Effects of extracellular calcium depletion on membrane topography and occluding junctions of mammary epithelial cells in culture.

Abstract

Ca 2+ dependence of occluding junction structure and permeability, well documented in explanted or cultured epithelial sheets, presumably reflects inherent control mechanisms. As an approach to identification of these mechanisms, we induced disassembly of zonulae occludentes in confluent monolayers of mouse mammary epithelial cells by exposure to low concentrations of the chelators, EGTA or sodium citrate. Stages in disassembly were monitored during treatment by phase-contrast microscopy and prepared for transmission and scanning electron microscopy. Cellular response included several events affecting occluding junctions: (a) Centripetal cytoplasmic contraction created tension on junction membranes and displaced intramembrane strands along lines determined by the axis of tension. (b) Destabilization of junction position, probably through increased membrane fluidity, augmented tensioninduced movement of strands, resulting in fragmentation of the junction belt. (c) Active ruffling and retraction of freed peripheral membranes remodeled cell borders to produce many filopodia, distally attached by occluding-junction fragments to neighboring cell membranes. Filopodia generally persisted until mechanically ruptured, when endocytosis of the junction and adhering cytoplasmic bleb ensued. Junction disassembly thus resulted from mechanical tensions generated by initial centripetal contraction and subsequent peripheral cytoskeletal activity, combined with destabilization of the junction's intramembrane strand pattern.

Regulation of mammary tight junctions through parathyroid hormone-related peptide-induced activation of apical calcium channels

“…The mammary epithelium is also a polarised epithelium and in the first experiment we confirmed that in mammary cells Ca sensitivity is indeed mediated only via the apical membrane. Moreover, we also confirmed earlier studies showing that extracellular Ca levels can influence TJ status (Neville & Peaker 1981, Pitelka et al 1983, Stelwagen et al 1995. Thus, for PTHrP to have an effect on mammary TJs it would have to act on the apical side of the cell.…”

“…Moreover, maintaining extracellular Ca levels, presumably to facilitate adequate intracellular levels of Ca, is a prerequisite for preventing mammary TJ breakdown in vitro and in vivo (Neville & Peaker 1981, Pitelka et al 1983, Stelwagen et al 1995. The role of PTHrP in maintaining cellular Ca homeostasis and Ca transport in epithelia, including that of the mammary gland, is well recognised (Seitz et al 1993, Philbrick et al 1996, Friedman 2000, Lafond et al 2001.…”

“…We also know that reducing the frequency of milk removal to once a day rapidly impairs the barrier function of mammary TJs in goats and cows (Stelwagen et al 1994(Stelwagen et al , 1997. Furthermore, from both in vitro and in vivo experiments it is clear that maintaining adequate extracellular Ca levels is critical to the integrity of mammary TJs (Pitelka et al 1983, Stelwagen et al 1995) and PTHrP appears to be an important hormone in maintaining cellular Ca homeostasis and epithelial Ca transport in various tissues, including the mammary gland (Seitz et al 1993, Philbrick et al 1996, Friedman 2000, Lafond et al 2001.…”

Histamine and thrombin modulate endothelial focal adhesion through centripetal and centrifugal forces.

“…Pitelka et al observed centripetal movement of the cortex of epithelial cells when centrifugal tethering was disrupted by chelation of extracellular calcium (11). Kolodney and Wysolmerski observed that thrombin increased centripetal isometric tension in cultured human umbilical vein endothelial cells (HUVEC) 1 (6).…”

The control of tissue architecture over nuclear organization is crucial for epithelial cell fate

“…To confirm that the absence of apical polarity primes cells to enter the cell cycle regardless of the method used to alter apical polarity, we performed similar experiments with S1 acini treated with EGTA. In different cell models, it has been shown that chelation of Ca 2+ by EGTA induces the loss of tight junction organization and thus, apical polarity (Pitelka et al, 1983;Bhat et al, 1993). Treatment of S1 acini with 1.5 mM EGTA efficiently altered apical polarity as shown by the absence of apical location of ZO-1 in the majority of the acini population after 24 hours of incubation (Fig.…”