Dexamethasone-Mediated Repression of MUC5AC Gene Expression in

Glucocorticoids regulate gene expression via binding of the ligand-activated glucocorticoid receptor (GR) to glucocorticoid responsive cis-elements (GRE) in target gene promoters. The MUC5AC gene, which encodes the protein backbone of an abundant secreted airway mucin, has several putative GRE cis-elements in its 5 ' sequence. Mechanism(s) whereby glucocorticoids regulate mucin genes have not previously been described. In this study, the glucocorticoid dexamethasone (Dex) decreased MUC5AC mRNA abundance in A549, NCI-H292 cell lines and primary differentiated normal bronchial epithelial cells by 50-80%, suggesting a common mechanism of MUC5AC gene repression in human lung epithelial cells. Kinetic analyses showed that MUC5AC mRNA was not significantly decreased until 6h following Dex exposure, and that nuclear translocation of GR was biphasic, suggesting that Dex-mediated cis-repression of MUC5AC gene expression was a delayed response of GR translocation. Transfection analyses demonstrated that Dex transcriptionally repressed the MUC5AC promoter. Electrophoretic mobility shift assays with wild type and mutant oligonucleotide probes showed that GR bound to two GRE cis-sites (nucleotides-930 to-912 and-369 to-351) in the MUC5AC promoter. Analyses of mutated MUC5AC promoter constructs demonstrated that NF[[KAPPA]]B cis-sites wer

Urokinase Receptor mRNA Stability Involves Tyrosine Phosphorylation

Interaction between urokinase-type plasminogen activator (uPA) and its receptor (uPAR) localizes cellular proteolysis and promotes cellular proliferation and migration, effects that may contribute to the pathogenesis of lung inflammation and neoplasia. Enhanced uPAR expression as well as stabilization of uPAR mRNA by transforming growth factor- � and phorbol myristate acetate (PMA) shares a common mechanism involving phosphorylation and dephosphorylation of a uPAR mRNA-binding protein (uPAR mRNABp). PMA-induced tyrosine phosphorylation of the uPAR mRNABp inhibited the uPAR mRNA–uPAR mRNABp interaction, stabilized uPAR mRNA and enhanced uPAR protein expression. Downregulation of the uPAR mRNA and uPAR mRNABp interaction by PMA and transforming growth factor- � can be reversed by pretreatment of cells with herbimycin which in turn inhibits expression of uPAR protein via a decrease in uPAR mRNA stability

Functional Analysis of Human MUC7 Mucin Gene 5�-Flanking Region in

The human MUC7 gene encodes a low-molecular-mass mucin glycoprotein that functions in modulation of microbial flora in the oral cavity and respiratory tracts. MUC7 gene expression is tissue- and cell-specific, with dominant expression in salivary gland acinar cells. To begin to understand the molecular mechanisms responsible for controlling MUC7 gene expression, we analyzed the promoter activity of MUC7 5�-flanking region in a human lung epithelial cell line A549. We demonstrated that MUC7 gene is expressed constitutively in this cell line and is upregulated by TNF- � stimulation. The promoter activities of a 2,762-bp fragment of the human genomic DNA (�2,732/�30 bp) and its deletion series, subcloned into a luciferase reporter vector, were characterized at the basal level and under stimulation by TNF-�. The results indicated that the minimal functional MUC7 promoter is in the region of �138/�30 bp. This region also revealed the greatest increase in the promoter activity upon TNF- � stimulation. Two putative AP1-binding elements and one NF-�B–binding element were identified within the proximal promoter. Further analyses demonstrated that mutations of these elements dramatically reduced specific DNA-protein binding ability and reporter gene expression. AP1 elements played an essential role in the constitutive expression, while the NF-�B element was crucially important in the response to TNF- � stimulation, demonstrating that TNF- � activates MUC7 transcription via NF-�B signaling pathway

Polarity of Human Parainfluenza Virus Type 3 Infection

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Role of Nucleolin in Human Parainfluenza Virus Type 3 Infection

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Oxidative-Nitrosative Stress and Post- Translational Protein Modifications: Implications to Lung Structure–Function Relations Asymmetric Dimethylarginine Induces Oxidative and

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced by epithelial and inflammatory cells are key mediators of the chronic airway inflammation of asthma. Low L-arginine levels can result in the uncoupling of nitric oxide synthase (NOS) leading to production of both ROS and RNS. Asymmetric dimethylarginine (ADMA) is a competitive endogenous inhibitor of all NOS isoforms and has been demonstrated to inhibit NO formation and increase oxidative stress in vascular endothelial and smooth muscle cells. The effect of ADMA on inducible NOS (iNOS) activity in epithelial cells has not been explored. In this study, we investigated whether addition of exogenous ADMA alters the generation of NO and superoxide anion (O 2), leading to peroxynitrite (ONOO) formation in a mouse epithelial cell line. In stimulated LA-4 cells, ADMA dosedependently inhibited nitrite accumulation after 24 h of treatment. In addition, ADMA concentrations as low as 10 �M induced rapid increases in O2 � production as measured by dihydroethidium oxidation. Furthermore, using dihydrorhodamine to monitor ONOO � formation, ADMA caused a dose-dependent increase in ONOO � after treatment for 24 h. Similar effects of ADMA were seen using purified iNOS protein in a cell-free system. Together, these data indicate that elevated ADMA may contribute to the production of ROS and RNS in airway inflammation

Environmental Health Perspectives

The presence of neuroendocrinelike epithelial cells in the lung of numerous species has been demonstrated by light and electron microscopy. Histochemical methods used to identify these cells have included staining with silver, amine-type fluorescence (APUD cell), periodic acid Schiff (PAS)-lead hematoxylin, and immunohistochemical localization of neuron-specific enolase. Cytoplasmic dense core vesicles (70-200 nm in diameter) have served as the major ultrastructural characteristic. Lung neuroendocrinelike cells have been shown to occur in fetal and adult mammals as solitary-type cells or as distinct organoids known as neuroepithelial bodies (NEBs). Although the frequency of both populations is considered low, solitary-type cells with dense-core granules can be found in as high as 5 % of epithelial cells in the cricoid region of the guinea-pig larynx. The solitary cells can be found throughout the airways of mammals, whereas the NEBs are confined to the intrapulmonary airways. Unmyelinated fibers have been traced from the lamina propria and into the NEB, where they ramified between the component cells of the NEB. The function of lung neuroendocrinelike cells is not known, but morphological and cytochemical studies suggest that the NEBs are intrapulmonary chemoreceptors that can respond to changes in airway gas composition. Hypoxia or hypercapnia has been shown to decrease the amine cytofluorescence in these organoids and apparently to increase the exocytosis of dense core vesicles from the basal region of the cell

Loss of Proliferation and Antigen Presentation Activity following Internalization of Polydispersed Carbon

Interactions between poly-dispersed acid functionalized single walled carbon nanotubes (AF-SWCNTs) and primary lung epithelial (PLE) cells were studied. Peritoneal macrophages (PMs, known phagocytic cells) were used as positive controls in this study. Recovery of live cells from cultures of PLE cells and PMs was significantly reduced in the presence of AF-SWCNTs, in a time and dose dependent manner. Both PLE cells as well as PMs could take up fluorescence tagged AF-SWCNTs in a time dependent manner and this uptake was significantly blocked by cytochalasin D, an agent that blocks the activity of acto-myosin fibers and therefore the phagocytic activity of cells. Confocal microscopic studies confirmed that AF-SWCNTs were internalized by both PLE cells and PMs. Intra-trachially instilled AF-SWCNTs could also be taken up by lung epithelial cells as well as alveolar macrophages. Freshly isolated PLE cells had significant cell division activity and cell cycling studies indicated that treatment with AF-SWCNTs resulted in a marked reduction in S-phase of the cell cycle. In a previously standardized system to study BCG antigen presentation by PLE cells and PMs to sensitized T helper cells, AF-SWCNTs could significantly lower the antigen presentation ability of both cell types. These results show that mouse primary lung epithelial cells can efficiently internalize AF-SWCNTs and the uptake of nanotubes interfered with biological functions of PLE cell

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Interaction between urokinase-type plasminogen activator (uPA) and its receptor (uPAR) localizes cellular proteolysis and promotes cellular proliferation and migration, effects that may contribute to the pathogenesis of lung inflammation and neoplasia. Enhanced uPAR expression as well as stabilization of uPAR mRNA by TGF-β and PMA shares a common mechanism involving phosphorylation and dephosphorylation of a uPAR mRNA binding protein (uPAR mRNABp). PMA-induced tyrosine phosphorylation of the uPAR mRNABp inhibited the uPAR mRNA-uPAR mRNABp interaction, stabilized uPAR mRNA and enhanced uPAR protein expression. Down regulation of the uPAR mRNA and uPAR mRNABp interaction by PMA and TGF-β can be reversed by pre-treatment of cells with herbimycin which in turn inhibits expression of uPAR protein via a decrease in uPAR mRNA stability. Our experiments indicate that posttranscriptional regulation of uPAR expression requires activation of tyrosine kinases. Cytokines can regulate uPAR expression of lung-derived epithelial cells at the posttranscriptional level by tyrosine phosphorylation of the uPAR mRNA binding protein and may thereby influence tissue remodeling in lung injury or neoplasia

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