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1.
Am J Respir Cell Mol Biol ; 66(3): 271-282, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34807800

RESUMO

Orai1 is a plasma membrane Ca2+ channel that mediates store-operated Ca2+ entry (SOCE) and regulates inflammation. Short palate lung and nasal epithelial clone 1 (SPLUNC1) is an asthma gene modifier that inhibits Orai1 and SOCE via its C-terminal α6 region. SPLUNC1 levels are diminished in asthma patient airways. Thus, we hypothesized that inhaled α6 peptidomimetics could inhibit Orai1 and reduce airway inflammation in a murine asthma model. To evaluate α6-Orai1 interactions, we used fluorescent assays to measure Ca2+ signaling, Förster resonance energy transfer, fluorescent recovery after photobleaching, immunostaining, total internal reflection microscopy, and Western blotting. To test whether α6 peptidomimetics inhibited SOCE and decreased inflammation in vivo, wild-type and SPLUNC1-/- mice were exposed to house dust mite (HDM) extract with or without α6 peptide. We also performed nebulization, jet milling, and scanning electron microscopy to evaluate α6 for inhalation. SPLUNC1-/- mice had an exaggerated response to HDM. In BAL-derived immune cells, Orai1 levels increased after HDM exposure in SPLUNC1-/- but not wild-type mice. Inhaled α6 reduced Orai1 levels in mice regardless of genotype. In HDM-exposed mice, α6 dose-dependently reduced eosinophilia and neutrophilia. In vitro, α6 inhibited SOCE in multiple immune cell types, and α6 could be nebulized or jet milled without loss of function. These data suggest that α6 peptidomimetics may be a novel, effective antiinflammatory therapy for patients with asthma.


Assuntos
Asma , Peptidomiméticos , Animais , Asma/tratamento farmacológico , Cálcio/metabolismo , Glicoproteínas , Humanos , Inflamação , Pulmão/metabolismo , Camundongos , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Fosfoproteínas
2.
Int J Mol Sci ; 21(7)2020 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-32230828

RESUMO

Multipotent mesenchymal stem cells (MSCs) possess regenerative properties and have been shown to improve outcomes and survival in acute and chronic lung diseases, but there have been some safety concerns raised related to MSC-based therapy. Subsequent studies have demonstrated that many of the regenerative effects of MSCs can be attributed to the MSC-derived secretome, which contains soluble factors and extracellular vesicles (EVs). MSC-derived extracellular vesicles (MSC-derived EVs) replicate many of the beneficial effects of MSCs and contain a variety of bioactive factors that are transferred to recipient cells, mediating downstream signaling. MSC-derived EV therapy holds promise as a safe and effective treatment for pulmonary disease, but there remain many scientific and clinical questions that will need to be addressed before EVs are widely applied as a therapy. To date, the use of MSC-derived EVs as a treatment for lung disease has been conducted primarily in in vitro or pre-clinical animal models. In this review, we will discuss the current published research investigating the use of EVs as a potential therapeutic for acute lung injury/acute respiratory distress syndrome (ALI/ARDS), bronchopulmonary dysplasia (BPD), idiopathic pulmonary fibrosis (IPF), pulmonary arterial hypertension (PAH), asthma, and silicosis.


Assuntos
Vesículas Extracelulares , Pneumopatias/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Lesão Pulmonar Aguda/terapia , Animais , Asma/terapia , Humanos , Células-Tronco Mesenquimais , Síndrome do Desconforto Respiratório/terapia
3.
Proc Natl Acad Sci U S A ; 109(41): 16528-33, 2012 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-23012413

RESUMO

MUC5AC, a major gel-forming mucin expressed in the lungs, is secreted at increased rates in response to infectious agents, implying that mucins exert a protective role against inhaled pathogens. However, epidemiological and pathological studies suggest that excessive mucin secretion causes airways obstruction and inflammation. To determine whether increased MUC5AC secretion alone produces airway obstruction and/or inflammation, we generated a mouse model overexpressing Muc5ac mRNA ~20-fold in the lungs, using the rCCSP promoter. The Muc5ac cDNA was cloned from mouse lungs and tagged internally with GFP. Bronchoalveolar lavage fluid (BALF) analysis demonstrated an approximate 18-fold increase in Muc5ac protein, which formed high-molecular-weight polymers. Histopathological studies and cell counts revealed no airway mucus obstruction or inflammation in the lungs of Muc5ac-transgenic (Muc5ac-Tg) mice. Mucus clearance was preserved, implying that the excess Muc5ac secretion produced an "expanded" rather than more concentrated mucus layer, a prediction confirmed by electron microscopy. To test whether the larger mucus barrier conferred increased protection against pathogens, Muc5ac-Tg animals were challenged with PR8/H1N1 influenza viruses and showed significant decreases in infection and neutrophilic responses. Plaque assay experiments demonstrated that Muc5ac-Tg BALF and purified Muc5ac reduced infection, likely via binding to α2,3-linked sialic acids, consistent with influenza protection in vivo. In conclusion, the normal mucus transport and absence of a pulmonary phenotype in Muc5ac-Tg mice suggests that mucin hypersecretion alone is not sufficient to trigger luminal mucus plugging or airways inflammation/goblet cell hyperplasia. In contrast, increased Muc5ac secretion appears to exhibit a protective role against influenza infection.


Assuntos
Modelos Animais de Doenças , Vírus da Influenza A Subtipo H1N1/imunologia , Pulmão/imunologia , Mucina-5AC/imunologia , Infecções por Orthomyxoviridae/imunologia , Animais , Sequência de Bases , Western Blotting , Líquido da Lavagem Broncoalveolar/química , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Pulmão/metabolismo , Pulmão/virologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Microscopia Eletrônica , Dados de Sequência Molecular , Mucina-5AC/genética , Mucina-5AC/metabolismo , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
4.
FASEB J ; 26(2): 533-45, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21990373

RESUMO

Cigarette smoke (CS) exposure induces mucus obstruction and the development of chronic bronchitis (CB). While many of these responses are determined genetically, little is known about the effects CS can exert on pulmonary epithelia at the protein level. We, therefore, tested the hypothesis that CS exerts direct effects on the CFTR protein, which could impair airway hydration, leading to the mucus stasis characteristic of both cystic fibrosis and CB. In vivo and in vitro studies demonstrated that CS rapidly decreased CFTR activity, leading to airway surface liquid (ASL) volume depletion (i.e., dehydration). Further studies revealed that CS induced internalization of CFTR. Surprisingly, CS-internalized CFTR did not colocalize with lysosomal proteins. Instead, the bulk of CFTR shifted to a detergent-resistant fraction within the cell and colocalized with the intermediate filament vimentin, suggesting that CS induced CFTR movement into an aggresome-like, perinuclear compartment. To test whether airway dehydration could be reversed, we used hypertonic saline (HS) as an osmolyte to rehydrate ASL. HS restored ASL height in CS-exposed, dehydrated airway cultures. Similarly, inhaled HS restored mucus transport and increased clearance in patients with CB. Thus, we propose that CS exposure rapidly impairs CFTR function by internalizing CFTR, leading to ASL dehydration, which promotes mucus stasis and a failure of mucus clearance, leaving smokers at risk for developing CB. Furthermore, our data suggest that strategies to rehydrate airway surfaces may provide a novel form of therapy for patients with CB.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Pulmão/metabolismo , Fumar/efeitos adversos , Fumar/metabolismo , Adulto , Idoso , Animais , Sequência de Bases , Transporte Biológico Ativo , Água Corporal/metabolismo , Bronquite Crônica/etiologia , Bronquite Crônica/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Cricetinae , Fibrose Cística/etiologia , Fibrose Cística/metabolismo , Fibrose Cística/terapia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Primers do DNA/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Depuração Mucociliar , Mucosa Respiratória/metabolismo , Solução Salina Hipertônica/farmacologia , Fumaça/efeitos adversos , Solubilidade
5.
J Biol Chem ; 286(2): 1381-8, 2011 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-21056985

RESUMO

Members of the Anoctamin (Ano)/TMEM16A family have recently been identified as essential subunits of the Ca(2+)-activated chloride channel (CaCC). For example, Ano1 is highly expressed in multiple tissues including airway epithelia, where it acts as an apical conduit for transepithelial Cl(-) secretion and helps regulate lung liquid homeostasis and mucus clearance. However, little is known about the oligomerization of this protein in the plasma membrane. Thus, utilizing mCherry- and eGFP-tagged Ano1 constructs, we conducted biochemical and Förster resonance energy transfer (FRET)-based experiments to determine the quaternary structure of Ano1. FRET and co-immunoprecipitation studies revealed that tagged Ano1 subunits directly associated before they reached the plasma membrane. This association was not altered by changes in cytosolic Ca(2+), suggesting that this is a fixed interaction. To determine the oligomeric structure of Ano1, we performed chemical cross-linking, non-denaturing PAGE, and electromobility shift assays, which revealed that Ano1 exists as a dimer. These data are the first to probe the quaternary structure of Ano1. Understanding the oligomeric nature of Ano1 is an essential step in the development of therapeutic drugs that could be useful in the treatment of cystic fibrosis.


Assuntos
Cálcio/metabolismo , Canais de Cloreto , Fibrose Cística/metabolismo , Citoesqueleto de Actina/fisiologia , Animais , Anoctamina-1 , Biofísica , Membrana Celular/metabolismo , Canais de Cloreto/química , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Dimerização , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Camundongos , Técnicas de Patch-Clamp , Domínios e Motivos de Interação entre Proteínas/fisiologia , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Mucosa Respiratória/citologia , Transfecção
6.
FASEB J ; 25(9): 2996-3003, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21628448

RESUMO

Adenosine (ADO) is an extracellular signaling molecule that is an important regulator of innate lung defense. On binding ADO, the A2B receptor (A2BR) stimulates cAMP production to activate the CFTR Cl(-) channel, increase ciliary beating, and initiate cytokine secretion. We tested the hypothesis that CFTR served as a positive regulator of the A2BRs. We found that A2BR and CFTR coimmunoprecipitated. They also underwent ADO-dependent Förster resonance energy transfer (FRET), which increased from 5% in the absence of agonist to 18% with 100 µM ADO (EC50 1.7 µM), suggesting that they dynamically associate in the plasma membrane. In contrast, despite colocalization, no FRET was observed between CFTR and GAP43. The interaction between A2BR and CFTR had some specificity: A2BR-stimulated but not forskolin-stimulated cAMP production was ~50% greater in the presence of CFTR, due to a CFTR-dependent increase in plasma membrane A2BR levels. These CFTR-dependent increases in A2BR levels and cAMP production resulted in significantly enhanced ciliary beating and increased cytokine secretion in normal compared to cystic fibrosis airway epithelia. Thus, we hypothesize that CFTR regulates A2BR levels in the plasma membrane to modulate cell signaling and to enhance selective components of the innate lung defense system.


Assuntos
Adenosina/farmacologia , AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Mucosa Respiratória/metabolismo , Adenosina/metabolismo , Animais , Comunicação Celular , Células Cultivadas , Cílios/fisiologia , AMP Cíclico/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Transferência Ressonante de Energia de Fluorescência , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Receptor A2B de Adenosina/metabolismo , Transdução de Sinais
7.
Physiol Rep ; 10(10): e15306, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35581745

RESUMO

Orai1 is a ubiquitously-expressed plasma membrane Ca2+ channel that is involved in store-operated Ca2+ entry (SOCE): a fundamental biological process that regulates gene expression, the onset of inflammation, secretion, and the contraction of airway smooth muscle (ASM). During SOCE, Ca2+ leaves the endoplasmic reticulum, which then stimulates a second, amplifying wave of Ca2+ influx through Orai1 into the cytoplasm. Short Palate LUng and Nasal epithelial Clone 1 (SPLUNC1; gene name BPIFA1) is a multi-functional, innate defense protein that is highly abundant in the lung. We have previously reported that SPLUNC1 was secreted from epithelia, where it bound to and inhibited Orai1, leading to reduced SOCE and ASM relaxation. However, the underlying mechanism of action is unknown. Here, we probed the SPLUNC1-Orai1 interactions in ASM and HEK293T cells using biochemical and imaging techniques. We observed that SPLUNC1 caused a conformational change in Orai1, as measured using Forster resonance energy transfer (FRET). SPLUNC1 binding also led to Nedd4-2 dependent ubiquitination of Orai1. Moreover, SPLUNC1 internalized Orai1 to lysosomes, leading to Orai1 degradation. Thus, we conclude that SPLUNC1 is an allosteric regulator of Orai1. Our data indicate that SPLUNC1-mediated Orai1 inhibition could be utilized as a therapeutic strategy to reduce SOCE.


Assuntos
Glicoproteínas/metabolismo , Pulmão , Músculo Liso , Fosfoproteínas/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Membrana Celular/metabolismo , Células HEK293 , Humanos , Pulmão/metabolismo , Músculo Liso/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo
8.
JCI Insight ; 7(6)2022 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-35315358

RESUMO

The chloride channel dysfunction caused by deleterious cystic fibrosis transmembrane conductance regulator (CFTR) variants generally correlates with severity of cystic fibrosis (CF). However, 3 adults bearing the common severe variant p.Phe508del (legacy: F508del) and a deletion variant in an ivacaftor binding region of CFTR (p.Phe312del; legacy: F312del) manifested only elevated sweat chloride concentration (sw[Cl-]; 87-105 mEq/L). A database review of 25 individuals with F312del and a CF-causing variant revealed elevated sw[Cl-] (75-123 mEq/L) and variable CF features. F312del occurs at a higher-than-expected frequency in the general population, confirming that individuals with F312del and a CF-causing variant do not consistently develop overt CF features. In primary nasal cells, CFTR bearing F312del and F508del generated substantial chloride transport (66.0% ± 4.5% of WT-CFTR) but did not respond to ivacaftor. Single-channel analysis demonstrated that F312del did not affect current flow through CFTR, minimally altered gating, and ablated the ivacaftor response. When expressed stably in CF bronchial epithelial (CFBE41o-) cells, F312del-CFTR demonstrated residual function (50.9% ± 3.3% WT-CFTR) and a subtle decrease in forskolin response compared with WT-CFTR. F312del provides an exception to the established correlation between CFTR chloride transport and CF phenotype and informs our molecular understanding of ivacaftor response.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Aminofenóis/farmacologia , Aminofenóis/uso terapêutico , Cloretos/metabolismo , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Fenótipo , Quinolonas
9.
Methods Mol Biol ; 742: 77-92, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21547727

RESUMO

The healthy human respiratory tract is lined with a pseudostratified epithelia composed of ∼80% ciliated cells and ∼20% goblet cells. These cells produce and are bathed by a layer of airway surface liquid (ASL), which plays a critical role in lung defense by helping to maintain the sterility of the lung. This layer is composed of two phases: the mucus layer which functions to trap particulates, bacteria, and viruses, and the underlying periciliary liquid layer (PCL), which provides hydration, enabling mucus transport and clearance. This chapter describes the methods used to measure the structure and height of the ASL by XZ confocal microscopy and mucus transport rates using epifluorescent microscopy in live airway cultures. Furthermore, we also demonstrate that these methods are also applicable in novel ways to probe the ultrastructure of the airways including the establishment of pH gradients and the ability of the apical membrane glycocalyx in excluding larger molecules from the cell surface.


Assuntos
Glicocálix/ultraestrutura , Imagem Molecular/métodos , Depuração Mucociliar , Muco/metabolismo , Mucosa Respiratória/ultraestrutura , Água/metabolismo , Células Cultivadas , Desidratação/metabolismo , Desidratação/fisiopatologia , Fluorocarbonos/química , Fluorocarbonos/metabolismo , Glicocálix/metabolismo , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Mucosa Respiratória/metabolismo , Água/análise
10.
J Biol Chem ; 278(40): 39143-54, 2003 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-12878596

RESUMO

The sequence of Vibrio cholerae genome revealed three genes belonging to the photolyase/cryptochrome blue-light photoreceptor family. The proteins encoded by the three genes were purified and characterized. All three proteins contain folate and flavin cofactors and have absorption peaks in the range of 350-500 nm. Only one of the three, VcPhr, is a photolyase specific for cyclobutane pyrimidine dimers. The other two are cryptochromes and were designated VcCry1 and VcCry2, respectively. Mutation of phr abolishes photoreactivation of UV-induced killing, whereas mutations in cry1 and cry2 do not affect photorepair activity. VcCry1 exhibits some unique features. Of all cryptochromes characterized to date, it is the only one that contains stoichiometric amounts of both chromophores and retains its flavin cofactor in the two-electron reduced FADH2 form. In addition, VcCry1 exhibits RNA binding activity and co-purifies with an RNA of 60-70 nucleotides in length.


Assuntos
Desoxirribodipirimidina Fotoliase/química , Proteínas de Drosophila , Proteínas do Olho , Flavina-Adenina Dinucleotídeo/análogos & derivados , Flavoproteínas/química , Células Fotorreceptoras de Invertebrados , Células Fotorreceptoras/química , Vibrio cholerae/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Criptocromos , Desoxirribodipirimidina Fotoliase/isolamento & purificação , Dimerização , Relação Dose-Resposta à Radiação , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Flavina-Adenina Dinucleotídeo/química , Flavoproteínas/isolamento & purificação , Temperatura Alta , Ligantes , Luz , Dados de Sequência Molecular , Mutação , Plasmídeos/metabolismo , RNA/metabolismo , Receptores Acoplados a Proteínas G , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência , Espectrofotometria , Raios Ultravioleta
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