Browsing by Subject "CFTR"

Now showing 1 - 3 of 3
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    Adrenergic regulation of CFTR-dependent anion secretion and cell migration in airway epithelial cells
    (2014-12) Peitzman, Elizabeth Ruth
    The overall goal of this thesis was to understand the mechanisms underlying beta-adrenergic receptor (beta-AR) regulation of anion secretion and epithelial cell migration on mucociliary clearance and epithelial repair in human airways. The first chapter of the thesis presents an overview of the literature with the purpose of providing background information of mucociliary clearance, airway epithelial cell restitution and specific inflammatory disease conditions where these processes contribute to the innate defense of the airways. Chapters 2 and 3 describe the methods, results and a discussion of experiments that were performed to address the overall goal of the thesis. The final chapter presents a general discussion of the major findings of these studies in the context of what is presently known about the role of beta-AR regulation of epithelial function in airways. Initial experiments presented in Chapter 2 so looked at the effects of growth conditions on beta-AR localization in a human airway epithelial cell model system (Calu-3 cells). The results from these experiments showed that under air-liquid interface (ALI) conditions basolateral stimulation with 1uM epinephrine produced a significant increase in CFTR dependent anion secretion, whereas cells that were grown under liquid liquid interface (LLI) had a much smaller increase secretion when stimulated with the same concentration of epinephrine. These results indicated that basolateral expression of beta-ARs is increased when cells were grown under ALI. Furthermore Calu-3 cells that were apically stimulated with the beta2-AR selective agonist salbutamol produced an increase in anion secretion similar to what was observed with 8cpt-cAMP, a non-metabolizable analog of cyclic AMP. Additionally, when cells were treated with carvedilol, an inverse agonist acting at beta2-ARs, an initial decrease in basal Isc occurred and carvedilol treatment after stimulation with 8cpt-cAMP inhibited anion secretion. Cells pretreated with nocotazole, an agent that disrupts microtubule assembly, blocked the inhibitory effects of carvedilol on anion secretion, suggesting that endocytosis was necessary in order to observe the inhibitory effects of carvedilol. Finally, western blot analysis of apical membrane proteins showed that when cells were treated with carvedilol there was reduced expression of CFTR in the apical membrane, which was not observed following stimulation with epinephrine.Experiments in chapter 3 were designed to investigate the effects of beta-AR agonists on epithelial cell migration. Stimulation of Normal Human Bronchial Epithelial (NHBE) cells and Calu-3 cells), with a beta2-AR agonist produced a significant increase in time to wound closure compared to untreated control cells. Moreover, agonist stimulated cells were rescued when pretreated with beta-AR antagonists propranolol or ICI-118551. The addition of beta-AR agonists epinephrine or salbutamol to CFTR silenced cells (shCFTR) or cells where CFTR was inhibited with 20uM CFTRinh-172 showed no further decrease in migration rate suggesting that inhibition due to a change in CFTR expression or activity. Furthermore beta-AR agonists reduced lamellipodia protrusion similar to what was observed after CFTR inhibition. Overall these results suggest that treatment of airway epithelial cells with beta-AR agonists causes a decrease in migration rate leading to a significant reduction in the time required for complete wound closure. Additionally these results suggest that stimulating cells with carvedilol causes inhibition of cAMP-stimulated anion secretion by promoting retrieval and internalization of CFTR from the apical membrane. These results signify that the chronic use of beta-AR agonists can lead to increased risk for infection in individuals with obstructive airway disease and the use of bias ligands that promote Gs signaling while blocking beta-arrestin signaling may be the key to effectively treating these patients. These drugs would potentially promote wound repair and mucociliary clearance while limiting the risk of increased exacerbations and infection.
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    Regulation of airway epithelial cell migration by the cystic fibrosis transmembrane conductance regulator.
    (2010-05) Schiller, Katherine Rebecca
    The airway epithelium is a critical barrier between the noxious particles in inspired air and the lung tissue. Mucociliary clearance is an essential function of the airway epithelium to protect against infection and airway damage. By this process, inhaled particles are trapped in the mucus lining the airway and propelled out of the airways by ciliary movement. Transport of Cl- and Na+ ions controls fluid secretion in the airways and the depth and viscosity of the periciliary liquid layer essential for cilia movement. The ion channel cystic fibrosis transmembrane conductance regulator (CFTR), transports Cl- and HCO3 - in the airways and other tissues, and controls the depth of the periciliary liquid layer. Cystic fibrosis is a fatal genetic disease in which CFTR is dysfunctional. As a result, mucociliary clearance is impaired and airways become chronically infected with microorganisms. Microorganism colonization results in recurrent inflammation and cycles of damage to the epithelial barrier followed by wound repair, eventually resulting in airway remodeling and ultimately respiratory failure. It has recently been demonstrated that the loss of CFTR function impairs epithelial restitution in the absence of infection. This suggests the CFTR plays a direct role in airway wound repair. To test this hypothesis, the role of CFTR in the initial step of epithelial wound repair-cell migration- was assessed. An impedance based assay was used to objectively measure cell migration rates of human bronchial epithelial cells. Inhibition of CFTR transport and silencing of CFTR protein expression were used to assess the effect of the loss of functional CFTR on cell migration rate. The migration rate of an airway epithelial cell line isolated from a cystic fibrosis patient was also compared to that of an airway epithelial cell line isolated from a healthy lung. A functional effect of CFTR on the process of lamellipodia protrusion during cell migration was also assessed morphometrically. Time lapse video images were also captured during wound closure of airway epithelial cells to assess how cell migration occurs. Additionally, the dependence of airway epithelial cell migration on Cl- and HCO3 - transport was assessed using ion substitution and selective inhibitors of CFTR and other transporters of these ions. Further, the effect of changes in extracellular pH on migration rate was assessed. The results of these studies revealed a role for CFTR transport of Cl- and HCO3 - to regulate airway epithelial cell migration. These studies contribute to the basic understanding of the repair process of the airways following damage and may potentially be important for the design of new treatments to limit airway damage in cystic fibrosis.
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    The roles of KCNQ1 (potassium voltage-gated channel, KQT-like subfamily, Member 1) and CFTR (cystic fibrosis transmembrane conductance regulator) in mouse and human GI cancers
    (2013-09) Than, Bich (Elaine) Le Ngoc
    The ion-channel genes Kcnq1 and Cftr were identified as gastrointestinal (GI) tract cancer susceptibility genes in multiple Sleeping Beauty DNA transposon-based forward genetic screens in mice. Kcnq1 encodes for the pore-forming alpha subunit of a voltage-gated potassium channel and Cftr encodes for the chloride conductance channel. These ion channels act together to maintain ion homeostasis in the cellular and extracellular environment. To confirm that Kcnq1 and Cftr have a functional role in GI tract cancer, mouse models in which targeted mutant alleles of Kcnq1 and Cftr were intogressed into the intestinal tumor susceptible ApcMin strain of mice. Results demonstrated that Kcnq1 mutant mice developed significantly more intestinal tumors, especially in the proximal small intestine and colon, with some of these tumors in the proximal small intestine progressing to adenocarcinomas. Gross tissue abnormalities and neoplasia were also observed in the rectum, pancreas and stomach. Similarly, Cftr mutant mice developed significantly more intestinal tumors, both in the colon and the entire small intestine. Colon organoid formation was significantly increased in organoids created from Kcnq1 mutant and Cftr mutant mice compared with wildtype littermate controls, suggesting a role for Kcnq1 and Cftr in regulation of the intestinal crypt stem cell compartment. To identify gene expression changes due to loss of Kcnq1 and Cftr, we carried out microarray studies in the colon and proximal small intestine. We identified an overlapping set of altered genes involved in innate immune responses, goblet and Paneth cell function, ion channels, intestinal stem cells, EGFR and other growth regulatory signaling pathways. We also found genes implicated in inflammation and in cellular detoxification. Pathway analysis using Ingenuity Pathway Analysis (IPA) and gene set enrichment analysis (GSEA) confirmed the importance of these gene clusters and further identified significant overlap with genes regulated by MUC2, another important regulator of intestinal homeostasis. To investigate the role of KCNQ1 in human colorectal cancer (CRC) we measured protein levels of KCNQ1 by immunohistochemistry in tissue microarrays containing samples from CRC patients with liver metastases who had undergone hepatic resection. Results showed that low expression of KCNQ1 expression was significantly associated with poor overall survival (OS). Our results indicate that both KCNQ1 and CFTR are potent tumor suppressor genes in GI cancer. Defining the mechanisms of action of KCNQ1 and CFTR, and elucidating the nature of their interactions in GI cancer can lead to their use as prognostic biomarkers and potential therapeutic targets for human cancers.