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The signaling cross talk between the tracheal mesenchyme and epithelium has not been researched extensively, leaving a substantial gap of knowledge in the mechanisms dictating embryonic development of the proximal airways by the adjacent mesenchyme. Recently, we reported that embryos lacking mesenchymal expression of Sox9 did not develop tracheal cartilage rings and showed aberrant differentiation of the tracheal epithelium. Here, we propose that tracheal cartilage provides local inductive signals responsible for the proper differentiation, metabolism, and inflammatory status regulation of the tracheal epithelium. The tracheal epithelium of mesenchyme-specific Sox9 Δ/Δ mutant embryos showed altered mRNA expression of various epithelial markers such as Pb1fa1, surfactant protein B (Sftpb), secretoglobulin, family 1A, member 1 (Scgb1a1), and trefoil factor 1 (Tff1). In vitro tracheal epithelial cell cultures confirmed that tracheal chondrocytes secrete factors that inhibit club cell differentiation. Whole gene expression profiling and ingenuity pathway analysis showed that the tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and transforming growth factor-β (TGF-β) signaling pathways were significantly altered in the Sox9 mutant trachea. TNF-α and IFN-γ interfered with the differentiation of tracheal epithelial progenitor cells into mature epithelial cell types in vitro. Mesenchymal knockout of Tgf-β1 in vivo resulted in altered differentiation of the tracheal epithelium. Finally, mitochondrial enzymes involved in fat and glycogen metabolism, cytochrome c oxidase subunit VIIIb (Cox8b) and cytochrome c oxidase subunit VIIa polypeptide 1 (Cox7a1), were strongly upregulated in the Sox9 mutant trachea, resulting in increases in the number and size of glycogen storage vacuoles. Our results support a role for tracheal cartilage in modulation of the differentiation and metabolism and the expression of inflammatory-related genes in the tracheal epithelium by feeding into the TNF-α, IFN-γ, and TGF-β signaling pathways., (Copyright © 2017 the American Physiological Society.)

The causes underlying the self-perpetuating nature of idiopathic pulmonary fibrosis (IPF), a progressive and usually lethal disease, remain unknown. We hypothesised that alveolar soluble annexin V contributes to lung fibrosis, based on the observation that human IPF bronchoalveolar lavage fluid (BALF) containing high annexin V levels promoted fibroblast involvement in alveolar epithelial wound healing that was reduced when annexin V was depleted from the BALF. Conditioned medium from annexin V-treated alveolar epithelial type 2 cells (AEC2), but not annexin V per se, induced proliferation of human fibroblasts and contained pro-fibrotic, IPF-associated proteins, as well as pro-inflammatory cytokines that were found to correlate tightly (r>0.95) with annexin V levels in human BALF. ErbB2 receptor tyrosine kinase in AECs was activated by annexin V, and blockade reduced the fibrotic potential of annexin V-treated AEC-conditioned medium. In vivo, aerosol delivery of annexin V to mouse lung induced inflammation, fibrosis and increased hydroxyproline, with activation of Wnt, transforming growth factor-β, mitogen-activated protein kinase and nuclear factor-κB signalling pathways, as seen in IPF. Chronically increased alveolar annexin V levels, as reflected in increased IPF BALF levels, may contribute to the progression of IPF by inducing the release of pro-fibrotic mediators., (Copyright ©ERS 2015.)

Background: Reciprocal interactions between lung mesenchymal and epithelial cells play essential roles in lung organogenesis and homeostasis. Although the molecular markers and related animal models that target lung epithelial cells are relatively well studied, molecular markers of lung mesenchymal cells and the genetic tools to target and/or manipulate gene expression in a lung mesenchyme-specific manner are not available, which becomes a critical barrier to the study of lung mesenchymal biology and the related pulmonary diseases., Results: We have identified a mouse Tbx4 gene enhancer that contains conserved DNA sequences across many vertebrate species with lung or lung-like gas exchange organ. We then generate a mouse line to express rtTA/LacZ under the control of the Tbx4 lung enhancer, and therefore a Tet-On inducible transgenic system to target lung mesenchymal cells at different developmental stages. By combining a Tbx4-rtTA driven Tet-On inducible Cre expression mouse line with a Cre reporter mouse line, the spatial-temporal patterns of Tbx4 lung enhancer targeted lung mesenchymal cells were defined. Pulmonary endothelial cells and vascular smooth muscle cells were targeted by the Tbx4-rtTA driver line prior to E11.5 and E15.5, respectively, while other subtypes of lung mesenchymal cells including airway smooth muscle cells, fibroblasts, pericytes could be targeted during the entire developmental stage., Conclusions: Developmental lung mesenchymal cells can be specifically marked by Tbx4 lung enhancer activity. With our newly created Tbx4 lung enhancer-driven Tet-On inducible system, lung mesenchymal cells can be specifically and differentially targeted in vivo for the first time by controlling the doxycycline induction time window. This novel system provides a unique tool to study lung mesenchymal cell lineages and gene functions in lung mesenchymal development, injury repair, and regeneration in mice.

Mechanical forces are critical for normal fetal lung development. However, the mechanisms regulating this process are not well-characterized. We hypothesized that strain-induced release of HB-EGF and TGF-α is mediated via integrin-ADAM17/TACE interactions. Employing an in vitro system to simulate mechanical forces in fetal lung development, we showed that mechanical strain of fetal epithelial cells actives TACE, releases HB-EGF and TGF-α, and promotes differentiation. In contrast, in samples incubated with the TACE inhibitor IC-3 or in cells isolated from TACE knock-out mice, mechanical strain did not release ligands or promote cell differentiation, which were both rescued after transfection of ADAM17. Cell adhesion assay and co-immunoprecipitation experiments in wild-type and TACE knock-out cells using several TACE constructs demonstrated not only that integrins α6 and β1 bind to TACE via the disintegrin domain but also that mechanical strain enhances these interactions. Furthermore, force applied to these integrin receptors by magnetic beads activated TACE and shed HB-EGF and TGF-α. The contribution of integrins α6 and β1 to differentiation of fetal epithelial cells by strain was demonstrated by blocking their binding site with specific antibodies and by culturing the cells on membranes coated with anti-integrin α6 and β1 antibodies. In conclusion, mechanical strain releases HB-EGF and TGF-α and promotes fetal type II cell differentiation via α6β1 integrin-ADAM17/TACE signaling pathway. These investigations provide novel mechanistic information on how mechanical forces promote fetal lung development and specifically differentiation of epithelial cells. This information could be also relevant to other tissues exposed to mechanical forces.

The miR-17 family of microRNAs has recently been recognized for its importance during lung development. The transgenic overexpression of the entire miR-17-92 cluster in the lung epithelium led to elevated cellular proliferation and inhibition of differentiation, while targeted deletion of miR-17-92 and miR-106b-25 clusters showed embryonic or early post-natal lethality. Herein we demonstrate that miR-17 and its paralogs, miR-20a, and miR-106b, are highly expressed during the pseudoglandular stage and identify their critical functional role during embryonic lung development. Simultaneous downregulation of these three miRNAs in explants of isolated lung epithelium altered FGF10 induced budding morphogenesis, an effect that was rescued by synthetic miR-17. E-Cadherin levels were reduced, and its distribution was altered by miR-17, miR-20a and miR-106b downregulation, while conversely, beta-catenin activity was augmented, and expression of its downstream targets, including Bmp4 as well as Fgfr2b, increased. Finally, we identified Stat3 and Mapk14 as key direct targets of miR-17, miR-20a, and miR-106b and showed that simultaneous overexpression of Stat3 and Mapk14 mimics the alteration of E-Cadherin distribution observed after miR-17, miR-20a, and miR-106b downregulation. We conclude that the mir-17 family of miRNA modulates FGF10-FGFR2b downstream signaling by specifically targeting Stat3 and Mapk14, hence regulating E-Cadherin expression, which in turn modulates epithelial bud morphogenesis in response to FGF10 signaling.

The mechanisms by which mechanical forces promote fetal lung development are not fully understood. Here, we investigated differentiation of fetal type II epithelial cells via the epidermal growth factor receptor (EGFR) in response to mechanical strain. First, we showed that incubation of embryonic day (E) 19 fetal type II cells with recombinant heparin-binding EGF-like growth factor (HB-EGF) or transforming growth factor (TGF)-alpha, but not with amphiregulin (AR), betacellulin (BTC) or epiregulin (EPR), increased fetal type II cell differentiation, as measured by surfactant protein B/C mRNA and protein levels. Next, we demonstrated that 5% cyclic stretch of E19 monolayers transfected with plasmid encoding alkaline phosphatase (AP)-tagged ligands shed mature HB-EGF and TGF-alpha into the supernatant and promoted type II cell differentiation. Release of these ligands was also observed in E19 cells subjected to higher degrees of cyclic strain, but not in cells exposed to continuous stretch. Interestingly, the addition of fibroblasts to type II cell cultures did not enhance release of HB-EGF. Whereas HB-EGF shedding was also detected in E18 cells exposed to 5% cyclic stretch, release of this ligand after 2.5% sustained stretch was restricted to cells isolated on E18 of gestation. In addition, mechanical stretch released EGF, AR and BTC. We conclude that mechanical stretch promotes fetal type II cell differentiation via ectodomain shedding of HB-EGF and TGF-alpha. The magnitude of shedding varied depending on gestational age, ligand, and strain protocol. These studies provide novel mechanistic information potentially relevant to fetal lung development and to mechanical ventilation-induced lung injury.

A new source of stem cells has recently been isolated from amniotic fluid; these amniotic fluid stem cells have significant potential for regenerative medicine. These cells are multipotent, showing the ability to differentiate into cell types from each embryonic germ layer. We investigated the ability of human amniotic fluid stem cells (hAFSC) to integrate into murine lung and to differentiate into pulmonary lineages after injury. Using microinjection into cultured mouse embryonic lungs, hAFSC can integrate into the epithelium and express the early human differentiation marker thyroid transcription factor 1 (TTF1). In adult nude mice, following hyperoxia injury, tail vein-injected hAFSC localized in the distal lung and expressed both TTF1 and the type II pneumocyte marker surfactant protein C. Specific damage of Clara cells through naphthalene injury produced integration and differentiation of hAFSC at the bronchioalveolar and bronchial positions with expression of the specific Clara cell 10-kDa protein. These results illustrate the plasticity of hAFSC to respond in different ways to different types of lung damage by expressing specific alveolar versus bronchiolar epithelial cell lineage markers, depending on the type of injury to recipient lung. Disclosure of potential conflicts of interest is found at the end of this article.

Bone Morphogenetic Proteins are key signaling molecules in vertebrate development. Little is known about Bmp gene regulation in any organ. In Drosophila, the Bmp gene, dpp is regulated by Dorsal, the invertebrate homologue of Rel-NF-kB. In this study we examined whether TNF-alpha, which activates NF-kB, can regulate Bmp4 gene expression. TNF-alpha reduced Bmp4 mRNA in lung adenocarcinoma A549 cells and repressed transcriptional activity of the human Bmp4 promoter in a dose-dependent manner. Similar repression was observed when the Bmp4 promoter was co-transfected with a p65 (RelA) expression vector in the absence of TNF-alpha treatment, suggesting that RelA mediates the effect of TNF-alpha. In support of this finding, the repressor effect of TNF-alpha on Bmp4 was abrogated by a co-transfected dominant negative mutant of IkB (S32A/S36A). The human Bmp4 promoter contains 3 putative consensus binding sites for NF-kB. Surprisingly, only one of the latter binding sites was capable of binding NF-kB. Repressor effect of NF-kB was not dependent on any of the three binding sites, but localized to a 122 bp fragment which bound both RelA and SP1. SP1 stimulated transcription, whereas increasing doses of RelA opposed this effect. In vivo, TNF-alpha inhibited branching morphogenesis and LacZ gene expression in Bmp4-lacz transgenic lungs. These data support a model in which TNF-alpha-induced RelA interacts with SP1 to bring about transcriptional repression of Bmp4 gene. These findings provide a mechanistic paradigm for interactions between mediators of inflammation and morphogenesis with relevant implications for normal lung development and pathogenesis of disease.

Epithelial-mesenchymal interactions that govern the development of the colon from the primitive gastrointestinal tract are still unclear. In this study, we determine the temporal-spatial expression pattern of Fibroblast growth factor 10 (Fgf10), a key developmental gene, in the colon at different developmental stages. We found that Fgf10 is expressed in the mesenchyme of the distal colon, while its main receptor Fgfr2-IIIb is expressed throughout the entire intestinal epithelium. We demonstrate that Fgf10 inactivation leads to decreased proliferation and increased cell apoptosis in the colonic epithelium at E10.5, therefore resulting in distal colonic atresia. Using newly described Fgf10 hypomorphic mice, we show that high levels of FGF10 are dispensable for the differentiation of the colonic epithelium. Our work unravels for the first time the pivotal role of FGF10 in the survival and proliferation of the colonic epithelium, biological activities which are essential for colonic crypt formation.

Burkholderia cepacia is a prevalent pulmonary pathogen in patients with cystic fibrosis (CF). The lung pathology observed in patients with CF is postulated to be due to an overexpression of chemokines. This study investigated the induction of the neutrophil chemoattractant chemokine IL-8 and the signaling pathways activated by B. cepacia-infected human lung epithelial A549 (HLE) cells. Cells were infected with B. cepacia (genomovar III of the B. cepacia complex), and reverse transcriptase-PCR and ELISA for the cytokines were performed. B. cepacia (multiplicity of infection > or =4:1) induced HLE cells to significantly secrete IL-8 in a more potent manner than the predominant CF pathogen Pseudomonas aeruginosa (multiplicity of infection > or =64:1). IL-8 secretion by B. cepacia-infected HLE cells was abrogated by the gene transcription inhibitor actinomycin D and the protein translation inhibitor cycloheximide, confirming that B. cepacia-induced IL-8 secretion was mediated through de novo protein synthesis. Treatment of B. cepacia with proteinase K failed to down-regulate IL-8 secretion; furthermore, IL-8 secretion by B. cepacia-infected HLE cells was abrogated by > or =80% in the presence of anti-CD14 [specific lipopolysaccharide (LPS) receptor] antibody, thus suggesting that the IL-8-inducing component of B. cepacia was LPS and therefore dependent on CD14. The p38 mitogen-activated protein kinase (MAPK) inhibitor and the extracellular signal-regulated kinase MAPK inhibitor significantly abrogated IL-8 secretion by B. cepacia-infected HLE cells (SB203580, > or =80% inhibition; PD98059, > or =30% inhibition). In conclusion, B. cepacia-induced IL-8 secretion in A549 airway epithelial cells is more potent than P. aeruginosa; is mediated through LPS, which is CD14 dependent; and involves activation of the p38 and ERK MAPK pathways.

Congenital pulmonary airway malformation (CPAM) is the most common congenital lesion detected in the neonatal lung, which may lead to respiratory distress, infection, and pneumothorax. CPAM is thought to result from abnormal branching morphogenesis during fetal lung development, arising from different locations within the developing respiratory tract. However, the pathogenic mechanisms are unknown, and previous studies have focused on abnormalities in airway epithelial cells. We have analyzed 13 excised lung specimens from infants (age

Dynamic regulation of intestinal cell differentiation is crucial for both homeostasis and the response to injury or inflammation. Sprouty2, an intracellular signaling regulator, controls pathways including PI3K and MAPKs that are implicated in differentiation and are dysregulated in inflammatory bowel disease. Here, we ask whether Sprouty2 controls secretory cell differentiation and the response to colitis. We report that colonic epithelial Sprouty2 deletion leads to expanded tuft and goblet cell populations. Sprouty2 loss induces PI3K/Akt signaling, leading to GSK3β inhibition and epithelial interleukin (IL)-33 expression. In vivo, this results in increased stromal IL-13+ cells. IL-13 in turn induces tuft and goblet cell expansion in vitro and in vivo. Sprouty2 is downregulated by acute inflammation; this appears to be a protective response, as VillinCre;Sprouty2F/F mice are resistant to DSS colitis. In contrast, Sprouty2 is elevated in chronic colitis and in colons of inflammatory bowel disease patients, suggesting that this protective epithelial-stromal signaling mechanism is lost in disease. Dynamic regulation of colonic secretory cell numbers is a critical component of the response to intestinal injury and inflammation. Here, the authors show that loss of the intracellular signalling regulator Sprouty2 in the intestinal epithelial cells is a protective response to injury that leads to increased secretory cell numbers, thus limiting colitis severity. [ABSTRACT FROM AUTHOR]

Epithelial organs are almost universally secretory. The lung secretes mucus of extremely variable consistency. In the early prenatal period, the secretions are of largely unknown composition, consistency, and flow rates. In addition to net outflow from secretion, the embryonic lung exhibits transient reversing flows from peristalsis. Airway peristalsis (AP) begins as soon as the smooth muscle forms, and persists until birth. Since the prenatal lung is liquid-filled, smooth muscle action can transport fluid far from the immediately adjacent tissues. The sensation of internal fluid flows has been shown to have potent morphogenetic effects, as has the transport of morphogens. We hypothesize that these effects play an important role in lung morphogenesis. To test these hypotheses in a quantitative framework, we analyzed the fluid-structure interactions between embryonic tissues and lumen fluid resulting from peristaltic waves that partially occlude the airway. We found that if the airway is closed, fluid transport is minimal; by contrast, if the trachea is open, shear rates can be very high, particularly at the stenosis. We performed a parametric analysis of flow characteristics' dependence on tissue stiffnesses, smooth muscle force, geometry, and fluid viscosity, and found that most of these relationships are governed by simple ratios. We measured the viscosity of prenatal lung fluid with passive bead microrheology. This paper reports the first measurements of the viscosity of embryonic lung lumen fluid. In the range tested, lumen fluid can be considered Newtonian, with a viscosity of 0.016 ± 0.008 Pa-s. We analyzed the interaction between the internal flows and diffusion and conclude that AP has a strong effect on flow sensing away from the tip and on transport of morphogens. These effects may be the intermediate mechanisms for the enhancement of branching seen in occluded embryonic lungs. [ABSTRACT FROM AUTHOR]

Background Reciprocal interactions between lung mesenchymal and epithelial cells play essential roles in lung organogenesis and homeostasis. Although the molecular markers and related animal models that target lung epithelial cells are relatively well studied, molecular markers of lung mesenchymal cells and the genetic tools to target and/or manipulate gene expression in a lung mesenchyme-specific manner are not available, which becomes a critical barrier to the study of lung mesenchymal biology and the related pulmonary diseases. Results We have identified a mouse Tbx4 gene enhancer that contains conserved DNA sequences across many vertebrate species with lung or lung-like gas exchange organ. We then generate a mouse line to express rtTA/LacZ under the control of the Tbx4 lung enhancer, and therefore a Tet-On inducible transgenic system to target lung mesenchymal cells at different developmental stages. By combining a Tbx4-rtTA driven Tet-On inducible Cre expression mouse line with a Cre reporter mouse line, the spatial-temporal patterns of Tbx4 lung enhancer targeted lung mesenchymal cells were defined. Pulmonary endothelial cells and vascular smooth muscle cells were targeted by the Tbx4-rtTA driver line prior to E11.5 and E15.5, respectively, while other subtypes of lung mesenchymal cells including airway smooth muscle cells, fibroblasts, pericytes could be targeted during the entire developmental stage. Conclusion Developmental lung mesenchymal cells can be specifically marked by Tbx4 lung enhancer activity. With our newly created Tbx4 lung enhancer-driven Tet-On inducible system, lung mesenchymal cells can be specifically and differentially targeted in vivo at the first time by controlling the doxycycline induction time window. This novel system provides a unique tool to study lung mesenchymal cell lineages and gene functions in lung mesenchymal development, injury repair, and regeneration in mice. [ABSTRACT FROM AUTHOR]

Little is known about the regulatory mechanisms underlying lung epithelial tight junction (TJ) assembly, which is inextricably linked to the preservation of epithelial polarity, and is highly coordinated by proteins that regulate epithelial cell polarity, such as aPKCζ. We recently reported that Eya1 phosphatase functions through aPKCζ-Notch1 signaling to control cell polarity in the lung epithelium. Here, we have extended these observations to TJ formation to demonstrate that Eya1 is crucial for the maintenance of TJ protein assembly in the lung epithelium, probably by controlling aPKCζ phosphorylation levels, aPKCζ-mediated TJ protein phosphorylation and Notch1- Cdc42 activity. Thus, TJs are disassembled after interfering with Eya1 function in vivo or during calcium-induced TJ assembly in vitro. These effects are reversed by reintroduction of wild-type Eya1 or partially inhibiting aPKCζ in Eya1siRNA cells. Moreover, genetic activation of Notch1 rescues Eya1-/- lung epithelial TJ defects. These findings uncover novel functions for the Eya1-aPKCζ-Notch1- Cdc42 pathway as a crucial regulatory mechanism of TJ assembly and polarity of the lung epithelium, providing a conceptual framework for future mechanistic and translational studies in this area. [ABSTRACT FROM AUTHOR]

Introduction or background The adult lung is a complex organ whose large surface area interfaces extensively with both the environment and circulatory system. Yet, in spite of the high potential for exposure to environmental or systemic harm, epithelial cell turnover in adult lung is comparatively slow. Moreover, loss of lung function with advancing age is becoming an increasingly costly healthcare problem. Cell-based therapies stimulating endogenous stem/progenitor cells or supplying exogenous ones have therefore become a prime translational goal. Alternatively when lung repair becomes impossible, replacement with tissue-engineered lung is an attractive emerging alternative using a decellularized matrix or bioengineered scaffold. Sources of data Endogenous and exogenous stem cells for lung therapy are being characterized by defining developmental lineages, surface marker expression, functions within the lung and responses to injury and disease. Seeding decellularized lung tissue or bioengineered matrices with various stem and progenitor cells is an approach that has already been used to replace bronchus and trachea in human patients and awaits further development for whole lung tissue. Areas of agreement Cellular therapies have clear potential for respiratory disease. However, given the surface size and complexity of lung structure, the probability of a single cellular population sufficing to regenerate the entire organ, as in the bone marrow, remains low. Hence, lung regenerative medicine is currently focused around three aims: (i) to identify and stimulate resident cell populations that respond to injury or disease, (ii) to transplant exogenous cells which can ameliorate disease and (iii) to repopulate decellularized or bioengineered lung matrix creating a new implantable organ. Areas of controversy Lack of consensus on specific lineage markers for lung stem and progenitor cells in development and disease constrains transferability of research between laboratories and sources of cellular therapy. Furthermore, effectiveness of individual cellular therapies to correct gas exchange and provide other critical lung functions remains unproven. Finally, feasibility of autologous whole organ replacement has not been confirmed as a durable therapy. Growing points Cellular therapies for lung regeneration would be enhanced by better lineage tracing within the lung, the ability to direct differentiation of exogenous stem or progenitor cells, and the development of functional assays for cellular viability and regenerative properties. Whether endogenous or exogeneous cells will ultimately play a greater therapeutic role remains to be seen. Reducing the need for lung replacement via endogenous cell-mediated repair is a key goal. Thereafter, improving the potential of donor lungs in transplant recipients is a further area where cell-based therapies may be beneficial. Ultimately, lung replacement with autologous tissue-engineered lungs is another goal for cell-based therapy. Areas timely for developing research Defining ‘lung stem or progenitor cell’ populations in both animal models and human tissue may help. Additionally, standardizing assays for assessing the potential of endogenous or exogenous cells within the lung is important. Understanding cell–matrix interactions in real time and with biomechanical insight will be central for lung engineering. Cautionary note Communicating the real potential for cell-based lung therapy needs to remain realistic, given the keen expectations of patients with end-stage lung disease. [ABSTRACT FROM AUTHOR]

Epithelial to mesenchymal transition (EMT) is a key process during embryonic development and disease development and progression. During EMT, epithelial cells lose epithelial features and express mesenchymal cell markers, which correlate with increased cell migration and invasion. Transforming growth factor-β (TGF-β) is a multifunctional cytokine that induces EMT in multiple cell types. The TGF-β pathway is regulated by microRNAs (miRNAs), which are small non-coding RNAs regulating the translation of specific messenger RNAs. Herein, we identified mir-99a and mir-99b as two novel TGF-β target miRNA genes, the expression of which increased during TGF-β induced EMT of NMUMG cells. Mir-99a and mir-99b inhibition decreased TGF-β activity by inhibiting SMAD3 phosphorylation, resulting in decreased migration and increased proliferation in response to TGF-β. However, mir-99a and mir-99b inhibition was insufficient to block TGF-β induced EMT of NMUMG cells. Mir-99a and mir-99b over-expression in epithelial NMUMG cells resulted in increased proliferation, migration and fibronectin expression, while E-cadherin and ZO-1 expression were negatively regulated. In conclusion, we identified mir- 99a and mir-99b as two novel modulators of TGF-β pathway that alter SMAD3 phosphorylation, in turn altering cell migration and adhesion of mesenchymal NMUMG cells. The effect of mir-99a and mir-99b over-expression on NMUMUG proliferation is dependent upon the epithelial or mesenchymal status of the cells. Our study suggests that mir-99a and mir- 99b may function as modulators within a complex network of factors regulating TGF-β induced breast epithelial to mesenchymal transition, as well as proliferation and migration of breast cancer cells, providing a possible target for future translationally oriented studies in this area. [ABSTRACT FROM AUTHOR]

Acute Tubular Necrosis (ATN) causes severe damage to the kidney epithelial tubular cells and is often associated with severe renal dysfunction. Stem-cell based therapies may provide alternative approaches to treating of ATN. We have previously shown that clonal c-kitpos stem cells, derived from human amniotic fluid (hAFSC) can be induced to a renal fate in an ex-vivo system. Herein, we show for the first time the successful therapeutic application of hAFSC in a mouse model with glycerol-induced rhabdomyolysis and ATN. When injected into the damaged kidney, luciferase-labeled hAFSC can be tracked using bioluminescence. Moreover, we show that hAFSC provide a protective effect, ameliorating ATN in the acute injury phase as reflected by decreased creatinine and BUN blood levels and by a decrease in the number of damaged tubules and apoptosis therein, as well as by promoting proliferation of tubular epithelial cells. We show significant immunomodulatory effects of hAFSC, over the course of ATN. We therefore speculate that AFSC could represent a novel source of stem cells that may function to modulate the kidney immune milieu in renal failure caused by ATN. [ABSTRACT FROM AUTHOR]

Abstract: Purpose: Duodenal obstruction occurs in 1 of 6000 live births and requires urgent surgical intervention. Duodenal atresia previously has been ascribed to a developmental failure of luminal recanalization; however, the cause of duodenal atresia remains incompletely understood. Although familial intestinal atresias have been described and syndromic associations are known, no specific genetic link has been established. Fibroblast growth factor-10 (Fgf10) is a known regulatory molecule relevant to mesenchymal-epithelial interactions, and mice deficient in Fgf10 demonstrate congenital anomalies in several organ systems including the gastrointestinal tract. The authors hypothesized that Fgf10 could serve a regulatory role in establishing normal duodenal development. Methods: Wild-type mice with β-galactosidase under the control of the Fgf10 promoter were harvested from timed-pregnancy mothers. The expression of Fgf10 in the duodenum during development was evaluated by developing the embryos in X-Gal solution. Wild-type and mutant Fgf10 −/− embryos were harvested from timed-pregnancy mothers at 18.5 days postconception (near term) and were analyzed for duodenal morphology (Institutional Animal Care and Use Committee–approved protocol 32-02). Photomicrographs were reviewed. Results: Fibroblast growth factor-10 is active in the duodenum at a late stage of development. The Fgf10 −/− mutants demonstrate duodenal atresia with a variable phenotype similar to clinical findings. The duodenum fails to develop luminal continuity and has proximal dilation. The phenotype occurs in an autosomal recessive pattern with incomplete penetrance (38%). Conclusions: Fibroblast growth factor-10 serves as a regulator in normal duodenal growth and development. Its deletion leads to duodenal atresia and challenges traditionally accepted theories of pathogenesis. This novel, genetically mediated duodenal malformation reflects an animal model that will allow further evaluation of the pathogenesis of this surgically correctable disease. By studying the mechanism of Fgf10 function in foregut development, the authors hope to better understand these anomalies and to explore possible therapeutic alternatives. [Copyright &y& Elsevier]

Alveolar epithelial type 2 cells (AEC2) isolated from hyperoxia-treated animals exhibit increases in both proliferation and DNA damage in response to culture. AEC2 express the zonula adherens proteins E-cadherin, α-, β- and γ-catenin, desmoglein, and pp120, as demonstrated by Western blotting. Immunohistochemical analysis of cultured AEC2 showed expression of E-cadherin on cytoplasmic membranes varying from strongly to weakly staining. When cultured AEC2 placed in suspension were labeled with fluorescent-tagged antibodies to E-cadherin, cells could be sorted into at least two subpopulations, either dim or brightly staining for this marker. With the use of antibody to E-cadherin bound to magnetic beads, cells were physically separated into E-cadherinpositive and -negative subpopulations, which were then analyzed for differences in proliferation and DNA damage. The E-cadherin-positive subpopulation contained the majority of damaged cells, was quiescent, and expressed low levels of telomerase activity, whereas the E-cadherin-negative subpopulation was undamaged, proliferative, and expressed high levels of telomerase activity. [ABSTRACT FROM AUTHOR]

Autophagy is a Greek-derived concept that means "self-eating" and is increasingly recognized as an important regulator of homeostasis and disease. In this issue of the JCI, Yeganeh et al. report the important finding that intrinsic autophagy is required for normal progression of lung development. Conditional deletion of the beclin 1-encoding gene (Becn1) specifically within lung epithelial cells of embryonic mice resulted in neonatal lethal respiratory distress that was associated with negative impacts on airway branching and differentiation of airway epithelial cell lineages. The authors draw speculative parallels with the alveolar simplification phenotype of bronchopulmonary dysplasia in premature human infants and suggest that stimulation of autophagy by cAMP-dependent kinase activation might conceivably rescue these phenotypes. [ABSTRACT FROM AUTHOR]

Abstract Postnatal organ-specific stem and progenitor cells are an attractive potential donor cell for tissue-engineering because they can be harvested autologous from the recipient and have sufficient potential to regenerate the tissue of interest with less risk for ectopic growth or tumor formation compared to donor cells from embryonic or fetal sources. We describe the generation of tissue-engineered larynx and trachea (TELT) from human and mouse postnatal organoid units (OU) as well as from human fetal OU. Mouse TELT contained differentiated respiratory epithelium lining large lumens, cartilage and smooth muscle. In contrast, human postnatal TE trachea, formed small epithelial lumens with rare differentiation, in addition to smooth muscle and cartilage. Human fetal TELT contained the largest epithelial lumens with all differentiated cell types as well as smooth muscle and cartilage. Increased epithelial cytokeratin 14 was identified in both human fetal and postnatal TELT compared to native trachea, consistent with regenerative basal cells. Cilia in TELT epithelium also demonstrated function with beating movements. While both human postnatal and fetal progenitors have the potential to generate TELT, there is more epithelial growth and differentiation from fetal progenitors, highlighting fundamental differences in these cell populations. Highlights • Human fetal TELT forms differentiated respiratory epithelium, cartilage and smooth muscle. • Cytokeratin 14 is upregulated in TELT epithelium. • Cilia in TELT epithelium have beating movement. [ABSTRACT FROM AUTHOR]

Mechanical forces are critical for normal fetal lung development. However, the mechanisms regulating this process are not well-characterized. We hypothesized that strain-induced release of HB-EGF and TGF-α is mediated via integrin-ADAM17/TACE interactions. Employing an in vitro system to simulate mechanical forces in fetal lung development, we showed that mechanical strain of fetal epithelial cells actives TACE, releases HB-EGF and TGF-α, and promotes differentiation. In contrast, in samples incubated with the TACE inhibitor IC-3 or in cells isolated from TACE knock-out mice, mechanical strain did not release ligands or promote cell differentiation, which were both rescued after transfection of ADAM17. Cell adhesion assay and co-immunoprecipitation experiments in wild-type and TACE knock-out cells using several TACE constructs demonstrated not only that integrins α6 and β1 bind to TACE via the disintegrin domain but also that mechanical strain enhances these interactions. Furthermore, force applied to these integrin receptors by magnetic beads activated TACE and shed HB-EGF and TGF-α. The contribution of integrins α6 and β1 to differentiation of fetal epithelial cells by strain was demonstrated by blocking their binding site with specific antibodies and by culturing the cells on membranes coated with anti-integrin α6 and β1 antibodies. In conclusion, mechanical strain releases HB-EGF and TGF-α and promotes fetal type II cell differentiation via α6β1 integrin-AD-AM17/TACE signaling pathway. These investigations provide novel mechanistic information on how mechanical forces promote fetal lung development and specifically differentiation of epithelial cells. This information could be also relevant to other tissues exposed to mechanical forces. [ABSTRACT FROM AUTHOR]

The Grainyhead family of transcription factors controls morphogenesis and differentiation of epithelial cell layers in multicellular organisms by regulating cell junction- and proliferation- related genes. Grainyhead-like 2 (Grhl2) is expressed in developing mouse lung epithelium and is required for normal lung organogenesis. The specific epithelial cells expressing Grhl2 and the genes regulated by Grhl2 in normal lungs are mostly unknown. In these studies we identified the NK2-homeobox 1 transcription factor (Nkx2-1) as a direct transcriptional target of Grhl2. By binding and transcriptional assays and by confocal microscopy we showed that these two transcription factors form a positive feedback loop in vivo and in cell lines and are co-expressed in lung bronchiolar and alveolar type II cells. The morphological changes observed in flattening lung alveolar type II cells in culture are associated with down-regulation of Grhl2 and Nkx2-1. Reduction of Grhl2 in lung epithelial cell lines results in lower expression levels of Nkx2-1 and of known Grhl2 target genes. By microarray analysis we identified that in addition to Cadherin1 and Claudin4, Grhl2 regulates other cell interaction genes such as semaphorins and their receptors, which also play a functional role in developing lung epithelium. Impaired collective cell migration observed in Grhl2 knockdown cell monolayers is associated with reduced expression of these genes and may contribute to the altered epithelial phenotype reported in Grhl2 mutant mice. Thus, Grhl2 functions at the nexus of a novel regulatory network, connecting lung epithelial cell identity, migration, and cell-cell interactions. [ABSTRACT FROM AUTHOR]

Abstract: Six1 is a member of the six-homeodomain family of transcription factors. Six1 is expressed in multiple embryonic cell types and plays important roles in proliferation, differentiation and survival of precursor cells of different organs, yet its function during lung development was hitherto unknown. Herein we show that Six1 −/− lungs are severely hypoplastic with greatly reduced epithelial branching and increased mesenchymal cellularity. Six1 is expressed at the distal epithelial tips of branching tubules as well as in the surrounding distal mesenchyme. Six1 −/− lung epithelial cells show increased expression of differentiation markers, but loss of progenitor cell markers. Six1 overexpression in MLE15 lung epithelial cells in vitro inhibited cell differentiation, but increases the expression of progenitor cell markers. In addition, Six1 −/− embryos and newborn mice exhibit mesenchymal overproliferation, decreased Fgf10 expression and severe defects in the smooth muscle component of the bronchi and major pulmonary vessels. These defects lead to rupture of major vessels in mutant lungs after birth. Treatment of Six1 −/− epithelial explants in culture with recombinant Fgf10 protein restores epithelial branching. As Shh expression is abnormally increased in Six1 −/− lungs, we also treated mutant mesenchymal explants with recombinant Shh protein and found that these explants were competent to respond to Shh and continued to grow in culture. Furthermore, inhibition of Shh signaling with cyclopamine stimulated Six1 −/− lungs to grow and branch in culture. This study provides the first evidence for the requirement of Six1 in coordinating Shh–Fgf10 signaling in embryonic lung to ensure proper levels of proliferation and differentiation along the proximodistal axis of epithelial, mesenchymal and endothelial cells. These findings uncover novel and essential functions for Six1 as a critical coordinator of Shh–Fgf10 signaling during embryonic lung development. We propose that Six1 is hence critical for coordination of proper lung epithelial, mesenchymal and vascular development. [Copyright &y& Elsevier]

Abstract: The proper level of proliferation and differentiation along the proximodistal axis is crucial for lung organogenesis. Elucidation of the factors that control these processes will therefore provide important insights into embryonic lung development and regeneration. Eya1 is a transcription factor/protein phosphatase that regulates cell lineage specification and proliferation. Yet its functions during lung development are unknown. In this paper we show that Eya1 −/− lungs are severely hypoplastic with reduced epithelial branching and increased mesenchymal cellularity. Eya1 is expressed at the distal epithelial tips of branching tubules as well as in the surrounding distal mesenchyme. Eya1 −/− lung epithelial cells show loss of progenitor cell markers with increased expression of differentiation markers and cell cycle exit. In addition, Eya1 −/− embryos and newborn mice exhibit severe defects in the smooth muscle component of the bronchi and major pulmonary vessels with decreased Fgf10 expression. These defects lead to rupture of the major vessels and hemorrhage into the lungs after birth. Treatment of Eya1 −/− epithelial explants in culture with recombinant Fgf10 stimulates epithelial branching. Since Shh expression and activity are abnormally increased in Eya1 −/− lungs, we tested whether genetically lowering Shh activity could rescue the Eya1 −/− lung phenotype. Indeed, genetic reduction of Shh partially rescues Eya1 −/− lung defects while restoring Fgf10 expression. This study provides the first evidence that Eya1 regulates Shh signaling in embryonic lung, thus ensuring the proper level of proliferation and differentiation along the proximodistal axis of epithelial, mesenchymal and endothelial cells. These findings uncover novel functions for Eya1 as a critical upstream coordinator of Shh–Fgf10 signaling during embryonic lung development. We conclude, therefore, that Eya1 function is critical for proper coordination of lung epithelial, mesenchymal and vascular development. [Copyright &y& Elsevier]

Abstract: Branching morphogenesis of many organs, including the embryonic lung, is a dynamic process in which growth factor mediated tyrosine kinase receptor activation is required, but must be tightly regulated to direct ramifications of the terminal branches. However, the specific regulators that modulate growth factor signaling downstream of the tyrosine kinase receptor remain to be determined. Herein, we demonstrate for the first time an important function for the intracellular protein tyrosine phosphatase Shp2 in directing embryonic lung epithelial morphogenesis. We show that Shp2 is specifically expressed in embryonic lung epithelial buds, and that loss of function by the suppression of Shp2 mRNA expression results in a 53% reduction in branching morphogenesis. Furthermore, by intra-tracheal microinjection of a catalytically inactive adenoviral Shp2 construct, we provide direct evidence that the catalytic activity of Shp2 is required for proper embryonic lung branch formation. We demonstrate that Shp2 activity is required for FGF10 induced endodermal budding. Furthermore, a loss of Shp2 catalytic activity in the embryonic lung was associated with a reduction in ERK phosphorylation and epithelial cell proliferation. However, epithelial cell differentiation was not affected. Our results show that the protein tyrosine phosphatase Shp2 plays an essential role in modulating growth factor mediated tyrosine kinase receptor activation in early embryonic lung branching morphogenesis. [Copyright &y& Elsevier]

Presents a study which examines the characteristics of the apoptosis and deoxyribonucleic acid (DNA) damage in connection with the type 2 alveolar epithelial cells cultured from hyperoxic rats. Definition of apoptosis; Characterization of the sublethal hyperoxic injury in rodents; Exposure of the adult male sprague-dawley rats.