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01.12.2018 | Research article | Ausgabe 1/2018 Open Access

BMC Gastroenterology 1/2018

Cobalt chloride compromises transepithelial barrier properties of CaCo-2 BBe human gastrointestinal epithelial cell layers

Zeitschrift:
BMC Gastroenterology > Ausgabe 1/2018
Autoren:
K. M. DiGuilio, M. C. Valenzano, E. Rybakovsky, J. M. Mullin
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi: https://​doi.​org/​10.​1186/​s12876-017-0731-5) contains supplementary material, which is available to authorized users.

Abstract

Background

Elevation of the transcription factor HIF-1 is a prominent mediator of not only processes that accompany hypoxia, but also the tumor microenvironment and tissue regeneration. This study uses mediators of “chemical hypoxia” to ask the question whether HIF-1α elevation in a healthy epithelial cell layer leads to leakiness in its tight junctional seals.

Methods

Transepithelial electrical resistance and transepithelial diffusion of 14C–D-mannitol and other radiolabeled probes are used as indicators of transepithelial barrier function of CaCo-2 BBe human gastrointestinal epithelial cell layers cultured on permeable supports. Western immunoblot analyses of integral tight junctional proteins (occludin and claudins) are used as further indicators of barrier function change.

Results

Cobalt, an inhibitor of the prolyl hydroxylase enzymes governing HIF-1α breakdown in the cell, induces transepithelial leakiness in CaCo-2 BBe cell layers in a time and concentration-dependent manner. This increased leakiness is accompanied by significant changes in certain specific integral tight junctional (TJ) proteins such as a decreased level of occludin and increased level of claudin-5. Similar results regarding barrier function compromise also occur with other chemical inhibitors of HIF-1α breakdown, namely ciclopiroxolamine (CPX) and dimethyloxalylglycine (DMOG). The increased leak is manifested by both decreased transepithelial electrical resistance (Rt) and increased paracellular diffusion of D-mannitol (Jm). The induced transepithelial leak shows significant size selectivity, consistent with induced effects on TJ permeability. Less-differentiated cell layers were significantly more affected than well-differentiated cell layers regarding induced transepithelial leak. A genetically modified CaCo-2 variant with reduced levels of HIF-1β, showed reduced transepithelial leak in response to cobalt exposure, further indicating that elevation of HIF-1α levels induced by agents of “chemical hypoxia” is responsible for the compromised barrier function of the CaCo-2 BBe cell layers.

Conclusions

Exposure to inducers of chemical hypoxia elevated HIF-1α levels and increased transepithelial leak. The degree of epithelial differentiation has significant effects on this action, possibly explaining the varying effects of HIF-1 modulation in epithelial and endothelial barrier function in different physiological and pathophysiological conditions.
Zusatzmaterial
Additional file 1: Representative western immunoblot probed for Claudin-5, showing effect of cobalt exposure of CACO-2 cell layers. (TIFF 1975 kb)
12876_2017_731_MOESM1_ESM.tif
Additional file 2: Representative western immunoblot probed for Claudin-7, showing effect of cobalt exposure of CACO-2 cell layers. (TIFF 2624 kb)
12876_2017_731_MOESM2_ESM.tif
Additional file 3: Representative western immunoblot probed for HIF-1a, showing effect of cobalt exposure of CACO-2 cell layers. (TIFF 2014 kb)
12876_2017_731_MOESM3_ESM.tif
Additional file 4: Representative western immunoblot probed for HIF-1b, comparing CACO-2 control cell layers vs CACO-2 HIF-1b knockdown cell layers. (TIFF 1975 kb)
12876_2017_731_MOESM4_ESM.tif
Additional file 5: Representative western immunoblot probed for Occludin, showing effect of cobalt exposure of CACO-2 cell layers. (TIFF 2080 kb)
12876_2017_731_MOESM5_ESM.tif
Literatur
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