Cholesterol efflux stimulates metalloproteinase-mediated cleavage of occludin and release of extracellular membrane particles containing its C-terminal fragments.

That changes in membrane lipid composition alter the barrier function of tight junctions illustrates the importance of the interactions between tetraspan integral tight junction proteins and lipids of the plasma membrane. Application of methyl-beta-cyclodextrin to both apical and basolateral surfaces of MDCK cell monolayers for 2 h, results in an approximately 80% decrease in cell cholesterol, a fall in transepithelial electrical resistance, and a 30% reduction in cell content of occludin, with a smaller reduction in levels of claudins-2, -3, and -7. There were negligible changes in levels of actin and the two non-tight junction membrane proteins GP-135 and caveolin-1. While in untreated control cells breakdown of occludin, and probably other tight junction proteins, is mediated by intracellular proteolysis, our current data suggest an alternative pathway whereby in a cholesterol-depleted membrane, levels of tight junction proteins are decreased via direct release into the intercellular space as components of membrane-bound particles. Occludin, along with two of its degradation products and several claudins, increases in the basolateral medium after incubation with methyl-beta-cyclodextrin for 30 min. In contrast caveolin-1 is detected only in the apical medium after adding methyl-beta-cyclodextrin. Release of occludin and its proteolytic fragments continues even after removal of methyl-beta-cyclodextrin. Sedimentation and ultrastructural studies indicate that the extracellular tight junction proteins are associated with the membrane-bound particles that accumulate between adjacent cells. Disruption of the actin filament network by cytochalasin D did not diminish methyl-beta-cyclodextrin-induced release of tight junction proteins into the medium, suggesting that the mechanism underlying their formation is not actin-dependent. The 41- and 48-kDa C-terminal occludin fragments formed during cholesterol depletion result from the action of a GM6001-sensitive metalloproteinase(s) at some point in the path leading to release of the membrane particles.

Cholesterol depletion alters detergent-specific solubility profiles of selected tight junction proteins and the phosphorylation of occludin.

Differential centrifugation of Triton X-100 or CHAPS lysates from control and cholesterol (CH)-depleted MDCK II cells, segregated integral tight junction (TJ) proteins associated with detergent-resistant membranes (DRMs) into two groups. Group A proteins (occludin, claudin-2 and -3) were detected in large, intermediate and small aggregates in both detergents, whereas group B proteins (claudin-1, -4 and -7) were observed in small aggregates in TX-100 and in intermediate and small aggregates in CHAPS. Depletion of CH altered the distribution of group A and B proteins among the three size categories in a detergent-specific manner. In lysates produced with octyl glucoside, a detergent that selectively extracts proteins from DRMs, group A proteins were undetectable in large aggregates and CH depletion did not alter the distribution of either group A or B proteins in intermediate or small aggregates. Neither occludin (group A) nor claudin-1 (group B) was in intimate enough contact with CH to be cross-linked to [(3)H]-photo-cholesterol. However, antibodies to either TJ protein co-immunoprecipitated caveolin-1, a CH-binding protein. Unlike claudins, occludin's presence in TJs and DRMs did not require palmitoylation. Equilibrium density centrifugation on discontinuous OptiPrep gradients revealed detergent-related differences in the densities of TJ-bearing DRMs. There was little or no change in those densities after CH depletion. Removing CH from the plasma membrane increased tyrosine and threonine phosphorylation of occludin, and transepithelial electrical resistance (TER) within 30 min. After 2 h of CH efflux, phospho-occludin levels and TER fell below control values. We conclude that the association of integral TJ proteins with DRMS, pelleted at low speeds, is partially CH-dependent. However, the buoyant density of TJ-associated DRMs is a function of the detergent used and is insensitive to decreases in CH.

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

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.