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1.  Respiratory epithelial cells orchestrate pulmonary innate immunity 
Nature immunology  2014;16(1):27-35.
The epithelial surfaces of the lungs are in direct contact with the environment and are subjected to dynamic physical forces as airway tubes and alveoli are stretched and compressed during ventilation. Mucociliary clearance in conducting airways, reduction of surface tension in the alveoli, and maintenance of near sterility have been accommodated by the evolution of a multi-tiered innate host-defense system. The biophysical nature of pulmonary host defenses are integrated with the ability of respiratory epithelial cells to respond to and ‘instruct’ the professional immune system to protect the lungs from infection and injury.
PMCID: PMC4318521  PMID: 25521682
2.  Diseases of Pulmonary Surfactant Homeostasis 
Annual review of pathology  2015;10:371-393.
Advances in physiology and biochemistry have provided fundamental insights into the role of pulmonary surfactant in the pathogenesis and treatment of preterm infants with respiratory distress syndrome. Identification of the surfactant proteins, lipid transporters, and transcriptional networks regulating their expression has provided the tools and insights needed to discern the molecular and cellular processes regulating the production and function of pulmonary surfactant prior to and after birth. Mutations in genes regulating surfactant homeostasis have been associated with severe lung disease in neonates and older infants. Biophysical and transgenic mouse models have provided insight into the mechanisms underlying surfactant protein and alveolar homeostasis. These studies have provided the framework for understanding the structure and function of pulmonary surfactant, which has informed understanding of the pathogenesis of diverse pulmonary disorders previously considered idiopathic. This review considers the pulmonary surfactant system and the genetic causes of acute and chronic lung disease caused by disruption of alveolar homeostasis.
PMCID: PMC4316199  PMID: 25621661
alveolar proteinosis; interstitial lung disease; respiratory distress syndrome; alveolar capillary dysplasia; pulmonary fibrosis; pulmonary alveolar microlithiasis
3.  Foxa2 Regulates Leukotrienes to Inhibit Th2-mediated Pulmonary Inflammation 
Foxa2 is a member of the Forkhead family of nuclear transcription factors that is highly expressed in respiratory epithelial cells of the developing and mature lung. Foxa2 is required for normal airway epithelial differentiation, and its deletion causes goblet-cell metaplasia and Th2-mediated pulmonary inflammation during postnatal development. Foxa2 expression is inhibited during aeroallergen sensitization and after stimulation with Th2 cytokines, when its loss is associated with goblet-cell metaplasia. Mechanisms by which Foxa2 controls airway epithelial differentiation and Th2 immunity are incompletely known. During the first 2 weeks after birth, the loss of Foxa2 increases the production of leukotrienes (LTs) and Th2 cytokines in the lungs of Foxa2 gene–targeted mice. Foxa2 expression inhibited 15-lipoxygenase (Alox15) and increased Alox5 transcription, each encoding key lipoxygenases associated with asthma. The inhibition of the cysteinyl LT (CysLT) signaling pathway by montelukast inhibited IL-4, IL-5, eotaxin-2, and regulated upon activation normal T cell expressed and presumably secreted expression in the developing lungs of Foxa2 gene–targeted mice. Montelukast inhibited the expression of genes regulating mucus metaplasia, including Spdef, Muc5ac, Foxa3, and Arg2. Foxa2 plays a cell-autonomous role in the respiratory epithelium, and is required for the suppression of Th2 immunity and mucus metaplasia in the developing lung in a process determined in part by its regulation of the CysLT pathway.
PMCID: PMC3931118  PMID: 23822876
Foxa2; leukotriene; Th2 inflammation; mucous metaplasia
4.  SPDEF Inhibits Prostate Carcinogenesis by Disrupting a Positive Feedback Loop in Regulation of the Foxm1 Oncogene 
PLoS Genetics  2014;10(9):e1004656.
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.
Author Summary
Development of prostate cancer is a multistep process that involves the loss of tumor suppressor functions and activation of oncogenes. SPDEF transcription factor is expressed in normal prostate epithelium and its expression changes during prostate carcinogenesis (PCa). Since the role of SPDEF in PCa remains controversial, we generated transgenic mice with loss- and gain-of-function of SPDEF to demonstrate that SPDEF functions as a tumor suppressor in PCa. In animal models, the loss of SPDEF promoted PCa and increased the levels of Foxm1, a well-known oncogenic protein. Overexpression of SPDEF in prostate epithelium decreased PCa and reduced Foxm1 levels. Proliferation defects in SPDEF-containing tumor cells were corrected by re-expression of Foxm1, providing direct evidence that SPDEF inhibits tumor cell proliferation through Foxm1. We further showed that SPDEF directly bound to Foxm1 promoter and prevented its auto-regulatory activation. In prostate cancer patients, the low SPDEF and high Foxm1 were found in most aggressive prostate tumors that were associated with poor prognosis. The combined two-gene signature of low SPDEF and high Foxm1 was a strong predictor of survival in prostate cancer patients. The present study identified novel molecular mechanism of prostate cancer progression, providing a crosstalk between SPDEF tumor suppressor and Foxm1 oncogene.
PMCID: PMC4177813  PMID: 25254494
6.  Orphan G Protein–Coupled Receptor GPR116 Regulates Pulmonary Surfactant Pool Size 
Pulmonary surfactant levels within the alveoli are tightly regulated to maintain lung volumes and promote efficient gas exchange across the air/blood barrier. Quantitative and qualitative abnormalities in surfactant are associated with severe lung diseases in children and adults. Although the cellular and molecular mechanisms that control surfactant metabolism have been studied intensively, the critical molecular pathways that sense and regulate endogenous surfactant levels within the alveolus have not been identified and constitute a fundamental knowledge gap in the field. In this study, we demonstrate that expression of an orphan G protein–coupled receptor, GPR116, in the murine lung is developmentally regulated, reaching maximal levels 1 day after birth, and is highly expressed on the apical surface of alveolar type I and type II epithelial cells. To define the physiological role of GPR116 in vivo, mice with a targeted mutation of the Gpr116 locus, Gpr116Δexon17, were generated. Gpr116Δexon17 mice developed a profound accumulation of alveolar surfactant phospholipids at 4 weeks of age (12-fold) that was further increased at 20 weeks of age (30-fold). Surfactant accumulation in Gpr116Δexon17 mice was associated with increased saturated phosphatidylcholine synthesis at 4 weeks and the presence of enlarged, lipid-laden macrophages, neutrophilia, and alveolar destruction at 20 weeks. mRNA microarray analyses indicated that P2RY2, a purinergic receptor known to mediate surfactant secretion, was induced in Gpr116Δexon17 type II cells. Collectively, these data support the concept that GPR116 functions as a molecular sensor of alveolar surfactant lipid pool sizes by regulating surfactant secretion.
PMCID: PMC3824053  PMID: 23590306
pulmonary surfactant; G protein–coupled receptors; GPR116; surfactant metabolism; alveolar epithelium
7.  Natural or engineered mutations in Surfactant Protein-D alter allergic asthmatic responses in mice and man 
Surfactant protein D (SP-D) has been proposed to be protective in allergic airway responses.
We aimed to determine the effect of SP-D deficiency on murine and human airway allergy.
Immunological responses of SP-D gene deficient mice (Sftpd−/−) at baseline and following four Aspergillus fumigatus exposures were assessed. In addition, the significance of a single nucleotide polymorphism (Met11Thr) in the human SP-D gene (known to decrease SP-D function) on asthma susceptibility was investigated.
Macrophage BALF levels and lung CD-4+ T-cells were increased in naive Sftpd−/− mice in association with increased lung CCL17 levels. Th2-associated antibody levels (IgG1 and IgE) were significantly lower in 4–6 week old Sftpd−/− mice (p<0.05). Accordingly, naive Sftpd−/− splenocytes released significantly less IL-4 and IL-13 upon anti-CD3/CD28 stimulation (p<0.01). Following intranasal allergen exposures, a modest decrease in BALF eosinophilia was observed in Sftpd−/− mice compared to wild type mice (p<0.01). Translational studies in a pediatric population of Caucasians with asthma revealed that a single nucleotide polymorphism in the SFTPD gene, affecting SP-D levels and pathogen binding, was associated with lower asthma susceptibility (p<0.05).
Sftpd−/− mice have an impaired systemic Th2 response at baseline and modestly reduced pulmonary eosinophilia following allergen exposure. Translational studies revealed that a mutation in the SFTPD gene was associated with lower asthma susceptibility in Caucasians. Taken together, these results support the hypothesis that SP-D-dependent innate immunity influences atopy and asthma.
Clinical implications
SP-D deficiency results in increased pulmonary inflammation and decreased susceptibility to asthma, perhaps related to impaired endotoxin and pathogen clearance.
PMCID: PMC4145593  PMID: 18355911
allergy; lung; surfactant protein D; polymorphism; eosinophil; IL-13; Aspergillus; endotoxin
8.  Diffuse Lung Disease in Children 
Pediatric pulmonology  2013;49(4):400-409.
A multi-disciplinary scientific conference focused on diffuse and interstitial lung diseases in children was held in La Jolla, CA in June 2012. The conference brought together clinicians (including Pediatric and Adult Pulmonologists, Neonatologists, Pathologists, and Radiologists), clinical researchers, basic scientists, government agency representatives, patient advocates, as well as children affected by diffuse lung disease (DLD) and their families, to review recent scientific developments and emerging concepts in the pathophysiology of childhood DLD. Invited speakers discussed translational approaches, including genetics and proteomics, epigenetics and epigenomics, models of DLD, including animal models and induced pluripotent stem cells, and regenerative medicine approaches. The presentations of the invited speakers are summarized here.
PMCID: PMC4145861  PMID: 23798474
diffuse lung disease; childhood interstitial lung disease; genetics; epigenetics; stem cell
9.  Alveolar Surfactant Homeostasis and the Pathogenesis of Pulmonary Disease 
Annual review of medicine  2010;61:105-119.
The alveolar region of the lung creates an extensive epithelial surface that mediates the transfer of oxygen and carbon dioxide required for respiration after birth. Maintenance of pulmonary function depends on the function of type II epithelial cells that synthesize and secrete pulmonary surfactant lipids and proteins, reducing the collapsing forces created at the air-liquid interface in the alveoli. Genetic and acquired disorders associated with the surfactant system cause both acute and chronic lung disease. Mutations in the ABCA3, SFTPA, SFTPB, SFTPC, SCL34A2, and TERT genes disrupt type II cell function and/or surfactant homeostasis, causing neonatal respiratory failure and chronic interstitial lung disease. Defects in GM-CSF receptor function disrupt surfactant clearance, causing pulmonary alveolar proteinosis. Abnormalities in the surfactant system and disruption of type II cell homeostasis underlie the pathogenesis of pulmonary disorders previously considered idiopathic, providing the basis for improved diagnosis and therapies of these rare lung diseases.
PMCID: PMC4127631  PMID: 19824815
alveolar proteinosis; interstitial lung disease; respiratory distress syndrome; pulmonary fibrosis; pulmonary alveolar microlithiasis
10.  Foxa3 Induces Goblet Cell Metaplasia and Inhibits Innate Antiviral Immunity 
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.
PMCID: PMC3977731  PMID: 24392884
rhinovirus; IFN; transcription factors; mucus
Developmental biology  2013;379(1):38-52.
Wntless (Wls), a gene highly conserved across the animal kingdom, encodes for a transmembrane protein that mediates Wnt ligand secretion. Wls is expressed in developing lung, wherein Wnt signaling is necessary for pulmonary morphogenesis. We hypothesize that Wls plays a critical role in modulating Wnt signaling during lung development and therefore affects processes critical for pulmonary morphogenesis. We generated conditional Wls mutant mice utilizing Shh-Cre and Dermo1-Cre mice to delete Wls in the embryonic respiratory epithelium and mesenchyme, respectively. Epithelial deletion of Wls disrupted lung branching morphogenesis, peripheral lung development and pulmonary endothelial differentiation. Epithelial Wls mutant mice died at birth due to respiratory failure caused by lung hypoplasia and pulmonary hemorrhage. In the lungs of these mice, VEGF and Tie2-angiopoietin signaling pathways, which mediate vascular development, were downregulated from early stages of development. In contrast, deletion of Wls in mesenchymal cells of the developing lung did not alter branching morphogenesis or early mesenchymal differentiation. In vitro assays support the concept that Wls acts in part via Wnt5a to regulate pulmonary vascular development. We conclude that epithelial Wls modulates Wnt ligand activities critical for pulmonary vascular differentiation and peripheral lung morphogenesis. These studies provide a new framework for understanding the molecular mechanisms underlying normal pulmonary vasculature formation and the dysmorphic pulmonary vasculature development associated with congenital lung disease.
PMCID: PMC3699333  PMID: 23523683
Wntless; Wnt; lung development; endothelium; epithelium
12.  Epithelial SCAP/INSIG/SREBP Signaling Regulates Multiple Biological Processes during Perinatal Lung Maturation 
PLoS ONE  2014;9(5):e91376.
Pulmonary surfactant is required for lung function at birth and throughout postnatal life. Defects in the surfactant system are associated with common pulmonary disorders including neonatal respiratory distress syndrome and acute respiratory distress syndrome in children and adults. Lipogenesis is essential for the synthesis of pulmonary surfactant by type II epithelial cells lining the alveoli. This study sought to identify the role of pulmonary epithelial SREBP, a transcriptional regulator of cellular lipid homeostasis, during a critical time period of perinatal lung maturation in the mouse. Genome wide mRNA expression profiling of lung tissue from transgenic mice with epithelial-specific deletions of Scap (ScapΔ/Δ, resulting in inactivation of SREBP signaling) or Insig1 and Insig2 (Insig1/2Δ/Δ, resulting in activation of SREBP signaling) was assessed. Differentially expressed genes responding to SREBP perturbations were identified and subjected to functional enrichment analysis, pathway mapping and literature mining to predict upstream regulators and transcriptional networks regulating surfactant lipid homeostasis. Through comprehensive data analysis and integration, time dependent effects of epithelial SCAP/INSIG/SREBP deletion and defined SCAP/INSIG/SREBP-associated genes, bioprocesses and downstream pathways were identified. SREBP signaling influences epithelial development, cell death and cell proliferation at E17.5, while primarily influencing surfactant physiology, lipid/sterol synthesis, and phospholipid transport after birth. SREBP signaling integrated with the Wnt/β-catenin and glucocorticoid receptor signaling pathways during perinatal lung maturation. SREBP regulates perinatal lung lipogenesis and maturation through multiple mechanisms by interactions with distinct sets of regulatory partners.
PMCID: PMC4012993  PMID: 24806461
13.  Molecular Determinants of Lung Development 
Development of the pulmonary system is essential for terrestrial life. The molecular pathways that regulate this complex process are beginning to be defined, and such knowledge is critical to our understanding of congenital and acquired lung diseases. A recent workshop was convened by the National Heart, Lung, and Blood Institute to discuss the developmental principles that regulate the formation of the pulmonary system. Emerging evidence suggests that key developmental pathways not only regulate proper formation of the pulmonary system but are also reactivated upon postnatal injury and repair and in the pathogenesis of human lung diseases. Molecular understanding of early lung development has also led to new advances in areas such as generation of lung epithelium from pluripotent stem cells. The workshop was organized into four different topics, including early lung cell fate and morphogenesis, mechanisms of lung cell differentiation, tissue interactions in lung development, and environmental impact on early lung development. Critical points were raised, including the importance of epigenetic regulation of lung gene expression, the dearth of knowledge on important mesenchymal lineages within the lung, and the interaction between the developing pulmonary and cardiovascular system. This manuscript describes the summary of the discussion along with general recommendations to overcome the gaps in knowledge in lung developmental biology.
PMCID: PMC3955361  PMID: 23607856
lung development; lung cell fate; lung cell differentiation; tissue interaction; environmental impact
14.  Kruppel-like factor 5 Controls Villus Formation and Initiation of Cytodifferentiation in the Embryonic Intestinal Epithelium 
Developmental biology  2012;375(2):128-139.
Kruppel-like factor 5 (Klf5) is a transcription factor expressed by embryonic endodermal progenitors that form the lining of the gastrointestinal tract. A Klf5 floxed allele was efficiently deleted from the intestinal epithelium by a Cre transgene under control of the Shh promoter resulting in the inhibition of villus morphogenesis and epithelial differentiation. Although proliferation of the intestinal epithelium was maintained, the expression of Elf3, Pparγ, Atoh1, Ascl2, Neurog3, Hnf4α, Cdx1, and other genes associated with epithelial cell differentiation was inhibited in the Klf5-deficient intestines. At E18.5, Klf5Δ/Δ fetuses lacked the apical brush border characteristic of enterocytes, and a loss of goblet and enteroendocrine cells was observed. The failure to form villi was not attributable to the absence of HH or PDGF signaling, known mediators of this developmental process. Klf5-deletion blocked the decrease in FoxA1 and Sox9 expression that accompanies normal villus morphogenesis. KLF5 directly inhibited activity of the FoxA1 promoter, and in turn FOXA1 inhibited Elf3 gene expression in vitro, linking the observed loss of Elf3 with the persistent expression of FoxA1 observed in Klf5-deficient mice. Genetic network analysis identified KLF5 as a key transcription factor regulating intestinal cell differentiation and cell adhesion. These studies indicate a novel requirement for KLF5 to initiate morphogenesis of the early endoderm into a compartmentalized intestinal epithelium comprised of villi and terminally differentiated cells.
PMCID: PMC3582784  PMID: 23266329
Elf3; FoxA1; intestine development; cell adhesion
15.  Molecular Determinants of Lung Development 
Development of the pulmonary system is essential for terrestrial life. The molecular pathways that regulate this complex process are beginning to be defined, and such knowledge is critical to our understanding of congenital and acquired lung diseases. A recent workshop was convened by the National Heart, Lung, and Blood Institute to discuss the developmental principles that regulate the formation of the pulmonary system. Emerging evidence suggests that key developmental pathways not only regulate proper formation of the pulmonary system but are also reactivated upon postnatal injury and repair and in the pathogenesis of human lung diseases. Molecular understanding of early lung development has also led to new advances in areas such as generation of lung epithelium from pluripotent stem cells. The workshop was organized into four different topics, including early lung cell fate and morphogenesis, mechanisms of lung cell differentiation, tissue interactions in lung development, and environmental impact on early lung development. Critical points were raised, including the importance of epigenetic regulation of lung gene expression, the dearth of knowledge on important mesenchymal lineages within the lung, and the interaction between the developing pulmonary and cardiovascular system. This manuscript describes the summary of the discussion along with general recommendations to overcome the gaps in knowledge in lung developmental biology.
PMCID: PMC3955361  PMID: 23607856
lung development; lung cell fate; lung cell differentiation; tissue interaction; environmental impact
16.  CDC42 is Required for Structural Patterning of the Lung During Development 
Developmental biology  2012;374(1):46-57.
The formation of highly branched epithelial structures is critical for the development of many essential organs, including lung, liver, pancreas, kidney and mammary glands. Elongation and branching of these structures require precise control of complex morphogenetic processes that are dependent upon coordinate regulation of cell shape, apical-basal polarity, proliferation, migration, and interactions among multiple cell types. Herein, we demonstrate that temporal-spatial regulation of epithelial cell polarity by the small GTPase, CDC42, is essential for branching morphogenesis of the developing lung. Epithelial cell-specific deletion of CDC42 in fetal mice disrupted epithelial cell polarity, the actin cytoskeleton, intercellular contacts, directional trafficking of proteins, proliferation and mitotic spindle orientation, impairing the organization and patterning of the developing respiratory epithelium and adjacent mesenchyme. Transition from a pseudostratified to a simple columnar epithelium was impaired, consistent with coordinate dysregulation of epithelial cell polarity, mitotic spindle orientation, and repositioning of mitotic cells within the epithelium during cell cycle progression. Expression of sonic hedgehog and its receptor, patched-1, was decreased, while fibroblast growth factor 10 expression in the mesenchyme was expanded, resulting in disruption of branching morphogenesis and bronchiolar smooth muscle formation in this model. CDC42 is required for spatial positioning of proliferating epithelial cells, as well as signaling interactions between the epithelium and mesenchyme and is, therefore, essential for formation and maintenance of the respiratory tract during morphogenesis of the fetal lung.
PMCID: PMC3549046  PMID: 23219958
Mouse Development; Branching Morphogenesis; Rho GTPase; Targeted Deletion; Intercellular Junctions; Pulmonary Hypoplasia
17.  The Pulmonary Collectins and Respiratory Syncytial Virus: Is There a Clinical Link? 
The Journal of pediatrics  2010;156(3):10.1016/j.jpeds.2009.11.040.
PMCID: PMC3870860  PMID: 20176182
18.  Foxm1 Transcription Factor Is Critical for Proliferation and Differentiation of Clara Cells during Development of Conducting Airways 
Developmental biology  2012;370(2):198-212.
Respiratory epithelial cells are derived from cell progenitors in the foregut endoderm that subsequently differentiate into the distinct cell types lining the conducting and alveolar regions of the lung. To identify transcriptional mechanisms regulating differentiation and maintenance of respiratory epithelial cells, we conditionally deleted Foxm1 transcription factor from the conducting airways of the developing mouse lung. Conditional deletion of Foxm1 from Clara cells, controlled by the Scgb1a1 promoter, dramatically altered airway structure and caused peribronchial fibrosis, resulting in airway hyperreactivity in adult mice. Deletion of Foxm1 inhibited proliferation of Clara cells and disrupted the normal patterning of epithelial cell differentiation in the bronchioles, causing squamous and goblet cell metaplasia, and the loss of Clara and ciliated cells. Surprisingly, conducting airways of Foxm1-deficient mice contained highly differentiated cuboidal type II epithelial cells that are normally restricted to the alveoli. Lineage tracing studies showed that the ectopic alveolar type II cells in Foxm1-deficient airways were derived from Clara cells. Deletion of Foxm1 inhibited Sox2 and Scgb1a1, both of which are critical for differentiation and function of Clara cells. In co-transfection experiments, Foxm1 directly bound to and induced transcriptional activity of Scgb1a1 and Sox2 promoters. Foxm1 is required for differentiation and maintenance of epithelial cells lining conducting airways.
PMCID: PMC3449302  PMID: 22885335
Foxm1; Clara cells; airway epithelium; type II cells; Sox2; airway development
19.  CCAAT/Enhancer Binding Protein–α Regulates the Protease/Antiprotease Balance Required for Bronchiolar Epithelium Regeneration 
Many transcription factors that regulate lung morphogenesis during development are reactivated to mediate repairs of the injured adult lung. We hypothesized that CCAAT/enhancer binding protein–α (C/EBPα), a transcription factor critical for perinatal lung maturation, regulates genes required for the normal repair of the bronchiolar epithelium after injury. Transgenic CebpαΔ/Δ mice, in which Cebpa was conditionally deleted from Clara cells and Type II cells after birth, were used in this study. Airway injury was induced in mice by the intraperitoneal administration of naphthalene to ablate bronchiolar epithelial cells. Although the deletion of C/EBPα did not influence lung structure and function under unstressed conditions, C/EBPα was required for the normal repair of terminal bronchiolar epithelium after naphthalene injury. To identify cellular processes that are influenced by C/EBPα during repair, mRNA microarray was performed on terminal bronchiolar epithelial cells isolated by laser-capture microdissection. Normal repair of the terminal bronchiolar epithelium was highly associated with the mRNAs regulating antiprotease activities, and their induction required C/EBPα. The defective deposition of fibronectin in CebpαΔ/Δ mice was associated with increased protease activity and delayed differentiation of FoxJ1-expressing ciliated cells. The fibronectin and ciliated cells were restored by the intratracheal treatment of CebpαΔ/Δ mice with the serine protease inhibitor. In conclusion, C/EBPα regulates the expression of serine protease inhibitors that are required for the normal increase of fibronectin and the restoration of ciliated cells after injury. Treatment with serine protease inhibitor may aid in the recovery of injured bronchiolar epithelial cells, and prevent common chronic lung diseases.
PMCID: PMC3488626  PMID: 22652201
C/EBPα; antiprotease; Spink5; naphthalene; Scgb1a1
20.  Inhibition of the Growth Factor MDK/Midkine by a Novel Small Molecule Compound to Treat Non-Small Cell Lung Cancer 
PLoS ONE  2013;8(8):e71093.
Midkine (MDK) is a heparin-binding growth factor that is highly expressed in many malignant tumors, including lung cancers. MDK activates the PI3K pathway and induces anti-apoptotic activity, in turn enhancing the survival of tumors. Therefore, the inhibition of MDK is considered a potential strategy for cancer therapy. In the present study, we demonstrate a novel small molecule compound (iMDK) that targets MDK. iMDK inhibited the cell growth of MDK-positive H441 lung adenocarcinoma cells that harbor an oncogenic KRAS mutation and H520 squamous cell lung cancer cells, both of which are types of untreatable lung cancer. However, iMDK did not reduce the cell viability of MDK-negative A549 lung adenocarcinoma cells or normal human lung fibroblast (NHLF) cells indicating its specificity. iMDK suppressed the endogenous expression of MDK but not that of other growth factors such as PTN or VEGF. iMDK suppressed the growth of H441 cells by inhibiting the PI3K pathway and inducing apoptosis. Systemic administration of iMDK significantly inhibited tumor growth in a xenograft mouse model in vivo. Inhibition of MDK with iMDK provides a potential therapeutic approach for the treatment of lung cancers that are driven by MDK.
PMCID: PMC3745462  PMID: 23976985
21.  FOXM1 Promotes Allergen-Induced Goblet Cell Metaplasia and Pulmonary Inflammation 
Molecular and Cellular Biology  2013;33(2):371-386.
Chronic airway disorders, including chronic obstructive pulmonary disease (COPD), cystic fibrosis, and asthma, are associated with persistent pulmonary inflammation and goblet cell metaplasia and contribute to significant morbidity and mortality worldwide. While the molecular pathogenesis of these disorders is actively studied, little is known regarding the transcriptional control of goblet cell differentiation and mucus hyperproduction. Herein, we demonstrated that pulmonary allergen sensitization induces expression of FOXM1 transcription factor in airway epithelial and inflammatory cells. Conditional deletion of the Foxm1 gene from either airway epithelium or myeloid inflammatory cells decreased goblet cell metaplasia, reduced lung inflammation, and decreased airway resistance in response to house dust mite allergen (HDM). FOXM1 induced goblet cell metaplasia and Muc5AC expression through the transcriptional activation of Spdef. FOXM1 deletion reduced expression of CCL11, CCL24, and the chemokine receptors CCR2 and CX3CR1, resulting in decreased recruitment of eosinophils and macrophages to the lung. Deletion of FOXM1 from dendritic cells impaired the uptake of HDM antigens and decreased cell surface expression of major histocompatibility complex II (MHC II) and costimulatory molecule CD86, decreasing production of Th2 cytokines by activated T cells. Finally, pharmacological inhibition of FOXM1 by ARF peptide prevented HDM-mediated pulmonary responses. FOXM1 regulates genes critical for allergen-induced lung inflammation and goblet cell metaplasia.
PMCID: PMC3554115  PMID: 23149934
22.  Thy-1 Signals through PPARγ to Promote Lipofibroblast Differentiation in the Developing Lung 
Thy-1 is a glycosylphosphytidylinositol-linked cell-surface glycoprotein present on a subset of lung fibroblasts, which plays an important role in postnatal alveolarization. In the present study, we define the role of Thy-1 in pulmonary lipofibroblast differentiation and in the regulation of lipid homeostasis via peroxisome proliferator–activated receptor–γ (PPARγ). Thy-1 was associated with interstitial cells containing lipid droplets in vivo. The transfection of Thy-1 into Thy-1 (−) fibroblasts increased triglyceride content, fatty-acid uptake, and the expression of the lipofibroblast marker adipocyte differentiation–related protein. Thy-1 (+) fibroblasts exhibited 2.4-fold higher PPARγ activity, and the inhibition or activation of PPARγ reduced and increased triglyceride content, respectively. Thy-1 (−) fibroblasts were not responsive to either of the PPARγ agonists ciglitazone or prostaglandin J2, supporting the importance of Thy-1 in signaling via PPARγ. Thy-1 (+) fibroblasts expressed significantly higher concentrations of fatty-acid transporter protein–3 mRNA, and demonstrated higher rates of fatty-acid uptake and increased triglyceride content. The inhibition of fatty-acid transporter protein function reduced Thy-1 (+) fibroblast lipid content. The expression of Thy-1 in C57BL/6 lung fibroblasts increased during the neonatal period, coinciding with the onset of alveolarization. Thy-1 promoted lipofibroblast differentiation via the expression of PPARγ, stimulated lipid accumulation via fatty-acid esterification, and enhanced the fatty-acid uptake mediated by fatty-acid transporter proteins. Thy-1 is important in the regulation of lipofibroblast differentiation in the developing lung.
PMCID: PMC3380285  PMID: 22268140
Thy-1; lipofibroblast; PPARγ; lipid metabolism
23.  Foxm1 Mediates Cross Talk between Kras/Mitogen-Activated Protein Kinase and Canonical Wnt Pathways during Development of Respiratory Epithelium 
Molecular and Cellular Biology  2012;32(19):3838-3850.
While Kras/mitogen-activated protein kinase (MAPK) and canonical Wnt/β-catenin are critical for lung morphogenesis, mechanisms integrating these important signaling pathways during lung development are unknown. Herein, we demonstrate that the Foxm1 transcription factor is a key downstream target of activated KrasG12D. Deletion of Foxm1 from respiratory epithelial cells during lung formation prevented structural abnormalities caused by activated KrasG12D. Kras/Foxm1 signaling inhibited the activity of canonical Wnt signaling in the developing lung in vivo. Foxm1 decreased T-cell factor (TCF) transcriptional activity induced by activated β-catenin in vitro. Depletion of Foxm1 by short interfering RNA (siRNA) increased nuclear localization of β-catenin, increased expression of β-catenin target genes, and decreased mRNA and protein levels of the β-catenin inhibitor Axin2. Axin2 mRNA was reduced in distal lung epithelium of Foxm1-deficient mice. Foxm1 directly bound to and increased transcriptional activity of the Axin2 promoter region. Foxm1 is required for Kras signaling in distal lung epithelium and provides a mechanism integrating Kras and canonical Wnt/β-catenin signaling during lung development.
PMCID: PMC3457538  PMID: 22826436
24.  KrasG12D and Nkx2-1 haploinsufficiency induce mucinous adenocarcinoma of the lung  
The Journal of Clinical Investigation  2012;122(12):4388-4400.
Mucinous adenocarcinoma of the lung is a subtype of highly invasive pulmonary tumors and is associated with decreased or absent expression of the transcription factor NK2 homeobox 1 (NKX2-1; also known as TTF-1). Here, we show that haploinsufficiency of Nkx2-1 in combination with oncogenic KrasG12D, but not with oncogenic EGFRL858R, caused pulmonary tumors in transgenic mice that were phenotypically similar to human mucinous adenocarcinomas. Gene expression patterns distinguished tumor goblet (mucous) cells from nontumorigenic airway and intestinal goblet cells. Expression of NKX2-1 inhibited urethane and oncogenic KrasG12D-induced tumorigenesis in vivo. Haploinsufficiency of Nkx2-1 enhanced KrasG12D-mediated tumor progression, but reduced EGFRL858R-mediated progression. Genome-wide analysis of gene expression demonstrated that a set of genes induced in mucinous tumors was shared with genes induced in a nontumorigenic chronic lung disease, while a distinct subset of genes was specific to mucinous tumors. ChIP with massively parallel DNA sequencing identified a direct association of NKX2-1 with the genes induced in mucinous tumors. NKX2-1 associated with the AP-1 binding element as well as the canonical NKX2-1 binding element. NKX2-1 inhibited both AP-1 activity and tumor colony formation in vitro. These data demonstrate that NKX2-1 functions in a context-dependent manner in lung tumorigenesis and inhibits KrasG12D-driven mucinous pulmonary adenocarcinoma.
PMCID: PMC3533546  PMID: 23143308
25.  Kruppel-like factor 5 is Required for Formation and Differentiation of the Bladder Urothelium 
Developmental biology  2011;358(1):79-90.
Kruppel-like transcription factor 5 (Klf5) was detected in the developing and mature murine bladder urothelium. Herein we report a critical role of KLF5 in the formation and terminal differentiation of the urothelium. The ShhGfpCre transgene was used to delete the Klf5floxed alleles from bladder epithelial cells causing prenatal hydronephrosis, hydroureter, and vesicoureteric reflux. The bladder urothelium failed to stratify and did not express terminal differentiation markers characteristic of basal, intermediate, and umbrella cells including keratins 20, 14, and 5, and the uroplakins. The effects of Klf5 deletion were unique to the developing bladder epithelium since maturation of the epithelium comprising the bladder neck and urethra were unaffected by the lack of KLF5. mRNA analysis identified reductions in Pparγ, Grhl3, Elf3, and Ovol1expression in Klf5 deficient fetal bladders supporting their participation in a transcriptional network regulating bladder urothelial differentiation. KLF5 regulated expression of the mGrhl3 promoter in transient transfection assays. The absence of urothelial Klf5 altered epithelial-mesenchymal signaling leading to the formation of an ectopic alpha smooth muscle actin positive layer of cells subjacent to the epithelium and a thinner detrusor muscle that was not attributable to disruption of SHH signaling, a known mediator of detrusor morphogenesis. Deletion of Klf5 from the developing bladder urothelium blocked epithelial cell differentiation, impaired bladder morphogenesis and function causing hydroureter and hydronephrosis at birth.
PMCID: PMC3180904  PMID: 21803035
bladder; vesicoureteral reflux; KLF5; GRHL3; urothelium; micro-CT

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