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Cholesterol depletion reduces apical transport capacity in epithelial Madin-Darby canine kidney cells.

ABSTRACT: Reduction of the cholesterol level in membranes of epithelial Madin-Darby canine kidney (MDCK) cells reverses the apical-to-basolateral transport ratio of the apical membrane marker protein influenza virus haemagglutinin and the secreted glycoprotein gp80. At the same time, basolateral transport of the vesicular stomatitis virus G protein is unaffected [Keller and Simons (1998) J. Cell Biol. 140, 1357-1367]. To investigate whether cholesterol depletion influences apical sorting mechanisms specifically, or apical transport capacity more generally, we studied the effect of cholesterol depletion on the secretion of three different classes of molecules from the apical and basolateral surfaces of MDCK cell layers: glycoprotein gp80, sulphated proteoglycans and proteins, and non-glycosylated rat growth hormone. In each case, cholesterol depletion reduced the fraction secreted to the apical medium and increased the fraction secreted basolaterally. The fact that this was observed for all sulphated proteins and proteoglycans and for the non-glycosylated rat growth hormone, which is randomly secreted in untreated cells, indicates that cholesterol depletion reduces the apical transport capacity, rather than interfering with specific recognition and sorting processes.

SUBMITTER: Prydz K

PROVIDER: S-EPMC1221922 | biostudies-other | 2001 Jul

REPOSITORIES: biostudies-other

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Project description:Madin-Darby canine kidney (MDCK) cells were cultured on polycarbonate filters to study the synthesis and sorting of proteoglycans in polarized epithelial cells. Two strains of MDCK cells were used. MDCK I cells resemble distal tubule epithelial cells, and MDCK II cells share some characteristics with proximal tubule cells. Both strains were grown to confluency and labelled with [35S]sulphate for 24 h. The apical and basolateral media and the cell fractions were harvested and analysed by DEAE ion-exchange chromatography. A large portion of the [35S]sulphate-labelled macromolecules bound strongly to the ion-exchange columns, and could be eluted in three distinct peaks. The latest eluting peak was demonstrated to contain almost exclusively chondroitin sulphate, whereas peak 2 contained mostly heparan sulphate, demonstrated by using chondroitinase ABC and nitrous acid (pH 1.5) respectively to depolymerize the [35S]glycosaminoglycan chains. Peak 1 contained negligible amounts of proteoglycans. Large differences could be observed in proteoglycan sorting in MDCK I and II cells. Strain I secreted approx. 67% of the proteoglycans to the apical side and 17% to the basolateral side. The cell fraction contained 17% of the proteoglycans after 24 h of labelling. In contrast, 19% of the proteoglycans were sorted to the apical side of MDCK II cells and 61% to the basolateral side, whereas the cell fraction contained 20%. Furthermore, the level of [35S]proteoglycan biosynthesis (apical and basolateral media and cell fraction total) was higher in MDCK I cells than in strain II. Based on the amount of material degraded by chondroitinase ABC and nitrous acid respectively, and the total amounts of [35S]proteoglycans recovered from the cells, it was calculated that the MDCK I strain synthesized approx. 56% chondroitin sulphate and 44% heparan sulphate. In contrast, the MDCK II strain synthesized 69% heparan sulphate and 31% chondroitin sulphate. To further identify the [35S]proteoglycans synthesized by MDCK I and II cells, antibodies against perlecan, versican and syndecan were used. The antibody against mouse syndecan did not cross-react with any of the proteoglycans produced in MDCK I or II cells. Both MDCK I and II cells expressed perlecan; 57-61% could be recovered from the basolateral fractions and 18-34% from the apical medium. Versican was also found in both MDCK I and II cells. Compared with perlecan, a larger percentage of versican (43-53%) was found in the cell fractions.

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Cholesterol depletion reduces apical transport capacity in epithelial Madin-Darby canine kidney cells.

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