| For medical practitioners and the general public - Cholesterol Journal Article Catalog. |
| First Page | Previous Page | Next Page | Last Page |
| Cholesterol depletion and modification of COOH-terminal targeting sequence of the prion protein inhibit formation of the scrapie isoform Taraboulos, A., M. Scott, et al. (1995), J Cell Biol 129(1): 121-32. Abstract: After the cellular prion protein (PrPC) transits to the cell surface where it is bound by a glycophosphatidyl inositol (GPI) anchor, PrPC is either metabolized or converted into the scrapie isoform (PrPSc). Because most GPI-anchored proteins are associated with cholesterol-rich membranous microdomains, we asked whether such structures participate in the metabolism of PrPC or the formation of PrPSc. The initial degradation of PrPC involves removal of the NH2 terminus of PrPC to produce a 17-kD polypeptide which was found in a Triton X-100 insoluble fraction. Both the formation of PrPSc and the initial degradation of PrPC were diminished by lovastatin-mediated depletion of cellular cholesterol but were insensitive to NH4Cl. Further degradation of the 17-kD polypeptide did occur within an NH4Cl-sensitive, acidic compartment. Replacing the GPI addition signal with the transmembrane and cytoplasmic domains of mouse CD4 rendered chimeric CD4PrPC soluble in cold Triton X-100. Both CD4PrPC and truncated PrPC without the GPI addition signal (Rogers, M., F. Yehieley, M. Scott, and S. B. Prusiner. 1993. Proc. Natl. Acad. Sci. USA. 90:3182-3186) were poor substrates for PrPSc formation. Thus, it seems likely that both the initial degradation of PrPC to the 17-kD polypeptide and the formation of PrPSc occur within a non-acidic compartment bound by cholesterol-rich membranes, possibly glycolipid-rich microdomains, where the metabolic fate of PrPC is determined. The pathway remains to be identified by which the 17-kD polypeptide and PrPSc are transported to an acidic compartment, presumably endosomes, where the 17-kD polypeptide is hydrolyzed and limited proteolysis of PrPSc produces PrP 27-30. |
| Cholesterol depletion blocks redistribution of lipid raft components and insulin-mimetic signaling by glimepiride and phosphoinositolglycans in rat adipocytes Muller, G., N. Hanekop, et al. (2002), Mol Med 8(3): 120-36. Abstract: Glycosylphosphatidylinositol-anchored plasma membrane (GPI) proteins, such as Gce1, the dually acylated nonreceptor tyrosine kinases (NRTKs), such as pp59(Lyn), and the membrane protein, caveolin, together with cholesterol are typical components of detergent/carbonate-insoluble glycolipid-enriched raft domains (DIGs) in the plasma membrane of most eucaryotes. Previous studies demonstrated the dissociation from caveolin and concomitant redistribution from DIGs of Gce1 and pp59(Lyn) in rat adipocytes in response to four different insulin-mimetic stimuli, glimepiride, phosphoinositolglycans, caveolin-binding domain peptide, and trypsin/NaCl-treatment. We now characterized the structural basis for this dynamic of DIG components. MATERIALS AND METHODS: Carbonate extracts from purified plasma membranes of basal and stimulated adipocytes were analyzed by high-resolution sucrose gradient centrifugation. RESULTS: This process revealed the existence of two distinct species of detergent/carbonate-insoluble complexes floating at higher buoyant density and harboring lower amounts of cholesterol, caveolin, GPI proteins, and NRTKs (lcDIGs) compared to typical DIGs of high cholesterol content (hcDIGs). The four insulin-mimetic stimuli decreased by 40-70% and increased by 2.5- to 5-fold the amounts of GPI proteins and NRTKs at hcDIGs and lcDIGs, respectively. Cholesterol depletion of adipocytes per se by incubation with methyl-beta-cyclodextrin or cholesterol oxidase also caused translocation of GPI proteins and NRTKs from hcDIGs to lcDIGs and their release from caveolin in reversible fashion without concomitant induction of insulin-mimetic signaling. Cholesterol depletion, however, reduced by 50-60% the stimulus-induced translocation as well as dissociation from hcDIGs-associated caveolin of GPI proteins and NRTKs, activation of NRTKs as well as insulin-mimetic signaling and metabolic action. In contrast, insulin-mimetic signaling induced by vanadium compounds was not significantly diminished by cholesterol depletion. CONCLUSIONS: The data provide evidence that insulin-mimetic signaling in rat adipocytes provoked by glimepiride, phosphoinositolglycans, caveolin-binding domain peptide, and trypsin/NaCl-treatment, but not vanadium compounds, relies on the dynamics of DIGs-the translocation of certain GPI proteins and NRTKs from hcDIGs to lcDIGs mediated by a trypsin/NaCl-sensitive cell surface component. The resultant stimulation of pp59(Lyn) in course of its dissociation from caveolin and incorporation into lcDIGs in combination with an lcDIGs-independent signal seems to substitute for activation of the insulin receptor tyrosine kinase. |
| Cholesterol depletion by methyl-beta-cyclodextrin blocks cholera toxin transport from endosomes to the Golgi apparatus in hippocampal neurons Shogomori, H. and A. H. Futerman (2001), J Neurochem 78(5): 991-9. Abstract: We recently demonstrated that although cholera toxin (CT) is found in detergent-insoluble domains/rafts at the cell surface of cultured hippocampal neurons, it is internalized via a raft-independent mechanism. Thus, cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) did not affect the rate of CT internalization from the plasma membrane, but did affect the rate of CT degradation, which occurs in lysosomes. In the current study, we analyze which step of CT intracellular transport is inhibited by MbetaCD. Whereas pre-incubation with MbetaCD completely blocked CT degradation, it had no effect on the degradation of wheat germ agglutinin (WGA) or bovine serum albumin (BSA), which are internalized by receptor-mediated and fluid phase endocytosis, respectively. Brefeldin A also completely blocked CT degradation but had no effect on WGA or BSA degradation. In contrast, MbetaCD did not affect CT degradation, or CT-mediated cAMP generation, when added to neurons after CT had been transported to the Golgi apparatus. We conclude that CT transport from endosomes to the Golgi apparatus is cholesterol-dependent, whereas CT transport from the Golgi apparatus to lysosomes is cholesterol-independent. |
| Cholesterol depletion by methyl-beta-cyclodextrin enhances myoblast fusion and induces the formation of myotubes with disorganized nuclei Mermelstein, C. S., D. M. Portilho, et al. (2005), Cell Tissue Res 319(2): 289-97. Abstract: The formation of a skeletal muscle fiber begins with the withdrawal of committed mononucleated precursors from the cell cycle. These myoblasts elongate while aligning with each other, guided by recognition between their membranes. This step is followed by cell fusion and the formation of long striated multinucleated myotubes. We used methyl-beta-cyclodextrin (MCD) in primary cultured chick skeletal muscle cells to deplete membrane cholesterol and investigate its role during myogenesis. MCD promoted a significant increase in the expression of troponin T, enhanced myoblast fusion, and induced the formation of large multinucleated myotubes with nuclei being clustered centrally and not aligned at the cell periphery. MCD myotubes were striated, as indicated by sarcomeric alpha-actinin staining, and microtubule and desmin filament distribution was not altered. Pre-fusion MCD-treated myoblasts formed large aggregates, with cadherin and beta-catenin being accumulated in cell adhesion contacts. We also found that the membrane microdomain marker GM1 was not present as clusters in the membrane of MCD-treated myoblasts. Our data demonstrate that cholesterol is involved in the early steps of skeletal muscle differentiation. |
| Cholesterol depletion delocalizes phosphatidylinositol bisphosphate and inhibits hormone-stimulated phosphatidylinositol turnover Pike, L. J. and J. M. Miller (1998), J Biol Chem 273(35): 22298-304. Abstract: Caveolae and detergent-insoluble, glycosphingolipid-enriched domains (DIGs) are cholesterol-enriched membrane domains that have been implicated in signal transduction because a variety of signaling proteins as well as phosphatidylinositol bisphosphate (PtdInsP2) are compartmentalized in these domains. We report here that depletion of cellular cholesterol leads to the inhibition of epidermal growth factor- and bradykinin-stimulated PtdIns turnover in A431 cells. This is associated with the loss of compartmentalization of epidermal growth factor receptors, Gq, and PtdInsP2 in the low density membrane domains. Replacement of cellular cholesterol leads to the reorganization of signaling molecules in the low density domains and the reestablishment of hormone-stimulated PtdIns hydrolysis. Oxysterol derivatives show a variable ability to functionally replace the cholesterol in this system. These data are consistent with the hypothesis that localization of signaling proteins and lipids to cholesterol-enriched domains is required for the proper function of hormone-stimulated PtdIns turnover. |
| Cholesterol depletion disrupts caveolae and differentially impairs agonist-induced arterial contraction Dreja, K., M. Voldstedlund, et al. (2002), Arterioscler Thromb Vasc Biol 22(8): 1267-72. Abstract: OBJECTIVE: This study assessed the role of cholesterol-rich membrane regions, including caveolae, in the regulation of arterial contractility. Methods and Results- Rat tail artery devoid of endothelium was treated with the cholesterol acceptor methyl-beta-cyclodextrin, and the effects on force and Ca2+ handling were evaluated. In cholesterol-depleted preparations, the force responses to alpha1-adrenergic receptors, membrane depolarization, inhibition of myosin light chain phosphatase, and activation of G proteins with a mixture of 20 mmol/L NaF and 60 micro mol/L AlCl3 were unaffected. In contrast, responses to 5-hydroxytryptamine (5-HT), vasopressin, and endothelin were reduced by >50%. The rise in global intracellular free Ca2+ concentration in response to 5-HT was attenuated, as was the generation of Ca2+ waves at the cellular level. By electron microscopy, cholesterol depletion was found to disrupt caveolae. The 5-HT response could be restored by exogenous cholesterol, which also restored caveolae. Western blots showed that the levels of 5-HT2A receptor and of caveolin-1 were unaffected by cholesterol extraction. Sucrose gradient centrifugation showed enrichment of 5-HT2A receptors, but not alpha1-adrenergic receptors, in the caveolin-1-containing fractions, suggesting localization of the former to caveolae. CONCLUSIONS: These results show that a subset of signaling pathways that regulate smooth muscle contraction depends specifically on cholesterol. Furthermore, the cholesterol-dependent step in serotonergic signaling occurs early in the pathway and depends on the integrity of caveolae. |
| Cholesterol depletion disrupts caveolae and insulin receptor signaling for metabolic control via insulin receptor substrate-1, but not for mitogen-activated protein kinase control Parpal, S., M. Karlsson, et al. (2001), J Biol Chem 276(13): 9670-8. Abstract: Insulin exerts its cellular control through receptor binding in caveolae in plasmalemma of target cells (Gustavsson, J., Parpal, S., Karlsson, M., Ramsing, C., Thorn, H., Borg, M., Lindroth, M., Peterson, K. H., Magnusson, K.-E., and Stralfors, P. (1999) FASEB. J. 13, 1961-1971). We now report that a progressive cholesterol depletion of 3T3-L1 adipocytes with beta-cyclodextrin gradually destroyed caveolae structures and concomitantly attenuated insulin stimulation of glucose transport, in effect making cells insulin-resistant. Insulin access to or affinity for the insulin receptor on rat adipocytes was not affected as determined by (125)I-insulin binding. By immunoblotting of plasma membranes, total amount of insulin receptor and of caveolin remained unchanged. Receptor autophosphorylation in response to insulin was not affected by cholesterol depletion. Insulin treatment of isolated caveolae preparations increased autophosphorylation of receptor before and following cholesterol depletion. Insulin-increased tyrosine phosphorylation of an immediate downstream signal transducer, insulin receptor substrate-1, and activation of the further downstream protein kinase B were inhibited. In contrast, insulin signaling to mitogenic control as determined by control of the extracellular signal-related kinases 1/2, mitogen-activated protein kinase pathway was not affected. Insulin did not control Shc phosphorylation, and Shc did not control extracellular signal-related kinases 1/2, whereas cholesterol depletion constitutively phosphorylated Shc. In conclusion, caveolae are critical for propagating the insulin receptor signal to downstream targets and have the potential for sorting signal transduction for metabolic and mitogenic effects. |
| Cholesterol depletion disrupts lipid rafts and modulates the activity of multiple signaling pathways in T lymphocytes Kabouridis, P. S., J. Janzen, et al. (2000), Eur J Immunol 30(3): 954-63. Abstract: Lipid rafts are specialized plasma membrane microdomains, in which glycosphingolipids and cholesterol are major structural components. In T lymphocytes, several signaling proteins are associated with lipid rafts including the protein tyrosine kinase LCK and the adapter protein LAT. To investigate their importance in T cell signaling, lipid rafts were disrupted by depleting cholesterol with methyl-beta-cyclodextrin (MbetaCD). This transiently induced tyrosine phosphorylation of multiple proteins, including the ZAP-70 tyrosine kinase, its associated T cell antigen receptor zeta chain, LAT and phospholipase Cgamma1. Tyrosine phosphorylation was dependent on expression of LCK in lipid rafts. Depletion of cholesterol also resulted in activation of the Ras-ERK pathway. This was largely dependent on phorbol ester-sensitive protein kinase C (PKC) and the PKC-theta isoform translocated to the plasma membrane following MbetaCD treatment. MbetaCD did not stimulate intracellular Ca2+ fluxes; however, consistent with its ability to stimulate Ras, MbetaCD synergized with a Ca2+ ionophore to induce formation of the transcription factor NF-AT. These data indicate a crucial role for cholesterol in the regulation of signaling pathways in T cells, which is likely to reflect its importance in the formation of plasma membrane lipid rafts. |
| Cholesterol depletion from the plasma membrane triggers ligand-independent activation of the epidermal growth factor receptor Chen, X. and M. D. Resh (2002), J Biol Chem 277(51): 49631-7. Abstract: We recently demonstrated that depletion of plasma membrane cholesterol with methyl-beta-cyclodextrin (MbetaCD) caused activation of MAPK (Chen, X., and Resh, M. D. (2001) J. Biol. Chem. 276, 34617-34623). MAPK activation was phosphatidylinositol 3-kinase (PI3K)-dependent and involved increased tyrosine phosphorylation of the p85 subunit of PI3K. We next determined whether MbetaCD treatment induced tyrosine phosphorylation of other cellular proteins. Here we report that cholesterol depletion of serum-starved COS-1 cells with MbetaCD or filipin caused an increase in Tyr(P) levels of a 180-kDa protein that was identified as the epidermal growth factor receptor (EGFR). Cross-linking experiments showed that MbetaCD induced dimerization of EGFR, indicative of receptor activation. Reagents that block release of membrane-bound EGFR ligands did not affect MbetaCD-induced tyrosine phosphorylation of EGFR, indicating that MbetaCD activation of EGFR is ligand-independent. Moreover, MbetaCD treatment resulted in increased tyrosine phosphorylation of EGFR downstream targets and Ras activation. Incubation of cells with the specific EGFR inhibitor AG4178 blocked MbetaCD-induced phosphorylation of EGFR, SHC, phospholipase C-gamma, and Gab-1 as well as MAPK activation. We conclude that cholesterol depletion from the plasma membrane by MbetaCD causes ligand-independent activation of EGFR, resulting in MAPK activation by PI3K and Ras-dependent mechanisms. Moreover, these studies reveal a novel mode of action of MbetaCD, in addition to its ability to disrupt membrane rafts. |
| Cholesterol depletion impairs vascular reactivity to endothelin-1 by reducing store-operated Ca2+ entry dependent on TRPC1 Bergdahl, A., M. F. Gomez, et al. (2003), Circ Res 93(9): 839-47. Abstract: The reactivity of the vascular wall to endothelin-1 (ET-1) is influenced by cholesterol, which is of possible importance for the progression of atherosclerosis. To elucidate signaling steps affected, the cholesterol acceptor methyl-beta-cyclodextrin (mbetacd, 10 mmol/L) was used to manipulate membrane cholesterol and disrupt caveolae in intact rat arteries. In endothelium-denuded caudal artery, contractile responsiveness to 10 nmol/L ET-1 (mediated by the ETA receptor) was reduced by mbetacd and increased by cholesterol. Neither ligand binding nor colocalization of ETA and caveolin-1 was affected by mbetacd. Ca2+ inflow via store-operated channels after depletion of intracellular Ca2+ stores was reduced in mbetacd-treated caudal arteries, as shown by Mn2+ quench rate and intracellular Ca2+ response. Expression of TRPC1, 3, and 6 was detected by reverse transcriptase-polymerase chain reaction, and colocalization of TRPC1 with caveolin-1 was reduced by mbetacd, as seen by immunofluorescence. Part of the contractile response to ET-1 was inhibited by Ni2+ (0.5 mmol/L) and by a TRPC1 blocking antibody. In the basilar artery, exhibiting less store-operated channel activity than the caudal artery, ET-1-induced contractions were insensitive to the TRPC1 blocking antibody and to mbetacd. Increased store-operated channel activity in basilar arteries after organ culture correlated with increased sensitivity of ET-1 contraction to mbetacd. These results suggest that cholesterol influences vascular reactivity to ET-1 by affecting the caveolar localization of TRPC1. |
| Cholesterol depletion increases membrane stiffness of aortic endothelial cells Byfield, F. J., H. Aranda-Espinoza, et al. (2004), Biophys J 87(5): 3336-43. Abstract: This study has investigated the effect of cellular cholesterol on membrane deformability of bovine aortic endothelial cells. Cellular cholesterol content was depleted by exposing the cells to methyl-beta-cyclodextrin or enriched by exposing the cells to methyl-beta-cyclodextrin saturated with cholesterol. Control cells were treated with methyl-beta-cyclodextrin-cholesterol at a molar ratio that had no effect on the level of cellular cholesterol. Mechanical properties of the cells with different cholesterol contents were compared by measuring the degree of membrane deformation in response to a step in negative pressure applied to the membrane by a micropipette. The experiments were performed on substrate-attached cells that maintained normal morphology. The data were analyzed using a standard linear elastic half-space model to calculate Young elastic modulus. Our observations show that, in contrast to the known effect of cholesterol on membrane stiffness of lipid bilayers, cholesterol depletion of bovine aortic endothelial cells resulted in a significant decrease in membrane deformability and a corresponding increase in the value of the elastic coefficient of the membrane, indicating that cholesterol-depleted cells are stiffer than control cells. Repleting the cells with cholesterol reversed the effect. An increase in cellular cholesterol to a level higher than that of normal cells, however, had no effect on the elastic properties of bovine aortic endothelial cells. We also show that although cholesterol depletion had no apparent effect on the intensity of F-actin-specific fluorescence, disrupting F-actin with latrunculin A abrogated the stiffening effect. We suggest that cholesterol depletion increases the stiffness of the membrane by altering the properties of the submembrane F-actin and/or its attachment to the membrane. |
| Cholesterol depletion induces large scale domain segregation in living cell membranes Hao, M., S. Mukherjee, et al. (2001), Proc Natl Acad Sci U S A 98(23): 13072-7. Abstract: Local inhomogeneities in lipid composition play a crucial role in regulation of signal transduction and membrane traffic. Nevertheless, most evidence for microdomains in cells remains indirect, and the nature of membrane inhomogeneities has been difficult to characterize. We used lipid analogs and lipid-anchored proteins with varying fluidity preferences to examine the effect of modulating cellular cholesterol on domain formation. We show that lowering cholesterol levels induces formation of visible micrometer-scale domains in the plasma membrane of several mammalian cell types with complementary distributions of fluorescent lipid analogs with preferences for fluid or ordered domains. A uniform distribution is restored by cholesterol repletion. Unexpectedly, cholesterol depletion does not visibly alter the distribution of a crosslinked or uncrosslinked glycosylphosphatidylinositol-anchored protein (the folate receptor). We also examined the effect of varying cholesterol content on the cold Triton X-100 solubility of several membrane constituents. Although a cholesterol analog, dehydroergosterol, and a glycosylphosphatidylinositol-anchored protein are largely retained after extraction, a lipid analog with saturated 16-carbon acyl chains is largely removed when the cellular cholesterol level is lowered. This result indicates that after cholesterol depletion molecules in the more ordered domains can be extracted differentially by cold nonionic detergents. |
| Cholesterol depletion induces PKA-mediated basolateral-to-apical transcytosis of the scavenger receptor class B type I in MDCK cells Burgos, P. V., C. Klattenhoff, et al. (2004), Proc Natl Acad Sci U S A 101(11): 3845-50. Abstract: Cholesterol-based membrane microdomains, or lipid rafts, are believed to play important, yet poorly defined, roles in protein trafficking and signal transduction. In polarized epithelial cells, the current view is that rafts are involved in apical but not in basolateral protein transport from the trans-Golgi network (TGN). We report here that cholesterol is required in a post-TGN mechanism of basolateral regionalization. Permanently transfected Madin-Darby canine kidney cells segregated the caveolae/raft-associated high-density lipoprotein scavenger receptor class B type I (SR-BI) predominantly to the basolateral domain where it was constitutively internalized and recycled basolaterally. Acute cholesterol depletion did not significantly alter SR-BI internalization, implying a cholesterol depletion-insensitive endocytic process but instead induced its transcytosis through a protein kinase A (PKA)- and microtubule-dependent mechanism. Forskolin also elicited SR-BI transcytosis. The basolateral distribution of endogenous epidermal growth factor receptor remained unaffected. Strikingly, cholesterol depletion induced PKA activity without increasing the cAMP levels. Thus, our results are consistent with a scenario in which cholesterol-based rafts promote internalization and basolateral recycling of internalized SR-BI whereas a PKA pool sensitive to cholesterol depletion mediates SR-BI transcytosis. Regulated transcytosis of SR-BI may provide an additional mechanism to control cholesterol homeostasis. These results disclose relationships between cholesterol-based rafts and PKA activity operating in a post-TGN mechanism of regulated apical-to-basolateral cell surface protein distribution. |
| Cholesterol depletion inhibits epidermal growth factor receptor transactivation by angiotensin II in vascular smooth muscle cells: role of cholesterol-rich microdomains and focal adhesions in angiotensin II signaling Ushio-Fukai, M., L. Hilenski, et al. (2001), J Biol Chem 276(51): 48269-75. Abstract: Angiotensin II (Ang II) induces transactivation of the epidermal growth factor (EGF) receptor (EGF-R), which serves as a scaffold for various signaling molecules in vascular smooth muscle cells (VSMCs). Cholesterol and sphingomyelin-enriched lipid rafts are plasma membrane microdomains that concentrate various signaling molecules. Caveolae are specialized lipid rafts that are organized by the cholesterol-binding protein, caveolin, and have been shown to be associated with EGF-Rs. Angiotensin II stimulation promotes a rapid movement of AT(1) receptors to caveolae; however, their functional role in angiotensin II signaling has not been elucidated. Here we show that cholesterol depletion by beta-cyclodextrin disrupts caveolae structure and concomitantly inhibits tyrosine phosphorylation of the EGF-R and subsequent activation of protein kinase B (PKB)/Akt induced by angiotensin II. Similar inhibitory effects were obtained with other cholesterol-binding agents, filipin and nystatin. In contrast, EGF-R autophosphorylation and activation of Akt/PKB in response to EGF are not affected by cholesterol depletion. The early Ang II-induced upstream signaling events responsible for transactivation of the EGF-R, such as the intracellular Ca(2+) increase and c-Src activation, also remain intact. The EGF-R initially binds caveolin, but these two proteins rapidly dissociate following angiotensin II stimulation during the time when EGF-R transactivation is observed. The activated EGF-R is localized in focal adhesions together with tyrosine-phosphorylated caveolin. These findings suggest that 1) a scaffolding role of caveolin is essential for EGF-R transactivation by angiotensin II and 2) cholesterol-rich microdomains as well as focal adhesions are important signal-organizing compartments required for the spatial and temporal organization of angiotensin II signaling in VSMCs. |
| Cholesterol depletion inhibits store-operated calcium currents and exocytotic membrane fusion in RBL-2H3 cells Kato, N., M. Nakanishi, et al. (2003), Biochemistry 42(40): 11808-14. Abstract: The effects of cholesterol depletion from the plasma membrane with methyl-beta-cyclodextrin (MbetaCD) on exocytotic processes were investigated in rat basophil leukemia cells (RBL-2H3 cells). Pretreatment of the cells with MbetaCD inhibited antigen-evoked exocytotic release dose-dependently. To elucidate the mechanism of this inhibition, we performed experiments on the effects of MbetaCD on exocytotic membrane fusion and mobilization of Ca(2+) and on the localization of the tyrosine kinase Lyn. Inhibition of degranulation by MbetaCD was observed even under stimulation with the phorbol ester and calcium ionophore. Therefore, MbetaCD affected a process downstream of Ca(2+) influx, or membrane fusion between the granule and the plasma membrane. Intracellular calcium measurements revealed that MbetaCD inhibited the Ca(2+) increase induced by antigen. Furthermore, we found that MbetaCD significantly inhibited Ca(2+) influx from the extracellular medium through the store-operated calcium channel (SOC) but did not affect Ca(2+) release from the intracellular Ca(2+) store. Fluorescent image analysis of cells expressing Lyn-YFP showed that treatment with MbetaCD scarcely affected the localization and lateral mobility of Lyn in the plasma membrane. These results suggest that cholesterol depletion by MbetaCD decreases degranulation mainly by inhibiting the SOC and membrane fusion between the secretory granules and the plasma membrane in mast cells. |
| Cholesterol depletion inhibits the generation of beta-amyloid in hippocampal neurons Simons, M., P. Keller, et al. (1998), Proc Natl Acad Sci U S A 95(11): 6460-4. Abstract: The amyloid precursor protein (APP) plays a crucial role in the pathogenesis of Alzheimer's disease. During intracellular transport APP undergoes a series of proteolytic cleavages that lead to the release either of an amyloidogenic fragment called beta-amyloid (Abeta) or of a nonamyloidogenic secreted form consisting of the ectodomain of APP (APPsec). It is Abeta that accumulates in the brain lesions that are thought to cause the disease. By reducing the cellular cholesterol level of living hippocampal neurons by 70% with lovastatin and methyl-beta-cyclodextrin, we show that the formation of Abeta is completely inhibited while the generation of APPsec is unperturbed. This inhibition of Abeta formation is accompanied by increased solubility in the detergent Triton X-100 and is fully reversible by the readdition of cholesterol to previously depleted cells. Our results show that cholesterol is required for Abeta formation to occur and imply a link between cholesterol, Abeta, and Alzheimer's disease. |
| Cholesterol depletion of caveolae causes hyperactivation of extracellular signal-related kinase (ERK) Furuchi, T. and R. G. Anderson (1998), J Biol Chem 273(33): 21099-104. Abstract: Previously we showed that activation of Erk in quiescent cells occurs in the caveolae fraction isolated from fibroblasts. Since the structure and function of caveolae is sensitive to the amount of cholesterol in the membrane, it might be that a direct link exists between the concentration of membrane cholesterol and mitogen-activated protein (MAP) kinase activation. We acutely lowered the cholesterol level of the caveolae fraction by incubating Rat-1 cells in the presence of either cyclodextrin or progesterone. Cholesterol-depleted caveolae had a reduced amount of several key protein components of the MAP kinase complex, including Ras, Grb2, Erk2, and Src. Incubation of these cells in the presence of epidermal growth factor (EGF) caused a rapid loss of EGF receptor from the caveolae fraction, but the usual recruitment of c-Raf was markedly inhibited. Despite the reduced amount of c-Raf and Erk2 in the cholesterol-depleted caveolae fraction, EGF caused a hyperactivation of the remaining caveolae Erk isoenzymes. This was followed by an increase in the amount of active Erk in the cytoplasm. The increased amount of activated Erk produced under these conditions was linked to a 2-fold higher level of EGF-stimulated DNA synthesis. Even cholesterol depletion by itself stimulated Erk activation and DNA synthesis. These results suggest that the MAP kinase pathway can connect the cholesterol level of caveolae membrane to the control of cell division. |
| Cholesterol depletion of enterocytes. Effect on the Golgi complex and apical membrane trafficking Hansen, G. H., L. L. Niels-Christiansen, et al. (2000), J Biol Chem 275(7): 5136-42. Abstract: Intestinal brush border enzymes, including aminopeptidase N and sucrase-isomaltase, are associated with "rafts" (membrane microdomains rich in cholesterol and sphingoglycolipids). To assess the functional role of rafts in the present work, we studied the effect of cholesterol depletion on apical membrane trafficking in enterocytes. Cultured mucosal explants of pig small intestine were treated for 2 h with the cholesterol sequestering agent methyl-beta-cyclodextrin and lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase. The treatment reduced the cholesterol content >50%. Morphologically, the Golgi complex/trans-Golgi network was partially transformed into numerous 100-200 nm vesicles. By immunogold electron microscopy, aminopeptidase N was localized in these Golgi-derived vesicles as well as at the basolateral cell surface, indicating a partial missorting. Biochemically, the rates of the Golgi-associated complex glycosylation and association with rafts of newly synthesized aminopeptidase N were reduced, and less of the enzyme had reached the brush border membrane after 2 h of labeling. In contrast, the basolateral Na(+)/K(+)-ATPase was neither missorted nor raft-associated. Our results implicate the Golgi complex/trans-Golgi network in raft formation and suggest a close relationship between this event and apical membrane trafficking. |
| Cholesterol depletion of human immunodeficiency virus type 1 and simian immunodeficiency virus with beta-cyclodextrin inactivates and permeabilizes the virions: evidence for virion-associated lipid rafts Graham, D. R., E. Chertova, et al. (2003), J Virol 77(15): 8237-48. Abstract: Recent evidence suggests that human immunodeficiency virus type 1 (HIV-1) particles assemble and bud selectively through areas in the plasma membrane of cells that are highly enriched with glycosylphosphatidylinositol-anchored proteins and cholesterol, called lipid rafts. Since cholesterol is required to maintain lipid raft structure and function, we proposed that virion-associated cholesterol removal with the compound 2-hydroxy-propyl-beta-cyclodextrin (beta-CD) might be disruptive to HIV-1 and simian immunodeficiency virus (SIV). We examined the effect of beta-CD on the structure and infectivity of cell-free virions. We found that beta-CD inactivated HIV-1 and SIV in a dose-dependent manner and permeabilized the viral membranes, resulting in the loss of mature Gag proteins (capsid, matrix, nucleocapsid, p1, and p6) without loss of the envelope glycoproteins. SIV also lost reverse transcriptase (RT), integrase (IN), and viral RNA. IN appeared to be only slightly diminished in HIV-1, and viral RNA, RT, matrix, and nucleocapsid proteins were retained in HIV-1 but to a much lesser degree. Host proteins located internally in the virus (actin, moesin, and ezrin) and membrane-associated host proteins (major histocompatibility complex classes I and II) remained associated with the treated virions. Electron microscopy revealed that under conditions that permeabilized the viruses, holes were present in the viral membranes and the viral core structure was perturbed. These data provide evidence that an intact viral membrane is required to maintain mature virion core integrity. Since the viruses were not fixed before beta-CD treatment and intact virion particles were recovered, the data suggest that virions may possess a protein scaffold that can maintain overall structure despite disruptions in membrane integrity. |
| Cholesterol depletion perturbs calcium handling by rat submandibular glands Garcia-Marcos, M., S. Tandel, et al. (2005), J Cell Physiol 203(2): 429-38. Abstract: The sensitivity to cholesterol depletion of calcium handling by rat submandibular glands was investigated. The glands were digested with collagenase. After homogenization, the lysate was extracted at 4 degrees C with 0.5% Triton X-100 and the extract was submitted to an ultracentrifugation in a sucrose discontinuous gradient. A population of detergent-resistant membranes (DRM) was collected at the 5%-35% interface. The DRM had a higher content of cholesterol, saturated and long-chain fatty acids. Caveolin-1 and alpha(q/11) were located in these membranes. They were more ordered than vesicles from total cellular lysate as determined by anisotropy measurement. They disappeared after cholesterol extraction with methyl-beta-cyclodextrin (MCD). Exposure of the cellular suspension with MCD nearly abolished the response to carbachol, epinephrine, and substance P and inhibited the activation of phospholipase C (PLC) by these agonists and by sodium fluoride. MCD did not affect the mobilization of intracellular pools of calcium by thapsigargin. It increased the uptake of extracellular calcium or barium and did not inhibit the uptake of calcium after depletion of the intracellular stores of this ion. From these results, it is concluded that Triton X-100 can extract a fraction of membrane resistant to detergents. Treatment of the cells with MCD disrupts these membranes. The coupling between the heterotrimeric GTP-binding protein G(q/11) and poly-phosphoinositide-specific PLC is affected by disruption of these membrane fractions. At the opposite, the store-operated calcium channel (SOCC) is not affected by DRM-disruption. |