AUTHOR=JanssenDuijghuijsen Lonneke M. , Grefte Sander , de Boer Vincent C. J. , Zeper Lara , van Dartel Dorien A. M. , van der Stelt Inge , Bekkenkamp-Grovenstein Melissa , van Norren Klaske , Wichers Harry J. , Keijer Jaap 

TITLE=Mitochondrial ATP Depletion Disrupts Caco-2 Monolayer Integrity and Internalizes Claudin 7

JOURNAL=Frontiers in Physiology

VOLUME=8

YEAR=2017

URL=https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2017.00794

DOI=10.3389/fphys.2017.00794

ISSN=1664-042X

ABSTRACT=<p><bold>Objective:</bold><italic>In vivo</italic> studies suggest that intestinal barrier integrity is dependent on mitochondrial ATP production. Here, we aim to provide mechanistic support, using an <italic>in vitro</italic> model mimicking the oxidative <italic>in vivo</italic> situation.</p><p><bold>Methods:</bold> Human Caco-2 cells were cultured for 10 days in culture flasks or for 14 days on transwell inserts in either glucose-containing or galactose-containing medium. Mitochondria were visualized and cellular respiration and levels of oxidative phosphorylation (OXPHOS) proteins were determined. Mitochondrial ATP depletion was induced using CCCP, rotenone, or piericidin A (PA). Monolayer permeability was assessed using transepithelial electrical resistance (TEER) and fluorescein flux. Gene expression and cellular distribution of tight junction proteins were analyzed.</p><p><bold>Results:</bold> Caco-2 cells cultured in galactose-containing, but not in glucose-containing, medium showed increased mitochondrial connectivity, oxygen consumption rates and levels of OXPHOS proteins. Inhibition of mitochondrial ATP production using CCCP, rotenone or PA resulted in a dose-dependent increase in Caco-2 monolayer permeability. In-depth studies with PA showed a six fold decrease in cellular ATP and revealed increased gene expression of tight junction proteins (<italic>TJP</italic>) 1 and 2, occludin, and claudin 1, but decreased gene expression of claudin 2 and 7. Of these, claudin 7 was clearly redistributed from the cellular membrane into the cytoplasm, while the others were not (TJP1, occludin) or slightly (claudin 2, actin) affected. <italic>In vivo</italic> studies suggest that intestinal barrier integrity is dependent on mitochondrial ATP production. Here, we aim to provide mechanistic support, using an <italic>in vitro</italic> model mimicking the oxidative <italic>in vivo</italic> situation.</p><p><bold>Conclusions:</bold> Well-functioning mitochondria are essential for maintaining cellular energy status and monolayer integrity of galactose grown Caco-2 cells. Energy depletion-induced Caco-2 monolayer permeability may be facilitated by changes in the distribution of claudin 7.</p>