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1.
Am J Physiol Cell Physiol ; 307(2): C195-207, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24898584

RESUMO

Vasoactive intestinal peptide (VIP), a neuropeptide, controls multiple functions in exocrine tissues, including inflammation, and relaxation of airway and vascular smooth muscles, and regulates CFTR-dependent secretion, which contributes to mucus hydration and local innate defense of the lung. We had previously reported that VIP stimulates the VPAC1 receptor, PKCϵ signaling cascade, and increases CFTR stability and function at the apical membrane of airway epithelial cells by reducing its internalization rate. Moreover, prolonged VIP stimulation corrects the molecular defects associated with F508del, the most common CFTR mutation responsible for the genetic disease cystic fibrosis. In the present study, we have examined the impact of the absence of VIP on CFTR maturation, cellular localization, and function in vivo using VIP knockout mice. We have conducted pathological assessments and detected signs of lung and intestinal disease. Immunodetection methods have shown that the absence of VIP results in CFTR intracellular retention despite normal expression and maturation levels. A subsequent loss of CFTR-dependent chloride current was measured in functional assays with Ussing chamber analysis of the small intestine ex vivo, creating a cystic fibrosis-like condition. Interestingly, intraperitoneal administration of VIP corrected tissue abnormalities, close to the wild-type phenotype, as well as associated defects in the vital CFTR protein. The results show in vivo a primary role for VIP chronic exposure in CFTR membrane stability and function and confirm in vitro data.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/fisiologia , Peptídeo Intestinal Vasoativo/metabolismo , Animais , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulação da Expressão Gênica/fisiologia , Intestino Delgado/patologia , Pulmão/patologia , Camundongos , Camundongos Knockout , Mucosa Respiratória/citologia , Traqueia/citologia , Peptídeo Intestinal Vasoativo/genética
2.
Am J Physiol Cell Physiol ; 307(1): C107-19, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24788249

RESUMO

Vasoactive intestinal peptide (VIP) is a topical airway gland secretagogue regulating fluid secretions, primarily by stimulating cystic fibrosis transmembrane conductance regulator (CFTR)-dependent chloride secretion that contributes to the airways innate defense mechanism. We previously reported that prolonged VIP stimulation of pituitary adenylate cyclase-activating peptide receptors (VPAC1) in airway cells enhances CFTR function by increasing its membrane stability. In the present study, we identified the key effectors in the VIP signaling cascade in the human bronchial serous cell line Calu-3. Using immunocytochemistry and in situ proximity ligation assays, we found that VIP stimulation increased CFTR membrane localization by promoting its colocalization and interaction with the scaffolding protein Na(+)/H(+) exchange factor 1 (NHERF1), a PDZ protein known as a positive regulator for CFTR membrane localization. VIP stimulation also increased phosphorylation, by protein kinase Cε of the actin-binding protein complex ezrin/radixin/moesin (ERM) and its interaction with NHERF1 and CFTR complex. On the other hand, it reduced intracellular CFTR colocalization and interaction with CFTR associated ligand, another PDZ protein known to compete with NHERF1 for CFTR interaction, inducing cytoplasmic retention and lysosomal degradation. Reducing NHERF1 or ERM expression levels by specific siRNAs prevented the VIP effect on CFTR membrane stability. Furthermore, iodide efflux assays confirmed that NHERF1 and P-ERM are necessary for VIP regulation of the stability and sustained activity of membrane CFTR. This study shows the cellular mechanism by which prolonged VIP stimulation of airway epithelial cells regulates CFTR-dependent secretions.


Assuntos
Brônquios/enzimologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/enzimologia , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Proteína Quinase C-épsilon/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Brônquios/citologia , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas da Matriz do Complexo de Golgi , Humanos , Proteínas de Membrana Transportadoras , Fosfoproteínas/genética , Fosforilação , Ligação Proteica , Interferência de RNA , Transdução de Sinais , Trocadores de Sódio-Hidrogênio/genética , Fatores de Tempo , Transfecção
3.
Am J Physiol Cell Physiol ; 301(1): C53-65, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21411725

RESUMO

The most common cystic fibrosis causing mutation F508del induces early degradation and reduced trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels to the apical membrane of epithelial cells. In the human nasal epithelial cells JME/CF15, we previously reported that vasoactive intestinal peptide (VIP) exposure corrects trafficking and membrane insertion of functional F508del-CFTR channels at 37°C. Correction of trafficking was PKA dependent, whereas enhanced membrane localization involved PKC. In the present study, we have identified PKCε as the isoform involved in VIP-dependent F508del-CFTR membrane insertion. Iodide effluxes were used to monitor the presence of VIP-rescued functional F508del-CFTR channels at the surface of JME/CF15 cells maintained at 37°C. Iodide efflux peaks measured in response to stimulation with forskolin were insensitive to PKC α, ß, γ, δ, ζ inhibitors. In contrast, efflux peaks were completely inhibited by pretreatment with the PKCε inhibitor peptide EAVSLKPT with an IC(50) of 4.9 µM or by PKCε small interfering RNA (siRNA). Immunostaining and confocal microscopy confirmed that membrane localization of F508del-CFTR induced by VIP was abolished in the presence of EAVSLKPT but not with other isoform inhibitors. In recombinant baby hamster kidney cells, endogenously expressing PKCε but no VIP receptor, wild-type, and F508del-CFTR sensitivity to cpt-cAMP stimulation was increased by PMA treatment. Biotinylation assays and immunoblots confirmed that PMA (0.5-2 h) induced a greater than threefold increase in membrane CFTR, whereas forskolin had no effect. The PMA effect was abolished by specifically inhibiting PKCε (EAVSLKPT IC(50) = 5.7 µM) but not other PKC isoforms. Taken together, these results indicate that stimulating PKCε by VIP or PMA increases membrane insertion and activity of WT- and F508del-CFTR.


Assuntos
Membrana Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Proteína Quinase C-épsilon/metabolismo , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Biotinilação , Linhagem Celular , Cricetinae , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/metabolismo , Humanos , Immunoblotting , Iodetos/metabolismo , Microscopia Confocal , Mutação , Reação em Cadeia da Polimerase , Isoformas de Proteínas , Proteína Quinase C-épsilon/antagonistas & inibidores , Interferência de RNA , RNA Interferente Pequeno , Receptores de Peptídeo Intestinal Vasoativo/genética , Receptores de Peptídeo Intestinal Vasoativo/metabolismo , Acetato de Tetradecanoilforbol/farmacologia
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