Search

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.

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.

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.

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.

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.

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.

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.

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.

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.

The homeodomain transcription factor Nkx2-1 is essential for normal lung development and homeostasis. In lung tumors, it is considered a lineage survival oncogene and prognostic factor depending on its expression levels. The target genes directly bound by Nkx2-1, that could be the primary effectors of its functions in the different cellular contexts where it is expressed, are mostly unknown. In embryonic day 11.5 (E11.5) mouse lung, epithelial cells expressing Nkx2-1 are predominantly expanding, and in E19.5 prenatal lungs, Nkx2-1-expressing cells are predominantly differentiating in preparation for birth. To evaluate Nkx2-1 regulated networks in these two cell contexts, we analyzed genome-wide binding of Nkx2-1 to DNA regulatory regions by chromatin immunoprecipitation followed by tiling array analysis, and intersected these data to expression data sets. We further determined expression patterns of Nkx2-1 developmental target genes in human lung tumors and correlated their expression levels to that of endogenous NKX2-1. In these studies we uncovered differential Nkx2-1 regulated networks in early and late lung development, and a direct function of Nkx2-1 in regulation of the cell cycle by controlling the expression of proliferation-related genes. New targets, validated in Nkx2-1 shRNA transduced cell lines, include E2f3, Cyclin B1, Cyclin B2, and c-Met. Expression levels of Nkx2-1 direct target genes identified in mouse development significantly correlate or anti-correlate to the levels of endogenous NKX2-1 in a dosage-dependent manner in multiple human lung tumor expression data sets, supporting alternative roles for Nkx2-1 as a transcriptional activator or repressor, and direct regulator of cell cycle progression in development and tumors.