Inhibitors of glycosphingolipid biosynthesis reduce transepithelial electrical resistance in MDCK I and FRT cells

Lawrence W. Leung1, Ruben G. Contreras2, Catalina Flores-Maldonado2, Marcelino Cereijido2, and Enrique Rodriguez-Boulan1,3

1 Margaret M. Dyson Vision Research Institute, Department of Ophthalmology, and 3 Department of Cell Biology, Weill Medical College of Cornell University, New York, New York 10021; and 2 Department of Physiology, Center for Research and Advanced Studies, 07000 Mexico City DF, Mexico


    ABSTRACT
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Madin-Darby canine kidney (MDCK) I and Fisher rat thyroid (FRT) cells exhibit transepithelial electrical resistance (TER) values in excess of 5,000 Omega  · cm2. When these cells were incubated in the presence of various inhibitors of sphingolipid biosynthesis, a >5-fold reduction of TER was observed without changes in the gate function for uncharged solutes or the fence function for apically applied fluorescent lipids. The localization of ZO-1 and occludin was not altered between control and inhibitor-treated cells, indicating that the tight junction was still intact. Furthermore, the complexity of tight junction strands, analyzed by freeze-fracture microscopy, was not reduced. Once the inhibitor was removed and the cells were allowed to synthesize sphingolipids, a gradual recovery of the TER was observed. Interestingly, these inhibitors did not attenuate the TER of MDCK II cells, a cell line that typically exhibits values below 800 Omega  · cm2. These results suggest that glycosphingolipids play a role in regulating the electrical properties of epithelial cells.

lipid microdomains; caveolin; claudin; occludin


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GLYCOSPHINGOLIPIDS are composed of a hydrophobic ceramide backbone covalently linked to a polar carbohydrate group. If classified solely by their carbohydrate groups, there are more than 150 different glycosphingolipids (GSL) expressed in humans (31). Approximately one-third of this number contain sialic acid and are known commonly as gangliosides (29). Although the consequences of sphingolipid accumulation in pathologies such as Gaucher's and Tay-Sach's disease are well known (3, 12), there are few reports detailing the physiological function of sphingolipids, despite the fact that the first GSLs were isolated and characterized over a century ago. In contrast, phosphatidylinositols, which have less than a dozen different headgroups, are known to be critical for such diverse functions as calcium release, protein kinase C activation, vesicle budding, and endocytic recycling (21, 25, 36, 40). The diversity of GSLs suggests they could mediate a large number of biological functions.

Interest in the cell biology of GSLs was sparked when it was theorized that GSLs could associate with cholesterol and form a membrane domain that was distinct from bulk phospholipids (44). Subsequently, the in vivo association of certain integral membrane proteins with these GSL- and cholesterol-rich membranes was inferred from the observation that insoluble material from cells extracted with 1% Triton X-100 was highly enriched with GSLs, cholesterol, and glycosylphosphatidylinositol-anchored proteins (5). Proteins such as caveolin, G proteins, and src family kinases can be found in these detergent-insoluble membranes, which are often referred to as Triton-insoluble floating fractions (TIFF) (9, 18, 30, 35, 37, 46). Interestingly, two proteins of the tight junction (TJ), ZO-1 and occludin, can also be isolated from TIFF (26). Cholesterol depletion from T84 cells using lovastatin caused a reduction in transepithelial electrical resistance (TER) from 1,000 to 400 Omega  · cm2 within 24 h, as well as the mislocalization of occludin from the lateral membrane to the cytoplasm. Moreover, disassembly of the TJ via calcium chelation caused the dissociation of occludin and ZO-1 from TIFF, leading to the conclusion that the TJ is a specialized lipid microdomain. A role for cholesterol in modulating TJ electrical properties has been demonstrated in Madin-Darby canine kidney (MDCK) cells. In contact-naive MDCK cells, cholesterol depletion results in accelerated Ca2+-induced TJ formation (39), whereas in fully polarized MDCK cells, cholesterol depletion results in a transient increase in TER, which can be reversed once cholesterol is restored to the cells (10). In both cases, the mechanism was attributed to altered signaling through lipid second messengers.

Studies demonstrating the effect of cholesterol depletion in mammalian tissue culture cells are possible because of the availability of cholesterol biosynthesis inhibitors such as compactin and lovastatin. In the case of GSLs, the other putative component of TIFF, fumonisin-beta 1 (FB1), an inhibitor of ceramide biosynthesis, has been shown to reduce the steady-state levels of glucosylceramide in MDCK cells (22). However, FB1 can also cause an increase in sphingoid bases, potential lipid second messengers (12, 13). Other inhibitors, such as 2-amino-3,4-dihydroxy-2-hydroxymethyl-14-oxo-6-eicosenoic acid (ISP1) and 1-phenyl-2-hexadecanoylamino-3-morphilino-1-propanol-HCl (PPMP), have been shown to reduce the levels of complex sphingolipids in melanoma cells (16) and MDCK cells (1), respectively. These inhibitors do not directly inhibit the formation of ceramide and thus should not increase the levels of sphingoid bases, making them potentially valuable tools in determining the function of GSLs.

While studying the properties of these GSL biosynthesis inhibitors, we observed that these inhibitors attenuated the TER in MDCK I cells by nearly an order of magnitude compared with control cells. Fisher rat thyroid cells (FRT), a cell line that also displays high TER, demonstrated the same susceptibility to these inhibitors. Under these conditions, the localization of ZO-1 and occludin was not altered between control and lipid-depleted cells, indicating that the TJ was not grossly altered. The presence of claudin-2, which was shown to reduce TER when expressed in MDCK I cells, was not observed (11). Finally, loss of GSLs did not cause a dissociation of occludin from TIFF. These results show that GSLs are involved in regulating the electrical properties of epithelial cells.


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Cell culture. MDCK (I and II) or FRT cells from a confluent 75-cm2 flask were trypsinized and seeded onto 12- or 25-mm-diameter Transwell filters (Corning, Rochester, NY) for TER measurements or onto 35-mm-diameter dishes for lipid analysis. Cells were cultured in DMEM supplemented with 5% fetal bovine serum. TERs were measured using an ohmeter (World Precision Instruments, Bradenton, FL).

Inhibition of sphingolipid biosynthesis. All inhibitors were added to 0.2% fat-free BSA in DMEM. PPMP (Matreya, Pleasant Gap, PA), FB1 (Sigma, St. Louis, MO), and ISP1 (Cayman, Ann Arbor, MI) were added from concentrated stocks in absolute ethanol. The final ethanol concentration did not exceed 0.1% (vol/vol). After 5 days at confluency, monolayers of MDCK or FRT cells were rinsed with DMEM and switched to media containing the indicated inhibitor. Control cells received 0.2% BSA and ethanol (0.1% vol/vol) only. In some studies, the PPMP-containing medium was replaced with 0.2% fat-free BSA in DMEM + 0.1% ethanol after 48 h.

Lipid analysis. GSLs from cultured cells were isolated following a published method (2). Briefly, cells grown on 35-mm dishes were rinsed twice with ice-cold phosphate-buffered saline and scraped into methanol/acetic acid (98:2; vol/vol, 1 ml) and transferred to a 13 × 100 mm test tube. Chloroform (0.5 ml) and water (1 ml) were added, and the solution was vortexed. Phase separation was achieved by the addition of 1 ml each of chloroform and water. The upper phase was discarded, and the organic phase evaporated under reduced pressure. Glycerophospholipids were removed by saponification in alkaline methanol followed by reextraction and evaporation. Aliquots of sphingolipids were spotted onto a thin-layer chromatography (TLC) plate (Keiselguhr 60, Merck) and developed in chloroform/methanol/water (60:35:4; vol/vol/vol). Lipids were visualized after spraying with hydroxytetralone in sulfuric acid followed by heating in a TLC oven (110°C, 20 min) using a Storm 860 phosphorimager in the red fluorescent mode.

Confocal immunofluorescence microscopy. MDCK I cells grown on filters in the presence or absence of lipid biosynthesis inhibitors were processed for immunofluorescence microscopy. Briefly, filters were washed twice in phosphate-buffered saline containing 0.1 mM CaCl2 and 1.0 mM MgCl2 (PBS-CM) and fixed in 3% paraformaldehyde for 30 min. After quenching with 75 mM glycine and 50 mM NH4Cl in PBS-CM for 15 min, the filters were rinsed in PBS-CM and incubated in PBS-CM containing 0.2% BSA and 0.075% saponin (buffer A) for 30 min. The filters were excised from their plastic holders and incubated in buffer A containing 2.0 µg/ml of rat anti-ZO-1 (Chemicon, Temecula, CA) and 5.0 µg/ml of rabbit anti-occludin (Zymed, San Francisco, CA). After 1 h, the filters were rinsed 3 × in buffer A and incubated in buffer A containing 5.0 µg/ml each of FITC-labeled donkey anti-rat IgG and Texas Red-labeled goat anti-rabbit IgG (Jackson Immunoresearch, West Grove, PA). After 30 min, the filters were rinsed, mounted, and examined with a Zeiss LSM 510 confocal microscope. Images were acquired and processed using the Zeiss Image Browser software.

Flotation gradients. The method described by Harder et al. (15) was used with the following modifications. Briefly, MDCK or FRT cells were grown on 25-mm-diameter Transwell filters in the absence or presence of PPMP. Two filters were used for each gradient. After 2 days, the filters were rinsed with ice-cold Hanks buffer (without Mg2+ and Ca2+), and the cells were scraped into 500 µl of the same buffer and centrifuged at 200 g for 2 min at 4°C. The supernatant was removed and the cell pellet was lysed in 200 µl of buffer B (25 mM Tris · HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% sucrose, 2% Triton X-100, 10 µg/ml each of aprotinin, leupeptin, and pepstatin, and 1 mM PMSF) at 4°C. Once the cell pellet was dissolved, 400 µl of ice-cold 60% Optiprep was added. The mixture was agitated gently and transferred to a SW 50 centrifuge tube. Additional 600-µl gradient steps consisting of 35, 30, 25, 20, and 0% Optiprep in buffer B were overlaid. Gradients were centrifuged for 6 h at 42,000 g at 4°C. Six fractions (600 µl each) were removed from the top of each tube, and the proteins in each fraction precipitated. The precipitated proteins were analyzed by SDS-PAGE and Western blotting. For Western blotting, rabbit anti-occludin, rabbit anti-claudin-1, and rabbit anti-claudin-2 antibodies were purchased from Zymed. Rabbit anti-claudin-4 was a generous gift from S. Tsukita (Kyoto Univ., Japan).

"Fence" function: diffusion of BODIPY-sphingomyelin. Sphingomyelin/BSA complexes (5 nmol/ml) were prepared in P buffer [145 mM NaCl, 10 mM HEPES (pH 7.4), 1.0 mM Na-pyruvate, 10 mM glucose, 3 mM CaCl2] using BODIPY-FL-sphingomyelin (Molecular Probes, Eugene, OR) and defatted BSA. Filter-grown MDCK cells were labeled from the apical side with BODIPY-sphingomyelin/BSA complexes for 10 min on ice. Cells were washed with P buffer and incubated for 0 or 1 h on ice and then analyzed by confocal microscopy Z-sectioning. In wild-type MDCK cells, polar lipid staining was stable for at least 20 min, and the lateral appearance of BODIPY-sphingomyelin was preceded by internalization. All pictures shown, however, were generated within the first 5 min of analysis.

"Gate" function: paracellular flux of 3-kDa dextran. FITC-labeled dextran (10 mg/ml in P buffer) was added to the apical side of filter-grown MDCK cells and incubated at 37°C. After 1 h, the medium from the basolateral chamber was collected, and FITC-dextran fluorescence was measured with a fluorometer (excitation 492 nm; emission 520 nm).

Freeze fracture analysis. For freeze fracture analysis, cell monolayers grown in flasks (Falcon Plastics, Cockeysvillen, MD) were fixed with 2.5% glutaraldehyde in PBS for 30 min at 37oC, washed three times with PBS, and cryoprotected by successive incubations in 10, 20, and 30% glycerol, for 30, 30, and 60 min, respectively. They were then detached from the substratum as a sheet by gently scraping with a rubber policeman, placed on gold specimen holders, and rapidly frozen in the liquid phase of partially solidified Freon 22 cooled with liquid nitrogen. Freeze fractures were performed in a Blazers BAF 400 (Balzers, Liechtenstein) at -150°C and 5 × 10-9 bar. Fractured faces were shadowed with platinum and carbon at 45 and 90°, respectively. Replicas were cleaned with chromic mixture and washed in distilled water, placed on 300-mesh copper grids, and examined in an electron microscope (JEM-2000EX: JEOL, Tokyo, Japan).

Morphometric analysis was performed on micrographs of freeze fracture replicas, printed at a magnification of ×50,000. A line parallel to the main axis of the TJs was traced, and a series of perpendicular lines was drawn (1 every 300 nm). The number of strands in a given segment of TJs was defined as the number of these intersections with the perpendicular line.


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Sphingolipid biosynthesis inhibitors reduce TER in MDCK I. Several commercially available inhibitors of sphingolipid biosynthesis, ISP1, PPMP, and FB1 (Fig. 1), were tested for their ability to reduce the amounts of GSLs in MDCK cells. For PPMP and ISP1, we determined the minimum concentration of each inhibitor that inhibited the activity of glucosylceramide or serine palmitoyltransferase by at least 90%, respectively (data not shown). In the case of FB1, we used the previously reported concentration (22). Unexpectedly, these inhibitors reduced the TER in MDCK I cells, but not in MDCK II cells (Fig. 2, A and B). Although all three inhibitors were effective in reducing the amount of glucosylceramide and lactosylceramide in MDCK I cells (Fig. 2C), PPMP (20 µM) and ISP1 (12 µM) were the most effective in reducing TER. After 48 h, the resistance values were ~1,000 Omega  · cm2. While significantly lower than in control MDCK I cells, the TER in drug-treated cells was still higher than that in MDCK II cells. In MDCK II cells, the same inhibitors had little effect on the TER or the level of lactosylceramide, but there was a small decrease in the amount of glucosylceramide (Fig. 2D).


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Fig. 1.   Inhibitors of mammalian sphingolipid biosynthesis. The fungal metabolites 2-amino-3,4-dihydroxy-2-hydroxymethyl-14-oxo-6-eicosenoic acid (ISP1) and fumonisin-beta 1 (FB1) act at early steps in the synthesis of sphingolipids. All 3 inhibitors result in reduced synthesis of glucosylceramide, the precursor of more complex glycosphingolipids (GSLs) such as gangliosides. Note that FB1 inhibits the synthesis of ceramide, whereas 1-phenyl-2-hexadecanoylamino-3-morphilino-1-propanol-HCl (PPMP) will cause ceramide to accumulate. PC, phosphatidylcholine.



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Fig. 2.   Inhibitors of GSL biosynthesis reduce transepithelial electrical resistance (TER) in Madin-Darby canine kidney (MDCK) I cells but not MDCK II cells. Filter-grown MDCK I (A) or MDCK II (B) were incubated in 0.2% BSA + DMEM in the presence or absence of various inhibitors. After 48 h, cells treated with ISP1, PPMP, and FB1 all exhibited TER values lower than that found in cells treated with BSA alone. The inhibitors significantly reduced the amount of glucosylceramide (glc-cer) and lactosylceramide (lac-cer) in MDCK I cells (C) but only slightly reduced the amount of these lipids in MDCK II cells (D). Lanes: a, 0.2% BSA; b, 12 µM ISP1; c, 20 µM PPMP; d, 28 µM FB1; e, bovine brain sphingomyelin; f, GSL standards; g, monosialogangliosides. gal-cer, Galactosylceramide; GM3, monosialoganglioside GM3.

We also tested to see if the effect of PPMP was reversible. After reducing the TER of MDCK I by incubation in the presence of 20 µM PPMP, removal of the inhibitor resulted in a gradual increase of TER that approached control levels after 20 h (Fig. 3A). In a separate experiment, we examined whether the return of TER was accompanied by a restoration of glucosylceramide. MDCK I cells were grown to confluency on 25-mm-diameter filters and treated in the presence (n = 6) or absence (n = 3) of 20 µM PPMP. After 2 days, the PPMP-treated cells exhibited TER lower than control cells (1,695 ± 49 vs. 5,891 ± 34 Omega  · cm2). At this point, the control filters and one-half of the PPMP were subjected to GSL extraction. The remaining PPMP-treated filters were rinsed in 0.2% BSA in DMEM and cultured in the same medium for an additional 2 days, at which point the TER resembled that of control cells (6,072 ± 315 Omega  · cm2). These cells were then subjected to GSL extraction. All three sets, control, PPMP, and PPMP + washout, were analyzed for glucosylceramide levels using TLC (Fig. 3B). The loss of TER in PPMP-treated cells was correlated with a loss in glucosylceramide levels, and the restoration of TER in the washout cells correlated with a return of glucosylceramide.


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Fig. 3.   A: PPMP attenuation of TER is reversible. After culturing MDCK I cells in the presence () or absence () of 20 µM PPMP for 2 days, the media were replaced with 0.2% BSA in DMEM. TER was measured periodically over 32 h. During this time, the TER of PPMP-treated cells returned to near normal levels after removal of the inhibitor. B: TER restoration correlates with glucosylceramide levels. MDCK cells were treated as in A. After 2 days, the TER and glucosylceramide levels from PPMP-treated filter-grown MDCK cells (PPMP) were significantly lower (see RESULTS) than those of control cells (control). PPMP-treated cells that were switched back to 0.2% BSA in DMEM for an additional 2 days (washout) eventually regained TER and glucosylceramide levels comparable to that of control cells.

The correlation between total GSL levels and TER could indicate an alteration of an ion channel/pump activity in the plasma membrane or it could indicate an alteration of the TJ, but there may be other explanations for the results we observed. In addition to reducing GSLs, PPMP and FB1 can increase the intracellular concentration of ceramide and sphinganine, respectively (Fig. 1). Although these bioactive molecules may act as signal transducers, it is unlikely that PPMP and FB1 reduce TER through these mechanisms. PPMP and FB1 have opposite effects on ceramide concentration, while ISP1 and FB1 have opposite effects on sphinganine concentration (Fig. 1). Therefore, the loss of TER in MDCK I cells correlates with a loss of GSLs and not an accumulation of second messenger lipids.

TJ remains intact while TER is reduced. To determine if the loss of lipids may have altered the TJ structure, we stained control and drug-treated cells for two TJ proteins, ZO-1 and occludin (Fig. 4). Treatment with 20 µM PPMP resulted in a fourfold decrease in TER of MDCK I cells, but occludin and ZO-1 were still localized to the apical-most portion of lateral plasma membrane. The inhibitors ISP1 and FB1 also reduced TER without altering the overall appearance of the TJ proteins. In control and drug-treated cells, the TJ was located between 2 and 3 µm from the apical membrane plasma membrane. Drug-treated cells retained their cuboidal shape and were similar from control cells. A small amount of intracellular occludin can be seen in the PPMP-treated cells, but this is absent in the ISP1- and FB1-treated cells, leading us to conclude that the appearance of this small pool of occludin in PPMP is not the cause of attenuated TER. In conclusion, the inhibitors reduced the TER without causing significant alterations to the appearance of the TJ at the level of light microscopy.


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Fig. 4.   GSL biosynthesis inhibitors do not alter the localization of tight junction (TJ) proteins. MDCK I cells grown on polycarbonate filters in the presence of PPMP (row B), ISP1 (row C), or FB1 (row D) show occludin and ZO-1 staining similar to that of control cells (row A). The TER of the cells was measured before fixation. Left column, occludin; middle column, ZO-1. Bar, 20 µm.

In addition to impeding the flow of ions through the paracellular space, TJs are also known to 1) restrict the diffusion of membrane components from the apical to lateral membrane (fence function), and 2) control the permeability of small noncharged solutes through the paracellular space (part of the gate function). In cells with intact TJs, fluorescent lipids applied to the outer leaflet of the apical plasma membrane are restricted from traversing to the lateral surface (8) unless they are capable of first "flipping" into the inner leaflet. In contrast, when a COOH-terminally truncated chicken occludin is expressed in MDCK cells, apical lipids are able to diffuse into the lateral membranes, indicating that the physical barrier to membrane diffusion is lost (2). At the same time, these cells are not able to restrict the flow of fluorescent dextrans through the paracellular space. Therefore, we examined the fence and gate function of PPMP-treated MDCK I cells.

To measure the fence function, confluent, filter-grown MDCK I cells were treated in the presence or absence of PPMP for 2 days and then incubated with BODIPY-sphingomyelin on the apical surface for 10 min at 4°C. The filters were washed and processed for confocal microscopy after 0 or 1 h. For both control and PPMP-treated MDCK cells, z-sections through the filters detected only an apical staining pattern for the fluorescent lipid after 0 h (Fig. 5, top). After incubating for 1 h at 4°C, there was no lateral lipid staining detectable in either control or PPMP-treated MDCK cells, indicating that PPMP did not interfere with the fence function of the TJ.


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Fig. 5.   PPMP-induced TER reduction does not cause a loss of TJ "fence" or "gate" function. Filter-grown MDCK cells were treated with or without PPMP for 2 days. While the TER in MDCK I cells was attenuated, the cells still prevented the basolateral diffusion of an apically applied fluorescent lipid. (top). At the same time, the cells with attenuated TER still acted as a barrier to the flux of FITC-dextran (JDEX) through the TJ (bottom).

To test whether PPMP altered the gate function of MDCK cells, we measured the diffusion of FITC-labeled 3-kDa dextran across cell monolayers grown on filters. Confluent, filter-grown MDCK cells were treated in the presence or absence of PPMP for 2 days. FITC-dextran was added to the apical compartment, and the cells were incubated at 37°C for 1 h. At the end of this time, medium from the basolateral compartment was removed and the fluorescence was measured using a spectrophotometer. Although TER was attenuated in cells receiving PPMP, there was no significant difference in the apical-to-basolateral dextran flux between control and PPMP-treated MDCK cells (Fig. 5, bottom), indicating that GSL depletion does not impair this aspect of the TJ gate function.

Effect of PPMP on the number of TJ strands. Because the previous experiments ruled out gross structural alteration of the TJ as a mechanism for the action of PPMP, we examined the possibility that the TJ ultrastructure might have been subtly modified. In freeze-fracture replicas, TJs appear as a network of interconnected strands. In some tissues, a high TER is correlated with a higher number of strands, leading to the hypothesis that increased electrical resistance is proportional to the number of strands (6). Therefore, one possible mechanism for the action of PPMP we considered was the reduction of the number of strands in MDCK I cells. When we examined freeze-fracture replicas of MDCK I cells grown under control and PPMP treatment, the mean number of strands in PPMP-treated cells were not lower than those of control cell. PPMP slightly alters the complexity of the TJ network compared with control cells (Fig. 6), increasing the mean strand number (2.77 for control vs. 3.06 for PPMP). Therefore, PPMP does not reduce TER by reducing the complexity of the strands.


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Fig. 6.   PPMP does not reduce the number of TJ strands in MDCK I. The number of strands was counted in segments of the TJ every 200 nM. Control and PPMP segments refer to the no. of segments with 1,2, ..., n strands in monolayers under control conditions or treated with PPMP, respectively.

Effect of PPMP on the expression of claudins 2 and 4. Clostridium perfringens enterotoxin (CPE) has been shown to specifically remove claudin-4 from the TJ of MDCK I cells, resulting in a reduction of TER within 48 h (38). Although ZO-1 and occludin were still localized to the TJ, there was a twofold reduction in the mean strand number of the TJ as seen by freeze fracture EM. After the toxin was washed out, the TER returned to near normal values. Sonoda et al. (38) speculated that epithelial cells may regulate the flow of ions through the paracellular space by controlling the expression of TJ components. Recently, it was shown that the expression of claudin-2 in MDCK I cells caused a decrease in TER. Ordinarily, claudin-2 is expressed in MDCK II, but not MDCK I (11). With this in mind, we tested whether the GSL inhibitors caused claudin-2 expression in MDCK I cells (Fig. 7, top). Confluent, filter-grown MDCK cells were treated with GSL inhibitors for 2 days, resulting in a reduction in TER. The cells were lysed and examined for the presence of claudin-2 by Western blot. In MDCK II cells, claudin-2 is quite prevalent, but the protein is absent in control and drug-treated MDCK I cells. Therefore, the attenuated TER is not due to the expression of claudin-2 in MDCK I. At the same time, we did not observe a reduction in the levels of claudin-4 by Western blot (data not shown). As mentioned earlier, claudin-4 removal results in a decrease in the complexity of the TJ, an effect not seen with PPMP.


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Fig. 7.   Top: PPMP does not cause the expression of claudin-2. MDCK I cells were treated with PPMP for 2 days, resulting in TER attenuation. Western blot analysis of the cells revealed the presence of claudin-2 (band at 22 kDa) in MDCK II lysates but not in any of the MDCK I cells. Triplicate filters are shown for control MDCK I, PPMP-treated MDCK I, and control MDCK II. Bottom: TER of Fisher rat thyroid (FRT) cells is sensitive to PPMP. FRT cells were grown to confluency on polycarbonate filters in normal media. After 3-4 days, the media were replaced with 0.2% BSA in DMEM alone () or 0.2% BSA in DMEM containing 10 µM PPMP (). The TER was measured periodically. After 48 h, the TER of the PPMP-treated cells was dramatically lower than the control cells.

Lipid microdomains and the TJ. Recently, it was reported that reduction of cholesterol levels in T84 cells (using lovastatin, an inhibitor of cholesterol biosynthesis), causes a reduction of TER (26). At the same time, hyperphosphorylated occludin and ZO-1 were removed from TIFF, leading the authors to conclude that the TJ is composed of cholesterol-rich, detergent-resistant membrane microdomains. These microdomains are thought to result from the hydrophobic interaction between the saturated fatty acyl chains of sphingolipids and cholesterol and are hypothesized to recruit specific membrane-associated proteins, such as caveolin, a protein that acts as a scaffold for several kinases involved in signal transduction (28, 34, 37). Nusrat et al. (26) went on to demonstrate that occludin and caveolin-1 could be colocalized by immunofluorescence microscopy and suggested the possibility that caveolin was playing a role in regulating TJ function.

Given that GSLs are putative components of lipid microdomains, it could be that PPMP reduces the levels of a lipid essential for caveolin function, and this in turn alters TER. We examined this possibility by testing the effect of PPMP on FRT cells, a cell line that does not express caveolin-1. Although FRT cells do express caveolin-2, it is located at the Golgi and not at the plasma membrane (24). Control FRT cells exhibit TER greater than 5,000 Omega  · cm2, similar to MDCK I. Within 48 h of treatment with 10 µM PPMP, FRT cells showed a dramatic loss of TER (Fig. 7, bottom). This result indicates that the effect of PPMP is not limited to MDCK I cells and, more importantly, that caveolin-1 is not involved in the reduction of TER caused by PPMP.

Because occludin was shown to reside in TIFF, we examined the effect of PPMP on the Triton insolubility of occludin. Using an Optiprep density gradient, we confirmed that hyperphosphorylated occludin associates with TIFF. However, occludin from both MDCK I and FRT cells is still found in TIFF when the TER is attenuated with PPMP (Fig. 8), suggesting that the loss of TER is not due to removal of occludin from a detergent-resistant lipid microdomain. At the same time, we observe that in MDCK I cells, only a small amount of claudin-1 associates with the TIFF under control conditions. In MDCK II cells, a slightly larger amount of claudin-1 can be seen in TIFF, but the majority is found in the high-density fractions. (Fig. 9).


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Fig. 8.   Occludin remains associated with lipid rafts while TER is reduced. Triton-insoluble floating fractions (TIFF) were isolated from FRT, MDCK I, and MDCK II cells treated with or without PPMP (10 µM PPMP for FRT, 20 µM PPMP for MDCK I and II). After 2 days, the TER was measured and the cells were analyzed for the presence of occludin in TIFF as described in METHODS. In the cells exhibiting high TER, the hyperphosphorylated form of occludin was found predominantly in the TIFF fraction. Treatment with PPMP reduces the TER but did not cause a redistribution of this TJ protein to the Triton-soluble fractions. In MDCK II cells, very little phosphorylated occludin could be seen in any of the fractions. PO4 occ, hyperphosphorylated occludin.



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Fig. 9.   Claudin-1 is found in Triton-soluble fractions. Triton-insoluble floating fractions (TIFF) were isolated from MDCK I and MDCK II cells treated with or without PPMP (20 µM). After 2 days, the TER was measured and the cells were analyzed for the presence of occludin in TIFF as described in METHODS. Only a small amount of claudin-1 can be found in the Triton-insoluble fractions in either cell line. Treatment with PPMP did not cause a significant redistribution of this protein from the Triton-soluble fractions.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our results demonstrate that reducing GSLs in MDCK I and FRT cells reduces TER in a novel, specific manner. The inhibitor of glucosylceramide synthase, PPMP, significantly reduces TER without 1) rearranging the strands, 2) impairing the barrier to small uncharged solutes, or 3) disrupting the fence function of the TJ. Other procedures have previously been reported to reduce TER, but none of them have the same properties as PPMP. Cholesterol removal in T-84 cells (~1,000 Omega  · cm2) causes a drastic (10-fold) reduction of TER but induces redistribution of occludin to an intracellular pool (26). In MDCK II cells, the rapid removal of cholesterol caused a reversible decrease in TER that was accompanied by a decrease in the occludin and ZO-1 at the TJ (10). The actin depolymerization agents cytochalasin B and mycalolide B both reduce TER in MDCK II cells (23, 41), and in the latter case, mycalolide B also caused increased permeability of the TJ to inulin, a small, nonionic molecule (41). Energy (ATP) depletion with 2-deoxyglucose in MDCK II cells also doubles the permeability to inulin and causes a slight modification in the structure of the strands (19). Incubation with CPE causes removal of claudin 4 from MDCK I, which is accompanied by loss of gate function for small solutes and reduction in the complexity of the strands (38). Addition of claudin-2 to MDCK I cells, which normally lacks it, has a more drastic effect than PPMP on the TER (reduction to MDCK II levels) but like PPMP has no effect on uncharged solute permeability or fence function (11). However, like ATP depletion, exogenous claudin-2 expression introduces slight changes in the morphology of the strands such as the appearance of particles in the E face and small discontinuities in the P face. Sonoda et al. (38) have shown that CPE specifically binds to an extracellular loop of claudin-4 in MDCK I cells, causing internalization and degradation of the claudin. This resulting decrease in TER, however, was accompanied by an increase in paracellular flux, as well as a reduction in the size and complexity of the TJ strands (38).

There are several scenarios that could explain how inhibitors of GSL biosynthesis might attenuate TER. Sphingolipids such as sphinganine and ceramide act as second messengers (13, 27, 45), and altering their concentration might result in the activation of signaling pathways, leading to a loss of TER. As discussed in RESULTS, PPMP may increase the concentration of ceramide, but its concentration is reduced by ISP1 and FB1, which argues against the involvement of ceramide in the loss of TER. By the same logic, we consider it unlikely that the TER attenuation is caused by an increase in sphinganine (in the cases of PPMP and FB1) because ISP1 will reduce the concentration of sphinganine.

Another scenario is that GSLs are structural components of the TJ. We tested this hypothesis by analyzing the lipid composition of occludin immunoprecipitates from MDCK I cells. However, we were not able to detect any GSLs in these preparations. In addition, we attempted immunofluorescence microscopy of MDCK I cells after paraformaldehyde fixation to visualize specific lipids at the TJ but were not successful. These negative results are not conclusive because the lipids could have been extracted during the washing steps in the immunoprecipitation. Furthermore, unlike proteins, GSLs are not fixed with paraformaldehyde and are still capable of diffusing after the cells are chemically fixed (32, 33).

Because sphingolipid depletion has been shown to alter the transport of some transmembrane proteins in MDCK cells to the plasma membrane (22), another possibility it that the inhibitors altered the steady-state localization of TJ protein components. Although this does not appear to be the case for ZO-1 and occludin (Fig. 3), we cannot exclude the possibility that other TJ proteins are mislocalized as a result of sphingolipid depletion. Work by Tsukita and colleagues (42, 43) indicates that TER depends on the presence of specific claudins at the TJ. Removal of claudin-4 using CPE caused a loss of TER in MDCK I cells, accompanied by a reduction in the number of strands in the TJ, as well as an increase in permeability to dextran. While the loss of TER is similar to our results, it is unlikely that PPMP is acting directly on claudin-4 because we do not observe similar alterations in the TJ morphology or an increase in paracellular flux. Recent work by Furuse et al. (11) shows that the addition of claudin-2 to MDCK I cells (which do not normally express claudin-2) causes a loss of TER without altering the fence role or permeability to noncharged solute gate properties, but the reduction of GSLs in our hands does not cause expression of claudin-2 in MDCK I cells.

Finally, it is possible that the reduction in TER is mediated by increased flow through ion channels located in the plasma membrane. Recent work in amphibian A6 cells demonstrates that the amiloride-sensitive epithelial sodium channel (ENaC) is located in TIFF (17). Furthermore, various potassium channels have also been found in these lipid microdomains (4, 7, 20). Although expression of rat ENaC in MDCK cells shows that the protein is not located in TIFF (14), it is conceivable that the loss of GSLs altered the location and/or the activity of one or more ion channels. In summary, we observe that a loss of GSLs causes a loss of TER in high-resistance epithelial cells but cannot determine if this effect is due to a subtle alteration of the TJ or an alteration of ion transport activity at the plasma membrane.


    FOOTNOTES

Address for reprint requests and other correspondence: E. Rodriguez-Boulan, Margaret M. Dyson Vision Research Institute, Box 233, Weill Medical College of Cornell Univ., 1300 York Ave., New York, NY 10021 (E-mail: boulan{at}med.cornell.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published December 21, 2002;10.1152/ajpcell.00149.2002

Received 3 April 2002; accepted in final form 6 December 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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