Retromer controls epithelial cell polarity by trafficking the apical determinant crumbs

The evolutionarily conserved apical determinant Crumbs (Crb) is essential for maintaining apicobasal polarity and integrity of many epithelial tissues [1]. Crb levels are crucial for cell polarity and homeostasis, yet strikingly little is known about its trafficking or the mechanism of its apical localization. Using a newly established, liposome-based system described here, we determined Crb to be an interaction partner and cargo of the retromer complex. Retromer is essential for the retrograde transport of numerous transmembrane proteins from endosomes to the trans-Golgi network (TGN) and is conserved between plants, fungi, and animals [2]. We show that loss of retromer function results in a substantial reduction of Crb in Drosophila larvae, wing discs, and the follicle epithelium. Moreover, loss of retromer phenocopies loss of crb by preventing apical localization of key polarity molecules, such as atypical protein kinase C (aPKC) and Par6 in the follicular epithelium, an effect that can be rescued by overexpression of Crb. Additionally, loss of retromer results in multilayering of the follicular epithelium, indicating that epithelial integrity is severely compromised. Our data reveal a mechanism for Crb trafficking by retromer that is vital for maintaining Crb levels and localization. We also show a novel function for retromer in maintaining epithelial cell polarity.

High permeability of the anionic form restricts accumulation of indomethacin by cultured gastric surface epithelial cells exposed to low apical pH

The 'ion-trapping' hypothesis suggests that the intracellular concentration of acidic non-steroidal anti-inflammatory drugs in gastric epithelial cells could be much higher than in the gastric lumen, and that such accumulation could contribute to their gastrotoxicity. Our aim was to examine the effect of the pH of the apical medium on the apical to basal transfer of the acidic drug indomethacin (pK a 4.5) across a gastric mucous epithelial cell monolayer, and to determine whether indomethacin accumulated in cells exposed to a low apical pH. Guinea-pig gastric mucous epithelial cells were grown on porous membrane culture inserts (Transwells®) for 72 h. Transfer and accumulation of [ 14C] indomethacin were assessed by scintillation counting. Transfer of [ 3H]mannitol and measurement of trans-epithelial electrical resistance were used to assess integrity of the monolayer. Distribution of [ 14C] urea was used to estimate the intracellular volume of the monolayer. The monolayer was not disrupted by exposure of the apical face to media of pH ≥ 3, or by indomethacin. Transfer of indomethacin (12 μM) to the basal medium increased with decreasing apical medium pH. The apparent permeability of the undissociated acid was estimated to be five times that of the anion. The intracellular concentration of indomethacin was respectively 5.3, 4.1 and 4.3 times that in the apical medium at pH 5.5, 4.5 and 3.0. In conclusion, this study represents the first direct demonstration that indomethacin accumulates in gastric epithelial cells exposed to low apical pH. However, accumulation of indomethacin was moderate and the predictions of the ion-trapping hypothesis were not met, probably due to the substantial permeability of anionic indomethacin across membranes. © 2006 Elsevier B.V. All rights reserved.

Bifunctional role for VEGF-induced heme oxygenase-1 in vivo:induction of angiogenesis and inhibition of leukocytic infiltration

Heme-oxygenases (HOs) catalyze the conversion of heme into carbon monoxide and biliverdin. HO-1 is induced during hypoxia, ischemia/reperfusion, and inflammation, providing cytoprotection and inhibiting leukocyte migration to inflammatory sites. Although in vitro studies have suggested an additional role for HO-1 in angiogenesis, the relevance of this in vivo remains unknown. We investigated the involvement of HO-1 in angiogenesis in vitro and in vivo. Vascular endothelial growth factor (VEGF) induced prolonged HO-1 expression and activity in human endothelial cells and HO-1 inhibition abrogated VEGF-driven angiogenesis. Two murine models of angiogenesis were used: (1) angiogenesis initiated by addition of VEGF to Matrigel and (2) a lipopolysaccharide (LPS)-induced model of inflammatory angiogenesis in which angiogenesis is secondary to leukocyte invasion. Pharmacologic inhibition of HO-1 induced marked leukocytic infiltration that enhanced VEGF-induced angiogenesis. However, in the presence of an anti-CD18 monoclonal antibody (mAb) to block leukocyte migration, VEGF-induced angiogenesis was significantly inhibited by HO-1 antagonists. Furthermore, in the LPS-induced model of inflammatory angiogenesis, induction of HO-1 with cobalt protoporphyrin significantly inhibited leukocyte invasion into LPS-conditioned Matrigel and thus prevented the subsequent angiogenesis. We therefore propose that during chronic inflammation HO-1 has 2 roles: first, an anti-inflammatory action inhibiting leukocyte infiltration; and second, promotion of VEGF-driven noninflammatory angiogenesis that facilitates tissue repair.