MbetaCD Treatment Research Articles

Nongenomic Steroid- and Ceramide-Induced Maturation in Amphibian Oocytes Involves Functional Caveolae-Like Microdomains Associated with a Cytoskeletal Environment1

Stimulation of full-grown amphibian oocytes with progesterone initiates a nontranscriptional signaling pathway that converges in the activation of Cdc2/cyclin B and reentry into meiosis. We observed that cholesterol depletion mediated by methyl-beta-cyclodextrin (MbetaCD) inhibited meiotic maturation, suggesting involvement of membrane rafts. In the present study, we further characterized caveolae-like membranes from Rhinella arenarum oocytes biochemically and functionally. The identification by mass spectrometry of a nonmuscle myosin heavy-chain associated with caveolar membranes showed evidence of direct involvement of the underlying cytoskeletal environment in the structure of oocyte rafts. Biophysical analysis using the fluorescent probe Laurdan revealed that MbetaCD-mediated cholesterol depletion affected membrane lipid order. In line with this finding, cholesterol removal also affected the localization of the raft marker lipid GM1. Results demonstrated that ceramide is an effective inducer of maturation that alters the distribution of the raft markers caveolin-1, SRC, and GM1, while progesterone seems not to affect membrane microdomain integrity. Cholesterol depletion had a greater effect on ceramide-induced maturation, thus suggesting that ceramide is an inducer more vulnerable to changes in the plasma membrane. MbetaCD treatment delayed tyrosine phosphorylation and MAPK activation in progesterone-induced maturation. Functional studies regarding tyrosine phosphorylation raise the possibility that the hormone receptor is located in the nonraft membrane in the absence of ligand and that it translocates to the caveola when it binds to progesterone. The presence of raft markers and the finding of signaling molecules from MAPK cascade functionally associated to oocyte light membranes suggest that this caveolae-rich fraction efficiently recreates, in part, maturation signaling.

Methyl-β-cyclodextrin Suppresses Hyaluronan Synthesis by Down-regulation of Hyaluronan Synthase 2 through Inhibition of Akt

Hyaluronan synthases (HAS1-3) are integral plasma membrane proteins that synthesize hyaluronan, a cell surface and extracellular matrix polysaccharide necessary for many biological processes. It has been shown that HAS is partly localized in cholesterol-rich lipid rafts of MCF-7 cells, and cholesterol depletion with methyl-beta-cyclodextrin (MbetaCD) suppresses hyaluronan secretion in smooth muscle cells. However, the mechanism by which cholesterol depletion inhibits hyaluronan production has remained unknown. We found that cholesterol depletion from MCF-7 cells by MbetaCD inhibits synthesis but does not decrease the molecular mass of hyaluronan, suggesting no major influence on HAS stability in the membrane. The inhibition of hyaluronan synthesis was not due to the availability of HAS substrates UDP-GlcUA and UDP-GlcNAc. Instead, MbetaCD specifically down-regulated the expression of HAS2 but not HAS1 or HAS3. Screening of signaling proteins after MbetaCD treatment revealed that phosphorylation of Akt and its downstream target p70S6 kinase, both members of phosphoinositide 3-kinase-Akt pathway, were inhibited. Inhibitors of this pathway suppressed hyaluronan synthesis and HAS2 expression in MCF-7 cells, suggesting that the reduced hyaluronan synthesis by MbetaCD is due to down-regulation of HAS2, mediated by the phosphoinositide 3-kinase-Akt-mTOR-p70S6K pathway.

HIF-1 is induced via EGFR activation and mediates resistance to anoikis-like cell death under lipid rafts/caveolae-disrupting stress

The plasma membrane microdomains, lipid rafts, are involved in regulation of cellular functions such as cell survival and adhesion. Cholesterol is a critical component of lipid rafts in terms of their integrity and functions and rafts disruption by cholesterol depletion can induce detachment-induced cell death. Hypoxia inducible factor-1 (HIF-1) alpha is stabilized in hypoxia and transactivates numerous genes required for cellular adaptation to hypoxia. It is also induced by non-hypoxic stimuli and contributes to cell survival. Because hypoxia inhibits cholesterol synthesis and HIF-1alpha plays a role in this process, we here explored a possible connection between lipid rafts and HIF-1alpha. We investigated whether HIF-1alpha is regulated during cholesterol depletion/rafts disruption in A431 cells in normoxic conditions. Methyl-beta cyclodextrin (MbetaCD), which induces cholesterol depletion, upregulated HIF-1alpha even under normoxic conditions and this upregulation required epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase 1 and 2 activation, but not Akt activation. MbetaCD treatment induced HIF-1alpha upregulation at both the transcriptional and translational levels but not at the posttranslational levels. In addition, MbetaCD robustly induced vascular endothelial growth factor production and stimulated an hypoxia response element-driven luciferase reporter activity under normoxic conditions, indicating that MbetaCD-induced HIF-1alpha is functionally activated. Both EGFR activity and HIF-1alpha expression were higher in the attached cells than in the detached cells after MbetaCD treatment. Furthermore, inhibition of HIF-1alpha by RNA interference accelerated cell detachment, thus increasing cell death, indicating that HIF-1alpha expression attenuates MbetaCD-induced anoikis-like cell death. These data suggest that, depending on cholesterol levels, lipid rafts or membrane fluidity are probably to regulate HIF-1alpha expression in normoxia by modulating rafts protein activities such as EGFR, and this connection between lipid rafts and HIF-1alpha regulation may provide cell survival under membrane-disturbing stress.

Differential Effect of Membrane Cholesterol Removal on μ- and δ-Opioid Receptors: A PARALLEL COMPARISON OF ACUTE AND CHRONIC SIGNALING TO ADENYLYL CYCLASE

According to the lipid raft theory, the plasma membrane contains small domains enriched in cholesterol and sphingolipid, which may serve as platforms to organize membrane proteins. Using methyl-beta-cyclodextrin (MbetaCD) to deplete membrane cholesterol, many G protein-coupled receptors have been shown to depend on putative lipid rafts for proper signaling. Here we examine the hypothesis that treatment of HEK293 cells stably expressing FLAG-tagged mu-opioid receptors (HEK FLAG-mu) or delta-opioid receptors (HEK FLAG-delta) with MbetaCD will reduce opioid receptor signaling to adenylyl cyclase. The ability of the mu-opioid agonist [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin to acutely inhibit adenylyl cyclase or to cause sensitization of adenylyl cyclase following chronic treatment was attenuated with MbetaCD. These effects were due to removal of cholesterol, because replenishment of cholesterol restored [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin responses back to control values, and were confirmed in SH-SY5Y cells endogenously expressing mu-opioid receptors. The effects of MbetaCD may be due to uncoupling of the mu receptor from G proteins but were not because of decreases in receptor number and were not mimicked by cytoskeleton disruption. In contrast to the results in HEK FLAG-mu cells, MbetaCD treatment of HEK FLAG-delta cells had no effect on acute inhibition or sensitization of adenylyl cyclase by delta-opioid agonists. The differential responses of mu- and delta-opioid agonists to cholesterol depletion suggest that mu-opioid receptors are more dependent on cholesterol for efficient signaling than delta receptors and can be partly explained by localization of mu- but not delta-opioid receptors in cholesterol- and caveolin-enriched membrane domains.

The “Caveolae Brake Hypothesis” and the Epidermal Barrier

Epidermal permeability barrier formation depends upon lamellar body (LB) secretion/fusion with the apical plasma membrane (APM) of outermost stratum granulosum (SG) cell, creating cholesterol/glycosphingolipid-enriched lipid rafts-like domains. We found that the dimensions of these domains are comparable to lipid raft in other cell types; and that acute barrier disruption regulates their size and dynamics. To assess the function of these LB-derived raft-like domains, we assessed APM dynamics and barrier recovery in methyl-beta-cyclodextrin (MbetaCD)-treated hairless mice and caveolin-1 knockouts (cav-1(-/-)). MbetaCD treatment impaired APM raft-like domain formation and barrier recovery. Accelerated barrier recovery is observed in cav-1(-/-) in parallel with expansion of raft-like domains. Barrier abrogation of normal epidermis resulted in translocation of cav-1 from the cytoplasm to raft-like membrane domains, restricting further raft-like domain formation and initiating terminal differentiation. Inhibition of LB secretion by monensin and absence of cav-1 delayed terminal differentiation. Furthermore, cav-1(-/-) mice exhibited an increased propensity to develop experimentally induced epidermal hyperplasia correlating with lipid raft persistence. Finally, the epidermal hyperplasia in psoriasis and Netherton syndrome is paralleled by increased lipid raft formation. These studies demonstrate that cav-1 delivery to the APM by LB trafficking to APM "brakes" further LB secretion, signals terminal differentiation, and regulates epidermal hyperproliferation.

Cholesterol depletion induces anoikis‐like apoptosis via FAK down‐regulation and caveolae internalization

Caveolae (lipid rafts), microdomains of the plasma membrane, are known to contain various signalling molecules and consequently are involved in the regulation of many biological functions. To investigate the role of the caveolae in cell survival and adhesion, we disrupted the caveolae by depletion of cholesterol, a major lipid component of the caveolae, with methyl-beta cyclodextrin (MbetaCD) treatment of A431 cells. We found that cholesterol depletion induced an anoikis-like cell death involving actin reorganization, resulting in a decrease in cell spreading and an increase in cell detachment, which was reversed by cholesterol addition. Disruption of caveolae led to the down-regulation of FAK, Src activation, tyrosine phosphorylation of caveolin-1 and mobilization of caveolae markers, GM1 and caveolin-1, from the cell surface to the cytoplasm, which were also recovered by cholesterol addition. The expression of dominant-active FAK was able to delay caveolae internalization and apoptosis and attenuated Akt inactivation by MbetaCD, whereas dominant-negative FAK expression resulted in enhanced apoptosis. Moreover, FAK down-regulation by si-RNA resulted in Akt inactivation and thus increased cell death by MbetaCD treatment. Our results suggest that the cholesterol content and/or surface levels of the caveolae affect the activity of FAK, which in turn regulates caveolae internalization and cell survival.

The role of membrane microdomains in shaping β2-adrenergic receptor-mediated cAMP dynamics

Recently, membrane rafts and caveolae have received much attention for their role as signaling platforms, particularly due to their involvement in the pathogenesis of a number of diseases, including HIV as well as neurological and cardiovascular conditions. Signaling mediated by the beta-adrenergic receptor (beta-AR), a member of the large family of G-protein coupled receptors (GPCRs) that transduce extracellular messages via the ubiquitous second messenger, cAMP, has been a focus of raft studies since multiple components of the pathway are compartmentalized by these membrane microdomains. However, how these membrane rafts behave and regulate signaling dynamics in a cellular context is poorly understood. Here, we describe a live-cell assay based on single-cell, real-time fluorescence imaging, via an improved FRET-based cAMP biosensor, to monitor raft regulation of second messenger dynamics. Upon cholesterol depletion with methyl-beta-cyclodextrin (MbetaCD), beta(2)-AR-mediated cAMP accumulation was enhanced and prolonged in HEK-293 cells, demonstrating that membrane raft integrity helps shape beta-AR signaling. Single-cell imaging in parallel with fractionation studies reveal that the enhancement and change of dynamics are mediated by the receptor and correlated with its redistribution. Finally, the effect of cholesterol depletion is receptor-type specific as MbetaCD treatment did not show the same effect when the raft-excluded prostaglandin E receptor was stimulated. This study highlights the potential of a live-cell, real-time imaging assay for studying membrane rafts, including high sensitivity and spatiotemporal resolution, to achieve a better understanding of the nuances of membrane microdomains in both healthy and diseased states.

S1P1 Receptor Localization Confers Selectivity for Gi-mediated cAMP and Contractile Responses

Adult mouse ventricular myocytes express S1P(1), S1P(2), and S1P(3) receptors. S1P activates Akt and ERK in adult mouse ventricular myocytes through a pertussis toxin-sensitive (G(i/o)-mediated) pathway. Akt and ERK activation by S1P are reduced approximately 30% in S1P(3) and 60% in S1P(2) receptor knock-out myocytes. With combined S1P(2,3) receptor deletion, activation of Akt is abolished and ERK activation is reduced by nearly 90%. Thus the S1P(1) receptor, while present in S1P(2,3) receptor knock-out myocytes, is unable to mediate Akt or ERK activation. In contrast, S1P induces pertussis toxin-sensitive inhibition of isoproterenol-stimulated cAMP accumulation in both WT and S1P(2,3) receptor knock-out myocytes demonstrating that the S1P(1) receptor can functionally couple to G(i). An S1P(1) receptor selective agonist, SEW2871, also decreased cAMP accumulation but failed to activate ERK or Akt. To determine whether localization of the S1P(1) receptor mediates this signaling specificity, methyl-beta-cyclodextrin (MbetaCD) treatment was used to disrupt caveolae. The S1P(1) receptor was concentrated in caveolar fractions, and associated with caveolin-3 and this localization was disrupted by MbetaCD. S1P-mediated activation of ERK or Akt was not diminished but inhibition of cAMP accumulation by S1P and SEW2871 was abolished by MbetaCD treatment. S1P inhibits the positive inotropic response to isoproterenol and this response is also mediated through the S1P(1) receptor and lost following caveolar disruption. Thus localization of S1P(1) receptors to caveolae is required for the ability of this receptor to inhibit adenylyl cyclase and contractility but compromises receptor coupling to Akt and ERK.

Disruption of Lipid Rafts Induces Gonadotropin Release in Ovine Pituitary and LbetaT2 Gonadotroph Cells1

In order to better understand the cellular mechanisms underlying LH and FSH secretion, we have addressed the contribution of lipid rafts to the secretion of gonadotropins. We used methyl-beta-cyclodextrin (MbetaCD), a cholesterol-sequestering agent, on an LbetaT2 murine gonadotroph cell line and on primary cultures of ovine pituitary cells. We found that in both systems, cholesterol depletion by MbetaCD induced a fast and substantial release of LH in the absence of natural stimulation by GnRH. In ovine pituitary cells, MbetaCD-mediated LH release was shown to be independent of protein synthesis. Twenty-four hours after MbetaCD treatment, there was no loss of cell viability and full recovery of LH secretory capabilities, as determined by GnRH or MbetaCD treatment. In addition, our data suggest the existence of a pool of LH that is not released by GnRH treatment but that is released by MbetaCD treatment. Finally, in ovine pituitary cells, MbetaCD treatment induced FSH secretion. Importantly, these in vitro data are supported by in vivo studies, because MbetaCD injected into the pituitary glands of anaesthetized sheep reproducibly induced a peak of LH release.

Lipid Rafts Establish Calcium Waves in Hepatocytes

Polarity is critical for hepatocyte function. Ca(2+) waves are polarized in hepatocytes because the inositol 1,4,5-trisphosphate receptor (InsP3R) is concentrated in the pericanalicular region, but the basis for this localization is unknown. We examined whether pericanalicular localization of the InsP3R and its action to trigger Ca(2+) waves depends on lipid rafts. Experiments were performed using isolated rat hepatocyte couplets and pancreatic acini, plus SkHep1 cells as nonpolarized controls. The cholesterol depleting agent methyl-beta-cyclodextrin (mbetaCD) was used to disrupt lipid rafts. InsP3R isoforms were examined by immunoblot and immunofluorescence. Ca(2+) waves were examined by confocal microscopy. Type II InsP3Rs initially were localized to only some endoplasmic reticulum fractions in hepatocytes, but redistributed into all fractions in mbetaCD-treated cells. This InsP3R isoform was concentrated in the pericanalicular region, but redistributed throughout the cell after mbetaCD treatment. Vasopressin-induced Ca(2+) signals began as apical-to-basal Ca(2+) waves, and mbetaCD slowed the wave speed and prolonged the rise time. MbetaCD had a similar effect on Ca(2+) waves in acinar cells but did not affect Ca(2+) signals in SkHep1 cells, suggesting that cholesterol depletion has similar effects among polarized epithelia, but this is not a nonspecific effect of mbetaCD. Lipid rafts are responsible for the pericanalicular accumulation of InsP3R in hepatocytes, and for the polarized Ca(2+) waves that result. Signaling microdomains exist not only in the plasma membrane, but also in the nearby endoplasmic reticulum, which in turn, helps establish and maintain structural and functional polarity.

Na+/H+ Exchanger NHE3 Activity and Trafficking Are Lipid Raft-dependent

A previous study showed that approximately 25-50% of rabbit ileal brush border (BB) Na(+)/H(+) exchanger NHE3 is in lipid rafts (LR) (Li, X., Galli, T., Leu, S., Wade, J. B., Weinman E. J., Leung, G., Cheong, A., Louvard, D., and Donowitz, M. (2001) J. Physiol. (Lond.) 537, 537-552). Here, we examined the role of LR in NHE3 transport activity using a simpler system: opossum kidney (OK) cells (a renal proximal tubule epithelial cell line) containing NHE3. approximately 50% of surface (biotinylated) NHE3 in OK cells distributed in LR by density gradient centrifugation. Disruption of LR with methyl-beta-cyclodextrin (MbetaCD) decreased NHE3 activity and increased K'(H+)(i), but K(m)((Na+)) was not affected. The MbetaCD effect was completely reversed by repletion of cholesterol, but not by an inactive analog of cholesterol (cholestane-3beta,5alpha,6beta-triol). The MbetaCD effect was specific for NHE3 activity because it did not alter Na(+)-dependent l-Ala uptake. MbetaCD did not alter OK cell BB topology and did not change the surface amount of NHE3, but greatly reduced the rate of NHE3 endocytosis. The effects of inhibiting phosphatidylinositol 3-kinase and of MbetaCD on NHE3 activity were not additive, indicating a common inhibitory mechanism. In contrast, 8-bromo-cAMP and MbetaCD inhibition of NHE3 was additive, indicating different mechanisms for inhibition of NHE3 activity. Approximately 50% of BB NHE3 and only approximately 11% of intracellular NHE3 in polarized OK cells were in LR. In summary, the BB pool of NHE3 in LR is functionally active because MbetaCD treatment decreased NHE3 basal activity. The LR pool is necessary for multiple kinetic aspects of normal NHE3 activity, including V(max) and K'(H+)(i), and also for multiple aspects of NHE3 trafficking, including at least basal endocytosis and phosphatidylinositol 3-kinase-dependent basal exocytosis. Because the C-terminal domain of NHE3 is necessary for its regulation and because the changes in NHE3 kinetics with MbetaCD resemble those with second messenger regulation of NHE3, these results suggest that the NHE3 C terminus may be involved in the MbetaCD sensitivity of NHE3.

Cyclodextrins but not Compactin Inhibit the Lateral Diffusion of Membrane Proteins Independent of Cholesterol

Cholesterol and glycosphingolipid-enriched membrane domains, termed lipid rafts, were proposed to play important roles in trafficking and signaling events. These functions are inhibited following putative disruption of rafts by cholesterol depletion, commonly induced by treatment with methyl-beta-cyclodextrin (MbetaCD). However, several studies showed that the lateral diffusion of membrane proteins is inhibited by MbetaCD, suggesting that it may have additional effects on membrane organization unrelated to cholesterol removal. Here, we investigated this possibility by comparison of the effects of cholesterol depletion by MbetaCD and by metabolic inhibition (compactin), and of treatment with alpha-CD, which does not bind cholesterol. The studies employed two series of proteins (Ras and influenza hemagglutinin), each containing as internal controls related mutants that differ in raft association. Mild MbetaCD treatment retarded the lateral diffusion of both raft and non-raft mutants, whereas similar cholesterol reduction (30-33%) by metabolic inhibition enhanced selectively the diffusion of the raft-associated mutants. Moreover, alpha-CD also inhibited the diffusion of raft and non-raft mutants, despite its lack of effect on cholesterol content. These findings suggest that the widely used treatment with CD to reduce cholesterol has additional, cholesterol-independent effects on membrane protein mobility, which do not necessarily distinguish between raft and non-raft proteins.

Caveolae and sarcoplasmic reticular coupling in smooth muscle cells of pressurised arteries: The relevance for Ca2+ oscillations and tone

A close association of caveolae and sarcoplasmic reticulum (SR) has been suggested to be important for contractile activation of smooth muscle. Here, we investigate the presence of such arrangements in pressurised resistance arteries and examine the influence of two agents purported to disrupt caveolae and/or SR conformations by different mechanisms of action. Rat mesenteric small arteries (RMSA) were mounted on a pressure myograph and the functional (lumen diameter and Ca(2+) oscillations) and ultrastructural effects of the phosphatase inhibitor calyculin-A (cal-A), or the cholesterol binding agent methyl-beta-cyclodextrin (mbetacd), examined by light and electron microscopy. Smooth muscle cells of RMSA exhibited a prominent peripheral SR that often encircled individual caveolae. The peripheral SR on occasion was observed to make contact with centrally located SR allowing for a structural association of caveoale-SR-myofilaments. Cal-A maximally constricted RMSA and disrupted the regular SR-caveolae appearance such that concentrated swirls of SR not enveloping caveolae were evident. Mbetacd treatment, in contrast, inhibited agonist contractility and reduced the appearance of caveolae whilst peripheral SR apposition to the plasmalemma could still be observed. Treatment with either agent inhibited agonist-mediated smooth muscle Ca(2+) oscillations. We present data that supports a structural arrangement of caveolae and underlying peripheral SR in smooth muscle cells of pressurised resistance arteries that serves to regulate Ca(2+) oscillations and contractile activation.

Enteropathogenic Escherichia coli Tir translocation and pedestal formation requires membrane cholesterol in the absence of bundle-forming pili

Enteropathogenic Escherichia coli (EPEC) is a significant cause of paediatric diarrhoea worldwide. Virulence requires adherence to intestinal epithelial cells, mediated in part through type IV bundle-forming pili (BFP), and the EPEC protein Tir. Tir is inserted into the enterocyte plasma membrane (PM), resulting in the formation of actin-rich pedestals. Tir is translocated by the type III secretion system (TTSS), through a pore comprised of EPEC proteins inserted into the PM. Here, we demonstrate that in the absence of BFP, EPEC adherence, effector translocation and pedestal formation are dependent on lipid rafts. Lipid raft disruption using methyl-beta-cyclodextrin (MbetaCD) decreased adherence by an EPEC BFP-deficient strain from 85% to 1%. Translocation of the effectors Tir and EspF was blocked by MbetaCD treatment, although the TTSS pore still formed. MbetaCD treatment after Tir delivery decreased pedestal formation by EPEC from 40% to 5%, but not by the related pathogen E. coli O157:H7 which uses a different Tir-based mechanism. In contrast, EPEC expressing the BFP can circumvent the requirement for membrane cholesterol. This suggests that lipid rafts play a role in virulence of this medically important pathogen.

Membrane cholesterol extraction decreases Na+ transport in A6 renal epithelia

In this study, we have investigated the dependence of Na+ transport regulation on membrane cholesterol content in A6 renal epithelia. We continuously monitored short-circuit current (Isc), transepithelial conductance (GT), and transepithelial capacitance (CT) to evaluate the effects of cholesterol extraction from the apical and basolateral membranes in steady-state conditions and during activation with hyposmotic shock, oxytocin, and adenosine. Cholesterol extraction was achieved by perfusing the epithelia with methyl-beta-cyclodextrin (mbetaCD) for 1 h. In steady-state conditions, apical membrane cholesterol extraction did not significantly affect the electrophysiological parameters; in contrast, marked reductions were observed during basolateral mbetaCD treatment. However, apical mbetaCD application hampered the responses of Isc and GT to hypotonicity, oxytocin, and adenosine. Analysis of the blocker-induced fluctuation in Isc demonstrated that apical mbetaCD treatment decreased the epithelial Na+ channel (ENaC) open probability (Po) in the steady state as well as after activation of Na+ transport by adenosine, whereas the density of conducting channels was not significantly changed as confirmed by CT measurements. Na+ transport activation by hypotonicity was abolished during basolateral mbetaCD treatment as a result of reduced Na+/K+ pump activity. On the basis of the findings in this study, we conclude that basolateral membrane cholesterol extraction reduces Na+/K+ pump activity, whereas the reduced cholesterol content of the apical membranes affects the activation of Na+ transport by reducing ENaC Po.

EFFECT OF THE MODULATION OF THE MEMBRANE LIPID COMPOSITION ON THE LOCALIZATION AND FUNCTION OF P-GLYCOPROTEIN IN MDR1-MDCK CELLS

Multidrug resistance (MDR) is a major obstacle in cancer therapy. It results from different mechanisms; among them is P-glycoprotein (P-gp)-mediated drug efflux out of cells. The mechanism of action remains elusive. The membrane lipid surrounding of P-gp, especially cholesterol, has been postulated to play an important role. To determine the effect of cholesterol depletion on P-gp, Madin Darby canine kidney (MDCK) cells, transfected with the mdr1 gene (MDR1-MDCK cells), were treated with methyl-beta-cyclodextrin (MbetaCD). The localization and function of P-gp were analyzed using confocal laser scanning microscopy. Treatment with 100 mM MbetaCD did not affect viability but altered the structural appearance of the cells and abolished efflux of rhodamine 123, a P-gp substrate. The MbetaCD treatment released P-gp from intact cells into the supernatant and reduced the amount of P-gp in total membrane preparations. The P-gp was shifted from the raft fractions (1% Triton X-100, 4 degrees C) to higher density fractions in MbetaCD-treated cells. The amount of cholesterol was significantly decreased in the raft fractions. Treatment of cells with 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, a glucosylceramide synthase inhibitor, also led to a shift of P-gp to higher density fractions. These results show that removal of cholesterol modulates the membrane lipid composition, changes the localization of P-gp, and results in loss of P-gp function.

Cholesterol Depletion Upregulates Involucrin Expression in Epidermal Keratinocytes Through Activation of p38

Cholesterol has been recently suggested to regulate the early steps of keratinocyte differentiation through lipid rafts. In many cell types, depletion of cholesterol activates signaling proteins like epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), or extracellular signal-regulated kinase (ERK) known to affect cell differentiation. In this study, we explored the effects of cholesterol depletion on the phenotype of cultured keratinocytes, using a treatment with methyl-beta-cyclodextrin (MbetaCD) to extract cholesterol and a treatment with lovastatin to inhibit cholesterol neosynthesis. Analysis of the expression of differentiation marker genes in early differentiating confluent cultures reveals that cholesterol depletion induces downregulation of keratin 14 (K14) and keratin 10 (K10) and upregulation of involucrin. MbetaCD treatment induces phosphorylation of EGFR, HER2, and ERK, but not HER3. Inhibition of EGFR with PD153035 impairs the MbetaCD-induced phosphorylation of EGFR, HER2, and ERK, but does not impair the alteration of K14, K10, or involucrin gene expression, indicating that other signaling proteins regulate this phenomenon. p38 has been suggested to regulate the expression of involucrin during keratinocyte differentiation. We found that MbetaCD treatment induces a prolonged phosphorylation of p38 in general and p38alpha in particular. An inhibition of p38 with PD169316 impairs the upregulation of involucrin mRNAs by a treatment with MbetaCD, but not by a p38delta-activating TPA treatment, which might suggest that cholesterol depletion alters involucrin gene expression through activation of p38alpha/beta.

Membrane cholesterol modulates dihydropyridine receptor function in mice fetal skeletal muscle cells.

Caveolae and transverse (T-) tubules are membrane structures enriched in cholesterol and glycosphingolipids. They play an important role in receptor signalling and myogenesis. The T-system is also highly enriched in dihydropyridine receptors (DHPRs), which control excitation-contraction (E-C) coupling. Recent results have shown that a depletion of membrane cholesterol alters caveolae and T-tubules, yet detailed functional studies of DHPR expression are lacking. Here we studied electrophysiological and morphological effects of methyl-beta-cyclodextrin (MbetaCD), a cholesterol-sequestering drug, on freshly isolated fetal skeletal muscle cells. Exposure of fetal myofibres to 1-3 mM MbetaCD for 1 h at 37 degrees C led to a significant reduction in caveolae and T-tubule areas and to a decrease in cell membrane electrical capacitance. In whole-cell voltage-clamp experiments, the L-type Ca(2+) current amplitude was significantly reduced, and its voltage dependence was shifted approximately 15 mV towards more positive potentials. Activation and inactivation kinetics were slower in treated cells than in control cells and stimulation by a saturating concentration of Bay K 8644 was enhanced. In addition, intramembrane charge movement and Ca(2+) transients evoked by a depolarization were reduced without a shift of the midpoint, indicating a weakening of E-C coupling. In contrast, T-type Ca(2+) current was not affected by MbetaCD treatment. Most of the L-type Ca(2+) conductance reduction and E-C coupling weakening could be explained by a decrease of the number of DHPRs due to the disruption of caveolae and T-tubules. However, the effects on L-type channel gating kinetics suggest that membrane cholesterol content modulates DHPR function. Moreover, the significant shift of the voltage dependence of L-type current without any change in the voltage dependence of charge movement and Ca(2+) transients suggests that cholesterol differentially regulates the two functions of the DHPR.

Initiation and Transduction of Stretch-induced RhoA and Rac1 Activation through Caveolae: CYTOSKELETAL REGULATION OF ERK TRANSLOCATION

The Rho family small GTPases play a crucial role in mediating cellular responses to stretch. However, it remains unclear how force is transduced to Rho signaling pathways. We investigated the effect of stretch on the activation and caveolar localization of RhoA and Rac1 in neonatal rat cardiomyocytes. In unstretched cardiomyocytes, RhoA and Rac1 were detected in both caveolar and non-caveolar fractions as assessed using detergent-free floatation analysis. Stretching myocytes for 4 min activated RhoA and Rac1. By 15 min of stretch, RhoA and Rac1 had dissociated from caveolae, and there was decreased coprecipitation of RhoA and Rac1 with caveolin-3. To determine whether compartmentation of RhoA and Rac1 within caveolae was necessary for stretch signaling, we disrupted caveolae with methyl beta-cyclodextrin (MbetaCD). Treatment with 5 mm MbetaCD for 1 h dissociated both RhoA and Rac1 from caveolae. Under this condition, stretch failed to activate RhoA or Rac1. Stretch-induced actin cytoskeletal organization was concomitantly impaired. Interestingly the ability of stretch to activate extracellular signal-regulated kinase (ERK) was unaffected by MbetaCD treatment, but ERK translocation to the nucleus was impaired. Stretch-induced hypertrophy was also inhibited. Actin cytoskeletal disruption with cytochalasin-D also prevented stretch from increasing nuclear ERK, whereas actin polymerization with jasplakinolide restored nuclear translocation of activated ERK in the presence of MbetaCD. We suggest that activation of RhoA or Rac1, localized in a caveolar compartment, is essential for sensing externally applied force and transducing this signal to the actin cytoskeleton and ERK translocation.

Cholesterol Depletion from the Plasma Membrane Triggers Ligand-independent Activation of the Epidermal Growth Factor Receptor

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.