Abstract
Abstract: :
Purpose: In principal, the permeability of tight junctions in the RPE can be changed by modulating the composition of the junction or by altering its structure. This study examines whether retinal secretions, alone or in collaboration with basal factors, alter the expression of claudins, proteins that determine the properties of tight junctional strands. Methods: The mRNA of claudins expressed by RPE, in vivo and in culture, was examined by real time, RT–PCR. RPE was isolated from chicken embryos at various stages of development and cultured on filters that separated the apical and basal medium chambers. The basal chamber contained a base medium (SF3), SF3+bovine pituitary extract or SF3+medium conditioned by chick embryonic fibroblasts (CEF). The apical chamber contained SF3 or SF3 that was conditioned by embryonic day 14 (E14) neural retinas (rcSF3). For RT–PCR, 18S rRNA was used as an internal standard in multiplex reactions using Tagman reporters. The transepithelial electrical resistance (TER) was used to monitor function. Results: Of the 10 claudins identified in chick, RPE expressed claudins 1, 2, 5, 12 and AL, a claudin–A–like protein, in vivo and in culture. Claudin 5 was the first to be expressed on embryonic day 7 (E7). Claudin 5 mRNA reached maximum expression on E10. Claudin 5 mRNA was surpassed in copy number by claudin 1 on E10 and Claudin AL on E18. On E18, claudin 1 constituted 83% of the total claudin mRNA, claudin AL 9% and claudin 5 6%. Claudins 2 and 12 constituted minor components (1% each) and appeared on E18. Culture on laminin–coated filters resulted in an over–expression of claudins 5 and 12. This was unaffected by either basal medium supplement. With basal CEF medium, apical rcSF3 had minimal effects on the expression of claudins despite increasing the TER. By contrast, rcSF3 modulated claudin expression when combined with pituitary extract. In E7 cultures, rcSF3 increased the expression of claudins 1, 5 and AL. However in E14 cultures, rcSF3 decreased the expression of claudin 12, while increasing the expression of claudins 1, 2 and AL. Conclusions: The change in the relative amounts of claudins expressed by RPE indicates that paracellular permeability might change during normal development. Although the culture models are imperfect, the cultures with pituitary extract are responsive to retinal factors in a way that is consistent with the normal pattern of development. Because other studies demonstrate that cultured E7 and E14 RPE have junctions with similar fine–structure, the differences in claudin expression may be used to determine the effects of individual claudins on permeability properties.
Keywords: pump/barrier function • cell adhesions/cell junctions • retinal pigment epithelium