RESUMEN
The rat lung epithelial cell line SV40-T2 was used to develop a cellular biosensing system to assay for environmental toxicants. The novel approach on which this system is based involves direct attachment of cultured rat or human cells onto a cell-adhesive matrix on the device through which shear horizontal surface acoustic waves (SH-SAW) are transmitted using 50 MHz SAW resonator. This novel design enables sensitive monitoring of changes of the electrophysical characteristics of cells, such as their conductivity and relative permittivity. A time-dependent change of phase of SAW and change of insertion loss (change of amplitude) were observed when the cells were treated with 0.5 or 1.0 mM H2O2. The change of insertion loss was biphasic, with an early phase (1-3 h) and a late phase (3-6 h). The late phase coincided with the destruction of cell-cell tight junctions detected by measurement of the transepithelial electrical resistance and paracellular permeability; in contrast, the early phase coincided with the destruction of intracellular actin filaments by H2O2. The early-phase effect of H2O2 on phase shift may be attributable to the change of intracellular permittivity by a change of cellular polarity. Immunofluorescence microscopy showed the disappearance of zonula occludens protein 1 from the region of cell-cell contact. These results suggest the correlation between the change of insertion loss as an SAW parameter and the destruction of tight junctions of the cells on the SH-SAW device in the late phase.
Asunto(s)
Acústica/instrumentación , Técnicas Biosensibles/instrumentación , Células Epiteliales/efectos de los fármacos , Células Epiteliales/diagnóstico por imagen , Peróxido de Hidrógeno/toxicidad , Actinas/efectos de los fármacos , Animales , Línea Celular , Células Cultivadas , Impedancia Eléctrica , Microscopía Fluorescente , Ratas , Resistencia al Corte , Uniones Estrechas/diagnóstico por imagen , Uniones Estrechas/efectos de los fármacos , UltrasonografíaRESUMEN
BACKGROUND: The aryl hydrocarbon (Ah) receptor is one of the best known ligand-activated transcription factors. The present study has focused on the wound-healing process on Ah receptor function. METHODS: Depletion of calcium from culture medium of Caco-2 human colon carcinoma cells by transfer to Minimal Essential Medium (Spinner Modification; S-MEM) destroyed adherens junctions and the cells were used as the model of wound-healing process. RESULTS: Calcium depletion induced both nuclear translocation of the Ah receptor, and increased expression of CYP1A1 and Slug mRNAs in Caco-2 cells. However, expression of Slug mRNA was not significantly induced by treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Knockdown of the Ah receptor and treatment with Ah receptor antagonists decreased level of CYP1A1 mRNA. The fragment of E-cadherin released by gamma-secretase was not involved in induction of CYP1A1 mRNA following S-MEM treatment. Knockdown of beta-catenin increased levels of Ah receptor mRNA, which may be attributable to direct or indirect involvement of beta-catenin in suppression of the Ah receptor gene. CONCLUSIONS: Our results suggest that mRNA induction of some genes by destruction of adherens junctions depends on the Ah receptor. beta-Catenin, one of the components of the adherens junction, was released from the E-cadherin complex, which resulted in its increased interaction with the Ah receptor, and was translocated into the nucleus, and consequently the target genes would be transcribed. GENERAL SIGNIFICANCE: Our observations suggest that some aspects of the molecular mechanism of wound healing involve the Ah receptor.
Asunto(s)
Uniones Adherentes/patología , Citocromo P-450 CYP1A1/genética , Receptores de Hidrocarburo de Aril/genética , Transducción de Señal/genética , beta Catenina/genética , Uniones Adherentes/efectos de los fármacos , Uniones Adherentes/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Células CACO-2 , Cadherinas/genética , Cadherinas/metabolismo , Calcio/deficiencia , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Medios de Cultivo , Citocromo P-450 CYP1A1/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Modelos Biológicos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dibenzodioxinas Policloradas/farmacología , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Receptores de Hidrocarburo de Aril/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Cicatrización de Heridas/efectos de los fármacos , Cicatrización de Heridas/genética , beta Catenina/metabolismoRESUMEN
Patulin is a mycotoxin and its contamination of food has been reported to cause gastrointestinal inflammation, ulcers, and bleeding. The toxicity of patulin is thought to be due to the destruction of tight junctions (TJs) in gastrointestinal tissues. However, the precise mechanism has not been clarified. Here, we investigated the phosphorylation of TJ components. The transepithelial electrical resistance (TER) of Caco-2 human colon cancer cells decreased gradually during the first 24h of treatment with 50µM patulin. Immunofluorescence microscopy showed that the TJ proteins ZO-1 and claudin-4, but not occludin, had decreased after 24h and decreased from the cell-cell contact regions of TJs after 48h of patulin treatment. Western blotting showed that the level of ZO-1 decreased after 48h of patulin treatment, but the levels of claudin-4 and occludin remained at the initial level until 72h. Phosphorylation of ZO-1 was detected by 24h and increased markedly after 72h of patulin treatment. However, phosphorylation of claudin-4 and occludin was not detected by probing with anti-phosphotyrosine antibody. Immunoprecipitation showed that interaction of ZO-1 with claudin-4 had decreased after 48h and was completely absent after 72h. These results suggest that phosphorylation caused the degradation of ZO-1 protein and the decrease in TER induced by patulin treatment of Caco-2 cells.
