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ABSTRACT: Cystic fibrosis transmembrane conductance regulator (CFTR) is an apical membrane chloride channel critical to the regulation of fluid, chloride, and bicarbonate transport in epithelia and other cell types. The most common cause of cystic fibrosis (CF) is the abnormal trafficking of CFTR mutants. Therefore, understanding the cellular machineries that transit CFTR from the endoplasmic reticulum to the cell surface is important. Vasoactive intestinal polypeptide (VIP) plays an important role in CFTR-dependent chloride transport. The present study was designed to observe the affection of VIP on the trafficking of CFTR, and channel gating in human bronchial epithelium cells (HBEC). Confocal microscopy revealed CFTR immunofluorescence extending from the apical membrane deeply into the cell cytoplasm. After VIP treatment, apical extension of CFTR immunofluorescence into the cell was reduced and the peak intensity of CFTR fluorescence shifted towards the apical membrane. Western blot showed VIP increased cell surface and total CFTR. Compared with the augmented level of total CFTR, the surface CFTR increased more markedly. Immunoprecipitation founded that the mature form of CFTR had a marked increase in HBEC treated with VIP. VIP led to a threefold increase in Cl(-) efflux in HBEC. Glibenclamide-sensitive and DIDS-insensitive CFTR Cl(-) currents were consistently observed after stimulation with VIP (10(-8) mol/L). The augmentation of CFTR Cl(-) currents enhanced by VIP (10(-8) mol/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 participate in the VIP-promoted CFTR Cl(-) currents.
Journal of Cellular Biochemistry 03/2011; 112(3):902-8. · 2.87 Impact Factor
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ABSTRACT: 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 checked to 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.
Chemico-biological interactions 12/2008; 179(2-3):219-26. · 2.46 Impact Factor
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ABSTRACT: To probe the mechanisms by which respiratory syncytial virus (RSV) infection in early life forms an important risk factor for the development of chronic asthma, an airway hyper-responsiveness (AHR) animal model of guinea-pigs with persistent RSV infection was established by intranasal instillation of 2 x 10(5) plaque-forming units RSV. On days 0, 7, 28, 42 and 60 postinoculation, the RSV copy numbers, airway function and peptidergic innervation were measured in the peripheral airways. The results showed that the virus was persistent in the lungs. During persistent infection (days 42 and 60), the lung resistance and the total cells, neutrophils and eosinophils of infected guinea-pigs increased significantly; the airway showed signs of chronic inflammation; and the substance P- and calcitonin gene-related peptide-positive fibres increased, but vasoactive intestinal polypeptide-positive fibres decreased. These results suggest that persistent RSV infection can cause long-term chronic airway inflammation and persistent airway neural network abnormality, which may be related to the occurrence of AHR.
Experimental Physiology 08/2008; 93(12):1284-91. · 3.21 Impact Factor
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ABSTRACT: Adhesion molecules play vital roles in airway hyperresponsiveness (AHR) or airway inflammation. Our previous study indicated that adhesion molecule catenin alpha-like 1 (CTNNAL1) is relevant closely to asthma susceptibility, but its biological function or significance is still unclear. In the present study, we observed the temporal and spatial distribution of CTNNAL1 expression in mouse lung tissue with the OVA-sensitized asthma model and found that the level of CTNNAL1 mRNA showed a prominent negative correlation with pulmonary resistance (R(L)). To study the function of CTNNAL1 in airway, effects of CTNNAL1 on proliferation and wound repair activity of human bronchial epithelial cells (HBEC) was investigated with antisense oligonucleotide (ASO) technique. The results showed that: (1) CTNNAL1 ASO could decelerate the repairing velocity and proliferation of HBEC; (2) CTNNAL1 expression was increased on the edge cells of mechanic wounded area in culture; (3) extracellular matrix component fibronectin (Fn) obviously promoted wound repair activity and proliferation of HBEC, which could be blocked by CTNNAL1 ASO; (4) Western blot showed that Fn could promote FAK phosphorylation, which also be inhibited by CTNNAL1 ASO. In conclusion, the level of CTNNAL1 mRNA expression is highly correlated to airway resistance; CTNNAL1 may contribute to the wound repair and proliferation of HBEC. Furthermore, it may serve to Fn mediated cell-extracellular adhesion and its signal transduction.
Journal of Cellular Biochemistry 03/2008; 103(3):920-30. · 2.87 Impact Factor
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ABSTRACT: Respiratory syncytial virus (RSV) infection causes bronchiolitis in infants and children, which is an important risk factor for the development of chronic asthma. To probe the underlying mechanisms that RSV infection increases the susceptibility of asthma, this present study was designed to establish a RSV persistent infection animal model by cyclophosphamide (CYP) pretreatment that more closely mimic human RSV infection. CYP is an immunosuppressant, which induced deficiency in cellular and humoral immunity. Pulmonary RSV titers, airway function and peptidergic innervation were measured on 7d, 28 d, 42 d and 60 d postinfection. The results showed that during RSV persistent infection, the lungs of RSV-inoculated animals pretreated with CYP showed higher RSV titers and exhibited obvious chronic inflammation. The results also showed that protein gene product 9.5 (PGP9.5), substance P (SP) and calcitonin gene-related peptide (CGRP)-immunoreactive fibers increased and vasoactive intestinal peptide (VIP)-immunoreactive fibers decreased during RSV persistent infection. These results demonstrate that RSV persistent infection induces significant alterations in the peptidergic innervation in the airways, which may be associated with the development of altered airway function.
Peptides 02/2008; 29(1):47-56. · 2.43 Impact Factor
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ABSTRACT: Airway re-modelling in asthma usually results in an irreversible weakness of pulmonary ventilation, however, its initiating or controlling mechanism remains unclear. In this study, we hypothesize that signal communication between airway epithelial cells and sub-mucosal fibroblast cells may play an important role in the maintenance of structure homeostasis in a physiologic condition and in initiation of airway remodelling in a stressed condition. To test the hypothesis, a co-cultured system of human bronchial epithelial cells (BEC) and human lung fibroblasts (HLF) were designed to observe the effects of BEC, in the normal state or in a BRS-3 activated state, on the proliferation and collagen synthesis of HLF. The results showed that the proliferation activities of both BEC and HLF inhibited each other under the normal state. BRS-3-activated BEC can transform the reciprocal inhibition into promoting effects. The secretion of TGF-beta1 increased and the synthesis of PGE2 decreased from BRS-3-activated BEC, which were correlated with the proliferation and collagen synthesis of HLF. The proliferation activities of HLF were weakened by co-culture with TGF-beta1 antisense oligonucleotides (ASO) treated BEC. It was concluded that, in the normal state, BEC inhibits the activities of fibroblasts through release of PGE2 to maintain the airway homeostasis; however when stressed, for example by BRS-3 activation, BEC promote the activities of fibroblasts mediated by TGF-beta1, thereby facilitating the airway re-modelling.
Cell Biology International 01/2008; 31(12):1495-500. · 1.48 Impact Factor
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ABSTRACT: It is commonly accepted that airway hyperresponsiveness (AHR) is a chronic airway inflammation although the exact mechanism of its pathogenesis is still unclear. In the past ten years, an epithelial defect hypothesis has gradually gained supports from the main stream. Airway epithelium is no longer considered only as a simple mechanic barrier but an active interface between the inner and outer environment. Bronchial epithelial cells play a critical role in maintenance of homeostasis in the airway local microenvironment through a wide range of physiologic functions including anti-oxidation, exocrine/endocrine secretions, mucus production and antigen presentation under health and stressed/inflamed/injured conditions. It is reasonably hypothesized that disruption of these functional processes or defects in airway epithelium integrity may be the initial steps leading to airway hyperresponsiveness such as in asthma and chronic obstructive pulmonary disease.
Sheng li xue bao: [Acta physiologica Sinica] 09/2007; 59(4):454-64.
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ABSTRACT: Previously, we found that bombesin receptor subtype 3 (BRS-3) significantly increased in an ozone-stressed airway hyperresponsiveness animal model and resulted in induced wound repair and protection from acute lung injury. In the present study, we determined molecular mechanisms of BRS-3 regulation in human BECs (bronchial epithelial cells) in response to ozone stress. Ten oligonucleotide probes corresponding to various regions of the BRS-3 promoter were used in EMSA (electrophoretic mobilityshift assays). Four were found to have an enhanced mobility shift with extracts from ozone-stressed cells. On the basis of the assay of mutated probes binding with extracts and antibody supershift, they were verified as MTF-1 (metal-regulatory-element-binding transcription factor-1), PPARalpha (peroxisome-proliferator-activated receptor alpha), AP-2alpha (activator protein 2alpha) and HSF-1 (heat-shock factor 1). Next, ChIP (chromatin immunoprecipitation) assay, site-directed mutagenesis technology and antisense oligonucleotide technology were used to observe these transcription factors associated with the BRS-3 promoter. Only AP-2alpha and PPARalpha increased ozone-inducible DNA binding on the BRS-3 promoter and BRS-3 expression. The time courses of AP-2alpha and PPARalpha activation, followed by BRS-3 expression, were also examined. It was shown that ozone-inducible BRS-3 expression and AP-2alpha- and PPARalpha-binding activity correlated over a 48 h period. The translocation of PPARalpha was observed by immunofluorescence assay, which showed that PPARalpha nuclear translocation increased after ozone exposure. Our data suggest that AP-2alpha and PPARalpha may be especially involved in this ozone-inducible up-regulation mechanism of BRS-3 expression.
Biochemical Journal 08/2007; 405(1):131-7. · 4.90 Impact Factor
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ABSTRACT: The present study was designed to investigate the role of bombesin receptor subtype 3 (BRS-3) in airway wound repair. The results showed that: (1) There was few expression of BRS-3 mRNA in the control group. In contrast, the expression of BRS-3 mRNA was gradually increased in the early 2 days, and peaked on the fourth day, and then decreased in the ozone-stressed AHR animal. BRS-3 mRNA was distributed in the ciliated columnar epithelium, monolayer columnar epithelium cells, scattered mesenchymal cells and Type II alveolar cells; (2) The wound repair and proliferation of bronchial epithelial cells (BECs) were accelerated in a concentration-dependent manner by BRS-3 activation with P3513, which could be inhibited by PKA inhibitor H89. The study demostrated that activation of BRS-3 may play an important role in wound repair of AHR.
Peptides 08/2006; 27(7):1852-8. · 2.43 Impact Factor
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ABSTRACT: To investigate the role and mechanism of bombesin receptor subtype 3 (BRS-3) in the proliferation and protection against injury of human brochial epithelial cells (HBECs).
Effect of P3513 (a specific agonist of BRS-3) on the proliferation of HBECs was observed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method; the release rate of 3H-Udr, and LDH activity, catalase activity, and the expression of cadherin and integrin beta1 were also analyzed under O3 stress with or without P3513 treatment.
The proliferation of HBECs was accelerated by P3513 in a concentration-dependent manner (10(-9) approximately 10(-7) mol/L). Ozone stress could promote the release rate of 3H-Udr, and LDH activity, which could be inhibited by P3513. P3513 could promote the activity of catalase. The effect of proliferation and protection against injury caused by P3513 could be inhibited by W7 (calmodulin inhibitor), PD98059 (tyrosin kinase inhibitor) and H89 (PKA inhibitor). P3513 could stimulate the expression of caderin and integrinbeta1 of ozone-stressed HBECs.
Activation of BRS-3 caused by P3513 may play an important role in protecting HBECs from oxidant injury, and the signal pathway is possibly relevant to Ca2+, MEK and PKA.
Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences 05/2006; 31(2):178-83.
