Hippo signaling promotes lung epithelial lineage commitment by curbing Fgf10 and ß-catenin signaling.

Organ growth and tissue homeostasis rely on the proliferation and differentiation of progenitor cell populations. In the developing lung, localized Fgf10 expression maintains distal Sox9-expressing epithelial progenitors and promotes basal cell differentiation in the cartilaginous airways. Mesenchymal Fgf10 expression is induced by Wnt signaling but inhibited by Shh signaling, and epithelial Fgf10 signaling activates ß-catenin signaling. The Hippo pathway is a well-conserved signaling cascade that regulates organ size and stem/progenitor cell behavior. Here, we show that Hippo signaling promotes lineage commitment of lung epithelial progenitors by curbing Fgf10 and ß-catenin signaling. Our findings show that both inactivation of the Hippo pathway (nuclear Yap) or ablation of Yap result in increased ß-catenin and Fgf10 signaling, suggesting a cytoplasmic role for Yap in epithelial lineage commitment. We further demonstrate redundant and non-redundant functions for the two nuclear effectors of the Hippo pathway, Yap and Taz, during lung development.

Resident mesenchymal vascular progenitors modulate adaptive angiogenesis and pulmonary remodeling via regulation of canonical Wnt signaling.

Adaptive angiogenesis is necessary for tissue repair, however, it may also be associated with the exacerbation of injury and development of chronic disease. In these studies, we demonstrate that lung mesenchymal vascular progenitor cells (MVPC) modulate adaptive angiogenesis via lineage trace, depletion of MVPC, and modulation of ß-catenin expression. Single cell sequencing confirmed MVPC as multipotential vascular progenitors, thus, genetic depletion resulted in alveolar simplification with reduced adaptive angiogenesis. Following vascular endothelial injury, Wnt activation in MVPC was sufficient to elicit an emphysema-like phenotype characterized by increased MLI, fibrosis, and MVPC driven adaptive angiogenesis. Lastly, activation of Wnt/ß-catenin signaling skewed the profile of human and murine MVPC toward an adaptive phenotype. These data suggest that lung MVPC drive angiogenesis in response to injury and regulate the microvascular niche as well as subsequent distal lung tissue architecture via Wnt signaling.

Protein Tyrosine Phosphatase-N13 Promotes Myofibroblast Resistance to Apoptosis in Idiopathic Pulmonary Fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a progressive, fibrotic interstitial lung disease characterized by (myo)fibroblast accumulation and collagen deposition. Resistance to Fas-induced apoptosis is thought to facilitate (myo)fibroblast persistence in fibrotic lung tissues by poorly understood mechanisms. OBJECTIVES: To test the hypothesis that PTPN13 (protein tyrosine phosphatase-N13) is expressed by IPF lung (myo)fibroblasts, promotes their resistance to Fas-induced apoptosis, and contributes to the development of pulmonary fibrosis. METHODS: PTPN13 was localized in lung tissues from patients with IPF and control subjects by immunohistochemical staining. Inhibition of PTPN13 function in primary IPF and normal lung (myo)fibroblasts was accomplished by: 1) downregulation with TNF-α (tumor necrosis factor-α)/IFN-γ, 2) siRNA knockdown, or 3) a cell-permeable Fas/PTPN13 interaction inhibitory peptide. The role of PTPN13 in the development of pulmonary fibrosis was assessed in mice with genetic deficiency of PTP-BL, the murine ortholog of PTPN13. MEASUREMENTS AND MAIN RESULTS: PTPN13 was constitutively expressed by (myo)fibroblasts in the fibroblastic foci of patients with IPF. Human lung (myo)fibroblasts, which are resistant to Fas-induced apoptosis, basally expressed PTPN13 in vitro. TNF-α/IFN-γ or siRNA-mediated PTPN13 downregulation and peptide-mediated inhibition of the Fas/PTPN13 interaction in human lung (myo)fibroblasts promoted Fas-induced apoptosis. Bleomycin-challenged PTP-BL-/- mice, while developing inflammatory lung injury, exhibited reduced pulmonary fibrosis compared with wild-type mice. CONCLUSIONS: These findings suggest that PTPN13 mediates the resistance of human lung (myo)fibroblasts to Fas-induced apoptosis and promotes pulmonary fibrosis in mice. Our results suggest that strategies aimed at interfering with PTPN13 expression or function may represent a novel strategy to reduce fibrosis in IPF.

Evidence for the involvement of fibroblast growth factor 10 in lipofibroblast formation during embryonic lung development.

Lipid-containing alveolar interstitial fibroblasts (lipofibroblasts) are increasingly recognized as an important component of the epithelial stem cell niche in the rodent lung. Although lipofibroblasts were initially believed merely to assist type 2 alveolar epithelial cells in surfactant production during neonatal life, recent evidence suggests that these cells are indispensable for survival and growth of epithelial stem cells during adulthood. Despite increasing interest in lipofibroblast biology, little is known about their cellular origin or the molecular pathways controlling their formation during embryonic development. Here, we show that a population of lipid-droplet-containing stromal cells emerges in the developing mouse lung between E15.5 and E16.5. This is accompanied by significant upregulation, in the lung mesenchyme, of peroxisome proliferator-activated receptor gamma (master switch of lipogenesis), adipose differentiation-related protein (marker of mature lipofibroblasts) and fibroblast growth factor 10 (previously shown to identify a subpopulation of lipofibroblast progenitors). We also demonstrate that although only a subpopulation of total embryonic lipofibroblasts derives from Fgf10(+) progenitor cells, in vivo knockdown of Fgfr2b ligand activity and reduction in Fgf10 expression lead to global reduction in the expression levels of lipofibroblast markers at E18.5. Constitutive Fgfr1b knockouts and mutants with conditional partial inactivation of Fgfr2b in the lung mesenchyme reveal the involvement of both receptors in lipofibroblast formation and suggest a possible compensation between the two receptors. We also provide data from human fetal lungs to demonstrate the relevance of our discoveries to humans. Our results reveal an essential role for Fgf10 signaling in the formation of lipofibroblasts during late lung development.

miR-142-3p balances proliferation and differentiation of mesenchymal cells during lung development.

The regulation of the balance between proliferation and differentiation in the mesenchymal compartment of the lung is largely uncharacterized, unlike its epithelial counterpart. In this study, we determined that miR-142-3p contributes to the proper proliferation of mesenchymal progenitors by controlling the level of WNT signaling. miR-142-3p can physically bind to adenomatous polyposis coli mRNA, functioning to regulate its expression level. In miR-142-3p loss-of-function experiments, proliferation of parabronchial smooth muscle cell progenitors is significantly impaired, leading to premature differentiation. Activation of WNT signaling in the mesenchyme, or Apc loss of function, can both rescue miR-142-3p knockdown. These findings show that in the embryonic lung mesenchyme, the microRNA machinery modulates the level of WNT signaling, adding an extra layer of control in the feedback loop between FGFR2C and ß-catenin-mediated WNT signaling.

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.

Progenitors of secondary crest myofibroblasts are developmentally committed in early lung mesoderm.

Development of the mammalian lung is predicated on cross-communications between two highly interactive tissues, the endodermally derived epithelium and the mesodermally derived pulmonary mesenchyme. While much attention has been paid for the lung epithelium, the pulmonary mesenchyme, partly due to lack of specific tractable markers remains under-investigated. The lung mesenchyme is derived from the lateral plate mesoderm and is the principal recipient of Hedgehog (Hh) signaling, a morphogenetic network that regulates multiple aspects of embryonic development. Using the Hh-responsive Gli1-cre(ERT2) mouse line, we identified the mesodermal targets of Hh signaling at various time points during embryonic and postnatal lung development. Cell lineage analysis showed these cells serve as progenitors to contribute to multiple lineages of mesodermally derived differentiated cell types that include parenchymal or interstitial myofibroblasts, peribronchial and perivascular smooth muscle as well as rare populations of cells within the mesothelium. Most importantly, Gli1-cre(ERT2) identified the progenitors of secondary crest myofibroblasts, a hitherto intractable cell type that plays a key role in alveolar formation, a vital process about which little is currently known. Transcriptome analysis of Hh-targeted progenitor cells transitioning from the pseudoglandular to the saccular phase of lung development revealed important modulations of key signaling pathways. Among these, there was significant downregulation of canonical WNT signaling. Ectopic stabilization of ß-catenin via inactivation of Apc by Gli1-cre(ERT2) expanded the Hh-targeted progenitor pools, which caused the formation of fibroblastic masses within the lung parenchyma. The Gli1-cre(ERT2) mouse line represents a novel tool in the analysis of mesenchymal cell biology and alveolar formation during lung development.

General parity between trio and pairwise breeding of laboratory mice in static caging.

Changes made in the 8th edition of the Guide for the Care and Use of Laboratory Animals included new recommendations for the amount of space for breeding female mice. Adopting the new recommendations required, in essence, the elimination of trio breeding practices for all institutions. Both public opinion and published data did not readily support the new recommendations. In response, the National Jewish Health Institutional Animal Care and Use Committee established a program to directly compare the effects of breeding format on mouse pup survival and growth. Our study showed an overall parity between trio and pairwise breeding formats on the survival and growth of the litters, suggesting that the housing recommendations for breeding female mice as stated in the current Guide for the Care and Use of Laboratory Animals should be reconsidered.

Wnt and FGF mediated epithelial-mesenchymal crosstalk during lung development.

BACKGROUND: The adaptation to terrestrial life required the development of an organ capable of efficient air-blood gas exchange. To meet the metabolic load of cellular respiration, the mammalian respiratory system has evolved from a relatively simple structure, similar to the two-tube amphibian lung, to a highly complex tree-like system of branched epithelial airways connected to a vast network of gas exchanging units called alveoli. The development of such an elaborate organ in a relatively short time window is therefore an extraordinary feat and involves an intimate crosstalk between mesodermal and endodermal cell lineages. RESULTS: This review describes the molecular processes governing lung development with an emphasis on the current knowledge on the role of Wnt and FGF signaling in lung epithelial differentiation. CONCLUSIONS: The Wnt and FGF signaling pathways are crucial for the dynamic and reciprocal communication between epithelium and mesenchyme during lung development. In addition, some of this developmental crosstalk is reemployed in the adult lung after injury to drive regeneration, and may, when aberrantly or chronically activated, result in chronic lung diseases. Novel insights into how the Wnt and FGF pathways interact and are integrated into a complex gene regulatory network will not only provide us with essential information about how the lung regenerates itself, but also enhance our understanding of the pathogenesis of chronic lung diseases, as well as improve the controlled differentiation of lung epithelium from pluripotent stem cells.

FGF10/FGFR2b signaling is essential for cardiac fibroblast development and growth of the myocardium.

The epicardium serves as a source of growth factors that regulate myocardial proliferation and as a source of epicardial-derived cells (EPDC), which give rise to interstitial cardiac fibroblasts and perivascular cells. These progenitors populate the compact myocardium to become part of the mature coronary vasculature and fibrous skeleton of the heart. Little is known about the mechanisms that regulate EPDC migration into the myocardium or the functions carried out by these cells once they enter the myocardium. However, it has been proposed that cardiac fibroblasts are important for growth of the heart during late gestation and are a source of homeostatic factors in the adult. Here, we identify a myocardial to epicardial fibroblast growth factor (FGF) signal, mediated by FGF10 and FGFR2b, that is essential for movement of cardiac fibroblasts into the compact myocardium. Inactivation of this signaling pathway results in fewer epicardial derived cells within the compact myocardium, decreased myocardial proliferation and a resulting smaller thin-walled heart.

Neutrophil transmigration triggers repair of the lung epithelium via beta-catenin signaling.

Injury to the epithelium is integral to the pathogenesis of many inflammatory lung diseases, and epithelial repair is a critical determinant of clinical outcome. However, the signaling pathways regulating such repair are incompletely understood. We used in vitro and in vivo models to define these pathways. Human neutrophils were induced to transmigrate across monolayers of human lung epithelial cells in the physiological basolateral-to-apical direction. This allowed study of the neutrophil contribution not only to the initial epithelial injury, but also to its repair, as manifested by restoration of transepithelial resistance and reepithelialization of the denuded epithelium. Microarray analysis of epithelial gene expression revealed that neutrophil transmigration activated ß-catenin signaling, and this was verified by real-time PCR, nuclear translocation of ß-catenin, and TOPFlash reporter activity. Leukocyte elastase, likely via cleavage of E-cadherin, was required for activation of ß-catenin signaling in response to neutrophil transmigration. Knockdown of ß-catenin using shRNA delayed epithelial repair. In mice treated with intratracheal LPS or keratinocyte chemokine, neutrophil emigration resulted in activation of ß-catenin signaling in alveolar type II epithelial cells, as demonstrated by cyclin D1 expression and/or reporter activity in TOPGAL mice. Attenuation of ß-catenin signaling by IQ-1 inhibited alveolar type II epithelial cell proliferation in response to neutrophil migration induced by intratracheal keratinocyte chemokine. We conclude that ß-catenin signaling is activated in lung epithelial cells during neutrophil transmigration, likely via elastase-mediated cleavage of E-cadherin, and regulates epithelial repair. This pathway represents a potential therapeutic target to accelerate physiological recovery in inflammatory lung diseases.

Cell competition drives bronchiolization and pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease arising from the maladaptive differentiation of lung stem cells into bronchial epithelial cells rather than into alveolar type 1 (AT1) cells, which are responsible for gas exchange. Here, we report that healthy lungs maintain their stem cells through tonic Hippo and ß-catenin signaling, which promote Yap/Taz degradation and allow for low level expression of the Wnt target gene Myc. Inactivation of upstream activators of the Hippo pathway in lung stem cells inhibits this tonic ß-catenin signaling and Myc expression and promotes their Taz mediated differentiation into AT1 cells. Vice versa, increased Myc in collaboration with Yap promotes the differentiation of lung stem cells along the basal and myoepithelial like lineages allowing them to invade and bronchiolize the lung parenchyma in a process reminiscent of submucosal gland development. Our findings indicate that stem cells exhibiting the highest Myc levels become supercompetitors that drive remodeling, whereas loser cells with lower Myc levels terminally differentiate into AT1 cells.

Protocol for the use of signal amplification by exchange reaction-fluorescence in situ hybridization on adult formalin-fixed paraffin-embedded mouse lung tissue.

Fluorescence in situ hybridization (FISH) is a useful tool for analyzing RNA expression, but difficulties arise with low-abundance RNA and in tissues that are formalin-fixed paraffin-embedded (FFPE) because reagents can be expensive. In this protocol, we adapt a previously designed FISH amplification protocol (SABER [signal amplification by exchange reaction]) for adult mouse FFPE lung sections by using probes that are extended and branched to amplify the signal. We combine FISH and immunostaining to identify cell-specific RNA. For complete details on the use and execution of this protocol, please refer to Kishi et al.1 and Lyu et al.2.

Hippo signaling impairs alveolar epithelial regeneration in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) consists of fibrotic alveolar remodeling and progressive loss of pulmonary function. Genetic and experimental evidence indicates that chronic alveolar injury and failure to properly repair the respiratory epithelium are intrinsic to IPF pathogenesis. Loss of alveolar type 2 (AT2) stem cells or mutations that either impair their self-renewal and/or impair their differentiation into AT1 cells can serve as a trigger of pulmonary fibrosis. Recent reports indicate increased YAP activity in respiratory epithelial cells in IPF lungs. Individual IPF epithelial cells with aberrant YAP activation in bronchiolized regions frequently co-express AT1, AT2, conducting airway selective markers and even mesenchymal or EMT markers, demonstrating 'indeterminate' states of differentiation and suggesting that aberrant YAP signaling might promote pulmonary fibrosis. Yet, Yap and Taz have recently also been shown to be important for AT1 cell maintenance and alveolar epithelial regeneration after Streptococcus pneumoniae-induced injury. To investigate how epithelial Yap/Taz might promote pulmonary fibrosis or drive alveolar epithelial regeneration, we inactivated the Hippo pathway in AT2 stem cells resulting in increased nuclear Yap/Taz, and found that this promotes their alveolar regenerative capacity and reduces pulmonary fibrosis following bleomycin injury by pushing them along the AT1 cell lineage. Vice versa, inactivation of both Yap1 and Wwtr1 (encoding Taz) or Wwtr1 alone in AT2 cell stem cells impaired alveolar epithelial regeneration and resulted in increased pulmonary fibrosis upon bleomycin injury. Interestingly, the inactivation of only Yap1 in AT2 stem cells promoted alveolar epithelial regeneration and reduced pulmonary fibrosis. Together, these data suggest that epithelial Yap promotes, and epithelial Taz reduces pulmonary fibrosis suggesting that targeting Yap but not Taz-mediated transcription might help promote AT1 cell regeneration and treat pulmonary fibrosis.

Osteoblasts derived from induced pluripotent stem cells form calcified structures in scaffolds both in vitro and in vivo.

Reprogramming somatic cells into an ESC-like state, or induced pluripotent stem (iPS) cells, has emerged as a promising new venue for customized cell therapies. In this study, we performed directed differentiation to assess the ability of murine iPS cells to differentiate into bone, cartilage, and fat in vitro and to maintain an osteoblast phenotype on a scaffold in vitro and in vivo. Embryoid bodies derived from murine iPS cells were cultured in differentiation medium for 8­12 weeks. Differentiation was assessed by lineage-specific morphology, gene expression, histological stain, and immunostaining to detect matrix deposition. After 12 weeks of expansion, iPS-derived osteoblasts were seeded in a gelfoam matrix followed by subcutaneous implantation in syngenic imprinting control region (ICR) mice. Implants were harvested at 12 weeks, histological analyses of cell and mineral and matrix content were performed. Differentiation of iPS cells into mesenchymal lineages of bone, cartilage, and fat was confirmed by morphology and expression of lineage-specific genes. Isolated implants of iPS cell-derived osteoblasts expressed matrices characteristic of bone, including osteocalcin and bone sialoprotein. Implants were also stained with alizarin red and von Kossa, demonstrating mineralization and persistence of an osteoblast phenotype. Recruitment of vasculature and microvascularization of the implant was also detected. Taken together, these data demonstrate functional osteoblast differentiation from iPS cells both in vitro and in vivo and reveal a source of cells, which merit evaluation for their potential uses in orthopedic medicine and understanding of molecular mechanisms of orthopedic disease.

Niche-mediated repair of airways is directed in an occupant-dependent manner.

In injured airways of the adult lung, epithelial progenitors are called upon to repair by nearby mesenchymal cells via signals transmitted through the niche. Currently, it is unclear whether repair is coordinated by the mesenchymal cells that maintain the niche or by the airway epithelial cells that occupy it. Here, we show that the spatiotemporal expression of Fgf10 by the niche is primarily orchestrated by the niche's epithelial occupants-both those that reside prior to, and following, injury. During homeostasis, differentiated airway epithelial cells secrete Sonic hedgehog (Shh) to inhibit Fgf10 expression by Gli1+ peribronchial mesenchymal cells in the niche. After injury, remaining epithelial cells produce Wnt7b to induce Fgf10 expression in airway smooth muscle cells in the niche. We find that this reliance on a common activator of airway epithelial stem cells also allows for the recruitment of remote stem cell populations when local populations have been exhausted.

Mesenchymal stromal cell aging impairs the self-organizing capacity of lung alveolar epithelial stem cells.

Multicellular organisms maintain structure and function of tissues/organs through emergent, self-organizing behavior. In this report, we demonstrate a critical role for lung mesenchymal stromal cell (L-MSC) aging in determining the capacity to form three-dimensional organoids or 'alveolospheres' with type 2 alveolar epithelial cells (AEC2s). In contrast to L-MSCs from aged mice, young L-MSCs support the efficient formation of alveolospheres when co-cultured with young or aged AEC2s. Aged L-MSCs demonstrated features of cellular senescence, altered bioenergetics, and a senescence-associated secretory profile (SASP). The reactive oxygen species generating enzyme, NADPH oxidase 4 (Nox4), was highly activated in aged L-MSCs and Nox4 downregulation was sufficient to, at least partially, reverse this age-related energy deficit, while restoring the self-organizing capacity of alveolospheres. Together, these data indicate a critical role for cellular bioenergetics and redox homeostasis in an organoid model of self-organization and support the concept of thermodynamic entropy in aging biology.

Many tissues in the body are capable of regenerating by replacing defective or worn-out cells with new ones. This process relies heavily on stem cells, which are precursor cells that lack a set role in the body and can develop into different types of cells under the right conditions. Tissues often have their own pool of stem cells that they use to replenish damaged cells. But as we age, this regeneration process becomes less effective. Many of our organs, such as the lungs, are lined with epithelial cells. These cells form a protective barrier, controlling what substances get in and out of the tissue. Alveoli are parts of the lungs that allow oxygen and carbon dioxide to move between the blood and the air in the lungs. And alveoli rely on an effective epithelial cell lining to work properly. To replenish these epithelial cells, alveoli have pockets, in which a type of epithelial cell, known as AEC2, lives. These cells can serve as stem cells, developing into a different type of cell under the right conditions. To work properly, AEC2 cells require close interactions with another type of cell called L-MSC, which supports the maintenance of other cells and also has the ability to differentiate into several other cell types. Both cell types can be found close together in these stem cell pockets. So far, it has been unclear how aging affects how these cells work together to replenish the epithelial lining of the alveoli. To investigate, Chanda et al. probed AEC2s and L-MSCs in the alveoli of young and old mice. The researchers collected both cell types from young (2-3 months) and aged (22-24 months) mice. Various combinations of these cells were grown to form 3D structures, mimicking how the cells grow in the lungs. Young L-MSCs formed normal 3D structures with both young and aged AEC2 cells. But aged L-MSCs developed abnormal, loose structures with AEC2 cells (both young and old cells). Aged L-MSCs were found to have higher levels of an enzyme (called Nox4) that produces oxidants and other 'pro-aging' factors, compared to young L-MSCs. However, reducing Nox4 levels in aged L-MSCs allowed these cells to form normal 3D structures with young AEC2 cells, but not aged AEC2 cells. These findings highlight the varying effects specific stem cells have, and how their behaviour is affected by pro-aging factors. Moreover, the pro-aging enzyme Nox4 shows potential as a therapeutic target ­ downregulating its activity may reverse critical effects of aging in cells.

miR-17 family of microRNAs controls FGF10-mediated embryonic lung epithelial branching morphogenesis through MAPK14 and STAT3 regulation of E-Cadherin distribution.

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.

Deletion of Pten expands lung epithelial progenitor pools and confers resistance to airway injury.

RATIONALE: Pten is a tumor-suppressor gene involved in stem cell homeostasis and tumorigenesis. In mouse, Pten expression is ubiquitous and begins as early as 7 days of gestation. Pten(-/-) mouse embryos die early during gestation indicating a critical role for Pten in embryonic development. OBJECTIVES: To test the role of Pten in lung development and injury. METHODS: We conditionally deleted Pten throughout the lung epithelium by crossing Pten(flox/flox) with Nkx2.1-cre driver mice. The resulting Pten(Nkx2.1-cre) mutants were analyzed for lung defects and response to injury. MEASUREMENTS AND MAIN RESULTS: Pten(Nkx2.1-cre) embryonic lungs showed airway epithelial hyperplasia with no branching abnormalities. In adult mice, Pten(Nkx2.1-cre) lungs exhibit increased progenitor cell pools composed of basal cells in the trachea, CGRP/CC10 double-positive neuroendocrine cells in the bronchi, and CC10/SPC double-positive cells at the bronchioalveolar duct junctions. Pten deletion affected differentiation of various lung epithelial cell lineages, with a decreased number of terminally differentiated cells. Over time, Pten(Nxk2.1-cre) epithelial cells residing in the bronchioalveolar duct junctions underwent proliferation and formed uniform masses, supporting the concept that the cells residing in this distal niche may also be the source of procarcinogenic stem cells. Finally, increased progenitor cells in all the lung compartments conferred an overall selective advantage to naphthalene injury compared with wild-type control mice. CONCLUSIONS: Pten has a pivotal role in lung stem cell homeostasis, cell differentiation, and consequently resistance to lung injury.

Temporospatial Expression of Fgfr1 and 2 During Lung Development, Homeostasis, and Regeneration.

Fgfr1 (Fibroblast growth factor receptor 1) and Fgfr2 are dynamically expressed during lung development, homeostasis, and regeneration. Our current analysis indicates that Fgfr2 is expressed in distal epithelial progenitors AT2, AT1, club, and basal cells but not in ciliated or neuroendocrine cells during lung development and homeostasis. However, after injury, Fgfr2 becomes upregulated in neuroendocrine cells and distal club cells. Epithelial Fgfr1 expression is minimal throughout lung development, homeostasis, and regeneration. We further find both Fgfr1 and Fgfr2 strongly expressed in cartilage progenitors and airway smooth muscle cells during lung development, whereas Fgfr1 but not Fgfr2 was expressed in lipofibroblasts and vascular smooth muscle cells. In the adult lung, Fgfr1 and Fgfr2 were mostly downregulated in smooth muscle cells but became upregulated after injury. Fgfr1 remained expressed in mesenchymal alveolar niche cells or lipofibroblasts with lower levels of expression in their descendant (alveolar) myofibroblasts during alveologenesis.