Concentrations Of MβCD Research Articles

INVERSE EXPRESSION BETWEEN TGF-β1 AND TWIST IN ORAL SQUAMOUS CELL CARCINOMA CELL LINE AFTER CHOLESTEROL DEPLETION

Objectives To evaluate the transforming growht factor-β1 and twist protein expression in oral squamous cell carcinoma (OSCC) cell line SCC-9 after cholesterol depletion with methyl-β-cyclodextrin (MβCD). Study Design SCC-9 cells were treated with MβCD at 7.5, 10, and 15 mM concentrations over 1 hour. After 24 hours of depletion, the gene expression of TGF-β1 and TWIST was accessed by TaqMan RT-qPCR. Results The TGF-β1 gene expression in SCC-9 treated by different concentrations of MβCD was upregulated upon cholesterol depletion at 7.5 mM compared to untreated SCC-9 (P = .06, 1-way analysis of variance [ANOVA]). Conversely, TWIST gene expression was uniform through the different concentrations of MβCD (P = .9, 1-way ANOVA). A significant difference between expressions of TGF-β1 treated with 7.5 mM (mean value = 1.9) and TWIST treated with 7.5 mM (mean value = 0.9) and 10 mM (mean value = 1.0) was noted (P = .01, 1-way ANOVA). No correlation was observed in the expression of TGF-β1 and TWIST in each concentration of MβCD (Pearson's correlation analysis). Conclusions Cholesterol depletion may affect TGF-β1 and TWIST expression. This finding should be associated with others related to epithelial-mesenchymal transition inducers, due to their potential in promoting carcinogenesis, in order to better understand OSCC biology. To evaluate the transforming growht factor-β1 and twist protein expression in oral squamous cell carcinoma (OSCC) cell line SCC-9 after cholesterol depletion with methyl-β-cyclodextrin (MβCD). SCC-9 cells were treated with MβCD at 7.5, 10, and 15 mM concentrations over 1 hour. After 24 hours of depletion, the gene expression of TGF-β1 and TWIST was accessed by TaqMan RT-qPCR. The TGF-β1 gene expression in SCC-9 treated by different concentrations of MβCD was upregulated upon cholesterol depletion at 7.5 mM compared to untreated SCC-9 (P = .06, 1-way analysis of variance [ANOVA]). Conversely, TWIST gene expression was uniform through the different concentrations of MβCD (P = .9, 1-way ANOVA). A significant difference between expressions of TGF-β1 treated with 7.5 mM (mean value = 1.9) and TWIST treated with 7.5 mM (mean value = 0.9) and 10 mM (mean value = 1.0) was noted (P = .01, 1-way ANOVA). No correlation was observed in the expression of TGF-β1 and TWIST in each concentration of MβCD (Pearson's correlation analysis). Cholesterol depletion may affect TGF-β1 and TWIST expression. This finding should be associated with others related to epithelial-mesenchymal transition inducers, due to their potential in promoting carcinogenesis, in order to better understand OSCC biology.

Membrane cholesterol regulates endocytosis and trafficking of the serotonin1A receptor: Insights from acute cholesterol depletion

Endocytosis and intracellular trafficking constitute important regulatory features associated with G protein-coupled receptor (GPCR) function. GPCR endocytosis involves several remodeling events at the plasma membrane orchestrated by a concerted interplay of a large number of proteins and membrane lipids. Although considerable literature exists on the protein framework underlying GPCR endocytosis, the role of membrane lipids in this process remains largely unexplored. In order to explore the role of membrane cholesterol (an essential and important lipid in higher eukaryotes) in GPCR endocytosis, we monitored the effect of acute cholesterol depletion using methyl-β-cyclodextrin (MβCD) on endocytosis and intracellular trafficking of the serotonin1A receptor, an important neurotransmitter GPCR. Our results show that the serotonin1A receptor exhibits agonist-induced clathrin-mediated endocytosis with a concentration-dependent inhibition in internalization with increasing concentrations of MβCD, which was restored upon cholesterol replenishment. Interestingly, subsequent to internalization under these conditions, serotonin1A receptors were re-routed toward lysosomal degradation, instead of endosomal recycling observed under normal conditions, thereby implicating membrane cholesterol in modulation of intracellular trafficking of the receptor. This raises the possibility of a novel cholesterol-dependent role of intracellular sorting proteins in GPCR trafficking. These results differ from our previous observations on the endocytosis of the serotonin1A receptor upon statin-induced chronic cholesterol depletion, in terms of endocytic pathway. We conclude that analysis of complex cellular trafficking events such as GPCR endocytosis under acute and chronic cholesterol depletion conditions should be carried out with caution due to fundamental differences underlying these processes.

Membrane Fluidity Sensing on the Single Virus Particle Level with Plasmonic Nanoparticle Transducers.

Viral membranes are nanomaterials whose fluidity depends on their composition, in particular, the cholesterol (chol) content. As differences in the membrane composition of individual virus particles can lead to different intracellular fates, biophysical tools capable of sensing the membrane fluidity on the single-virus level are required. In this manuscript, we demonstrate that fluctuations in the polarization of light scattered off gold or silver nanoparticle (NP)-labeled virus-like-particles (VLPs) encode information about the membrane fluidity of individual VLPs. We developed plasmonic polarization fluctuation tracking microscopy (PFTM) which facilitated the investigation of the effect of chol content on the membrane fluidity and its dependence on temperature, for the first time on the single-VLP level. Chol extraction studies with different methyl-β-cyclodextrin (MβCD) concentrations yielded a gradual decrease in polarization fluctuations as a function of time. The rate of chol extraction for individual VLPs showed a broad spread, presumably due to differences in the membrane composition for the individual VLPs, and this heterogeneity increased with decreasing MβCD concentration.

Experimental Analysis of Interacting HT22 Plasma Membrane Cholesterol and β-Amyloid

The peptide β-Amyloid (β-A) is known to be one of the primary factors causing neurodegeneration in the Alzheimer disease. Hence, one would like to know the factors that would increase or decrease the toxicity of β-Amyloid in the brain. One of the factors that are debated in the literature is cholesterol, where it is not clear if modulating the levels of cholesterol would affect the degree of toxicity of β-Amyloid on neuron cells in the brain. In order to investigate this problem, data were collected and analyzed for three types of experiments: 1) Correspondence between cholesterol and methyl-β-cyclodextrin (MβCD) measurements; 2) measurements of the relative fluorescence unit (RFU) with respect to MβCD concentration (with/without β-A); and 3) RFU measurements with respect to β-A concentration (with/without MβCD). HT22 hippocampal neurons immortalized with the simian virus SV-40 large T-antigen plasmid vector were used to conduct the experiments. Mito-ID Membrane potential cytotoxicity was used as a measure of mitochondrial potential change. The statistical analysis of the presented experimental results indicates that cholesterol has no statistically significant effect on the degree of toxicity of β-Amyloid.

Kinetic Analysis of the Methyl-β-cyclodextrin-Mediated Intervesicular Transfer of Pyrene-Labeled Phospholipids.

Methyl-β-cyclodextrin (MβCD) can transfer phospholipids between vesicles, and its transfer ability has been utilized for the preparation of asymmetric vesicle and lipid incorporation into culture cells. Nevertheless, a detailed kinetic analysis of the MβCD-mediated phospholipid transfer has not yet been carried out. We performed real-time monitoring of intervesicular lipid transfer by means of the fluorescence of pyrene-labeled phospholipids. Intermolecular excimer formation of the pyrene-labeled lipids in a membrane strongly depends on the local concentration of the fluorophore and decreases when the pyrene-labeled lipids are transferred from donor (fluorophore-containing) vesicles to acceptor (fluorophore-free) vesicles. We monitored the fluorescence intensity of the pyrene monomer and excimer simultaneously and found that the excimer/monomer ratio decreased in the presence of MβCD, pointing to MβCD-mediated lipid transfer. The transfer rate depended on the MβCD concentration but not on the lipid concentration, suggesting that dissociation from the membrane via extraction by MβCD is the rate-limiting step of the lipid transfer. Calibration of the excimer/monomer ratio to the molar fraction of the pyrene-labeled lipids enabled us to evaluate the dissociation rate constant correctly. From the temperature dependence of the transfer, we obtained the thermodynamic activation parameters, which revealed that the extraction of phosphatidylcholine by MβCD from membranes is less enthalpically unfavorable than that of phosphatidylglycerol and phosphatidylinositol.

The cholesterol-lowering agent methyl-β-cyclodextrin promotes glucose uptake via GLUT4 in adult muscle fibers and reduces insulin resistance in obese mice.

Insulin stimulates glucose uptake in adult skeletal muscle by promoting the translocation of GLUT4 glucose transporters to the transverse tubule (T-tubule) membranes, which have particularly high cholesterol levels. We investigated whether T-tubule cholesterol content affects insulin-induced glucose transport. Feeding mice a high-fat diet (HFD) for 8 wk increased by 30% the T-tubule cholesterol content of triad-enriched vesicular fractions from muscle tissue compared with triads from control mice. Additionally, isolated muscle fibers (flexor digitorum brevis) from HFD-fed mice showed a 40% decrease in insulin-stimulated glucose uptake rates compared with fibers from control mice. In HFD-fed mice, four subcutaneous injections of MβCD, an agent reported to extract membrane cholesterol, improved their defective glucose tolerance test and normalized their high fasting glucose levels. The preincubation of isolated muscle fibers with relatively low concentrations of MβCD increased both basal and insulin-induced glucose uptake in fibers from controls or HFD-fed mice and decreased Akt phosphorylation without altering AMPK-mediated signaling. In fibers from HFD-fed mice, MβCD improved insulin sensitivity even after Akt or CaMK II inhibition and increased membrane GLUT4 content. Indinavir, a GLUT4 antagonist, prevented the stimulatory effects of MβCD on glucose uptake. Addition of MβCD elicited ryanodine receptor-mediated calcium signals in isolated fibers, which were essential for glucose uptake. Our findings suggest that T-tubule cholesterol content exerts a critical regulatory role on insulin-stimulated GLUT4 translocation and glucose transport and that partial cholesterol removal from muscle fibers may represent a useful strategy to counteract insulin resistance.

In vitro drug release of natamycin from β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin-functionalized contact lens materials

Purpose: The antifungal agent natamycin can effectively form inclusion complexes with beta-cyclodextrin (β-CD) and 2-hydroxypropyl-β-cyclodextrin (HP-βCD) to improve the water solubility of natamycin by 16-fold and 152-fold, respectively (Koontz, J. Agric. Food. Chem. 2003). The purpose of this study was to develop contact lens materials functionalized with methacrylated β-CD (MβCD) and methacrylated HP-βCD (MHP-βCD), and to evaluate their ability to deliver natamycin in vitro. Methods: Model conventional hydrogel (CH) materials were synthesized by adding varying amounts of MβCD and MHP-βCD (0, 0.22, 0.44, 0.65, 0.87, 1.08% of total monomer weight) to a monomer solution containing 2-hydroxyethyl methacrylate (HEMA). Model silicone hydrogel (SH) materials were synthesized by adding similar concentrations of MβCD and MHP-βCD to N,N-dimethylacrylamide (DMAA)/10% 3-methacryloxypropyltris(trimethylsiloxy)silane (TRIS). The gels were cured with UV light, washed with ethanol and then, hydrated for 24 h (h). The model materials were then incubated with 2 mL of 100 μg/mL of natamycin in phosphate buffered saline (PBS) pH 7.4 for 48 h at room temperature. The release of natamycin from these materials in 2 mL of PBS, pH 7.4 at 32 ± 2 °C was monitored using UV–vis spectrophotometry at 304 nm over 24 h. Results: For both CH and SH materials, functionalization with MβCD and MHP-βCD improved the total amount of drugs released up to a threshold loading concentration, after which further addition of methacrylated CDs decreased the amount of drugs released (p < 0.05). The addition of CDs did not extend the drug release duration; the release of natamycin by all model materials reached a plateau after 12 h (p < 0.05). Overall, DMAA/10% TRIS materials released significantly more drug than HEMA materials (p < 0.05). The addition of MHP-βCD had a higher improvement in drug release than MβCD for both HEMA and DMAA/10% TRIS gels (p < 0.05). Conclusions: A high loading concentration of methacrylated CDs decreases overall drug delivery efficiency, which likely results from an unfavorable arrangement of the CDs within the polymer network leading to reduced binding of natamycin to the CDs. HEMA and DMAA/10% TRIS materials functionalized with MHP-βCD are more effective than those functionalized with MβCD to deliver natamycin.

Sarcolemmal cholesterol and caveolin-3 dependence of cardiac function, ischemic tolerance, and opioidergic cardioprotection.

Cholesterol-rich caveolar microdomains and associated caveolins influence sarcolemmal ion channel and receptor function and protective stress signaling. However, the importance of membrane cholesterol content to cardiovascular function and myocardial responses to ischemia-reperfusion (I/R) and cardioprotective stimuli are unclear. We assessed the effects of graded cholesterol depletion with methyl-β-cyclodextrin (MβCD) and lifelong knockout (KO) or overexpression (OE) of caveolin-3 (Cav-3) on cardiac function, I/R tolerance, and opioid receptor (OR)-mediated protection. Langendorff-perfused hearts from young male C57Bl/6 mice were untreated or treated with 0.02-1.0 mM MβCD for 25 min to deplete membrane cholesterol and disrupt caveolae. Hearts were subjected to 25-min ischemia/45-min reperfusion, and the cardioprotective effects of morphine applied either acutely or chronically [sustained ligand-activated preconditioning (SLP)] were assessed. MβCD concentration dependently reduced normoxic contractile function and postischemic outcomes in association with graded (10-30%) reductions in sarcolemmal cholesterol. Cardioprotection with acute morphine was abolished with ≥20 μM MβCD, whereas SLP was more robust and only inhibited with ≥200 μM MβCD. Deletion of Cav-3 also reduced, whereas Cav-3 OE improved, myocardial I/R tolerance. Protection via SLP remained equally effective in Cav-3 KO mice and was additive with innate protection arising with Cav-3 OE. These data reveal the membrane cholesterol dependence of normoxic myocardial and coronary function, I/R tolerance, and OR-mediated cardioprotection in murine hearts (all declining with cholesterol depletion). In contrast, baseline function appears insensitive to Cav-3, whereas cardiac I/R tolerance parallels Cav-3 expression. Novel SLP appears unique, being less sensitive to cholesterol depletion than acute OR protection and arising independently of Cav-3 expression.

Membrane cholesterol modulates the hyaluronan-binding ability of CD44 in T lymphocytes and controls rolling under shear flow

The adhesion of circulating lymphocytes to the surface of vascular endothelial cells is important for their recruitment from blood to secondary lymphoid organs and to inflammatory sites. CD44 is a key adhesion molecule for this interaction and its ligand-binding ability is tightly regulated. Here we show that the hyaluronan-binding ability of CD44 in T cells is upregulated by the depletion of membrane cholesterol with methyl-β-cyclodextrin (MβCD), which disintegrates lipid rafts, i.e. cholesterol- and sphingolipid-enriched membrane microdomains. Increasing concentrations of MβCD led to a dose-dependent decrease in cellular cholesterol content and to upregulation of hyaluronan binding. Additionally, a cholesterol-binding agent filipin also increased hyaluronan binding. Cholesterol depletion caused CD44 to be dispersed from cholesterol-enriched membrane microdomains. Cholesterol depletion also increased the number of cells undergoing rolling adhesion under physiological flow conditions. Our results suggest that the ligand-binding ability of CD44 is governed by its cholesterol-dependent allocation to membrane microdomains at the cell surface. These findings provide novel insight into the regulation of T cell adhesion under blood flow.

Lower concentrations of methyl-β-cyclodextrin combined with interleukin-2 can preferentially induce activation and proliferation of natural killer cells in human peripheral blood

Previous studies have demonstrated that high concentrations of methyl-β-cyclodextrin (MβCD, 10–15 mM) can interfere with the formation of lipid rafts and inhibit activation of lymphocytes. In this report, we determined that lower concentrations of MβCD (1–4 mM) could accelerate the proliferation of lymphocytes in human peripheral blood mononuclear cells (PBMCs). In the expanded cells, CD3 -CD56 + natural killer (NK) cells were the dominant subpopulation, and a significant dose–effect relationship existed between the proportion of NK cells and the concentration of MβCD. In the groups treated with 3–4 mM MβCD, the proportions of NK cells reached a level of more than 60%. When PBMCs were treated with MβCD, CD69 was more preferentially expressed on CD3 -CD56 + cells than on CD3 + cells at 48 and 72 hours. The expression of CD25 had no distinct difference at 48 hours, but when recombinant human interleukin-2 (IL-2) was added for a further 24 hours, it was also preferentially expressed on NK cells. MβCD and IL-2 synergistically could also induce interferon-γ (IFN-γ) production in CD56 + human PBMCs. Mechanistic studies revealed that IFN-γ production in response to MβCD plus IL-2 was IL-12 independent but depended on endogenous IL-18 and IL-1β, and CD56 +CD14 + dendritic cell-like cells and B cells might mediate the ability of MβCD to activate NK cells. The MβCD-activated NK cells also had high cytotoxicity against the natural killer cell–sensitive K562 cells or lymphokine-activated killer cell–sensitive DAUDI cells in vitro. These studies indicated that lower concentrations of MβCD combined with IL-2 can preferentially induce activation and proliferation of NK cells in PBMCs.

Methyl-β-Cyclodextrins Preferentially Remove Cholesterol from the Liquid Disordered Phase in Giant Unilamellar Vesicles

Methyl-β-cyclodextrins (MβCDs) are molecules that are extensively used to remove and to load cholesterol (Chol) from artificial and natural membranes; however, the mechanism of Chol extraction by MβCD from pure lipids or from complex mixtures is not fully understood. One of the outstanding questions in this field is the capability of MβCD to remove Chol from lipid domains having different packing. Here, we investigated the specificity of MβCD to remove Chol from coexisting macrodomains with different lipid packing. We used giant unilamellar vesicles (GUVs) made of 1,2-dioleoylphosphatidylcholine:1,2-dipalmitoylphatidylcholine:free cholesterol, 1:1:1 molar ratio at 27°C. Under these conditions, individual GUVs present Chol distributed into l o and l d phases. The two phases can be distinguished and visualized using Laurdan generalized polarization and two-photon excitation fluorescence microscopy. Our data indicate that MβCD removes Chol preferentially from the more disordered phase. The process of selective Chol removal is dependent on the MβCD concentration. At high concentrations, MβCD also removes phospholipids.

Tuning of the Outer Hair Cell Motor by Membrane Cholesterol

Cholesterol affects diverse biological processes, in many cases by modulating the function of integral membrane proteins. We observed that alterations of cochlear cholesterol modulate hearing in mice. Mammalian hearing is powered by outer hair cell (OHC) electromotility, a membrane-based motor mechanism that resides in the OHC lateral wall. We show that membrane cholesterol decreases during maturation of OHCs. To study the effects of cholesterol on hearing at the molecular level, we altered cholesterol levels in the OHC wall, which contains the membrane protein prestin. We show a dynamic and reversible relationship between membrane cholesterol levels and voltage dependence of prestin-associated charge movement in both OHCs and prestin-transfected HEK 293 cells. Cholesterol levels also modulate the distribution of prestin within plasma membrane microdomains and affect prestin self-association in HEK 293 cells. These findings indicate that alterations in membrane cholesterol affect prestin function and functionally tune the outer hair cell.

Lipid raft disruption by cholesterol depletion enhances influenza A virus budding from MDCK cells.

Lipid rafts play critical roles in many aspects of the influenza A virus life cycle. Cholesterol is a critical structural component of lipid rafts, and depletion of cholesterol leads to disorganization of lipid raft microdomains. In this study, we have investigated the effect of cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) treatment on influenza virus budding. When virus-infected Madin-Darby canine kidney cells were treated with MbetaCD at the late phase of infection for a short duration, budding of virus particles, as determined by protein analysis and electron microscopy, increased with increasing concentrations and lengths of treatment. However, infectious virus yield varied, depending on the concentration and duration of MbetaCD treatment. Low concentrations of MbetaCD increased infectious virus yield throughout the treatment period, but higher concentrations caused an initial increase of infectious virus titer followed by a decrease with a longer duration. Relative infectivity of the released virus particles, on the other hand, decreased with increasing concentrations and durations of MbetaCD treatment. Loss of infectivity of virus particles is due to multiple effects of MbetaCD-mediated cholesterol depletion causing disruption of lipid rafts, changes in structural integrity of the viral membrane, leakage of viral proteins, a nick or hole on the viral envelope, and disruption of the virus structure. Exogenous cholesterol increased lipid raft integrity, inhibited particle release, and partially restored the infectivity of the released virus particles. These data show that disruption of lipid rafts by cholesterol depletion caused an enhancement of virus particle release from infected cells and a decrease in the infectivity of virus particles.

Interaction between Fluorocarbon End-Capped Poly(ethylene oxide) and Cyclodextrins

Well-defined fluorocarbon end-capped poly(ethylene oxide) (PEO), known as F-HEUR, were synthesized by varying both fluorocarbon end groups and PEO chain length. Their solution properties were studied and compared with those of hydrogenated carbon end-capped PEO. Variation of the end-capped functional group from −C6F13 to −C8F17 gave rise to a significant shift in the solution rheological properties. The interaction between different cyclodextrins and these fluorocarbon derivatives of PEO was systematically examined by rheological, dynamic light scattering (DLS), isothermal titration calorimetric (ITC), and 19F NMR techniques. The viscosity of F-HEUR solution was drastically reduced in the presence of methylated β-cyclodextrin (m-βCD) due to the disruption of intermolecular hydrophobic associations, but not observed for α- and γ-cyclodextrins. The destruction of F-HEUR network structure in the presence of m-βCD was reflected by the large reduction in the plateau modulus ( ). The activation energies (Ea) determined from zero-shear viscosity decreased with m-βCD concentration until a molar ratio of 2; thereafter it remained constant. At high m-βCD concentration, subtle difference was observed for the complexes produced by −C6F13 and −C8F17 hydrophobic moieties. The inclusion complex formation between m-βCD and different fluorocarbon groups derived from ITC thermograms revealed a stoichiometry of 1:1 for C6F13 but 1:1 and 2:1 inclusion complexes for C8F17 at different m-βCD concentrations. DLS results showed a different mechanism for C6F13 and C8F17 fluorocarbon hydrophobes, which were in agreement with the results deduced from activation energies and 19F NMR spectra. A mathematic model was adopted to describe the relationship between and m-βCD concentration.

Characterization of methyl-β-cyclodextrin toxicity in NGF-differentiated PC12 cell death

Cyclodextrins (CDs) are used to deliver hydrophobic molecules in aqueous environments. Methyl-β-cyclodextrin (MβCD), a member of this family of molecules, has been proposed to be a good carrier to deliver fatty acids to cells in culture. This report focuses on studying the in vitro effects of MβCD on nerve growth factor-differentiated PC12 (NGFDPC12) cells, a tissue culture model to study neuronal survival and differentiation. The main findings are: (1) NGFDPC12 cells have normal viability when exposed to 0.12% MβCD but showed a significant loss in cell viability at higher concentrations; (2) NGFDPC12 cells exposed to 0.25% MβCD exhibit nuclear condensation, blebbing and apoptotic bodies, and whole cell lysates exhibited an increase in caspase-3-like activity and high levels of Bax and Bcl-X L protein expression compared to control. Cultures treated with 0.25% MβCD also showed cleavage of normal 21-kDa Bax protein into a 18-kDa fragment. (3) Experiments using 0.12% MβCD to deliver oleic acid did not affect cell viability, in contrast NGFDPC12 cultures in which 0.25% MβCD concentration is used exhibited similar loss of cell viability as observed with 0.25% MβCD alone. Treating these cultures with caspase-3 inhibitor z-VAD-fmk did not protect the cells from MβCD toxic effects. (4) Immortalized Schwann cells (iSC) exposed to MβCD 0.12% did not show loss of cell viability while 0.25% MβCD triggered a significant toxicity but with a different dose and time course dynamic than NGFDPC12 cells. Thus, NGFDPC12 or iSC cell cultures exposed to 0.12% MβCD exhibits normal viability while higher concentrations increase in cell death and apoptosis.

Shedding of Collagen XVII Ectodomain Depends on Plasma Membrane Microenvironment

Collagen XVII, a hemidesmosomal component, mediates the adhesion of epidermal keratinocytes to the underlying basement membrane. It exists as a full-length transmembrane protein and a soluble ectodomain that is proteolytically released from the cell surface by sheddases of a disintegrin and metalloproteinase (ADAM) family; TACE, the tumor necrosis factor-alpha-converting enzyme, is the major physiological proteinase. Because both collagen XVII and the ADAMs are transmembrane proteins, their plasma membrane microenvironment can influence shedding. Lipid rafts, assemblies of sphingolipids and cholesterol within the plasma membrane, are responsible for the separation of membrane proteins and are thought to regulate shedding of cell surface proteins. In this study we analyzed the influence of the cholesterol-depleting agent methyl-beta-cyclodextrin (MbetaCD), which disintegrates lipid rafts, on the shedding of collagen XVII in HaCaT keratinocytes and in transfected COS-7 cells. Increasing concentrations of MbetaCD led to a dose-dependent decrease of membrane cholesterol levels and to stimulation of collagen XVII shedding. The stimulation was completely inhibited by sheddase inhibitors, and experiments with COS-7 cells co-transfected with TACE and collagen XVII demonstrated that TACE mediated the low cholesterol-dependent shedding. Co-patching analysis by double immunofluorescence staining revealed co-localization of collagen XVII with the raft resident phosphatidylinositol-linked placental alkaline phosphatase and segregation from the non-raft protein human transferrin receptor, indicating that a majority of collagen XVII molecules was incorporated into lipid rafts. These data deliver the first evidence for the role of plasma membrane lipid organization in the regulation of collagen XVII shedding and, therefore, in the regulation of keratinocyte migration and differentiation.

Rheological properties of hydrophobic ethoxylated urethane (HEUR) in the presence of methylated β-cyclodextrin

Hydrophobic ethoxylated urethane (HEUR) is one type of hydrophobically modified polyethylene glycol, which could form a transient network structure in aqueous solution via intermolecular hydrophobic associations. The viscosity of HEUR solution is dependent on two parameters; namely the effective elastic chain density and the relaxation time. In the present study, different amounts of methylated β-cyclodextrin (m-βCD) were added into aqueous HEUR solutions, and significant reduction in the solution viscosity was observed when the m-βCD concentration exceeds a critical value. Oscillatory shear indicated that the plateau modulus does not decrease appreciably with the addition of m-βCD, however a large reduction in relaxation time was observed. At low m-βCD concentrations, addition of m-βCD does not decouple the hydrophobic association. The ring-shaped hydrophobic inner cores of m-βCD interact with the hydrophobic moiety of looping HEUR chains, leading to a reduction in the relaxation time, which weakens the transient network. However, at higher m-βCD concentrations, bridging chains are decoupled, thereby disrupting the transient network and produce smaller isolated aggregates.

Cholesterol modulates ligand binding and G-protein coupling to serotonin1A receptors from bovine hippocampus

The serotonin 1A (5-HT 1A ) receptor is an important member of the superfamily of seven-transmembrane domain G-protein-coupled receptors. We have examined the modulatory role of cholesterol on the ligand binding activity and G-protein coupling of the bovine hippocampal 5-HT 1A receptor by depleting cholesterol from native membranes using methyl-β-cyclodextrin (MβCD). Removal of cholesterol from bovine hippocampal membranes using varying concentrations of MβCD results in a concentration-dependent reduction in specific binding of the agonist 8-OH-DPAT to 5-HT 1A receptors. This is accompanied by alterations in binding affinity and sites obtained from analysis of binding data. Importantly, cholesterol depletion affected G-protein-coupling of the receptor as monitored by the GTP-γ-S assay. The concomitant changes in membrane order were reported by changes in fluorescence polarization of membrane probes such as DPH and TMA-DPH, which are incorporated at different locations (depths) in the membrane. Replenishment of membranes with cholesterol led to recovery of ligand binding activity as well as membrane order to a considerable extent. Our results provide evidence, for the first time, that cholesterol is necessary for ligand binding and G-protein coupling of this important neurotransmitter receptor. These results could have significant implications in understanding the influence of the membrane lipid environment on the activity and signal transduction of other G-protein-coupled transmembrane receptors.

Cholesterol modulates ligand binding and G-protein coupling to serotonin 1A receptors from bovine hippocampus

The serotonin 1A (5-HT 1A) receptor is an important member of the superfamily of seven-transmembrane domain G-protein-coupled receptors. We have examined the modulatory role of cholesterol on the ligand binding activity and G-protein coupling of the bovine hippocampal 5-HT 1A receptor by depleting cholesterol from native membranes using methyl-β-cyclodextrin (MβCD). Removal of cholesterol from bovine hippocampal membranes using varying concentrations of MβCD results in a concentration-dependent reduction in specific binding of the agonist 8-OH-DPAT to 5-HT 1A receptors. This is accompanied by alterations in binding affinity and sites obtained from analysis of binding data. Importantly, cholesterol depletion affected G-protein-coupling of the receptor as monitored by the GTP-γ-S assay. The concomitant changes in membrane order were reported by changes in fluorescence polarization of membrane probes such as DPH and TMA-DPH, which are incorporated at different locations (depths) in the membrane. Replenishment of membranes with cholesterol led to recovery of ligand binding activity as well as membrane order to a considerable extent. Our results provide evidence, for the first time, that cholesterol is necessary for ligand binding and G-protein coupling of this important neurotransmitter receptor. These results could have significant implications in understanding the influence of the membrane lipid environment on the activity and signal transduction of other G-protein-coupled transmembrane receptors.