RESUMO
Claudin proteins are major constituents of epithelial and endothelial tight junctions (TJs) that regulate paracellular permeability to ions and solutes. Claudin 18, a member of the large claudin family, is highly expressed in lung alveolar epithelium. To elucidate the role of claudin 18 in alveolar epithelial barrier function, we generated claudin 18 knockout (C18 KO) mice. C18 KO mice exhibited increased solute permeability and alveolar fluid clearance (AFC) compared with wild-type control mice. Increased AFC in C18 KO mice was associated with increased ß-adrenergic receptor signaling together with activation of cystic fibrosis transmembrane conductance regulator, higher epithelial sodium channel, and Na-K-ATPase (Na pump) activity and increased Na-K-ATPase ß1 subunit expression. Consistent with in vivo findings, C18 KO alveolar epithelial cell (AEC) monolayers exhibited lower transepithelial electrical resistance and increased solute and ion permeability with unchanged ion selectivity. Claudin 3 and claudin 4 expression was markedly increased in C18 KO mice, whereas claudin 5 expression was unchanged and occludin significantly decreased. Microarray analysis revealed changes in cytoskeleton-associated gene expression in C18 KO mice, consistent with observed F-actin cytoskeletal rearrangement in AEC monolayers. These findings demonstrate a crucial nonredundant role for claudin 18 in the regulation of alveolar epithelial TJ composition and permeability properties. Increased AFC in C18 KO mice identifies a role for claudin 18 in alveolar fluid homeostasis beyond its direct contributions to barrier properties that may, at least in part, compensate for increased permeability.
Assuntos
Claudinas/metabolismo , Células Epiteliais/metabolismo , Alvéolos Pulmonares/metabolismo , Junções Íntimas/metabolismo , Animais , Células Cultivadas , Claudina-3/metabolismo , Claudina-4/metabolismo , Claudina-5/metabolismo , Claudinas/deficiência , Claudinas/genética , Citoesqueleto/metabolismo , Modelos Animais de Doenças , Impedância Elétrica , Genótipo , Homeostase , Humanos , Transporte de Íons , Camundongos , Camundongos Knockout , Ocludina/metabolismo , Permeabilidade , Fenótipo , Alvéolos Pulmonares/fisiopatologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Lesão Pulmonar Induzida por Ventilação Mecânica/genética , Lesão Pulmonar Induzida por Ventilação Mecânica/metabolismo , Lesão Pulmonar Induzida por Ventilação Mecânica/fisiopatologiaRESUMO
Peroxisome proliferator activated receptor γ (PPARγ) agonists are effective antifibrotic agents in a number of tissues. Effects of these agents on epithelial-mesenchymal transition (EMT) of primary alveolar epithelial cells (AEC) and potential mechanisms underlying effects on EMT have not been well delineated. We examined effects of troglitazone, a synthetic PPARγ agonist, on transforming growth factor (TGF)-ß1-induced EMT in primary rat AEC and an alveolar epithelial type II (AT2) cell line (RLE-6TN). TGF-ß1 (2.5 ng/mL) induced EMT in both cell types, as evidenced by acquisition of spindle-like morphology, increased expression of the mesenchymal marker α-smooth muscle actin (α-SMA) and downregulation of the tight junctional protein zonula occludens-1 (ZO-1). Concurrent treatment with troglitazone (or rosiglitazone), ameliorated effects of TGF-ß1. Furthermore, following stimulation with TGF-ß1 for 6 days, troglitazone reversed EMT-related morphological changes and restored both epithelial and mesenchymal markers to control levels. Treatment with GW9662 (an irreversible PPARγ antagonist), or overexpression of a PPARγ dominant negative construct, failed to inhibit these effects of troglitazone in AEC. Troglitazone not only attenuated TGF-ß1-induced phosphorylation of Akt and glycogen synthase kinase (GSK)-3ß, but also inhibited nuclear translocation of ß-catenin, phosphorylation of Smad2 and Smad3 and upregulation of the EMT-associated transcription factor SNAI1. These results demonstrate inhibitory actions of troglitazone on TGF-ß1-induced EMT in AEC via a PPARγ-independent mechanism likely through inhibition of ß-catenin-dependent signaling downstream of TGF-ß1, supporting a role for interactions between TGF-ß and Wnt/ß-catenin signaling pathways in EMT.
Assuntos
Cromanos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , PPAR gama/metabolismo , Alvéolos Pulmonares/citologia , Tiazolidinedionas/farmacologia , Fator de Crescimento Transformador beta1/farmacologia , Anilidas/farmacologia , Animais , Western Blotting , Linhagem Celular , Células Cultivadas , Humanos , Masculino , Microscopia de Fluorescência , PPAR gama/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Troglitazona , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Evidence suggests epithelial-mesenchymal transition (EMT) as one potential source of fibroblasts in idiopathic pulmonary fibrosis. To assess the contribution of alveolar epithelial cell (AEC) EMT to fibroblast accumulation in vivo following lung injury and the influence of extracellular matrix on AEC phenotype in vitro, Nkx2.1-Cre;mT/mG mice were generated in which AECs permanently express green fluorescent protein (GFP). On days 17-21 following intratracheal bleomycin administration, ~4% of GFP-positive epithelial-derived cells expressed vimentin or α-smooth muscle actin (α-SMA). Primary AECs from Nkx2.1-Cre;mT/mG mice cultured on laminin-5 or fibronectin maintained an epithelial phenotype. In contrast, on type I collagen, cells of epithelial origin displayed nuclear localization of Smad3, acquired spindle-shaped morphology, expressed α-SMA and phospho-Smad3, consistent with activation of the transforming growth factor-ß (TGFß) signalling pathway and EMT. α-SMA induction and Smad3 nuclear localization were blocked by the TGFß type I receptor (TßRI, otherwise known as Alk5) inhibitor SB431542, while AEC derived from Nkx2.1-Cre;Alk5(flox/KO) mice did not undergo EMT on collagen, consistent with a requirement for signalling via Alk5 in collagen-induced EMT. Inability of a pan-specific TGFß neutralizing antibody to inhibit effects of collagen together with absence of active TGFß in culture supernatants is consistent with TGFß ligand-independent activation of Smad signalling. These results support the notion that AECs can acquire a mesenchymal phenotype following injury in vivo and implicate type I collagen as a key regulator of EMT in AECs through signalling via Alk5, likely in a TGFß ligand-independent manner.
Assuntos
Células Epiteliais Alveolares/patologia , Colágeno Tipo I/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Fibrose Pulmonar/patologia , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Actinas/metabolismo , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Angiotensina II/metabolismo , Animais , Antibióticos Antineoplásicos/toxicidade , Benzamidas/farmacologia , Bleomicina/toxicidade , Células Cultivadas , Dioxóis/farmacologia , Modelos Animais de Doenças , Feminino , Ligantes , Masculino , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Transdução de Sinais , Vimentina/metabolismoRESUMO
Endoplasmic reticulum (ER) stress has been implicated in alveolar epithelial type II (AT2) cell apoptosis in idiopathic pulmonary fibrosis. We hypothesized that ER stress (either chemically induced or due to accumulation of misfolded proteins) is also associated with epithelial-mesenchymal transition (EMT) in alveolar epithelial cells (AECs). ER stress inducers, thapsigargin (TG) or tunicamycin (TN), increased expression of ER chaperone, Grp78, and spliced X-box binding protein 1, decreased epithelial markers, E-cadherin and zonula occludens-1 (ZO-1), increased the myofibroblast marker, α-smooth muscle actin (α-SMA), and induced fibroblast-like morphology in both primary AECs and the AT2 cell line, RLE-6TN, consistent with EMT. Overexpression of the surfactant protein (SP)-C BRICHOS mutant SP-C(ΔExon4) in A549 cells increased Grp78 and α-SMA and disrupted ZO-1 distribution, and, in primary AECs, SP-C(ΔExon4) induced fibroblastic-like morphology, decreased ZO-1 and E-cadherin and increased α-SMA, mechanistically linking ER stress associated with mutant SP to fibrosis through EMT. Whereas EMT was evident at lower concentrations of TG or TN, higher concentrations caused apoptosis. The Src inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4]pyramidine) (PP2), abrogated EMT associated with TN or TG in primary AECs, whereas overexpression of SP-C(ΔExon4) increased Src phosphorylation, suggesting a common mechanism. Furthermore, increased Grp78 immunoreactivity was observed in AT2 cells of mice after bleomycin injury, supporting a role for ER stress in epithelial abnormalities in fibrosis in vivo. These results demonstrate that ER stress induces EMT in AECs, at least in part through Src-dependent pathways, suggesting a novel role for ER stress in fibroblast accumulation in pulmonary fibrosis.
Assuntos
Retículo Endoplasmático/metabolismo , Epitélio/patologia , Regulação da Expressão Gênica , Mesoderma/patologia , Alvéolos Pulmonares/metabolismo , Proteína C Associada a Surfactante Pulmonar/química , Animais , Apoptose , Chaperona BiP do Retículo Endoplasmático , Fibroblastos/metabolismo , Humanos , Masculino , Camundongos , Mutação , Desnaturação Proteica , Fibrose Pulmonar/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
To explore mechanisms of nanoparticle interactions with and trafficking across lung alveolar epithelium, we utilized primary rat alveolar epithelial cell monolayers (RAECMs) and an artificial lipid bilayer on filter model (ALBF). Trafficking rates of fluorescently labeled polystyrene nanoparticles (PNPs; 20 and 100 nm, carboxylate (negatively charged) or amidine (positively charged)-modified) in the apical-to-basolateral direction under various experimental conditions were measured. Using confocal laser scanning microscopy, we investigated PNP colocalization with early endosome antigen-1, caveolin-1, clathrin heavy chain, cholera toxin B, and wheat germ agglutinin. Leakage of 5-carboxyfluorescein diacetate from RAECMs, and trafficking of (22)Na and (14)C-mannitol across ALBF, were measured in the presence and absence of PNPs. Results showed that trafficking of positively charged PNPs was 20-40 times that of negatively charged PNPs across both RAECMs and ALBF, whereas translocation of PNPs across RAECMs was 2-3 times faster than that across ALBF. Trafficking rates of PNPs across RAECMs did not change in the presence of EGTA (which decreased transepithelial electrical resistance to zero) or inhibitors of endocytosis. Confocal laser scanning microscopy revealed no intracellular colocalization of PNPs with early endosome antigen-1, caveolin-1, clathrin heavy chain, cholera toxin B, or wheat germ agglutinin. Leakage of 5-carboxyfluorescein diacetate from alveolar epithelial cells, and sodium ion and mannitol flux across ALBF, were not different in the presence or absence of PNPs. These data indicate that PNPs translocate primarily transcellularly across RAECMs, but not via known major endocytic pathways, and suggest that such translocation may take place by diffusion of PNPs through the lipid bilayer of cell plasma membranes.
Assuntos
Células Epiteliais Alveolares/metabolismo , Nanopartículas , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/efeitos dos fármacos , Animais , Transporte Biológico/efeitos dos fármacos , Cátions , Ácido Egtázico/farmacologia , Endocitose/efeitos dos fármacos , Fluoresceínas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/metabolismo , Masculino , Manitol/metabolismo , Peptídeos/farmacologia , Permeabilidade/efeitos dos fármacos , Poliestirenos/metabolismo , Ratos , Ratos Sprague-Dawley , Sódio/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Aglutininas do Germe de Trigo/metabolismoRESUMO
We investigated the influence of extracellular matrix on transport properties of mouse alveolar epithelial cell (AEC) monolayers (MAECM) and transdifferentiation of isolated mouse alveolar epithelial type II (AT2) cells into an alveolar epithelial type I (AT1) cell-like phenotype. Primary mouse AT2 cells plated on laminin 5-coated polycarbonate filters formed monolayers with transepithelial resistance (R(T)) and equivalent short-circuit current (I(EQ)) of 1.8 kOmega.cm(2) and 5.3 microA/cm(2), respectively, after 8 days in culture. Amiloride (10 microM), ouabain (0.1 mM), and pimozide (10 microM) decreased MAECM I(EQ) to 40%, 10%, and 65% of its initial value, respectively. Sequential addition of pimozide and amiloride, in either order, revealed that their inhibitory effects are additive, suggesting that cyclic nucleotide-gated channels contribute to amiloride-insensitive active ion transport across MAECM. Ussing chamber measurements of unidirectional ion fluxes across MAECM under short-circuit conditions indicated that net absorption of Na(+) in the apical-to-basolateral direction is comparable to net ion flux calculated from the observed short-circuit current: 0.38 and 0.33 microeq.cm(-2).h(-1), respectively. Between days 1 and 9 in culture, AEC demonstrated increased expression of aquaporin-5 protein, an AT1 cell marker, and decreased expression of pro-surfactant protein-C protein, an AT2 cell marker, consistent with transition to an AT1 cell-like phenotype. These results demonstrate that AT1 cell-like MAECM grown on laminin 5-coated polycarbonate filters exhibit active and passive transport properties that likely reflect the properties of intact mouse alveolar epithelium. This mouse in vitro model will enhance the study of AEC derived from mutant strains of mice and help define important structure-function correlations.
Assuntos
Células Epiteliais/citologia , Células Epiteliais/fisiologia , Mucosa Respiratória/citologia , Mucosa Respiratória/fisiologia , Amilorida/farmacologia , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/fisiologia , Broncodilatadores/farmacologia , Técnicas de Cultura de Células , Células Cultivadas , Cloretos/metabolismo , Cultura em Câmaras de Difusão , Antagonistas de Dopamina/farmacologia , Impedância Elétrica , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos , Ouabaína/farmacologia , Fenótipo , Pimozida/farmacologia , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/fisiologia , Sódio/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Terbutalina/farmacologiaRESUMO
Inhaled nanoparticles have been reported to contribute to deleterious effects on human health. In this study, we investigated the effects of ultrafine ambient particulate suspensions (UAPS), polystyrene nanoparticles (PNP; positively and negatively charged; 20, 100, 120 nm), quantum dots (QD; positively and negatively charged; 30 nm) and single-wall carbon nanotubes (SWCNT) on alveolar epithelial cell barrier properties. Transmonolayer resistance (R(t)) and equivalent short-circuit current (I(eq)) of primary rat alveolar epithelial monolayers were measured in the presence and absence of varying concentrations of apical nanoparticles. In some experiments, apical-to-basolateral fluxes of radiolabeled mannitol or inulin were determined with or without apical UAPS exposure and lactate dehydrogenase (LDH) release was analyzed after UAPS or SWCNT exposure. Results revealed that exposure to UAPS decreased R(t) and I(eq) significantly over 24 h, although neither mannitol nor inulin fluxes changed. Positively charged QD decreased R(t) significantly (with subsequent recovery), while negatively charged QD did not. R(t) decreased significantly after SWCNT exposure (with subsequent recovery). On the other hand, PNP exposure had no effects on R(t) or I(eq). No significant increases in LDH release were observed after UAPS or SWCNT exposure. These data indicate that disruption of alveolar epithelial barrier properties due to apical nanoparticle exposure likely involves alteration of cellular transport pathways and is dependent on specific nanoparticle composition, shape and/or surface charge.
Assuntos
Células Epiteliais/efeitos dos fármacos , Pulmão/citologia , Nanotubos de Carbono/toxicidade , Pontos Quânticos , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Ratos , Fatores de TempoRESUMO
Vascular endothelial growth factor (VEGF) increases microvascular permeability and has been implicated in the development of numerous pathologies including diabetic retinopathy (DR), hypoxia/ischemia, and tumor biology. The transport pathways by which water and solutes cross the endothelium in response to VEGF, however, are not completely understood. We measured, in real time, bovine retinal endothelial cell (BREC) hydraulic conductivity (Lp), 70 kDa dextran permeability (Pe), and the solvent-drag reflection coefficient (sigma) before and after addition of 50 ng/ml VEGF. The diffusional permeability coefficient for dextran (Pd) was measured before pressure gradient application. The sudden application of a 10-cm H2O hydrostatic pressure gradient induced water and solute fluxes that decayed to steady-state values after approximately 2 h. Subsequently, the addition of VEGF significantly increased Lp and Pe by 4.3-fold +/- 0.7-fold and 3.0-fold +/- 0.3-fold, respectively, after 110 min; however, the reflection coefficient remained approximately constant throughout the experiment (approximately 0.8). These observations suggest that water and dextran utilize common paracellular channels across BREC monolayers. Furthermore, the addition of VEGF increases the number or availability of channels but does not alter the selectivity of the monolayer to 70 kDa dextran.