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Distinct members of the Ets family of transcription factors act as positive or negative regulators of genes involved in cellular proliferation, development, and tumorigenesis. In human lung cancer, increased ETS1 expression is associated with poor prognosis and metastasis. We tested whether ETS1 contributes to lung tumorigenesis by binding to Twist1, a gene involved in tumor cell motility and dissemination. We used a mouse lung cancer model with metastasis driven by conditionally activated Kras and concurrent tumor suppressor Lkb1 loss (KrasG12D/ Lkb1−/− model) and a similar model of lung cancer that does not metastasize, driven by conditionally activated Kras alone (KrasG12D model). We show that Ets1 and Twist1 gene expression differs between KrasG12D tumors (low Ets1 and Twist1 expression) and KrasG12D/Lkb1−/− tumors (high Ets1 and Twist1 expression). In human lung tumors, ETS1 and TWIST1 expression positively correlates and low combined ETS1 and TWIST1 levels are associated with improved survival compared to high levels. Using mouse cell lines derived from KrasG12D and KrasG12D/Lkb1−/− mouse models and the human lung cancer (A549) cell line, we show that ETS1 regulates Twist1 expression. Chromatin immunoprecipitation assays confirm binding of ETS1 to the Twist1 promoter. Overexpression studies show that ETS1 transactivates Twist1 promoter activity in mouse and human cells. Silencing endogenous Ets1 by siRNA in mouse cell lines decreases Twist1 mRNA levels, decreases invasion, and increases cell growth. Ets1 and Twist1 are at the crossroad of several signaling pathways in cancer. Understanding their regulation may inform the development of therapies to impair lung tumor metastasis.

Cellular senescence is a biological process by which cells lose their capacity to proliferate yet remain metabolically active. Although originally considered a protective mechanism to limit the formation of cancer, it is now appreciated that cellular senescence also contributes to the development of disease, including common respiratory ailments such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. While many factors have been linked to the development of cellular senescence, mitochondrial dysfunction has emerged as an important causative factor. In this study, we uncovered that the mitochondrial biogenesis pathway driven by the mammalian target of rapamycin/peroxisome proliferator-activated receptor-γ complex 1α/β (mTOR/PGC-1α/β) axis is markedly upregulated in senescent lung epithelial cells. Using two different models, we show that activation of this pathway is associated with other features characteristic of enhanced mitochondrial biogenesis, including elevated number of mitochondrion per cell, increased oxidative phosphorylation, and augmented mitochondrial reactive oxygen species (ROS) production. Furthermore, we found that pharmacological inhibition of the mTORC1 complex with rapamycin not only restored mitochondrial homeostasis but also reduced cellular senescence to bleomycin in lung epithelial cells. Likewise, mitochondrial-specific antioxidant therapy also effectively inhibited mTORC1 activation in these cells while concomitantly reducing mitochondrial biogenesis and cellular senescence. In summary, this study provides a mechanistic link between mitochondrial biogenesis and cellular senescence in lung epithelium and suggests that strategies aimed at blocking the mTORC1/PGC-1α/β axis or reducing ROS-induced molecular damage could be effective in the treatment of senescence-associated lung diseases.

The function of most long noncoding RNAs (lncRNAs) is unknown. However, recent studies reveal important roles of lncRNAs in regulating cancer-related pathways. Human antisense lncRNA-NKX2-1-AS1 partially overlaps the NKX2-1/TTF1 gene within chromosomal region 14q13.3. Amplification of this region and/or differential expression of genes therein are associated with cancer progression. Herein we show higher levels of NKX2-AS1 and NKX2-1 in lung adenocarcinomas relative to non-tumor controls but no correlation between NKX2-1-AS1 and NKX2-1 levels across specimens, or with amplification of the 14q13.3 region, suggesting that NKX2-1-AS1 and NKX2-1 are independently regulated. Loss-and-gain of function experiments showed that NKX2-1-AS1 does not regulate NKX2-1 expression, or nearby genes, but controls genes in trans. Genes up-regulated by NKX2-1-AS1-knockdown belong to cell adhesion and PD-L1/PD-1 checkpoint pathways. NKX2-1-AS1 negatively regulates endogenous CD274/PD-L1, a known target of NKX2-1, and the transcriptional activity of -1kb-CD274 promoter-reporter construct. Furthermore, NKX2-1-AS1 interferes with NKX2-1 protein binding to the CD274-promoter, likely by NKX2-1 protein-NKX2-1-AS1 interactions. Finally, NKX2-1-AS1 negatively regulates cell migration and wound healing, but not proliferation or apoptosis. These findings support potential roles of NKX2-1-AS1 in limiting motility and immune system evasion of lung carcinoma cells, highlighting a novel mechanism that may influence tumorigenic capabilities of lung epithelial cells.

Distinct members of the Ets family of transcription factors act as positive or negative regulators of genes involved in cellular proliferation, development, and tumorigenesis. In human lung cancer, increased ETS1 expression is associated with poor prognosis and metastasis. We tested whether ETS1 contributes to lung tumorigenesis by binding to Twist1, a gene involved in tumor cell motility and dissemination. We used a mouse lung cancer model with metastasis driven by conditionally activated Kras and concurrent tumor suppressor Lkb1 loss (Kras

Podoplanin (PDPN) is a transmembrane glycoprotein that promotes tumor cell migration, invasion, and cancer metastasis. In fact, PDPN expression is induced in many types of cancer. Thus, PDPN has emerged as a functionally relevant cancer biomarker and chemotherapeutic target. PDPN contains 2 intracellular serine residues that are conserved between species ranging from mouse to humans. Recent studies indicate that protein kinase A (PKA) can phosphorylate PDPN in order to inhibit cell migration. However, the number and identification of specific residues phosphorylated by PKA have not been defined. In addition, roles of other kinases that may phosphorylate PDPN to control cell migration have not been investigated. We report here that cyclin dependent kinase 5 (CDK5) can phosphorylate PDPN in addition to PKA. Moreover, results from this study indicate that PKA and CDK5 cooperate to phosphorylate PDPN on both intracellular serine residues to decrease cell motility. These results provide new insight into PDPN phosphorylation dynamics and the role of PDPN in cell motility. Understanding novel mechanisms of PDPN intracellular signaling could assist with designing novel targeted chemotherapeutic agents and procedures.

Summary Induced pluripotent stem cells (iPSCs) provide an inexhaustible source of cells for modeling disease and testing drugs. Here we develop a bioinformatic approach to detect differences between the genomic programs of iPSCs derived from diseased versus normal human cohorts as they emerge during in vitro directed differentiation. Using iPSCs generated from a cohort carrying mutations (PiZZ) in the gene responsible for alpha-1 antitrypsin (AAT) deficiency, we find that the global transcriptomes of PiZZ iPSCs diverge from normal controls upon differentiation to hepatic cells. Expression of 135 genes distinguishes PiZZ iPSC-hepatic cells, providing potential clues to liver disease pathogenesis. The disease-specific cells display intracellular accumulation of mutant AAT protein, resulting in increased autophagic flux. Furthermore, we detect beneficial responses to the drug carbamazepine, which further augments autophagic flux, but adverse responses to known hepatotoxic drugs. Our findings support the utility of iPSCs as tools for drug development or prediction of toxicity., Graphical Abstract, Highlights • Patient iPSC-hepatic cells model disease features that normalize with gene editing • Disease-specific transcriptomic signature emerges only upon hepatic differentiation • Patient iPSC-hepatic cells are used to test response to drugs or toxicity in vitro, In this report, Kotton and colleagues examine the global transcriptomic and epigenomic programs of iPSCs derived from diseased (AAT-deficient PiZZ) or normal human cohorts during directed differentiation. The PiZZ transcriptome diverges from controls upon reaching hepatic stage, when AAT is first expressed, providing potential clues to liver disease pathogenesis. PiZZ iPSC-hepatic cells model key liver disease features and demonstrate both therapeutic drug responsiveness and sensitivity to toxic agents.

SummaryTwo populations of Nkx2-1+ progenitors in the developing foregut endoderm give rise to the entire postnatal lung and thyroid epithelium, but little is known about these cells because they are difficult to isolate in a pure form. We demonstrate here the purification and directed differentiation of primordial lung and thyroid progenitors derived from mouse embryonic stem cells (ESCs). Inhibition of TGFβ and BMP signaling, followed by combinatorial stimulation of BMP and FGF signaling, can specify these cells efficiently from definitive endodermal precursors. When derived using Nkx2-1GFP knockin reporter ESCs, these progenitors can be purified for expansion in culture and have a transcriptome that overlaps with developing lung epithelium. Upon induction, they can express a broad repertoire of markers indicative of lung and thyroid lineages and can recellularize a 3D lung tissue scaffold. Thus, we have derived a pure population of progenitors able to recapitulate the developmental milestones of lung/thyroid development.

Epigenetic regulation of transcription plays an important role in cell-specific gene expression by altering chromatin structure and access of transcriptional regulators to DNA binding sites. Surfactant protein B (Sftpb) is a developmentally regulated lung epithelial gene critical for lung function. Thyroid transcription factor 1 (Nkx2-1) regulates Sftpb gene expression in various species. We show that Nkx2-1 binds to the mouse Sftpb (mSftpb) promoter in the lung. In a mouse lung epithelial cell line (MLE-15), Nkx2-1 knockdown reduces Sftpb expression, and mutation of Nkx2-1 cis-elements significantly reduces mSftpb promoter activity. Whether chromatin structure modulates Nkx2-1 regulation of Sftpb transcription is unknown. We found that DNA methylation of the mSftpb promoter inversely correlates with known patterns of Sftpb expression in vivo. The mSftpb promoter activity can be manipulated by altering its cytosine methylation status in vitro. Nkx2-1 activation of the mSftpb promoter is impaired by DNA methylation. The unmethylated Sftpb promoter shows an active chromatin structure enriched in the histone modification H3K4me3 (histone 3-lysine 4 trimethylated). The ATP-dependent chromatin remodeling protein Brg1 is recruited to the Sftpb promoter in Sftpb-expressing, but not in non-expressing tissues and cell lines. Brg1 knockdown in MLE-15 cells greatly decreases H3K4me3 levels at the Sftpb promoter region and expression of the Sftpb gene. Brg1 can be co-immunoprecipitated with Nkx2-1 protein. Last, Nkx2-1 and Brg1 with intact ATPase activity are required for mSftpb promoter activation in vitro. Our findings suggest that DNA methylation and chromatin modifications cooperate with Nkx2-1 to regulate Sftpb gene cell specific expression.

Caveolin-1 protein has been called a ‘conditional tumor suppressor’ because it can either suppress or enhance tumor progression depending on cellular context. Caveolin-1 levels are dynamic in non-small-cell lung cancer, with increased levels in metastatic tumor cells. We have shown previously that transactivation of an erythroblastosis virus-transforming sequence (ETS) cis-element enhances caveolin-1 expression in a murine lung epithelial cell line. Based on high sequence homology between the murine and human caveolin-1 promoters, we proposed that ETS proteins might regulate caveolin-1 expression in human lung tumorigenesis. We confirm that caveolin-1 is not detected in well-differentiated primary lung tumors. Polyoma virus enhancer activator 3 (PEA3), a pro-metastatic ETS protein in breast cancer, is expressed at low levels in well-differentiated tumors and high levels in poorly differentiated tumors. Conversely, Net, a known ETS repressor, is expressed at high levels in the nucleus of well-differentiated primary tumor cells. In tumor cells in metastatic lymph node sites, caveolin-1 and PEA3 are highly expressed, whereas Net is now expressed in the cytoplasm. We studied transcriptional regulation of caveolin-1 in two human lung cancer cell lines, Calu-1 (high caveolin-1 expressing) and NCI-H23 (low caveolin-1 expressing). Chromatin immunoprecipitation-binding assays and small interfering RNA experiments show that PEA3 is a transcriptional activator in Calu-1 cells and that Net is a transcriptional repressor in NCI-H23 cells. These results suggest that Net may suppress caveolin-1 transcription in primary lung tumors and that PEA3 may activate caveolin-1 transcription in metastatic lymph nodes.

The Division of Lung Diseases of the National Heart, Lung, and Blood Institute (NHLBI) recently held a workshop to identify gaps in our understanding and treatment of childhood lung diseases and to define strategies to enhance translational research in this field. Leading experts with diverse experience in both laboratory and patient-oriented research reviewed selected areas of pediatric lung diseases, including perinatal programming and epigenetic influences; mechanisms of lung injury, repair, and regeneration; pulmonary vascular disease; sleep and control of breathing; and the application of novel translational methods to enhance personalized medicine. This report summarizes the proceedings of this workshop and provides recommendations for emphasis on targeted areas for future investigation. The priority areas identified for research in pediatric pulmonary diseases included: (1) epigenetic and environmental influences on lung development that program pediatric lung diseases; (2) injury, regeneration, and repair in the developing lung; (3) pulmonary vascular disease in children; (4) development and adaptation of ventilatory responses to postnatal life; (5) nonatopic wheezing: aberrant large airway development or injury?; (6) strategies to improve assessment, diagnosis, and treatment of pediatric respiratory diseases; and (7) predictive and personalized medicine for children.

Mucin-type O-glycans (O-glycans) are highly expressed in vascular ECs. However, it is not known whether they are important for vascular development. To investigate the roles of EC O-glycans, we generated mice lacking T-synthase, a glycosyltransferase encoded by the gene C1galt1 that is critical for the biosynthesis of core 1-derived O-glycans, in ECs and hematopoietic cells (termed here EHC T-syn(-/-) mice). EHC T-syn(-/-) mice exhibited embryonic and neonatal lethality associated with disorganized and blood-filled lymphatic vessels. Bone marrow transplantation and EC C1galt1 transgene rescue demonstrated that lymphangiogenesis specifically requires EC O-glycans, and intestinal lymphatic microvessels in EHC T-syn(-/-) mice expressed a mosaic of blood and lymphatic EC markers. The level of O-glycoprotein podoplanin was significantly reduced in EHC T-syn(-/-) lymphatics, and podoplanin-deficient mice developed blood-filled lymphatics resembling EHC T-syn(-/-) defects. In addition, postnatal inactivation of C1galt1 caused blood/lymphatic vessel misconnections that were similar to the vascular defects in the EHC T-syn(-/-) mice. One consequence of eliminating T-synthase in ECs and hematopoietic cells was that the EHC T-syn(-/-) pups developed fatty liver disease, because of direct chylomicron deposition via misconnected portal vein and intestinal lymphatic systems. Our studies therefore demonstrate that EC O-glycans control the separation of blood and lymphatic vessels during embryonic and postnatal development, in part by regulating podoplanin expression.

To identify genes expressed during initiation of lung organogenesis, we generated transcriptional profiles of the prospective lung region of the mouse foregut (mid-foregut) microdissected from embryos at three developmental stages between embryonic day 8.5 (E8.5) and E9.5. This period spans from lung specification of foregut cells to the emergence of the primary lung buds. We identified a number of known and novel genes that are temporally regulated as the lung bud forms. Genes that regulate transcription, including DNA binding factors, co-factors, and chromatin remodeling genes, are the main functional groups that change during lung bud formation. Members of key developmental transcription and growth factor families, not previously described to participate in lung organogenesis, are expressed in the mid-foregut during lung bud induction. These studies also show early expression in the mid-foregut of genes that participate in later stages of lung development. This characterization of the mid-foregut transcriptome provides new insights into molecular events leading to lung organogenesis.

We previously identified an Ets cis-element in the mouse caveolin-1 promoter that is selectively activated in lung epithelial (E10), but not lung endothelial murine lung endothelial cell line (MFLM-4), cell lines and therefore appears important for differential, cell-specific caveolin-1 transcription. In the present study, we demonstrate that immunostaining of adult mouse lung detects the ETS protein Ets-related molecule (ERM PEA3) in distal lung epithelium in alveolar type I and II cells, but not in bronchial epithelium or lung endothelial cells. We tested ERM and polyomavirus enhancer activator 3 (PEA3) for their ability to increase endogenous caveolin-1 transcripts and to activate caveolin-1 promoter fragments containing the -865 Ets cis-element. Chromatin immunoprecipitation (ChIP) assays show that both ERM and PEA3 bind to the caveolin-1 promoter in murine E10, but not MFLM-4, cells. Normalized luciferase activities show that only ERM activates the caveolin-1 promoter in E10 cells, but neither protein enhances promoter activity in MFLM-4 cells. Mutation of the Ets site blocks ERM-mediated promoter activation in E10 cells. Furthermore, overexpression of ERM increases the cellular content of caveolin-1 mRNA and protein, in E10, but not MFLM-4, cells. The effects of PEA3 on the cellular content of endogenous caveolin-1 expression are variable. These results demonstrate that ERM is involved in caveolin-1 regulation in a murine lung epithelial, but not lung endothelial cell line. We conclude that transcriptional regulation of caveolin-1 differs markedly between lung epithelial and endothelial cell lines, perhaps explaining why the onset of caveolin-1 expression differs in epithelial and endothelial cells during lung development.

Background The transcription factor NK2 homeobox 1 (Nkx2-1) plays essential roles in epithelial cell proliferation and differentiation in mouse and human lung development and tumorigenesis. A better understanding of genes and pathways downstream of Nkx2-1 will clarify the multiple roles of this critical lung factor. Nkx2-1 regulates directly or indirectly numerous protein-coding genes; however, there is a paucity of information about Nkx2-1-regulated microRNAs (miRNAs). Methods and results By miRNA array analyses of mouse epithelial cell lines in which endogenous Nkx2-1 was knocked-down, we revealed that 29 miRNAs were negatively regulated including miR-200c, and 39 miRNAs were positively regulated by Nkx2-1 including miR-1195. Mouse lungs lacking functional phosphorylated Nkx2-1 showed increased expression of miR-200c and alterations in the expression of other top regulated miRNAs. Moreover, chromatin immunoprecipitation assays showed binding of NKX2-1 protein to regulatory regions of these miRNAs. Promoter reporter assays indicated that 1kb of the miR-200c 5′ flanking region was transcriptionally active but did not mediate Nkx2-1- repression of miR-200c expression. 3′UTR reporter assays support a direct regulation of the predicted targets Nfib and Myb by miR-200c. Conclusions These studies suggest that Nkx2-1 controls the expression of specific miRNAs in lung epithelial cells. In particular, we identified a regulatory link between Nkx2-1, the known tumor suppressor miR-200c, and the developmental and oncogenic transcription factors Nfib and Myb, adding new players to the regulatory mechanisms driven by Nkx2-1 in lung epithelial cells that may have implications in lung development and tumorigenesis. Electronic supplementary material The online version of this article (doi:10.1186/s12931-015-0186-6) contains supplementary material, which is available to authorized users.

In the lung, caveolin-1 is expressed in both type I alveolar epithelial and endothelial cells where it is hypothesized to modulate molecular signaling activities and progression of tumorigenesis. Developmentally, caveolin-1α is expressed in fetal lung endothelial, but not epithelial, cells; in adult lung, both cell types express caveolin-1α. To test the hypothesis that caveolin-1 transcription is differentially regulated in type I and endothelial cells, we characterized the proximal promoter of the mouse caveolin-1 gene in lung cell lines to identify factors that control its cell-specific expression. We show that caveolin-1 expression is regulated by an Ets cis-element in a lung epithelial cell line, but not a lung endothelial cell line, and that three ETS family members, ETS-1, PEA3, and ERM, recognize and bind the Ets site in the epithelial cell line. Based on these findings, we have identified the Ets cis-element as a region that accounts for differential transcriptional regulation of caveolin-1 in lung epithelial and endothelial cells.

Within the vascular system, the mucin-type transmembrane glycoprotein T1alpha/podoplanin is predominantly expressed by lymphatic endothelium, and recent studies have shown that it is regulated by the lymphatic-specific homeobox gene Prox1. In this study, we examined the role of T1alpha/podoplanin in vascular development and the effects of gene disruption in mice. T1alpha/podoplanin is first expressed at around E11.0 in Prox1-positive lymphatic progenitor cells, with predominant localization in the luminal plasma membrane of lymphatic endothelial cells during later development. T1alpha/podoplanin(-/-) mice die at birth due to respiratory failure and have defects in lymphatic, but not blood vessel pattern formation. These defects are associated with diminished lymphatic transport, congenital lymphedema and dilation of lymphatic vessels. T1alpha/podoplanin is also expressed in the basal epidermis of newborn wild-type mice, but gene disruption did not alter epidermal differentiation. Studies in cultured endothelial cells indicate that T1alpha/podoplanin promotes cell adhesion, migration and tube formation, whereas small interfering RNA-mediated inhibition of T1alpha/podoplanin expression decreased lymphatic endothelial cell adhesion. These data identify T1alpha/podoplanin as a novel critical player that regulates different key aspects of lymphatic vasculature formation.

T1alpha, a differentiation gene of lung alveolar epithelial type I cells, is developmentally regulated and encodes an apical membrane protein of unknown function. Morphological differentiation of type I cells to form the air-blood barrier starts in the last few days of gestation and continues postnatally. Although T1alpha is expressed in the foregut endoderm before the lung buds, T1alpha mRNA and protein levels increase substantially in late fetuses when expression is restricted to alveolar type I cells. We generated T1alpha null mutant mice to study the role of T1alpha in lung development and differentiation and to gain insight into its potential function. Homozygous null mice die at birth of respiratory failure, and their lungs cannot be inflated to normal volumes. Distal lung morphology is altered. In the absence of T1alpha protein, type I cell differentiation is blocked, as indicated by smaller airspaces, many fewer attenuated type I cells, and reduced levels of aquaporin-5 mRNA and protein, a type I cell water channel. Abundant secreted surfactant in the narrowed airspaces, normal levels of surfactant protein mRNAs, and normal patterns and numbers of cells expressing surfactant protein-B suggest that differentiation of type II cells, also alveolar epithelial cells, is normal. Anomalous proliferation of the mesenchyme and epithelium at birth with unchanged numbers of apoptotic cells suggests that loss of T1alpha and/or abnormal morphogenesis of type I cells alter the proliferation rate of distal lung cells, probably by disruption of epithelial-mesenchymal signaling.

SUMMARY Caveolin-1 is a scaffolding protein component of caveolae, membrane invaginations involved in endocytosis, signal transduction, trans- and intracellular trafficking, and protein sorting. In adult lung, caveolae and caveolin-1 are present in alveolar endothelium and Type I epithelial cells but rarely in Type II cells. We have analyzed patterns of caveolin-1 expression during mouse lung development. Two caveolin-1 mRNAs, full-length and a 5 � variant that will translate mainly into caveolin-1 � and – � isoforms, are detected by RT-PCR at embryonic day 12 (E12) and afterwards in the developing and adult lung. Immunostaining analysis, starting at E10, shows caveolin-1 � localized in primitive blood vessels of the forming lung, in an overlapping pattern to the endothelial marker PECAM-1, and later in all blood vessels. Caveolin-1 � is not detected in fetal or neonatal lung epithelium but is detected in adult epithelial Type I cells. Caveolin-1 was previously shown to be expressed in alveolar Type I cells. These data suggest that expression of caveolin-1 isoforms is differentially regulated in endothelial and epithelial cells during lung development. Caveolin-1 � is an early marker for lung vasculogenesis, primarily expressed in developing blood vessels. When the lung is fully differentiated postnatally, caveolin-1 � is also expressed in alveolar Type I cells. (J Histochem Cytochem 50:33–42, 2002)

Parathyroid hormone-related peptide (PTHrP) and the parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) receptor are important developmental regulators of cell growth and differentiation in some organs. In lung, both the peptide and the receptor are expressed early in development and in alveolar cells in adults. In adult alveolar cells, PTHrP appears to promote the alveolar type II cell phenotype in vitro. Mice carrying null mutations in genes for either receptor or ligand die at birth of respiratory failure. To determine if absence of the PTH/PTHrP receptor alters morphogenesis or cellular differentiation of the distal lung, we analyzed the morphology and gene expression patterns in PTH/PTHrP receptor null mutant mice right before birth and compared them with wild-type and heterozygous null littermates. Using semiquantitative Northern blots, we observed that messenger RNA (mRNA) for aquaporin-5, the type I cell-specific water channel, was markedly decreased. The abundance of other marker mRNAs for type I and type II cell phenotypes, including T1alpha, surfactant proteins, and others, was unaltered. Gross morphology and lung pattern, assessed by in situ hybridization for surfactant protein C, were normal. We conclude therefore that, although signaling through this receptor may influence expression of specific lung genes, it does not play a major role in the general regulation of lung development and growth.

The T1α gene is one of few markers for the type I cell phenotype in the adult mammalian lung. Type I cells form a large, thin epithelial layer that facilitates gas exchange and transport of fluids between the air spaces and capillaries. The T1α gene has a complex pattern of developmental expression in lung and brain; in vitro studies indicate that expression is regulated in part by thyroid transcription factor 1, forkhead proteins, and Sp1/Sp3 proteins. To explore the mechanisms that confine T1α expression in intact adult animals to alveolar type I and choroid plexus epithelial cells, we generated mice bearing a 1.3-kb T1α promoter-chloramphenicol acetyltransferase (CAT) gene. In situ hybridization and RNase protection assays show that the 1.3-kb promoter confers a pattern of CAT expression that largely matches the endogenous T1α in embryos and mid-term fetuses in lung and central nervous system. However, the 1.3-kb promoter lacks elements important for perinatal up-regulation of T1α in the lung and maintenance of that expression in the adult lung and brain. The final adult pattern of T1α expression may be directed by elements outside the 1.3-kb fragment, perhaps those 5' to the 1.3-kb fragment as we show herein, or in 3' and intronic regions. Dev Dyn 1999;215:319–331. © 1999 Wiley-Liss, Inc.

Chronic injury of alveolar lung epithelium leads to epithelial disintegrity in idiopathic pulmonary fibrosis (IPF). We had reported earlier that Grhl2, a transcriptional factor, maintains alveolar epithelial cell integrity by directly regulating components of adherens and tight junctions and thus hypothesized an important role of GRHL2 in pathogenesis of IPF. Comparison of GRHL2 distribution at different stages of human lung development showed its abundance in developing lung epithelium and in adult lung epithelium. However, GRHL2 is detected in normal human lung mesenchyme only at early fetal stage (week 9). Similar mesenchymal reexpression of GRHL2 was also observed in IPF. Immunofluorescence analysis in serial sections from three IPF patients revealed at least two subsets of alveolar epithelial cells (AEC), based on differential GRHL2 expression and the converse fluorescence intensities for epithelial vs. mesenchymal markers. Grhl2 was not detected in mesenchyme in intraperitoneal bleomycin-induced injury as well as in spontaneously occurring fibrosis in double-mutant HPS1 and HPS2 mice, whereas in contrast in a radiation-induced fibrosis model, with forced Forkhead box M1 (Foxm1) expression, an overlap of Grhl2 with a mesenchymal marker was observed in fibrotic regions. Grhl2's role in alveolar epithelial cell plasticity was confirmed by altered Grhl2 gene expression analysis in IPF and further validated by in vitro manipulation of its expression in alveolar epithelial cell lines. Our findings reveal important pathophysiological differences between human IPF and specific mouse models of fibrosis and support a crucial role of GRHL2 in epithelial activation in lung fibrosis and perhaps also in epithelial plasticity.

Podoplanin (PDPN) is a transmembrane receptor that affects the activities of Rho, ezrin, and other proteins to promote tumor cell motility, invasion, and metastasis. PDPN is found in many types of cancer and may serve as a tumor biomarker and chemotherapeutic target. The intracellular region of PDPN contains only two serines, and these are conserved in mammals including mice and humans. We generated cells from the embryos of homozygous null Pdpn knock-out mice to investigate the relevance of these serines to cell growth and migration on a clear (PDPN-free) background. We report here that one or both of these serines can be phosphorylated by PKA (protein kinase A). We also report that conversion of these serines to nonphosphorylatable alanine residues enhances cell migration, whereas their conversion to phosphomimetic aspartate residues decreases cell migration. These results indicate that PKA can phosphorylate PDPN to decrease cell migration. In addition, we report that PDPN expression in fibroblasts causes them to facilitate the motility and viability of neighboring melanoma cells in coculture. These findings shed new light on how PDPN promotes cell motility, its role in tumorigenesis, and its utility as a functionally relevant biomarker and chemotherapeutic target.

We describe the effect of an inositol phosphoglycan (IPG) purified from Trypanosoma cruzi on the stimulation of aldosterone and cAMP production by ACTH in calf adrenocortical cells. T. cruzi IPG has two galactofuranose residues (Galf) which are not frequent in other IPGs. The effect of IPG with galactofuranose residues (IPG Galf) and IPG without these residues (IPG) was investigated. It was found that IPG Galf slightly decreased the stimulation of aldosterone and cAMP production by ACTH, whereas IPG significantly inhibited ACTH-mediated accumulation of both aldosterone and cAMP. The inhibition of aldosterone content in ACTH-treated cells by IPG was dose dependent. It was also found that the pretreatment of calf adrenocortical cells with IPG inhibited the accumulation of aldosterone provoked by ACTH and dibutyryladenosine-3′,5′-cyclic monophosphate (db-cAMP). On the other hand, the activation of a GPI (glycosyl phosphatidylinositol)-phospholipase C by ACTH was evaluated. First it was found that the release of ceramide from a GPI-like molecule: a glycoinositol-phosphoceramide (LPPG) purified from T. cruzi is increased in ACTH-treated cells. Second, the release of alkaline phosphatase, a GPI-anchored enzyme, to the extracellular medium was increased in these cells by ACTH. These data suggest that ACTH activates a phospholipase C in calf adrenocortical cells, releasing IPG, which in turn may inhibit, or modulate ACTH action.

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.

Pneumonia results from bacteria in the alveoli. The alveolar epithelium consists of type II cells, which secrete surfactant and associated proteins, and type I cells, which constitute 95% of the surface area and meet anatomic and structural needs. Other than constitutively expressed surfactant proteins, it is unknown whether alveolar epithelial cells have distinct roles in innate immunity. Because innate immunity gene induction depends on NF-κB RelA (also known as p65) during pneumonia, we generated a murine model of RelA mutated throughout the alveolar epithelium. In response to LPS, only 2 of 84 cytokine transcripts (CCL20 and CXCL5) were blunted in lungs of mutants, suggesting that a very limited subset of immune mediators is selectively elaborated by the alveolar epithelium. Lung CCL20 induction required epithelial RelA regardless of stimulus, whereas lung CXCL5 expression depended on RelA after instillation of LPS but not pneumococcus. RelA knockdown in vitro suggested that CXCL5 induction required RelA in type II cells but not type I cells. Sorted cell populations from mouse lungs revealed that CXCL5 was induced during pneumonia in type I cells, which did not require RelA. TLR2 and STING were also induced in type I cells, with RelA essential for TLR2 but not STING. To our knowledge, these data are the first direct demonstration that type I cells, which constitute the majority of the alveolar surface, mount innate immune responses during bacterial infection. These are also, to our knowledge, the first evidence for entirely RelA-independent pathways of innate immunity gene induction in any cell during pneumonia.

Cholesterol 7 alpha-hydroxylase (7 alpha-hydroxylase) is the rate-limiting enzyme in bile acid biosynthesis. It is subject to a feedback control, whereby high levels of bile acids suppress its activity, and cholesterol exerts a positive control. It has been suggested that posttranscriptional control plays a major part in that regulation. We have studied the mechanisms by which cholesterol and bile acids regulate expression of the 7 alpha-hydroxylase gene and found it to be solely at the transcriptional level by using two different approaches. First, using a tissue culture system, we localized a liver-specific enhancer located 7 kb upstream of the transcriptional initiation site. We also showed that low-density lipoprotein mediates transcriptional activation of chimeric genes, containing either the 7 alpha-hydroxylase or the albumin enhancer in front of the 7 alpha-hydroxylase proximal promoter, to the same extent as the in vivo cholesterol-mediated regulation of 7 alpha-hydroxylase mRNA. In a second approach, using transgenic mice, we have found that expression of an albumin enhancer-7 alpha-hydroxylase-lacZ fusion gene is restricted to the liver and is regulated by cholesterol and bile acids in a manner quantitatively similar to that of the endogenous gene. We also found, that a liver-specific enhancer is necessary for expression of the rat 7 alpha-hydroxylase gene, in agreement with the tissue culture experiments. Together, these results demonstrate that cholesterol and bile acids regulate the expression of the 7 alpha-hydroxylase gene solely at the transcriptional level.

The lipopeptidophosphoglycan is the major cell surface glycoconjugate of the epimastigote forms of the parasitic protozoan Trypanosoma cruzi. A detailed partial structure for this molecule has been reported (Previato, J. O., Gorin, P. A. J., Mazurek, M., Xavier, M. T., Fournet, B., Wieruszesk, J. M., and Mendonca-Previato, L. (1990) J. Biol. Chem. 265, 2518-2526). In this study, we complete the primary structure assignments and describe the microheterogeneity found in the lipopeptidophosphoglycan glycan, using a combination of 1H and 31P NMR, fast atom bombardment mass spectrometry, methylation linkage analysis, and exoglycosidase sequencing. The lipopeptidophosphoglycan is a glycosylated inositol-phosphoceramide with striking homology to glycosylphosphatidylinositol membrane anchors found attached to a wide variety of plasma membrane proteins throughout the eukaryotes.

The lipopeptidophosphoglycan (LPPG) from Trypanosoma cruzi, a major constituent of the plasma membrane of epimastigote forms, has been now extracted with butanol/water from delipidated cells and purified by hydrophobic chromatography. We have found that the LPPG undergoes two reactions, characteristic of the glycosylphosphatidylinositol anchors: (a) cleavage of the ceramide by phosphatidylinositol-specific phospholipase C (PtdIns-specific phospholipase C) from Bacillus thuringiensis, (b) nitrous acid deamination of the non-N-acylated glucosamine. Palmitoylsphinganine, palmitoylsphingosine, lignoceroylsphinganine and, as minor components, the stearoylceramides were identified by gas liquid chromatography/mass spectrometry. The presence of cross reacting determinant (CRD) epitopes in the glycophosphoinositol released by PtdIns-specific phospholipase C was investigated by direct and inhibition ELISA. A sample of glycophosphoinositol containing 5 micrograms carbohydrate caused 60% inhibition of the binding of anti-CRD antibodies raised against the soluble form of variant surface glycoprotein.

Angiotensin converting enzyme (ACE) 2 is a carboxypeptidase that shares 42% amino acid homology with ACE. Little is known about the regulation or pattern of expression of ACE2 in the mouse lung, including its definitive cellular distribution or developmental changes. Based on Northern blot and RT‐PCR data, we report two distinct transcripts of ACE2 in the mouse lung and kidney and describe a 5′ exon 1a previously unidentified in the mouse. Western blots show multiple isoforms of ACE2, with predominance of a 75–80 kDa protein in the mouse lung versus a 120 kDa form in the mouse kidney. Immunohistochemistry localizes ACE2 protein to Clara cells, type II cells, and endothelium and smooth muscle of small and medium vessels in the mouse lung. ACE2 mRNA levels peak at embryonic day 18.5 in the mouse lung, and immunostaining demonstrates protein primarily in the bronchiolar epithelium at that developmental time point. In murine cell lines ACE2 is strongly expressed in the Clara cell line mtCC, as opposed to the low mRNA expression detected in E10 (type I‐like alveolar epithelial cell line), MLE‐15 (type II alveolar epithelial cell line), MFLM‐4 (fetal pulmonary vasculature cell line), and BUMPT‐7 (renal proximal tubule cell line). In summary, murine pulmonary ACE2 appears to be primarily epithelial, is developmentally regulated, and has two transcripts that include a previously undescribed exon. J. Cell. Biochem. 101:1278–1291, 2007. © 2007 Wiley‐Liss, Inc.

Cancer is a leading cause of death. In fact, cancer killed over 8 million people around the world in 2012. Over 90% of these cancer deaths are due to metastasis, which results from tumor cell migration and invasion. Specific cancer biomarkers need to be identified in order to effectively target these motile cells. The transmembrane glycoprotein receptor podoplanin (PDPN) promotes tumor cell motility and metastasis in many aggressive cancers. Indeed, PDPN has emerged as a prime cancer biomarker and chemotherapeutic target. Here, we describe how PDPN can be targeted to inhibit the growth and motility of melanoma and oral cancer cells. PDPN has a short intracellular domain of 9 amino acids which include two conserved serine residues. PDPN also has a large extracellular domain that is extensively O-glycosylated with α2,3-sialic acid linked to galactose. We are developing novel methods to target the intracellular and extracellular domains of PDPN to combat cancer progression. For example, we have found that some activators of protein kinase A can induce phosphorylation of the intracellular serine residues of PDPN to inhibit tumor cell migration. In addition, we have found that Maackia amurensis seed lectin (MASL) can target the extracellular domain of PDPN to inhibit tumor cell growth, migration, and tumor progression in cell culture and animal models. Furthermore, we utilized live cell imaging to find that PDPN expression can be modulated in cancer associated fibroblasts to inhibit neighboring tumor cell migration and survival. Thus, reagents can be used to target PDPN from inside of the cell and outside of the cell to inhibit tumor cell migration and combat cancer progression. This work illuminates novel strategies designed to exploit PDPN as a functionally relevant biomarker and chemotherapeutic target. Citation Format: Harini Krishnan, Jhon Ochoa-alvarez, Yongquan Shen, Evan Nevel, David Kephart, Angels Nguyen, Min Han, Nimish Acharya, Robert Nagele, Maria Ramirez, W. Todd Miller, Evelyne Kalyoussef, Soly Baredes, Mahnaz Fatahzadeh, Lasse Jensen, Alan Shienbaum, Gary Goldberg. Podoplanin (PDPN): novel biomarker and chemotherapeutic target. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4375. doi:10.1158/1538-7445.AM2015-4375

Developmentally important genes have recently been linked to tissue regeneration and epithelial cell repair in neonatal and adult animals in several organs, including liver, skin, prostate, and musculature. We hypothesized that developmentally important genes play roles in lung injury repair in adult mice. Although there is considerable information known about these processes, the specific molecular pathways that mediate injury and regulate tissue repair are not fully elucidated. Using a hyperoxic injury model to study these mechanisms of lung injury and tissue repair, we selected the following genes based upon their known or putative roles in lung development and organogenesis: TTF-1, FGF9, FGF10, BMP4, PDGF-A, VEGF, Ptc, Shh, Sca-1, BCRP, CD45, and Cyclin-D2. Our findings demonstrate that several developmentally important genes (Sca-1, Shh, PDGF-A, VEGF, BCRP, CD45, BMP4, and Cyclin-D2) change during hyperoxic injury and normoxic recovery in mice, suggesting that adult lung may reactivate key developmental regulatory pathways for tissue repair. The mRNA for one gene (TTF-1), unchanged during hyperoxia, was upregulated late in recovery phase. These novel findings provide the basis for testing the efficacy of post-injury lung repair in animals genetically modified to inactivate or express individual molecules.

Background Development of lung alveolar sacs of normal structure and size at late gestation is necessary for the gas exchange process that sustains respiration at birth. Mice lacking the lung differentiation gene T1α [T1α(-/-)] fail to form expanded alveolar sacs, resulting in respiratory failure at birth. Since little is known about the molecular pathways driving alveolar sacculation, we used expression microarrays to identify genes altered in the abnormal lungs and, by inference, may play roles in normal lung morphogenesis. Results Altered expression of genes related to cell-cell interaction, such as ephrinA3, are observed in T1α(-/-) at E18.5. At term, FosB, Egr1, MPK-1 and Nur77, which can function as negative regulators of the cell-cycle, are down-regulated. This is consistent with the hyperproliferation of peripheral lung cells in term T1α (-/-) lungs reported earlier. Biochemical assays show that neither PCNA nor p21 are altered at E18.5. At term in contrast, PCNA is increased, and p21 is decreased. Conclusion This global analysis has identified a number of candidate genes that are significantly altered in lungs in which sacculation is abnormal. Many genes identified were not previously associated with lung development and may participate in formation of alveolar sacs prenatally.

Both the acquisition and the extinction of memories leave short- and long-term mnemonic traces. Here, we show that in male Wistar rats, the short-term memory for a step-down inhibitory avoidance task (IA) is resistant to extinction, and that its expression does not influence retrieval or extinction of long-term memory. It has been known for some time that short- and long-term inhibitory avoidance memory involve separate and parallel processes. Here we show that, instead, short-term extinction of IA long-term memory is the first step towards its long-term extinction, and that this link requires functional NMDA receptors and protein synthesis in the CA1 region of the dorsal hippocampus at the time of the first CS-no US presentation.

Although retinoic acid (RA) has been shown to be critical for lung development, little is known about when RA is required and the role of individual RA receptors (RAR) in this process. Previously reported data from an RA responsive element RARE-lacZ reporter mouse show that when epithelial tubules are branching and differentiating RA signaling becomes markedly down-regulated in the epithelium. It is unclear why this down-regulation occurs and what role it might play in the developing lung. Here we analyze the effects of preventing potential progenitors of the distal lung from turning off RA signaling by locally expressing constitutively activated RARalpha or RARbeta chimeric receptors (RARVP16) in branching airways of transgenic mice. Continued RA activation resulted in lung immaturity in both cases, but the phenotypes were remarkably different. RARalphaVP16 lungs did not expand to form saccules or morphologically identifiable type I cells. High levels of surfactant protein C (Sp-C), thyroid transcription factor-1 (Ttf1), and Gata6, but not Sp-A or Sp-B in the epithelium at birth suggested that in these lungs differentiation was arrested at an early stage. These alterations were not observed in RARbetaVP16 lungs, which showed relatively less severe changes. Our data suggest a model in which activation of RAR signaling at the onset of lung development establishes an initial program that assigns distal cell fate to the prospective lung epithelium. Down-regulation of RA signaling, however, is required to allow completion of later steps of this differentiation program that ultimately form mature type I and II cells.

The transcription factor Sp1 plays an important regulatory role in transactivation of the lung type I cell differentiation gene T1alpha. Like other lung cells, type I cells may encounter changes in oxygen concentration during the lifetime of the organism. We found that exposure of mice to hyperoxia rapidly increases expression of T1alpha and other type I cell genes, and that returning the mice to normoxia quickly decreases expression. Likewise hyperoxia increases both endogenous T1alpha expression in lung epithelial cell lines and transcription of luciferase (Luc) from T1alpha promoter deletion constructs. Using wild-type promoter fragments and gel shift assays, we determined that Sp1/Sp3 and a key Sp cis-element in the proximal promoter mediate the hyperoxic response. Mutations of this element and inhibition of Sp-DNA binding by mithramycin block the hyperoxic response. Western analyses of cell homogenates show that the overall abundance of Sp1 and Sp3 proteins is not altered by hyperoxia. However, the abundance of nuclear Sp1 increases after short hyperoxic exposures, suggesting that signaling pathways activated by hyperoxia lead to Sp protein translocation, perhaps as a result of increased Sp phosphorylation.

Alveolar type I epithelial cells form the major surface for gas exchange in the lung. To explore how type I cells differ in gene expression from their progenitor alveolar type II cells, we analyzed transcriptional regulation of T1alpha, a gene expressed by adult type I but not type II cells. In vivo developmental patterns of T1alpha expression in lung and brain suggest active gene regulation. We cloned and sequenced 1.25 kilobase pairs of the T1alpha promoter that can drive reporter expression in lung epithelial cell lines. Deletion analyses identified regions important for lung cell expression. The base pair (bp) -100 to -170 fragment conferred differential regulation in lung epithelial cells compared with fibroblasts. Sequence alignment of this fragment with type II-specific surfactant protein B and C promoters shows similar consensus elements arranged in a different order. Gel retardation studies with alveolar epithelial cell line nuclear extracts, thyroid transcription factor I (TTF-1) homeodomain, hepatic nuclear factor (HNF)-3beta, or Sp1 proteins, and supershift assays were used to characterize TTF-1, HNF-3 (TGT3), and Sp1/Sp3 binding sites. The TGT3 site binds factors with binding properties similar to HNF-3/Fkh (hepatic nuclear factor-3/forkhead) proteins but different from HNF-3alpha or HNF-3beta. Co-transfection with a TTF-1 expression vector moderately transactivated the -170 bp-reporter construct. Mutational analysis of these three binding sites showed reduced transcriptional activity of the -170 bp promoter. Therefore, several regulatory sequences involved in type II cell gene regulation are also present in the T1alpha promoter, suggesting that genes of the peripheral lung epithelium may be regulated by similar factors.