Masumi Kurasawa

Effect of RNA interference of tight junction-related molecules on intercellular barrier function in cultured human keratinocytes

Accumulating evidence shows that tight junctions (TJs) in the granular layer contribute to the epidermal barrier, suggesting that the regulation of TJ assembly in keratinocytes may provide a clue to understanding the role of barrier formation in epidermis. In this study, we investigated the behavior of TJ-related molecules in cultured human keratinocytes during keratinization induced by transfer to high-calcium medium, and the effect of RNA interference of TJ-related molecules on intercellular permeability and morphological features. The expression of TJ-related molecules and transepithelial electrical resistance were increased by transfer to high-calcium medium. In cells under the same conditions, we observed by freeze-fracture electron microscopy that TJ strands developed on the apposing cell membranes. In contrast, the transepithelial electrical resistance was clearly suppressed when the expression of claudin-1 or occludin was blocked by RNA interference. The morphological features of these knock-down cells were the same as those of MOCK cells, except for a marked decrease in the number of TJ strands. Furthermore, claudin-1 suppression inhibited occludin localization at the cell membrane, whereas suppression of occludin did not influence the localization of claudin-1. These results suggest that claudin-1 plays a crucial role in recruiting occludin to TJs, and that occludin is involved in intercellular barrier function.

Tight junction regulates epidermal calcium ion gradient and differentiation.

It is well known that calcium ions (Ca(2+)) induce keratinocyte differentiation. Ca(2+) distributes to form a vertical gradient that peaks at the stratum granulosum. It is thought that the stratum corneum (SC) forms the Ca(2+) gradient since it is considered the only permeability barrier in the skin. However, the epidermal tight junction (TJ) in the granulosum has recently been suggested to restrict molecular movement to assist the SC as a secondary barrier. The objective of this study was to clarify the contribution of the TJ to Ca(2+) gradient and epidermal differentiation in reconstructed human epidermis. When the epidermal TJ barrier was disrupted by sodium caprate treatment, Ca(2+) flux increased and the gradient changed in ion-capture cytochemistry images. Alterations of ultrastructures and proliferation/differentiation markers revealed that both hyperproliferation and precocious differentiation occurred regionally in the epidermis. These results suggest that the TJ plays a crucial role in maintaining epidermal homeostasis by controlling the Ca(2+) gradient.

Regulation of tight junction permeability by sodium caprate in human keratinocytes and reconstructed epidermis

Tight junctions (TJs) restrict paracellular flux of water and solutes in epithelia and endothelia. In epidermis, the physiological role of TJs is not fully understood. In this study, sodium caprate (C10), which dilates intestinal TJs, was applied to cultured human epidermal keratinocytes and reconstructed human epidermis to investigate the effects of C10 on epidermal TJs. C10 treatment decreased transepithelial electrical resistance and increased paracellular permeability, although Western blots showed that the expression of TJ-related transmembrane proteins was not decreased. The effects of C10 were reversible. Immunofluorescence microscopy and immuno-replica electron microscopy showed that the localization of TJ strands were disintegrated, concomitant with the dispersion and/or disappearance of TJ-related molecules from the cell surface. These findings suggest that C10 impairs barrier function by physically disrupting TJ conformation in the epidermis. Furthermore, these results also show that proper localization of the molecules on the cellular membrane is important for TJ barrier function.

Perturbation of lamellar granule secretion by sodium caprate implicates epidermal tight junctions in lamellar granule function

BACKGROUND Polarized secretion of lamellar granules (LGs) delivers various lipids, proteases, and protease inhibitors into the stratum corneum (SC) of the epithelium. Disruption of LGs is associated with severe cutaneous diseases, but the mechanism of their polarized secretion is not known. On the other hand, recent study shows epidermal tight junctions (TJs) localize in stratum granulosum (SG), and TJs are involved in polarized molecule secretion. Thus, we hypothesized epidermal TJs relate to polarized LGs secretion. OBJECTIVE To assess the possibility that epidermal TJs are involved in polarized LGs secretion. METHODS In order to examine LGs secretion, we used fluorescent ceramide (BODIPY FL C(5)-ceramide) and a natural LG cargo, lympho-epithelial Kazal-type-related inhibitor (LEKTI), in cultured normal human epidermal keratinocytes and a reconstructed human epidermis. We investigated their alteration using the medium-chain fatty acid sodium caprate (C10), TJs inhibitor. In addition, LG distribution was observed by electron microscopy. RESULTS C10 significantly inhibited secretion of both fluorescent ceramide and LEKTI in cultured normal human epidermal keratinocytes and a reconstructed human epidermis. C10 also disturbed the polarized localization of fluorescent ceramide and LEKTI in the reconstructed epidermis. Electron microscopy revealed that a large number of LGs remained in corneocytes in the C10-treated epidermis, rather than being secreted. CONCLUSION Our data indicate that C10 perturbs the polarized secretion of LGs. Our study therefore suggests that epidermal TJs are possibly involved in polarized LG secretion and provides new insights into potential of treatments for skin diseases caused by abnormal LG secretion.