RESUMO
Therapeutic options to treat virus-induced asthma exacerbations are limited and urgently needed. Therefore, we tested Pim1 kinase as potential therapeutic target in human rhinovirus (HRV) infections. We hypothesised that inhibition of Pim1 kinase reduces HRV replication by augmenting the interferon-induced anti-viral response due to increased activity of the janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway.Air-liquid interface (ALI) cultures of primary bronchial epithelial cells (PBECs) from healthy individuals and moderate-to-severe asthmatic volunteers were infected with HRV-16 with or without a specific Pim1 inhibitor; viral replication and induction of anti-viral responses were measured using RT-qPCR. Viral titres were measured by 50% tissue culture infective dose and release of interferon-γ-induced protein 10 (IP-10) and RANTES protein assessed by ELISA. Phosphorylation of STAT-1 was determined using western blotting.Viral replication was reduced in ALI cultures of healthy and asthmatic PBECs treated with the Pim1 inhibitor. Using cultures from healthy donors, enhanced STAT-1 phosphorylation upon inhibition of Pim1 kinase activity resulted in increased mRNA expression of interferon-ß, interleukin-29, IP-10 and RANTES 12â h after infection and increased protein levels of IP-10 and RANTES 24â h after infection.We have identified Pim1 kinase as novel target to reduce viral replication in ALI cultures of PBECs. This may open new avenues for therapeutic interventions in virus-induced asthma exacerbations.
Assuntos
Asma/virologia , Citocinas/metabolismo , Células Epiteliais/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Rhinovirus/fisiologia , Replicação Viral , Células Cultivadas , Quimiocina CCL5/metabolismo , Progressão da Doença , Humanos , Interferon beta/metabolismo , Interferon gama/metabolismoRESUMO
Single-channel electrophysiology with lipid bilayer systems requires ion channel expression, purification from cell culture, and reconstitution in proteoliposomes for delivery to a planar bilayer. Here we demonstrate that single-channel current measurements of the potassium channels KcsA and hERGS5-S6 can be obtained by direct insertion in interdroplet lipid bilayers from microliters of a cell-free expression medium.
Assuntos
Canais Iônicos/fisiologia , Bicamadas Lipídicas , Sistema Livre de Células , ProteolipídeosRESUMO
We show that interactions of fatty acids with the central cavity of potassium channel KcsA can be characterized using the fluorescence probe 11-dansylaminoundecanoic acid (Dauda). The fluorescence emission spectrum of Dauda bound to KcsA in bilayers of dioleoylphosphatidylcholine contains three components, which can be attributed to KcsA-bound and lipid-bound Dauda together with unbound Dauda. The binding of Dauda to KcsA was characterized by a dissociation constant of 0.47 ± 0.10 µM with 0.94 ± 0.06 binding site per KcsA tetramer. Displacement of KcsA-bound Dauda by the tetrabutylammonium (TBA) ion confirmed that the Dauda binding site was in the central cavity of KcsA. Dissociation constants for a range of fatty acids were determined by displacement of Dauda: binding of fatty acids increased in strength with an increasing chain length from C14 to C20 but then decreased in strength from C20 to C22. Increasing the number of double bonds in the chain from one to four had little effect on binding, dissociation constants for oleic acid and arachidonic acid, for example, being 2.9 ± 0.2 and 3.0 ± 0.4 µM, respectively. Binding of TBA to KcsA was very slow, whereas binding of Dauda was fast, suggesting that TBA can enter the cavity only through an open channel whereas Dauda can bind to the closed channel, presumably entering the cavity via the lipid bilayer.
Assuntos
Proteínas de Bactérias/metabolismo , Ácidos Graxos/metabolismo , Canais de Potássio/metabolismo , Álcool Desidrogenase , Sítios de Ligação , Compostos de Dansil/química , Compostos de Dansil/metabolismo , Ácidos Graxos/química , Fluorescência , Lipídeos/química , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Espectrofotometria AtômicaRESUMO
Interactions of fatty acids with the potassium channel KcsA were studied using Trp fluorescence quenching and electron paramagnetic resonance (EPR) techniques. The brominated analogue of oleic acid was shown to bind to annular sites on KcsA and to the nonannular sites at each protein-protein interface in the homotetrameric structure with binding constants relative to dioleoylphosphatidylcholine of 0.67 ± 0.04 and 0.87 ± 0.08, respectively. Mutation of the two Arg residues close to the nonannular binding sites had no effect on fatty acid binding. EPR studies with a spin-labeled analogue of stearic acid detected a high-affinity binding site for the fatty acid with strong immobilization. Fluorescence quenching studies with the spin-labeled analogue showed that the binding site detected in the EPR experiments could not be one of the annular or nonannular binding sites. Instead, it is proposed that the EPR studies detect binding to the central hydrophobic cavity of the channel, with a binding constant in the range of ~0.1-1 µM.
Assuntos
Ácidos Graxos/metabolismo , Canais de Potássio/metabolismo , Sítios de Ligação , Espectroscopia de Ressonância de Spin EletrônicaAssuntos
Apoptose/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , RNA Mensageiro/efeitos dos fármacos , RNA Viral/análise , Rhinovirus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Brônquios/citologia , Células Cultivadas , Quimiocina CCL5/efeitos dos fármacos , Quimiocina CCL5/genética , Quimiocina CXCL10/efeitos dos fármacos , Quimiocina CXCL10/genética , Células Epiteliais/virologia , Humanos , Técnicas In Vitro , Interferon beta/efeitos dos fármacos , Interferon beta/genética , Interferons , Interleucinas/genética , RNA Mensageiro/metabolismo , Carga Viral/efeitos dos fármacosRESUMO
Airborne particulate matter (PM) is a leading cause of mortality and morbidity. However, understanding of the range and mechanisms of effects of PM components is poor. PM generated in underground railways is rich in metals, especially iron. In the ultrafine (UFPM; <0.1 µm diameter) fraction, the combination of small size and metal enrichment poses an unknown health risk. This study aimed to analyse transcriptomic responses to underground UFPM in primary bronchial epithelial cells (PBECs), a key site of PM deposition. The oxidation state of iron in UFPM from an underground station was determined by X-ray absorption near edge structure (XANES) spectroscopy. Antioxidant response was assayed using a reporter cell line transfected with an antioxidant response element (ARE)-luciferase construct. Differentiated PBECs were exposed to UFPM for 6 h or 24 h for RNA-Seq and RT-qPCR analysis. XANES showed predominance of redox-active Fe3O4, with ROS generation confirmed by induction of ARE-luciferase expression. 6 h exposure of PBECs to UFPM identified 52 differentially expressed genes (DEGs), especially associated with epithelial maintenance, whereas 24 h exposure yielded 23 DEGs, particularly involved with redox homeostasis and metal binding. At both timepoints, there was upregulation of members of the metallothionein family, low molecular weight proteins with antioxidant activity whose main function is binding and homeostasis of zinc and copper ions, but not iron ions. This upregulation was partially inhibited by metal chelation or ROS scavenging. These data suggest differential regulation of responses to metal-rich UFPM depending on exposure period, and highlight novel pathways and markers of PM exposure, with the role of metallothioneins warranting further investigation.
Assuntos
Metalotioneína/metabolismo , Antioxidantes/química , Antioxidantes/metabolismo , Cobre/metabolismo , Metalotioneína/química , Oxirredução , Material Particulado/química , Espécies Reativas de Oxigênio/metabolismo , Espectroscopia por Absorção de Raios X , Zinco/metabolismoRESUMO
One of the most important actions of insulin is the stimulation of the uptake of glucose into fat and muscle cells. Crucial to this response is the translocation of GLUT4 (glucose transporter-4) to the plasma membrane. The insulin-stimulated GLUT4 vesicle docking at the plasma membrane requires an interaction between VAMP-2 (vesicle-associated membrane protein-2) on the GLUT4 vesicle and syntaxin-4 in the plasma membrane. In the basal state, munc18c is thought to preclude GLUT4 vesicle docking by inhibiting this interaction. Here, we have used FCS (fluorescence correlation spectroscopy) in single living cells to show that munc18c binds to syntaxin-4 in both the basal and insulin-stimulated states. We show that munc18c contains two binding sites for syntaxin-4, one of which is disrupted by insulin, while the other is activated by insulin. Insulin-triggered repositioning of munc18c on syntaxin-4 in this way in turn allows syntaxin-4 to adopt its 'open' conformation and bind VAMP-2, resulting in the docking of the GLUT4 vesicle at the cell surface. The results also demonstrate the utility of using FCS in intact single living cells to elucidate cell signalling events.
Assuntos
Transportador de Glucose Tipo 4/fisiologia , Insulina/farmacologia , Proteínas Munc18/fisiologia , Proteínas Qa-SNARE/fisiologia , Células 3T3 , Adipócitos/fisiologia , Animais , Células CHO , Cricetinae , Cricetulus , Eletroporação , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Camundongos , Microscopia Confocal , Proteínas Recombinantes/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
Development of biocompatible and functional scaffolds for tissue engineering is a major challenge, especially for development of polarised epithelia that are critical structures in tissue homeostasis. Different in vitro models of the lung epithelial barrier have been characterized using non-degradable polyethylene terephthalate membranes which limits their uses for tissue engineering. Although poly-L-lactic acid (PLLA) membranes are biodegradable, those prepared via conventional Diffusion Induced Phase Separation (DIPS) lack open-porous geometry and show limited permeability compromising their use for epithelial barrier studies. Here we used PLLA membranes prepared via a modification of the standard DIPS protocol to control the membrane surface morphology and permeability. These were bonded to cell culture inserts for use in barrier function studies. Pulmonary epithelial cells (H441) readily attached to the PLLA membranes and formed a confluent cell layer within two days. This was accompanied by a significant increase in trans-epithelial electrical resistance and correlated with the formation of tight junctions and vectorial cytokine secretion in response to TNFα. Our data suggest that a structurally polarized and functional epithelial barrier can be established on PLLA membranes produced via a non-standard DIPS protocol. Therefore, PLLA membranes have potential utility in lung tissue engineering applications requiring bio-absorbable membranes.
Assuntos
Células Epiteliais/citologia , Células Epiteliais/fisiologia , Pulmão/citologia , Pulmão/fisiologia , Poliésteres/química , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Implantes Absorvíveis , Materiais Biocompatíveis/química , Adesão Celular/fisiologia , Técnicas de Cultura de Células/métodos , Linhagem Celular , Citocinas/metabolismo , Impedância Elétrica , Humanos , Teste de Materiais , Membranas Artificiais , Polietilenotereftalatos/química , Junções Íntimas/fisiologiaRESUMO
Insulin triggers the translocation of glucose transporter GLUT4 to the plasma membrane. To understand the nature of the missing links between upstream insulin activated kinases and proteins of the GLUT4 translocation apparatus, the role of 80K-H was examined to test if it was one such missing link in live cells. Fluorescence correlation spectroscopy showed that the mobility of 80K-H was significantly decreased by insulin stimulation. This was dependent on the presence of PKCzeta and an intact binding site for PKCzeta. Insulin also increased the mobility of munc18c in an 80K-H- and PKCzeta dependent manner. These results indicate that insulin induces dynamic associations between PKCzeta, 80K-H, and munc18c and that 80K-H may act as a key signaling link between PKCzeta and munc18c in live cells.
Assuntos
Transportador de Glucose Tipo 4/metabolismo , Glucosidases/metabolismo , Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Munc18/metabolismo , Proteína Quinase C/metabolismo , Animais , Células CHO , Proteínas de Ligação ao Cálcio , Cricetinae , Cricetulus , Glucosidases/genética , Humanos , Insulina/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Munc18/genética , Proteína Quinase C/genética , Transdução de SinaisRESUMO
The bronchial epithelium and underlying fibroblasts form an epithelial mesenchymal trophic unit (EMTU) which controls the airway microenvironment. We hypothesized that cell-cell communication within the EMTU propagates and amplifies the innate immune response to respiratory viral infections. EMTU co-culture models incorporating polarized (16HBE14o-) or differentiated primary human bronchial epithelial cells (HBECs) and fibroblasts were challenged with double-stranded RNA (dsRNA) or rhinovirus. In the polarized EMTU model, dsRNA affected ionic but not macromolecular permeability or cell viability. Compared with epithelial monocultures, dsRNA-stimulated pro-inflammatory mediator release was synergistically enhanced in the basolateral compartment of the EMTU model, with the exception of IL-1α which was unaffected by the presence of fibroblasts. Blockade of IL-1 signaling with IL-1 receptor antagonist (IL-1Ra) completely abrogated dsRNA-induced basolateral release of mediators except CXCL10. Fibroblasts were the main responders to epithelial-derived IL-1 since exogenous IL-1α induced pro-inflammatory mediator release from fibroblast but not epithelial monocultures. Our findings were confirmed in a differentiated EMTU model where rhinovirus infection of primary HBECs and fibroblasts resulted in synergistic induction of basolateral IL-6 that was significantly abrogated by IL-1Ra. This study provides the first direct evidence of integrated IL-1 signaling within the EMTU to propagate inflammatory responses to viral infection.
Assuntos
Comunicação Celular , Microambiente Celular , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Interleucina-1alfa/metabolismo , Mucosa Respiratória/metabolismo , Permeabilidade Capilar , Linhagem Celular , Células Cultivadas , Quimiocina CXCL10/metabolismo , Células Epiteliais/virologia , Fibroblastos/virologia , Humanos , Mucosa Respiratória/citologia , Mucosa Respiratória/virologia , Rhinovirus/patogenicidade , Transdução de SinaisRESUMO
BACKGROUND: The asthma gene PCDH1 encodes Protocadherin-1, a putative adhesion molecule of unknown function expressed in the airway epithelium. Here, we characterize the localization, differential expression, homotypic adhesion specificity and function of PCDH1 in airway epithelial cells in asthma. METHODS: We performed confocal fluorescence microscopy to determine subcellular localization of PCDH1 in 16HBE cells and primary bronchial epithelial cells (PBECs) grown at air-liquid interface. Next, to compare PCDH1 expression and localization in asthma and controls we performed qRT-PCR and fluorescence microscopy in PBECs and immunohistochemistry on airway wall biopsies. We examined homotypic adhesion specificity of HEK293T clones overexpressing fluorescently tagged-PCDH1 isoforms. Finally, to evaluate the role for PCDH1 in epithelial barrier formation and repair, we performed siRNA knockdown-studies and measured epithelial resistance. RESULTS: PCDH1 localized to the cell membrane at cell-cell contact sites, baso-lateral to adherens junctions, with increasing expression during epithelial differentiation. No differences in gene expression or localization of PCDH1 isoforms expressing the extracellular domain were observed in either PBECs or airway wall biopsies between asthma patients and controls. Overexpression of PCDH1 mediated homotypic interaction, whereas downregulation of PCDH1 reduced epithelial barrier formation, and impaired repair after wounding. CONCLUSIONS: In conclusion, PCDH1 is localized to the cell membrane of bronchial epithelial cells baso-lateral to the adherens junction. Expression of PCDH1 is not reduced nor delocalized in asthma even though PCDH1 contributes to homotypic adhesion, epithelial barrier formation and repair.