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Author(s): Gerd P. Pfeifer Author Affiliations: Smoke signals in the DNA of normal lung cells According to the World Health Organization, there are 1.1 billion smokers worldwide and an estimated [...], Healthy cells in smokers' lungs have a high burden of mutations, similar to the mutational profile of lung cancer. Surprisingly, ex-smokers' lungs have a large fraction of healthy cells with nearly normal profiles. Ex-smokers' lungs have a large fraction of cells with few mutations.

Introduction Polycystic kidney diseases (PKD) are a heterogeneous group of disorders characterized by the development of multiple fluid-filled cysts that gradually lead to end-stage renal failure. The most common genetic [...], Mutations in polycystin-1, polycystin-2, or fibrocystin account for autosomal dominant or recessive polycystic kidney disease. Renal cystogenesis is linked to abnormal localization and function of these cystoproteins in renal primary cilia. They are also expressed in extrarenal tissues in which their functions are unclear. Here we found that human type-II alveolar epithelial A549, airway submucosal Calu-3 cells, and rat bronchioles contain primary or multiple cilia in which we detected these cystoproteins. At sub-confluency, polycystin-1 was expressed on plasma membrane, while polycystin-2 was localized to the ER of resting cells. Both polycystins were detected on the spindle and mid-body of mitotic cells, while fibrocystin was on centrosome throughout cell cycle. Polycystins and fibrocystin may participate in regulating mucociliary sensing and transport within pulmonary airways. Key words: polycystin, fibrocystin, primary cilium, lung, A549, Calu-3. Les mutations sur la polycystine-1, la polycystine-2 ou la fibrocystine sont la cause de la maladie polykystique des reins, une maladie autosomique dominante ou recessive. La formation des kystes est liee a une localisation et a une fonction anormales de ces cystoproteines dans les cils primaires renaux. Elles sont aussi exprimees dans des tissus extra-renaux, dans lesquels leurs fonctions ne sont pas claires. Les auteurs ont trouve ici que les cellules d'epithelium alveolaire humain de type IIA549, les cellules des voies respiratoires sous-muqueuses Calu-3 et les bronchioles de rat, contiennent des cils primaires ou multiples dans lesquels ils ont detecte ces cystoproteines. A sous-confluence, la polycystine-1 etait exprimee a la membrane plasmique alors que la polycystine-2 etait localisee dans le RE des cellules en quiescence. Les deux polycystines ont ete detectees sur le fuseau et le corps intermediaire des cellules en mitose, alors que la fibrocystine se trouvait au centrosome durant tout le cycle cellulaire. Les polycystines et la fibrocystine peuvent participer a la regulation de la detection muco-ciliaire et du transport dans les voies respiratoires pulmonaires. [Traduit par la Redaction] Mots-cles: polycystine, fibrocystine, cils primaires, poumon, A549, Calu-3.

Introduction Over the past two decades, nearly all aspects of avian biology from behaviour to systematics had benefited from the application of molecular methods (Lerner and Fleischer 2010). Genomic approaches [...], The jungle crow (Corvus macrorhynchos) belongs to the order Passeriformes of bird species and is important for avian ecological and evolutionary genetics studies. However, there is limited information on the transcriptome data of this species. In the present study, we report the characterization of the lung transcriptome of the jungle crow using GS FLX Titanium XLR70. Altogether, 1510 303 high-quality sequence reads with 581198 230 bases was de novo assembled into 22 169 isotigs (isotig represents an individual transcript) and 784 009 singletons. Using these isotigs and 581681 length-filtered (greater than 300 bp) singletons, 20 010 unique protein-coding genes were identified by BLASTx comparison against a nonredundant (nr) protein sequence database. Comparative analysis revealed that 46 604 (70.29%) and 51 642 (72.48%) of the assembled transcripts have significant similarity to zebra finch and chicken RefSeq proteins, respectively. As determined by GO annotation and KEGG pathway mapping, functional annotation of the unigenes recovered diverse biological functions and processes. Transcripts putatively involved in the immune response were identified. Furthermore, 20 599 single nucleotide polymorphisms (SNPs) and 7525 simple sequence repeats (SSRs) were retrieved from the assembled transcript database. This resource should lay an important base for future ecological, evolutionary, and conservation genetic studies on this species and in other related species. Key words: jungle crow, Corvus macrorhynchos, transcriptome, 454 pyrosequencing, single nucleotide polymorphisms, simple sequence repeats. Le corbeau a gros bec (Corvus macrorhynchos) est un oiseau qui appartient a l'ordre des Passeriformes et qui est important pour les etudes en ecologie et en genetique evolutive. Cependant, il existe peu de donnees transcriptomiques pour cette espece. Dans le present travail, les auteurs rapportent la caracterisation du transcriptome pulmonaire du corbeau a gros bec a l'aide d'un appareil GS FLX Titanium XLR70. Au total, 1 510 303 sequences de haute qualite, totalisant 581 198 230 bases, ont ete assemblees de novo pour former 22 169 isotigs (chaque isotig representant un transcrit individuel) et 784 009 singletons. En analysant ces isotigs et 581 681 singletons retenus (mesurant au moins 300 nucleotides), 20 010 genes codant pour des proteines ont ete identifies par analyse BLASTx contre une base de donnees non-redondante (nr) de sequences proteiques. Des analyses comparatives ont revele que 46 603 (70,29 %) et 51 642 (72,48%) des transcrits assembles presentaient respectivement une similarite significative avec des proteines RefSeq du diamant mandarin et du poulet. Au terme d'une annotation GO et d'une cartographie des sentiers KEGG, l'annotation fonctionnelle de ces unigenes a mis en evidence plusieurs fonctions et processus biologiques. Des transcrits vraisemblablement impliques dans la reponse immunitaire ont ete identifies. De plus, 20 599 polymorphismes mononucleotidiques (SNP) et 7525 sequences simples repetees (SSR) ont ete identifies au sein du transcriptome assemble. Cette ressource devrait constituer une assise importante pour de futures etudes genetiques en ecologie, en evolution et en conservation chez cette espece et des especes apparentees. [Traduit par la Redaction] Mots-cles : corbeau a gros bec, Corvus macrorhynchos, transcriptome, pyrosequencage 454, polymorphismes mononucleotidiques, sequences simples repetees.

The mammalian lung is a highly branched network in which the distal regions of the bronchial tree transform during development into a densely packed honeycomb of alveolar air sacs that [...]

Alveolar type II cells have multiple functions, including surfactant production and fluid clearance, which are critical for lung function. Differentiation of type II cells occurs in cultured fetal lung epithelial cells treated with dexamethasone plus cAMP and isobutylmethylxanthine (DCI) and involves increased expression of 388 genes. In this study, type II cells of human adult lung were isolated at ~95% purity, and gene expression was determined (Affymetrix) before and after culturing 5 days on collagen-coated dishes with or without DCI for the final 3 days. In freshly isolated cells, highly expressed genes included SFTPA/B/C, SCGB1A, IL8, CXCL2, and SFN in addition to ubiquitously expressed genes. Transcript abundance was correlated between fetal and adult cells (r = 0.88), with a subset of 187 genes primarily related to inflammation and immunity that were expressed > 10-fold higher in adult cells. During control culture, expression increased for 8.1% of expressed genes and decreased for ~4% including 118 immune response and 10 surfactant-related genes. DCI treatment promoted lamellar body production and increased expression of ~3% of probed genes by [less than or equal to] l.5-fold; 40% of these were also induced in fetal cells. Highly induced genes ([less than or equal to] 10-fold) included PGC, ZBTB16, DUOX1, PLUNC, CIT, and CRTAC1. Twenty-five induced genes, including six genes related to surfactant (SFTPA/B/C, PGC, CEBPD, and ADFP), also had decreased expression during control culture and thus are candidates for hormonal regulation in vivo. Our results further define the adult human type II cell molecular phenotype and demonstrate that a subset of genes remains hormone responsive in cultured adult cells. expression profiling; dexamethasone; cAMP; surfactant; pepsinogen C doi: 10.1152/ajplung.00427.2009.

Based largely on studies in xenograft models, lipocalin-2 (Lcn2) has been implicated in the progression of multiple types of human tumors, including breast cancer. Here we examine the role of Lcn2 in mammary tumorigenesis and lung metastasis using an in vivo molecular genetics approach. We crossed a well-characterized transgenic mouse model of breast cancer, the MMTV-PyMT (mouse mammary tumor virus-polyoma middle T antigen) mouse, with two independent gene-targeted [Lcn2.sup.-/-] mouse strains of the 129/ Ola or C57BL/6 genetic background. The onset and progression of mammary tumor development and lung metastasis in the female progeny of these crosses were monitored over a 20-week period. Female [Lcn2.sup.-/-]MMTV-PyMT mice of the 129/Ola background ([Lcn2.sup.-/-][PyMT.sup.129]) showed delayed onset of mammary tumors, and both [Lcn2.sup.-/-][PyMT.sup.129] mice and [Lcn2.sup.-/-]MMTV-PyMT mice of the C57BL/6 background ([Lcn2.sup.-/-][PyMT.sup.B6]) exhibited significant decreases in multiplicity and tumor burden (~2- to 3-fold), as measured by total tumor weight and volume. At the molecular level, mammary tumors derived from [Lcn2.sup.-/-][PyMT.sup.B6] females showed reduced matrix metalloproteinase-9 (MMP-9) activity and a lack of high molecular weight MMP activity. However, although increased MMP-9 activity has been linked to tumor progression, neither [Lcn2.sup.-/-][PyMT.sup.B6] nor [Lcn2.sup.-/-][PyMT.sup.129] female mice showed a reduction in lung metastases compared to [Lcn2.sup.+/+]PyMT controls. Our results demonstrate, using an in vivo animal model approach, that Lcn2 is a potent inducer of mammary tumor growth but not a significant promoter of lung metastasis. breast cancer | lipocalin-2 | matrix metalloproteinase-9 | mouse mammary tumor virus-polyoma middle T antigen www.pnas.org/cgi/doi/10.1073/pnas.1000101107

Lung arteriovenous (AV) shunts or malformations cause significant morbidity and mortality in several distinct clinical syndromes. For most patients with lung AV shunts, there is still no optimal treatment. The underlying molecular and cellular etiology for lung AV shunts remains elusive, and currently described animal models have insufficiently addressed this problem. Using a tetracycline-repressible system, we expressed constitutively active Notch4 ([Notch4.sup.*]) specifically in the endothelium of adult mice. More than 90% of mice developed lung hemorrhages and respiratory insufficiency and died by 6-7 wk after gene expression began. Vascular casting and fluorescent microsphere analysis showed evidence of lung AV shunts in affected mice. Cessation of [Notch4.sup.*] expression reversed these pathophysiological effects. Assessment of the vascular morphology revealed enlarged, tortuous vessels in the lungs that resembled arteriovenous malformations. By using whole lung organ culture, we demonstrated the effects of constitutively active Notch4 on the lung vasculature to be a primary lung phenomenon. Together, our results indicate the importance of Notch signaling in maintaining the lung vasculature and offer a new, reliable model with which to study the pathobiology of lung arteriovenous shunts and malformations. vascular; pulmonary; endothelium; Notch doi: 10.1152/ajplung.00188.2009

Extracellular signal-regulated kinase 3 (Erk3) is an atypical member of the mitogen-activated protein (MAP) kinase family. No function has yet been ascribed to this MAP kinase. Here we show that targeted disruption of the Mapk6 gene (encoding Erk3) leads to intrauterine growth restriction, associated with marked pulmonary hypoplasia, and early neonatal death during the first day of life. Around 40% of [Erk3.sup.-/-] neonates die within minutes after birth from acute respiratory failure. Erk3-deficient mice have normal lung-branching morphogenesis, but show delayed lung maturation characterized by decreased sacculation, atelectasis, and defective type II pneumocyte differentiation. Interestingly, in utero administration of glucocorticoid promoted fetal lung maturity and rescued differentiation of type II cells, but failed to alter the neonatal lethality. We observed that loss of Erk3 retards intrauterine growth, as reflected by a marked reduction in fetal lung, heart, and liver weights, and by low body weight at birth. Importantly, we found that insulin-like growth factor (IGF)-2 levels are decreased in the serum of Erk3-deficient mice. Our findings reveal a critical role for Erk3 in the establishment of fetal growth potential and pulmonary function in the mouse. gene targeting | MAP kinase | lung | IUGR

Trachealess (Trh) is a PAS domain transcription factor regulating Drosophila tracheogenesis. No other Trh homolog has been associated with a respiratory phenotype. Seeking homolog(s) regulating lung development, we screened murine genomic DNA using trh oligonucleotides, identifying only Npas3. Npas3 mRNA peaks in lung from E10.5 to E13.5, verified by sequencing, with immuno-staining in airway epithelial cells. Npas3-null mice have reduced lung branching morphogenesis but are viable prenatally. Npas3-null newborns die in respiratory distress, with diminished alveolarization, decreased Shh, Fgf9, Fgf10, and Bmp4 mRNAs, and increased Spry2, consistent with reduced FGF signaling. Exogenous FGF10 rescues branching morphogenesis in Npas3-null lungs. In promoter reporter assays, NPAS3 directly upregulates Shh and represses Spry2. [Npas3.sup.+1-] mice have a milder lung phenotype, surviving postnatally, but develop emphysema and airways hyperreactivity. Therefore, absence of a developmentally expressed transcription factor can alter downstream gene expression and multiple signaling pathways in organogenesis. NPAS3 haplo-insufficiency may also lead to emphysema and asthma. branching morphogenesis | emphysema | fibroblast growth factors | sonic hedgehog | transcription factors

We have investigated the effects of cigarette smoke exposure in three different strains of mice. DBA/2 and C57BL/6J are susceptible to smoke and develop different lung changes in response to chronic exposure, whereas ICR mice are resistant to smoke and do not develop emphysema. The present study was carried out to determine early changes in the gene expression profile of mice exposed to cigarette smoke with either a susceptible or resistant phenotype. The three strains of mice were exposed to smoke from three cigarettes per day, 5 days/wk, for 4 wk. Microarray analysis was carried out on total RNA extracted from the lung using the Affymetrix platform. Cigarette smoke modulates several clusters of genes (i.e., proemphysematous, acute phase response, and cell adhesion) in smoke-sensitive DBA/2 or C57BL/6J strains, but the same genes are not altered by smoke in ICR resistant mice. Only a few genes were commonly modulated by smoke in the three strains of mice. This pattern of gene expression suggests that the response to smoke is strain-dependent and may involve different molecular signaling pathways. Real-time quantitative PCR was used to verify the pattern of modulation of selected genes and their potential biological relevance. We conclude that gene expression response to smoke is highly dependent on the mouse genetic background. We speculate that the definition of gene clusters associated, to various degrees, with mouse susceptibility or resistance to smoke may be instrumental in defining the molecular basis of the individual response to smoke-induced lung injury in humans. emphysema; antioxidant defenses; cigarette smoke susceptibility; mouse strains; microarrays

We focused on the regulation of inflammatory mediator expression by adenovirus E1A in lung epithelial cells and the role of this viral protein in the pathogenesis of chronic obstructive pulmonary disease (COPD). We previously reported that E1A, a well-known regulator of host genes, increased ICAM-1 expression in human bronchial epithelial (HBE) and A549 cells in response to LPS stimulation. In this report, we clarified the mechanism of this regulation. We found NF-[kappa]B translocation to the nucleus after LPS stimulation in both E1A-positive and -negative HBE cells. ICAM-1 promoter reporter constructs revealed that a mutation in the proximal NF-[kappa]B binding site completely inhibited increased transcription, whereas the mutation in a distal site did not. We analyzed the participation of E1A in transcriptional complex formation at this promoter using chromatin immunoprecipitation. In E1A-positive HBE and A549 cells, LPS stimulation increased ICAM-1 promoter immunoprecipitation by NF-[kappa]B p65 and p300 but not activator protein-1 antibodies with a concomitant increase by the E1A antibody. No increase was found in E1A-negative cells except in HBE cells with p65 antibody. The association of E1A with the increased promoter immunoprecipitation with p300 was also observed after TNF-[alpha] stimulation of A549 cells. These results suggest that adenovirus E1A regulates the ICAM-1 promoter through its proximal NF-[kappa]B binding site, most likely by interacting with the transcriptional complex that forms at this site. E1A regulation of the LPS response may play a role in acute exacerbations as a consequence of bacterial infections in COPD. gene regulation; inflammation; nuclear factor-[kappa]B

One hundred Hampshire x Duroc crossbred pigs and 100 Nebraska Index line pigs were infected with porcine reproductive and respiratory syndrome virus (PRRSV) and evaluated for resistance and susceptibility. Controls (100/line) were uninfected littermates to infected pigs. Viremia (V), BW change (WT[DELTA]), and rectal temperature at 0, 4, 7, and 14 d postinfection were recorded. Lung, bronchial lymph node (BLN), and blood tissue were collected at necropsy (14 d postinfection). Infected pigs were classified as low or high responders to PRRSV based on the first principal component from principal component analyses of all variables. Low responders to PRRSV (low PRRSV burden) and their uninfected littermates were assigned to the low (L) class. High responders to PRRSV (high PRRSV burden) and their uninfected littermates were assigned to the high (H) class. Infected pigs in the L class had large WT[DELTA], low V, and few lung lesions; H-class pigs had small WT[DELTA], high V, and many lung lesions. Ribonucleic acid was extracted from lung and BLN tissue of the 7 highest and 7 lowest responders per line and from each of their control littermates. A control reference design was used, and cDNA from each reference sample tissue was prepared from pooled RNA extracted from 2 control pigs from each line whose infected littermates had a principal component value of 0. Design variables in data analyses were line (Index vs. Hampshire x Duroc), class (H vs. L), treatment (infected vs. uninfected controls), and slide/pig as error. Oligo differential expression was based on P < 0.01 occurring in both lung and BLN. Line and treatment effects were significant for 38 and 541 oligos, respectively, in both lung and BLN. Line x class interaction existed for expression of thymosin [beta]-4, DEAD box RNA helicase 3, acetyl-cholinesterase, and Homo sapiens X (inactive)-specific transcript in both tissues. Treatment x class existed for expression of CCAAT/enhancer-binding [delta] protein, nuclear factor of [kappa] light polypeptide gene enhancer in B cells inhibitor [alpha], thioredoxin-interacting protein, major facilitator superfamily domain containing 1, and unknown sequences SS00012040 and SS00012343. Line x treatment and line x treatment x class interactions were not significant. Possible important genetic associations for fine-mapping candidate genes related to response to PRRSV and determining causative alleles were revealed. Key words: gene expression, microarray, pig, porcine reproductive and respiratory syndrome virus resistance

Neuregulin is an important growth factor in fetal surfactant synthesis, and downregulation of its receptor, ErbB4, impairs fetal surfactant synthesis. We hypothesized that pulmonary ErbB4 deletion will affect the developing lung leading to an abnormal postnatal lung function. ErbB4-deleted lungs of I l- to 14-wk-old adult [HER4.sup.heart] mice, rescued from their lethal cardiac defects, were studied for the effect on lung function, alveolarization, and the surfactant system. ErbB4 deletion impairs lung function and structure in [HER4.sup.heart] mice resulting in a hyperreactive airway system and alveolar simplification, as seen in preterm infants with bronchopulmonary dysplasia. It also leads to a downregulation of surfactant protein D expression and an underlying chronic inflammation in these lungs. Our findings suggest that this animal model could be used to further study the pathogenesis of bronchopulmonary dysplasia and might help design protective interventions. surfactant; lung development; alveolar simplification; hyperreactive airway system

Chronic obstructive pulmonary disease (COPD) is a smoking-related disease that lacks effective therapies due partly to the poor understanding of disease pathogenesis. The aim of this study was to identify molecular pathways that could be responsible for the damaging consequences of smoking. To do this, we employed Gene Set Enrichment Analysis to analyze differences in global gene expression, which we then related to the pathological changes induced by cigarette smoke (CS). Sprague-Dawley rats were exposed to whole body CS for 1 day and for various periods up to 8 too. Gene Set Enrichment Analysis of microarray data identified that metabolic processes were most significantly increased early in the response to CS. Gene sets involved in stress response and inflammation were also upregulated. CS exposure increased neutrophil chemokines, cytokines, and proteases (MMP-12) linked to the pathogenesis of COPD. After a transient acute response, the CS-exposed rats developed a distinct molecular signature after 2 wk, which was followed by the chronic phase of the response. During this phase, gene sets related to immunity and defense progressively increased and predominated at the later time points in smoke-exposed rats. Chronic CS inhalation recapitulated many of the phenotypic changes observed in COPD patients including oxidative damage to macrophages, a slowly resolving inflammation, epithelial damage, mucus hypersecretion, airway fibrosis, and emphysema. As such, it appears that metabolic pathways are central to dealing with the stress of CS exposure; however, over time, inflammation and stress response gene sets become the most significantly affected in the chronic response to CS. chronic obstructive pulmonary disease; metabolic pathways; oxidant stress

Mechanical ventilation (MV) with [O.sub.2]-rich gas offers life-saving treatment for extremely premature infants with respiratory failure but often leads to neonatal chronic lung disease (CLD), characterized by defective formation of alveoli and blood vessels in the developing lung. We discovered that MV of 2- to 4-day-old mice with 40% [O.sub.2] for 8 h, compared with unventilated control pups, reduced lung expression of genes that regulate lung septation and angiogenesis (VEGF-A and its receptor, VEGF-R2; PDGF-A; and tenascin-C). MV with air for 8 h yielded similar results for PDGF-A and tenascin-C but did not alter lung mRNA expression of VEGF or VEGF-R2. MV of 4- to 6-day-old mice with 40% [O.sub.2] for 24 h reduced lung protein abundance of VEGF-A, VEGF-R2, PDGF-A, and tenascin-C and resulted in lung structural abnormalities consistent with evolving CLD. After MV with 40% [O.sub.2] for 24 h, lung volume was similar to unventilated controls, whereas distal air space size, assessed morphometrically, was greater in lungs of ventilated pups, indicative of impaired septation. Immunostaining for vimentin, which is expressed in myofibroblasts, was reduced in distal lung after 24 h of MV with 40% [O.sub.2]. These molecular, cellular, and structural changes occurred without detectable lung inflammation as evaluated by histology and assays for proinflammatory cytokines, myeloperoxidase activity, and water content in lung. Thus lengthy MV of newborn mice with [O.sub.2]-rich gas reduces lung expression of genes and proteins that are critical for normal lung growth and development. These changes yielded lung structural defects similar to those observed in evolving CLD. bronchopulmonary dysplasia; neonatal chronic lung disease; lung growth and development; vascular endothelial growth factor; VEGF receptor 2 (fetal liver kinase-1); platelet-derived growth factor; tenascin-C; pulmonary inflammation

Male rats aged 3 months, 12 months and 20 months were subjected to breathing 8% oxygen for 6 hours. Lung preproadrenomedullin (AM) messenger RNA (mRNA) levels were measured by solution hybridization-RNase protection assay while AM was measured by radioimmunoassay. The binding of hypoxia-inducible factor-1[alpha] (HIF-1[alpha]) to DNA was determined by electrophoretic mobility shift. There was an age-related increase in basal levels of preproAM mRNA and AM and of the binding of hypoxia-inducible factor (HIF) to DNA. Upon hypoxic stimulation, HIF binding to DNA increased in the young and middle-aged rats, but not in the old rats. AM gene expression increased in response to hypoxia in rats of all ages, but the increase was much less in the old rats. AM peptide levels in the lung decreased with age in hypoxia. In a separate experiment, male rats aged 3 months and 20 months were subjected to hypoxia as described above. PreproAM, calcitonin receptor-like receptor (CRLR), receptor activity modifying protein (RAMP) mRNA, HIF-1 and peptidyl-glycine-amidating monooxygenase (PAM) mRNA levels were measured by reverse transcription--polymerase chain reaction. All except PAM showed a decrease in basal levels and a diminished response to hypoxia in the old rats. Polysome profiling demonstrated decreases in the percentages of translatable preproAM mRNA in response to hypoxia, with a greater decrease in the old than the young rats. It is concluded that an agedependent decrease in the hypoxic response of the AM system in the lung was associated with high basal levels of H1F activity and AM expression in the old rats, and a lower proportion of translatable preproAM mRNA in the old rats in response to hypoxia. Thus, the HIF-AM pathway may be impaired in the aged lung, and other mechanisms may be present to maintain an AM response to hypoxia.

The lung develops from the endoderm through a process of branching morphogenesis. This process is highly active during the pseudoglandular stage of lung development and continues into the canalicular stage, resulting in the formation of terminal sacs. CCAAT/enhancer binding proteins (C/EBPs) are transcription factors regulating central aspects of differentiation and proliferation. We report here the developmental expression of C/EBP[alpha], [beta], and -[delta] in the lung. C/EBP[alpha] exhibits a dynamic expression pattern and is first detected during the late pseudoglandular stage. At this stage, expression is observed in a subset of epithelial cells in the distal parts of the branching tubules. The expression of C/EBP[alpha] is confined to nonproliferating cells. To examine the role of C/EBP[alpha] in lung development, we generated transgenic mice ectopically expressing C/EBP[alpha] in the lung epithelium using the human surfactant protein C promoter. Lungs from these mice were of normal size but exhibited a phenotype characterized by fewer and larger developing epithelial tubules, indicating that the branching process was affected. No effects on overall proliferation or cellular differentiation were observed. When this phenotype was compared with that of mice carrying a targeted mutation of the Cebpa gene, the [Cebpa.sup.-/-] mice exhibited a similar developmental phenotype. In conclusion, our results show a role for C/EBP[alpha] in lung development and suggest a function in the later stages of lung branching morphogenesis. CCAAT/enhancer binding proteins; transcription factors; expression pattern; branching morphogenesis; cellular differentiation

The symposium addressed the burgeoning interest in fundamental mechanisms underlying the onset of pneumonia. Bacteria exploit the lung's innate immune mechanism, resulting in pathophysiological cell signaling. As a consequence inflammation develops, leading to pneumonia. New mechanisms have been identified by which bacteria or bacterial products in the airway induce cross-compartmental signaling that leads to inflammatory consequences. The speakers addressed activation of the transcription factor, NF-[kappa]B occurring as a consequence of bacterial interactions with specific receptors, such as the Toll-like receptors and the TNF receptor 1 (Prince), or as a consequence of cytokine induction (Mizgerd). Also considered were mechanisms of bacterial virulence in the clinical setting (Wiener-Kronish) and the role of alveolar-capillary signaling mechanisms in the initiation of lung inflammation. doi:10.1152/ajplung.00138.2006

Using a study on mice, the influence of signaling activity of retinoic acid receptors on initial lung development is examined.

The role of WNT signaling and its interactions with other morphogenetic pathways were investigated during lung development. Previously, we showed that targeted disruption of Wnt5a results in over-branching of the epithelium and thickening of the interstitium in embryonic lungs. In this study, we generated and characterized transgenic mice with lung-specific over-expression of Wnt5a from the SpC promoter. Over-expression of Wnt5a interfered with normal epithelial-mesenchymal interactions resulting in reduced epithelial branching and dilated distal airways. During early lung development, over-expression of Wnt5a in the epithelium resulted in increased Fgf10 in the mesenchyme and decreased Shh in the epithelium. Both levels and distribution of SHH receptor, Ptc were reduced in SpC-Wnt5a transgenic lungs and were reciprocally correlated to changes of Fgf10 in the mesenchyme, suggesting that SHH signaling is decreased by over-expression of Wnt5a. Cultured mesenchyme-free epithelial explants from SpC-Wnt5a transgenic lungs responded abnormally to recombinant FGF 10 supplied uniformly in the Matrigel with dilated branch tips that mimic the in vivo phenotype. In contrast, chemotaxis of transgenic epithelial explants towards a directional FGF10 source was inhibited. These suggest that over-expression of Wnt5a disrupts epithelial-response to FGF10. In conclusion, Wnt5a regulates SHH and FGF10 signaling during lung development. Keywords: Wnt5a; Shh; Fgf10; Lung development; Morphogenesis

It is well established that exposure to high levels of oxygen (hyperoxia) injures and kills microvascular endothelial and alveolar type I epithelial cells. In contrast, significant death of airway and type II epithelial cells is not observed at mortality, suggesting that these cell types may express genes that protect against oxidative stress and damage. During a search for genes induced by hyperoxia, we previously reported that airway and alveolar type II epithelial cells uniquely express the growth arrest and DNA damage (Gadd)45a gene. Because Gadd45a has been implicated in protection against genotoxic stress, adult Gadd45a (+/+) and Gadd45a (-/-) mice were exposed to hyperoxia to investigate whether it protected epithelial ceils against oxidative stress. During hyperoxia, Gadd45a deficiency did not affect loss of airway epithelial expression of Clara cell secretory protein or type II epithelial cell expression of pro-surfactant protein C. Likewise, Gadd45a deficiency did not alter recruitment of inflammatory cells, edema, or overall mortality. Consistent with Gadd45a not affecting the oxidative stress response, [p21.sup.Cip1/WAF1] and heme oxygenase-1 were comparably induced in Gadd45a (+/+) and Gadd45a (-/-) mice. Additionally, Gadd45a deficiency did not affect oxidative DNA damage or apoptosis as assessed by oxidized guanine and terminal deoxyneucleotidyl transferase-mediated dUTP nick-end labeling staining. Overexpression of Gadd45a in human lung adenocarcinoma cells did not affect viability or survival during exposure, whereas it was protective against UV-radiation. We conclude that increased tolerance of airway and type II epithelial cells to hyperoxia is not attributed solely to expression of Gadd45a. DNA damage; lung; mice; in situ nick-end labeling; 8-hydroxy-2'deoxoguanosine

The Notch/Notch-ligand pathway regulates cell fate decisions and patterning in various tissues. Several of its components are expressed in the developing lung, suggesting that this pathway is important for airway cellular patterning. Fringe proteins, which modulate Notch signaling, are crucial for defining morphogenic borders in several organs. Their role in controlling cellular differentiation along anterior-posterior axis of the airways is unknown. Herein, we report the temporal-spatial expression patterns of Lunatic fringe (Lfng) and Notch-regulated basic helix-loop-helix factors, Hes1 and Mash-1, during murine lung development. Lfng was only expressed during early development in epithelial cells lining the larger airways. Those epithelial cells also expressed Hes1, but at later gestation Hes1 expression was confined to epithelium lining the terminal bronchioles. Mash-1 displayed a very characteristic expression pattern. It followed neural crest migration in the early lung, whereas at later stages Mash-1 was expressed in lung neuroendocrine cells. To clarify whether Lfng influences airway cell differentiation, Lfng was overexpressed in distal epithelial cells of the developing mouse lung. Overexpression of Lfng did not affect spatial or temporal expression of Hes1 and Mash-1. Neuroendocrine CGRP and protein gene product 9.5 expression was not altered by Lfng overexpression. Expression of proximal ciliated ([beta]-tubulin IV), nonciliated (CCSP), and distal epithelial cell (SP-C, T1[alpha]) markers also was not influenced by Lfng excess. Overexpression of Lfng had no effect on mesenchymal cell marker ([alpha]-sma, vWF, PECAM-1) expression. Collectively, the data suggest that Lunatic fringe does not play a significant role in determining cell fate in fetal airway epithelium. Notch signaling pathway; lung development; epithelial differentiation

Genomic DNA from the mouse pulmonary surfactant protein C (SP-C) gene was analyzed in transgenic mice to identify DNA essential for alveolar type II cell-specific expression. SP-C promoter constructs extending either 13 or 4.8 kb upstream of the transcription start site directed lung-specific expression of the bacterial chloramphenicol acetyl transferase (CAT) reporter gene. In situ hybridization analysis demonstrated alveolar cell-specific expression in the lungs of adult transgenic mice, and the pattern of 4.8 SP-C-CAT expression during development paralleled that of the endogenous SP-C gene. With the use of deletion constructs, lung-specific, low-level CAT activity was detected in tissue assays of SP-C-CAT transgenic mice retaining 318 bp of the promoter, In transient and stable cell transfection experiments, the 4.8-kb SP-C promoter was 90-fold more active as a stably integrated gene. These findings indicate that l) the 4.8-kb SP-C promoter is sufficient to direct cell-specific and developmental expression, 2) an enhancer essential for lung-specific expression maps to the proximal 318-bp promoter, and 3) the activity of the 4.8-kb SP-C promoter construct is highly dependent on its chromatin environment. surfactant protein C; type II cells

Members of the Dickkopf (Dkk) family of secreted proteins are potent inhibitors of Wnt/[beta]-catenin signaling. In this study we show that Dkk1, -2, and -3 are expressed distally in the epithelium, while Kremen1, the needed co-receptor, is expressed throughout the epithelium of the developing lung. Using TOPGAL mice [DasGupta, R., Fuchs, E., 1999. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation. Development 126, 4557-4568] to monitor the Wnt pathway, we show that canonical Wnt signaling is dynamic in the developing lung and is active throughout the epithelium and in the proximal smooth muscle cells (SMC) until E12.5. However, from E13.5 onwards, TOPGAL activity is absent in the SMC and is markedly reduced in the distal epithelium coinciding with the onset of Dkk-1 expression in the distal epithelium. To determine the role of Wnt signaling in early lung development, E11.5 organ cultures were treated with recombinant DKK1. Treated lungs display impaired branching, characterized by failed cleft formation and enlarged terminal buds, and show decreased [alpha]-smooth muscle actin ([alpha]-SMA) expression as well as detects in the formation of the pulmonary vasculature. These defects coincide with a pattern of decreased fibronectin (FN) deposition. DKK1-induced morphogenetic defects can be mimicked by inhibition of FN and overcome by addition of exogenous FN, suggesting an involvement of FN in Wnt-regulated morphogenetic processes. Keywords: Lung; Dickkopf-1; Wnt signaling; Fibronectin; Branching morphogenesis; Smooth muscle; Vascular development

During development, the proximal and distal regions of respiratory tract undergo distinct processes that ultimately give rise to conducting airways and alveoli. To gain insights into the genetic pathways differentially activated in these regions when branching morphogenesis is initiating, we characterized their transcriptional profiles in murine rudiments isolated at embryonic (E) day 11.5. By using oligonucleotide microarrays, we identified 83 and 128 genes preferentially expressed in branching and non-branching regions, respectively. The majority of these genes (85%) had not been previously described in the lung, or in other organs. We report restricted expression patterns of 22 of these genes were by in situ hybridization. Among them in the lung potential components of the Wnt, TGF beta, FGF and retinoid pathways identified in other systems, and uncharacterized genes, such as translocases, small GTPases and splicing factors. In addition, we provide a more detailed analysis of the expression pattern and regulation of a representative gene from the distal (transforming growth factor, beta induced) and proximal (WW domain-containing protein 2) regions. Our data suggest that these genes may regulate focal developmental events specific of each of these regions during respiratory tract formation. Keywords: Lung development; Microarrays; Branching morphogenesis; Respiratory tract; Growth factors; TGF beta: Tgfbi; Wwp2; Ubiquitination; Retinoic acid

The forkhead box (Fox) fl transcription factor is expressed in the mouse splanchnic (visceral) mesoderm, which contributes to development of the liver, gallbladder, lung, and intestinal tract. Pulmonary hemorrhage and peripheral microvascular defects were found in approximately half of the newborn Foxfl(+/-) mice, which expressed low levels of lung Foxfl mRNA [low-Foxfl(+/-) mice]. Microvascular development was normal in the surviving newborn high-Foxfl(+/-) mice, which compensated for pulmonary Foxfl haploinsufficiency and expressed wild-type Foxfl levels. To identify expression of genes regulated by Foxfl, we used Affymetrix microarrays to determine embryonic lung RNAs influenced by Foxfl haploinsufficiency. Embryonic Foxfl(+/-) lungs exhibited diminished expression of hepatocyte growth factor receptor c-Met, myosin VI, the transcription factors SP-3, BMI-1, ATF-2, and glucocorticoid receptor, and cell cycle inhibitors p53, [p21.sup.Cip1], retinoblastoma, and p107. Furthermore, Notch-2 signaling was decreased in embryonic Foxfl (+/-) lungs, as evidenced by significantly reduced levels of the Notch-2 receptor and the Notch-2 downstream target hairy enhancer of split-1. The severity of the Notch-2-signaling defect in 18-day postcoitus Foxfl(+/-) lungs correlated with Foxfl mRNA levels. Disruption of pulmonary Notch-2 signaling continued in newborn low-Foxfl(+/-) mice, which died of lung hemorrhage and failed to compensate for Foxfl haploinsufficiency. In contrast, in newborn high-Foxfl(+/-) lungs, Notch-2 signaling was restored to the level found in wild-type mice, which was associated with normal microvascular formation and survival. Foxfl haploinsufficiency disrupted pulmonary expression of genes in the Notch-2-signaling pathway and resulted in abnormal development of lung microvasculature. winged helix DNA-binding domain; Notch-2 receptor; microarray analysis; forkhead box transcription factor; hepatocyte nuclear factor/ forkhead homolog-8; forkhead-related activator-1

During the development of the mouse lung, the expression of a number of genes, including those encoding growth factors and components of their downstream signaling pathways, is enriched in the epithelium and/or mesenchyme of the distal buds. In this location, they regulate processes such as cell proliferation, branching morphogenesis, and the differentiation of specialized cell types. Here, we report that the expression of Pea3 and Erm (or Etv5, Ets variant gene 5), which encode Pea3 subfamily ETS domain transcription factors, is initially restricted to the distal buds of the developing mouse lung. Erm is transcribed exclusively in the epithelium, while Pea3 is expressed in both epithelium and mesenchyme. Erm/Pea3 are downstream of FGF signaling from the mesenchyme, but their responses toward different FGFs are not the same. The functions of the two proteins were investigated by transgenic expression of a repressor form of Erm specifically in the embryonic lung epithelium. When examined at E18.5, the distal epithelium of transgenic lungs is composed predominantly of immature type II cells, while no mature type I cells are observed. In contrast, the differentiation of proximal epithelial cells, including ciliated cells and Clara cells, appears to be unaffected. A model is proposed for the role of Pea3/Erm during the dynamic process of lung bud outgrowth and proximal-distal differentiation, in response to FGF signaling. Our results provide the first functional evidence that Pea3 subfamily members play a role in epithelial-mesenchymal interactions during lung organogenesis. Keywords: Lung; FGF; Erm; Pea3; ETS domain proteins

Hermansky-Pudlak syndrome (HPS) is a genetically heterogeneous inherited disease causing hypopigmentation and prolonged bleeding times. An additional serious clinical problem of HPS is the development of lung pathology, which may lead to severe lung disease and premature death. No cure for the disease exists, and previously, no animal model for the HPS lung abnormalities has been reported. A mouse model of HPS, which is homozygously recessive for both the Hps1 (pale ear) and Hps2 (pearl) genes, exhibits striking abnormalities of lung type II cells. Type II cells and lamellar bodies of this mutant are greatly enlarged, and the lamellar bodies are engorged with surfactant. Mutant lungs accumulate excessive autofluorescent pigment. The air spaces of mutant lungs contain age-related elevations of inflammatory cells and foamy macrophages. In vivo measurement of lung hysteresivity demonstrated aberrant lung function in mutant mice. All these features are similar to the lung pathology described in HPS patients. Morphometry of mutant lungs indicates a significant emphysema. These mutant mice provide a model to further investigate the lung pathology and therapy of HPS. We hypothesize that abnormal type II cell lamellar body structure/function may predict future lung pathology in HPS. inherited lung dysfunction; inflammation; surfactant protein; lamellar body; secretion

[Na.sup.+]-[K.sup.+]-ATPase plays an essential role in active alveolar epithelial fluid resorption. In fetal and adult alveolar epithelial cells, glucocorticoids (GC) increase [Na.sup.+]-[K.sup.+]-ATPase activity and mRNA levels. We sought to define the mechanism of [Na.sup.+]-[K.sup.+]-ATPase gene upregulation by GC. In a rat alveolar epithelial cell line (RLE), dexamethasone (Dex) increased [[beta].sup.1]-subunit [Na.sup.+]-[K.sup.+]-ATPase mRNA expression two- to threefold within 3 h after exposure to the GC. The increased gene expression was due to increased transcription as demonstrated by nuclear run-on assays, whereas mRNA stability remained unchanged. Transient transfection of 5' deletion mutants of a [[beta].sub.1] promoter-reporter construct demonstrated a 1.5- to 2.2-fold increase in promoter activity by Dex. All of the 5' deletion constructs contained partial or palindromic GC regulatory elements (GRE) and responded to GC. The increased expression of promoter reporter was inhibited by RU-486, a GC receptor (GR) antagonist, suggesting the involvement of GR. The palindromic GRE at -631 demonstrated Dex induction in a heterologous promoter construct. Gel mobility shift assays using RLE nuclear extracts demonstrated specific binding to this site and the presence of GR. We conclude that GC directly stimulate transcription of [Na.sup.+]-[K.sup.+]-ATPase [[beta].sub.1] gene expression in adult rat lung epithelial cells through a GR-dependent mechanism that can act at multiple sites. sodium pump; glucocorticoid receptor; glucocorticoid response element; promoter; alveolar fluid; ion transport; type II cells

Impaired lung function in severe acute pancreatitis is the primary cause of morbidity and mortality in this condition. Preprotachykinin-A (PPT-A) gene products substance P and neurokinin (NK)-A have been shown to play important roles in neurogenic inflammation. Substance P acts primarily (but not exclusively) via the NK1 receptor. NKA acts primarily via the NK2 receptor. Earlier work has shown that knockout mice deficient in NK1 receptors are protected against acute pancreatitis and associated lung injury. NK1 receptors, however, bind other peptides in addition to substance P, not all of which are derived from the PPT-A gene. To examine the role of PPT-A gene products in acute pancreatitis, the effect of PPT-A gene deletion on the severity of acute pancreatitis and the associated lung injury was investigated. Deletion of PPT-A almost completely protected against acute pancreatitis-associated lung injury, with a partial protection against local pancreatic damage. These results show that PPT-A gene products are critical proinflammatory mediators in acute pancreatitis and the associated lung injury. multiple organ dysfunction syndrome; caerulein; substance P; neurogenic inflammation; systemic inflammatory response syndrome

T1[alpha], 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 T1[alpha] is expressed in the foregut endoderm before the lung buds, T1[alpha] mRNA and protein levels increase substantially in late fetuses when expression is restricted to alveolar type I cells. We generated T1[alpha] null mutant mice to study the role of T1[alpha] 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 T1[alpha] 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 T1[alpha] and/or abnormal morphogenesis of type I cells alter the proliferation rate of distal lung cells, probably by disruption of epithelial-mesenchymal signaling. Keywords: Lung; Development; Epithelium; Alveoli; Type I cell; Knockout; Differentiation; Proliferation

Transgenic mice expressing platelet-derived growth factor A chain (PDGF-A) in the distal lung epithelium from the surfactant protein C (SPC) promoter were generated to investigate the role of this growth factor in lung development. Expression of the SPC-PDGFA transgene resulted in an enlarged, nonfunctional lung and perinatal lethality caused by failure to initiate ventilation. Histologic analysis of embryonic day (E) 16.5 lungs revealed increased mesenchymal cells and acinar buds and decreased bronchioles and dilated airspaces in SPC-PDGFA transgenic mice. At E18.5, nontransgenic lungs exhibited lung morphology typical of the saccular stage of lung development, including dilated airspaces, thin respiratory epithelium and mesenchyme, and elastin fiber deposition in primary septa. In contrast, E18.5 transgenic lungs retained many features of the canalicular stage of lung development, including undilated airspaces, cuboidal respiratory epithelium, thickened mesenchyme, and lack of parenchymal elastin deposition. These results indicate that PDGF-A is a potent growth factor for mesenchymal cells in the developing lung and that the downregulation of PDGF-A expression that normally occurs in the lung during late gestation is required for transition from the canalicular to the saccular stage of lung development.

The transcriptional repressor, CCAAT displacement protein (CDP) (Cutl1), has been found to be necessary for epithelial cell differentiation of the hair follicle and the lung. The C-terminal Cut repeat 3 and homeodomain exons were replaced with an in-frame lacZ gene by targeted mutagenesis in mice for the study.

Molecular regulation of lung development is discussed. Evidence suggesting that formation of the tracheobronchial tree and alveoli comes from heterogeneity of the epithelial-mesenchymal interactions is growing in volume. Various pattern regulators and how they may be involved in initiation, branching and differentiation of the respiratory system are considered. They include signaling molecules along the proximal--distal axis, with prominent roles apparently played by fibroblast growth factors, Sonic hedgehog, transforming growth factors and retinoids.

The Na+ channel and Na+-K+-ATPase are essential for the perinatal removal of alveolar solute and fluid. It was hypothesized that maternal glucocorticoids (GCs) increased Na+-K+-ATPase gene expression in a fetal lung epithelial cell line because Na+-pump mRNA and activity increase before birth. The results showed that GCs increased Na+-K+-ATPase transcription in a fetal lung epithelial cell line. The findings suggest important therapeutic implications, one of which is optimizing treatment of premature infants by maternal GCs.

A purine-box transcriptional regulator complex which exists in the nucleus of 16HBE (human bronchial epithelial) cells has been studied. These cells are located at the interface between the host and the external environment and regulate the airway inflammatory response to noxious stimuli and pathogens. The purine-box regulator binds with its target DNA sequence in nonstimulated 16HBE cells and is transcriptionally silent. Stimulated 16HBE cells have greater purine-box DNA-binding activity, induces transcription and triggers IL-2 protein secretion.

A study was conducted to examine the action of glucocorticoid on glucocorticoid receptor (GR) mRNA expression and protein elaboration in rat lung cells. An area of rat GR cDNA open reading frame was excised between the Xba I and Pst I restriction sites of the pRdN93 construct while rats were injected daily with the solvent dimethyl sulfoxide. Results indicated that glucocorticoids and androgens support opposite effects on fetal lung GR.

A study was conducted to examine the mechanism associated with the secretagogue-induced surfactant protein A (SP-A) binding to rat lung epithelial cells. Type II cels were isolated from Sprague-Dawley rats after perfusion through the pulmonery artery. The cells were then plated on 24-mm inserts of microporous membranes. Results indicated that secretagogues influence SP-A binding sites through the recruitment of receptors to the cell surface.

A study was conducted to characterize genes associated in the regulation of rat lung cell proliferation during the early postpneumonectomy interval. Reverse transcriptase polymerase chain reaction (PCR) was carried out with 200 ng of total RNA as the template and oligonucleotide as a primer. A series of experiments were also performed to obtain the optimal annealing temperatures for the PCR reactions. Results indicated various expression of zinc finger genes after pneumonectomy.

A study was conducted to examine the functions of mechanical signals in the induction of immediate-early gene (IEG) expression after pneumonectomy. Total RNA was determined from whole lung tissue with the TriReagent equipment while RNA samples were denatured and size fractionated on 1.2% agarose gels supporting 0.4M formaldehyde. Results suggested that elevated IEG expression may support the initiation of commpensatory lung growth.

Research was conducted to test whether the platelet-derived growth factor receptor-alpha in rat myofibroblasts is influenced in vivo during pulmonary fibrogenesis. Bronchoalveolar lavage was utilized to determine alveolar macrophages from Sprague-Dawley rats while rat lung fibroblasts were seeded in plates and grown to confluence. Results suggested that interleukin-1-beta-mediated induction of PDGF-R-alpha in vivo is important to lung myofibroblast hyperplasia during fibrogenesis.

Research was conducted to study the ontogeny and cellular localization of cyclooxygenase-1 (COX) and COX-2 gene expression in lungs from lambs. Lung tissues were analyzed from three groups of mixed-breed lambs while mean values were compared using analysis of variance with Newman-Keuls post hoc testing. Results indicated the delineation of the ontogeny of COX gene expression in the lungs of fetal and newborn sheep. They also suggested that COX-1 gene expression may also be under hormonal influence.

Research was conducted to study the relationship between dexamethasone (Dex), vitamin A and lung development by examining the effects of retinoic acid (RA) and Dex on retinoic acid receptor and surfactant protein-C gene expression. Fetal lung explants and lung adenocarcinoma cells were utilized to examine the effect of RA while cells were treated with Dex or trans-RA that were dissolved in dimethyl sulfoxide. Results indicated that the effect of RA and Dex on each specific mRNA may involve separate promoter regions in the gene with separate transcription factors.

Research shows that the genetic expression of matrix gla protein (MGP) in early embryonic mouse lung culture is more pronounced in the presence of exogenous transforming growth factor-beta1 (TGF-beta1). MGP messenger ribonucleic acid expression under the influence of TGF-beta1 changes over time and in varying concentrations of TGF-beta1. Experimental evidence suggests that MGP may play a crucial role in lung morphogenesis, possibly via endogenous TGF-beta signaling.

The effects of retinoic acid on the stability of fatty-acid synthase messenger RNA (mRNA) were analyzed in cultured fetal rat lungs after prolonged incubation in a dexamethasone-containing medium. Northern blot analysis of the extracted RNA from dexamethasone-incubated cells indicated the presence of enhanced FAS mRNA stability compared to cells that were not cultured in dexamethasone. Furthermore, the hormone induced the transcription of FAS gene in dexamethasone-cultured explants of fetal rat lungs.

The expression of gamma-glutamyltransferase (gamma-GT) in bronchiolar Clara cells and alveolar type II cells was analyzed to determine the ontogeny of the single-copy gene in fetal and early postnatal rat lungs. Analysis of gamma-GT ontogeny in bronchiolar Clara cells and alveolar type II cells indicated the developmental regulation of the gene. Furthermore, gamma-GT was produced exclusively in alveolar type II cells of perinatal rat lungs.

A novel reverse-transcriptase polymerase-chain reaction (RT-PCR) technique was utilized for the quantitative analysis of airway mucins such as MUC2 and MUC5AC in normal human tracheobronchial epithelial cells (NHTBE). MUC2 and MUC5Ac were isolated by amplification of ribonucleic acid (RNA) from NHTBE cultures. Analysis of mucin gene expression in NHTBE cells indicated an increase in MUC2 and MUC5AC levels after treatment with retinoic acid or retinol due to a 50-fold increase in mucin messenger RNA levels.

The role of fibronectins (FN) in tissue repair during alveolar injury was analyzed in rabbit fetal lung, normal adult lung and adult lung after oxidative stress. The type III FN splice variant, EIIIB and EIIA exons, were detected in rabbit fetal, normal adult lung and adult lung exposed to oxidative damage. Recovery from alveolar injury due to oxidative stress is also characterized by increased EIIIB messenger ribonucleic acid expression. Furthermore, alveolar cells treated with transforming growth factors exhibited increased expression of FN EIIIA and EIIIB splice variants.