Abstract
Increased endothelial permeability induced by inflammatory stimuli promotes atherosclerosis. Tight junction that is composed of transmembrane proteins, including claudin, and peripheral membrane proteins is a major barrier of endothelial permeability, whereas roles of claudin in the regulation of endothelial TJ permeability are not yet clear. We demonstrate that claudin-1 is dominantly expressed in cultured human endothelial cells by immonofluorescence and immunoblotting. To elucidate the physiological roles of claudin-1 in endothelial permeability, we depleted claudin-1 by RNA interference in endothelial cells. Depletion of claudin-1 protein expression significantly increased TJ permeability measured by transendothelial electrical resistance compared to control cells, indicating that claudin-1 is a crucial regulator of TJ permeability in endothelial cells. Ubiquitin-proteasome system has been implicated in the regulation of degradation and intracellular localization of proteins. We show that ubiquitin-proteasome system regulates claudin-1 localization from plasma membrane to cytoplasm using a proteasome inhibitor N-acetyl-Leu-Leu-Nle-CHO (ALLN) in endothelial cells. On the other hand, members of the small GTPase Rab protein family play important roles in intracellular vesicle transport and localization of membrane proteins. We examined colocalization of claudin-1 and Rab5a by confocal microscopy and studied the involvement of proteasome in the localization and the Rab-mediated transport of claudin-1 in endothelial cells. In control cells, claudin-1 was localized at cell-cell contact sites. In contrast, caludin-1 localization was observed at perinuclear regions in cytoplasm in association with Rab5a and EEA-1, a marker of early endosome, in ALLN-treated cells. Depletion of Rab5a by RNA interference reversed the localization of claudin-1 in ALLN-treated cells. These data suggest that Rab5a in early endosome is implicated in proteasome-mediated localization at cell-cell contacts of claudin-1 in endothelial cells. Rab5a-mediated intracellular transport of claudin-1 that is regulated by proteasome may play a role in the regulation of tight junction permeability in endothelial cells.
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