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    The Effects of Antenatal Corticosteroids on Short- and Long-Term Outcomes in Small-for-Gestational-Age Infants. Ishikawa Hiroshi,Miyazaki Ken,Ikeda Tomoaki,Murabayashi Nao,Hayashi Kazutoshi,Kai Akihiko,Ishikawa Kaoru,Miyamoto Yoshihiro,Nishimura Kunihiro,Kono Yumi,Kusuda Satoshi,Fujimura Masanori, International journal of medical sciences AIM:To evaluate the effect of antenatal corticosteroids (ANS) on short- and long-term outcomes in small-for-gestational age (SGA) infants. METHODS:A retrospective database analysis was performed. A total of 1,931 single infants (birth weight <1,500 g) born at a gestational age between 22 weeks and 33 weeks 6 days who were determined to be SGA registered in the Neonatal Research Network Database in Japan between 2003 and 2007 were evaluated for short-term outcome and long-term outcome. RESULTS:ANS was administered to a total of 719 infants (37%) in the short-term outcome evaluation group and 344 infants (36%) in the long-term outcome evaluation group. There were no significant differences between the ANS group and the no-ANS group for primary short-term outcome (adjusted odds ratio (OR) 0.73; 95% confidence interval (CI) 0.45-1.20; P-value 0.22) or primary long-term outcome (adjusted OR 0.69; 95% CI 0.40-1.17; P-value 0.17). CONCLUSIONS:Our results show that ANS does not affect short- or long-term outcome in SGA infants when the birth weight is less than 1500 g. This study strongly suggests that administration of ANS resulted in few benefits for preterm FGR fetuses. 10.7150/ijms.11523
    The biguanide metformin alters phosphoproteomic profiling in mouse brain. Khang Rin,Park ChiHu,Shin Joo-Ho Neuroscience letters Metformin, a potent antihyperglycemic agent is recommended as the first-line oral therapy for type 2 diabetes (T2D). Recently, metformin has been reported to be beneficial to neurodegenerative disease models. However, the putative mechanisms underlying the neuroprotective effects of metformin in disease models are unknown. Thus, we applied LC-MS/MS-based pattern analysis and two-dimensional electrophoresis (2DE)-based proteomic approach to understand the global phosphoproteomic alteration in the brain of metformin-administrated mice. Collectively, LC-MS/MS-based pattern analysis reveals that 41 phosphoproteins were upregulated and 22 phosphoproteins were downregulated in the brain of metformin-administrated mice. In addition, 5 differentially expressed phosphoproteins were identified upon metformin administration by 2DE coupled with mass spectrometry. The phosphorylation status of metabolic enzymes was decreased while that of mitochondrial proteins was increased by metformin. Interestingly, phosphorylated α-synuclein was significantly decreased by metformin administration. Taken together, our results might provide potential pathways to understand the pharmacological effect of metformin on neuroprotection. 10.1016/j.neulet.2014.07.029
    Genomic determinants implicated in the glucocorticoid-mediated induction of KLF9 in pulmonary epithelial cells. Mostafa Mahmoud M,Bansal Akanksha,Michi Aubrey N,Sasse Sarah K,Proud David,Gerber Anthony N,Newton Robert The Journal of biological chemistry Ligand-activated glucocorticoid receptor (GR) elicits variable glucocorticoid-modulated transcriptomes in different cell types. However, some genes, including Krüppel-like factor 9 (KLF9), a putative transcriptional repressor, demonstrate conserved responses. We show that glucocorticoids induce KLF9 expression in the human airways in vivo and in differentiated human bronchial epithelial (HBE) cells grown at air-liquid interface (ALI). In A549 and BEAS-2B pulmonary epithelial cells, glucocorticoids induce KLF9 expression with similar kinetics to primary HBE cells in submersion culture. A549 and BEAS-2B ChIP-seq data reveal four common glucocorticoid-induced GR binding sites (GBSs). Two GBSs mapped to the 5'-proximal region relative to KLF9 transcription start site (TSS) and two occurred at distal sites. These were all confirmed in primary HBE cells. Global run-on (GRO) sequencing indicated robust enhancer RNA (eRNA) production from three of these GBSs in BEAS-2B cells. This was confirmed in A549 cells, plus submersion, and ALI culture of HBE cells. Cloning each GBS into luciferase reporters revealed glucocorticoid-induced activity requiring a glucocorticoid response element (GRE) within each distal GBS. While the proximal GBSs drove modest reporter induction by glucocorticoids, this region exhibited basal eRNA production, RNA polymerase II enrichment, and looping to the TSS, plausibly underlying constitutive KLF9 expression. Post glucocorticoid treatment, interactions between distal and proximal GBSs and the TSS correlated with KLF9 induction. CBP/P300 silencing reduced proximal GBS activity, but negligibly affected KLF9 expression. Overall, a model for glucocorticoid-mediated regulation of KLF9 involving multiple GBSs is depicted. This work unequivocally demonstrates that mechanistic insights gained from cell lines can translate to physiologically relevant systems. 10.1074/jbc.RA120.015755
    Glucocorticoid signalling drives reduced versican levels in the fetal mouse lung. Short Kelly L,Bird A Daniel,Seow Bennet K L,Ng Judy,McDougall Annie R A,Wallace Megan J,Hooper Stuart B,Cole Timothy J Journal of molecular endocrinology Glucocorticoid (GC) signaling via the glucocorticoid receptor (GR) is essential for lung maturation in mammals. Previous studies using global or conditional mouse model knockouts of the GR gene have established that GR-mediated signaling in the interstitial mesenchyme of the fetal lung is critical for normal lung development. Screens for downstream GC-targets in conditional mesenchymal GR deficient mouse lung (GRmesKO) identified Versican (Vcan), an important extracellular matrix component and cell proliferation regulator, as a potential GR-regulated target. We show that, of the five major VCAN isoforms, the VCAN-V1 isoform containing the GAGβ domain is the predominant VCAN isoform in the fetal mouse lung distal mesenchyme at both E16.5 and E18.5, whereas the GAGα-specific VCAN-V2 isoform was only localized to the smooth muscle surrounding proximal airways. Both Vcan-V1 mRNA and protein levels were strongly overexpressed in the GRmesKO lung at E18.5. Finally, we investigated the GC regulation of the ECM protease ADAMTS 12 and showed that Adamts 12 mRNA levels were markedly reduced at E18.5 in GRmesKO fetal mouse lung and were strongly induced by both cortisol and betamethasone in cultures of primary rat fetal lung fibroblasts. ADAMTS12 protein immunoreactivity was also strongly increased in the distal lung at E18.5, after dexamethasone treatment in utero. In summary, glucocorticoid signaling via GR represses GAGβ domain-containing VCAN isoforms in distal lung mesenchyme in vivo by repressing Vcan gene expression and, in part, by inducing the ECM protease ADAMTS12, thereby contributing to the control of ECM remodelling and lung cell proliferation prior to birth. 10.1530/JME-19-0235
    Glucocorticoid regulates mesenchymal cell differentiation required for perinatal lung morphogenesis and function. Bridges James P,Sudha Parvathi,Lipps Dakota,Wagner Andrew,Guo Minzhe,Du Yina,Brown Kari,Filuta Alyssa,Kitzmiller Joseph,Stockman Courtney,Chen Xiaoting,Weirauch Matthew T,Jobe Alan H,Whitsett Jeffrey A,Xu Yan American journal of physiology. Lung cellular and molecular physiology While antenatal glucocorticoids are widely used to enhance lung function in preterm infants, cellular and molecular mechanisms by which glucocorticoid receptor (GR) signaling influences lung maturation remain poorly understood. Deletion of the glucocorticoid receptor gene () from fetal pulmonary mesenchymal cells phenocopied defects caused by global deletion, while lung epithelial- or endothelial-specific deletion did not impair lung function at birth. We integrated genome-wide gene expression profiling, ATAC-seq, and single cell RNA-seq data in mice in which GR was deleted or activated to identify the cellular and molecular mechanisms by which glucocorticoids control prenatal lung maturation. GR enhanced differentiation of a newly defined proliferative mesenchymal progenitor cell (PMP) into matrix fibroblasts (MFBs), in part by directly activating extracellular matrix-associated target genes, including , , and and by modulating VEGF, JAK-STAT, and WNT signaling. Loss of mesenchymal GR signaling blocked fibroblast progenitor differentiation into mature MFBs, which in turn increased proliferation of SOX9+ alveolar epithelial progenitor cells and inhibited differentiation of mature alveolar type II (AT) and AT cells. GR signaling controls genes required for differentiation of a subset of proliferative mesenchymal progenitors into matrix fibroblasts, in turn, regulating signals controlling AT/AT progenitor cell proliferation and differentiation and identifying cells and processes by which glucocorticoid signaling regulates fetal lung maturation. 10.1152/ajplung.00459.2019
    Levels of surfactant-associated protein messenger ribonucleic acids in rabbit lung during perinatal development and after hormonal treatment. Connelly I H,Hammond G L,Harding P G,Possmayer F Endocrinology The levels of messenger RNAs for the surfactant-associated proteins, SP-A, SP-B, and SP-C, have been examined in the developing rabbit lung in vivo. Northern blot analysis detected SP-C mRNA by day 22 of gestation (term 31 days) and SP-A mRNA and SP-B mRNA on day 26, while solution hybridization assays detected all three mRNAs on day 22 of gestation. Both techniques revealed that the mRNA levels increased rapidly during the last quarter of gestation. The mRNA levels determined by solution hybridization were highly correlated during development, with average molar ratios of 1.0:1.1:2.1 for SP-A, SP-B, and SP-C, respectively. We also examined the effect of accelerating fetal pulmonary maturation by maternal administration of either 17 beta-estradiol or betamethasone (9 alpha-fluoro-16 beta-methylprednisolone) on day 26 of gestation. These treatments increased SP-A mRNA levels 8- to 12-fold, resulting in levels 3- to 4-fold greater than in the adult. SP-B mRNA levels increased by approximately 2-fold to near adult levels, while SP-C mRNA was lowered somewhat by 17 beta-estradiol and significantly to less than half by betamethasone. No differences in the levels of surfactant apoprotein mRNAs or in choline incorporation into total or disaturated phosphatidylcholine were noted between male and female fetuses. These observations are consistent with the accepted view that the genes for the surfactant-associated proteins are independently regulated. However, the various factors affecting these mRNAs result in a coordination of mRNA levels during normal perinatal development. 10.1210/endo-129-5-2583
    TGF-beta1 inhibits surfactant component expression and epithelial cell maturation in cultured human fetal lung. Beers M F,Solarin K O,Guttentag S H,Rosenbloom J,Kormilli A,Gonzales L W,Ballard P L The American journal of physiology Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine shown to play a critical role in organ morphogenesis, development, growth regulation, cellular differentiation, gene expression, and tissue remodeling after injury. We examined the effect of exogenously administered TGF-beta1 on the expression of surfactant proteins (SPs) and lipids, fatty acid synthetase, and ultrastructural morphology in human fetal lung cultured for 5 days with and without dexamethasone (10 nM). Expression of the type II cell-specific marker surfactant proprotein C (proSP-C), studied by [35S]Met incorporation and immunoprecipitation, increased sevenfold with dexamethasone treatment. TGF-beta1 (0.1-100 ng/ml) in the presence of dexamethasone inhibited 21-kDa proSP-C expression in a dose-dependent manner (maximal inhibition 31% of control level at 100 ng/ml). There was no change in [35S]Met incorporation into total protein in any of the treatment groups vs. the control group. In immunoblotting experiments, TGF-beta1 blocked culture-induced accumulation of SP-A and SP-B. Under the same conditions, TGF-beta1 reduced mRNA content for SP-A, SP-B, and SP-C to 20, 38, and 41%, respectively, of matched control groups but did not affect levels of beta-actin mRNA. SP transcription rates after 24 h of exposure to TGF-beta1 were reduced to a similar extent (20-50% of control level). In both control and dexamethasone-treated explants, TGF-beta1 (10 ng/ml) also decreased fatty acid synthetase mRNA, protein, and enzyme activity and the rate of [3H]choline incorporation into phosphatidylcholine. By electron microscopy, well-differentiated type II cells lining potential air spaces were present in explants cultured with dexamethasone, whereas exposure to TGF-beta1 with or without dexamethasone resulted in epithelial cells lacking lamellar bodies. We conclude that exogenous TGF-beta1 disrupts culture-induced maturation of fetal lung epithelial cells and inhibits expression of surfactant components through effects on gene transcription. 10.1152/ajplung.1998.275.5.L950
    Direct effects of corticotropin-releasing hormone and thyrotropin-releasing hormone on fetal lung explants. Emanuel R L,Torday J S,Asokananthan N,Sunday M E Peptides Fetal lung produces corticotropin-releasing hormone (CRH) without known direct effects. We tested the hypothesis that CRH can directly regulate lung development. In baboon fetal lung explants, CRH strongly induces surfactant phospholipid synthesis and SP-C immunostaining, plus [(3)H]thymidine incorporation. CRH receptor mRNA was detected in lung from multiple baboons at e125. Testing thyrotropin (TRH) as a specificity control, we did demonstrate different direct effects with only modest stimulation of surfactant phospholipid synthesis and strong induction of cytidylyltransferase gene expression. Therefore, CRH, similar to ACTH and glucocorticoids, is a potent inducer of cell differentiation in fetal lung.
    Regulation of surfactant proteins A and B by TNF-alpha and phorbol ester independent of NF-kappa B. Pryhuber G S,Khalak R,Zhao Q The American journal of physiology Acute lung inflammation is complicated by altered pulmonary surfactant phospholipid and protein composition. The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) inhibit expression of surfactant-associated proteins A and B (SP-A and SP-B), both important for normal surfactant function. The transcription factor nuclear factor-kappa B (NF-kappa B) frequently mediates regulation of gene expression by TPA and TNF-alpha. In the present study, electrophoretic mobility shift assays (EM-SAs) and pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappa B activation, were utilized to determine the role of NF-kappa B activation in TPA and TNF-alpha inhibition of the surfactant proteins in NCI-H441 cells. Pentoxifylline (PTX), which inhibits TNF-alpha cellular effects without preventing NF-kappa B activation, was also tested. By EMSA, TPA and TNF-alpha increased nuclear NF-kappa B binding activity in temporally distinct patterns. PDTC decreased TPA- and TNF-alpha-induced NF-kappa B binding activity but did not limit their inhibition of SP-A and SP-B mRNAs. PDTC independently decreased both SP-A and SP-B mRNAs. PTX partially reversed TNF-alpha-but not TPA-mediated inhibition of SP-A and SP-B mRNAs without altering NF-kappa B binding. The effects of PDTC and PTX on NF-kappa B and the surfactant proteins suggest that NF-kappa B activation does not mediate TPA or TNF-alpha inhibition of SP-A and SP-B mRNA accumulation. 10.1152/ajplung.1998.274.2.L289
    The combined effects of insulin and cortisol on surfactant protein mRNA levels. Dekowski S A,Snyder J M Pediatric research Infants of diabetic mothers are frequently hyperinsulinemic and have an increased incidence of neonatal respiratory distress syndrome, a disease caused by a deficiency in the production of pulmonary surfactant by alveolar type II cells. It has been hypothesized that insulin inhibits fetal lung type II cell differentiation. We have shown previously that insulin inhibits the accumulation of surfactant protein (SP)-A and SP-B mRNA and has no effect on SP-C mRNA levels in human fetal lung tissue maintained in vitro. We hypothesized that treatment with glucocorticoids, which are used clinically to accelerate human fetal lung maturation, would overcome the inhibitory effects of insulin on human fetal lung development. In the present study, human fetal lung explants were maintained in the presence or absence of cortisol added alone, or in insulin plus cortisol added together. Cortisol significantly decreased SP-A mRNA levels by approximately 50% at the 100 nM concentration and significantly increased levels by approximately 20% at the 1 nM concentration. Cortisol increased SP-B and SP-C mRNA levels in a dose-dependent fashion (5- and 45-fold at 100 nM cortisol, respectively). The combination of 1 nM cortisol and insulin resulted in inhibition of mRNA levels for SP-A, SP-B, and SP-C at the high insulin concentrations (approximately 50% inhibition for SP-A and SP-B and approximately 25% inhibition of SP-C mRNA levels, in the presence of 40 pmol/L x 10(-3) insulin). Surprisingly, 100 nM cortisol plus inhibitory concentrations of insulin increased SP-A mRNA levels (2-fold at 40 pmol/L x 10(-3).(ABSTRACT TRUNCATED AT 250 WORDS) 10.1203/00006450-199510000-00007
    Human surfactant proteins A1 and A2 are differentially regulated during development and by soluble factors. Scavo L M,Ertsey R,Gao B Q The American journal of physiology An RT-PCR method for the relative quantitation of the mRNAs for human surfactant protein (SP) A1 and SP-A2 was developed, verified, and then utilized to determine the relative levels of these mRNAs in fetal and adult lung samples in vivo, as well as in cultured human fetal lung explants and H441 cells. For the cultured tissue and cells, we assessed the effects of a variety of soluble factors known to modulate total SP-A. Comprehensive analysis revealed many significant findings, including the following: both mRNAs were expressed as early as 15 wk of gestation; throughout midgestation, SP-A1 was present at higher levels than SP-A2, with an average ratio of 30:1. In the adult lung, SP-A1 mRNA was present at lower levels than SP-A2, with a ratio of 0.4:1, whereas in H441 cells, the ratio was 0.85:1. In fetal lung cultured for 4 days, both mRNAs increased, with a greater increase in SP-A2 (97-fold) than in SP-A1 (15-fold), resulting in a final ratio of 4:1. Differential regulation was demonstrated for 8-(4-chlorophenylthio)-cAMP, interferon (IFN)-gamma, tumor necrosis factor-alpha, and transforming growth factor (TGF)-beta in the human fetal lung explant system, with SP-A2 being more affected, and for IFN-gamma and TGF-beta in the H441 cells, where SP-A1 showed greater regulation. Of the soluble factors tested, IFN-gamma and TGF-beta had the most potent and consistent effects in both systems. 10.1152/ajplung.1998.275.4.L653
    Lipidomics of cellular and secreted phospholipids from differentiated human fetal type II alveolar epithelial cells. Postle Anthony D,Gonzales Linda W,Bernhard Wolfgang,Clark Graeme T,Godinez Marye H,Godinez Rodolfo I,Ballard Philip L Journal of lipid research Maturation of fetal alveolar type II epithelial cells in utero is characterized by specific changes to lung surfactant phospholipids. Here, we quantified the effects of hormonal differentiation in vitro on the molecular specificity of cellular and secreted phospholipids from human fetal type II epithelial cells using electrospray ionization mass spectrometry. Differentiation, assessed by morphology and changes in gene expression, was accompanied by restricted and specific modifications to cell phospholipids, principally enrichments of shorter chain species of phosphatidylcholine (PC) and phosphatidylinositol, that were not observed in fetal lung fibroblasts. Treatment of differentiated epithelial cells with secretagogues stimulated the secretion of functional surfactant-containing surfactant proteins B and C (SP-B and SP-C). Secreted material was further enriched in this same set of phospholipid species but was characterized by increased contents of short-chain monounsaturated and disaturated species other than dipalmitoyl PC (PC16:0/16:0), principally palmitoylmyristoyl PC (PC16:0/14:0) and palmitoylpalmitoleoyl PC (PC16:0/16:1). Mixtures of these PC molecular species, phosphatidylglycerol, and SP-B and SP-C were functionally active and rapidly generated low surface tension on compression in a pulsating bubble surfactometer. These results suggest that hormonally differentiated human fetal type II cells do not select the molecular composition of surfactant phospholipid on the basis of saturation but, more likely, on the basis of acyl chain length. 10.1194/jlr.M600054-JLR200
    Immunoglobulin therapy ameliorates the phenotype and increases lifespan in the severely affected dystrophin-utrophin double knockout mice. Nunes Bruno Ghirotto,Loures Flávio Vieira,Bueno Heloisa Maria Siqueira,Cangussu Erica Baroni,Goulart Ernesto,Coatti Giuliana Castello,Caldini Elia Garcia,Condino-Neto Antonio,Zatz Mayana European journal of human genetics : EJHG Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder, caused by mutations in the dystrophin gene, affecting 1:3500-5000 boys worldwide. The lack of dystrophin induces degeneration of muscle cells and elicits an immune response characterized by an intensive secretion of pro-inflammatory cytokines. Immunoglobulins modulate the inflammatory response through several mechanisms and have been widely used as an adjuvant therapy for autoimmune diseases. Here we evaluated the effect of immunoglobulin G (IG) injected intraperitoneally in a severely affected double knockout (dko) mouse model for Duchenne muscular dystrophy. The IG dko treated mice were compared regarding activity rates, survival and histopathology with a control untreated group. Additionally, dendritic cells and naïve lymphocytes from these two groups and WT mice were obtained to study in vitro the role of the immune system associated to DMD pathophysiology. We show that IG therapy significantly enhances activity rate and lifespan of dko mice. It diminishes muscle tissue inflammation by decreasing the expression of costimulatory molecules MHC, CD86 and CD40 and reducing Th1-related cytokines IFN-γ, IL-1β and TNF-α release. IG therapy dampens the effector immune responses supporting the hypothesis according to which the immune response accelerates DMD progression. As IG therapy is already approved by FDA for treating autoimmune disorders, with less side-effects than currently used glucocorticoids, our results may open a new therapeutic option aiming to improve life quality and lifespan of DMD patients. 10.1038/s41431-017-0017-y
    Surfactant apoprotein in adult rat lung compartments is increased by dexamethasone. Young S L,Ho Y S,Silbajoris R A The American journal of physiology The distribution of the major surfactant apoprotein (SP-A) in adult rat lung was determined in order to gain insight into its metabolism, including packaging of SP-A into lamellar bodies. The effect of glucocorticoid treatment on surfactant apoprotein was studied to test whether regulation of surfactant apoprotein genes, which has been described for the fetal lung, can be demonstrated in the adult animal. We measured the amounts of immunoreactive SP-A in several lung tissue compartments and lavage fractions from control animals and from the lungs of rats given dexamethasone for 1 wk. Protein and phospholipids were measured, SP-A was quantitated with a noncompetitive enzyme-linked immunoabsorbent assay (ELISA) and SP-A, SP-B, and SP-C mRNAs were estimated by Northern blotting. We found an 85-fold concentration of SP-A in a lamellar body-rich fraction compared with lung tissue homogenate and we calculated that as much as one-half of all the tissue SP-A might be accounted for by a lamellar body pool. After 1 wk of dexamethasone treatment, there was an increase in adult rat lung SP-A, SP-B, and SP-C mRNA and a substantial increase in tissue and lavage fluid immunoreactive SP-A pools. Lamellar body fraction SP-A content per lung was 1.4-fold higher after dexamethasone, and there was a fivefold increase in the lavage SP-A pool, much of which was inseparable from the alveolar macrophages. We conclude that SP-A is concentrated in the lamellar bodies of type II cells, that dexamethasone treatment increased all surfactant mRNAs, and that it increased SP-A content in adult rat lung. 10.1152/ajplung.1991.260.2.L161
    Primary cell culture of human type II pneumonocytes: maintenance of a differentiated phenotype and transfection with recombinant adenoviruses. Alcorn J L,Smith M E,Smith J F,Margraf L R,Mendelson C R American journal of respiratory cell and molecular biology Studies of the regulation of surfactant lipoprotein metabolism and secretion and surfactant protein gene expression have been hampered by the lack of a cell culture system in which the phenotypic properties of type II cells are maintained. We have developed a primary culture system that facilitates the maintenance of a number of morphologic and biochemical properties of type II pneumonocytes for up to 2 wk. Cells were isolated by collagenase digestion of midgestation human fetal lung tissue that had been maintained in organ culture in the presence of dibutyryl cyclic AMP (Bt2cAMP) for 5 days. The isolated cells were enriched for epithelial components by treatment with DEAE-dextran, plated on an extracellular matrix (ECM) derived from Madin-Darby canine kidney (MDCK) cells, and incubated at an air/liquid interface in a minimal amount of culture medium containing Bt2cAMP. The cell cultures were comprised of islands of round epithelial-like cells containing numerous dense osmiophilic granules, surrounded by sparse spindle-shaped cells with the appearance of fibroblasts. Ultrastructural examination revealed that the osmiophilic granules had the appearance of lamellar bodies, the distinguishing feature of type II pneumonocytes. Additionally, the cultures maintained elevated levels of SP-A gene expression for up to 2 wk. The expression of mRNAs encoding SP-A, SP-B, and SP-C were regulated in the cultured cells by glucocorticoids and cyclic AMP in a manner similar to that observed in fetal lung tissue in organ culture. The differentiated phenotype was most apparent when the cells were cultured at an air/liquid interface. In order to utilize the cultured type II cells for study of the effects of overexpression of various proteins and for promoter analysis, it is of essence to transfect DNA constructs into these cells with high efficiency. Unfortunately, we found the cells to be refractory to efficient transfer of DNA using conventional methods (i.e., lipofection, electroporation, or calcium phosphate-mediated transfection). However, replication-defective recombinant human adenoviruses were found to provide a highly efficient means of introducing DNA into the type II pneumonocytes. Furthermore, we observed in type II cell-enriched cultures infected with recombinant adenoviruses containing the lacZ gene under control of a cytomegalovirus promoter, that beta-galactosidase was expressed uniformly in the islands of type II cells and surrounding fibroblasts. By contrast, in cultures infected with recombinant adenoviruses containing the human growth hormone (hGH) gene under control of the SP-A gene promoter and 5'-flanking region, hGH was expressed only in the type II cells. Thus, this culture system provides an excellent means for identifying genomic elements that mediate type II cell-specific gene expression. 10.1165/ajrcmb.17.6.2858
    Regulation of surfactant protein D in human fetal lung. Dulkerian S J,Gonzales L W,Ning Y,Ballard P L American journal of respiratory cell and molecular biology Surfactant protein D (SP-D) is a collagenous glycoprotein, produced by lung type II cells, that has structural and functional similarities with SP-A. In this study we postulated that SP-D and SP-A gene expression are regulated in a similar fashion to provide a coordinated local immune defense response to pulmonary infection. We determined content of SP-D protein and mRNA in second-trimester fetal lung and in postnatal tissue by protein blotting and hybridization analyses. Low levels of SP-D mRNA and protein were detected at 16 wk gestation, before appearance of SP-A, and levels increased during gestation. The content of SP-D did not change during 5 days of explant culture, whereas SP-A increased manyfold. Dexamethasone treatment during culture increased SP-D mRNA and protein about 2-fold with maximal response after 1 to 3 days' exposure to 100 nM steroid; under the same conditions SP-A mRNA content is inhibited. There was no significant change in SP-D mRNA after treatment of explants with adenosine 3',5'-monophosphate (cAMP) analog or interferon-gamma, agents which increase SP-A gene expression, nor after exposure to phorbol ester, tumor necrosis factor-alpha, or lipopolysaccharide at concentrations that reduced levels of SP-A mRNA by approximately 50%. We conclude that SP-D in the human lung is under developmental and glucocorticoid regulation occurring at a pretranslational level. SP-D is not influenced by inflammatory mediators that regulate SP-A, suggesting that these two proteins are not coordinately regulated in response to lung infection. 10.1165/ajrcmb.15.6.8969273
    Lung fibroblasts improve differentiation of rat type II cells in primary culture. Shannon J M,Pan T,Nielsen L D,Edeen K E,Mason R J American journal of respiratory cell and molecular biology Epithelial-mesenchymal interactions mediate prenatal lung morphogenesis and differentiation, yet little is known about their effects in the adult. In this study we have examined the influence of cocultured lung fibroblasts on rat alveolar type II cell differentiation in primary culture. Type II cells that were co-cultured with lung fibroblasts showed significant increases in messenger RNA (mRNA) levels of surfactant protein (SP)-A, SP-B, SP-C, and SP-D. Metabolic labeling and immunohistochemistry demonstrated that these mRNAs were translated and processed. Addition of 10(-7) M dexamethasone (DEX) to cocultures antagonized the effects of the fibroblasts on SP-A and SP-C, but significantly augmented the effects on SP-B; expression of SP-D was unaffected. Coculture of type II cells with lung fibroblasts also increased acetate incorporation into phospholipids 10-fold, which was antagonized by DEX. Keratinocyte growth factor (KGF) mimicked the effects of lung fibroblasts on SP gene expression, but KGF neutralizing antibodies only partially reduced the effects of lung fibroblasts. KGF increased acetate incorporation into surfactant phospholipids, and the addition of DEX augmented this response. Together, our observations suggest that epithelial--mesenchymal interactions affect type II cell differentiation in the adult lung, and that these effects are partially mediated by KGF. 10.1165/ajrcmb.24.3.4302
    Protection of insulin-producing cells against toxicity of dexamethasone by catalase overexpression. Roma Leticia P,Bosqueiro Jose R,Cunha Daniel A,Carneiro Everardo M,Gurgul-Convey Ewa,Lenzen Sigurd,Boschero Antonio C,Souza Kleber L A Free radical biology & medicine Pancreatic beta cells are very sensitive to reactive oxygen species (ROS) and this might play an important role in beta cell death in diabetes. Dexamethasone is a synthetic diabetogenic glucocorticoid, which impairs pancreatic beta cell function. Therefore we investigated the toxicity of dexamethasone in RINm5F insulin-producing cells and its dependence on the expression level of the antioxidant enzyme catalase, which inactivates hydrogen peroxide. This was correlated with oxidative stress and cell death. An increased generation of ROS was observed in dexamethasone-treated cells together with an increase in caspase-3 activity and apoptosis rate. Interestingly, exposure to dexamethasone increased the cytosolic superoxide dismutase Cu/ZnSOD protein expression and activity, whereas the mitochondrial MnSOD isoform was not affected by the glucocorticoid. Catalase overexpression in insulin-producing cells prevented all the cytotoxic effects of dexamethasone. In conclusion, dexamethasone-induced cell death in insulin-producing cells is ROS mediated. Increased levels of expression and activity of the Cu/ZnSOD might favor the generation of hydrogen peroxide in dexamethasone-treated cells. Increased ROS scavenging capacity in insulin-producing cells, through overexpression of catalase, prevents a deleterious increase in hydrogen peroxide generation and thus prevents dexamethasone-induced apoptosis. 10.1016/j.freeradbiomed.2009.08.010
    Partial deficiency of surfactant protein B in an infant with chronic lung disease. Ballard P L,Nogee L M,Beers M F,Ballard R A,Planer B C,Polk L,deMello D E,Moxley M A,Longmore W J Pediatrics OBJECTIVE:To evaluate components of pulmonary surfactant and identify mutations in the surfactant protein B gene (SP-B) of a term infant with severe respiratory distress and chronic lung disease. PATIENT AND TESTING: Respiratory distress developed in an infant delivered at term, and he required extracorporeal bypass support for 2 weeks. Until his unexpected death at 9.5 months, he was ventilator and oxygen dependent and required continual dexamethasone therapy. Tracheobronchial lavage samples were analyzed for content of surfactant proteins (SPs), and DNA from blood samples were sequenced and analyzed by polymerase chain reaction restriction analysis for the presence of SP-B gene mutations. Surfactant lipid composition and function, the contents of SPs and their messenger RNAs (mRNAs), and the immunostaining pattern for SPs were determined in postmortem lung tissue. RESULTS:The lavage sample contained SP-A but not SP-B, and DNA restriction analysis indicated that the patient and his mother were heterozygous for the previously described 121ins2 mutation of SP-B. Postmortem lung tissue contained normal levels of SP-A and its mRNA, a low but detectable level of SP-B, and near normal content of SP-B mRNA. SP-C was abundant on staining, and some 6-kd precursor was present in tissue. A surfactant fraction was deficient in phosphatidylglycerol and was not surface active. On DNA sequencing, a point mutation was found in exon 7 of the patient's SP-B gene allele without the 121ins2 mutation, resulting in a cysteine for arginine substitution, and the father was a carrier for the same mutation. CONCLUSIONS:We describe a patient who is a compound heterozygote with a new mutation and only a partial deficiency of SP-B. Some forms of inherited SP-B deficiency may have low expression of immunoreactive and possibly functional SP-B with milder lung disease and longer survival. These infants may benefit from glucocorticoid therapy and may not develop antibodies to SP-B after either lung transplant or gene therapy.
    Molecular and cellular processing of lung surfactant. Rooney S A,Young S L,Mendelson C R FASEB journal : official publication of the Federation of American Societies for Experimental Biology Pulmonary surfactant, a complex material that lines the alveolar surface of the lung, is synthesized in the type II pneumocyte. Surfactant consists largely of phospholipids, of which phosphatidylcholine is by far the most abundant component, and is mainly responsible for surface activity. Surfactant also contains four unique proteins, surfactant protein (SP)-A, SP-B, SP-C, and SP-D, which are synthesized in a lung-specific manner. SP-A and SP-D are glycoproteins (M(r) approximately 30,000-40,000) whereas SP-B and SP-C are small (M(r) approximately 5,000-18,000), extremely hydrophobic proteolipids released from large precursors by proteolysis. Synthesis of surfactant lipids and proteins is developmentally regulated in fetal lung and can be accelerated by glucocorticoids and other hormones. Developing fetal lung in vivo and in organ culture has been used extensively to study regulation of surfactant synthesis and gene expression. Glucocorticoids stimulate the rate of fetal lung phosphatidylcholine biosynthesis and the activity of the rate-regulatory enzyme, cholinephosphate cytidylyltransferase (CYT). The hormone, however, does not increase the amount of CYT; there is evidence that the increase in activity is mediated by increased fatty biosynthesis due to enhanced expression of the fatty acid synthase gene. Glucocorticoids also regulate expression of the SP-A, SP-B, and SP-C genes in the late gestation fetal lung. Hormone response elements and other cis-acting regulatory elements have been identified in the 5'-flanking regions of the SP-A, SP-B, and SP-C genes. Surfactant phospholipids are stored in lamellar bodies, secretory granules in the type II cell, and secreted by exocytosis. Lamellar bodies are also rich in SP-B and SP-C but there are conflicting data on the cellular distribution of SP-A. Secretion of SP-A may be constitutive and occur independently of lamellar bodies. Phosphatidylcholine secretion is a regulated process, and in isolated type II cells it can be stimulated by physiological and other agents that act via at least three signal-transduction mechanisms. After secretion, surfactant is transformed into tubular myelin, and the lipid and protein components are separated as the lipid is inserted into a monolayer at the air-liquid interface. The majority of surfactant is removed from the alveolar space by reuptake into the type II cell by mechanisms that may include receptor-mediated endocytosis. Some components of surfactant are directly recycled into new surfactant whereas other components are degraded. 10.1096/fasebj.8.12.8088461
    Dexamethasone enhances surfactant protein gene expression in streptozotocin-induced immature rat lungs. Rayani H H,Warshaw J B,Floros J Pediatric research Because surfactant protein (SP) mRNA levels in rat fetuses are increased by maternal dexamethasone (dex) treatment and decreased in streptozotocin-induced diabetic (STZ-DB) pregnancy, we investigated the in vivo effects of dex on SP gene expression in STZ-DB pregnancy. The mRNA levels of SP (SP-A, SP-B, SP-C) were assessed in d 18 and 20 fetuses by Northern blot analysis, and nuclear run-on assays were performed with lung nuclei from d 20 fetuses (term = 22 d). Our findings indicate: 1) dex causes a greater increase in SP-A and SP-B mRNA levels in d 18 (12-16-fold) compared with day 20 (4-6-fold) fetuses (p < 0.05) in normal and STZ-DB pregnancy; 2) a 2-3-fold increase in SP-C mRNA levels was observed in response to dex in d 18 and 20 fetuses; 3) the increase in transcription of SP-A and SP-B in d 20 fetuses after dex is 68 and 60%, respectively, of the increase in their mRNA levels whereas in STZ-DB, the decrease in transcription compared with mRNA levels is 3.67-fold for SP-A and 2.42 fold SP-B; and 4) changes in SP-C transcription in either in vivo model, dex-treated or STZ-DB, correspond well with changes in mRNA levels. Together, these findings indicate that dex can enhance SP expression in STZ-DB immature lungs and support differential regulation of fetal SP genes in the models studied. 10.1203/00006450-199512000-00008
    Regulation of messenger RNAs for the hydrophobic surfactant proteins in human lung. Liley H G,White R T,Warr R G,Benson B J,Hawgood S,Ballard P L The Journal of clinical investigation The pulmonary surfactant proteins SP-B (8,000 D) and SP-C (4,000 D) accelerate surface film formation by surfactant phospholipids. We used cDNA probes to examine regulation of these proteins in human fetal lung. The mRNAs were detectable at 13 wk gestation and increased to approximately 50% (SP-B) and approximately 15% (SP-C) of adult levels at 24 wk. The mRNAs were detected only in lung of 11 dog tissues examined. When human fetal lung was cultured as explants without hormones, SP-B mRNA increased and SP-C mRNA decreased. Exposure for 48 h to glucocorticoids, but not other steroids, increased both SP-B mRNA (approximately 4-fold) and SP-C mRNA (approximately 30-fold) vs. controls. Half-maximal stimulation occurred with 1 nM dexamethasone and 300 nM cortisol for SP-B mRNA and at three- to fivefold higher concentrations for SP-C mRNA. Both stimulation and its reversal on removal of hormone were more rapid for SP-B than for SP-C. Terbutaline and forskolin increased SP-B mRNA but not SP-C mRNA. Levels of both mRNAs were much higher in type II cells than fibroblasts prepared from explants. Thus, the genes for SP-B and SP-C are expressed in vivo before synthesis of both SP-A (28,000-36,000 D) and surfactant lipids. Glucocorticoid induction of SP-B and SP-C mRNAs in type II cells appears to be receptor mediated but may involve different mechanisms. 10.1172/JCI114000
    Bronchial mucosal immunoreactivity of sensory neuropeptides in severe airway diseases. Chanez P,Springall D,Vignola A M,Moradoghi-Hattvani A,Polak J M,Godard P,Bousquet J American journal of respiratory and critical care medicine Neuropeptides act on most of the components of the bronchial environment. They influence bronchomotor tone and bronchial vascular caliber and permeability. To investigate the nonadrenergic, noncholinergic system within the airways in asthma and chronic bronchitis, we performed endobronchial biopsies in 16 normal human volunteers, 49 patients with asthma of varying severity, including 16 patients treated with oral corticosteroids, and 13 patients with chronic bronchitis. Frozen sections of biopsies stained with specific antibodies against the neural marker PGP 9.5, vasoactive intestinal peptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP), and neuropeptide Y (NPY) were analyzed for the presence of nerves through indirect immunofluorescence. Nerves were present in most of the biopsies and were found within and below the epithelium and adjacent to smooth muscle, glands, and blood vessels. By comparison with those in normal subjects, the numbers of VIP-immunoreactive nerves were not significantly decreased in patients with asthma and chronic bronchitis, but NPY-immunoreactive nerves were significantly decreased in the smooth muscle of these latter two groups of patients (p < 0.005). There was no correlation between disease severity and the number of nerves found in the biopsies. This study does not confirm previous findings in autopsy material of some defects in sensory and VIP-containing nerves in severe asthma. 10.1164/ajrccm.158.3.9608104
    Transcription and mRNA stability regulate developmental and hormonal expression of rabbit surfactant protein B gene. Margana R K,Boggaram V The American journal of physiology Surfactant protein B (SP-B), a hydrophobic protein of pulmonary surfactant, is essential for the surface tension-reducing properties of surfactant. In the present study, we isolated and characterized cDNAs encoding rabbit SP-B, and used transcription run-on assays and Northern blot analysis to investigate the role of transcriptional and posttranscriptional mechanisms in the developmental and cAMP and dexamethasone induction of SP-B mRNA. We found two forms of SP-B cDNAs that differed by an insertion of 69 nucleotides in the 3' untranslated regions. We found that transcription across the SP-B gene is nonequimolar and the 3' end of the gene has high levels of antisense transcription. SP-B gene transcription and SP-B mRNA levels increased during fetal lung development. However, increased SP-B mRNA levels could not be accounted for primarily on the basis of increased transcription. These results suggested that enhanced SP-B gene transcription and enhanced SP-B mRNA stability mediate developmental induction of SP-B gene. In rabbit fetal lung in vitro, both dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and dexamethasone increased SP-B mRNA levels. DBcAMP-dependent increase in SP-B mRNA levels resulted from increased SP-B gene transcription, whereas dexamethasone-dependent increase resulted from combined effects on increased SP-B gene transcription and SP-B mRNA stability. In tissues treated with dexamethasone the half-life (t1/2) of SP-B mRNA increased > 2.5-fold (t1/2 control = 9 h; t1/2 dex-treated = 25 h). These data show that both transcription and mRNA stability regulate induction of SP-B gene expression during fetal lung development and by cAMP and dexamethasone in fetal lung in vitro. 10.1152/ajplung.1995.268.3.L481
    Characterization of the rat pulmonary surfactant protein A promoter. Lacaze-Masmonteil T,Fraslon C,Bourbon J,Raymondjean M,Kahn A European journal of biochemistry The expression of the pulmonary surfactant protein A (SP-A) is developmentally regulated and controlled by several hormones. In an attempt to characterize cis-acting elements involved in the regulation of SP-A expression, we have cloned the 5' flanking sequence of the rat SP-A gene. The promoter region contains a TATA box but no CAAT box. The transcription start site has been identified by anchored polymerase chain reaction and S1 nuclease mapping of the mature and precursor transcripts. S1 mapping of precursor transcripts has confirmed the stimulating effect of glucocorticoids on SP-A rat gene transcription in vivo. This hormonal effect may be mediated by a putative glucocorticoid responsive element located 140 bp upstream from the initiation site and protected against DNase 1 digestion in footprinting experiments. In vitro transcription of a G-free reporter cassette linked to the 212-bp 5' flanking DNA fragment has established that this putative promoter region is functional. Efficient transcription of the G-free reporter cassette was obtained with cell-free fetal lung extracts, whereas no transcript was detectable with cell-free liver extracts. Comparative analysis of the human and rat 5' flanking sequences shows the presence of strongly conserved motifs, unrelated to previously known consensus sequences. Some of these motifs, specifically protected in DNase 1 footprinting studies, could therefore be involved in the regulation of SP-A gene expression. 10.1111/j.1432-1033.1992.tb16966.x
    Mechanisms of induction of airway smooth muscle hyperplasia by transforming growth factor-beta. Xie Shaoping,Sukkar Maria B,Issa Razao,Khorasani Nadia M,Chung Kian Fan American journal of physiology. Lung cellular and molecular physiology Airway smooth muscle (ASM) hyperplasia is a characteristic feature of the asthmatic airway, but the underlying mechanisms that induce ASM hyperplasia remain unknown. Because transforming growth factor (TGF)-beta is a potent regulator of ASM cell proliferation, we determined its expression and mitogenic signaling pathways in ASM cells. We obtained ASM cells by laser capture microdissection of bronchial biopsies and found that ASM cells from asthmatic patients expressed TGF-beta1 mRNA and protein to a greater extent than nonasthmatic individuals using real-time RT-PCR and immunohistochemistry, respectively. TGF-beta1 stimulated the growth of nonconfluent and confluent ASM cells either in the presence or absence of serum in a time- and concentration-dependent manner. The mitogenic activity of TGF-beta1 on ASM cells was inhibited by selective inhibitors of TGF-beta receptor I kinase (SD-208), phosphatidylinositol 3-kinase (PI3K, LY-294002), ERK (PD-98059), JNK (SP-600125), and NF-kappaB (AS-602868). On the other hand, p38 MAPK inhibitor (SB-203580) augmented TGF-beta1-induced proliferation. To study role of the Smads, we transduced ASM cells with an adenovirus vector-expressing Smad4, Smad7, or dominant-negative Smad3 and found no involvement of these Smads in TGF-beta1-induced proliferation. Dexamethasone caused a dose-dependent inhibition in TGF-beta1-induced proliferation. Our findings suggest that TGF-beta1 may act in an autocrine fashion to induce ASM hyperplasia, mediated by its receptor and several kinases including PI3K, ERK, and JNK, whereas p38 MAPK is a negative regulator. NF-kappaB is also involved in the TGF-beta1 mitogenic signaling, but Smad pathway does not appear important. 10.1152/ajplung.00068.2007
    TGF-beta 3 inhibits the increased gene expressions of pulmonary surfactant proteins induced by dexamethasone in fetal rat lung in vitro. Wang X,Jin Q,Xu J,Shen W,Wang J,Post M Chinese medical journal OBJECTIVE:To determine the effects of transforming growth factor-beta 3 (TGF-beta 3) on expressions of surfactant proteins (SPs). METHODS:Northern blot hybridization and reverse transcriptase polymerase chain reaction (RT-PCR) were employed to determine the SP-A, SP-B and SP-C mRNA levels of cultured lung explants and type II cells from fetal rat lungs. RESULTS:TGF-beta 3 alone did not affect the expressions of SPs either in explants or in type II cells. However, TGF-beta 3 did inhibit the increase of SP-B, SP-C mRNA levels caused by dexamethasone (100 nmol/L), in a dose-dependent manner. Furthermore, TGF-beta 3 had no direct inhibition on the increase of SP-B, SP-C mRNA induced by dexamethasone in type II cells, but had an indirect inhibitory effect mediated by fibroblasts. CONCLUSION:TGF-beta 3 plays an important inhibitory role in the development of pulmonary surfactant proteins. The present study may provide a possible explanation for the unideal effect of glucocorticoids in the prevention of neonatal respiratory distress syndrome.
    Surfactant protein C: hormonal control of SP-C mRNA levels in vitro. Veletza S V,Nichols K V,Gross I,Lu H,Dynia D W,Floros J The American journal of physiology We have studied hormonal regulation of the surfactant protein C (SP-C) in fetal 18-dah rat lung explants. SP-C mRNA was detected in Northern blots with a specific rat SP-C cDNA probe and quantified by densitometry. Treatment of the explants with dexamethasone resulted in a dose-dependent increase of the SP-C mRNA level. Transcriptional assays have shown that the regulation of SP-C mRNA by dexamethasone involves a transcriptional step. Administration of the cAMP analogues, 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), produced a dose-dependent increase of SP-C mRNA levels, with maximum stimulation observed at 200 microM. The thyroid hormone T3 had no effect on SP-C mRNA levels, whether administered alone or in combination with dexamethasone. Variation in the effects of the above hormones on three surfactant protein mRNAs, SP-A, SP-B and SP-C, indicates that the hormonal regulation of the surfactant proteins is a complex process and that each gene is, in part, differentially regulated. 10.1152/ajplung.1992.262.6.L684
    Regulation of mRNA levels for pulmonary surfactant-associated proteins in developing rabbit lung. Xu J,Yao L J,Possmayer F Biochimica et biophysica acta Gene transcriptional activities and steady-state mRNA levels have been examined for the surfactant-associated proteins SP-A, SP-B and SP-C in developing rabbit lung. It was observed SP-C mRNA levels increase early in gestation, while SP-A and SP-B mRNA levels increase rapidly between 26 and 30 days gestation. Transcriptional activities for all three surfactant apoproteins increase between 26 and 30 days. Studies conducted with fetal lung explants of 26 days gestation demonstrated exposure to low doses of dexamethasone increases SP-A and SP-C mRNA levels, while high doses stimulate transcription, although this only significant for SP-C. Time course studies revealed different temporal patterns and glucocorticoid responses for SP-A and SP-C mRNAs. SP-A and SP-C mRNA production and steady-state levels were reduced after treatment with cycloheximide. In contrast, SP-B gene transcription was selectively stimulated, suggesting involvement of a labile negative regulatory factory. It is concluded that expression of the three surfactant apoproteins is independently regulated. Early in gestation, SP-C mRNA levels may be regulated in vivo through message stabilization. Glucocorticoids can affect SP-A and SP-C mRNA levels in culture at both transcriptional and post-transcriptional levels. The ability of glucocorticoids to influence these processes declines during fetal development. 10.1016/0005-2760(94)00191-z
    Regulation of pulmonary surfactant protein synthesis in fetal lung: a major role of glucocorticoids and cyclic AMP. Mendelson C R,Boggaram V Trends in endocrinology and metabolism: TEM Augmented synthesis of the lipoprotein, pulmonary surfactant, is initiated in fetal lung toward the end of-gestation. Inadequate surfactant synthesis by the lungs of premature infants can result in respiratory distress syndrome, the leading cause of neonatal morbidity and mortality in developed countries. The surfactant-associated proteins act with surfactant glycerophospholipids to reduce alveolar surface tension, and mediate the reutilization of secreted surfactant components by type II cells. Genes encoding the surfactant proteins SP-A, SP-B, and SP-C have been isolated and characterized. Recent findings suggest that surfactant protein gene expression in fetal lung is under multifactortal control and is regulated by glucocorticoids, cAMP, growth factors, and insulin. 10.1016/1043-2760(89)90025-8
    Hormonal effects on the surfactant protein B (SP-B) mRNA in cultured fetal rat lung. Floros J,Gross I,Nichols K V,Veletza S V,Dynia D,Lu H W,Wilson C M,Peterec S M American journal of respiratory cell and molecular biology Glucocorticoids, triiodothyronine (T3), and cyclic adenosine monophosphate (cAMP) have been shown previously to modulate phosphatidylcholine and surfactant protein A (SP-A) synthesis in fetal rat lung explant cultures. In this report, we have examined the hormonal regulation of the rat surfactant protein B (SP-B) mRNA to determine whether SP-B expression is coordinately regulated with the surfactant phospholipids or with SP-A. Dexamethasone (1 to 200 nM) and cAMP (200 microM) had a stimulatory effect on SP-B mRNA levels, whereas T3 tended to inhibit the accumulation of SP-B mRNA. In combination experiments, treatment with dibutyryl-cAMP (200 microM) and dexamethasone (100 nM) resulted in about a 22-fold increase, whereas dexamethasone or dibutyryl-cAMP alone produced 18- and 2-fold increases, respectively. When the cAMP analogue 8-bromo-cAMP (200 microM) was used in combination with dexamethasone, there was no significant difference between the combined effect and that of dexamethasone alone. T3 treatment, however, resulted in a significant reduction of the dexamethasone-induced stimulation from about a 22-fold to a 14-fold increase. Tissue in situ hybridization showed that dexamethasone stimulated the levels of SP-B mRNA in cells from both the alveolar and bronchiolar epithelium. These data indicate that there are differences in the hormonal regulation of the components of surfactant, suggesting that they are independently regulated. 10.1165/ajrcmb/4.5.449
    Rabbit surfactant protein C: cDNA cloning and regulation of alternatively spliced surfactant protein C mRNAs. Boggaram V,Margana R K The American journal of physiology Surfactant protein C (SP-C), a hydrophobic protein of pulmonary surfactant is essential for surfactant function. Toward elucidating molecular mechanisms that mediate regulation of SP-C gene expression in rabbit lung, we isolated and characterized cDNAs encoding rabbit SP-C and studied the regulation of SP-C gene expression during fetal lung development and by adenosine 3',5'-cyclic monophosphate (cAMP) and dexamethasone in fetal lung tissues in vitro. We found that rabbit SP-C is highly homologous to SP-C of other species and is encoded by two mRNAs that differ by an insertion of 31 nucleotides in the 3' untranslated regions. SP-C mRNAs were classified into two types based on the nucleotide sequence; type I represents RNA without the 31 nucleotide insert and comprises approximately 80-90% of total SP-C mRNA content, whereas type II represents RNA containing the insert and comprises approximately 10-20% of total SP-C mRNA content. SP-C mRNAs were induced in a coordinate manner during fetal lung development and by cAMP and dexamethasone in fetal lung tissues in vitro. Southern hybridization analysis of genomic DNA suggested that SP-C mRNAs are encoded by a single gene. Polymerase [corrected] chain reaction-amplification of genomic DNA with oligonucleotide primers flanking the insertional sequence and sequence analysis of amplified DNA showed that SP-C mRNAs are produced by alternative use of 3' splice sites of intron 5 of SP-C gene. 10.1152/ajplung.1992.263.6.L634
    Regulation of surfactant protein D expression by glucocorticoids in vitro and in vivo. Deterding R R,Shimizu H,Fisher J H,Shannon J M American journal of respiratory cell and molecular biology SP-D is a recently described lung-associated protein that is produced by alveolar type II cells and may function in pulmonary host defenses. Since little is known regarding the hormonal regulation of SP-D, and since the other surfactant proteins (SP-A, SP-B, and SP-C) are known to be regulated by glucocorticoids, we sought to determine the effects of glucocorticoids on SP-D mRNA and protein expression, both in vitro and in vivo, in the fetal rat lung. In vitro experiments were performed on lung explants from fetuses on gestational day 15 or 18. Explants were cultured in serum-free conditions with or without hydrocortisone for 3 days. SP-D mRNA expression was evaluated by Northern blot analysis. SP-D protein expression was analyzed using a polyclonal antibody against SP-D and standard immunohistochemical techniques. The expression of SP-D mRNA increased in fetal day 15 explants but remained unchanged in fetal day 18 explants cultured without the addition of hydrocortisone, compared with in vivo controls. The addition of hydrocortisone resulted in increases in SP-D mRNA expression at both gestational ages. This pattern of SP-D mRNA expression was compared with the expression of the other surfactant proteins and found to be most similar to that of SP-B. In vivo experiments were performed using maternal administration of dexamethasone (1 mg/kg) or an equal volume of saline on fetal days 15, 16, and 17 or on fetal day 17 with sacrifice on fetal day 18.(ABSTRACT TRUNCATED AT 250 WORDS) 10.1165/ajrcmb.10.1.8292379
    Characterization of the promoter of human pulmonary surfactant protein B gene. Venkatesh V C,Planer B C,Schwartz M,Vanderbilt J N,White R T,Ballard P L The American journal of physiology Pulmonary surfactant protein B (SP-B) is required for normal surfactant function and for survival at birth. To further study SP-B gene expression, we sequenced genomic clones and examined promoter activity of SP-B DNA fragments by transient transfection. A plasmid construct containing human SP-B fragment -1039/+431 linked to chloramphenicol acetyltransferase (CAT) reporter gene was readily expressed in H441 cells, which are derived from a human lung adenocarcinoma, but was < 4% as active in Hep G2, HeLa, and Calu 6 cell lines. SP-B promoter activity in H441 cells was orientation dependent and increased by linked Rous sarcoma virus (RSV) enhancer and was stronger than for thymidine kinase (tk) and RSV promoters. Deletional mapping of the 5' flanking region with exonuclease III suggested nonspecific negative (-811/-1039)- and positive (-453/-641)-control regions and a cell-specific enhancer region at -208 to -54. When a fragment from -403 to -35 base pairs (bp) was placed upstream or downstream of tkCAT, in either orientation, expression in H441 cells but not other cell lines was increased 4- to 28-fold relative to tkCAT. Deletional analysis of the 3' terminus indicated a requirement for at least 7 bp 3' of the transcription start site. Promoter activity was strongly inhibited in a dose-dependent fashion by phorbol ester, with responsiveness mapped to bp -208/-54, but was not responsive to glucocorticoid.(ABSTRACT TRUNCATED AT 250 WORDS) 10.1152/ajplung.1995.268.4.L674
    Hormonal control of the surfactant system in fetal lung. Mendelson C R,Boggaram V Annual review of physiology The synthesis of surfactant glycerophospholipids and proteins is under multifactorial control and is regulated by a number of hormones and factors, including glucocorticoids, prolactin, insulin, growth factors, estrogens, androgens, thyroid hormones and catecholamines acting through beta-adrenergic receptors, and cAMP. In studies with human fetal lung in organ culture, glucocorticoids, in combination with prolactin and/or insulin, were found to increase the rate of lamellar body PC synthesis and increase the molar ratio of surfactant PG to PI to a value similar to that of surfactant secreted by the human fetal lung at term. Recognition of the potential importance of the surfactant proteins SP-A, SP-B, and SP-C in the reduction of alveolar surface tension and in endocytosis and reutilization of secreted surfactant by type II cells has stimulated rapid advancement of knowledge concerning the structures of these proteins and their genes, as well as their developmental and hormonal regulation in fetal lung tissue. The genes encoding the surfactant proteins are expressed in a lung-specific manner and appear to be regulated independently during fetal development. SP-A gene expression is initiated in fetal lung tissue after 75-85% of gestation is completed in all mammalian species studied to date. In the human fetus, however, expression of the SP-B and SP-C genes is detectable prior to mid-gestation. In situ hybridization studies of human lung tissue indicate that the SP-A gene is expressed only in type II cells, whereas SP-B gene expression is detectable in bronchioalveolar epithelial cells as well. Cyclic AMP and glucocorticoids have pronounced effects on the regulation of SP-A gene expression in human and rabbit fetal lung in culture. In human fetal lung in vitro, the effects of cAMP are primarily at the level of gene transcription. By contrast, glucocorticoids have stimulatory effects on SP-A gene transcription and inhibitory effects on SP-A mRNA stability. Furthermore, the combined effects of cAMP and glucocorticoids on SP-A gene transcription in human fetal lung in vitro are synergistic. Glucocorticoids appear to be of primary importance in the regulation of the genes encoding SP-B and SP-C. Elucidation of the molecular mechanisms involved in the regulation of expression of the surfactant protein genes in developing fetal lung will be of fundamental importance to our understanding of the developmental and tissue-specific regulation of eukaryotic gene expression. 10.1146/annurev.ph.53.030191.002215
    Recovery of rat type II cell surfactant components during primary cell culture. Bates Sandra R,Gonzales Linda W,Tao Jian-Qin,Rueckert Peter,Ballard Philip L,Fisher Aron B American journal of physiology. Lung cellular and molecular physiology A culture system designed to maintain the differentiated characteristics of rat type II cells based on protocols used for human fetal lung pneumocytes was investigated. Type II cells were isolated either from adult rats with elastase (adult type II cells) or from young rats (4-11 days postnatal) with collagenase and trypsin (young type II cells) and were incubated with dexamethasone (Dex, 10 nM) and cAMP (0.1 mM). By day 4 of culture with hormone treatment, the mRNA levels in adult type II cells were less than 3% of day 0 values, whereas surfactant protein (SP)-A protein content was 26%. However, young type II cells maintained lamellar bodies and microvilli and secreted phospholipid in response to ATP. SP-A, -B, and -C mRNA levels were elevated to 159, 350, and 39%, respectively, of day 0 values with a synergistic response to Dex and cAMP, whereas SP-A protein content rose to 119%. Surfactant mRNA and protein did not recover in cells cultured without hormones. This cell culture system restored surfactant components in rat type II cells. 10.1152/ajplung.00227.2001
    A non-BRICHOS surfactant protein c mutation disrupts epithelial cell function and intercellular signaling. Woischnik Markus,Sparr Christiane,Kern Sunčana,Thurm Tobias,Hector Andreas,Hartl Dominik,Liebisch Gerhard,Mulugeta Surafel,Beers Michael F,Schmitz Gerd,Griese Matthias BMC cell biology BACKGROUND:Heterozygous mutations of SFTPC, the gene encoding surfactant protein C (SP-C), cause sporadic and familial interstitial lung disease (ILD) in children and adults. The most frequent SFTPC mutation in ILD patients leads to a threonine for isoleucine substitution at position 73 (I73T) of the SP-C preprotein (proSP-C), however little is known about the cellular consequences of SP-CI73T expression. RESULTS:To address this, we stably expressed SP-CI73T in cultured MLE-12 alveolar epithelial cells. This resulted in increased intracellular accumulation of proSP-C processing intermediates, which matched proSP-C species recovered in bronchial lavage fluid from patients with this mutation. Exposure of SP-CI73T cells to drugs currently used empirically in ILD therapy, cyclophosphamide, azathioprine, hydroxychloroquine or methylprednisolone, enhanced expression of the chaperones HSP90, HSP70, calreticulin and calnexin. SP-CI73T mutants had decreased intracellular phosphatidylcholine level (PC) and increased lyso-PC level without appreciable changes of other phospholipids. Treatment with methylprednisolone or hydroxychloroquine partially restored these lipid alterations. Furthermore, SP-CI73T cells secreted into the medium soluble factors that modulated surface expression of CCR2 or CXCR1 receptors on CD4+ lymphocytes and neutrophils, suggesting a direct paracrine influence of SP-CI73T on neighboring cells in the alveolar space. CONCLUSION:We show that I73T mutation leads to impaired processing of proSP-C in alveolar type II cells, alters their stress tolerance and surfactant lipid composition, and activates cells of the immune system. In addition, we show that some of the mentioned cellular aspects behind the disease can be modulated by application of pharmaceutical drugs commonly applied in the ILD therapy. 10.1186/1471-2121-11-88
    Effects of maternal dexamethasone on expression of SP-A, SP-B, and SP-C in the fetal rat lung. Schellhase D E,Shannon J M American journal of respiratory cell and molecular biology Prenatal administration of glucocorticoids has been shown to enhance surfactant production in the fetus. Since the surfactant proteins play an important role in surfactant function and secretion, we wished to determine the effects of maternal glucocorticoid administration on their fetal expression and appearance. Daily dexamethasone (DEX) (1 mg/kg/day) or 0.9% saline was administered to timed-pregnant rats on gestational days 14 through 16 or on day 16 with sacrifice on day 17 (term day 22), and on gestational days 14 through 18, or days 16 through 18, or day 18 with sacrifice on day 19. SP-A content was determined in lung homogenates from treated and control male and female fetal rats by an enzyme-linked in lung homogenates from treated and control male and female fetal rats by an enzyme-linked immunosorbent assay. The abundance of mRNAs for SP-A, SP-B, and SP-C per fixed amount of total cellular RNA was also determined in lungs from treated and control male and female fetal rats by Northern blot analysis. In litters sacrificed on day 17, DEX administered on days 14 through 16 and on day 16 resulted in significant increases in SP-A content. Expression of SP-A mRNA, which was not detectable in control fetuses on day 17, became clearly apparent after either 1 or 3 d of DEX treatment. The abundance of mRNAs for SP-B and SP-C also increased in day-17 fetuses after either 1 or 3 d of DEX treatment.(ABSTRACT TRUNCATED AT 250 WORDS) 10.1165/ajrcmb/4.4.304
    Developmental and hormonal regulation of surfactant protein C (SP-C) gene expression in fetal lung. Role of transcription and mRNA stability. Boggaram V,Margana R K The Journal of biological chemistry Pulmonary surfactant protein C (SP-C) gene expression is developmentally and hormonally regulated in fetal lung. In the present study, we investigated the role of transcriptional and posttranscriptional mechanisms in the developmental, cAMP, and dexamethasone induction of SP-C mRNA. We found that developmental induction of SP-C mRNA was not coincident with induction of SP-C gene transcription. SP-C mRNA levels reached approximately 90% of levels in adult lung on day 24 of gestation, whereas SP-C gene transcription was only approximately 4% of level in adult lung and did not increase until day 28 of gestation (term in rabbit = 31 days). Treatment of fetal lung tissues in vitro with dibutyryl cyclic AMP (Bt2cAMP) and dexamethasone increased SP-C mRNA accumulation by different mechanisms. Increase in SP-C mRNA accumulation by Bt2cAMP was the result of increased SP-C gene transcription, whereas increased SP-C mRNA accumulation by dexamethasone was due to stabilization of RNA. In control tissues the SP-C mRNA half-life (t1/2) was 11.2 h, and after dexamethasone treatment it increased to 30 h. These data show that both transcriptional and mRNA stabilization mechanisms regulate induction of SP-C gene expression during fetal lung development and by cAMP and dexamethasone in fetal lung in vitro.
    Leptin Matures Aspects of Lung Structure and Function in the Ovine Fetus. De Blasio Miles J,Boije Maria,Kempster Sarah L,Smith Gordon C S,Charnock-Jones D Stephen,Denyer Alice,Hughes Alexandra,Wooding F B Peter,Blache Dominique,Fowden Abigail L,Forhead Alison J Endocrinology In human and ovine fetuses, glucocorticoids stimulate leptin secretion, although the extent to which leptin mediates the maturational effects of glucocorticoids on pulmonary development is unclear. This study investigated the effects of leptin administration on indices of lung structure and function before birth. Chronically catheterized singleton sheep fetuses were infused iv for 5 days with either saline or recombinant ovine leptin (0.5 mg/kg · d leptin (LEP), 0.5 LEP or 1.0 mg/kg · d, 1.0 LEP) from 125 days of gestation (term ∼145 d). Over the infusion, leptin administration increased plasma leptin, but not cortisol, concentrations. On the fifth day of infusion, 0.5 LEP reduced alveolar wall thickness and increased the volume at closing pressure of the pressure-volume deflation curve, interalveolar septal elastin content, secondary septal crest density, and the mRNA abundance of the leptin receptor (Ob-R) and surfactant protein (SP) B. Neither treatment influenced static lung compliance, maximal lung volume at 40 cmH2O, lung compartment volumes, alveolar surface area, pulmonary glycogen, protein content of the long form signaling Ob-Rb or phosphorylated signal transducers and activators of transcription-3, or mRNA levels of SP-A, C, or D, elastin, vascular endothelial growth factor-A, the vascular endothelial growth factor receptor 2, angiotensin-converting enzyme, peroxisome proliferator-activated receptor γ, or parathyroid hormone-related peptide. Leptin administration in the ovine fetus during late gestation promotes aspects of lung maturation, including up-regulation of SP-B. 10.1210/en.2015-1729
    Natural protection from apoptosis by surfactant protein A in type II pneumocytes. White M K,Baireddy V,Strayer D S Experimental cell research Surfactant-associated protein A (SP-A) is a component of pulmonary surfactant that binds to a specific receptor (SPAR) on the surface of type II alveolar cells of the lung and regulates gene expression and surfactant secretion. Previously we have shown that activation of SPAR by SP-A binding initiates a signal through pathways that involve tyrosine phosphorylation, include IRS-1, and entail activation of phosphatidylinositol 3-kinase (PI3K). In other cell types, cytokines that activate the PI3K signaling pathway promote cell survival. Therefore we investigated whether there was an effect of SP-A on apoptosis as measured by DNA laddering, FACS analysis, TUNEL assay, and annexin V binding. SP-A protected primary cultures of rat type II alveolar cells against the apoptotic effects of etoposide and UV light and also protected the H441 human Clara lung tumor cell line against staurosporine-induced apoptosis. The protective effects of SP-A were abrogated by inhibition of either tyrosine-specific protein kinase activity or PI3K. SP-A/SPAR interaction thus initiates a signaling pathway that regulates apoptosis in type II cells. These findings may be important in understanding the pathogenesis of acute lung injury and pulmonary tumorigenesis and may suggest new therapeutic options. 10.1006/excr.2000.5120
    Tumor necrosis factor-alpha and embryonic mouse lung morphogenesis. Jaskoll T,Boyer P D,Melnick M Developmental dynamics : an official publication of the American Association of Anatomists The ontogeny of the embryonic and fetal lung involves complex interactions between epithelial and mesenchymal primordia which require a specific program of gene regulation and signal transduction. Past studies in our laboratory using congenic mouse strains indicate that one or more genes which map to the H-2 region of chromosome 17 regulate the rate of lung morphogenesis, defined in this context as differentiative heterochrony among strains. Since hormones and growth factors are the messengers of morphogenesis, it was logical to propose that tumor necrosis factor-alpha (TNF-alpha), a well-characterized cytokine whose gene maps to the D-region of the H-2 complex, is a putative mediator of lung morphogenesis. We investigated this proposition using immunochemical methods and a serumless, chemically defined in vitro model system. Our results demonstrate that: (1) TNF-alpha has a specific spatiotemporal localization, in vivo and in vitro; (2) TNF-alpha receptor, in vivo and in vitro, is localized throughout the embryonic lung; (3) TNF-alpha supplementation in vitro of embryonic lung primordia has a marked dose-dependent, stimulatory effect on branching morphogenesis and surfactant-associated protein (SP-A) expression; (4) multiple immunoreactive proteins, including 17, 26, and 68 kDa species, are expressed during development in vivo, and a subset of these are expressed in vitro; and (5) both time- and glucocorticoid-dependent changes occur in the in vivo expression pattern of TNF-alpha immunoreactive proteins after 4 and 7 days in vitro, including the up-regulation of a novel 40 kDa protein. Given that glucocorticoids (CORT) regulate TNF-alpha expression and TNF-alpha's ability to stimulate pulmonary morphodifferentiation and histodifferentiation, we conclude that TNF-alpha is an autocrine/paracrine pulmonary cytokine, probably a component of the lung morphogenesis pathway regulated by CORT. 10.1002/aja.1002010205
    rhKGF stimulates lung surfactant production in neonatal rats in vivo. Gesche Jens,Fehrenbach Heinz,Koslowski Roland,Ohler Florian M,Pynn Christopher J,Griese Matthias,Poets Christian F,Bernhard Wolfgang Pediatric pulmonology Surfactant deficiency and bronchopulmonary dysplasia (BPD), major obstacles in preterm infants, are addressed with pre- and postnatal glucocorticoids which also evoke harmful catabolic side-effects. Keratinocyte growth factor (KGF) accelerates surfactant production in fetal type II pneumocytes (PN-II), protects epithelia from injury and is deficient in lungs developing BPD, highlighting its potential efficacy in neonates. Neonatal rats were treated with recombinant human (rh)KGF, betamethasone, or their combination for 48 hr prior to sacrifice after which body weight, surfactant, and tissue phosphatidylcholines (PC) were investigated at postnatal d3, d7, d15, and d21. Pneumocyte proliferation, surfactant protein (SP) expression and SP-B/C in lung lavage fluid (LLF) were also determined at d7 and d21 to identify broader surfactant changes occurring at the beginning and end of the initial alveolarization phase. While all treatments increased secreted surfactant PC, BM compromised animal growth whereas rhKGF did not. At d3 rhKGF was more effective in male compared to female rats. Single treatments became less effective towards d21. Neither treatment altered PC composition in LLF. BM inhibited PN-II proliferation and increased surfactant PCs at the expense of tissue PCs. rhKGF however increased surfactant PCs without decreasing other PC species. Whereas SP-B/C gene expression was induced by all treatments, the changes in secreted SP-B/C mirrored those observed for surfactant PC. Our results encourage investigation of the mechanisms by which rhKGF improves surfactant homoeostasis, and detailed examination of its efficacy in neonatal lung injury models with a view to implementing it as a non-catabolic surfactant-increasing therapeutic in neonatal intensive care. 10.1002/ppul.21443
    Glucocorticoids both stimulate and inhibit production of pulmonary surfactant protein A in fetal human lung. Liley H G,White R T,Benson B J,Ballard P L Proceedings of the National Academy of Sciences of the United States of America Pulmonary surfactant is a mixture of phospholipids and proteins which stabilizes lung alveoli and prevents respiratory failure. The surfactant-associated protein of Mr = 28,000-36,000 (SP-A) influences the structure, function (film formation), and metabolism of surfactant. We have characterized glucocorticoid regulation of SP-A and SP-A mRNA in explants of fetal human lung. The time course of response to dexamethasone was biphasic, with early stimulation and later inhibition of SP-A accumulation. Maximal induction of SP-A occurred with 3-10 nM dexamethasone and approximately 300 nM cortisol for 72 hr, and stimulation diminished at higher concentrations. SP-A mRNA accumulation was maximally stimulated at 24-48 hr of exposure to dexamethasone (10 nM) and was generally inhibited by 4-6 days. Stimulation was also observed with cortisone and corticosterone but not with sex steroids, suggesting a receptor-mediated process. When explants were exposed to cortisol for only 24 hr, SP-A content was transiently increased above the level in continuously treated tissue and subsequently was similar to control. The content of SP-A and its mRNA was also increased by dibromo-cAMP, terbutaline, and forskolin, and effects were approximately additive with those of dexamethasone. However, elevated in tracellular cAMP did not alter the biphasic time course or dose-response patterns of dexamethasone. We propose that glucocorticoids have both stimulatory and inhibitory effects on SP-A gene expression. This biphasic regulation is not consistent with generalized toxic effects, product-feedback inhibition, or receptor down-regulation, and it appears to be specific for SP-A among the various surfactant components. 10.1073/pnas.85.23.9096
    Prostaglandins regulate surfactant protein A (SP-A) gene expression in human fetal lung in vitro. Acarregui M J,Snyder J M,Mitchell M D,Mendelson C R Endocrinology We previously have observed that dexamethasone has a biphasic effect on surfactant protein A (SP-A) mRNA levels in human fetal lung in vitro. At concentrations of 10(-10)-10(-9) M, dexamethasone increases the levels of SP-A mRNA, whereas, at concentrations greater than 10(-8) M, the steroid is markedly inhibitory. In studies to define the molecular mechanisms for these effects, we observed that dexamethasone causes a dose-dependent stimulation of SP-A gene transcription, but paradoxically causes a dose-dependent inhibition of SP-A mRNA stability. In light of the well-characterized inhibitory effect of glucocorticoids on prostaglandin (PG) synthesis in a number of tissues, it was our objective in the present study to investigate the role of PGs on SP-A gene expression in human fetal lung in vitro and to determine whether the action of dexamethasone (greater than 10(-8) M) to reduce SP-A mRNA levels could be mediated by its effect to inhibit PG synthesis. We found that dexamethasone 10(-7) M) caused a marked decrease in the secreted levels of the PGE2 and PGF2 alpha, the prostacyclin metabolite, 6-keto-PGF1 alpha, and the thromboxane A2 metabolite, thromboxane B2. Indomethacin, which also caused a pronounced reduction in the levels of these secreted prostanoids, had a marked effect to reduce SP-A mRNA levels in human fetal lung in vitro. The inhibitory effects of indomethacin were associated with an 73% reduction in cAMP formation by the fetal lung in culture, and were prevented by simultaneous incubation with dibutyryl cAMP or with PGE2. PGE2 markedly increased cAMP formation by the human fetal lung tissue incubated in the absence or presence of indomethacin. Inhibitory effects of dexamethasone and indomethacin also were observed on two morphological indices of lung differentiation, alveolar lumenal volume density, and lamellar body volume density. PGE2 significantly increased lumenal volume density of the human fetal lung explants. The finding that the inhibitory action of dexamethasone (10(-7) M) on SP-A mRNA levels could not be prevented by simultaneous incubation with either PGE2 or dibutyryl cAMP and that dexamethasone had no apparent effect on cAMP formation by the fetal lung in vitro is suggestive that the action of dexamethasone (greater than or equal to 10(-8) M) to reduce SP-A mRNA levels is mediated at least in part by actions alternative to its inhibitory effects on PG synthesis.(ABSTRACT TRUNCATED AT 400 WORDS) 10.1210/endo-127-3-1105
    Antagonistic effects of dexamethasone and retinoic acid on rat lung morphogenesis. Oshika E,Liu S,Singh G,Michalopoulos G K,Shinozuka H,Katyal S L Pediatric research We have reported that dexamethasone (DEX) treatment of early embryonic rat lungs in culture induced features of both distorted and accelerated maturation. In this report, we investigated the effects of retinoids on normal and DEX-induced lung development in vitro. Lung maturation was assessed by examining the morphology and the expression of genes related to epithelial differentiation (surfactant proteins, SP-A, SP-B and SP-C and Clara cell protein, CC10) and growth [keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF)]. We cultured d 14 and 15 fetal rat lungs in the presence of DEX (1-1000 nM) and/or all-trans-retinoic acid (RA) (10(-7)-10(-5) M) for 4 d. RA at 10(-6) and 10(-5) M inhibited branching and dilated the distal tubules, and at 10(-5) M caused dilatation of the proximal tubules destined to form the trachea and the main bronchi. The adverse effects of DEX, such as distorted branching, tubular dilatation, and suppression of both lung growth and epithelial cell proliferation, were all prevented by RA. In addition, RA inhibited several features of DEX-induced accelerated maturation, such as: 1) the increased levels of SP-A, SP-B, and CC10 mRNAs; 2) the attenuation of mesenchymal tissue; and 3) the mature distribution of cells expressing SP-C mRNA. In contrast, RA potentiated the increase of KGF and decrease of HGF transcripts induced by DEX. In conclusion, the study shows antagonism by RA of DEX-induced effects on lung morphology and gene expression. We postulate that normal lung development requires a balanced action of endogenous retinoids and glucocorticoids. 10.1203/00006450-199804001-01874
    Modulation of surfactant protein D expression by glucocorticoids in fetal rat lung. Mariencheck W,Crouch E American journal of respiratory cell and molecular biology The production of pulmonary surfactant protein D (SP-D) increases abruptly during late gestation, and the accumulation of this protein in lung tissue is increased following the exposure of fetal rats to glucocorticoids in utero. To examine the regulation of these events, we administered dexamethasone (Dex; 1 mg/kg/day intramuscularly for 3 days), or saline, to timed-pregnant rats and harvested the lungs on days 19 to 21 of gestation. Samples of pooled fetal lungs were analyzed for SP-D protein, mRNA, and gene transcription by immunoblot, Northern hybridization, and nuclear run-off transcription assays. Lungs from 19 day controls showed barely detectable levels of SP-D gene transcription and negligible accumulation of SP-D message. However, SP-D transcription and the accumulation of SP-D mRNA and protein were readily detected in lungs from 19 day Dex-treated rats. Dexamethasone also caused dose- and time-dependent increases in SP-D protein and mRNA accumulation in 19 day fetal lung explants. Immunohistochemistry of control 19 day lung using antibodies to rat SP-D showed only weak labeling of a small number of airway epithelial cells. By contrast, Dex-exposed rats showed strong staining of columnar and cuboidal epithelial cells lining airways and epithelial tubules and cuboidal cells lining primitive air sacs. In situ hybridization assays showed similar alterations in the number, intensity, and distribution of labeled epithelial cells in 19 day Dex-exposed lungs and demonstrated labeling of alveolar type II and nonciliated columnar cells in adult lung. These data indicate that the accelerated lung maturation accompanying glucocorticoid exposure in utero is associated with a precocious increase in SP-D gene transcription and protein production by pulmonary epithelial cells. 10.1165/ajrcmb.10.4.8136157
    Transcriptional regulation of the gene encoding the major surfactant protein (SP-A) in rabbit fetal lung. Boggaram V,Mendelson C R The Journal of biological chemistry The expression of the major protein of rabbit pulmonary surfactant (SP-A), a glycoprotein of Mr congruent to 29,000-36,000, is regulated during development and by hormones. In the present study, utilizing a cDNA insert complementary to mRNA coding for SP-A and nuclear transcription elongation assays, we have investigated the developmental and hormonal regulation of transcription of the SP-A gene in rabbit fetal lung tissue. The relative rates of transcription of SP-A mRNA increased as a function of the gestational age of the fetus. The rate of transcription reached a maximum level in lung tissues of 28-day gestational age fetuses and declined slightly in those of neonatal rabbits. The relative rate of transcription of SP-A mRNA increased in rabbit fetal lung explants maintained in organ culture in control medium as a function of incubation time. Dibutyryl cyclic AMP (Bt2cAMP) treatment of fetal lung explants increased the rate of transcription of SP-A mRNA over that of control tissues by several-fold; after 12 h of incubation in the presence of Bt2cAMP, there was greater than 4-fold increase in the rate of transcription of SP-A mRNA as compared to control lung explants. In contrast, glucocorticoids had a rapid effect to decrease the rate of SP-A mRNA transcription. The rapid effect of glucocorticoids to inhibit the transcription of SP-A mRNA was transient; in fetal lung explants incubated in the presence of dexamethasone for greater than 24 h, there was an increase in the rate of transcription of SP-A mRNA over that of control explants. Cycloheximide caused an inhibition of both basal as well as Bt2cAMP-stimulated rates of transcription of SP-A mRNA in the rabbit fetal lung tissue in vitro. This finding is suggestive of a role of labile protein factor(s) in mediating transcription of the SP-A gene as well as its induction by Bt2cAMP. The magnitude of changes in the relative rates of transcription of SP-A mRNA during development of rabbit fetal lung in vitro as well as those effected by hormones in vitro were similar to changes in the steady-state levels of SP-A mRNA, suggestive that the regulation of the levels of SP-A mRNA in fetal rabbit lung tissue both in vivo and in vitro occurs primarily at the transcriptional level.
    The surfactant protein C mutation A116D alters cellular processing, stress tolerance, surfactant lipid composition, and immune cell activation. Zarbock Ralf,Woischnik Markus,Sparr Christiane,Thurm Tobias,Kern Sunčana,Kaltenborn Eva,Hector Andreas,Hartl Dominik,Liebisch Gerhard,Schmitz Gerd,Griese Matthias BMC pulmonary medicine BACKGROUND:Surfactant protein C (SP-C) is important for the function of pulmonary surfactant. Heterozygous mutations in SFTPC, the gene encoding SP-C, cause sporadic and familial interstitial lung disease (ILD) in children and adults. Mutations mapping to the BRICHOS domain located within the SP-C proprotein result in perinuclear aggregation of the proprotein. In this study, we investigated the effects of the mutation A116D in the BRICHOS domain of SP-C on cellular homeostasis. We also evaluated the ability of drugs currently used in ILD therapy to counteract these effects. METHODS:SP-CA116D was expressed in MLE-12 alveolar epithelial cells. We assessed in vitro the consequences for cellular homeostasis, immune response and effects of azathioprine, hydroxychloroquine, methylprednisolone and cyclophosphamide. RESULTS:Stable expression of SP-CA116D in MLE-12 alveolar epithelial cells resulted in increased intracellular accumulation of proSP-C processing intermediates. SP-CA116D expression further led to reduced cell viability and increased levels of the chaperones Hsp90, Hsp70, calreticulin and calnexin. Lipid analysis revealed decreased intracellular levels of phosphatidylcholine (PC) and increased lyso-PC levels. Treatment with methylprednisolone or hydroxychloroquine partially restored these lipid alterations. Furthermore, SP-CA116D cells secreted soluble factors into the medium that modulated surface expression of CCR2 or CXCR1 receptors on CD4+ lymphocytes and neutrophils, suggesting a direct paracrine effect of SP-CA116D on neighboring cells in the alveolar space. CONCLUSIONS:We show that the A116D mutation leads to impaired processing of proSP-C in alveolar epithelial cells, alters cell viability and lipid composition, and also activates cells of the immune system. In addition, we show that some of the effects of the mutation on cellular homeostasis can be antagonized by application of pharmaceuticals commonly applied in ILD therapy. Our findings shed new light on the pathomechanisms underlying SP-C deficiency associated ILD and provide insight into the mechanisms by which drugs currently used in ILD therapy act. 10.1186/1471-2466-12-15
    The glucocorticoid-glucocorticoid receptor signal transduction pathway, transforming growth factor-beta, and embryonic mouse lung development in vivo. Jaskoll T,Choy H A,Melnick M Pediatric research Lung morphogenesis has been shown to be regulated by glucocorticoids (CORT). Because CORT has been primarily thought to affect fetal lung development, previous studies have focused on the role of CORT receptor (GR)-mediated regulation of fetal lung development. Although endogenous CORT increases during embryonic and fetal stages and exogenous CORT treatment in vivo and in vitro clearly accelerates embryonic lung development, little is known about the morphoregulatory role of the embryonic CORT-GR signal transduction pathway during lung development. In this study, we characterize the embryonic mouse CORT-GR pathway and demonstrate: stage-specific in situ patterns of GR immunolocalization; similarity in GR relative mobility with progressive (E13 --> E17) development; that embryonic GR can be activated to bind a GR response element (GRE); significantly increasing levels of functional GR with increasing lung maturation; and the presence of heat shock protein (hsp) 70 and hsp90 from early (E13) to late (E17) developmental stages. These results support the purported importance of the embryonic CORT-GR signal transduction pathway in progressive lung differentiation. To demonstrate that the embryonic CORT-GR directed pathway plays a role in lung development, early embryonic (E12) lungs were exposed to CORT in utero and surfactant-associated protein A (SP-A) expression was analyzed; CORT treatment up-regulates SP-A mRNA expression and spatiotemporal protein distribution. Finally, to determine whether CORT-GR-directed pulmonary morphogenesis in vivo involves the modulation of growth factors, we studied the effect of CORT on TGF-beta gene expression. Northern analysis of TGF-beta 1, TGF-beta 2, and TGF-beta 3 transcript levels in vivo indicates that CORT regulates the rate of lung morpho- and histodifferentiation by down-regulating TGF-beta 3 gene expression. 10.1203/00006450-199605000-00002
    Characterization of two baboon surfactant protein A genes. Gao E,Wang Y,McCormick S M,Li J,Seidner S R,Mendelson C R The American journal of physiology The gene encoding surfactant protein A (SP-A) is expressed in type II pneumonocytes and is developmentally and hormonally regulated in fetal lung tissue. SP-A is encoded by a single-copy gene in rabbits, dogs, rats, and mice. By contrast, the human genome contains two similar genes, hSP-A1 and hSP-A2, which are differentially regulated during development and differentially regulated by adenosine 3',5'-cyclic monophosphate (cAMP) and glucocorticoid treatment of human fetal lung in culture. In the present study, we have isolated and characterized baboon genomic clones containing two highly similar SP-A genes. Restriction mapping of these clones, together with Southern analysis of genomic DNA, indicates that these comprise two distinct baboon SP-A genes. Sequence comparison of DNA upstream of the transcription initiation sites and within the 3'-untranslated regions encoded by exon VI indicates that one of the baboon SP-A genes (bSP-A1) is more similar to hSP-A1, whereas the other (bSP-A2) is more similar to hSP-A2. Primer extension analysis of baboon lung mRNA indicates that both baboon SP-A genes utilize conserved transcription initiation sites. Reverse transcriptase-polymerase chain reaction analysis of RNA isolated from lung tissues of fetal baboons of 160 days gestational age indicates that both bSP-A1 and bSP-A2 are expressed in baboon fetal lung and that mRNA transcripts of bSP-A1 and bSP-A2 genes are primarily comprised of sequences encoded by exons I and III-VI. However, minor transcripts of the bSP-A1 gene containing exon II and exon II plus an extension also were detected. The presence of two SP-A genes in the baboon suggests that duplication of the SP-A gene occurred > 26.5 million years ago, before divergence of the baboon lineage from the man-gorilla-chimpanzee clade. 10.1152/ajplung.1996.271.4.L617
    Glucocorticoids induce the accumulation of novel angiotensinogen gene transcripts. Ben-Ari E T,Lynch K R,Garrison J C The Journal of biological chemistry Previous work from this laboratory has shown that glucocorticoids and (Sp)-cAMPS, a cyclic AMP analogue, stimulate the accumulation of angiotensinogen mRNA in isolated hepatocytes. The present study demonstrated that glucocorticoids stimulate the accumulation of a novel, 2.25-kilobase transcript of the angiotensinogen gene, both in isolated hepatocytes and in the intact liver. (Sp)-cAMPS was synergistic with dexamethasone in causing the accumulation of the larger species of RNA in hepatocytes, but had no effect by itself. Primer extension analysis and S1 mapping experiments proved that the 2.25-kilobase angiotensinogen RNA consisted of two larger forms of angiotensinogen RNA extended at their 5' ends. The novel transcripts are generated by the use of two new transcription initiation sites in the angiotensinogen gene, located at nucleotide positions -328 and -386 relative to the start site at position +1 used in the absence of hormone. A consensus TATA box is found at the expected position 25-30 nucleotides upstream from the start site at position -328, and another TATA-like sequence is found 25-30 nucleotides upstream from the start site at -386. In addition, two consensus glucocorticoid response elements occur upstream from the initiation sites. The larger angiotensinogen RNAs do not appear to code for a novel form of the angiotensinogen protein. It appears that glucocorticoids can direct transcription from a second promoter in the angiotensinogen gene and that this promoter is absolutely dependent on the hormone. To date, this situation appears to be unique to the angiotensinogen gene.
    In vitro quantification of dexamethasone-induced surfactant protein B expression in human lung cells. Vidaeff A C,Ramin S M,Gilstrap L C,Alcorn J L The journal of maternal-fetal & neonatal medicine : the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstetricians OBJECTIVE:To determine whether the effect of a single 48-h exposure to dexamethasone in human lung cells is limited to 7-8 days. STUDY DESIGN:We used the NCI-H441 cell line, in which stability can be maintained beyond 7 days. The outcome was the stimulatory effect of dexamethasone on surfactant protein B (SP-B) gene transcription as expressed by SP-B mRNA accumulation. The experiment was conducted five times, in parallel with control. SP-B mRNA was determined at baseline, 48 h after dexamethasone exposure, and at 48-h intervals thereafter, up to 14 days, by quantitative reverse transcription polymerase chain reaction. Comparisons were made by the Mann-Whitney test. RESULTS:In conditions of our experiment, the inductive profile of SP-B mRNA after exposure to dexamethasone demonstrated maximal stimulation at 48 h (13-fold over control). Subsequently, there was a decline in mRNA, with return to near control levels by day 8, suggesting reversibility of dexamethasone action. CONCLUSION:Our data support the view that the surfactant-inducing properties of corticosteroids are limited to 7-8 days. 10.1080/14767050410001668248
    Nitric oxide inhibits surfactant protein B gene expression in lung epithelial cells. Salinas Darrell,Sparkman Loretta,Berhane Kiflu,Boggaram Vijayakumar American journal of physiology. Lung cellular and molecular physiology Surfactant protein B (SP-B) is an essential constituent of pulmonary surfactant. In a number of inflammatory diseases of the lung, elevated nitric oxide (NO) levels are associated with decreased SP-B levels, suggesting that reduced SP-B levels contribute to lung injury. In this study, we investigated the effects of NO on SP-B gene expression in H441 and MLE-12 cells, cell lines with characteristics of bronchiolar (Clara) and alveolar type II epithelial cells, respectively. Results show that NO donors decreased SP-B mRNA levels in a concentration- and time-dependent manner in H441 and MLE-12 cells. The NO donors also antagonized dexamethasone induction of SP-B mRNA in H441 cells. NO donor inhibition of SP-B mRNA was blocked by the transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranozyl-benzimidazole. NO donors decreased luciferase expression from a reporter plasmid containing -911/+41 bp of human SP-B 5'-flanking DNA in H441 and MLE-12 cells, indicating inhibitory effects on SP-B promoter activity. NO inhibition of SP-B mRNA levels was not blocked by LY-83583 and KT-5823, inhibitors of soluble guanylate cyclase and protein kinase G, respectively. Furthermore, cell-permeable cGMP analog 8-bromo-cGMP had no effect on SP-B mRNA levels. These data indicate that elevated NO levels negatively regulate SP-B gene expression by inhibiting gene transcription and that NO inhibits SP-B gene expression independently of cGMP levels. These data imply that reduced SP-B expression due to elevated NO levels can contribute to lung injury. 10.1152/ajplung.00084.2003
    Inflammatory and anti-inflammatory responsiveness of surfactant proteins in fetal and neonatal rabbit lung. Vayrynen Outi,Glumoff Virpi,Hallman Mikko Pediatric research Spontaneous preterm birth due to intrauterine infection is associated with increased concentrations of cytokines in amniotic fluid and in the airways at birth. Intra-amniotic IL-1 induces fetal lung maturity, consistent with the decrease in the incidence of respiratory distress syndrome (RDS) in intrauterine inflammation. On the other hand, antenatal corticosteroid decreases the incidence of RDS in infants born prematurely. The aim of the present study was to investigate the interaction between IL-1 and glucocorticoid in the expression of the surfactant proteins SP-A, -B, and -C. Lung explants from rabbit fetuses at 22 (immature), 27 (transitional), and 30 (mature) d of gestation (term, 30-31 d) and on d 1 after term birth were cultured with dexamethasone (Dx), IL-1alpha, or vehicle in the presence or absence of actinomycin D. According to the present results, IL-1alpha and Dx additively increased the expression of SP-A and SP-B on d 22. Later in gestation, SP-B and SP-C were suppressed by IL-1, whereas glucocorticoid tended to increase the expression of SP-B and SP-C and prevented the IL-1-induced suppression of SP. IL-1alpha and steroid interactively increased the stability of SP mRNA compared with the single agonist, possibly explaining the additive effects on the SP mRNA levels. The present results reveal beneficial additive effects of glucocorticoid and cytokine on lung surfactant. They may explain some of the acute beneficial effects of glucocorticoid therapy in chorioamnionitis before premature birth and in inflammatory lung disease after birth. 10.1203/01.PDR.0000100462.41671.C6
    Maintenance of surfactant protein A and D secretion by rat alveolar type II cells in vitro. Mason Robert J,Lewis Michele C,Edeen Karen E,McCormick-Shannon Kathleen,Nielsen Larry D,Shannon John M American journal of physiology. Lung cellular and molecular physiology Secretion of surfactant proteins A and D (SP-A and SP-D) has been difficult to study in vitro because a culture system for maintaining surfactant secretion has been difficult to establish. We evaluated several growth factors, corticosteroids, rat serum, and a fibroblast feeder layer for the ability to produce and maintain a polarized epithelium of type II cells that secretes SP-A and SP-D into the apical medium. Type II cells were plated on a filter insert coated with an extracellular matrix and were cultured at an air-liquid interface. Keratinocyte growth factor (KGF) stimulated type II cell proliferation and secretion of SP-A and SP-D more than fibroblast growth factor-10 (FGF-10), hepatocyte growth factor (HGF), or heparin-binding epidermal-like growth factor (HB-EGF). Cells cultured in the presence of KGF and rat serum with or without fibroblasts had high surfactant protein mRNA levels and exhibited a high level of SP-A and SP-D secretion. Dexamethasone inhibited type II cell proliferation but increased expression of SP-B. In the presence of KGF, rat serum, and dexamethasone, the mRNAs for the surfactant proteins were maintained at high levels. Secretion of SP-A and SP-D was found to be independent of phospholipid secretion. 10.1152/ajplung.00027.2001
    Mechanism of all trans-retinoic acid and glucocorticoid regulation of surfactant protein mRNA. George T N,Miakotina O L,Goss K L,Snyder J M The American journal of physiology The surfactant proteins (SPs) are required for the normal function of pulmonary surfactant, a lipoprotein substance that prevents alveolar collapse at end expiration. We characterized the effects of cortisol and all trans-retinoic acid (RA) on SP-A and SP-B gene expression in H441 cells, a human pulmonary adenocarcinoma cell line. Cortisol, at 10(-6) M, caused a significant inhibition of SP-A mRNA to levels that were 60-70% of controls and a five- to sixfold increase in the levels of SP-B mRNA. RA alone (10(-6) M) had no effect on SP-A mRNA levels and modestly reduced the inhibitory effect of cortisol. RA alone and the combination of cortisol and RA both significantly increased SP-B mRNA levels. RA had no effect on the rate of SP-A gene transcription or on SP-A mRNA stability. Cortisol alone and the combination of cortisol and RA significantly inhibited the rate of SP-A gene transcription but had no effect on SP-A mRNA half-life. RA at 10(-6) M had no effect on the rate of SP-B gene transcription but prolonged SP-B mRNA half-life. Cortisol alone and the combination of cortisol and RA caused a significant increase in the rate of SP-B gene transcription and also caused a significant increase in SP-B mRNA stability. We conclude that RA has no effect on SP-A gene expression and increases SP-B mRNA levels by an effect on SP-B mRNA stability and not on the rate of SP-B gene transcription. In addition, the effects of the combination of RA and cortisol were generally similar to those of cortisol alone. 10.1152/ajplung.1998.274.4.L560
    [Regulation of epidermal growth factor, thyrotropin releasing hormone and dexamethasone on the expression of fetal lung SP mRNAs in rabbits]. Wang Feng-ying,Wang Yu-mei,Cao Hong-wei,Zhang Chan-yu,Zhang Ning,Shi Chang-xu Zhonghua fu chan ke za zhi OBJECTIVE:To compare the effect of epidermal growth factor (EGF), thyrotropin releasing hormone (TRH) and dexamethasone (Dex) on the expression of surfactant protein (SP)-A, SP-B and SP-C mRNA in fetal rabbit lungs. METHODS:Sixty-four Japanese white pregnant rabbits, put into four different groups (16 rabbits each), were given EGF (EGF group), TRH (TRH group), Dex (Dex group) or normal saline (control group) intravenously from gestational day 22(nd) (5 rabbits in each group) or 24(th) (11 rabbits in each group) for three days before killing. The levels of SP-A, SP-B and SP-C mRNAs in the fetal rabbit lungs were detected by reverse transcription polymerase chain reaction (RT-PCR) and Southern blotting. RESULTS:In the 27(th) gestational day, SP-B mRNA levels in the fetal rabbit lungs in all treated groups were found significantly higher than those in the control group (P < 0.05). In the 25(th) day, there had a higher expression of SP-A and SP-C mRNAs in the fetal lungs in all groups, but SP-B mRNA could be tested only in the Dex group. SP-B mRNA level was lower than that of SP-A mRNA and SP-C mRNA in the same fetal lungs in the 27(th) gestationed day. CONCLUSION:The time of detectable SP-B mRNA in rabbit fetal lungs appeared later than that of the SP-A and SP-C mRNAs. The expression of SP-B mRNA is lower in comparison with those of SP-A mRNA and SP-C mRNA in the same samples. Prenatal maternal treatment with intravenously Dex, EGF and TRH could increase the expression of SP-B mRNA in fetal rabbit lungs, and the effect of Dex may be more significant at earlier stage of gestation.
    Genomic elements involved in transcriptional regulation of the rabbit surfactant protein-A gene. Alcorn J L,Gao E,Chen Q,Smith M E,Gerard R D,Mendelson C R Molecular endocrinology (Baltimore, Md.) Expression of the surfactant protein-A (SP-A) gene is lung specific and is developmentally and hormonally regulated in fetal lung tissue. Cyclic AMP analogs and glucocorticoids stimulate transcriptional activity of the SP-A gene in fetal rabbit lung tissue in culture; an additive effect is observed when the agents are added in combination. To analyze the genomic regions that regulate SP-A promoter activity, fusion genes comprised of -1766, -991, -378, and -47 basepairs (bp) of DNA flanking the 5'-end of the SP-A gene, the transcription initiation site, and 20 bp of exon I linked to the human GH (hGH) structural gene were subcloned into a replication-defective human adenovirus vector and transfected into differentiated rat type II cells in primary culture. SP-A promoter activity was analyzed by RIA of hGH protein in the culture medium. In type II cells transfected with SP-A-1766:hGH and SP-A-991:hGH fusion genes, hGH production was induced 30- to 40-fold by (Bu)2AMP (Bt2cAMP; 1 mM). When type II cells were transfected with the SP-A-378:hGH fusion gene, basal levels of expression were reduced by more than 50%; however, Bt2cAMP caused an 11-fold increase in hGH production. In type II cells transfected with the SP-A-47:hGH fusion gene, basal levels of hGH production were essentially undetectable, and no stimulatory effect of Bt2cAMP was apparent. Cyclic AMP stimulation of expression of the SP-A-1766:hGH, SP-A-991:hGH, and SP-A-378:hGH fusion genes was limited to type II pneumonocytes in primary culture and was absent in two lung adenocarcinoma cell lines (NCl-H358 and A549), which do not express SP-A, and in cAMP-responsive adrenal Y1 cells. Mutations of a putative cAMP-responsive element (TGACCTCA) at -261 bp revealed its functional importance in mediating cAMP regulation of SP-A gene expression. Unexpectedly, dexamethasone (Dex; 10(-7) M) antagonized the stimulatory effect of Bt2cAMP on expression of SP-A:hGH fusion genes containing from -378 to -1766 bp of 5'-flanking DNA as well as that of a fusion gene construct containing -991 bp of 5'-flanking DNA, the first exon, the first intron, and 20 bp of the second exon (SP-A-991+670:hGH). The inhibitory effect of Dex was dose dependent, with half-maximal inhibition occurring at a Dex concentration of 8 x 10(-10) M. The inhibitory effect of Dex was prevented by the glucocorticoid receptor antagonist RU486.(ABSTRACT TRUNCATED AT 400 WORDS) 10.1210/mend.7.8.8232306
    Structure and organization of the gene encoding human pulmonary surfactant proteolipid SP-B. Pilot-Matias T J,Kister S E,Fox J L,Kropp K,Glasser S W,Whitsett J A DNA (Mary Ann Liebert, Inc.) Human pulmonary surfactant proteolipid SP-B arises by proteolytic processing of a 42,000-dalton precursor. The active proteolipid SP-B is one of two small hydrophobic proteins identified in surfactant that impart surface-active properties to surfactant phospholipids. We report the isolation and characterization of complete SP-B cDNA from a human lung cDNA library. The cDNA was used to isolate the gene encoding the SP-B precursor from a lambda EMBL3 library of human embryonic kidney DNA. The entire SP-B gene was sequenced and is approximately 9.5 kb long, with 11 exons and 10 introns including a large 823-nucleotide 3' untranslated exon. The sequence derived from the exons differs from the cDNA sequence at 3 positions out of 2001, only one of which is in the translated region. Direct RNA sequencing indicated that the 5' untranslated region is only 14 nucleotides long. A number of putative regulatory elements were found upstream of the SP-B gene, including a GC box and several putative cAMP and glucocorticoid receptor binding sites. Several Alu repeats and a region of potential Z-DNA formation were found in the introns. Southern blotting of human genomic DNA probed with SP-B cDNA indicated the presence of only one SP-B gene in the human genome, and the gene was localized to chromosome 2. 10.1089/dna.1.1989.8.75
    Gene profiling studies in the neonatal ovine lung show enhancing effects of VEGF on the immune response. Sow Fatoumata B,Gallup Jack M,Meyerholz David K,Ackermann Mark R Developmental and comparative immunology Preterm and young neonates have an increased predisposition to respiratory distress syndrome (RDS) associated with an immature development of lung surfactant. Glucocorticoids (GCs) are the major immunomodulatory agents used to increase lung development and reduce the mortality and morbidity of preterm infants with RDS. However, their safety remains uncertain, and the precise mechanisms by which they improve lung function are unclear. In previous studies, we found that vascular endothelial growth factor (VEGF) enhances the innate immune response by respiratory epithelial cells, causes a monocytic infiltration into the lung, and reduces the severity of infection by respiratory syncytial virus (RSV), a respiratory pathogen known to affect preterm infants at a high prevalence. The purpose of this study is to measure the effects of VEGF administration on local immune responses in neonatal lambs, as the ovine lung is well suited for comparison to the human lung, due to similarities in alveolar development, immune responses, and RSV susceptibility. We hypothesized that VEGF induces the expression of genes necessary for host immune responses. We analyzed global gene expression profiles in the lungs of neonate lambs treated with VEGF by real-time qPCR. We report that VEGF induced the expression of chemokines (IL-8, RANTES, MCP-1), cytokines (IFN-gamma, IL-6, TNF-alpha, GMCSF), Toll-like receptor (TLR)-4, complement family members (C3, CFB, CFH) and collectins (SP-A, SP-D). These results suggest that VEGF can regulate local immune gene expression in vivo and should be further explored as a potential exogenous therapy for various lung diseases. 10.1016/j.dci.2009.01.004
    Analysis of genomic regions involved in regulation of the rabbit surfactant protein A gene in transgenic mice. Alcorn J L,Hammer R E,Graves K R,Smith M E,Maika S D,Michael L F,Gao E,Wang Y,Mendelson C R The American journal of physiology The gene encoding surfactant protein (SP) A, a developmentally regulated pulmonary surfactant-associated protein, is expressed in a lung-specific manner, primarily in pulmonary type II cells. SP-A gene transcription in the rabbit fetal lung is increased by cAMP. To delineate the genomic regions involved in regulation of SP-A gene expression, lines of transgenic mice carrying fusion genes composed of various amounts of 5'-flanking DNA from the rabbit SP-A gene linked to the human growth hormone structural gene as a reporter were established. We found that as little as 378 bp of 5'-flanking DNA was sufficient to direct appropriate lung cell-selective and developmental regulation of transgene expression. The same region was also sufficient to mediate cAMP induction of transgene expression. Mutagenesis or deletion of either of two DNA elements, proximal binding element and a cAMP response element-like sequence, previously found to be crucial for cAMP induction of SP-A promoter activity in transfected type II cells, did not affect lung-selective or temporal regulation of expression of the transgene; however, overall levels of fusion gene expression were reduced compared with those of wild-type transgenes. 10.1152/ajplung.1999.277.2.L349
    [Comparison of the effect of ambroxol and dexamethasone on the expression of pulmonary surfactant proteins in the fetal rat lungs]. Fu Xue-ming,Yu Jia-lin,Liu Guan-xin,Deng Bing Zhonghua er ke za zhi = Chinese journal of pediatrics OBJECTIVE:To investigate the effects of maternally administered dexamethasone and ambroxol on the mRNA levels of surfactant proteins (SP-A, SP-B and SP-C) expression in fetal rat lungs at gestational age day 19. METHODS:A 19-day fetal rat lung model was employed. In situ hybridization was used to detect the expression of SP-B mRNA in alveolar type II cell, and the levels of SP-A, SP-B and SP-C mRNAs were detected by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS:(1) SP-B mRNA was detected in situ in alveolar type II cells in fetal rat lung of day 19 gestational age; (2) In the late developmental period of fetal rat lungs, alveolar type II cells were also found around bronchus; (3) Comparing to beta-actin mRNA, the relative values of SP-A, SP-B and SP-C mRNAs were 0.81 +/- 0.26, 0.97 +/- 0.20 and 0.88 +/- 0.11 in fetal lung in the control group. The relative values of mRNAs of SP-A, SP-B and SP-C to beta-actin were 1.04 +/- 0.16, 1.28 +/- 0.29, 1.09 +/- 0.25 in fetal lungs of the ambroxol injected rats, and were 1.08 +/- 0.25, 1.23 +/- 0.35, 1.21 +/- 0.25 in fetal lungs of the dexamethasone injected rats, respectively. Both ambroxol and dexamethasone-treated rats had significantly higher mRNA expression of surfactant proteins compared to the control saline injected animals (P < 0.05). (4) There were no significant differences between ambroxol and dexamethasone in the effects of increasing expressions of surfactant protein mRNAs (P > 0.05). CONCLUSION:Antepartum administration of both ambroxol and dexamethasone can significantly increase fetal lung SP-A, SP-B and SP-C mRNAs expression.
    Developmental and hormonal regulation of SP-A gene expression in baboon fetal lung. Seidner S R,Smith M E,Mendelson C R The American journal of physiology In the present study, we found that surfactant protein A (SP-A) mRNA levels, which are barely detectable in baboon fetal lung at midgestation (92 days), are increased approximately four fold between 125 and 140 days gestation, approximately 7-fold between 140 and 160 days, and approximately 1.5-fold between 160 and 174 days gestation. We also investigated the effects of dibutyryl-adenosine 3',5'-cyclic monophosphate (DB-cAMP) and dexamethasone (Dex) on SP-A gene expression in lung explants from fetal baboons at 92, 125, 140, 160, and 174 days of gestation (term = 184 days). SP-A mRNA levels, which were barely detectable in lung tissues from 92- and 125-day fetal baboons before culture, were induced after incubation for 5 days in serum-free medium and were markedly stimulated by DBcAMP. Dex caused a dose-dependent inhibition of SP-A mRNA levels and antagonized the stimulatory effect of DBcAMP. SP-A mRNA was detectable in lung tissues from 140-day fetal baboons before culture; the levels were further induced after culture and were increased greatly by DBcAMP. Again, Dex antagonized the induction of SP-A mRNA by DBcAMP. The stimulatory effects of DBcAMP and inhibitory effects of Dex on SP-A mRNA levels in lung tissues of 92- to 140-day gestational age fetal baboons were highly similar to those observed in studies using lung explants of midgestation human abortuses. By contrast, SP-A mRNA was present in relatively high levels in lung tissues of 160- and 174-day fetal baboons before culture and was relatively unaffected after incubation for 5 days in control medium. In lung explants from 160- and 174-day fetal baboons, the stimulatory effect of DBcAMP and inhibitory effect of Dex on SP-A mRNA levels were relatively modest compared with the effects of these agents on SP-A mRNA in fetal lung tissues from 92-, 125-, and 140-day gestational age fetuses. These findings suggest that, with increased lung maturation and the developmental induction of SP-A gene expression, there is a decrease in responsiveness of the fetal lung to the stimulatory effects of cAMP and inhibitory effects of glucocorticoids on SP-A gene expression. 10.1152/ajplung.1996.271.4.L609
    Characterization of mRNA transcripts and organization of human SP-A1 and SP-A2 genes. McCormick S M,Boggaram V,Mendelson C R The American journal of physiology In the present study, we have characterized the mRNA transcripts and intron-exon organization of the human surfactant protein (SP)A1 and SP-A2 genes. By primer extension analysis of mRNA isolated from human fetal lung explants using an oligonucleotide primer to exon II (as delineated in the SP-A1 gene), a minimum of nine primer extended transcripts was observed. Rapid amplification of cDNA ends was used to amplify the primer extended transcripts for sequence analysis. Sequence analysis of 47 full-length primer extended cDNAs and comparison with the sequences of the genes encoding SP-A1 and SP-A2 revealed four different classes of transcripts of the SP-A2 gene and five different classes of transcripts of the gene encoding SP-A1. A major difference between SP-A2 and SP-A1 mRNA transcripts is that SP-A2 transcripts are always comprised of sequences contained within six exons; the extra exon in SP-A2 (exon II of VI) encodes additional 5'-untranslated sequence and is located between exons I and II of SP-A1. By contrast, the majority of transcripts of the SP-A1 gene are comprised of sequences contained within five exons. In the cases of both SP-A1 and SP-A2 genes, a small proportion of the mRNA transcripts contain sequences present in alternate exons. In addition, the majority of the SP-A1 mRNA transcripts are initiated 5 bp downstream of the transcription initiation site of SP-A2. In our companion paper [McCormick and Mendelson. Am. J. Physiol. 266 (Lung Cell. Mol. Physiol. 10): L367-L374, 1994], we report that the SP-A1 and SP-A2 genes are differentially regulated during development and by adenosine 3',5'-cyclic monophosphate and glucocorticoids in human fetal lung in culture. 10.1152/ajplung.1994.266.4.L354
    Surfactant protein B in human fetal lung: developmental and glucocorticoid regulation. Beers M F,Shuman H,Liley H G,Floros J,Gonzales L W,Yue N,Ballard P L Pediatric research Pulmonary surfactant protein B (SP-B) enhances phospholipid film formation in vitro and is essential for normal surfactant function in vivo. We examined human fetal lung before and during explant culture for content and cellular localization of SP-B mRNA and protein. SP-B mRNA was low in preculture specimens (18-20 wk) but hybridization signal increased over epithelial cells during culture and was enhanced by dexamethasone treatment (10 nM). SP-B immunofluorescence was very low in preculture specimens, increased during culture, and was uniformly intense in epithelial cells of dexamethasone-treated tissue. With a newly developed immunoassay, SP-B protein was undetectable in preculture lung (< 2% of adult), appeared during culture (26% of adult), and was further increased approximately 3-fold by dexamethasone treatment (86% of adult); lung tissue of two newborn infants contained 7-9-fold more SP-B than is found in the adult. Using Western blot with enhanced chemiluminescence, mature SP-B was undetectable in 16-wk specimens but was present in 19-24-wk preculture tissue at 0.2-2.9% of the adult level. By comparison, SP-B mRNA content is 14 and 50% of adult level in 19- and 24-wk lung tissue, respectively; levels increase 3-fold during culture and a further 3-fold with dexamethasone. Based on these observed differences between mRNA and protein content, we conclude that basal SP-B gene expression in epithelial cells of human fetal lung is regulated primarily at the level of translation or protein stability, whereas glucocorticoids act transcriptionally. We speculate that SP-B protein accumulates only as type II cells differentiate and acquire lamellar bodies for processing and storage of SP-B. 10.1203/00006450-199511000-00007
    Expression and glucocorticoid regulation of surfactant protein C in human fetal lung. Solarin K O,Ballard P L,Guttentag S H,Lomax C A,Beers M F Pediatric research The hydrophobic surfactant protein C (SP-C) is known to modulate the biophysical properties of surfactant phospholipid. Although SP-C mRNA has been demonstrated in human fetal lung, there is limited information regarding developmental expression and processing of proSP-C protein. Two epitope-specific human proSP-C antisera, anti-hCPROSP-C (His59-Ser72) and anti-hCTERMSP-C (Gly162-Gly175), were generated to complement previously produced anti-NPROSP-C (Met10-Gln23) for the study of proSP-C expression in human fetal lung. Western blotting and immunocytochemistry detected expression of proSP-C protein by 12-16 wk of gestation. ProSP-C immunoreactivity of preculture lung, limited to expression of proSP-C21 in airway epithelial cells, was markedly enhanced by culture of lung explants in dexamethasone. To examine synthesis of proSP-C, homogenates from explants were labeled with 35S-Met/Cys for 0.5-4 h. Immunoprecipitation with anti-NPROSP-C detected 35S-proSP-C21 by 30 min and, after 2 h of labeling, there was a 15-fold increase in 35S-proSP-C21 in dexamethasone-treated lungs versus controls. Synthesis of proSP-C21 was followed by the appearance of a 24-kD form and smaller processing intermediates including 6-10-kD forms. Posttranslational processing of proSP-C21 was not observed in control explants. SP-C(6-10) were not recognized by either anti-CPROSP-C or anti-hCTERMSP-C. These results indicate that low level expression of proSP-C protein first occurs in epithelial cells early in the second trimester and that expression can be enhanced by dexamethasone. Initial posttranslational processing of human proSP-C involves modification of proSP-C21 to SP-C24 and subsequent proteolysis of C-terminal propeptide domains. We speculate that absence of low Mr intermediates in unstimulated second trimester fetal lung tissue reflects developmental and glucocorticoid dependent regulation of proSP-C21 synthesis and posttranslational processing. 10.1203/00006450-199709000-00017
    Regulation of expression of the gene encoding the major surfactant protein (SP-A) in human fetal lung in vitro. Disparate effects of glucocorticoids on transcription and on mRNA stability. Boggaram V,Smith M E,Mendelson C R The Journal of biological chemistry We previously observed that dexamethasone had a biphasic effect on the levels of mRNA encoding the major surfactant protein (SP-A) in human fetal lung in vitro; at concentrations of 10(-10) and 10(-9) M, dexamethasone caused an increase in the levels of SP-A mRNA, whereas at concentrations of greater than 10(-8) M, the steroid had a pronounced inhibitory effect on SP-A mRNA levels. It was also found that dexamethasone antagonized the stimulatory effect of dibutyryl cyclic AMP (Bt2cAMP) on SP-A mRNA levels in human fetal lung in vitro. It was our objective, in the present study, to characterize further the effects of dexamethasone and Bt2cAMP on SP-A mRNA levels in human fetal lung tissue and to determine whether such effects are associated with comparable changes in the transcriptional activity of the SP-A gene. We found that the action of dexamethasone (10(-7) M) to reduce the levels of SP-A mRNA in control and Bt2cAMP-treated fetal lung explants was evident within 2 h of its addition to the culture medium; SP-A mRNA was reduced to barely detectable levels in control and in Bt2cAMP-treated tissues after 24 h of dexamethasone treatment. The action of dexamethasone to reduce SP-A mRNA levels was not prevented by co-incubation with either actinomycin D or cycloheximide. In contrast to its dose-related biphasic effects on the levels of SP-A mRNA, we found that dexamethasone caused a dose-dependent stimulation of SP-A gene transcription. Bt2cAMP also increased the transcriptional activity of the SP-A gene in the human fetal lung in vitro. In fetal lung explants incubated in the presence of dexamethasone plus Bt2cAMP, a synergistic induction of SP-A gene transcription was observed at concentrations of dexamethasone of 10(-9)-10(-7) M. Our findings are indicative that the stimulatory effects of dexamethasone (10(-10)-10(-9) M) on SP-A mRNA levels are reflective of a stimulatory effect of the steroid on SP-A gene transcription, whereas the inhibitory effects of dexamethasone (10(-7) M) on SP-A mRNA levels are the result of a dominant effect of the steroid in elevated concentrations to reduce SP-A mRNA stability.
    Posttranscriptional regulation of surfactant protein-A messenger RNA in human fetal lung in vitro by glucocorticoids. Boggaram V,Smith M E,Mendelson C R Molecular endocrinology (Baltimore, Md.) Surfactant protein-A (SP-A), the major pulmonary surfactant-associated protein, is a developmentally and hormonally regulated sialoglycoprotein of about 35,000 mol wt. In previous studies we observed that dexamethasone has dose-dependent biphasic effects on the levels of SP-A and its mRNA in human fetal lung in vitro. At concentrations of 10(-10)-10(-9) M, dexamethasone increased the levels of SP-A and its mRNA over those of control tissues, whereas at concentrations greater than or equal to 10(-8) M, the steroid was markedly inhibitory. Our findings suggest that the inhibitory action of dexamethasone (greater than 10(-8) M) on SP-A mRNA levels was mediated by an effect to reduce SP-A mRNA stability, since the steroid caused a dose-dependent increase in the rate of transcription; however, an effect to increase transcription with premature termination leading to instability of mRNA transcripts could not be ruled out. In the present investigation we have studied in detail the mechanisms underlying the biphasic effects of glucocorticoids on SP-A mRNA levels in human fetal lung tissues in vitro. Our findings indicate that dexamethasone (10(-7) M) has no adverse effect on the elongation of nascent mRNA transcripts throughout the SP-A gene; elongation of SP-A mRNA transcripts in dexamethasone-treated tissue explants was similar to that observed in tissues incubated in control medium or medium containing (Bu)2cAMP. Therefore, premature termination of SP-A mRNA transcription leading to the instability of SP-A mRNA can be ruled out. On the other hand, we found that dexamethasone (10(-7) M) had a pronounced effect to reduce the apparent half-life of SP-A mRNA; in control explants maintained in the presence of actinomycin-D to block gene transcription, the SP-A mRNA half-life was estimated to be 11.4 h, whereas in tissues also treated with dexamethasone, the SP-A mRNA half-life was reduced by more than 60% to 5.0 h. Dexamethasone also was found to have dose-dependent effects on the degradation of SP-A mRNA. After 12 h of incubation in the presence of actinomycin-D and dexamethasone at 10(-9) and 10(-7) M, the levels of SP-A mRNA were reduced by 50% and 80%, respectively, compared to those in tissue incubated with actinomycin-D alone. The inhibitory effects of glucocorticoids on SP-A mRNA levels were completely reversible and were blocked by the glucocorticoid antagonist RU486.(ABSTRACT TRUNCATED AT 400 WORDS) 10.1210/mend-5-3-414
    Developmental and hormonal regulation of surfactant protein A (SP-A) gene expression in fetal lung. Mendelson C R,Acarregui M J,Odom M J,Boggaram V Journal of developmental physiology Pulmonary surfactant is a developmentally-regulated lipoprotein synthesized and secreted by the type II cells of the pulmonary alveolus where surfactant glycerophospholipids and proteins act to reduce surface tension at the alveolar air-liquid interface. Surfactant protein A (SP-A), the major surfactant-associated protein, appears to serve an important role in surfactant function and reutilization by type II cells. SP-A synthesis and gene expression are initiated in fetal lung tissue in concert with the developmental induction of surfactant glycerophospholipid synthesis. In studies using midtrimester human fetal lung explants maintained in organ culture, we have observed that cyclic AMP and glucocorticoids have pronounced effects on morphologic development and on the levels of SP-A gene expression. Cyclic AMP analogues act primarily to induce SP-A gene transcription; whereas, glucocorticoids have complex effects at both the transcriptional and posttranscriptional levels. We also have found that human fetal lung in vitro secretes into the culture medium relatively large amounts of prostaglandins (PG) PGE2 and PGF2 alpha and the PGI2 and thromboxane A2 metabolites, 6-keto-PGF1 alpha and TxB2, respectively. The prostaglandin synthesis inhibitor, indomethacin, markedly inhibits SP-A gene expression and cyclic AMP formation by human fetal lung in culture; the inhibitory effect of indomethacin on SP-A gene expression can be prevented by simultaneous incubation with either Bt2cAMP or PGE2. These findings are suggestive that prostaglandins acting through cyclic AMP also may serve an important role in the regulation of SP-A gene expression in human fetal lung tissue.
    Regulation of pulmonary surfactant apoprotein SP 28-36 gene in fetal human lung. Ballard P L,Hawgood S,Liley H,Wellenstein G,Gonzales L W,Benson B,Cordell B,White R T Proceedings of the National Academy of Sciences of the United States of America Pulmonary surfactant stabilizes lung alveoli, preventing respiratory failure and hyaline membrane disease in premature infants. In addition to lipids, surfactant contains apoproteins that are thought to be critical for normal surfactant function. We have examined the ontogeny and regulation of the major surfactant-associated protein of molecular mass 28-36 kDa (SP 28-36) in human fetal lung. SP 28-36 was not detected in tissue from second trimester abortuses by either immunoblot analysis or enzyme-linked immunosorbent assay (less than 0.02 microgram per mg of DNA). Levels of mRNA for SP 28-36, assayed by cDNA hybridization, were low or undetectable in all preculture specimens. The concentration of saturated phosphatidylcholine in lung tissue was 30% of the adult value with no apparent increase between 15 and 24 weeks gestation. SP 28-36 content increased during explant culture in the absence of serum and hormones, exceeding adult levels (3.2 +/- 1.0 micrograms per mg of DNA) after 5 days. In cultures treated with triiodothyronine (2 nM) and dexamethasone (10 nM), hormones that regulate phosphatidylcholine synthesis, the increase in SP 28-36 was accelerated (treated/control ratio was 7.1 and 3.4 at 3 and 5 days, respectively). Levels of mRNA for SP 28-36 also increased during culture and were stimulated by hormones (treated/control = 8.6 and 1.9 at 3 and 5 days, respectively). SP 28-36 and its mRNA increased similarly in the presence of dexamethasone alone, whereas triiodothyronine alone had no apparent effect. The molecular weight and charge pattern was similar for SP 28-36 of adult and cultured fetal tissue. These findings indicate that expression of the SP 28-36 gene is low during the second trimester, increases during explant culture, and is accelerated by glucocorticoid treatment. 10.1073/pnas.83.24.9527
    In vivo regulation of surfactant proteins by glucocorticoids. Fisher J H,McCormack F,Park S S,Stelzner T,Shannon J M,Hofmann T American journal of respiratory cell and molecular biology Surfactant proteins have key roles in regulating surfactant secretion, in recycling, and in the assembly of the surfactant monolayer but little is known about their regulation in vivo. Surfactant proteins SP-A, SP-B, and SP-C have been shown to be upregulated by glucocorticoids in vitro, but the role of glucocorticoids in the physiologic regulation of surfactant protein synthesis remains unknown. We have studied the effects of exogenously administered glucocorticoids on the regulation of steady-state surfactant protein mRNA accumulation. We have also studied the effects of adrenalectomy on the accumulation of the surfactant protein mRNAs. Surfactant protein genes appear to have quantitatively different responses to exogenously administered glucocorticoids, with SP-C mRNA increasing at the lowest dose, SP-A and SP-B mRNA increasing in response to similar glucocorticoids doses but with SP-B yielding the highest maximum response. Adrenalectomy, however, does not alter surfactant protein mRNA levels. These observations support a minor role for glucocorticoids in maintaining the steady-state accumulation of surfactant protein mRNA. Adrenalectomy decreases total pulmonary SP-A when compared to sham-operated animals in the absence of changes in its mRNA. Therefore, glucocorticoids may have translational or post-translational effects that regulate total pulmonary SP-A accumulation, but the effects appear to be minor. These findings support a potential role for the adrenal in the pulmonary response to stress and demonstrate for the first time differential accumulation of the surfactant protein mRNAs to glucocorticoids in vivo. 10.1165/ajrcmb/5.1.63
    Glucocorticoid enhances pulmonary surfactant protein B gene transcription. O'Reilly M A,Clark J C,Whitsett J A The American journal of physiology The effect of glucocorticoid on the regulation of pulmonary surfactant protein B (SP-B) synthesis was studied in a human pulmonary adenocarcinoma cell line. Northern blot analysis demonstrated a marked increase in SP-B mRNA expression after treatment with dexamethasone for 48 h. Actinomycin D, puromycin, or cycloheximide blocked the induction of SP-B mRNA by glucocorticoid. Nuclear run-on experiments demonstrated that the effects of dexamethasone on SP-B mRNA were due in part to increased transcription of the SP-B gene. However, during this time period, there was a discrepancy between SP-B gene transcription, which was increased only 2- to 4-fold, and SP-B mRNA, which increased 60- to 150-fold after treatment with dexamethasone. In the presence of actinomycin D, SP-B mRNA was relatively stable, decreasing slowly in the presence or absence of glucocorticoid. In contrast to the relative stability of SP-B mRNA in the presence of actinomycin D, SP-B mRNA was markedly decreased after exposure to puromycin, supporting the premise that continued protein synthesis, rather than transcription alone, is required for maintenance of SP-B mRNA levels. Induction of SP-B expression by glucocorticoids was dependent on enhanced SP-B gene transcription and was also dependent on continued protein synthesis. The discrepancy between the relative enhancement of SP-B transcription and SP-B mRNA suggests that posttranscriptional factors influence SP-B expression in this cell line. 10.1152/ajplung.1991.260.2.L37
    Regulation of translation by upstream translation initiation codons of surfactant protein A1 splice variants. Tsotakos Nikolaos,Silveyra Patricia,Lin Zhenwu,Thomas Neal,Vaid Mudit,Floros Joanna American journal of physiology. Lung cellular and molecular physiology Surfactant protein A (SP-A), a molecule with roles in lung innate immunity and surfactant-related functions, is encoded by two genes in humans: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The mRNAs from these genes differ in their 5'-untranslated regions (5'-UTR) due to differential splicing. The 5'-UTR variant ACD' is exclusively found in transcripts of SP-A1, but not in those of SP-A2. Its unique exon C contains two upstream AUG codons (uAUGs) that may affect SP-A1 translation efficiency. The first uAUG (u1) is in frame with the primary start codon (p), but the second one (u2) is not. The purpose of this study was to assess the impact of uAUGs on SP-A1 expression. We employed RT-qPCR to determine the presence of exon C-containing SP-A1 transcripts in human RNA samples. We also used in vitro techniques including mutagenesis, reporter assays, and toeprinting analysis, as well as in silico analyses to determine the role of uAUGs. Exon C-containing mRNA is present in most human lung tissue samples and its expression can, under certain conditions, be regulated by factors such as dexamethasone or endotoxin. Mutating uAUGs resulted in increased luciferase activity. The mature protein size was not affected by the uAUGs, as shown by a combination of toeprint and in silico analysis for Kozak sequence, secondary structure, and signal peptide and in vitro translation in the presence of microsomes. In conclusion, alternative splicing may introduce uAUGs in SP-A1 transcripts, which in turn negatively affect SP-A1 translation, possibly affecting SP-A1/SP-A2 ratio, with potential for clinical implication. 10.1152/ajplung.00058.2014
    Glucocorticoid regulation of surfactant-associated proteins in rabbit fetal lung in vivo. Durham P L,Wohlford-Lenane C L,Snyder J M The Anatomical record The effects of a maternally administered synthetic glucocorticoid, betamethasone, on the levels of mRNA for the surfactant proteins SP-A, SP-B, and SP-C and on the levels of SP-A protein were investigated in day 27 gestational age rabbit fetal lung tissue. Betamethasone administration to the pregnant rabbit caused approximately a twofold increase in the fetal lung level of SP-A protein and a threefold increase in fetal lung SP-A mRNA levels when compared to levels in fetuses obtained from saline-treated or uninjected animals. SP-B mRNA was increased fourfold in fetal lung tissue obtained from glucocorticoid-treated pregnant does when compared to levels in fetuses of uninjected pregnant does. However, SP-B mRNA levels in fetal lung tissue from saline-injected controls were also significantly elevated, approximately twofold, when compared to fetal lung SP-B mRNA levels in the uninjected control condition. SP-C mRNA levels in lung tissue of fetuses from both saline-injected and betamethasone-injected pregnant does were increased similarly, approximately twofold, over SP-C mRNA levels in fetal lung tissue obtained from uninjected control does. These data are suggestive that betamethasone treatment increases fetal lung SP-A and SP-B mRNA levels and that maternal stress alone can increase the expression of SP-B and SP-C mRNA in rabbit fetal lung tissue. Using in situ hybridization, SP-A mRNA was shown to be present primarily in alveolar type II cells in fetuses of control and saline-injected does. However, SP-A mRNA was easily detected in both alveolar type II cells and bronchiolar epithelial cells of rabbit fetal lung tissue following maternal betamethasone treatment. In contrast, SP-B and SP-C mRNA were present only in alveolar type II cells of lung tissue obtained from fetuses of control, saline, or betamethasone-treated does. Thus maternal administration of glucocorticoids increased SP-A protein as well as SP-A and SP-B mRNA levels in rabbit fetal lung tissue. SP-A mRNA was localized to both alveolar type II cells and in smaller amounts in bronchiolar epithelial cells of rabbit fetal lung tissue. However, SP-B and SP-C mRNA were detected only in alveolar type II cells. 10.1002/ar.1092370310
    Glucocorticoid-induced effects on pattern formation and epithelial cell differentiation in early embryonic rat lungs. Oshika E,Liu S,Ung L P,Singh G,Shinozuka H,Michalopoulos G K,Katyal S L Pediatric research In this study, we examined the effects of dexamethasone (DEX) on airway branching and subsequent lung maturation. DEX treatment of fetal rat lung explants was initiated during the early pseudoglandular stage of development. Day 14 fetal lung explants were cultured with and without DEX for 4 d. Explants treated with 10 nM or higher concentrations of DEX showed features of both distorted and accelerated maturation. DEX-treated lungs had growth retardation, distorted branching, dilated proximal tubules, and suppressed proliferation of epithelial cells of the distal tubules. Several biochemical and morphologic features of accelerated maturation were also observed: 1) the epithelial cells lining the distal tubules (prospective respiratory airways) were generally cuboidal or flattened; 2) the cuboidal cells often contained lamellar bodies and abundant glycogen; 3) rudimentary septa and large airspace were present; 4) mesenchymal tissue was attenuated and compressed between adjacent epithelial tubules; 5) the distribution of SP-C mRNA in distal tubules was more mature, with individual and clusters of cells expressing SP-C transcripts; and 6) the transcript levels of several genes related to epithelial growth [keratinocyte growth factor (KGF), KGF receptor, and hepatocyte growth factor receptor] and differentiation [surfactant proteins, SP-A, SP-B and SP-C and the Clara cell secretory protein, CC10] were precociously increased. These results show that DEX treatment of the lung during the early pseudoglandular stage accelerates the acquisition of several features of advanced maturation that normally accompany late stages of fetal development. We postulate that KGF mediates at least some effects of DEX on lung maturation and gene expression. 10.1203/00006450-199804001-01814
    Maternal low-protein diet affects epigenetic regulation of hepatic mitochondrial DNA transcription in a sex-specific manner in newborn piglets associated with GR binding to its promoter. Jia Yimin,Li Runsheng,Cong Rihua,Yang Xiaojing,Sun Qinwei,Parvizi Nahid,Zhao Ruqian PloS one Mitochondrial oxidative phosphorylation (OXPHOS) plays an important role in energy homeostasis by controlling electron transfer and ATP generation. Maternal malnutrition during pregnancy affects mitochondrial (mt) DNA-encoded OXPHOS activity in offspring, yet it is unknown whether epigenetic mechanism is involved in the transcriptional regulation of mtDNA-encoded OXPHOS genes. In this study, 14 primiparous purebred Meishan sows were fed either standard- (SP, 12% crude protein) or low-protein (LP; 6% crude protein) diets throughout gestation, and the hepatic expression and transcriptional regulation of mtDNA-encoded OXPHOS genes were analyzed in newborn piglets. Maternal low protein diet decreased hepatic mtDNA copy number in males, but not in females. LP male piglets had significantly higher hepatic AMP concentration and low energy charge, which was accompanied by enhanced mRNA expression of NADH dehydrogenase subunits 6, cytochrome c oxidase subunit 1, 2, 3 and cytochrome b, as well as increased cytochrome c oxidase enzyme activity. In contrast, LP female piglets showed significantly lower hepatic AMP concentrations and higher energy charge with no alterations in OXPHOS gene expression. Moreover, LP males demonstrated higher glucocorticoid receptor (GR) binding to the mtDNA promoter compared with SP males, which was accompanied by lower cytosine methylation and hydroxymethylation on mtDNA promoter. Interestingly, opposite changes were seen in females, which showed diminished GR binding and enriched cytosine methylation and hydroxymethylation on mtDNA promoter. These results suggest that maternal low protein diet during pregnancy causes sex-dependent epigenetic alterations in mtDNA-encoded OXPHOS gene expression, possibly GR is involved in mtDNA transcription regulation. 10.1371/journal.pone.0063855
    Glucocorticoids induce the expression of the leptin gene through a non-classical mechanism of transcriptional activation. De Vos P,Lefebvre A M,Shrivo I,Fruchart J C,Auwerx J European journal of biochemistry Leptin is a hormone which is produced in adipose tissue and which plays a role in the regulation of energy homeostasis. The expression of the ob gene, encoding leptin, is under multi-hormonal control. We have shown previously that high doses of glucocorticoids are positive regulators of leptin expression in rats and that they concomitantly reduce food intake and body mass gain in these animals. In the present report we analyse the molecular mechanism of this glucocorticoid regulation of leptin expression. In cultured rat adipocytes dexamethasone induces leptin mRNA levels, an effect not inhibited by the protein synthesis inhibitor cycloheximide. In addition, our data indicate that the induction of the expression of the leptin gene by dexamethasone is at least in part due to a transcriptional activation that is mediated by the glucocorticoid receptor. Deletion mapping of the human leptin promoter shows that cis-elements involved in the glucocorticoid effect are located between -55 and +31 relative to the transcription initiation site. Since this region does not contain a binding site for the glucocorticoid receptor, the effect does not rely on the classical molecular mechanism of glucocorticoid receptor action. A role of C/EBP and Sp-1 in mediating this glucocorticoid effect was furthermore excluded. Multiple nuclear factors from 3T3-L1 preadipocytes interact with this promoter region of the human leptin gene and may be potential mediators of the induction by glucocorticoids. 10.1046/j.1432-1327.1998.2530619.x
    Dexamethasone-induced prenatal alveolar wall thinning is associated with a decrease in EIIIA+ fibronectin isoform in the fetal rat lung. Arai Hirokazu,Kikuchi Wataru,Ishida Akira,Takada Goro Biology of the neonate BACKGROUND:Glucocorticoid hormones play an important role in architectural and biochemical lung maturation. Although much of the molecular mechanism of their action in the lung is not fully understood, glucocorticoids directly or indirectly regulate lung maturation. Indirect effects of glucocorticoids may involve the modulation of cell-cell or cell-matrix interactions. Fibronectin (FN) is the major constituent of the pulmonary extracellular matrix and exists in multiple isoforms arising from alternative RNA splicing. EIIIA is the major alternatively spliced segment, and its expression is regulated in a spatiotemporal and oncodevelopmental manner. OBJECTIVES:The present study focuses on the regulation of EIIIA-containing FN isoforms (referred to as EIIIA+ FN) by glucocorticoids in the developing lung. METHODS:Dexamethasone (DEX) or saline was injected daily into pregnant rats from day 15 of gestation (term = day 22) until 24 h before sacrifice. The expression of EIIIA+ FN and proliferating cell nuclear antigen (PCNA), a biochemical marker for cell proliferation, was investigated in the fetal rat lung. RESULTS:At day 20 of gestation (the canalicular stage), the DEX-treated lung showed a significant decrease in weight and saccular septal wall thickness, while the messenger RNA expression of the surfactant protein SP-B was increased in the DEX-treated lung, as compared with the control lung. The expression of EIIIA+ FN and PCNA around the distal airspaces was less extensive in the DEX-treated lung than in the control lung at day 20 of gestation. CONCLUSIONS:Given the finding in vitro that EIIIA+ FN regulated the cell cycle, our results suggest that the change of EIIIA+ FN expression in the DEX-treated lung affected pulmonary cell proliferation. 10.1159/000082119
    Activation of the GLP-1 Receptor by Liraglutide Increases ACE2 Expression, Reversing Right Ventricle Hypertrophy, and Improving the Production of SP-A and SP-B in the Lungs of Type 1 Diabetes Rats. Romaní-Pérez Marina,Outeiriño-Iglesias Verónica,Moya Christian M,Santisteban Pilar,González-Matías Lucas C,Vigo Eva,Mallo Federico Endocrinology Diabetes alters microvascular function in the vascular beds of organs, including the lungs. Cardiovascular complications of pulmonary vascular affectation may be a consequence of the overactivation of the vasoconstrictive and proliferative components of the renin-angiotensin system. We previously reported that pulmonary physiology and surfactant production is improved by the glucagon-like peptide 1 receptor (GLP-1R) agonist liraglutide (LIR) in a rat model of lung hypoplasia. Because we hypothesized that streptozotocin-induced diabetes rats would show deficiencies in lung function, including surfactant proteins, and develop an imbalance of the renin-angiotensin system in the lungs. This effect would in turn be prevented by long-acting agonists of the GLP-1R, such as LIR. The induction of diabetes reduced the surfactant protein A and B in the lungs and caused the vasoconstrictor component of the renin-angiotensin system to predominate, which in turn increased angiotensin II levels, and ultimately being associated with right ventricle hypertrophy. LIR restored surfactant protein levels and reversed the imbalance in the renin-angiotensin system in this type 1 diabetes mellitus rat model. Moreover, LIR provoked a strong increase in angiotensin-converting enzyme 2 expression in the lungs of both diabetic and control rats, and in the circulating angiotensin(1-7) in diabetic animals. These effects prompted complete reversion of right ventricle hypertrophy. The consequences of LIR administration were independent of glycemic control and of glucocorticoids, and they involved NK2 homeobox 1 signaling. This study demonstrates by first time that GLP-1R agonists, such as LIR, might improve the cardiopulmonary complications associated with diabetes. 10.1210/en.2014-1685
    Association of Surfactant Protein with Expression of and in Rabbit Fetal Lung. Chung Sung-Hoon,Bae Chong-Woo International journal of medical sciences genes regulate organ formation and identity of the embryo, and expressed in specific temporo-spatial patterns in the developing embryo. We compared the expression levels of the , , surfactant protein , and genes in immature and mature rabbit fetal lung tissues, and to uncover roles for Hoxa5, Hoxb5, SP-A, and SP-B. Cesarean sections were performed after rabbits were divided into two groups of 30-31 days of gestation (term group, n = 24) and 26-27 days of gestation (preterm group, n = 24). mRNA levels of , , , and were compared by quantitative reverse transcriptase polymerase chain reaction, and protein expression of Hoxa5 and Hoxb5 was compared by western blot analysis. Fetal lung tissue histology was observed by hematoxylin and eosin (H&E) staining. The relative expression ratios of and mRNA in the term to preterm groups were 2.45:1 and 2.94:1, respectively. mRNA and protein levels decreased in the term group, with a relative expression ratio of 0.48:1 and 0.50:1, however, mRNA and protein levels increased in the term group with a relative expression ration of 2.99:1 and 2.33:1, respectively, for the term to preterm groups. Moreover, a significant positive correlation was found between the expression of and , in the term group. gene may be essential for the expression of and in term rabbits. The gene may be an important factor for lung maturation in preterm rabbits. 10.7150/ijms.20721
    Mechanical strain and dexamethasone selectively increase surfactant protein C and tropoelastin gene expression. Nakamura T,Liu M,Mourgeon E,Slutsky A,Post M American journal of physiology. Lung cellular and molecular physiology Physical forces derived from fetal breathing movements and hormones such as glucocorticoids are implicated in regulating fetal lung development. To elucidate whether the different signaling pathways activated by physical and hormonal factors are integrated and coordinated at the cellular and transcriptional levels, organotypic cultures of mixed fetal rat lung cells were subjected to static culture or mechanical strain in the presence and absence of dexamethasone. Tropoelastin and collagen type I were used as marker genes for fibroblasts, whereas surfactant protein (SP) A and SP-C were used as marker genes for distal epithelial cells. Mechanical strain, but not dexamethasone, significantly increased SP-C mRNA expression. Tropoelastin mRNA expression was upregulated by both mechanical strain and dexamethasone. No additive or synergistic effect was observed when cells were subjected to mechanical stretch in the presence of dexamethasone. Neither mechanical strain nor dexamethasone alone or in combination had any significant effect on the expression of SP-A mRNA. Dexamethasone decreased collagen type I mRNA expression, whereas mechanical strain had no effect. The increases in tropoelastin and SP-C mRNA levels induced by mechanical strain and/or dexamethasone were accompanied by increases in their heterogeneous nuclear RNA. In addition, the stretch- and glucocorticoid-induced alterations in tropoelastin and SP-C mRNA expression were abrogated with 10 microg/ml actinomycin D. These findings suggest that tropoelastin and SP-C genes are selectively stimulated by physical and/or hormonal factors at the transcriptional level in fetal lung fibroblasts and distal epithelial cells, respectively. 10.1152/ajplung.2000.278.5.L974
    Exposure of rabbit fetal lung to glucocorticoids in vitro does not enhance transcription of the gene encoding pulmonary surfactant-associated protein-B (SP-B). Xu J,Possmayer F Biochimica et biophysica acta We have investigated the ontogeny and hormonal regulation of both synthesis rates and cellular accumulation of the mRNA for surfactant-associated protein B (SP-B) in rabbit fetal lung. The developmental pattern for SP-B mRNA synthesis increased as a function of gestational age and paralleled that for SP-B mRNA levels except on days 22-26 of gestation where relatively higher levels of gene transcription were observed. Time-course studies with explants from 26- and 30-day fetal lung maintained in culture revealed a gradual increase in mRNA levels and a much smaller increase in gene transcription relative to adult values. Within 48 h of exposure of 26-day explants to dexamethasone at 10(-8) M there was a rapid increase in SP-B mRNA levels to 7-fold adult levels. A similar overall although somewhat slower and attenuated pattern was observed with 30-day explants. Dexamethasone at 10(-8) M had no effect on SP-B gene transcription with explants of either gestational age. We conclude that the major effect of dexamethasone treatment in vitro on SP-B mRNA levels appears to be post-transcriptional and there are small but distinct differences in the effects of glucocorticoids on SP-B mRNA levels with explant cultures from early and late stages of fetal lung maturation. 10.1016/0005-2760(93)90199-j
    Sexually dimorphic effects of maternal dietary protein restriction on fetal growth and placental expression of 11β-HSD2 in the pig. Shang Yueli,Jia Yimin,Sun Qinwei,Shi Wei,Li Runsheng,Wang Song,Sui Shiyan,Zhao Ruqian Animal reproduction science Placental 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) inactivates glucocorticoids (GCs) to protect fetuses from over-exposure to maternal GCs, yet how maternal malnutrition affects placental 11β-HSD2 expression is unknown. In this study, Meishan sows were fed standard-protein (SP) or low-protein (LP, 50% of SP) diets and fetuses/newborn piglets were weighed and the corresponding placenta and umbilical cord blood were collected on gestational day 70 and the day of parturition. Significant growth retardation was observed in female, but not male, fetuses (P < 0.05) and the newborns (P < 0.01) of the LP group, which was accompanied by sexually dimorphic expression of 11β-HSD2 in placentas. Female fetuses in LP group showed significant decrease in placental 11β-HSD2 protein content (P < 0.05) and enzyme activity (P < 0.05), whereas male fetuses demonstrated significantly enhanced placental 11β-HSD2 activity (P < 0.05). Serum cortisol levels were significantly higher (P < 0.05) in male piglets compared to females, and the effects of maternal protein restriction on thyroid hormones (T3 and T4) in the umbilical cord blood were also sex dimorphic. Male piglets in LP group had significantly higher T3 (P < 0.01) and lower T4 (P < 0.01), whereas female piglets showed significantly lower T4 (P < 0.01) with no change in T3. As a result, male piglets in LP group exhibited significantly higher T3/T4 ratio compared to female counterparts. These results indicate that the effects of maternal protein restriction on placental 11β-HSD2 expression are gender-dependent in the pig, and thyroid hormones may be involved in such effects. 10.1016/j.anireprosci.2015.07.001
    Progesterone Antagonizes Dexamethasone-Regulated Surfactant Proteins In Vitro. Kunzmann Steffen,Ottensmeier Barbara,Speer Christian P,Fehrholz Markus Reproductive sciences (Thousand Oaks, Calif.) Pregnant women at risk of preterm labor routinely receive glucocorticoids (GCs) and frequently also progesterone. Administration of GCs accelerates intrauterine surfactant synthesis and lung maturation, thereby reducing the incidence of neonatal respiratory distress syndrome; progesterone has the potential to prevent preterm birth. Little is known about possible interactions of GCs and progesterone. Our aim was to clarify whether progesterone can affect dexamethasone (DXM)-regulated expression of surfactant protein A (SP-A), SP-B, and SP-D in lung epithelial cells. H441 cells were exposed to DXM and progesterone and expression of SPs was analyzed by quantitative real-time polymerase chain reaction and immunoblotting. Although progesterone had no direct effect on the expression of SP-B, DXM-mediated induction was inhibited dose dependently on the transcriptional (64 µM [ < .0001], 32 µM [ = .0005], 16 µM [ = .0019]) and the translational level. Furthermore, progesterone inhibited stimulatory effects of other GCs as well. While exogenous tissue growth factor β1 (TGF-β1) inhibited DXM-induced SP-B expression (messenger RNA [mRNA]: = .0014), progesterone itself did not influence TGF-β1 mRNA expression and/or TGF-β1/Smad signaling, demonstrating that TGF-β1 and/or Smad activation is not involved. The inhibitory effect of progesterone could be imitated by the GC and progesterone receptor (PR) antagonist RU-486, but not by the specific PR antagonist PF-02413873, indicating that progesterone acts as a competitive antagonist of DXM. The effect of progesterone on DXM-regulated genes was not specific for SP-B, as expression of SP-A and SP-D mRNAs was also antagonized. The present study highlights a new action of progesterone as a potential physiological inhibitor of GC-dependent SP expression in lung epithelial cells. The clinical relevance of this in vitro finding is currently unknown. 10.1177/1933719118804668
    TTF-1 response element is critical for temporal and spatial regulation and necessary for hormonal regulation of human surfactant protein-A2 promoter activity. Liu Dongyuan,Yi Ming,Smith Margaret,Mendelson Carole R American journal of physiology. Lung cellular and molecular physiology Expression of the human surfactant protein-A2 (hSP-A2) gene is lung specific, occurs in type II and Clara cells, and is developmentally and hormonally regulated in fetal lung. Using transfected human fetal type II cells, we previously observed that approximately 300 bp of 5'-flanking DNA mediated cAMP and interleukin-1 (IL-1) stimulation and dexamethasone (Dex) inhibition of hSP-A2 promoter activity. This region contains response elements for estrogen-related receptor alpha element (ERRE, -241 bp), thyroid transcription factor (TTF)-1/Nkx2.1 (TTF-binding protein, -171 bp), upstream stimulatory factor 1/2 (E-box, -80 bp), and stimulatory protein (Sp) 1 (G/T-box, -62 bp), which are essential for basal and cAMP induction of hSP-A2 expression. To define genomic regions necessary for developmental, hormonal, and tissue-specific regulation of hSP-A2 expression in vivo, we analyzed transgenic mice carrying hGH reporter genes comprised of 313 bp of hSP-A2 gene 5'-flanking DNA +/- mutation in the TBE or 175 bp of 5'-flanking DNA, containing TBE, E-box and G/T-box, but lacking ERRE. Transgenes containing 313 or 175 bp of hSP-A2 5'-flanking DNA were expressed in a lung cell-specific manner and developmentally regulated in concert with the endogenous mouse SP-A gene. In cultured lung explants from hSP-A(-313):hGH transgenic fetal mice, cAMP and IL-1 induced and Dex inhibited transgene expression. However, the 175-bp hSP-A2 genomic region was insufficient to mediate hormonal regulation of hSP-A2 promoter activity. The finding that expression of the hSP-A(-313TBEmut):hGH transgene was essentially undetectable in fetal lung and was not hormonally regulated in transgenic fetal lung explants underscores the critical importance of the TBE in lung cell-specific, developmental, and hormonal regulation of hSP-A2 gene expression. 10.1152/ajplung.00069.2008
    Regulation of expression of human SP-A1 and SP-A2 genes in fetal lung explant culture. Karinch A M,Deiter G,Ballard P L,Floros J Biochimica et biophysica acta Human pulmonary surfactant protein A (SP-A) is genetically complex and its regulation may also be complex, reflecting genotypic variability. Fetal lung explants were used to study the regulation of the SP-A genes, SP-A1 and SP-A2, by dexamethasone, interferon, gamma (IFN gamma), cyclic 3',-5' adenosine monophosphate (cAMP), and tumor necrosis factor alpha (TNF alpha). For comparison, the mRNA levels of surfactant protein B (SP-B) and its response to test substances were also examined. Results showed: (a) In control culture total SP-A mRNA varied widely among explants (C.V. = 0.70) compared with SP-B (C.V. = 0.26) (b) IFN gamma significantly increased total SP-A mRNA but there were marked differences among fetal lungs in response to all treatments. (c) SP-A1 mRNA concentration is higher than SP-A2 in both control and treated explants. (d) SP-A1 alleles are inhibited to a greater degree by dexamethasone than SP-A2 alleles. The relative effect of cAMP and IFN gamma on SP-A1 and SP-A2 mRNA varied widely among explants. We conclude that SP-A genotype may account in part for the marked differences in SP-A mRNA concentration among fetal lungs and that the SP-A genes and/or alleles may be differentially regulated. 10.1016/s0167-4781(98)00047-5
    Quantification of Hox and surfactant protein-B transcription during murine lung development. Grier David G,Thompson Alexander,Lappin Terence R J,Halliday Henry L Neonatology BACKGROUND:Genetic processes underlying fetal lung development and maturation are incompletely understood. Better knowledge of these processes would provide insights into the causes of lung malformations and prevention of respiratory distress syndrome and the potential adverse effects of glucocorticoids. Hox genes are involved in the lung branching morphogenesis and maturation of respiratory epithelium, but their expression pattern remains to be defined. OBJECTIVES:We hypothesized that genes involved in lung branching would be downregulated during early development, whereas those involved in maturation would be unchanged or upregulated. METHODS:TaqMan real-time primers and probes were designed for all 39 murine Hox genes, and the murine SP-B gene and transcription profiles of these genes were obtained from whole lungs isolated at e14.5, e16.5, e18.5, e19.5 and postnatal days 1 and 20. RESULTS:Hox genes in clusters A and B, specifically those between paralog groups 3 and 7, were the most represented, with Hoxa4 and Hoxa5 being the most highly transcribed. A wave of reduced transcription in 16 Hox genes, coincident with increased SP-B transcription, was observed with advancing gestation. Consistently high transcription of Hoxa5 from e14.5 to postnatal day 20 may indicate that sustained transcription is required for normal lung maturation. When e15.5 lungs were cultured with dexamethasone, Hoxb6, Hoxb7 and Hoxb8 levels were significantly upregulated, creating the potential for modulation of diverse downstream target genes. CONCLUSIONS:Improved understanding of the genetic processes underlying lung development afforded by our Q-PCR platform may allow development of more specific methods for inducing fetal lung maturation. 10.1159/000201739
    Proteasome dysfunction inhibits surfactant protein gene expression in lung epithelial cells: mechanism of inhibition of SP-B gene expression. Das Aparajita,Boggaram Vijayakumar American journal of physiology. Lung cellular and molecular physiology Surfactant proteins maintain lung function through their actions to reduce alveolar surface tension and control of innate immune responses in the lung. The ubiquitin proteasome pathway is responsible for the degradation of majority of intracellular proteins in eukaryotic cells, and proteasome dysfunction has been linked to the development of neurodegenerative, cardiac, and other diseases. Proteasome function is impaired in interstitial lung diseases associated with surfactant protein C (SP-C) mutation mapping to the BRICHOS domain located in the proSP-C protein. In this study we determined the effects of proteasome inhibition on surfactant protein expression in H441 and MLE-12 lung epithelial cells to understand the relationship between proteasome dysfunction and surfactant protein gene expression. Proteasome inhibitors lactacystin and MG132 reduced the levels of SP-A, SP-B, and SP-C mRNAs in a concentration-dependent manner in H441 and MLE-12 cells. In H441 cells, lactacystin and MG132 inhibition of SP-B mRNA was associated with similar decreases in SP-B protein, and the inhibition was due to inhibition of gene transcription. Proteasome inhibitors decreased thyroid transcription factor-1 (TTF-1)/Nkx2.1 DNA binding activity, and the reduced TTF-1 DNA binding activity was due to reduced expression levels of TTF-1 protein. These data indicated that the ubiquitin proteasome pathway is essential for the maintenance of surfactant protein gene expression and that disruption of this pathway inhibits surfactant protein gene expression via reduced expression of TTF-1 protein. 10.1152/ajplung.00103.2006
    Biphasic glucocorticoid regulation of pulmonary SP-A: characterization of inhibitory process. Iannuzzi D M,Ertsey R,Ballard P L The American journal of physiology Pulmonary surfactant, which is necessary for normal lung function, is under both developmental and hormonal regulation. Glucocorticoids induce all components of surfactant and have a unique biphasic effect on surfactant protein A (SP-A), either stimulating or inhibiting accumulation in cultured fetal lung depending on dose and time of exposure. In this study we further characterized glucocorticoid inhibition of SP-A in cultured explants of human fetal lung. Decreased content of SP-A mRNA was the dominant response to dexamethasone added either early or later during culture. Inhibition occurred at < or = 1 nM dexamethasone on prolonged exposure, was blocked by RU 486, and was observed with other glucocorticoids but not sex steroids. When cortisol was removed from the culture medium, inhibition was rapidly reversed. The immediate inhibitory effect of 100 nM dexamethasone on SP-A mRNA content was completely blocked in the presence of cycloheximide. SP-A gene transcription, measured by nuclear elongation assay, was decreased by 60% after 4- to 8-h exposure to 100 nM dexamethasone. Stability of SP-A mRNA, determined both by addition of actinomycin D and by label-chase experiments, was transiently decreased immediately after adding dexamethasone (t1/2 approximately 3 h). In tissue treated with dexamethasone for > or = 8 h the stability of SP-A mRNA in control and treated explants was not different (t1/2 approximately 8 h). Our findings indicate that inhibition of SP-A is the dominant response to glucocorticoid. This effect is receptor mediated and apparently involves induction of a labile protein(s) that decreases gene transcription and transiently reduces mRNA stability. 10.1152/ajplung.1993.264.3.L236
    Differential glucocorticoid regulation of the pulmonary hydrophobic surfactant proteins SP-B and SP-C. Venkatesh V C,Iannuzzi D M,Ertsey R,Ballard P L American journal of respiratory cell and molecular biology Glucocorticoids increase expression of the genes for the pulmonary surfactant-associated proteins SP-B and SP-C in fetal lung both in vivo and in vitro. To examine the mechanism of these effects, we studied induction of SP-B and SP-C mRNAs in human fetal lung cultured as explants. Both mRNA levels rose rapidly in response to 100 nM dexamethasone (Dex), with a faster response for SP-B: maximal levels of induction were achieved in < or = 12 h for SP-B (3.5-fold versus control) versus approximately 24 h for SP-C mRNA (35-fold versus control). Cycloheximide (2.5 micrograms/ml) did not affect glucocorticoid induction of SP-B mRNA but markedly decreased induction of SP-C mRNA. In control cultures, cycloheximide did not significantly reduce levels of either transcript. In nuclear run-on assays, Dex increased the rate of gene transcription for both SP-B (2.8 +/- 0.3-fold versus control, n = 4) and SP-C (10- to 30-fold). Using actinomycin D to assess mRNA stability, the t1/2 of SP-B mRNA was increased from 7.5 +/- 0.4 h to 18.8 +/- 2.9 h by Dex treatment (P < 0.05), whereas the t1/2 of SP-C mRNA was not affected (9.3 +/- 1.7 h versus 8.1 +/- 1.2 h; NS). A similar increase in SP-B mRNA t1/2 with Dex (from 6 h to 19 h) was observed in label-chase studies with [3H]uridine. We conclude that glucocorticoids regulate the hydrophobic surfactant proteins of alveolar type II cells by different mechanisms: induction of SP-B is a primary response and includes an increase in both transcription rate and mRNA stability, whereas induction of SP-C is a secondary process, requiring ongoing protein synthesis, involving increased transcription rate without a change in mRNA stability. 10.1165/ajrcmb/8.2.222
    Human SP-A1 and SP-A2 genes are differentially regulated during development and by cAMP and glucocorticoids. McCormick S M,Mendelson C R The American journal of physiology Expression of the surfactant protein A (SP-A) gene is lung specific, developmentally induced, and regulated by adenosine 3',5'-cyclic monophosphate (cAMP) and glucocorticoids. Humans have two highly similar genes encoding SP-A (SP-A1 and SP-A2). In the companion paper [S.M. McCormick, V. Boggaram, and C.R. Mendelson Am. J. Physiol. 266 (Lung Cell. Mol. Physiol. 10): L354-L366, 1994] we report that SP-A1 and SP-A2 RNA transcripts are alternatively spliced at their 5' ends, resulting in nine different primer-extended transcripts. In the present study, primer extension was used to assess the relative levels of expression of the SP-A1 and SP-A2 genes in human adult lung tissue and in fetal lung tissues maintained in organ culture in the absence or presence of dibutyryl (DB)cAMP (1 mM) and dexamethasone (Dex, 10(-4) M). Primer extension and Northern analysis were used to assess the effects of these agents on the levels of expression of these genes. In human adult lung tissue, 65% of the SP-A mRNA transcripts were derived from the SP-A2 gene, whereas only 35% were from SP-A1. On the other hand, in lung tissue from a 28-wk gestation neonate, only SP-A1 mRNA transcripts were detected, and, in midgestation fetal lung cultured in control medium, 65% of the SP-A mRNA was found to be SP-A1 and 35% was SP-A2.(ABSTRACT TRUNCATED AT 250 WORDS) 10.1152/ajplung.1994.266.4.L367
    Transcriptional regulation of human pulmonary surfactant proteins SP-B and SP-C by glucocorticoids. Ballard P L,Ertsey R,Gonzales L W,Gonzales J American journal of respiratory cell and molecular biology Expression of the pulmonary surfactant-associated proteins SP-B and SP-C is under both developmental and hormonal regulation. We used human fetal lung to investigate developmental changes and the mechanism of glucocorticoid stimulation of SP-B and SP-C gene expression. There were similar approximately 3-fold increases in SP-B cytoplasmic mRNA content and transcription rate comparing lung samples of 24 wk versus 16 wk gestation. During 5 days of lung explant culture without hormones, the transcription rate increased for SP-B and decreased for SP-C, paralleling changes in mRNA content. Treatment with 100 nM dexamethasone maximally increased transcription of the SP-B gene (approximately 3-fold) and SP-C gene (approximately 11-fold) after 2 and 8 h, respectively, similar to changes in mRNA content. In dose-response studies, the maximal increase in transcription rate occurred at approximately 10 nM dexamethasone for SP-B and at > or = 100 nM for SP-C. Induction of SP-B mRNA content and transcription rate were not affected by prior cycloheximide exposure, whereas induction of SP-C mRNA was decreased by as little as 1 h exposure to inhibitor. We conclude that glucocorticoids, acting directly in type II cells, regulate the SP-B and SP-C genes primarily at the level of transcription. Induction of SP-C, but not SP-B, requires ongoing protein synthesis which likely reflects involvement of a labile transcription factor. The difference in glucocorticoid sensitivity may indicate that the two surfactant protein genes contain glucocorticoid response elements with different affinities for receptor. 10.1165/ajrcmb.14.6.8652188
    Glucocorticoid inhibition of human SP-A1 promoter activity in NCI-H441 cells. Hoover R R,Thomas K H,Floros J The Biochemical journal Glucocorticoids have complex effects on human surfactant protein (SP) SP-A1 and SP-A2 gene expression that occur at both transcriptional and post-transcriptional levels. In the lung adenocarcinoma cell line NCI-H441, dexamethasone causes a dose-dependent decrease in total SP-A mRNA levels and inhibits SP-A gene transcription. In this study, a deletional analysis of the SP-A1 promoter was performed in order to identify cis-acting elements that mediate dexamethasone responsiveness in NCI-H441 cells. The region -32/+63 relative to the start of SP-A1 transcription mediated both basal promoter activity and dexamethasone repression of transcription. Removal of the region +18/+63 abolished dexamethasone responsiveness, indicating that sequences within this region are necessary for the inhibitory effect. Furthermore, the region -32/+63 formed a sequence-specific DNA-protein complex with NCI-H441 nuclear extract. This DNA-protein complex was induced by dexamethasone exposure and its formation was mediated partially by sequences within the region +26/+63.
    Characterization of the human surfactant protein D promoter: transcriptional regulation of SP-D gene expression by glucocorticoids. Rust K,Bingle L,Mariencheck W,Persson A,Crouch E C American journal of respiratory cell and molecular biology We have previously described the characterization of genomic clones encoding the entire translated sequence of human pulmonary surfactant protein D (SP-D). We now describe the characterization of a genomic fragment (H5E7) that encodes the entirety of the first translated exon (Exon 2), Intron 1, a short transcribed untranslated sequence (Exon 1; 39 bp), and approximately 4 kb of sequence upstream from the transcription initiation site. The start site was identified by 5'-RACE-PCR cloning and primer extension. A putative TATA box (CATAAATA) was identified approximately 30 bp upstream of the start site. Complete sequencing of a HindIII/SacI fragment (HS-1674) encoding approximately 1.7 kb of sequence 5' to the TATA demonstrated multiple potential cis-regulatory elements including half-site glucocorticoid response elements (GRE), a canonical AP-1 consensus, several AP-1 like sequences, E-box sequences, NF-IL-6 and PEA3 motifs, and putative interferon response elements. H441 lung adenocarcinoma cells, which express low levels of SP-D mRNA, and liver HepG2 cells, were transiently co-transfected with chloramphenicol acetyl transferase (CAT) reporter constructs containing up to 3,000 base pairs of upstream sequence, and with constructs encoding beta-gal. H441 cells transfected with constructs containing at least 161 bp of upstream sequence gave normalized levels of CAT activity greater than or equal to that obtained for parallel positive control transfections using pTK-CAT. Treatment of the cells for 48 h with 50 nM dexamethasone (Dex) gave a 2- to 5-fold increase in CAT activity. Interestingly, a 5'-deletion construct containing 161 bp of upstream sequence (pFS161-CAT) conferred both cell type-restricted and dexamethasone-responsive expression. These studies emphasize the potential complexity of SP-D gene regulation, and further support the hypothesis that the effects of glucocorticoids on SP-D production in vivo are regulated at the level of transcription. 10.1165/ajrcmb.14.2.8630261
    Developmental and glucocorticoid regulation of surfactant protein mRNAs in preterm lambs. Tan R C,Ikegami M,Jobe A H,Yao L Y,Possmayer F,Ballard P L The American journal of physiology Glucocorticoid treatment increases content of surfactant protein (SP) A and SP-B in lung tissue and lavage fluid of preterm lambs. To investigate this process, we determined the ontogeny and glucocorticoid induction of SP mRNAs. In separate treatment protocols, each with its own controls, sheep were injected with betamethasone 15 h, 48 h, or weekly for 1-4 doses before preterm delivery. Using ovine SP cDNAs, we found an increase equal to or more than threefold in basal levels of all three SP mRNAs between 125 days and term. After betamethasone treatment, SP-B and SP-C mRNA levels increased by 15 h and all SP mRNAs were elevated after 24 h (>/=2-fold); mRNA levels in fetuses delivered 1-3 wk after betamethasone were not different from control. We conclude that in vivo betamethasone rapidly induces a coordinated increase in SP mRNAs, which is fully reversible within 7 days despite repetitive doses of betamethasone. Similar increases in mRNA and protein contents for SP-A and SP-B suggest that glucocorticoid regulation of these SPs in vivo is primarily pretranslational. 10.1152/ajplung.1999.277.6.L1142
    Prenatal glucocorticoids improve lung morphology and partially restores surfactant mRNA expression in lambs with diaphragmatic hernia undergoing fetal tracheal occlusion. Davey Marcus G,Danzer Enrico,Schwarz Uwe,Robinson Lauren,Shegu Shincy,Adzick N Scott,Flake Alan W,Hedrick Holly L Pediatric pulmonology In fetal sheep with surgically created diaphragmatic hernia (DH), tracheal occlusion (TO) can restore lung growth but does not ameliorate the increase in inter-alveolar wall thickness (T(W)). We determined whether prenatal exposure to glucocorticoids (GC) could reduce T(w) in fetuses with DH undergoing TO. At 65 days of gestation, DH was created in 12 fetal sheep, and TO subsequently performed at 110 days (DH/TO). Six of these fetuses were exposed to betamethasone (DH/TO + GC; 0.5 mg/kg; maternal, IM) 48 hr before delivery; Sham operated fetuses (n = 7) served as controls. At 139 days, we measured alveolar surface density (S(V)), parenchymal tissue fraction, T(W), alveolar type 2 (AE2) cell density and lung surfactant protein (SP) mRNA expression. Prenatal GC decreased T(W) and S(V) by 33% and 27% respectively, and increased fixed lung volume (by 55%), AE2 cell density and partially restored SPmRNA expression. Our data indicate that prenatal exposure to GC can reverse some of the negative effects of prolonged fetal TO. We hypothesize that a GC-induced reduction in lung liquid volume during TO contributes, in part, to the observed increase in AE2 cell density and SPmRNA expression. 10.1002/ppul.20516
    Differential regulation of SP-A1 and SP-A2 genes by cAMP, glucocorticoids, and insulin. Kumar A R,Snyder J M The American journal of physiology In the human fetal lung, surfactant protein A (SP-A) is encoded by two highly similar genes, SP-A1 and SP-A2, which are developmentally and hormonally regulated. Using primer extension analysis, we evaluated the levels of SP-A1 and SP-A2 mRNA transcripts in human fetal lung explants and in a human adult lung adenocarcinoma cell line (H441 cells) cultured in the absence or presence of either dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP, 1 mM), dexamethasone (10(-7) M), or insulin (2.5 micrograms/ml). In the human fetal lung explants, the content of SP-A1 mRNA was approximately four times that of SP-A2 mRNA. DBcAMP increased SP-A1 mRNA levels by 100% and SP-A2 mRNA levels by 500%, thus reducing the ratio of SP-A1 mRNA to SP-A2 mRNA to approximately 1:1. Dexamethasone inhibited all of the SP-A1 and SP-A2 mRNA transcripts to the same extent, by approximately 70%, whereas insulin inhibited all SP-A mRNA transcripts by approximately 60%. The ratio of SP-A1 to SP-A2 mRNA in dexamethasone- or insulin-treated explants was the same as the ratio observed in controls. In the H441 cells, SP-A1 mRNA levels were approximately 1.5 times that of SP-A2 mRNA levels. DBcAMP increased both SP-A1 and SP-A2 mRNA levels by 100%. Dexamethasone inhibited SP-A1 mRNA levels in the cell line by 60%, whereas SP-A2 mRNA levels were not significantly affected. Insulin inhibited SP-A1 mRNA levels in the cell line by 40% without affecting SP-A2 mRNA levels. These findings suggest that the two human SP-A genes are regulated differently in the two model systems. 10.1152/ajplung.1998.274.2.L177
    Dexamethasone potentiates keratinocyte growth factor-stimulated SP-A and SP-B gene expression in alveolar epithelial cells. Mouhieddine-Gueddiche O Banine,Pinteur Claudie,Chailley-Heu Bernadette,Barlier-Mur Anne-Marie,Clement Annick,Bourbon Jacques R Pediatric research Keratinocyte growth factor (KGF, or fibroblast growth factor 7) was previously reported to enhance the synthesis of surfactant in alveolar type II cells. We investigated the possible interactions between KGF and a glucocorticoid, dexamethasone (Dex), on surfactant protein (SP) gene expression. In cultured fetal rat type II cells, KGF and Dex induced greater-than-additive stimulating effects on SP-A and SP-B expressions that were enhanced three-fold and 30-fold, respectively, but had only additive effects on SP-C expression. Using murine lung epithelial (MLE) cells, KGF increased SP-A, SP-B (up to two-fold), and SP-C (up to three-fold) mRNA levels in a dose-dependent way. Dex 10(-9) to 10(-7) M increased SP-A and SP-B mRNA 1.5-fold and SP-C mRNA two-fold. Consistent with type II cell findings, simultaneous treatment by KGF and Dex induced a synergistic increase of SP-A and SP-B transcripts (three-fold and 4.5-fold, respectively), but not of SP-C transcripts. SP-A protein was present in MLE-15 and was increased about three-fold by KGF plus Dex. Expression study of a reporter gene placed under either the SP-A or the SP-B gene regulatory sequences and transfected in MLE-15 cells indicated that the Dex-KGF synergy was achieved mainly through a transcriptional effect for SP-A, and both transcriptional and nontranscriptional effects for SP-B. For the latter, increased mRNA stability was evidenced with the aid of actinomycin D. The Dex-KGF synergy may have potential interest for diseases associated with surfactant deficiency. 10.1203/01.PDR.0000047840.77635.AE
    The effects of intrauterine growth restriction and antenatal glucocorticoids on ovine fetal lung development. Sutherland Amy E,Crossley Kelly J,Allison Beth J,Jenkin Graham,Wallace Euan M,Miller Suzanne L Pediatric research INTRODUCTION:Intrauterine growth restriction (IUGR) is associated with high rates of neonatal morbidity. IUGR babies are often born preterm and are, therefore, exposed to antenatal glucocorticoids. Antenatal glucocorticoids significantly improve overall survival rates of preterm infants, but there is a paucity of information about their effects on IUGR Infants. METHODS:We induced IUGR in sheep by single umbilical artery ligation (SUAL), or sham in control fetuses. To half the ewes, we administered betamethasone (BM) on d 5 (BM1) and 6 (BM2) following surgery, and collected fetal lung tissue on d 7. RESULTS:SUAL alone was associated with higher circulating fetal cortisol levels (2.8 ± 0.4 vs. 1.0 ± 0.4, P = 0.001) as compared with controls but not with changes in lung morphology or surfactant protein (SP) gene expression. BM was associated with a significant reduction in lung tissue density (P = 0.048). There were no significant differences between groups in lung DNA concentration or septal crest density. SP-A, SP-B, and SP-C gene expressions were significantly increased in control and SUAL fetuses that were administered BM. DISCUSSION:These results show that in SUAL fetuses, maternal BM is associated with acceleration of fetal lung structure, as occurs in normally grown fetuses, and that BM induces SP production, an effect not observed in SUAL-induced IUGR fetuses alone. 10.1038/pr.2012.19
    The protective effect of different airway humidification liquids to lung after tracheotomy in traumatic brain injury: The role of pulmonary surfactant protein-A (SP-A). Su Xinyang,Li Zefu,Wang Meilin,Li Zhenzhu,Wang Qingbo,Lu Wenxian,Li Xiaoli,Zhou Youfei,Xu Hongmei Gene The purpose of this study was to establish a rat model of a brain injury with tracheotomy and compared the wetting effects of different airway humidification liquids, afterward, the best airway humidification liquid was selected for the clinical trial, thus providing a theoretical basis for selecting a proper airway humidification liquid in a clinical setting. Rats were divided into a sham group, group A (0.9% NaCl), group B (0.45% NaCl), group C (0.9% NaCl+ambroxol) and group D (0.9% NaCl+Pulmicort). An established rat model of traumatic brain injury with tracheotomy was used. Brain tissue samples were taken to determine water content, while lung tissue samples were taken to determine wet/dry weight ratio (W/D), histological changes and expression levels of SP-A mRNA and SP-A protein. 30 patients with brain injury and tracheotomy were selected and divided into two groups based on the airway humidification liquid instilled in the trachea tube, 0.45% NaCl and 0.9% NaCl+ambroxol. Blood was then extracted from the patients to measure the levels of SP-A, interleukin-6 (IL-6), interleukin-8 (IL-8) and tumour necrosis factor-α (TNF-α). The difference between group C and other groups in lung W/D and expression levels of SP-A mRNA and SP-A protein was significant (P<0.05). In comparison, the histological changes showed that the lung tissue damage was smallest in group C compared to the three other groups. Aspect of patients, 0.45% NaCl group and 0.9% NaCl+ambroxol group were significantly different in the levels of SP-A, IL-6, IL-8 and TNF-α (P<0.01). In the present study, 0.9% NaCl+ambroxol promote the synthesis and secretion of pulmonary surfactant, and has anti-inflammatory and antioxidant effects, which inhibit the release of inflammatory factors and cytokines, making it an ideal airway humidification liquid. 10.1016/j.gene.2015.11.024
    The upstream region of the SP-B gene: intrinsic promoter activity and glucocorticoid responsiveness related to a new DNA-binding protein. Luzi P,Anceschi M,Strayer D S Gene We identified and cloned the rabbit SP-B gene, encoding the pulmonary surfactant-associated protein, and sequenced its upstream region from -2635 to +428, including a much larger fragment of the upstream region than has previously been reported for an SP-B for any species. Rabbit SP-B showed substantial homology to its human counterpart in the coding and noncoding regions immediately upstream from the TATAA box. Using a luciferase (Luc) reporter gene (luc) construct we measured promoter activity with a 212-bp fragment (SPB212) from nucleotides (nt) -41 to -252, inclusive. SPB212 functioned as an active promoter in this assay. Further, we identified, cloned and sequenced the cDNA encoding a unique DNA-binding protein, N, that bound SPB212 at approx. -195. When the N cDNA was cloned into the expression vector pKC4 and cotransfected with the luc reporter construct, N significantly enhanced Luc production, but only in the presence of dexamethasone. Therefore, we identified and sequenced a functional promoter region upstream from rabbit SP-B, and isolated and characterized a DNA-binding protein that confers enhanced glucocorticoid responsiveness on this promoter. 10.1016/0378-1119(95)00506-2
    Association between the surfactant protein A (SP-A) gene locus and respiratory-distress syndrome in the Finnish population. Rämet M,Haataja R,Marttila R,Floros J,Hallman M American journal of human genetics Respiratory-distress syndrome (RDS) in the newborn is a major cause of neonatal mortality and morbidity. Although prematurity is the most-important risk factor for RDS, the syndrome does not develop in many premature infants. The main cause of RDS is a deficiency of pulmonary surfactant, which consists of phospholipids and specific proteins. The genes underlying susceptibility to RDS are insufficiently known. The candidate-gene approach was used to study the association between the surfactant protein A (SP-A) gene locus and RDS in the genetically homogeneous Finnish population. In the present study, 88 infants with RDS and 88 control infants that were matched for degree of prematurity, prenatal glucocorticoid therapy, and sex were analyzed for SP-A genotypes. We show that certain SP-A1 alleles (6A2 and 6A3) and an SP-A1/SP-A2 haplotype (6A2/1A0) were associated with RDS. The 6A2 allele was overrepresented and the 6A3 allele was underrepresented in infants with RDS. These associations were particularly strong among small premature infants born at gestational age <32 wk. In infants protected from RDS (those that had no RDS, despite extreme prematurity and lack of glucocorticoid therapy), compared with infants that had RDS develop despite having received glucocorticoid therapy, the frequencies of 6A2 (.22 vs.71), 6A3 (.72 vs.17), 6A2/1A0 (.17 vs.68), 6A3/1A1 (.39 vs.10), and 6A3/1A2 (.28 vs.06) in the two groups, respectively, were strikingly different. According to the results of conditional logistic-regression analysis, diseases associated with premature birth did not explain the association between the odds of a particular homozygous SP-A1 genotype (6A2/6A2 and 6A3/6A3) and RDS. In the population evaluated in the present study, SP-B intron 4 variant frequencies were low and had no detectable association with RDS. We conclude that the SP-A gene locus is an important determinant for predisposition to RDS in premature infants. 10.1086/302906
    Down-regulation of thyroid transcription factor-1 gene expression in fetal lung hypoplasia is restored by glucocorticoids. Losada A,Tovar J A,Xia H M,Diez-Pardo J A,Santisteban P Endocrinology The thyroid transcription factor (TTF)-1 has an essential role in lung morphogenesis and development. It is involved in the transcription of surfactant proteins (SP), which are critical in respiratory function. Neonates with congenital diaphragmatic hernia die of respiratory failure caused by pulmonary hypoplasia with associated biochemical immaturity. To gain new insights into the causes of this disorder and the effect of prenatal hormonal treatment on reducing mortality in these infants, we evaluated the expression of TTF-1 as marker of lung morphogenesis and SP-B as marker of lung maturity. Using a rat model of lung immaturity, we show that TTF-1 and SP-B messenger RNA (mRNA) levels are drastically reduced in congenital lung hypoplasia. Interestingly, prenatal dexamethasone (Dex) treatment increased both TTF-1 and SP-B mRNAs over control levels when administered to rats with lung hypoplasia, but it had no effect on TTF-1 or a moderate effect on SP-B mRNA when administered to control rats. TRH alone also increases TTF-1 and SP-B mRNA levels but to a lesser extent than Dex. When administered together with Dex, TRH counteracts the induction observed with the glucocorticoid. The decrease in TTF-1 mRNA levels in lung hypoplasia is paralleled by a down-regulation of TTF-1 protein levels, as well as by a decrease in the TTF-1/DNA complex when the TTF-1-binding site of the SP-B promoter was used as a probe. Both parameters were reestablished after glucocorticoid treatment. Moreover, the regulation of TTF-1 gene expression described in this report is accompanied by the same regulation in its promoter activity, as demonstrated in transfection experiments performed in H-441 human lung-derived adenocarcinoma cells. In conclusion, our data demonstrate, for the first time, that lung hypoplasia and the associated respiratory dysfunction caused by SP-B deficiency are caused, in part, by down-regulation of TTF-1 gene expression. The observations that prenatal glucocorticoid treatment induces the expression of TTF-1 supports routine in utero glucocorticoid treatment of patients expected to have lung hypoplasia. 10.1210/endo.141.6.7522
    Targeted Synthesis and Characterization of a Gene Cluster Encoding NAD(P)H-Dependent 3α-, 3β-, and 12α-Hydroxysteroid Dehydrogenases from Eggerthella CAG:298, a Gut Metagenomic Sequence. Mythen Sean M,Devendran Saravanan,Méndez-García Celia,Cann Isaac,Ridlon Jason M Applied and environmental microbiology Gut metagenomic sequences provide a rich source of microbial genes, the majority of which are annotated by homology or unknown. Genes and gene pathways that encode enzymes catalyzing biotransformation of host bile acids are important to identify in gut metagenomic sequences due to the importance of bile acids in gut microbiome structure and host physiology. Hydroxysteroid dehydrogenases (HSDHs) are pyridine nucleotide-dependent enzymes with stereospecificity and regiospecificity for bile acid and steroid hydroxyl groups. HSDHs have been identified in several protein families, including medium-chain and short-chain dehydrogenase/reductase families as well as the aldo-keto reductase family. These protein families are large and contain diverse functionalities, making prediction of HSDH-encoding genes difficult and necessitating biochemical characterization. We located a gene cluster in sp. CAG:298 predicted to encode three HSDHs (CDD59473, CDD59474, and CDD59475) and synthesized the genes for heterologous expression in We then screened bile acid substrates against the purified recombinant enzymes. CDD59475 is a novel 12α-HSDH, and we determined that CDD59474 (3α-HSDH) and CDD59473 (3β-HSDH) constitute novel enzymes in an iso-bile acid pathway. Phylogenetic analysis of these HSDHs with other gut bacterial HSDHs and closest homologues in the database revealed predictable clustering of HSDHs by function and identified several likely HSDH sequences from bacteria isolated or sequenced from diverse mammalian and avian gut samples. Bacterial HSDHs have the potential to significantly alter the physicochemical properties of bile acids, with implications for increased/decreased toxicity for gut bacteria and the host. The generation of oxo-bile acids is known to inhibit host enzymes involved in glucocorticoid metabolism and may alter signaling through nuclear receptors such as farnesoid X receptor and G-protein-coupled receptor TGR5. Biochemical or similar approaches are required to fill in many gaps in our ability to link a particular enzymatic function with a nucleic acid or amino acid sequence. In this regard, we have identified a novel 12α-HSDH and a novel set of genes encoding an iso-bile acid pathway (3α-HSDH and 3β-HSDH) involved in epimerization and detoxification of harmful secondary bile acids. 10.1128/AEM.02475-17
    SP-A 3'-UTR is involved in the glucocorticoid inhibition of human SP-A gene expression. Hoover R R,Floros J The American journal of physiology The synthetic glucocorticoid dexamethasone has a major inhibitory effect on human surfactant protein A1 (SP-A1) and SP-A2 gene expression that occurs at both the transcriptional and posttranscriptional levels. Toward the identification of cis-acting elements that may be involved in the dexamethasone regulation of SP-A mRNA stability, chimeric chloramphenicol acetyltransferase (CAT) constructs that contained various portions of SP-A1 or SP-A2 cDNA in place of the native CAT 3'-untranslated region (UTR) were transiently transfected into the lung adenocarcinoma cell line NCI-H441. CAT activity was reduced in NCI-H441 cells by exposure to 100 nM dexamethasone only for the chimeric CAT constructs that contained the SP-A 3'-UTR. Moreover, the inhibitory response seen with dexamethasone was greater for the 3'-UTR derived from the SP-A1 allele 6A3 than with the 3'-UTR derived from either the SP-A1 allele 6A2 or SP-A2 allele 1A0, indicating differential regulation between SP-A genes and/or alleles. 10.1152/ajplung.1999.276.6.L917
    Glucocorticoid regulation of human pulmonary surfactant protein-B mRNA stability involves the 3'-untranslated region. Huang Helen W,Bi Weizhen,Jenkins Gaye N,Alcorn Joseph L American journal of respiratory cell and molecular biology Expression of pulmonary surfactant, a complex mixture of lipids and proteins that acts to reduce alveolar surface tension, is developmentally regulated and restricted to lung alveolar type II cells. The hydrophobic protein surfactant protein-B (SP-B) is essential in surfactant function, and insufficient levels of SP-B result in severe respiratory dysfunction. Glucocorticoids accelerate fetal lung maturity and surfactant synthesis both experimentally and clinically. Glucocorticoids act transcriptionally and post-transcriptionally to increase steady-state levels of human SP-B mRNA; however, the mechanism(s) by which glucocorticoids act post-transcriptionally is unknown. We hypothesized that glucocorticoids act post-transcriptionally to increase SP-B mRNA stability via sequence-specific mRNA-protein interactions. We found that glucocorticoids increase SP-B mRNA stability in isolated human type II cells and in nonpulmonary cells, but do not alter mouse SP-B mRNA stability in a mouse type II cell line. Deletion analysis of an artificially-expressed SP-B mRNA indicates that the SP-B mRNA 3'-untranslated region (UTR) is necessary for stabilization, and the region involved can be restricted to a 126-nucleotide-long region near the SP-B coding sequence. RNA electrophoretic mobility shift assays indicate that cytosolic proteins bind to this region in the absence or presence of glucocorticoids. The formation of mRNA:protein complexes is not seen in other regions of the SP-B mRNA 3'-UTR. These results indicate that a specific 126-nucleotide region of human SP-B 3'-UTR is necessary for increased SP-B mRNA stability by glucocorticoids by a mechanism that is not lung cell specific and may involve mRNA-protein interactions. 10.1165/rcmb.2007-0303oc
    Amplification of steroid-mediated SP-B expression by physiological levels of caffeine. Fehrholz Markus,Hütten Matthias,Kramer Boris W,Speer Christian P,Kunzmann Steffen American journal of physiology. Lung cellular and molecular physiology Factors positively influencing surfactant homeostasis in general and surfactant protein B (SP-B) expression in particular are considered of clinical importance regarding an improvement of lung function in preterm infants. The objective of this study was to identify effects of physiological levels of caffeine on glucocorticoid-mediated SP-B expression in vitro and in vivo. Levels of SP-B and pepsinogen C were quantified by quantitative real-time RT-PCR or immunoblotting in NCI-H441 cells daily exposed to caffeine and/or dexamethasone (DEX). In vivo, SP-B expression was analyzed in bronchoalveolar lavage (BAL) of preterm sheep exposed to antenatal DEX and/or postnatal caffeine. If DEX and caffeine were continuously present, SP-B mRNA and protein levels were increased for up to 6 days after induction (P < 0.05). Additionally, caffeine enhanced SP-B mRNA expression in DEX-pretreated cells (P < 0.05). Moreover, caffeine amplified DEX-induced pepsinogen C mRNA expression (P < 0.05). After short-term treatment with caffeine in vivo, only slightly higher SP-B levels could be detected in BAL of preterm sheep following antenatal DEX, combined with an increase of arterial oxygen partial pressure (P < 0.01). Our data demonstrated that the continuous presence of caffeine in vitro is able to amplify DEX-mediated SP-B expression. In contrast, short-term improvement of lung function in vivo is likely to be independent of altered SP-B transcription and translation. An impact of caffeine on release of surfactant reservoirs from lamellar bodies could, however, quickly affect SP-B content in BAL, which has to be further investigated. Our findings indicate that caffeine is able to amplify main effects of glucocorticoids that result from changes in surfactant production, maturation, and release. 10.1152/ajplung.00257.2013
    Glucocorticoid inhibition of SP-A gene expression in lung type II cells is mediated via the TTF-1-binding element. Alcorn Joseph L,Islam Kazi N,Young Pampee P,Mendelson Carole R American journal of physiology. Lung cellular and molecular physiology Induction of surfactant protein-A (SP-A) gene expression in fetal lung type II cells by cAMP and IL-1 is mediated by increased binding of thyroid transcription factor-1 (TTF-1) and NF-B proteins p50 and p65 to the TTF-1-binding element (TBE) at -183 bp. In type II cell transfections, dexamethasone (Dex) markedly inhibits cAMP-induced expression of rabbit SP-A:human growth hormone (hGH) fusion genes containing as little as 300 bp of the SP-A 5'-flanking sequence. Dex inhibition is blocked by RU-486, suggesting a role of the glucocorticoid receptor (GR). The present study was undertaken to define the mechanisms for GR inhibition of SP-A expression. Cotransfection of primary cultures of type II cells with a GR expression vector abrogated cAMP induction of SP-A promoter activity while, at the same time, causing a 60-fold induction of cotransfected mouse mammary tumor virus (MMTV) promoter. In lung cells transfected with a fusion gene containing three TBEs fused to the basal SP-A promoter, Dex prevented the stimulatory effect of IL-1 on TTF-1 induction of SP-A promoter activity, suggesting that the GR inhibits SP-A promoter activity through the TBE. In gel shift assays using nuclear extracts from human fetal type II cells cultured in the absence or presence of cAMP, Dex markedly reduced binding of nuclear proteins to the TBE and blocked the stimulatory effect of cAMP on TBE-binding activity. Our finding that Dex increased expression of the NF-kappaB inhibitory partner IkappaB-alpha suggests that the decrease in TBE-binding activity may be caused, in part, by GR inhibition of NF-kappaB interaction with this site. 10.1152/ajplung.00280.2003
    Glucocorticoids: extensive physiological actions modulated through multiple mechanisms of gene regulation. Zanchi Nelo Eidy,Filho Mário Alves de Siqueira,Felitti Vitor,Nicastro Humberto,Lorenzeti Fábio Medici,Lancha Antonio Herbert Journal of cellular physiology Glucocorticoid hormones are important regulators of several physiological processes. Despite having been initially named based on their role in glucose metabolism, glucocorticoids are also fundamental in the regulation of developmental, metabolic, and neurobiological processes, as well as several other biological functions. Due to their involvement in a diverse array of biological pathways, its wide spectrum of action, it is predicted that a wide range of genes may have their expression regulated by the activated glucocorticoid receptor (GR). In fact, it has been demonstrated that in addition to the regulation of several effectors genes, the expression of the gene encoding for GR itself is regulated by physiological stimuli and fine-tuning mechanisms. Importantly, such generalized effector responses and fine-tuning responses seem to be largely mediated by mechanisms of gene regulation. Therefore, this review aims to describe the mechanisms of gene regulation by glucocorticoid hormones, which are capable of regulating differential gene transcription, within a physiological context. From this discussion, we hope to shed light on how a single molecule that is capable of exerting such divergent effects is also capable of promoting such distinct responses in different target tissues. 10.1002/jcp.22141
    Synergistic effect of caffeine and glucocorticoids on expression of surfactant protein B (SP-B) mRNA. Fehrholz Markus,Bersani Iliana,Kramer Boris W,Speer Christian P,Kunzmann Steffen PloS one Administration of glucocorticoids and caffeine is a common therapeutic intervention in the neonatal period, but possible interactions between these substances are still unclear. The present study investigated the effect of caffeine and different glucocorticoids on expression of surfactant protein (SP)-B, crucial for the physiological function of pulmonary surfactant. We measured expression levels of SP-B, various SP-B transcription factors including erythroblastic leukemia viral oncogene homolog 4 (ErbB4) and thyroid transcription factor-1 (TTF-1), as well as the glucocorticoid receptor (GR) after administering different doses of glucocorticoids, caffeine, cAMP, or the phosphodiesterase-4 inhibitor rolipram in the human airway epithelial cell line NCI-H441. Administration of dexamethasone (1 µM) or caffeine (5 mM) stimulated SP-B mRNA expression with a maximal of 38.8±11.1-fold and 5.2±1.4-fold increase, respectively. Synergistic induction was achieved after co-administration of dexamethasone (1 mM) in combination with caffeine (10 mM) (206±59.7-fold increase, p<0.0001) or cAMP (1 mM) (213±111-fold increase, p = 0.0108). SP-B mRNA was synergistically induced also by administration of caffeine with hydrocortisone (87.9±39.0), prednisolone (154±66.8), and betamethasone (123±6.4). Rolipram also induced SP-B mRNA (64.9±21.0-fold increase). We detected a higher expression of ErbB4 and GR mRNA (7.0- and 1.7-fold increase, respectively), whereas TTF-1, Jun B, c-Jun, SP1, SP3, and HNF-3α mRNA expression was predominantly unchanged. In accordance with mRNA data, mature SP-B was induced significantly by dexamethasone with caffeine (13.8±9.0-fold increase, p = 0.0134). We found a synergistic upregulation of SP-B mRNA expression induced by co-administration of various glucocorticoids and caffeine, achieved by accumulation of intracellular cAMP. This effect was mediated by a caffeine-dependent phosphodiesterase inhibition and by upregulation of both ErbB4 and the GR. These results suggested that caffeine is able to induce the expression of SP-transcription factors and affects the signaling pathways of glucocorticoids, amplifying their effects. Co-administration of caffeine and corticosteroids may therefore be of benefit in surfactant homeostasis. 10.1371/journal.pone.0051575