Biochem. J. (2005) 387
(553–560) (Printed in Great Britain)
Depletion of Caco-2 cell cholesterol disrupts barrier function by altering the detergent solubility and distribution of specific tight-junction proteins
Daniel LAMBERT, Catherine A. O'NEILL and Philip J. PADFIELD1
Division of Gastrointestinal Science, University of Manchester, Hope Hospital, Salford M6 8HD, U.K.
In the present study, we have investigated the role of cholesterol in maintaining the barrier properties of the model intestinal cell line Caco-2. We have extracted membrane cholesterol using methyl-b-cyclodextrin and demonstrated that maximally, methyl-b-cyclodextrin lowered cell cholesterol levels by 40–45%. Depletion of cell cholesterol was accompanied by an 80–90% decrease in monolayer transepithelial electrical resistance and a significant increase in the paracellular permeability of dextrans of 4, 10 and 40 kDa. The increase in dextran permeability was most pronounced for the two lower molecular mass species. In addition to the decline in the barrier properties of the monolayers, extraction of cell cholesterol produced an increase in the Triton X-100 solubility of claudin 3, claudin 4 and occludin, and the loss of all three proteins from the plasma membrane (tight junctions). In contrast, removal of cholesterol had no detectable influence on the detergent solubility or morphological distribution of claudin 1. These results indicate that membrane cholesterol is a critical factor in maintaining the barrier property of epithelial monolayers. More specifically, cholesterol appears to stabilize the association of certain proteins with the tight junctions.
Key words: Caco-2 cell, cholesterol, claudin, occludin, tight junction.
Abbreviations used: MCD, methyl-b-cyclodextrin; MDCK, Madin–Darby canine kidney; MEM, modified Eagle's medium; PIP2, phosphatidylinositol 4,5-bisphosphate; TEER, transepithelial electrical resistance; TX100, Triton X-100.
1To whom correspondence should be addressed (email ppadfiel@fs1.ho.man.ac.uk).
Received 13 August 2004/7 October 2004; accepted 25 October 2004
Published as BJ Immediate Publication 25 October 2004, DOI 10.1042/BJ20041377
The Biochemical Society, London ©2005
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