ABSTRACT
Transcription factors (TFs) are dosage-sensitive master regulators of gene expression, with haploinsufficiency frequently leading to life-threatening disease. Numerous mechanisms have evolved to tightly regulate the expression and activity of TFs at the transcriptional, translational, and posttranslational levels. A subset of long noncoding RNAs (lncRNAs) is spatially correlated with transcription factors in the genome, but the regulatory relationship between these lncRNAs and their neighboring TFs is unclear. We identified a regulatory feedback loop between the TF Foxa2 and a downstream lncRNA, Falcor (Foxa2-adjacent long noncoding RNA). Foxa2 directly represses Falcor expression by binding to its promoter, while Falcor functions in cis to positively regulate the expression of Foxa2. In the lung, loss of Falcor is sufficient to lead to chronic inflammatory changes and defective repair after airway epithelial injury. Moreover, disruption of the Falcor-Foxa2 regulatory feedback loop leads to altered cell adhesion and migration, in turn resulting in chronic peribronchial airway inflammation and goblet cell metaplasia. These data reveal that the lncRNA Falcor functions within a regulatory feedback loop to fine-tune the expression of Foxa2, maintain airway epithelial homeostasis, and promote regeneration.
Subject(s)
Epithelial Cells/metabolism , Hepatocyte Nuclear Factor 3-beta/genetics , Lung/cytology , Lung/metabolism , RNA, Long Noncoding/genetics , Animals , Cell Adhesion , Cell Line , Cell Movement , Female , Gene Expression Regulation , Hepatocyte Nuclear Factor 3-beta/metabolism , Homeostasis , Humans , Male , Mice , Promoter Regions, Genetic , Regeneration , Transcription, GeneticABSTRACT
Bronchopulmonary dysplasia (BPD), a long-term respiratory morbidity of prematurity, is characterized by attenuated alveolar and vascular development. Supplemental oxygen and immature antioxidant defenses contribute to BPD development. Our group identified thioredoxin reductase-1 (TXNRD1) as a therapeutic target to prevent BPD. The present studies evaluated the impact of the TXNRD1 inhibitor aurothioglucose (ATG) on pulmonary responses and gene expression in newborn C57BL/6 pups treated with saline or ATG (25 mg/kg ip) within 12 h of birth and exposed to room air (21% O2) or hyperoxia (>95% O2) for 72 h. Purified RNA from lung tissues was sequenced, and differential expression was evaluated. Hyperoxic exposure altered ~2,000 genes, including pathways involved in glutathione metabolism, intrinsic apoptosis signaling, and cell cycle regulation. The isolated effect of ATG treatment was limited primarily to genes that regulate angiogenesis and vascularization. In separate studies, pups were treated as described above and returned to room air until 14 days. Vascular density analyses were performed, and ANOVA indicated an independent effect of hyperoxia on vascular density and alveolar architecture at 14 days. Consistent with RNA-seq analyses, ATG significantly increased vascular density in room air, but not in hyperoxia-exposed pups. These findings provide insights into the mechanisms by which TXNRD1 inhibitors may enhance lung development.
Subject(s)
Air , Aurothioglucose/pharmacology , Hyperoxia/pathology , Lung/blood supply , Lung/pathology , Neovascularization, Physiologic/drug effects , Acute Disease , Animals , Animals, Newborn , Apoptosis/drug effects , Apoptosis/genetics , DNA/biosynthesis , Glutathione/metabolism , Lung/drug effects , Lung/embryology , Mice, Inbred C57BL , Pulmonary Alveoli/drug effects , Pulmonary Alveoli/embryology , Pulmonary Alveoli/pathology , Signal Transduction/drug effects , Transcription, Genetic/drug effects , Transcriptome/genetics , Up-Regulation/drug effectsABSTRACT
KIF3A, the gene encoding kinesin family member 3A, is a susceptibility gene locus associated with asthma; however, mechanisms by which KIF3A might influence the pathogenesis of the disorder are unknown. In this study, we deleted the mouse Kif3a gene in airway epithelial cells. Both homozygous and heterozygous Kif3a gene-deleted mice were highly susceptible to aeroallergens from Aspergillus fumigatus and the house dust mite, resulting in an asthma-like pathology characterized by increased goblet cell metaplasia, airway hyperresponsiveness, and Th2-mediated inflammation. Deletion of the Kif3a gene increased the severity of pulmonary eosinophilic inflammation and expression of cytokines (Il-4, Il-13, and Il-17a) and chemokine (Ccl11) RNAs following pulmonary exposure to Aspergillus extract. Inhibition of Kif3a disrupted the structure of motile cilia and impaired mucociliary clearance, barrier function, and epithelial repair, demonstrating additional mechanisms by which deficiency of KIF3A in respiratory epithelial cells contributes to pulmonary pathology. Airway epithelial KIF3A suppresses Th2 pulmonary inflammation and airway hyperresponsiveness following aeroallergen exposure, implicating epithelial microtubular functions in the pathogenesis of Th2-mediated lung pathology.
Subject(s)
Allergens/immunology , Aspergillus fumigatus/immunology , Asthma/immunology , Epithelial Cells/immunology , Kinesins/immunology , Respiratory Mucosa/immunology , Th2 Cells/immunology , Animals , Asthma/chemically induced , Asthma/genetics , Asthma/pathology , Cytokines/genetics , Cytokines/immunology , Epithelial Cells/pathology , Kinesins/genetics , Lung/immunology , Lung/pathology , Mice , Mice, Transgenic , Respiratory Mucosa/pathology , Th2 Cells/pathologyABSTRACT
'LungGENS', our previously developed web tool for mapping single-cell gene expression in the developing lung, has been well received by the pulmonary research community. With continued support from the 'LungMAP' consortium, we extended the scope of the LungGENS database to accommodate transcriptomics data from pulmonary tissues and cells from human and mouse at different stages of lung development. Lung Gene Expression Analysis (LGEA) web portal is an extended version of LungGENS useful for the analysis, display and interpretation of gene expression patterns obtained from single cells, sorted cell populations and whole lung tissues. The LGEA web portal is freely available at http://research.cchmc.org/pbge/lunggens/mainportal.html.
Subject(s)
Computational Biology/methods , Gene Expression Profiling/methods , Gene Expression , Internet , Lung/growth & development , Animals , Chromosome Mapping , Humans , Mice , Software , Transcription FactorsABSTRACT
SAM-pointed domain-containing ETS transcription factor (SPDEF) is expressed in normal prostate epithelium. While its expression changes during prostate carcinogenesis (PCa), the role of SPDEF in prostate cancer remains controversial due to the lack of genetic mouse models. In present study, we generated transgenic mice with the loss- or gain-of-function of SPDEF in prostate epithelium to demonstrate that SPDEF functions as tumor suppressor in prostate cancer. Loss of SPDEF increased cancer progression and tumor cell proliferation, whereas over-expression of SPDEF in prostate epithelium inhibited carcinogenesis and reduced tumor cell proliferation in vivo and in vitro. Transgenic over-expression of SPDEF inhibited mRNA and protein levels of Foxm1, a transcription factor critical for tumor cell proliferation, and reduced expression of Foxm1 target genes, including Cdc25b, Cyclin B1, Cyclin A2, Plk-1, AuroraB, CKS1 and Topo2alpha. Deletion of SPDEF in transgenic mice and cultures prostate tumor cells increased expression of Foxm1 and its target genes. Furthermore, an inverse correlation between SPDEF and Foxm1 levels was found in human prostate cancers. The two-gene signature of low SPDEF and high FoxM1 predicted poor survival in prostate cancer patients. Mechanistically, SPDEF bound to, and inhibited transcriptional activity of Foxm1 promoter by interfering with the ability of Foxm1 to activate its own promoter through auto-regulatory site located in the -745/-660 bp Foxm1 promoter region. Re-expression of Foxm1 restored cellular proliferation in the SPDEF-positive cancer cells and rescued progression of SPDEF-positive tumors in mouse prostates. Altogether, SPDEF inhibits prostate carcinogenesis by preventing Foxm1-regulated proliferation of prostate tumor cells. The present study identified novel crosstalk between SPDEF tumor suppressor and Foxm1 oncogene and demonstrated that this crosstalk is required for tumor cell proliferation during progression of prostate cancer in vivo.
Subject(s)
Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Forkhead Transcription Factors/genetics , Gene Expression Regulation , Prostate/metabolism , Prostate/pathology , Proto-Oncogene Proteins c-ets/genetics , Proto-Oncogene Proteins c-ets/metabolism , Animals , Cell Line, Tumor , Disease Models, Animal , Forkhead Box Protein M1 , Gene Expression Regulation, Neoplastic , Gene Order , Gene Targeting , Humans , Male , Mice , Mice, Knockout , Prognosis , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/mortality , Prostatic Neoplasms/pathologyABSTRACT
The SRY-box containing transcription factor Sox17 is required for endoderm formation and vascular morphogenesis during embryonic development. In the lung, Sox17 is expressed in mesenchymal progenitors of the embryonic pulmonary vasculature and is restricted to vascular endothelial cells in the mature lung. Conditional deletion of Sox17 in splanchnic mesenchyme-derivatives using Dermo1-Cre resulted in substantial loss of Sox17 from developing pulmonary vascular endothelial cells and caused pulmonary vascular abnormalities before birth, including pulmonary vein varices, enlarged arteries, and decreased perfusion of the microvasculature. While survival of Dermo1-Cre;Sox17Δ/Δ mice (herein termed Sox17Δ/Δ) was unaffected at E18.5, most Sox17Δ/Δ mice died by 3 weeks of age. After birth, the density of the pulmonary microvasculature was decreased in association with alveolar simplification, biventricular cardiac hypertrophy, and valvular regurgitation. The severity of the postnatal cardiac phenotype was correlated with the severity of pulmonary vasculature abnormalities. Sox17 is required for normal formation of the pulmonary vasculature and postnatal cardiovascular homeostasis.
Subject(s)
HMGB Proteins/metabolism , Lung/blood supply , Lung/embryology , SOXF Transcription Factors/metabolism , Animals , Arteries/abnormalities , Cell Differentiation , Endothelial Cells/metabolism , Gene Deletion , HMGB Proteins/genetics , Mesoderm/cytology , Mesoderm/embryology , Mesoderm/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Pulmonary Veins/abnormalities , Repressor Proteins/genetics , SOXF Transcription Factors/genetics , Twist-Related Protein 1/geneticsABSTRACT
RATIONALE: Goblet cell metaplasia accompanies common pulmonary disorders that are prone to recurrent viral infections. Mechanisms regulating both goblet cell metaplasia and susceptibility to viral infection associated with chronic lung diseases are incompletely understood. OBJECTIVES: We sought to identify the role of the transcription factor FOXA3 in regulation of goblet cell metaplasia and pulmonary innate immunity. METHODS: FOXA3 was identified in airways from patients with asthma and chronic obstructive pulmonary disease. We produced transgenic mice conditionally expressing Foxa3 in airway epithelial cells and developed human bronchial epithelial cells expressing Foxa3. Foxa3-regulated genes were identified by immunostaining, Western blotting, and RNA analysis. Direct binding of FOXA3 to target genes was identified by chromatin immunoprecipitation sequencing correlated with RNA sequencing. MEASUREMENTS AND MAIN RESULTS: FOXA3 was highly expressed in airway goblet cells from patients with asthma and chronic obstructive pulmonary disease. FOXA3 was induced by either IL-13 or rhinovirus. Foxa3 induced goblet cell metaplasia and enhanced expression of a network of genes mediating mucus production. Paradoxically, FOXA3 inhibited rhinovirus-induced IFN production, IRF-3 phosphorylation, and IKKε expression and inhibited viral clearance and expression of genes required for antiviral defenses, including MDA5, RIG-I, TLR3, IRF7/9, and nuclear factor-κB. CONCLUSIONS: FOXA3 induces goblet cell metaplasia in response to infection or Th2 stimulation. Suppression of IFN signaling by FOXA3 provides a plausible mechanism that may serve to limit ongoing Th1 inflammation during the resolution of acute viral infection; however, inhibition of innate immunity by FOXA3 may contribute to susceptibility to viral infections associated with chronic lung disorders accompanied by chronic goblet cell metaplasia.
Subject(s)
Asthma/metabolism , Goblet Cells/pathology , Hepatocyte Nuclear Factor 3-gamma/metabolism , Immunity, Innate/physiology , Picornaviridae Infections/immunology , Pulmonary Disease, Chronic Obstructive/metabolism , Animals , Asthma/complications , Asthma/immunology , Asthma/pathology , Biomarkers/metabolism , Blotting, Western , Chromatin Immunoprecipitation , Disease Susceptibility , Goblet Cells/immunology , Goblet Cells/metabolism , Hepatocyte Nuclear Factor 3-gamma/immunology , Humans , Interferons/metabolism , Metaplasia , Mice , Mice, Transgenic , Picornaviridae Infections/etiology , Pulmonary Disease, Chronic Obstructive/complications , Pulmonary Disease, Chronic Obstructive/immunology , Pulmonary Disease, Chronic Obstructive/pathology , Rhinovirus , Sequence Analysis, RNA , Th1-Th2 BalanceABSTRACT
Airway mucus plays a critical role in clearing inhaled toxins, particles, and pathogens. Diverse toxic, inflammatory, and infectious insults induce airway mucus secretion and goblet cell metaplasia to preserve airway sterility and homeostasis. However, goblet cell metaplasia, mucus hypersecretion, and airway obstruction are integral features of inflammatory lung diseases, including asthma, chronic obstructive lung disease, and cystic fibrosis, which cause an immense burden of morbidity and mortality. These chronic lung diseases are united by susceptibility to microbial colonization and recurrent airway infections. Whether these twinned phenomena (mucous metaplasia, compromised host defenses) are causally related has been unclear. Here, we demonstrate that SAM pointed domain ETS factor (SPDEF) was induced by rhinoviral infection of primary human airway cells and that cytoplasmic activities of SPDEF, a transcriptional regulator of airway goblet cell metaplasia, inhibited Toll-like receptor (TLR) activation of epithelial cells. SPDEF bound to and inhibited activities of TLR signaling adapters, MyD88 and TRIF, inhibiting MyD88-induced cytokine production and TRIF-induced interferon ß production. Conditional expression of SPDEF in airway epithelial cells in vivo inhibited LPS-induced neutrophilic infiltration and bacterial clearance. SPDEF-mediated inhibition of both TLR and type I interferon signaling likely protects the lung against inflammatory damage when inciting stimuli are not eradicated. Present findings provide, at least in part, a molecular explanation for increased susceptibility to infection in lung diseases associated with mucous metaplasia and a mechanism by which patients with florid mucous metaplasia may tolerate microbial burdens that are usually associated with fulminant inflammatory disease in normal hosts.
Subject(s)
Epithelial Cells/metabolism , Proto-Oncogene Proteins c-ets/metabolism , Respiratory Mucosa/metabolism , Signal Transduction , Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Anti-Bacterial Agents/pharmacology , Blotting, Western , Doxycycline/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/virology , Gene Expression/drug effects , HEK293 Cells , Host-Pathogen Interactions , Humans , Immunity, Innate , Interleukin-13/pharmacology , Lipopolysaccharides/pharmacology , Lung Diseases/drug therapy , Lung Diseases/metabolism , Lung Diseases/pathology , Metaplasia , Mice , Microscopy, Confocal , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/metabolism , Protein Binding , Proto-Oncogene Proteins c-ets/genetics , Respiratory Mucosa/drug effects , Respiratory Mucosa/pathology , Reverse Transcriptase Polymerase Chain Reaction , Rhinovirus/physiology , Toll-Like Receptors/genetics , Toll-Like Receptors/metabolismABSTRACT
Differential chromatin accessibility accompanies and mediates transcriptional control of diverse cell fates and their differentiation during embryogenesis. While the critical role of NKX2-1 and its transcriptional targets in lung morphogenesis and pulmonary epithelial cell differentiation is increasingly known, mechanisms by which chromatin accessibility alters the epigenetic landscape and how NKX2-1 interacts with other co-activators required for alveolar epithelial cell differentiation and function are not well understood. Here, we demonstrate that the paired domain zinc finger transcriptional regulators PRDM3 and PRDM16 regulate chromatin accessibility to mediate cell differentiation decisions during lung morphogenesis. Combined deletion of Prdm3 and Prdm16 in early lung endoderm caused perinatal lethality due to respiratory failure from loss of AT2 cell function. Prdm3/16 deletion led to the accumulation of partially differentiated AT1 cells and loss of AT2 cells. Combination of single cell RNA-seq, bulk ATAC-seq, and CUT&RUN demonstrated that PRDM3 and PRDM16 enhanced chromatin accessibility at NKX2-1 transcriptional targets in peripheral epithelial cells, all three factors binding together at a multitude of cell-type specific cis-active DNA elements. Network analysis demonstrated that PRDM3/16 regulated genes critical for perinatal AT2 cell differentiation, surfactant homeostasis, and innate host defense. Lineage specific deletion of PRDM3/16 in AT2 cells led to lineage infidelity, with PRDM3/16 null cells acquiring partial AT1 fate. Together, these data demonstrate that NKX2-1-dependent regulation of alveolar epithelial cell differentiation is mediated by epigenomic modulation via PRDM3/16.
ABSTRACT
Ventilation is dependent upon pulmonary alveoli lined by two major epithelial cell types, alveolar type-1 (AT1) and 2 (AT2) cells. AT1 cells mediate gas exchange while AT2 cells synthesize and secrete pulmonary surfactants and serve as progenitor cells which repair the alveoli. We developed transgenic mice in which YAP was activated or deleted to determine its roles in alveolar epithelial cell differentiation. Postnatal YAP activation increased epithelial cell proliferation, increased AT1 cell numbers, and caused indeterminate differentiation of subsets of alveolar cells expressing atypical genes normally restricted to airway epithelial cells. YAP deletion increased expression of genes associated with mature AT2 cells. YAP activation enhanced DNA accessibility in promoters of transcription factors and motif enrichment analysis predicted target genes associated with alveolar cell differentiation. YAP participated with KLF5, NFIB, and NKX2-1 to regulate AGER. YAP plays a central role in a transcriptional network that regulates alveolar epithelial differentiation.
ABSTRACT
Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease causing fibrotic remodeling of the peripheral lung, leading to respiratory failure. Peripheral pulmonary epithelial cells lose normal alveolar epithelial gene expression patterns and variably express genes associated with diverse conducting airway epithelial cells, including basal cells. Single-cell RNA sequencing of pulmonary epithelial cells isolated from IPF lung tissue demonstrated altered expression of LncRNAs, including increased MEG3. MEG3 RNA was highly expressed in subsets of the atypical IPF epithelial cells and correlated with conducting airway epithelial gene expression patterns. Expression of MEG3 in human pulmonary epithelial cell lines increased basal cell-associated RNAs, including TP63, KRT14, STAT3, and YAP1, and enhanced cell migration, consistent with a role for MEG3 in regulating basal cell identity. MEG3 reduced expression of TP73, SOX2, and Notch-associated RNAs HES1 and HEY1, in primary human bronchial epithelial cells, demonstrating a role for MEG3 in the inhibition of genes influencing basal cell differentiation into club, ciliated, or goblet cells. MEG3 induced basal cell genes and suppressed genes associated with terminal differentiation of airway cells, supporting a role for MEG3 in regulation of basal progenitor cell functions, which may contribute to tissue remodeling in IPF.
Subject(s)
Cell Differentiation/physiology , Epithelial Cells/metabolism , Idiopathic Pulmonary Fibrosis/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Binding Sites , Biomarkers , Cell Differentiation/genetics , Cell Line , Cell Movement , Gene Expression Regulation , Humans , Idiopathic Pulmonary Fibrosis/genetics , Keratin-14/genetics , Lung/metabolism , Promoter Regions, Genetic , STAT3 Transcription Factor/genetics , Sequence Analysis, RNA , Transcription Factors/genetics , Tumor Suppressor Proteins/geneticsABSTRACT
Hippo/YAP signaling plays pleiotropic roles in the regulation of cell proliferation and differentiation during organogenesis and tissue repair. Herein we demonstrate increased YAP activity in respiratory epithelial cells in lungs of patients with idiopathic pulmonary fibrosis (IPF), a common, lethal form of interstitial lung disease (ILD). Immunofluorescence staining in IPF epithelial cells demonstrated increased nuclear YAP and loss of MST1/2. Bioinformatic analyses of epithelial cell RNA profiles predicted increased activity of YAP and increased canonical mTOR/PI3K/AKT signaling in IPF. Phospho-S6 (p-S6) and p-PTEN were increased in IPF epithelial cells, consistent with activation of mTOR signaling. Expression of YAP (S127A), a constitutively active form of YAP, in human bronchial epithelial cells (HBEC3s) increased p-S6 and p-PI3K, cell proliferation and migration, processes that were inhibited by the YAP-TEAD inhibitor verteporfin. Activation of p-S6 was required for enhancing and stabilizing YAP, and the p-S6 inhibitor temsirolimus blocked nuclear YAP localization and suppressed expression of YAP target genes CTGF, AXL, and AJUBA (JUB). YAP and mTOR/p-S6 signaling pathways interact to induce cell proliferation and migration, and inhibit epithelial cell differentiation that may contribute to the pathogenesis of IPF.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Epithelial Cells/metabolism , Idiopathic Pulmonary Fibrosis/metabolism , Lung Diseases, Interstitial/metabolism , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Carrier Proteins/metabolism , Cell Culture Techniques , Cell Differentiation , Cell Movement , Cell Proliferation , Epithelial Cells/pathology , Hepatocyte Growth Factor/metabolism , Hippo Signaling Pathway , Humans , Idiopathic Pulmonary Fibrosis/pathology , Lung/metabolism , Lung/pathology , Lung Diseases, Interstitial/pathology , Membrane Proteins/metabolism , Oncogene Protein v-akt/metabolism , Organogenesis , PTEN Phosphohydrolase/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphoproteins/antagonists & inhibitors , Phosphorylation , Proto-Oncogene Proteins/metabolism , Ribosomal Protein S6 Kinases , Serine-Threonine Kinase 3 , Signal Transduction/drug effects , Sirolimus/analogs & derivatives , Sirolimus/pharmacology , TOR Serine-Threonine Kinases/metabolism , Transcription Factors , Tumor Suppressor Proteins/metabolism , Verteporfin/pharmacology , YAP-Signaling ProteinsABSTRACT
Idiopathic pulmonary fibrosis (IPF) is a lethal interstitial lung disease characterized by airway remodeling, inflammation, alveolar destruction, and fibrosis. We utilized single-cell RNA sequencing (scRNA-seq) to identify epithelial cell types and associated biological processes involved in the pathogenesis of IPF. Transcriptomic analysis of normal human lung epithelial cells defined gene expression patterns associated with highly differentiated alveolar type 2 (AT2) cells, indicated by enrichment of RNAs critical for surfactant homeostasis. In contrast, scRNA-seq of IPF cells identified 3 distinct subsets of epithelial cell types with characteristics of conducting airway basal and goblet cells and an additional atypical transitional cell that contributes to pathological processes in IPF. Individual IPF cells frequently coexpressed alveolar type 1 (AT1), AT2, and conducting airway selective markers, demonstrating "indeterminate" states of differentiation not seen in normal lung development. Pathway analysis predicted aberrant activation of canonical signaling via TGF-ß, HIPPO/YAP, P53, WNT, and AKT/PI3K. Immunofluorescence confocal microscopy identified the disruption of alveolar structure and loss of the normal proximal-peripheral differentiation of pulmonary epithelial cells. scRNA-seq analyses identified loss of normal epithelial cell identities and unique contributions of epithelial cells to the pathogenesis of IPF. The present study provides a rich data source to further explore lung health and disease.
Subject(s)
Epithelial Cells/cytology , Idiopathic Pulmonary Fibrosis/pathology , Sequence Analysis, RNA , Gene Expression Profiling , Genetic Markers , Humans , Lung/cytology , Single-Cell Analysis , TranscriptomeABSTRACT
The Hippo/Yap pathway is a well-conserved signaling cascade that regulates cell proliferation and differentiation to control organ size and stem/progenitor cell behavior. Following airway injury, Yap was dynamically regulated in regenerating airway epithelial cells. To determine the role of Hippo signaling in the lung, the mammalian Hippo kinases, Mst1 and Mst2, were deleted in epithelial cells of the embryonic and mature mouse lung. Mst1/2 deletion in the fetal lung enhanced proliferation and inhibited sacculation and epithelial cell differentiation. The transcriptional inhibition of cell proliferation and activation of differentiation during normal perinatal lung maturation were inversely regulated following embryonic Mst1/2 deletion. Ablation of Mst1/2 from bronchiolar epithelial cells in the adult lung caused airway hyperplasia and altered differentiation. Inhibitory Yap phosphorylation was decreased and Yap nuclear localization and transcriptional targets were increased after Mst1/2 deletion, consistent with canonical Hippo/Yap signaling. YAP potentiated cell proliferation and inhibited differentiation of human bronchial epithelial cells in vitro. Loss of Mst1/2 and expression of YAP regulated transcriptional targets controlling cell proliferation and differentiation, including Ajuba LIM protein. Ajuba was required for the effects of YAP on cell proliferation in vitro. Hippo/Yap signaling regulates Ajuba and controls proliferation and differentiation of lung epithelial progenitor cells.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Lung/metabolism , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Stem Cells/cytology , Stem Cells/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Cell Cycle Proteins , Cell Differentiation/genetics , Cell Proliferation , Cluster Analysis , Gene Expression , Gene Expression Profiling , Gene Knockout Techniques , Hepatocyte Growth Factor/genetics , Hippo Signaling Pathway , Humans , Hyperplasia , LIM Domain Proteins/metabolism , Lung/embryology , Lung/pathology , Mice , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phosphoproteins/genetics , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins/genetics , Regeneration/genetics , Respiratory Mucosa/cytology , Respiratory Mucosa/embryology , Respiratory Mucosa/metabolism , Respiratory Mucosa/pathology , Serine-Threonine Kinase 3 , Transcription Factors/genetics , Transcription Factors/metabolism , YAP-Signaling ProteinsABSTRACT
RATIONALE: The cystic fibrosis transmembrane conductance regulator (CFTR) and Calcium-activated Chloride Conductance (CaCC) each play critical roles in maintaining normal hydration of epithelial surfaces including the airways and colon. TGF-beta is a genetic modifier of cystic fibrosis (CF), but how it influences the CF phenotype is not understood. OBJECTIVES: We tested the hypothesis that TGF-beta potently downregulates chloride-channel function and expression in two CF-affected epithelia (T84 colonocytes and primary human airway epithelia) compared with proteins known to be regulated by TGF-beta. MEASUREMENTS AND MAIN RESULTS: TGF-beta reduced CaCC and CFTR-dependent chloride currents in both epithelia accompanied by reduced levels of TMEM16A and CFTR protein and transcripts. TGF-beta treatment disrupted normal regulation of airway-surface liquid volume in polarized primary human airway epithelia, and reversed F508del CFTR correction produced by VX-809. TGF-beta effects on the expression and activity of TMEM16A, wtCFTR and corrected F508del CFTR were seen at 10-fold lower concentrations relative to TGF-beta effects on e-cadherin (epithelial marker) and vimentin (mesenchymal marker) expression. TGF-beta downregulation of TMEM16A and CFTR expression were partially reversed by Smad3 and p38 MAPK inhibition, respectively. CONCLUSIONS: TGF-beta is sufficient to downregulate two critical chloride transporters in two CF-affected tissues that precedes expression changes of two distinct TGF-beta regulated proteins. Our results provide a plausible mechanism for CF-disease modification by TGF-beta through effects on CaCC.