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.

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.

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.

Mesodermal ALK5 controls lung myofibroblast versus lipofibroblast cell fate.

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

Dihydrotestosterone potentiates EGF-induced ERK activation by inducing SRC in fetal lung fibroblasts.

Lung maturation is regulated by interactions between mesenchymal and epithelial cells, and is delayed by androgens. Fibroblast-Type II cell communications are dependent on extracellular signal-regulated kinases (ERK) 1/2 activation by the ErbB receptor ligands epidermal growth factor (EGF), transforming growth factor (TGF)-α, and neuregulin (Nrg). In other tissues, dihydrotestosterone (DHT) has been shown to activate SRC by a novel nontranscriptional mechanism, which phosphorylates EGF receptors to potentiate EGF-induced ERK1/2 activation. This study sought to determine if DHT potentiates EGFR signaling by a nontranscriptional mechanism. Embryonic day (E)17 fetal lung cells were isolated from dams treated with or without DHT since E12. Cells were exposed to 30 ng/ml DHT for periods of 30 minutes to 3 days before being stimulated with 100 ng/ml EGF, TGF-α, or Nrg for up to 30 minutes. Lysates were immunoblotted for ErbB and SRC pathway signaling intermediates. DHT increased ERK1/2 activation by EGF, TGF-α, and Nrg in fibroblasts and Type II cells. Characterization in fibroblasts showed that potentiation of the EGF pathway was significant after 60 minutes of DHT exposure and persisted in the presence of the translational inhibitor cycloheximide. SRC and EGF receptor phosphorylation was increased by DHT, as was EGF-induced SHC1 phosphorylation and subsequent association with GRB2. Finally, SRC silencing, SRC inhibition with PP2, and overexpression of a dominant-negative SRC each prevented DHT from increasing EGF-induced ERK1/2 phosphorylation. These results suggest that DHT activates SRC to potentiate the signaling pathway leading from the EGF receptor to ERK activation in primary fetal lung fibroblasts.

TGF-beta activates Erk MAP kinase signalling through direct phosphorylation of ShcA.

Erk1/Erk2 MAP kinases are key regulators of cell behaviour and their activation is generally associated with tyrosine kinase signalling. However, TGF-beta stimulation also activates Erk MAP kinases through an undefined mechanism, albeit to a much lower level than receptor tyrosine kinase stimulation. We report that upon TGF-beta stimulation, the activated TGF-beta type I receptor (TbetaRI) recruits and directly phosphorylates ShcA proteins on tyrosine and serine. This dual phosphorylation results from an intrinsic TbetaRI tyrosine kinase activity that complements its well-defined serine-threonine kinase function. TGF-beta-induced ShcA phosphorylation induces ShcA association with Grb2 and Sos, thereby initiating the well-characterised pathway linking receptor tyrosine kinases with Erk MAP kinases. We also found that TbetaRI is tyrosine phosphorylated in response to TGF-beta. Thus, TbetaRI, like the TGF-beta type II receptor, is a dual-specificity kinase. Recruitment of tyrosine kinase signalling pathways may account for aspects of TGF-beta biology that are independent of Smad signalling.

beta1 integrins modulate p66ShcA expression and EGF-induced MAP kinase activation in fetal lung cells.

ShcA proteins mediate Erk1/Erk2 activation by integrins and epidermal growth factor (EGF), and are expressed as p46ShcA, p52ShcA, and p66ShcA. Although p52ShcA and p46ShcA mediate Erk1/Erk2 activation, p66ShcA antagonizes Erk activation. p66ShcA is spatially regulated during lung development, leading us to hypothesize that integrin signaling regulates p66ShcA expression and, consequently, EGF signaling. Fetal lung mesenchymal cells were isolated from E16 Swiss-Webster mice, stimulated with oligopeptide extracellular matrix analogs or anti-integrin antibodies, and subjected to ShcA Western analyses and EGF-stimulated Erk1/Erk2 kinase assays. p66ShcA expression was decreased by anti-alpha1 integrin antibody and DGEA collagen analog, and increased by anti-beta1, anti-alpha4, and anti-alpha5 integrin antibodies and RGDS fibronectin analog. Paradoxically, beta1 integrin stimulation increased EGF-induced Erk activation while increasing expression of the inhibitory p66ShcA isoform. This paradox was resolved by demonstrating that Erk inhibition attenuates integrin-mediated p66ShcA induction. These results suggest that p66ShcA is up-regulated as inhibitory feedback on integrin-mediated Erk activation.

New role for nuclear hormone receptors and coactivators in regulation of BRCA1-mediated DNA repair in breast cancer cell lines.

INTRODUCTION: The breast cancer susceptibility gene BRCA1 is involved in the repair of double-strand breaks induced by ionizing radiation and chemotherapy drugs. BRCA1 interacts with coactivators such as p300 and CREB-binding protein (CBP) to activate target gene transcription. Estrogen and retinoic acid receptors (ER and RAR) also require coactivator proteins for their ligand-dependent functions. Few studies have suggested a role for nuclear hormone receptors in DNA repair. METHODS: DNA damage and repair activity were quantified with the use of single-cell gel electrophoresis and plasmid end-joining assays. Cell cycle progression and apoptosis were determined by bromodeoxyuridine and TdT-mediated dUTP nick end labelling assays. Stable transfection was accomplished with the lipofection procedure. Protein interaction and expression were determined by immunoprecipitation and western blotting. RESULTS: 17beta-estradiol (E2) and all-trans retinoic acid (RA) had opposing effects on DNA damage and breast cancer cell survival after double-strand break damage. Treatment with E2, but not with RA, resulted in complex formation between ERalpha, CBP, and BRCA1 in ER-positive cell lines. Mutant BRCA1 reduced the expression and activity of DNA damage repair proteins but did not block nuclear hormone-dependent effects. Mutant BRCA1 failed to form complexes with ERalpha and CBP, which correlated with its ability to exert E2-independent effects on DNA repair. Mutant BRCA1 inhibited cell cycle progression and produced increased survival in cells with double-strand breaks. Ectopic ERalpha expression reproduced the E2-mediated effects on DNA damage, repair, and survival. CONCLUSION: The present study proposes a new mechanism by which ER and RAR regulate BRCA1-mediated DNA repair by means of CBP.

Developmental regulation of p66Shc is altered by bronchopulmonary dysplasia in baboons and humans.

RATIONALE: The p66(Shc) adapter protein antagonizes mitogen-activated protein, or MAP, kinase, mediates oxidative stress, and is developmentally regulated in fetal mouse lungs. OBJECTIVES: To determine if p66(Shc) is similarly regulated in primates and in bronchopulmonary dysplasia (BPD), which results from oxidative injury to immature lungs. METHODS: Normal and injured lungs from humans and baboons were evaluated by Western analysis and immunohistochemistry. MEASUREMENTS AND MAIN RESULTS: In baboons, p66(Shc) decreased 80% between 125 and 175 days' gestation (p = 0.025), then doubled after term delivery at 185 days (p = 0.0013). In the hyperoxic 140-day fetal baboon BPD model, p66(Shc) expression persisted, and its localization shifted from the epithelium of gestational controls to the mesenchyme of diseased lungs, coincident with expression of proliferating cell nuclear antigen and cleaved poly(adenyl ribose) polymerase, a marker of apoptosis. Treatment with the antibombesin antibody 2A11 attenuated BPD, reduced cell proliferation, increased p66(Shc) expression 10.5-fold, and preserved epithelial p66(Shc) localization. p66(Shc) also decreased during normal human lung development, falling 87% between 18 and 24 weeks' gestation (p = 0.02). p66(Shc) was expressed throughout 18-week human lungs, became restricted to scattered epithelial cells by 24 weeks, and localized to isolated mesenchymal cells after term delivery. In contrast, p66(Shc) remained prominent in the epithelium of lungs with acute injury or mild BPD, and in the mesenchyme of lungs with severe disease. p66(Shc) localized to tissues expressing proliferating cell nuclear antigen and cleaved poly(adenyl ribose) polymerase. CONCLUSIONS: p66(Shc) expression, cell proliferation, and apoptosis are concomitantly altered during lung development and in BPD.