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

SummaryAlthough 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.

TRPV4 Regulates Tight Junctions and Affects Differentiation in a Cell Culture Model of the Corneal Epithelium

“…Initially, we determined whether TRPV4 was involved in the establishment of the TJ. We monitored the development of the transepithelial electric resistance as a measure of TJ formation (Cereijido et al, ; Ortiz‐Melo et al, ). RCE1(5T5) cells were seeded on polycarbonate transwells and on day 7, before the establishment of the TJ, the cells were treated either with the TRPV4 activator GSK101 at 10, 50, or 100 nM or with the specific TRPV4 blocker RN‐1734 at 10, 20, or 30 μM.…”

“…To evaluate the degree of TJ sealing, RCE1(5T5) corneal epithelial cells were plated into Costar® 24‐transwell, 4 μm polycarbonate cell culture inserts (Corning, Acton, MA) as described previously (Ortiz‐Melo et al, ). TER measurements were performed using an EVOM voltmeter (World Precision Instruments, Sarasota, FL).…”

Purple Potato Extract Promotes Intestinal Epithelial Differentiation and Barrier Function by Activating AMP‐Activated Protein Kinase

“…Retinoic acid enhances AP activity associated with upregulated mRNA of claudin‐2 in Caco‐2 cells . The mRNA expression of claudin‐2 and claudin‐4, and TEER increased significantly in corneal epithelial RCE1(5T5) cells once differentiation initiated . PPE stimulated intestinal differentiation, and enhanced tight junction assembly during intestinal development.…”

Diurnal Variation of Tight Junction Integrity Associates Inversely with Matrix Metalloproteinase Expression in Xenopus laevis Corneal Epithelium: Implications for Circadian Regulation of Homeostatic Surface Cell Desquamation

“…Junctional adhesion molecule (JAM) is also a component of tight junctions, but is also expressed at cell borders throughout all layers of the CE [52] , so was not a subject of this study. There are several types of claudin, and claudin-4 is one of the most consistently and highly-expressed members of the claudin family in CE [53] , [54] , so it was chosen for inclusion in this study. It is generally considered that occludin is ubiquitously expressed in all tight junctions, but that expression of various claudin types tends to be more tissue-specific [55] – [58] .…”