RESUMEN
In animal models of asthma, interleukin-13 (IL-13) induces goblet cell metaplasia, eosinophil infiltration of the bronchial mucosa, and bronchial hyperreactivity, but the basis of its effects on airway epithelia remain unknown. Lesions of the epithelial barrier, frequently observed in asthma and other chronic lung inflammatory diseases, are repaired through proliferation, migration, and differentiation of epithelial cells. An inflammatory process may then, therefore, influence epithelial regeneration. We have thus investigated the effect of IL-13 on mucociliary differentiation of human nasal epithelial cells in primary culture. We show that IL-13 alters ciliated cell differentiation and increases the proportion of secretory cells. IL-13 downregulates the actin-binding protein ezrin and other cytoskeletal components. IL-13 also impairs lateral cell contacts and interferes with the apical localization of ezrin seen in differentiated ciliated cells. In addition, an IL-4 antagonistic mutant protein (Y124D), which binds to the IL-4 receptor alpha subunit, a common chain of IL-4 and IL-13 receptors, inhibits IL-13's effects. IL-13 also decreases ciliary beat frequency in a time- and dose-dependent manner. These results suggest that, in human allergic asthmatic responses, IL-13 affects both ciliated and secretory cell differentiation, leading to airway damage and obstruction.
Asunto(s)
Asma/etiología , Bronquios/efectos de los fármacos , Interleucina-13/farmacología , Bronquios/citología , Diferenciación Celular/efectos de los fármacos , Polaridad Celular , Células Cultivadas , Cilios/efectos de los fármacos , Cilios/fisiología , Proteínas del Citoesqueleto , Relación Dosis-Respuesta a Droga , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Humanos , Interleucina-4/fisiología , Mucina 2 , Mucinas/genética , Membrana Mucosa/citología , Membrana Mucosa/efectos de los fármacos , Fosfoproteínas/análisisRESUMEN
Centrin protein is an ubiquitously expressed cytoskeletal component and is a member of the EF-hand superfamily of calcium-binding proteins. It was first discovered in the flagellar apparatus of unicellular green algae where it is involved in contraction of Ca(2+)-sensitive structures. Centrin protein is associated with centrosome-related structures such as spindle pole body in yeast, and centriole/basal bodies in flagellar and ciliated cells. Three centrin genes have been cloned in human cells. In this work, we have performed a comparative biochemical and functional analysis of centrin isoforms using a primary culture of human nasal epithelial cells which provides an efficient way to obtain a complete ciliated cell differentiation process. RT-PCR experiments show that the expression of the three human centrin genes increases during cell differentiation, and that only centrin 2 and 3 are expressed during cell proliferation. Using polyclonal antibodies raised against recombinant human centrin 2 and 3, we show a specific pattern of protein expression. Ultrastructural immunolocalization suggests that centrin proteins are involved in the early process of centriole assembly, as they are concentrated within the precursor structures of centriole/basal bodies. It also shows a differential localisation of centrin proteins in mature centriole/basal bodies, suggesting different functions for centrins 1/2 and centrin 3. This is also supported by functional analyses showing that centrin 1 and/or centrin 2 are involved in ciliary beating.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Células Epiteliales/metabolismo , Proteínas de Ciclo Celular , Diferenciación Celular , Células Cultivadas , Cilios/metabolismo , Cilios/ultraestructura , Células Epiteliales/citología , Células Epiteliales/ultraestructura , Humanos , Microscopía Electrónica , Isoformas de Proteínas/metabolismoRESUMEN
Tubulins are the major proteins within centriolar and axonemal structures. In all cell types studied so far, numerous alpha- and beta-tubulin isoforms are generated both by expression of a multigenic family and various post-translational modifications. We have developed a primary culture of human nasal epithelial cells where the ciliated cell differentiation process has been observed and quantified. We have used this system to study several properties concerning polyglutamylation and polyglycylation of tubulin. GT335, a monoclonal antibody directed against glutamylated tubulins, stained the centriole/basal bodies and the axonemes of ciliated cells, and the centrioles of non-ciliated cells. By contrast, axonemal but not centriolar tubulins were polyglycylated. Several polyglutamylated and polyglycylated tubulin isotypes were detected by two-dimensional electrophoresis, using GT335 and a specific monoclonal antibody (TAP952) directed against short polyglycyl chains. Immunoelectron microscopy experiments revealed that polyglycylation only affected axonemal tubulin. Using the same technical approach, polyglutamylation was shown to be an early event in the centriole assembly process, as gold particles were detected in fibrogranular material corresponding to the first cytoplasmic structures involved in centriologenesis. In a functional assay, GT335 and TAP952 had a dose-dependent inhibitory effect on ciliary beat frequency. TAP952 had only a weak effect while GT335 treatment led to a total arrest of beating. These results strongly suggest that in human ciliated epithelial cells, tubulin polyglycylation has only a structural role in cilia axonemes, while polyglutamylation may have a function both in centriole assembly and in cilia activity.
Asunto(s)
Diferenciación Celular/fisiología , Cilios/fisiología , Procesamiento Proteico-Postraduccional , Mucosa Respiratoria/citología , Mucosa Respiratoria/fisiología , Tubulina (Proteína)/metabolismo , Cilios/ultraestructura , Glicosilación , Humanos , Microtúbulos/fisiología , Microtúbulos/ultraestructura , Pólipos Nasales/patología , Ácido Poliglutámico/metabolismo , Isoformas de Proteínas/metabolismo , Mucosa Respiratoria/patología , Cornetes Nasales/patologíaRESUMEN
Primary cultures of rabbit tracheal epithelial (RbTE) cells have been performed in two different ways. Quantitative analysis of both proliferative capacities and ciliated differentiation process were carried out using epithelial cell cultures from tracheal explants and from dissociated tracheal epithelial cells in air-liquid interface conditions. We show that both alpha- and beta-tubulins from RbTE cells are polyglutamylated and that this posttranslational modification is restricted to cilia axonemes and centrioles of non-ciliated cells. A monoclonal antibody raised against polyglutamylated tubulins was used to quantify the proportion of ciliated cells. Even though epithelial cells from outgrowths obtained by the explant technique highly proliferated during the first days of culture, no ciliated differentiation occurred. On the other hand, using air-liquid interface conditions after proliferation of dissociated cells, we could observe and quantify a ciliated cell differentiation in vitro by both Western blot and flow cytometric analysis. The specific detection and quantification of ciliated cells open the way for the biochemical and molecular characterization of centriolar components during ciliated differentiation.