| Objectives:The change of mucosal fluid composition result in the decrease of mucosa cilia clearance rate,the increase abnormally of airway mucosal fluid secretory volume,a key pathophysiological process in airway hyperresponsive diseases such as asthma and bronchitis.Cystic fibrosis transmembrane conductance regulator(CFTR),functions as a cAMP-regulated Cl- channel which controls transepithelial electrolyte transport,fluid flow,ion concentrations in the airway.Loss of CFTR Cl-function caused a lack of fluid secretion with excessive fluid absorption, which led to watery component reduction in airway surface liquid,mucous thickening,blockage of submucosal gland ducts,and impairment of mucociliary clearance that were followed with infection,inflammation,and ultimately,the tissue destruction characteristic of bronchiectasis.on account of above part cue,we propose that the gating and the expression regulation of CFTR in BECs participate in the homeostasis of airway function,which the defect of CFTR function could be related with AHR.Contents:1.Ozone stress down-regulates the expression of cystic fibrosis transmembrane conductance regulator in human bronchial epithelial cellsWistar rat inhaled 1.5ppm ozone 1h per day for three days to establish a airway hyperresponsiveness animal model.BECs were cultured on slides in DMEM/F12 medium with or without 1.5ppm ozone for 30 minutes to establish oxitative stress model.To investigate abnormalities of cystic fibrosis transmembrane conductance regulator(CFTR) expression in chronic inflammatory airway diseases and its regulation mechanisms,the present study was designed to observe the expression of CFTR,CFTR chloride current and the possible relevant signal pathways in in vitro and in vivo bronchial epithelium by using real-time PCR,immunofluorescence, western blot and whole cell patch-clamp.The results demonstrated that CFTR staining was decreased in rat airway epithelium under ozone stress. Ozone stress also down-regulated CFTR protein and mRNA expression and CFTR chloride current in cultured human bronchial epithelial cells (HBEC).STAT1 signal pathway was checkedto investigate the signal mechanism.It was found that pretreatment with STAT1 inhibitor attenuated the down-regulated CFTR expression induced by ozone stress. We also observed that ozone stress accelerated the phosphorylation of STAT1 in HBEC,which could be influenced by some signaling molecules related to the early transduction of cellular stress.Furthermore,reactive oxygen species inhibitors N-acetylcysteine and nitric oxide synthase inhibitor aminoguanidine increased the expression of CFTR.Ozone stress could down-regulate the expression of CFTR and decrease CFTR chloride current in HBEC.The signal mechanism which referred to cascade events in cells included early oxidative stress signal transmission molecules,and subsequently transcription modulator STAT1.2.Activation of CFTR trafficking and gating by vasoactive intestinal peptide in Human bronchial epithelial cellsThe present study was designed to observe the trafficking of CFTR, and channel gating in Human bronchial epithelium cells(HBEC) by using confocal microscopy,western blot,immunoprecipitation and Whole-cell patch clamp.Confocal microscopy revealed CFTR immunofluorescence extending from the apical membrane deeply into the cell cytoplasm. During stimulation with VIP,apical extension of CFTR immunofluorescence into the cell was reduced significantly and the peak intensity of CFTR fluorescence shifted towards the apical membrane. Western blot showed VIP raised cell surface and total CFTR.Compare with the augmented level of total CFTR,the surface CFTR increased well than that the total CFTR.Immunoprecipitation founded that CFTR band C had an increase markedly in HBEC treated with VIP,compared with the control group.We also observed an increase in the CFTR band B,the immature form of CFTR,which the extent was lower than that band C. VIP led to 4-fold increases in Cl-effiux in HBEC.Glibenclamide-sensitive and DIDS-insensitive CFTR Cl- currents was consistently observed after stimulation with VIP(10-8mol/L).The augmention of CFTR Cl- currents enhanced by VIP(10-8mol/L) was reversed,at least in part,by the protein kinase A(PKA) inhibitor,H-89 and the protein kinase C(PKC) inhibitor, H-7,suggesting PKA and PKC participated in the VIP-promoted CFTR Cl-currents.3.Study gene expression modulation of CFTRTo probe the mechanisms of the induced expression of CFTR under ozone stress,six oligonucleotide probes corresponding to various regions of the CFTR promoter were used in EMSA studies.Two were found to have a decrease mobility shift with extracts from ozone-stressed cells. Based on the assay of antibody supershift,they were verified as Sp1 and ERα.By ChIP assay,Sp1 and ERαdecreaed the ozone-inducible DNA binding on the CFTR promoter.Next,site-directed mutagenesis technology and antisense oligonucleotide technology were used to observe the inhibitory effects of the two nuclear factors on CFTR promoter activation and expression.The results also showed that Sp1 and ERαdecreased the ozone-inducible DNA binding on the CFTR promoter and CFTR expression.The translocation of Sp1 was observed by immunofluorescence assay, which showed that Sp1 nuclear translocation was decreased after ozone exposure.The time courses of Sp1 and ERαactivation and inactivation, followed by CFTR expression were also examined.It was shown that ozone-depression CFTR expression and Sp1 and ERαbinding activity correlated during a-24 hour time course.In summary:In present study,we observed that ozone stress repressed the eapression and function of CFTR,which provided support for studies investigating CFTR function in inflammatory lung diseases other than cystic fibrosis.We also investigate that the production of NO,ROS were increased and participated in activation of STAT1 after ozone stress,which contributed to the inhibition of CFTR expression,the binding activity and the translocation were inhibited of Sp1,ERαafter ozone stress,which resulted in the repression of CFTR transcription.In addition,we revealed VIP activated the trafficking and gating,the CFTR channel was regulated by PKA and PKC pathway through the phosphorylation of CFTR regulatory domain,which offered a choose to cure inflammatory lung diseases.Conclusion:Ozone stress repressed the expression and function of CFTR in BECs. As follow were its mechanisms:(1) Ozone stress down-regulated the expression of CFTR.Its pathways had NO,ROS,JAK/STAT.Ozone stress depressed the binding activity and the translocation of Sp1,ERα,which resulted in the inhibition of CFTR transcription;(2) Ozone stress inhibitted CFTR cl- current,VIP activated CFTR cl-current.Its signal pathway had PKA and PKC;(3) VIP raised CFTR trafficking,which included enhance recycling of CFTR,augment the expression of mature CFTR and the protein expression of plasmalemma CFTR.
|
PDF Full Text Request