title: Expression of claudins -2 and -4 and cingulin is coordinated with the start of stratification and differentiation in corneal epithelial cells: retinoic acid reversibly disrupts epithelial barrier.
abstract: Although tight junctions (TJ) have been extensively studied in simple epithelial cells, it is still unknown whether their organization is coupled to cell differentiation in stratified epithelia. We studied the expression of TJ in RCE1(5T5) cells, an in vitro model which mimics the sequential steps of rabbit corneal epithelial differentiation. RCE1(5T5) cells expressed TJ components which were assembled once cells constituted differentiated epithelia, as suggested by the increase of transepithelial electrical resistance (TER) which followed a similar kinetic to the expression of the early differentiation marker Pax-6. TJ were functional as indicated by the establishment of an epithelial barrier nonpermeable to ruthenium red or a biotin tracer. In immunostaining experiments, TJ were located at the superficial cells from the suprabasal layers; Western blot and RT-PCR suggested that TJ were composed of claudins (cldn) -1, -2, -4, cingulin (cgn), occludin (ocln) and ZO-1. Semi-quantitative RT-PCR and TER measurements showed that TJ became organized when cells began to form a 3-5 layers stratified epithelium; TER increased once cells reached confluence, with a time course comparable to the raise in the expression of cgn, cldn-2 and -4. Nevertheless, cldn-1, -2, ZO-1 and ocln were present in the cells from the beginning of cultivation, suggesting that TER increases mainly depend on TJ assembly. While EGF increased epithelial barrier strength, retinoic acid disrupted it, increasing paracellular flux about 2-fold; this effect was concentration dependent and completely reversible. Our results suggest that TJ assembly is tightly linked to the expression of corneal epithelial terminal phenotype.
authors: Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional ,  Apartado Postal 14-740, México City 07000 ,  México ; Posgrado en Ciencias Biológicas, FES-Iztacala, Universidad Nacional Autónoma de México ,  México City 04510 ,  México.
authors: María Teresa
authors: MT
authors: Ortiz-Melo
authors: Erika
authors: E
authors: Sánchez-Guzmán
authors: Arturo
authors: A
authors: González-Robles
authors: Jesús
authors: J
authors: Valdés
authors: Eber
authors: E
authors: Gómez-Flores
authors: Federico
authors: F
authors: Castro-Muñozledo
keywords: Cell culture; Cell differentiation; Cingulin; Claudin; Corneal epithelium; Occludin; Retinoic acid; Tight junction