Occludin knockdown is not sufficient to induce transepithelial macromolecule passage

Abstract

Reiche (2019) Occludin knockdown is not sufficient to induce transepithelial macromolecule passage, Tissue Barriers, 7:2, 1612661, ABSTRACT Occludin, a tight junction protein, has been reported to regulate barrier functionparticularly the leak pathway for larger solutesin epithelia. Therefore, we aimed to precisely define its role in macromolecule passage at single cell-cell junctions. A combination of varying occludin expression by transient and stable knockdown including systematic seeding strategies was employed to achieve a broad and defined pattern of variance in occludin expression over epithelia. This variance model enabled us to examine occludin function in the leak pathway using global and local analysis, i.e. to analyze macromolecule flux across epithelia and macromolecule passage at single-cell level. Macromolecular flux was found not to correlate with occludin expression in intestinal epithelial cells. In fact, by spatially resolving macromolecular permeation sites using a recently developed method we uncovered leaky cell junctions at the edge of Transwells resulting in increased passage. This demonstrates that rare leaks can determine net flux of macromolecules across epithelia while the vast majority of cellular junctions do not contribute significantly. Hence, concomitant local analysis of macromolecule passage across epithelial barriers is indispensable for interpretation of global flux data. By combining this new approach with cell culture models of the leak pathway, we can present evidence that lack of occludin is not sufficient to stimulate the epithelial leak pathway. ARTICLE HISTORY

The Epithelial Cell Leak Pathway

“…Several groups have reported no effect of occludin knockdown on Leak Pathway permeability in MDCK II cells [ 79 , 130 , 131 , 132 ]. Occludin knockdown in Caco-2 cells was reported to increase Leak Pathway permeability [ 100 , 133 ], although this was later disputed [ 134 ]. Occludin overexpression did not alter Leak Pathway permeability in MDCK II cells [ 104 , 130 ].…”

The Leak Pathway: A Pathway in Flux

“…Van Itallie et al [88] reported that overexpression of occludin enhanced and occludin knockdown diminished the ability of cytokines to alter TER and permeability of large solutes in MDCK Type II renal epithelial cells. Lack of an effect of occludin content on the permeability of fluorescein-dextrans across Caco-2 intestinal epithelial cell monolayers was reported by Richter et al [105]. In contrast to the above findings, the paracellular permeability of 3 kDa fluorescein-dextran [106] and mannitol [107] in MDCK Type II renal epithelial cells was reported to be increased by over-expression of occludin protein.…”

“…Our speculative model of Leak Pathway permeability suggests that any stimulus which decreases the extent of interconnection among the tight junction proteins or, possibly, the interaction of tight junction proteins with the cytoskeleton could cause a "relaxing" of the macromolecular lattice leading to increased breaks in the claudin network and increased porosity (Figure 1b). If this occurs at a small number of sites within a cell population, as suggested by the studies of Richter et al [105,124] and Stephenson et al [125], this would lead to increased Leak Pathway permeability without altering Pore Pathway permeability. If these interactions are disrupted on a more global scale, however, this would lead to increases in the tight junction permeability to both macromolecules and small ions and solutes as these solutes cross the epithelium through these large disruptions of the tight junction structure.…”

“…These leaks were rapidly repaired (within ~5 minutes) by actomyosin-mediated concentration of tight junction proteins. Richter et al [105,124] have also reported data suggesting the Leak Pathway permeability represents movement of macromolecules through a limited number of sites within a cell population. These weakened areas of the tight junction correspond to sites of increased stress/tension on the tight junction structure.…”

Intestinal Barrier Function in Gluten-Related Disorders

“…However, Saitou et al [27] and Schulzke et al [28] have shown that occludin knockout mice present normal TJ strand formation and normal barrier function. Furthermore, the hypothesis that occludin expression might define the epithelial leak pathway for macromolecules was not confirmed [29].…”