Uncoupling of the Cholera Toxin-GM1 Ganglioside Receptor Complex from Endocytosis, Retrograde Golgi Trafficking, and Downstream Signal Transduction by Depletion of Membrane Cholesterol
2002; Elsevier BV; Volume: 277; Issue: 18 Linguagem: Inglês
10.1074/jbc.m109834200
ISSN1083-351X
AutoresAnne A. Wolf, Yukako Fujinaga, Wayne I. Lencer,
Tópico(s)Protein Structure and Dynamics
ResumoTo induce toxicity, cholera toxin (CT) must first bind ganglioside GM1 at the plasma membrane, enter the cell by endocytosis, and then traffic retrograde into the endoplasmic reticulum. We recently proposed that GM1provides the sorting motif necessary for retrograde trafficking into the biosynthetic/secretory pathway of host cells, and that such trafficking depends on association with lipid rafts and lipid raft function. To test this idea, we examined whether CT action in human intestinal T84 cells depends on membrane cholesterol. Chelation of cholesterol with 2-hydroxypropyl β-cyclodextrin or methyl β-cyclodextrin reversibly inhibited CT-induced chloride secretion and prolonged the time required for CT to enter the cell and induce toxicity. These effects were specific to CT, as identical conditions did not alter the potency or toxicity of anthrax edema toxin that enters the cell by another mechanism. We found that endocytosis and trafficking of CT into the Golgi apparatus depended on membrane cholesterol. Cholesterol depletion also changed the density and specific protein content of CT-associated lipid raft fractions but did not entirely displace the CT-GM1 complex from these lipid raft microdomains. Taken together these data imply that cholesterol may function to couple the CT-GM1 complex with raft domains and with other membrane components of the lipid raft required for CT entry into the cell. To induce toxicity, cholera toxin (CT) must first bind ganglioside GM1 at the plasma membrane, enter the cell by endocytosis, and then traffic retrograde into the endoplasmic reticulum. We recently proposed that GM1provides the sorting motif necessary for retrograde trafficking into the biosynthetic/secretory pathway of host cells, and that such trafficking depends on association with lipid rafts and lipid raft function. To test this idea, we examined whether CT action in human intestinal T84 cells depends on membrane cholesterol. Chelation of cholesterol with 2-hydroxypropyl β-cyclodextrin or methyl β-cyclodextrin reversibly inhibited CT-induced chloride secretion and prolonged the time required for CT to enter the cell and induce toxicity. These effects were specific to CT, as identical conditions did not alter the potency or toxicity of anthrax edema toxin that enters the cell by another mechanism. We found that endocytosis and trafficking of CT into the Golgi apparatus depended on membrane cholesterol. Cholesterol depletion also changed the density and specific protein content of CT-associated lipid raft fractions but did not entirely displace the CT-GM1 complex from these lipid raft microdomains. Taken together these data imply that cholesterol may function to couple the CT-GM1 complex with raft domains and with other membrane components of the lipid raft required for CT entry into the cell. cholera toxin ganglioside Galβ1–3GalNAcβ1–4(NeuAcα2–3)Galβ1–4Glcβ1 ceramide ganglioside NeuAcα2–3Galβ1–3GalNAcβ1–44Glcβ1 ceramide 2-hydroxypropyl β-cyclodextrin methyl β-cyclodextrin anthrax edema toxin vasoactive intestinal peptide short circuit current Hanks' balanced salt solution transepithelial resistance endoplasmic reticulum horseradish peroxidase glycosylphosphatidylinositol Vibrio cholerae causes worldwide epidemics of life-threatening secretory diarrhea by colonizing the intestinal lumen and producing cholera toxin (CT),1 a potent enterotoxin that invades the intestinal epithelial cell as a fully folded protein. Structurally, CT consists of two components. The pentameric B-subunit binds stoichiometrically to five GM1 gangliosides on the apical (lumenal) surface of intestinal epithelial cells, and the enzymatic A-subunit activates adenylyl cyclase inside the cell by catalyzing the ADP-ribosylation of the heterotrimeric GTPase Gs (1Lencer W.I. Hirst T.R. Holmes R.K. Biochim. Biophys. Acta. 1999; 1450: 177-190Crossref PubMed Scopus (222) Google Scholar). Activation of adenylyl cyclase in intestinal crypt epithelial cells leads to Cl− secretion, the fundamental transport event in secretory diarrhea. To induce disease, both A- and B-subunits must enter the host epithelial cell as a fully assembled holotoxin by moving retrograde through the biosynthetic pathway into the ER, 2Y. Fujinaga and W. I. Lencer, unpublished data.2Y. Fujinaga and W. I. Lencer, unpublished data. where the A-subunit unfolds, dissociates from the B-pentamer, and translocates to the cytosol, presumably by dislocation through the protein conducting channel sec61p (2Tsai B. Rodighiero C. Lencer W.I. Rapoport T. Cell. 2001; 104: 937-948Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). The time between CT binding to GM1 at the cell surface and induction of toxicity has been termed the "lag phase." The lag phase corresponds to the time required for trafficking CT into the ER, unfolding of the A-subunit by interaction with protein-disulfide isomerase, and finally dislocation of the A-subunit into the cytosol (2Tsai B. Rodighiero C. Lencer W.I. Rapoport T. Cell. 2001; 104: 937-948Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, 3Lencer W.I. Am. J. Physiol. 2001; 280: G781-G786PubMed Google Scholar). In the model polarized intestinal epithelial cell line T84, CT function depends on B-subunit binding to (and possibly clustering) ganglioside GM1. Binding to GM1 anchors CT to the host cell membrane and associates CT with cholesterol-rich detergent-insoluble membrane microdomains, termed DIGs (detergent-insolubleglycosphingolipid-rich membrane microdomains) or lipid rafts (4Wolf A.A. Jobling M.G. Wimer-Mackin S. Ferguson-Maltzman M. Madara J.L. Holmes R.K. Lencer W.I. J. Cell Biol. 1998; 141: 917-927Crossref PubMed Scopus (178) Google Scholar). GM1 exhibits specificity for CT action in human intestinal epithelial cells, as the ganglioside GD1a does not substitute for GM1 as a receptor for toxin action or for association with lipid rafts in this cell type (4Wolf A.A. Jobling M.G. Wimer-Mackin S. Ferguson-Maltzman M. Madara J.L. Holmes R.K. Lencer W.I. J. Cell Biol. 1998; 141: 917-927Crossref PubMed Scopus (178) Google Scholar, 5Badizadegan K. Dickinson B.L. Wheeler H.E. Blumberg R.S. Holmes R.K. Lencer W.I. Am. J. Physiol. 2000; 278: G895-G904Crossref PubMed Google Scholar). Chimeric toxins, for example, containing the A-subunit of CT assembled with the B-subunit of Escherichia coli toxin LTIIb, bound only to GD1a, did not associate with lipid rafts, and did not lead to a functional response (4Wolf A.A. Jobling M.G. Wimer-Mackin S. Ferguson-Maltzman M. Madara J.L. Holmes R.K. Lencer W.I. J. Cell Biol. 1998; 141: 917-927Crossref PubMed Scopus (178) Google Scholar). Based on these studies, we have recently proposed that the B-subunit-GM1 complex represents the sorting motif necessary for toxin trafficking retrograde into the Golgi apparatus and possibly ER of host eukaryotic cells. We have also proposed that such trafficking depends on association with lipid rafts and lipid raft function. To test this idea, we now examine whether CT action depends on membrane cholesterol. Lipid rafts are highly enriched in cholesterol, GPI-linked proteins, and glycosphingolipids, including ganglioside GM1, the receptor for CT. Abundant evidence indicates that raft structure and function in cellular metabolism, including certain forms of ligand-induced signal transduction, protein and lipid sorting, endocytosis, and transcytosis, depend critically on cholesterol (4Wolf A.A. Jobling M.G. Wimer-Mackin S. Ferguson-Maltzman M. Madara J.L. Holmes R.K. Lencer W.I. J. Cell Biol. 1998; 141: 917-927Crossref PubMed Scopus (178) Google Scholar,6Oh J.E. Schnitzer P. Pinney E. Allard J. J. Cell Biol. 1994; 127: 1217-1232Crossref PubMed Scopus (771) Google Scholar, 7Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2609) Google Scholar, 8Fiedler K. Kobayashi T. Kurzchalia T.V. Simons K. Biochemistry. 1993; 32: 6365Crossref PubMed Scopus (225) Google Scholar, 9Bagnat M. Keranen S. Shevchenko A. Shevchenko A. Simons K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3254-3259Crossref PubMed Scopus (502) Google Scholar, 10Grimmer S. Iversen T. van Deurs B. Sandvig K. Mol. Biol. Cell. 2000; 11: 4205-4216Crossref PubMed Scopus (81) Google Scholar, 11Brown D.A. London E. Biochem. Biophys. Res. Commun. 1997; 240: 1-7Crossref PubMed Scopus (462) Google Scholar, 12van Meer G. Stelzer E.H.K. Wijnaendts-van Resandt R.W. Simons K. J. Cell Biol. 1987; 105: 1623-1635Crossref PubMed Scopus (305) Google Scholar, 13Rock P. Allietta M. Young W.W. J Thompson T.E. Tillack T.W. Biochemistry. 1990; 29: 8484-8490Crossref PubMed Scopus (99) Google Scholar, 14Rock P. Allietta M. Young W.W.J. Thompson T.E. Tillack T.W. Biochemistry. 1991; 30: 19-25Crossref PubMed Scopus (78) Google Scholar, 15Keller P. Simons K. J. Cell Biol. 1998; 140: 1357-1367Crossref PubMed Scopus (471) Google Scholar). Demonstration of such sensitivity to membrane cholesterol has been taken widely as evidence for dependence on raft function in these cellular processes. With respect to CT and aerolysin toxin, another bacterial enterotoxin that binds membrane receptors located in lipid rafts, disruption of membrane cholesterol by the sterol-binding molecule filipin or by chelation with β-cyclodextrin has been shown to effect endocytosis and toxin action (16Abrami L. van der Goot F.G. J. Cell Biol. 1999; 147: 175-184Crossref PubMed Scopus (138) Google Scholar, 17Orlandi P.A. Fishman P.H. J. Cell Biol. 1998; 141: 905-915Crossref PubMed Scopus (638) Google Scholar). On the other hand, whether lipid rafts function directly in CT endocytosis has recently been questioned (18Shogomori H. Futerman A.H. J. Biol. Chem. 2001; 276: 9182-9188Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). In some cellular systems, clathrin-mediated endocytosis displays sensitivity to disruption of membrane cholesterol (19Rodal S.K. Skretting G. Garred O. Vilhardt F. van Deurs B. Sandvig K. Mol. Biol. Cell. 1999; 10: 961-974Crossref PubMed Scopus (824) Google Scholar, 20Subtil A.S. Giadarov I. Kobylarz K. Lampson M.A. Keen J.H. McGraw T.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6775-6780Crossref PubMed Scopus (486) Google Scholar, 21Parton R.G. J. Histochem. Cytochem. 1994; 42: 155-166Crossref PubMed Scopus (453) Google Scholar), and although CT binds GM1 located both in lipid rafts and clathrin-coated pits, the toxin enters hippocampal neurons and A431 epithelial cells by clathrin-mediated mechanisms (18Shogomori H. Futerman A.H. J. Biol. Chem. 2001; 276: 9182-9188Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 19Rodal S.K. Skretting G. Garred O. Vilhardt F. van Deurs B. Sandvig K. Mol. Biol. Cell. 1999; 10: 961-974Crossref PubMed Scopus (824) Google Scholar). In the current study, we utilize 2-hydroxy and methyl β-cyclodextrin to examine CT trafficking and function in model epithelial cells acutely depleted in cholesterol (22Kilsdonk E.P.C. Yancy P.G. Stoudt G. Bangerter F.W. Johnson W.J. Philips M.C. Rothblat G.H. J. Biol. Chem. 1995; 270: 17250-17256Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar, 23Christian A.E. Haynes M.P. Philips M.C. Rothblat G.H. J. Lipid Res. 1997; 38: 2264-2272Abstract Full Text PDF PubMed Google Scholar, 24Neufeld E.B. Cooney A.D. Pitha J. Davidowitz E.A. Dwyer N.K. Pentchev P.G. Blanchette-Mackie E.J. J. Biol. Chem. 1996; 271: 21604-21613Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). To control for general effects of cholesterol depletion on membrane dynamics, we utilize anthrax edema toxin (EdTx). Like CT, anthrax EdTx is a two component toxin. Both CT and EdTx enter polarized monolayers of intestinal T84 cells by receptor-mediated endocytosis, and both toxins induce an identical cAMP-dependent Cl− secretory response, but by different mechanisms. The enzymatic component of EdTx, termed edema factor, is itself a potent calmodulin-dependent adenylyl cyclase that, unlike CT, translocates to the cytosol directly across the endosome membrane. Membrane translocation of edema factor and the induction of toxicity depend critically on entry of EdTx into an acidic endosomal compartment. Retrograde transport into Golgi cisternae or ER is not required (25Beauregard K.E. Wimer-Mackin S. Collier R.J. Lencer W.I. Infect. Immun. 1999; 67: 3026-3030Crossref PubMed Google Scholar). In addition, unlike CT, the membrane-binding component of EdTx, termed protective antigen, recognizes a receptor on T84 cell membranes that displays strict basolateral polarity, and the EdTx-receptor complex does not fractionate with lipid rafts (25Beauregard K.E. Wimer-Mackin S. Collier R.J. Lencer W.I. Infect. Immun. 1999; 67: 3026-3030Crossref PubMed Google Scholar). Our data show that endocytosis and trafficking of CT into Golgi cisternae, as well as CT-induced Cl− secretion, depend on membrane cholesterol. Cholesterol depletion altered lipid raft structure and presumably function, and only partially displaced the CT-GM1 complex from lipid raft microdomains. Cholesterol depletion had no detectable affect on entry of anthrax EdTx into acidic endosomes, as assessed as an EdTx-induced Cl− secretory response. These data indicate that CT trafficking depends on toxin association with lipid rafts and imply that cholesterol may function in lipid raft structure to couple the CT-GM1 complex with raft domains and with other membrane components involved in membrane sorting or downstream signal transduction required for CT entry into the cell. Cholera toxin and cholera toxin B-subunit coupled to horseradish peroxidase (HRP) were obtained fromCalbiochem (San Diego, CA) or were recombinantly expressed. Anthrax edema toxin was a kind gift from Dr. R. John Collier (Department of Microbiology, Harvard Medical School, Boston, MA). 2-Hydroxy β-cyclodextrin, methyl β-cyclodextrin, cholesterol, and cholesterol analogues were obtained from Sigma. Mouse monoclonal antibody to pp60src was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY) rabbit polyclonal antibody to caveolin-1 from Santa Cruz Biotechnology (Santa Cruz, CA) and mouse monoclonal antibody to ecto-5′-nucleotidase from Linda Thompson (Oklahoma Medical Research Foundation, Oklahoma City, OK). Rabbit polyclonal antibodies to CT B-subunits were previously described (26Lencer W.I. Moe S. Rufo P.A. Madara J.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10094-10098Crossref PubMed Scopus (100) Google Scholar). All other chemicals were from Sigma unless otherwise stated. T84 cells obtained from American Type Culture Collection (Rockville, MD) were cultured and passaged as previously described (27Lencer W.I. Constable C. Moe S. Jobling M. Webb H.M. Ruston S. Madara J.L. Hirst T. Holmes R. J. Cell Biol. 1995; 131: 951-962Crossref PubMed Scopus (183) Google Scholar). Passages 80–92 were used for these studies. Monolayers used for electrophysiology and for toxin binding studies were grown on 0.33-cm2 polycarbonate filters (Costar, Corning, NY) and used 12–14 days after plating. Monolayers used for isolation of lipid rafts were grown on 45-cm2 filters and used 21 days after plating. Hanks' balanced salt solution (HBSS) to which 10 mm HEPES was added and pH adjusted to 7.4 was used for all assays unless otherwise noted. Initial studies showed that cyclodextrins induced a progressive concentration- and time-dependent decrease in transepithelial resistance. We thus defined conditions for acute cholesterol depletion that preserved monolayer resistance using either 16.5 mm2-hydroxypropyl-cyclodextrin (2OHβ-CD) or 4 mm methyl β-cyclodextrin (mβ-CD) applied to apical or basolateral cell surfaces of T84 cell monolayers for 1 h. To assay for cholesterol depletion, total cell lipids from T84 cell monolayers were extracted in chloroform, dried, and then resuspended in minimal volume (3 μl) of chloroform. Cholesterol content was analyzed per milligram of total cell protein by colorimetric assay using the Infinity cholesterol reagent according to the manufacture's directions (Sigma Diagnostics). Absorbance was read at 500 nm on a Spectronic21D spectrophotometer (Milton Roy Products). For assessment of Cl− secretion (measured as a short circuit current, Isc), cells were treated as above with the appropriate cyclodextrin analogue for 1 h at 37 °C before adding CT (20 or 1 nm) to apical or basolateral reservoirs, respectively, in the continued presence or absence of cyclodextrin. The time course of toxin-induced Cl− secretion (Isc) and transepithelial resistance were measured as previously described (28Lencer W.I. Delp C. Neutra M.R. Madara J.L. J. Cell Biol. 1992; 117: 1197-1209Crossref PubMed Scopus (121) Google Scholar). To provide two point calibration for all studies, the cAMP agonist vasoactive intestinal peptide (VIP, 10 nm) was applied to basolateral reservoirs at the end of each experiment (either 60 or 85 min after application of toxin). To replete monolayers with cholesterol, cholesterol and related sterols were complexed with mβ-CD as previously described and used at a final concentration of 0.2 mm sterol (29Pike L.J. Miller J.M. J. Biol. Chem. 1998; 273: 22298-22304Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). Specific toxin binding to apical membranes of T84 cell monolayers treated or not treated with 16.5 mm2OHβ-CD at 37 °C was measured. All incubations were done in buffer made of HBSS containing 0.1% bovine serum albumin. After pretreatment, monolayers were cooled to 4 °C, and incubated with buffer alone or 100 nm CT B-subunit (to measure nonspecific binding) in buffer for 20 min. Monolayers were then washed and incubated apically with 20 nm CT B-subunit-HRP in buffer for 30 min on ice. Unbound toxin was removed by three washes with ice-cold HBSS. Cell surface-bound CT B-HRP was assessed by colorimetric assay using 30% hydrogen peroxide in 1 mm2,2′-azino-di-3-ethylbenzthiazoline sulfonic acid in 100 mmcitrate-phosphate buffer, pH 4.2, as previously described (4Wolf A.A. Jobling M.G. Wimer-Mackin S. Ferguson-Maltzman M. Madara J.L. Holmes R.K. Lencer W.I. J. Cell Biol. 1998; 141: 917-927Crossref PubMed Scopus (178) Google Scholar). Specific binding of toxin to the cell surface was determined by subtracting nonspecific binding from total surface-bound toxin. T84 cells grown for 2 weeks on 0.33-cm2 filter supports were treated with 4 mmmβ-CD or buffer alone for 1 h at 37 °C, brought to 4 °C in HBSS, and incubated with 20 nm CT in the presence of 4 mm mβ-CD or buffer alone for 45 min at 4 °C. Monolayers were then warmed to 37 °C in the presence of apical 20 nm CT 16.5 mm mβ-CD or buffer for the indicated times, or kept at 4 °C for the duration of the experiment. All monolayers were then washed in ice-cold buffer to remove unbound toxin. Where indicated, cell surface-bound CT was removed by immersing each monolayer in HBSS, pH 7.4, at 37 °C for 10 s (to further release unbound toxin at the cell surface) and then immediately transferred to HBSS, pH 2.5, at 4 °C for 5 min. Each monolayer was then immersed for 5 min in HBSS, pH 7.4, at 4 °C, and incubated again in HBSS, pH 2.5, at 4 °C for an additional 5 min. 97.5% ± 16.5% (n = 4) of CT was stripped from the cell surface using this procedure. After removal of cell surface-bound toxin, monolayers were cut from their filter supports, immersed in 5% SDS, and boiled for analysis of total cell-associated CT B-subunit. Sucrose equilibrium density centrifugation was performed as described previously (4Wolf A.A. Jobling M.G. Wimer-Mackin S. Ferguson-Maltzman M. Madara J.L. Holmes R.K. Lencer W.I. J. Cell Biol. 1998; 141: 917-927Crossref PubMed Scopus (178) Google Scholar). One or two confluent 45-cm2 monolayers of T84 cells were used for isolation of detergent-insoluble membranes. All steps were performed at 4 °C. Cells were scraped into 2 ml of ice-cold 50 mm Tris-buffered saline containing 16.5 mm Tween 20 and a protease inhibitor tablet (containing EDTA, pancreas extract, Pronase, thermolysin, chymotrypsin, trypsin, papain) from Roche Molecular Biochemicals and homogenized by five strokes in a tight-fitting Dounce homogenizer on ice. The homogenate was adjusted to 40% sucrose by adding 2 ml of 80% sucrose in Tris-buffered saline containing Tween 20, layered under a continuous 5–30% sucrose gradient, and centrifuged at 39,000 rpm for 16–20 h in a swinging bucket rotor (model SW41; Beckman Instruments, Palo Alto, CA). The presence of a floating membrane fraction was noted visually, and 0.5-ml fractions were collected from the top. For step gradients, the cell homogenates in 4 ml of 40% sucrose were layered first under 5 ml of 30% sucrose, and then 5 ml of 5% sucrose was added to the top of the gradient. The step gradients were centrifuged as described above. Raft fractions were collected from the 30–5% interface and combined. Soluble material remained in the 40% sucrose fraction at the bottom of the gradient. For all gradients, fractions were normalized for protein content (Pierce BCA protein assay), analyzed by SDS-PAGE, Western-blotted for the indicated proteins as previously described (30Lencer W.I. Constable C. Moe S. Rufo P.A. Wolf A. Jobling M.G. Ruston S.P. Madara J.L. Holmes R.K. Hirst T.R. J. Biol. Chem. 1997; 272: 15562-15568Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), and quantified by densitometry using a Kodak Digital Science Image Station 440 CF (PerkinElmer Life Sciences). All signals were in the linear range. Raft and soluble fractions from some experiments were analyzed for 5′-nucleotidase (CD73) activity as previously described (31Resta R. Hooker S.W. Hansen K.R. Laurent A.B. Park J.L. Blackburn M.R. Knudsen T.B. Thompson L.F. Gene (Amst.). 1993; 133: 171-177Crossref PubMed Scopus (68) Google Scholar). CT holotoxin was engineered to contain the sulfation consensus motif SAEDYEYPS at the C terminus of its B-subunits.2 Recombinant toxins were produced and purified as previously described (32Rodighiero C. Aman A.T. Kenny M.J. Moss J. Lencer W.I. Hirst T.R. J. Biol. Chem. 1999; 274: 3962-3969Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). For in vivo sulfation experiments, T84 cells were first washed, incubated in sulfate-free HBSS for 30 min at 37 °C, and then incubated again for an additional 1 h in fresh sulfate-free HBSS. The monolayers were then incubated with 0.5 mCi/ml Na235SO4 in the same buffer for 30 min. The CT-variant toxin containing the sulfation motif was added to T84 cells both apically and basolaterally to a final concentration of 20 nm, incubated for 50 min at 37 °C, and then washed twice with ice-cold HBSS. Following total cell lysis, an immunoprecipitation using rabbit anti-CT-B antibodies as described previously (33Rodighiero C. Fujinaga Y. Hirst T.R. Lencer W.I. J. Biol. Chem. 2001; 276: 36939-36945Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) was performed. Samples were run on 10–20% denaturing Tris·HCl polyacrylamide gels (Bio-Rad), and analyzed with a PhosphorImager (Molecular Dynamics Inc, Sunnyvale, CA). Data were analyzed using Statview 512+ software (Brainpower, Inc., Calabasa, CA) We utilized the cyclodextrin analogues 2OHβ-CD and mβ-CD to determine whether the mechanism of CT action depends on membrane cholesterol. Initial studies showed that treatment of T84 cell monolayers with 2OHβ-CD caused a dose- and time-dependent loss of transepithelial resistance (TER), presumably because of direct effects of cholesterol depletion on tight junction structure (34Francis S.A. Kelly J.M. McCormack J. Rogers R.A. Lai J. Schneeberger E.E. Lynch R.D. Eur. J. Cell Biol. 1999; 78: 473-484Crossref PubMed Scopus (98) Google Scholar, 35Nusrat A. Parkos C.A. Verkade P. Foley C.S. Liang T.W. Innis-Whitehouse W. Eastburn K.K. Madara J.L. J. Cell Sci. 2000; 113: 1771-1781Crossref PubMed Google Scholar). Conditions were thus defined that depleted ∼50% of total cell cholesterol but maintained monolayer resistance intact throughout the time course of each experiment. Under optimal conditions, however, transepithelial resistance still fell progressively to 15% of initial values by the end of each time course (from 1035 ± 54 to 151 ± 22 Ω, n = 10), and the cAMP agonist VIP was used in all studies to calibrate measurement of Isc. Like CT, VIP increases intracellular cAMP in T84 cells by activating the heterotrimeric GTPase Gs. All subsequent events in the Cl− secretory response are identical. The alternative sterol-binding agent filipin could not be used in these experiments to deplete membrane cholesterol, as short 15-min incubations with filipin (7.6 μm) caused complete loss of monolayer resistance that prevented assessment of cell function by electrophysiology. T84 cells treated with 2OHβ-CD exhibited an attenuated response to apically applied CT. Fig. 1Ashows a representative time course of CT-induced Cl−secretion in monolayers depleted or not depleted in cholesterol. VIP was applied at 90 min to control for the effect of 2OHβ-CD on monolayer resistance as described above. 2OHβ-CD prolonged the "lag phase" by nearly 2-fold (Fig. 1, A and B) and diminished the apparent rate of toxin-induced Cl−secretion by >60% (ΔIsc = 0.98 ± 0.10 versus0.39 ± 0.08 μA/cm2/min, n = 8, control versus 2OHβ-CD-treated monolayers,p < 0.05). Peak Isc responses to apically applied CT were inhibited by 50% when calibrated against that induced by VIP (Fig. 1, A and C). 2OHβ-CD, however, had no detectable effect on the time course of Isc induced by basolaterally applied CT (data not shown). Two lines of evidence indicated that treatment with 2OHβ-CD did not inhibit CT action by depleting the toxin's receptor ganglioside GM1 from cell membranes. First, T84 monolayers treated or not with 2OHβ-CD exhibited nearly identical densities of apical membrane receptors for CT (Fig.2A). Second, the effects of 2OHβ-CD were reversed completely by treatment with cholesterol alone (Fig. 2B). For these studies, 2OHβ-CD-treated monolayers were washed free of 2OHβ-CD and incubated for an additional 90 min in the presence or absence of excess cholesterol or the cholesterol analogues 25-hydroxycholesterol or cholestane-3β,5α,6β-triol. Monolayers depleted in cholesterol and subsequently allowed to recover in buffer alone exhibited strongly attenuated CT-induced peak Isc values equal to that of T84 monolayers treated continuously with 2OHβ-CD (Fig. 2B, column 3 versus column 2). In contrast, monolayers pretreated with 2OHβ-CD and then allowed to recover in the presence of cholesterol exhibited peak secretory responses equal to that of control monolayers not treated with 2OHβ-CD (Fig.2B, column 6 versus column 1). Cholesterol-treated monolayers also exhibited recovery of the lag phase, consistent with the idea that depletion of membrane cholesterol affected a rate-limiting step required for toxin trafficking into the ER (lag phase = 31 ± 4 versus 48 ± 7 versus 37 ± 5 min, means ± S.E., n = 7–8, control versuscholesterol-depleted versus cholesterol-replete monolayers). Monolayers treated with the inactive cholesterol analogues cholestane-3β,5α,6β-triol or 25-hydroxycholesterol exhibited only partial or no recovery (Fig. 2B, columns 5 and 4 versus control columns 1 and 6). Thus, the effect of 2OHβ-CD on CT-induced Cl− secretion was fully reversible and specific to cholesterol. Nonspecific effects of 2OHβ-CD on GM1 receptor density, if any, cannot explain these results. Taken together, these initial studies indicate that CT action on T84 cells, presumably because of toxin entry into the cell or trafficking retrograde into Golgi cisternae and ER, depends on membrane cholesterol.Figure 2Effect of 2OHβ-CD on GM1 receptor density and reversibility of inhibition by treatment with exogenous cholesterol. Binding of CT to T84 monolayers treated (shaded bar) or not treated (solid bar) with 2OHβ-CD (means ± S.E.,n = 8, p ≤ 0.05). B, cholesterol, but not cholestane-3β,5α,6β-triol or 25-hydroxycholesterol, fully reversed the effect of 2OH-β-CD on CT-induced Cl− secretion. T84 cell monolayers were treated for 1 h in the absence (−) or presence (+) of 2OHβ-CD at 37 °C for 1 h, washed, and then treated with either buffer alone (lanes 1 and 3) or 2OHβ-CD (lane 2) containing 20 nm CT for the remainder of the time course or with 0.2 mm amounts of the indicated oxysterol (25-OH chol, 25-hydroxycholesterol; cholestane, cholestane-3β,5α,6β-triol). Results are normalized to the maximal Isc induced by VIP under identical conditions (means ± S.E.,n = 4, * indicates p ≤ 0.05 compared against lanes 1 and 6).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Effect of cholesterol depletion on the protein composition of lipid rafts. A, the CT-GM1 complex remains raft-associated following a 24-h treatment with 16.5 mm 2OHβ-CD. T84 cell monolayers were pretreated with 16.5 mm 2OHβ-CD (upper panel) or media alone (lower panel) for 24 h at 37 °C, after which they were brought to 4 °C, washed, and exposed to 20 nm CT. Monolayers were extracted in 1% Triton X-100 at 4 °C and layered under continuous sucrose gradients for equilibrium density centrifugation. Fractions were collected, and protein content and sucrose density were determined for each fraction. Fractions were analyzed by SDS-PAGE and Western blot loaded for equal protein (0.5 μg). *, 5 ng of CT loaded as control.B, protein content of raft fractions isolated from monolayers depleted (open bar) or not depleted (solid bar) in cholesterol. Results expressed as means ± S.E., n = 8. *, p ≤ 0.05. C, treatment with 2OHβ-CD decreases the apparent density of lipid rafts containing CT. T84 cell extracts were prepared from monolayers pretreated with 2OHβ-CD for 2 h. The extracts were layered under a 15–30% continuo
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