Knockdown of occludin expression leads to diverse phenotypic alterations in epithelial cells

Abstract

The function of occludin (Occ) in the tight junction is undefined. To gain insight into its role in epithelial cell biology, occludin levels in Madin-Darby canine kidney II cells were suppressed by stably expressing short interfering RNA. Suppression of occludin was associated with a decrease in claudins-1 and -7 and an increase in claudins-3 and -4. Claudin-2 levels were unaffected. The tight junction "fence" function was not impaired in suppressed Occ (Occ-) clones, as determined by BODIPY-sphingomyelin diffusion in the membrane. The most striking changes were those related to control of the cytoskeleton and the "gate" function of tight junctions. A reduced ability of Occ- clones to extrude apoptotic cells from the monolayers suggested that neighbors of apoptotic cells either failed to sense their presence or were unable to coordinate cytoskeletal activity necessary for their extrusion. To further test the extent to which actin cytoskeletal activity depends on the presence of occludin, Occ- and Occ+ monolayers were depleted of cholesterol. Previous studies showed that cholesterol depletion is associated with reorganization of the actin cytoskeleton and a fall in transepithelial electrical resistance. In contrast to control Occ (Occ+) cells, transepithelial electrical resistance did not fall significantly in cholesterol-depleted Occ- monolayers and they failed to generate Rho-GTP, one of the signaling molecules involved in regulating the actin cytoskeleton. While steady-state transepithelial electrical resistance was similar in all clones, tight junction permeability to mono- and divalent inorganic cations was increased in Occ- monolayers. In addition, there was a disproportionately large increase in permeability to monovalent organic cations, up to 6.96 A in diameter. Chloride permeability was unaffected and there was little change in mannitol flux. The data suggest that occludin transduces external (apoptotic cells) and intramembrane (rapid cholesterol depletion) signals via a Rho signaling pathway that, in turn, elicits reorganization of the actin cytoskeleton. Impaired signaling in the absence of occludin may also alter the dynamic behavior of tight junction strands, as reflected by an increase in permeability to large organic cations; the permeability of ion pores formed of claudins, however, is less affected.

Occludin is required for cytokine-induced regulation of tight junction barriers

“…We first confirmed the previously published behavior of occludin KD cells and compared the results with tricellulin knockdown cells generated in our laboratory. Consistent with previously published results (Yu et al, 2005) we found that KD of Ocln in MDCK cell monolayers does not alter the levels (Fig. 3A) or localization (Fig.…”

“…Occludin itself was shown to be a component of a detergent-resistant membrane microdomain (Nusrat et al, 2000;Lambert et al, 2005;Lynch et al, 2007) and several studies have implicated the importance of a cholesterol-rich membrane domain in tight junction function. For example, removal of cholesterol from wild-type MDCK cells results in decreased transepithelial electrical resistance (TER), but has no effect on TER in occludindepleted MDCK cells (Yu et al, 2005). In addition, occludin has also been demonstrated in co-immunoprecipitation experiments to interact with the cholesterol-binding protein caveolin-1 (Nusrat et al, 2000;Lynch et al, 2007).…”

Lipid Polarity Is Maintained in Absence of Tight Junctions

“…However, Yu et al (29) showed that knockdown of occludin did not affect lipid compartmentalization. Thus, the role of occludin in the barrier formation has remained controversial.…”

Biology of claudins

“…However, occludin and JAM-A do not form tight junction fibrils when expressed in fibroblasts that lack tight junctions (32,54). Neither paracellular permeability nor tight junction morphology is significantly affected by occludin knockout in mice (87) or knockdown in cell lines (117). Tricellulin is confined mostly to the vertically oriented tight junction strands of tricellular contacts (49).…”