhagem Celular , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Humanos , Camundongos , Modelos Biológicos , Fatores de Transcrição de p300-CBP/metabolismo
6.
Am J Physiol Lung Cell Mol Physiol ; 312(3): L371-L391, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28062486

RESUMO

Various pathophysiological conditions such as surfactant dysfunction, mechanical ventilation, inflammation, pathogen products, environmental exposures, and gastric acid aspiration stress lung cells, and the compromise of plasma membranes occurs as a result. The mechanisms necessary for cells to repair plasma membrane defects have been extensively investigated in the last two decades, and some of these key repair mechanisms are also shown to occur following lung cell injury. Because it was theorized that lung wounding and repair are involved in the pathogenesis of acute respiratory distress syndrome (ARDS) and idiopathic pulmonary fibrosis (IPF), in this review, we summarized the experimental evidence of lung cell injury in these two devastating syndromes and discuss relevant genetic, physical, and biological injury mechanisms, as well as mechanisms used by lung cells for cell survival and membrane repair. Finally, we discuss relevant signaling pathways that may be activated by chronic or repeated lung cell injury as an extension of our cell injury and repair focus in this review. We hope that a holistic view of injurious stimuli relevant for ARDS and IPF could lead to updated experimental models. In addition, parallel discussion of membrane repair mechanisms in lung cells and injury-activated signaling pathways would encourage research to bridge gaps in current knowledge. Indeed, deep understanding of lung cell wounding and repair, and discovery of relevant repair moieties for lung cells, should inspire the development of new therapies that are likely preventive and broadly effective for targeting injurious pulmonary diseases.


Assuntos
Membrana Celular/patologia , Pneumopatias/patologia , Cicatrização , Animais , Humanos , Lesão Pulmonar/patologia , Modelos Biológicos , Transdução de Sinais
7.
BMC Biol ; 14: 19, 2016 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-26984772

RESUMO

BACKGROUND: Epithelial-mesenchymal cross talk is centerpiece in the development of many branched organs, including the lungs. The embryonic lung mesoderm provides instructional information not only for lung architectural development, but also for patterning, commitment and differentiation of its many highly specialized cell types. The mesoderm also serves as a reservoir of progenitors for generation of differentiated mesenchymal cell types that include αSMA-expressing fibroblasts, lipofibroblasts, endothelial cells and others. Transforming Growth Factor ß (TGFß) is a key signaling pathway in epithelial-mesenchymal cross talk. Using a cre-loxP approach we have elucidated the role of the TGFß type I receptor tyrosine kinase, ALK5, in epithelial-mesenchymal cross talk during lung morphogenesis. RESULTS: Targeted early inactivation of Alk5 in mesodermal progenitors caused abnormal development and maturation of the lung that included reduced physical size of the sub-mesothelial mesoderm, an established source of specific mesodermal progenitors. Abrogation of mesodermal ALK5-mediated signaling also inhibited differentiation of cell populations in the epithelial and endothelial lineages. Importantly, Alk5 mutant lungs contained a reduced number of αSMA(pos) cells and correspondingly increased lipofibroblasts. Elucidation of the underlying mechanisms revealed that through direct and indirect modulation of target signaling pathways and transcription factors, including PDGFRα, PPARγ, PRRX1, and ZFP423, ALK5-mediated TGFß controls a process that regulates the commitment and differentiation of αSMA(pos) versus lipofibroblast cell populations during lung development. CONCLUSION: ALK5-mediated TGFß signaling controls an early pathway that regulates the commitment and differentiation of αSMA(pos) versus LIF cell lineages during lung development.


Assuntos
Pulmão/citologia , Pulmão/embriologia , Mesoderma/citologia , Mesoderma/embriologia , Miofibroblastos/citologia , Proteínas Serina-Treonina Quinases/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Células-Tronco/citologia , Animais , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/genética , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Pulmão/anormalidades , Pulmão/metabolismo , Mesoderma/anormalidades , Mesoderma/metabolismo , Camundongos Endogâmicos C57BL , Músculo Liso/anormalidades , Músculo Liso/citologia , Músculo Liso/embriologia , Músculo Liso/metabolismo , Miofibroblastos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/metabolismo
8.
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
9.
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 , 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
10.
Dev Biol ; 408(1): 56-65, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26460096

RESUMO

Pten is a multifunctional tumor suppressor. Deletions and mutations in the Pten gene have been associated with multiple forms of human cancers. Pten is a central regulator of several signaling pathways that influences multiple cellular functions. One such function is in cell motility and migration, although the precise mechanism remains unknown. In this study, we deleted Pten in the embryonic lung epithelium using Gata5-cre mice. Absence of Pten blocked branching morphogenesis and ERK and AKT phosphorylation at E12.5. In an explant model, Pten(Δ/Δ) mesenchyme-free embryonic lung endoderm failed to branch. Inhibition of budding in Pten(Δ/Δ) explants was associated with major changes in cell migration, while cell proliferation was not affected. We further examined the role of ERK and AKT in branching morphogenesis by conditional, endodermal-specific mutants which blocked ERK or AKT phosphorylation. MEK(DM/+); Gata5-cre (blocking of ERK phosphorylation) lung showed more severe phenotype in branching morphogenesis. The inhibition of budding was also associated with disruption of cell migration. Thus, the mechanisms by which Pten is required for early endodermal morphogenesis may involve ERK, but not AKT, mediated cell migration.


Assuntos
Endoderma/embriologia , Endoderma/enzimologia , Pulmão/embriologia , Sistema de Sinalização das MAP Quinases , Morfogênese , PTEN Fosfo-Hidrolase/metabolismo , Animais , Movimento Celular , Epitélio/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator de Transcrição GATA5/metabolismo , Deleção de Genes , Integrases/metabolismo , Camundongos , Modelos Biológicos , Especificidade de Órgãos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo
11.
J Biochem ; 158(5): 355-65, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26261051

RESUMO

The role of WNT signalling in metazoan organogenesis has been a topic of widespread interest. In the lung, while the role of canonical WNT signalling has been examined in some detail by multiple studies, the non-canonical WNT signalling has received limited attention. Reliable evidence shows that this important signalling mechanism constitutes a major regulatory pathway in lung development. In addition, accumulating evidence has also shown that the non-canonical WNT pathway is critical for maintaining lung homeostasis and that aberrant activation of this pathway may underlie several debilitating lung diseases. Functional analyses have further revealed that the non-canonical WNT pathway regulates multiple cellular activities in the lung that are dependent on the specific cellular context. In most cell types, non-canonical WNT signalling regulates canonical WNT activity, which is also critical for many aspects of lung biology. This review will summarize what is currently known about the role of non-canonical WNT signalling in lung development, homeostasis and pathogenesis of disease.


Assuntos
Pulmão/metabolismo , Modelos Biológicos , Organogênese , Mucosa Respiratória/metabolismo , Via de Sinalização Wnt , Animais , Maturidade dos Órgãos Fetais , Humanos , Pulmão/embriologia , Pulmão/crescimento & desenvolvimento , Pulmão/patologia , Pneumopatias/imunologia , Pneumopatias/metabolismo , Pneumopatias/patologia , Mucosa Respiratória/embriologia , Mucosa Respiratória/crescimento & desenvolvimento , Mucosa Respiratória/patologia
12.
Stem Cells ; 33(3): 999-1012, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25448080

RESUMO

Development of the mammalian lung is predicated on cross-communications between two highly interactive tissues, the endodermally derived epithelium and the mesodermally derived pulmonary mesenchyme. While much attention has been paid for the lung epithelium, the pulmonary mesenchyme, partly due to lack of specific tractable markers remains under-investigated. The lung mesenchyme is derived from the lateral plate mesoderm and is the principal recipient of Hedgehog (Hh) signaling, a morphogenetic network that regulates multiple aspects of embryonic development. Using the Hh-responsive Gli1-cre(ERT2) mouse line, we identified the mesodermal targets of Hh signaling at various time points during embryonic and postnatal lung development. Cell lineage analysis showed these cells serve as progenitors to contribute to multiple lineages of mesodermally derived differentiated cell types that include parenchymal or interstitial myofibroblasts, peribronchial and perivascular smooth muscle as well as rare populations of cells within the mesothelium. Most importantly, Gli1-cre(ERT2) identified the progenitors of secondary crest myofibroblasts, a hitherto intractable cell type that plays a key role in alveolar formation, a vital process about which little is currently known. Transcriptome analysis of Hh-targeted progenitor cells transitioning from the pseudoglandular to the saccular phase of lung development revealed important modulations of key signaling pathways. Among these, there was significant downregulation of canonical WNT signaling. Ectopic stabilization of ß-catenin via inactivation of Apc by Gli1-cre(ERT2) expanded the Hh-targeted progenitor pools, which caused the formation of fibroblastic masses within the lung parenchyma. The Gli1-cre(ERT2) mouse line represents a novel tool in the analysis of mesenchymal cell biology and alveolar formation during lung development.


Assuntos
Pulmão/embriologia , Mesoderma/citologia , Miofibroblastos/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular/fisiologia , Feminino , Perfilação da Expressão Gênica , Pulmão/citologia , Mesoderma/metabolismo , Camundongos , Miofibroblastos/metabolismo , Gravidez , Transdução de Sinais , Células-Tronco/metabolismo
13.
Am J Physiol Lung Cell Mol Physiol ; 306(4): L316-25, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24375794

RESUMO

Many signaling pathways are mediated by Shc adapter proteins that, in turn, are expressed as three isoforms with distinct functions. The p66(Shc) isoform antagonizes proliferation, regulates oxidative stress, and mediates apoptosis. It is highly expressed in the canalicular but not the later stages of mouse lung development, and its expression persists in bronchopulmonary dysplasia, a chronic disease associated with premature birth. These observations suggest that p66(Shc) has a developmental function. However, constitutive p66(Shc) deletion yields no morphological phenotype, and the structure of the Shc gene precludes its inducible deletion. To elucidate its function in lung development, we transfected p66(Shc) or nonsilencing small-interfering RNA (siRNA) into the epithelia of embryonic day 11 mouse lungs that were then cultured for 3 days and analyzed morphometrically. To assess cellular proliferation and epithelial differentiation, lung explants were immunostained and immunoblotted for p66(Shc), proliferating cell nuclear antigen (PCNA), the proximal airway differentiation antigens Clara cell 10-kDa protein (CC10) and thyroid transcription factor (TTF)-1, and the alveolar surfactant proteins (SP)-A, -B, and -C. Explants transfected with nonsilencing siRNA demonstrated specific epithelial uptake and normal morphological development relative to uninjected controls. In contrast, transfection with p66(Shc) siRNA significantly increased lumenal cross-sectional areas, decreased branching, and increased epithelial proliferation (P < 0.05 for all). Relative to controls, the expression of SP-B, SP-C, CC10, and TTF-1 was decreased by p66(Shc) knockdown. SP-A was not expressed in either control or treated lungs. These data suggest that p66(Shc) attenuates epithelial proliferation while promoting both distal and proximal epithelial maturation.


Assuntos
Células Epiteliais Alveolares/fisiologia , Pulmão/embriologia , Morfogênese , Proteínas Adaptadoras da Sinalização Shc/fisiologia , Células 3T3 , Animais , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Pulmão/citologia , Pulmão/metabolismo , Camundongos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína B Associada a Surfactante Pulmonar/metabolismo , RNA Interferente Pequeno/genética , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Técnicas de Cultura de Tecidos , Fatores de Transcrição , Uteroglobina/metabolismo
14.
Development ; 140(18): 3731-42, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23924632

RESUMO

Localized Fgf10 expression in the distal mesenchyme adjacent to sites of lung bud formation has long been thought to drive stereotypic branching morphogenesis even though isolated lung epithelium branches in the presence of non-directional exogenous Fgf10 in Matrigel. Here, we show that lung agenesis in Fgf10 knockout mice can be rescued by ubiquitous overexpression of Fgf10, indicating that precisely localized Fgf10 expression is not required for lung branching morphogenesis in vivo. Fgf10 expression in the mesenchyme itself is regulated by Wnt signaling. Nevertheless, we found that during lung initiation simultaneous overexpression of Fgf10 is not sufficient to rescue the absence of primary lung field specification in embryos overexpressing Dkk1, a secreted inhibitor of Wnt signaling. However, after lung initiation, simultaneous overexpression of Fgf10 in lungs overexpressing Dkk1 is able to rescue defects in branching and proximal-distal differentiation. We also show that Fgf10 prevents the differentiation of distal epithelial progenitors into Sox2-expressing airway epithelial cells in part by activating epithelial ß-catenin signaling, which negatively regulates Sox2 expression. As such, these findings support a model in which the main function of Fgf10 during lung development is to regulate proximal-distal differentiation. As the lung buds grow out, proximal epithelial cells become further and further displaced from the distal source of Fgf10 and differentiate into bronchial epithelial cells. Interestingly, our data presented here show that once epithelial cells are committed to the Sox2-positive airway epithelial cell fate, Fgf10 prevents ciliated cell differentiation and promotes basal cell differentiation.


Assuntos
Diferenciação Celular , Células Epiteliais/patologia , Fator 10 de Crescimento de Fibroblastos/metabolismo , Pulmão/embriologia , Pulmão/metabolismo , Morfogênese , Células-Tronco/patologia , Animais , Regulação para Baixo , Ativação Enzimática , Células Epiteliais/metabolismo , Fator 10 de Crescimento de Fibroblastos/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Deformidades Congênitas dos Membros/patologia , Pulmão/anormalidades , Pulmão/patologia , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Respiração , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Células-Tronco/enzimologia , Traqueia/patologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo
15.
PLoS One ; 8(4): e62215, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23646120

RESUMO

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 ß-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 ß-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.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Receptores Notch/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteínas Wnt/metabolismo , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Cílios , Feminino , Fatores de Transcrição Forkhead/metabolismo , Deleção de Genes , Inativação Gênica , Células Germinativas/metabolismo , Recombinação Homóloga , Humanos , Integrases/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1 , Pulmão/citologia , Pulmão/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Proteínas Serrate-Jagged , Espermatogênese/genética , Testículo/citologia , Testículo/metabolismo , beta Catenina/metabolismo
16.
Dev Biol ; 378(1): 13-24, 2013 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-23562608

RESUMO

Wnt signaling is critical for cell fate specification and cell differentiation in many organs, but its function in pulmonary neuroendocrine cell (PNEC) differentiation has not been fully addressed. In this study, we examined the role of canonical Wnt signaling by targeting the gene for Adenomatous Polyposis Coli (Apc), which controls Wnt signaling activity via mediating phosphorylation of beta-catenin (Ctnnb). Targeting the Apc gene in lung epithelial progenitors by Nkx2.1-cre stabilized Ctnnb and activated canonical Wnt signaling. Apc deficiency altered lung epithelial cell fate by inhibiting Clara and ciliated cell differentiation and activating Uchl1, a marker of neuroendocrine cells. Similar to PNEC in normal lung, Uchl1(positive) cells were innervated. In mice with targeted inactivation of Ctnnb by Nkx2.1-cre, PNEC differentiation was not interrupted. These indicate that, after lung primordium formation, Wnt signaling is not essential for PNEC differentiation; however, its over-activation promotes PNEC features. Interestingly, Nkx2.1 was extinguished in Apc deficient epithelial progenitors before activation of Uchl1. Examination of Nkx2.1 null lungs suggested that early deletion of Nkx2.1 inhibits PNEC differentiation, while late repression does not. Nkx2.1 was specifically inhibited in Apc deficient lungs but not in Ctnnb gain-of-function lungs indicating a functional difference between Apc deletion and Ctnnb stabilization, both of which activate Wnt signaling. Further analysis revealed that Apc deficiency led to increased TGF-beta signaling, which inhibited Nkx2.1 in cultured lung endodermal explants. In contrast, TGF-beta activity was not increased in Ctnnb gain-of-function lungs. Therefore, our studies revealed an important mechanism involving Apc and TGF-beta signaling in regulating the key transcriptional factor, Nkx2.1, for lung epithelial progenitor cell fate determination.


Assuntos
Proteína da Polipose Adenomatosa do Colo/fisiologia , Células Epiteliais/citologia , Pulmão/citologia , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Genes APC , Pulmão/embriologia , Camundongos , Modelos Biológicos , Morfogênese , Proteínas Nucleares/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/genética
17.
J Clin Invest ; 122(11): 3862-72, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23023706

RESUMO

Alveolar capillary dysplasia (ACD) is a congenital, lethal disorder of the pulmonary vasculature. Phosphatase and tensin homologue deleted from chromosome 10 (Pten) encodes a lipid phosphatase controlling key cellular functions, including stem/progenitor cell proliferation and differentiation; however, the role of PTEN in mesodermal lung cell lineage formation remains unexamined. To determine the role of mesodermal PTEN in the ontogeny of various mesenchymal cell lineages during lung development, we specifically deleted Pten in early embryonic lung mesenchyme in mice. Pups lacking Pten died at birth, with evidence of failure in blood oxygenation. Analysis at the cellular level showed defects in angioblast differentiation to endothelial cells and an accompanying accumulation of the angioblast cell population that was associated with disorganized capillary beds. We also found decreased expression of Forkhead box protein F1 (Foxf1), a gene associated with the ACD human phenotype. Analysis of human samples for ACD revealed a significant decrease in PTEN and increased activated protein kinase B (AKT). These studies demonstrate that mesodermal PTEN has a key role in controlling the amplification of angioblasts as well as their differentiation into endothelial cells, thereby directing the establishment of a functional gas exchange interface. Additionally, these mice could serve as a murine model of ACD.


Assuntos
Diferenciação Celular , Células Endoteliais/enzimologia , Pulmão/embriologia , Mesoderma/embriologia , PTEN Fosfo-Hidrolase/metabolismo , Síndrome da Persistência do Padrão de Circulação Fetal/embriologia , Animais , Linhagem da Célula , Células Endoteliais/patologia , Ativação Enzimática , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Pulmão/enzimologia , Pulmão/patologia , Mesoderma/enzimologia , Mesoderma/patologia , Camundongos , Camundongos Knockout , PTEN Fosfo-Hidrolase/genética , Síndrome da Persistência do Padrão de Circulação Fetal/enzimologia , Síndrome da Persistência do Padrão de Circulação Fetal/genética , Síndrome da Persistência do Padrão de Circulação Fetal/patologia , Fenótipo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Alvéolos Pulmonares/anormalidades , Alvéolos Pulmonares/embriologia , Alvéolos Pulmonares/enzimologia , Alvéolos Pulmonares/patologia
18.
Stem Cells ; 30(5): 946-55, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22331706

RESUMO

The airways of the mammalian lung are lined with highly specialized epithelial cell types that are the targets of airborne toxicants and injury. Notch signaling plays an important role in the ontogeny of airway epithelial cells, but its contributions to recruitment, expansion or differentiation of resident progenitor/stem cells, and repair and re-establishment of the normal composition of airway epithelium following injury have not been addressed. In this study, the role of a specific Notch receptor, Notch1, was investigated by targeted inactivation in the embryonic lung epithelium using the epithelial-specific Gata5-Cre driver line. Notch1-deficient mice are viable without discernible defects in pulmonary epithelial cell-fate determination and differentiation. However, in an experimental model of airway injury, activity of Notch1 is found to be required for normal repair of the airway epithelium. Absence of Notch1 reduced the ability of a population of cells distinguished by expression of PGP9.5, otherwise a marker of pulmonary neuroendocrine cells, which appears to serve as a reservoir for regeneration of Clara cells. Hairy/enhancer of split-5 (Hes5) and paired-box-containing gene 6 (Pax6) were found to be downstream targets of Notch1. Both Hes5 and Pax6 expressions were significantly increased in association with Clara cell regeneration in wild-type lungs. Ablation of Notch1 reduced Hes5 and Pax6 and inhibited airway epithelial repair. Thus, although dispensable in developmental ontogeny of airway epithelial cells, normal activity of Notch1 is required for repair of the airway epithelium. The signaling pathway by which Notch1 regulates the repair process includes stimulation of Hes5 and Pax6 gene expression.


Assuntos
Lesão Pulmonar/metabolismo , Receptor Notch1/metabolismo , Regeneração , Mucosa Respiratória/metabolismo , Transdução de Sinais , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas do Olho/biossíntese , Proteínas do Olho/genética , Regulação da Expressão Gênica/genética , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Lesão Pulmonar/genética , Lesão Pulmonar/patologia , Camundongos , Camundongos Knockout , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/biossíntese , Fatores de Transcrição Box Pareados/genética , Receptor Notch1/genética , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Mucosa Respiratória/lesões , Mucosa Respiratória/patologia , Ubiquitina Tiolesterase/biossíntese , Ubiquitina Tiolesterase/genética
19.
Respir Res ; 12: 134, 2011 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-21985298

RESUMO

BACKGROUND: Alveolar septation marks the beginning of the transition from the saccular to alveolar stage of lung development. Inflammation can disrupt this process and permanently impair alveolar formation resulting in alveolar hypoplasia as seen in bronchopulmonary dysplasia in preterm newborns. NF-κB is a transcription factor central to multiple inflammatory and developmental pathways including dorsal-ventral patterning in fruit flies; limb, mammary and submandibular gland development in mice; and branching morphogenesis in chick lungs. We have previously shown that epithelial overexpression of NF-κB accelerates lung maturity using transgenic mice. The purpose of this study was to test our hypothesis that targeted deletion of NF-κB signaling in lung epithelium would impair alveolar formation. METHODS: We generated double transgenic mice with lung epithelium-specific deletion of IKKß, a known activating kinase upstream of NF-κB, using a cre-loxP transgenic recombination strategy. Lungs of resulting progeny were analyzed at embryonic and early postnatal stages to determine specific effects on lung histology, and mRNA and protein expression of relevant lung morphoreulatory genes. Lastly, results measuring expression of the angiogenic factor, VEGF, were confirmed in vitro using a siRNA-knockdown strategy in cultured mouse lung epithelial cells. RESULTS: Our results showed that IKKß deletion in the lung epithelium transiently decreased alveolar type I and type II cells and myofibroblasts and delayed alveolar formation. These effects were mediated through increased alveolar type II cell apoptosis and decreased epithelial VEGF expression. CONCLUSIONS: These results suggest that epithelial NF-κB plays a critical role in early alveolar development possibly through regulation of VEGF.


Assuntos
Proteínas Reguladoras de Apoptose/deficiência , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Quinase I-kappa B/deficiência , Alvéolos Pulmonares/crescimento & desenvolvimento , Mucosa Respiratória/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Proteínas Reguladoras de Apoptose/genética , Quinase I-kappa B/genética , Pulmão/embriologia , Pulmão/crescimento & desenvolvimento , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Alvéolos Pulmonares/embriologia , Alvéolos Pulmonares/patologia , Mucosa Respiratória/embriologia , Mucosa Respiratória/crescimento & desenvolvimento , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/fisiologia
20.
Am J Respir Cell Mol Biol ; 44(6): 804-12, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20693404

RESUMO

Alveolar formation is hallmarked by the transition of distal lung saccules into gas exchange units through the emergence of secondary crests and an exponential increase in surface area. Several cell types are involved in this complex process, including families of epithelial cells that differentiate into alveolar type I and II cells. Subsets of cells expressing Clara cell secretory protein (CCSP) have been identified in both lung and bone marrow compartments, and are described as a progenitor/stem cell pool involved in airway regeneration and alveolar homeostasis. Whether these cells also participate in alveolar formation during postnatal development remains unknown. Based on their regenerative capacity, we asked whether these cells participate in alveogenesis. We used a previously described transgenic mouse model (CCSP-tk) in which Ganciclovir exposure selectively depletes all cells with CCSP promoter activity through intracellular generation of a toxic metabolite of thymidine kinase. Our results showed that Ganciclovir treatment in newborn CCtk mice depleted this cell population in lung airways and bone marrow, and was associated with alveolar hypoplasia and respiratory failure. Hypoplastic lungs had fewer alveolar type I and II cells, with impaired secondary crest formation and decreased vascular endothelial growth factor expression in distal airways. These findings are consistent with a model in which a unique population of cells with CCSP promoter activity that expresses vascular endothelial growth factor participates in alveolar development.


Assuntos
Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Alvéolos Pulmonares/crescimento & desenvolvimento , Uteroglobina/genética , Animais , Ganciclovir/farmacologia , Pulmão/metabolismo , Camundongos , Camundongos Transgênicos , Naftalenos/farmacologia , Células-Tronco/citologia , Timidina Quinase/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
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