Decoding the IGF1 signaling gene regulatory network behind alveologenesis from a mouse model of bronchopulmonary dysplasia.

Lung development is precisely controlled by underlying gene regulatory networks (GRN). Disruption of genes in the network can interrupt normal development and cause diseases such as bronchopulmonary dysplasia (BPD) - a chronic lung disease in preterm infants with morbid and sometimes lethal consequences characterized by lung immaturity and reduced alveolarization. Here, we generated a transgenic mouse exhibiting a moderate severity BPD phenotype by blocking IGF1 signaling in secondary crest myofibroblasts (SCMF) at the onset of alveologenesis. Using approaches mirroring the construction of the model GRN in sea urchin's development, we constructed the IGF1 signaling network underlying alveologenesis using this mouse model that phenocopies BPD. The constructed GRN, consisting of 43 genes, provides a bird's eye view of how the genes downstream of IGF1 are regulatorily connected. The GRN also reveals a mechanistic interpretation of how the effects of IGF1 signaling are transduced within SCMF from its specification genes to its effector genes and then from SCMF to its neighboring alveolar epithelial cells with WNT5A and FGF10 signaling as the bridge. Consistently, blocking WNT5A signaling in mice phenocopies BPD as inferred by the network. A comparative study on human samples suggests that a GRN of similar components and wiring underlies human BPD. Our network view of alveologenesis is transforming our perspective to understand and treat BPD. This new perspective calls for the construction of the full signaling GRN underlying alveologenesis, upon which targeted therapies for this neonatal chronic lung disease can be viably developed.

Hedgehog-responsive PDGFRa(+) fibroblasts maintain a unique pool of alveolar epithelial progenitor cells during alveologenesis.

The lung alveolus is lined with alveolar type 1 (AT1) and type 2 (AT2) epithelial cells. During alveologenesis, increasing demand associated with expanding alveolar numbers is met by proliferating progenitor AT2s (pAT2). Little information exists regarding the identity of this population and their niche microenvironment. We show that during alveologenesis, Hedgehog-responsive PDGFRa(+) progenitors (also known as SCMFs) are a source of secreted trophic molecules that maintain a unique pAT2 population. SCMFs are in turn maintained by TGFß signaling. Compound inactivation of Alk5 TßR2 in SCMFs reduced their numbers and depleted the pAT2 pool without impacting differentiation of daughter cells. In lungs of preterm infants who died with bronchopulmonary dysplasia, PDGFRa is reduced and the number of proliferative AT2s is diminished, indicating that an evolutionarily conserved mechanism governs pAT2 behavior during alveologenesis. SCMFs are a transient cell population, active only during alveologenesis, making them a unique stage-specific niche mesodermal cell type in mammalian organs.

Wnt5a Promotes AT1 and Represses AT2 Lineage-Specific Gene Expression in a Cell-Context-Dependent Manner.

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

FOXO1 Couples KGF and PI-3K/AKT Signaling to NKX2.1-Regulated Differentiation of Alveolar Epithelial Cells.

NKX2.1 is a master regulator of lung morphogenesis and cell specification; however, interactions of NKX2.1 with various transcription factors to regulate cell-specific gene expression and cell fate in the distal lung remain incompletely understood. FOXO1 is a key regulator of stem/progenitor cell maintenance/differentiation in several tissues but its role in the regulation of lung alveolar epithelial progenitor homeostasis has not been evaluated. We identified a novel role for FOXO1 in alveolar epithelial cell (AEC) differentiation that results in the removal of NKX2.1 from surfactant gene promoters and the subsequent loss of surfactant expression in alveolar epithelial type I-like (AT1-like) cells. We found that the FOXO1 forkhead domain potentiates a loss of surfactant gene expression through an interaction with the NKX2.1 homeodomain, disrupting NKX2.1 binding to the SFTPC promoter. In addition, blocking PI-3K/AKT signaling reduces phosphorylated FOXO-1 (p-FOXO1), allowing accumulated nuclear FOXO1 to interact with NKX2.1 in differentiating AEC. Inhibiting AEC differentiation in vitro with keratinocyte growth factor (KGF) maintained an AT2 cell phenotype through increased PI3K/AKT-mediated FOXO1 phosphorylation, resulting in higher levels of surfactant expression. Together these results indicate that FOXO1 plays a central role in AEC differentiation by directly binding NKX2.1 and suggests an essential role for FOXO1 in mediating AEC homeostasis.

WNT5a-ROR Signaling Is Essential for Alveologenesis.

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.

Grp78 Loss in Epithelial Progenitors Reveals an Age-linked Role for Endoplasmic Reticulum Stress in Pulmonary Fibrosis.

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.

The role of pulmonary mesenchymal cells in airway epithelium regeneration during injury repair.

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.

Secondary crest myofibroblast PDGFRα controls the elastogenesis pathway via a secondary tier of signaling networks during alveologenesis.

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.

Plasma membrane wounding and repair in pulmonary diseases.

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.

The 78-kD Glucose-Regulated Protein Regulates Endoplasmic Reticulum Homeostasis and Distal Epithelial Cell Survival during Lung Development.

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.

PTEN regulates lung endodermal morphogenesis through MEK/ERK pathway.

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.

The p66Shc adapter protein regulates the morphogenesis and epithelial maturation of fetal mouse lungs.

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.

Localized Fgf10 expression is not required for lung branching morphogenesis but prevents differentiation of epithelial progenitors.

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.

Tissue-dependent consequences of Apc inactivation on proliferation and differentiation of ciliated cell progenitors via Wnt and notch signaling.

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.

Apc deficiency alters pulmonary epithelial cell fate and inhibits Nkx2.1 via triggering TGF-beta signaling.

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.

Mesodermal Pten inactivation leads to alveolar capillary dysplasia- like phenotype.

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.

NOTCH1 is required for regeneration of Clara cells during repair of airway injury.

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.

Conditional deletion of epithelial IKKß impairs alveolar formation through apoptosis and decreased VEGF expression during early mouse lung morphogenesis.

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.

A subset of epithelial cells with CCSP promoter activity participates in alveolar development.

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.

Cell type-specific expression of adenomatous polyposis coli in lung development, injury, and repair.

Adenomatous polyposis coli (Apc) is critical for Wnt signaling and cell migration. The current study examined Apc expression during lung development, injury, and repair. Apc was first detectable in smooth muscle layers in early lung morphogenesis, and was highly expressed in ciliated and neuroendocrine cells in the advanced stages. No Apc immunoreactivity was detected in Clara or basal cells, which function as stem/progenitor cell in adult lung. In ciliated cells, Apc is associated mainly with apical cytoplasmic domain. In response to naphthalene-induced injury, Apc(positive) cells underwent squamous metaplasia, accompanied by changes in Apc subcellular distribution. In conclusion, both spatial and temporal expression of Apc is dynamically regulated during lung development and injury repair. Differential expression of Apc in progenitor vs. nonprogenitor cells suggests a functional role in cell-type specification. Subcellular localization changes of Apc in response to naphthalene injury suggest a role in cell shape and cell migration.