Use of cyclodextrins to manipulate plasma membrane cholesterol content: Evidence, misconceptions and control strategies

The physiological importance of cholesterol in the cell plasma membrane has attracted increased attention in recent years. Consequently, the use of methods of controlled manipulation of membrane cholesterol content has also increased sharply, especially as a method of studying putative cholesterol-enriched cell membrane domains (rafts). The most common means of modifying the cholesterol content of cell membranes is the incubation of cells or model membranes with cyclodextrins, a family of compounds, which, due to the presence of relatively hydrophobic cavity, can be used to extract cholesterol from cell membranes. However, the mechanism of this activity of cyclodextrins is not completely established. Moreover, under conditions commonly used for cholesterol extraction, cyclodextrins may remove cholesterol from both raft and non-raft domains of the membrane as well as alter the distribution of cholesterol between plasma and intracellular membranes. In addition, other hydrophobic molecules such as phospholipids may also be extracted from the membranes by cyclodextrins. We review the evidence for the specific and non-specific effects of cyclodextrins and what is known about the mechanisms for cyclodextrin-induced cholesterol and phospholipid extraction. Finally, we discuss useful control strategies that may help to verify that the observed effects are due specifically to cyclodextrin-induced changes in cellular cholesterol.     

Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies

The physiological importance of cholesterol in the cell plasma membrane has attracted increased attention in recent years. Consequently, the use of methods of controlled manipulation of membrane cholesterol content has also increased sharply, especially as a method of studying putative cholesterol-enriched cell membrane domains (rafts). The most common means of modifying the cholesterol content of cell membranes is the incubation of cells or model membranes with cyclodextrins, a family of compounds, which, due to the presence of relatively hydrophobic cavity, can be used to extract cholesterol from cell membranes. However, the mechanism of this activity of cyclodextrins is not completely established. Moreover, under conditions commonly used for cholesterol extraction, cyclodextrins may remove cholesterol from both raft and non-raft domains of the membrane as well as alter the distribution of cholesterol between plasma and intracellular membranes. In addition, other hydrophobic molecules such as phospholipids may also be extracted from the membranes by cyclodextrins. We review the evidence for the specific and non-specific effects of cyclodextrins and what is known about the mechanisms for cyclodextrin-induced cholesterol and phospholipid extraction. Finally, we discuss useful control strategies that may help to verify that the observed effects are due specifically to cyclodextrin-induced changes in cellular cholesterol.     

细胞内胆固醇运输

作为生物膜的重要成分,细胞内不同膜上胆固醇含量的高低直接影响生物膜的生物物理特性和细胞信号的传递,与细胞正常的生理功能密切相关。外源内吞的胆固醇和内源合成的胆固醇通过囊泡介导和非囊泡介导的胆固醇运输途径在不同细胞膜之间转运,从而维持了不同细胞器上胆固醇的浓度梯度。一系列胆固醇结合和转运蛋白在细胞内胆固醇的运输中发挥了重要作用。本文旨在总结细胞内胆固醇运输途径与参与胆固醇运输的重要分子及相关作用机制。     

Interaction of hormone-sensitive lipase with steroidogenic acute regulatory protein: facilitation of cholesterol transfer in adrenal

Hormone-sensitive lipase (HSL) is responsible for the neutral cholesteryl ester hydrolase activity in steroidogenic tissues. Through its action, HSL is involved in regulating intracellular cholesterol metabolism and making unesterified cholesterol available for steroid hormone production. Steroidogenic acute regulatory protein (StAR) facilitates the movement of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane and is a critical regulatory step in steroidogenesis. In the current studies we demonstrate a direct interaction of HSL with StAR using in vitro glutathione S-transferase pull-down experiments. The 37-kDa StAR is coimmunoprecipitated with HSL from adrenals of animals treated with ACTH. Deletional mutations show that HSL interacts with the N-terminal as well as a central region of StAR. Coexpression of HSL and StAR in Chinese hamster ovary cells results in higher cholesteryl ester hydrolytic activity of HSL. Transient overexpression of HSL in Y1 adrenocortical cells increases mitochondrial cholesterol content under conditions in which StAR is induced. It is proposed that the interaction of HSL with StAR in cytosol increases the hydrolytic activity of HSL and that together HSL and StAR facilitate cholesterol movement from lipid droplets to mitochondria for steroidogenesis.     

Cholesterol-rich intracellular membranes: a precursor to the plasma membrane

The disposition of newly synthesized sterols in cultured human fibroblasts has been examined in this study. We began by demonstrating that cholesterol mass and exogenously added [3H]cholesterol both are markers for the plasma membrane, perhaps better than 5'-nucleotidase. Cells were incubated with radioactive acetate to label their endogenous sterols biosynthetically, treated with cholesterol oxidase to convert plasma membrane cholesterol to cholestenone, and then homogenized and spun to equilibrium on sucrose gradients. The density gradient profiles of the various organelles were monitored using these markers: plasma membrane, radioactive cholestenone; smooth endoplasmic reticulum, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase); and Golgi apparatus, galactosyltransferase. The buoyant density profiles of radioactive intracellular cholesterol and lanosterol both had a peak at 1.12 g/cm3, similar to 5'-nucleotidase and galactosyltransferase but not to HMG-CoA reductase. This result suggests that cholesterol biosynthesis is not taken to completion in the endoplasmic reticulum. Digitonin treatment shifted the profiles of both plasma membrane and intracellular cholesterol to higher densities. Pretreatment of intact cells with cholesterol oxidase abolished the digitonin shift of plasma membranes but not the intracellular cholesterol, indicating that these two membrane pools are not entirely physically associated. Because intracellular cholesterol was shifted more than any of the organelle markers, it must reside in a separate membrane. Since digitonin selectively shifts the density of membranes rich in cholesterol, we infer that newly synthesized cholesterol accumulates in such membranes prior to its delivery to the plasma membrane. Taken together, these results suggest that cholesterol may be concentrated for delivery to the plasma membrane by being synthesized from a sterol precursor such as lanosterol in a discrete but undefined intracellular membrane.     

STARD4 knockdown in HepG2 cells disrupts cholesterol trafficking associated with the plasma membrane,ER, and ERC

STARD4, a member of the evolutionarily conserved START gene family, has been implicated in the nonvesicular intracellular transport of cholesterol. However, the direction of transport and the membranes with which this protein interacts are not clear. We present studies of STARD4 function using small hairpin RNA knockdown technology to reduce STARD4 expression in HepG2 cells. In a cholesterol-poor environment, we found that a reduction in STARD4 expression leads to retention of cholesterol at the plasma membrane, reduction of endoplasmic reticulum-associated cholesterol, and decreased ACAT synthesized cholesteryl esters. Furthermore, D4 KD cells exhibited a reduced rate of sterol transport to the endocytic recycling compartment after cholesterol repletion. Although these cells displayed normal endocytic trafficking in cholesterol-poor and replete conditions, cell surface low density lipoprotein receptor (LDLR) levels were increased and decreased, respectively. We also observed a decrease in NPC1 protein expression, suggesting the induction of compensatory pathways to maintain cholesterol balance. These data indicate a role for STARD4 in nonvesicular transport of cholesterol from the plasma membrane and the endocytic recycling compartment to the endoplasmic reticulum and perhaps other intracellular compartments as well.     

Pharmacological inhibition of the intracellular transport of low-density lipoprotein-derived cholesterol in Chinese hamster ovary cells

Mammalian cells, cultured in the presence of serum lipoproteins, acquire cholesterol necessary for growth from the uptake and lysosomal hydrolysis of low-density lipoproteins (LDL). The mechanism(s) of intracellular transport of LDL-derived cholesterol from lysosomes to other cellular sites is unknown. In this study, various pharmacological agents were assessed for their ability to inhibit the movement of LDL-cholesterol from lysosomes to the plasma membrane. The only pharmacological agent tested in these experiments that specifically inhibited LDL-cholesterol movement was U18666A. Ketoconazole impaired the intracellular transport of LDL-cholesterol; however, ketoconazole also had a general effect on cholesterol movement, since it impeded the desorption of endogenously synthesized cholesterol into the medium. Other drugs that affected cholesterol movement appeared to be nonspecific. Cholesterol transport from lysosomes to plasma membranes was not significantly altered by agents that affect lysosomal function or cytoskeletal organization, as well as energy poisons and cycloheximide.     

Cholesterol movement between the plasma membrane and the cholesteryl ester droplets of cultured Leydig tumour cells.

The present studies characterize the turnover of plasma membrane cholesterol in MA-10 Leydig tumour cells. Plasma membrane cholesterol of MA-10 cells was slowly internalized and converted into cholesteryl ester. Low-density lipoprotein (LDL) stimulated, in a dose- and time-dependent fashion, plasma membrane cholesterol conversion into intracellular esters. Stimulation of membrane internalization was not simply the consequence of accelerated uptake of membrane with LDL, since binding and internalization of epidermal growth factor and transferrin had no effect on turnover of plasma membrane cholesterol. The protein of LDL is unimportant as well, since delipidated LDL had no effect on membrane turnover. The action of LDL on cholesterol turnover was explained entirely by its contribution to cholesteryl ester stores. The degree of plasma membrane cholesterol internalization and esterification was directly proportional to the size of cellular ester stores.     

Intermediate filaments and lipoprotein cholesterol

The ability of cells to utilize cholesterol derived from lipoprotein is important in plasma membrane biosynthesis, steroidogenesis and the regulation of sterol synthesis. While the endocytosis of lipoprotein-derived cholesterol has been well characterized, the subsequent events that mediate its post-lysosomal intracellular transport are not understood. Recent studies have suggested that vimentin-type intermediate filaments may have a role in cholesterol transport. The mechanism by which vimentin filaments affect this process is not known, but future studies promise to provide new insights into both the post-lysosomal transport of cholesterol and the intracellular functions of intermediate filaments.     

Movement of zymosterol, a precursor of cholesterol, among three membranes in human fibroblasts

Where examined, cholesterol is synthesized in the endoplasmic reticulum; however, its precursor, zymosterol, is found mostly in the plasma membrane. The novel implication of these disparate findings is that zymosterol circulates within the cell. In tracing its movements, we have now established the following: (a) in human fibroblasts, zymosterol is converted to cholesterol solely in the rough ER. (b) Little or no zymosterol or cholesterol accumulates in the rough ER in vivo. (c) Newly synthesized zymosterol moves to the plasma membrane without a detectable lag and with a half-time of 9 min, about twice as fast as cholesterol. (d) The pool of radiolabeled zymosterol in the plasma membrane turns over rapidly, faster than does intracellular cholesterol. Thus, plasma membrane zymosterol is not stagnant. (e) [3H]Zymosterol pulsed into intact cells is initially found in the plasma membrane. It is rapidly internalized and is then converted to [3H] cholesterol. Half of the [3H]cholesterol produced returns to the plasma membrane within 30 min of the initial [3H]zymosterol pulse. (f) Nascent zymosterol accumulates in a buoyant sterol-rich intracellular membrane before it reaches the plasma membrane. This membrane also acquires nascent cholesterol, exogenous [3H]zymosterol pulsed into intact cells, and [3H]cholesterol synthesized from the exogenous [3H] zymosterol. These results suggest that at least one sterol moves rapidly and in both directions among the rough endoplasmic reticulum, a sterol-rich intracellular membrane bearing nascent cholesterol, and the plasma membrane.     

Binding of high density lipoproteins to cell receptors promotes translocation of cholesterol from intracellular membranes to the cell surface

Cultured cells have on their cell surface a specific high-affinity binding site (receptor) for high density lipoproteins (HDL) which appears to promote cholesterol efflux. In this study we characterized the cellular mechanisms involved in HDL receptor-mediated transport of cholesterol from cultured human fibroblasts and bovine aortic endothelial cells. HDL3, chemically modified by tetranitromethane (TNM-HDL3), is not recognized by this receptor and was used as a control for efflux not mediated by HDL receptor binding. HDL3 and TNM-HDL3 were found to be equally effective in causing efflux of plasma membrane cholesterol radiolabeled with [3H]cholesterol. However, HDL3 was much more effective than TNM-HDL3 in causing efflux of [3H]cholesterol associated with intracellular membranes. By measuring movement of endogenously synthesized [3H]cholesterol to the plasma membrane, and into the medium, we found that HDL3 induced a rapid movement of [3H]cholesterol from a preplasma membrane compartment to the plasma membrane that preceded [3H]cholesterol efflux. This effect was not observed with TNM-HDL3. Thus, receptor binding of HDL3 appears to facilitate removal of cellular cholesterol from specific intracellular pools by initiation of translocation of intracellular cholesterol to the plasma membrane.     

Cholesterol homeostasis in T cells. Methyl-beta-cyclodextrin treatment results in equal loss of cholesterol from Triton X-100 soluble and insoluble fractions

Methyl-beta-cyclodextrin (MBCD) is frequently used to acutely deplete cells of cholesterol. A widespread assumption is that MBCD preferentially targets cholesterol in lipid rafts and that sensitivity to MBCD is proof of lipid raft involvement in a cellular process. To analyse any MBCD preference systematically, progressive cholesterol depletion of Jurkat T cells was performed using MBCD and [3H]-cholesterol. It was found that at 37 degrees C, MBCD extracts similar proportions of cholesterol from the Triton X-100 resistant (lipid raft enriched) as it does from other cellular fractions and that the cells rapidly reestablish the relative differences in cholesterol concentration between different compartments. Moreover, cells restore the cholesterol level in the plasma membrane by mobilising cholesterol from intracellular cholesterol stores. Interestingly, mere incubation at 0 degrees C caused a loss of plasma membrane cholesterol with a concomitant increase in cholesteryl esters and adiposomes. Moreover, only 35% of total cholesterol could be extracted by MBCD at 0 degrees C and was accompanied by a complete loss of plasma membrane and endocytotic recycling centre filipin staining. This study clearly shows that MBCD does not specifically extract cholesterol from any cellular fraction, that cholesterol redistributes upon temperature changes and that intracellular cholesterol stores can be used to replenish plasma membrane cholesterol.     

Increased expression of LDL receptor-related protein 1 during human cytomegalovirus infection reduces virion cholesterol and infectivity

In response to virus infection, cells can alter protein expression to modify cellular functions and limit viral replication. To examine host protein expression during infection with human cytomegalovirus (HCMV), an enveloped DNA virus, we performed a semiquantitative, temporal analysis of the cell surface proteome in infected fibroblasts. We determined that resident low density lipoprotein related receptor 1 (LRP1), a plasma membrane receptor that regulates lipid metabolism, is elevated early after HCMV infection, resulting in decreased intracellular cholesterol. siRNA knockdown or antibody-mediated inhibition of LRP1 increased intracellular cholesterol and concomitantly increased the infectious virus yield. Virions produced under these conditions contained elevated cholesterol, resulting in increased infectivity. Depleting cholesterol from virions reduced their infectivity by blocking fusion of the virion envelope with the cell membrane. Thus, LRP1 restricts HCMV infectivity by controlling the availability of cholesterol for the virion envelope, and increased LRP1 expression is likely a defense response to infection.     

Secretory vesicular transport from the Golgi is altered during ATP-binding cassette protein A1 (ABCA1)-mediated cholesterol efflux

Apolipoprotein AI (apoAI)-mediated cholesterol efflux is a process by which cells export excess cellular cholesterol to apoAI to form high density lipoprotein. ATP-binding cassette protein A1 (ABCA1) has recently been identified as the key regulator of this process. The pathways of intracellular cholesterol transport during efflux are largely unknown nor is the molecular mechanism by which ABCA1 governs cholesterol efflux well understood. Here, we report that, in both macrophages and fibroblasts, the secretory vesicular transport changes in response to apoAI-mediated cholesterol efflux. Vesicular transport from the Golgi to the plasma membrane increased 2-fold during efflux. This increase in vesicular transport during efflux was observed in both raft-poor and raft-rich vesicle populations originated from the Golgi. Importantly, enhanced vesicular transport in response to apoAI is absent in Tangier fibroblasts, a cell type with deficient cholesterol efflux due to functional ABCA1 mutations. These findings are consistent with an efflux model whereby cholesterol is transported from the storage site to the plasma membrane via the Golgi. ABCA1 may influence cholesterol efflux in part by enhancing vesicular trafficking from the Golgi to the plasma membrane.     

Cholesterol domains in biological membranes

Membrane cholesterol is distributed asymmetrically both within the cell or within cellular membranes. Elaboration of intracellular cholesterol trafficking, targeting and intramembrane distribution has been spurred by both molecular and structural approaches. The expression of recombinant sterol carrier proteins in L-cell fibroblasts has been especially useful in demonstrating for the first time that such proteins actually elicit intracellular and intra-plasma membrane redistribution of sterol. Additional advances in the use of native fluorescent sterols allowed resolution of transbilayer and lateral cholesterol domains in plasma membranes from cultured fibroblasts, brain synaptosomes and erythrocytes. In all three cell surface membranes, cholesterol is enriched in the inner, cytofacial leaflet. Up to three different cholesterol domains have been identified in the lateral plane of the plasma membrane: a fast exchanging domain comprising less than 10% of cholesterol, a slowly exchanging domain comprising about 30% of cholesterol, and a very slowly or non-exchangeable sterol domain comprising 50–60.

Of plasma membrane cholesterol. Factors modulating plasma membrane cholesterol domains include polyunsaturated fatty acids, expression of intracellular sterol carrier proteins, drugs such as ethanol, and several membrane pathologies (systemic lupus erythematosus, sickle cell anaemia and aging). Disturbances in plasma membrane cholesterol domains after transbilayer fluidity gradients in plasma membranes. Such changes are associated with decreased Ca²⁺ -ATPase and Na ⁺, K⁺ -ATPase activity. Thus, the size, dynamics and distribution of cholesterol domains within membranes not only regulate cholesterol efflux/influx but also modulate plasma membrane protein functions and receptor-effector coupled systems.     

Essentially All Excess Fibroblast Cholesterol Moves from Plasma Membranes to Intracellular Compartments

It has been shown that modestly increasing plasma membrane cholesterol beyond its physiological set point greatly increases the endoplasmic reticulum and mitochondrial pools, thereby eliciting manifold feedback responses that return cell cholesterol to its resting state. The question arises whether this homeostatic mechanism reflects the targeting of cell surface cholesterol to specific intracellular sites or its general equilibration among the organelles. We now show that human fibroblast cholesterol can be increased as much as two-fold from 2-hydroxypropyl-β-cyclodextrin without changing the size of the cell surface pool. Rather, essentially all of the added cholesterol disperses rapidly among cytoplasmic membranes, increasing their overall cholesterol content by as much as five-fold. We conclude that the level of plasma membrane cholesterol is normally at capacity and that even small increments above this physiological set point redistribute essentially entirely to intracellular membranes, perhaps down their chemical activity gradients.     

Cholesterol homeostasis in T cells. Methyl-β-cyclodextrin treatment results in equal loss of cholesterol from Triton X-100 soluble and insoluble fractions

Methyl-β-cyclodextrin (MBCD) is frequently used to acutely deplete cells of cholesterol. A widespread assumption is that MBCD preferentially targets cholesterol in lipid rafts and that sensitivity to MBCD is proof of lipid raft involvement in a cellular process. To analyse any MBCD preference systematically, progressive cholesterol depletion of Jurkat T cells was performed using MBCD and [³H]-cholesterol. It was found that at 37 °C, MBCD extracts similar proportions of cholesterol from the Triton X-100 resistant (lipid raft enriched) as it does from other cellular fractions and that the cells rapidly reestablish the relative differences in cholesterol concentration between different compartments. Moreover, cells restore the cholesterol level in the plasma membrane by mobilising cholesterol from intracellular cholesterol stores. Interestingly, mere incubation at 0 °C caused a loss of plasma membrane cholesterol with a concomitant increase in cholesteryl esters and adiposomes. Moreover, only 35% of total cholesterol could be extracted by MBCD at 0 °C and was accompanied by a complete loss of plasma membrane and endocytotic recycling centre filipin staining. This study clearly shows that MBCD does not specifically extract cholesterol from any cellular fraction, that cholesterol redistributes upon temperature changes and that intracellular cholesterol stores can be used to replenish plasma membrane cholesterol.     

High density lipoprotein stimulates sterol translocation between intracellular and plasma membrane pools in human monocyte-derived macrophages

Binding of high density lipoprotein (HDL) to its receptor on cultured fibroblasts and aortic endothelial cells was previously shown to facilitate sterol efflux by initiation of translocation of intracellular sterol to the plasma membrane. After cholesterol-loaded human monocyte-derived macrophages were incubated with either [3H]mevalonolactone or lipoprotein-associated [3H]cholesteryl ester to radiolabel intracellular pools of sterol, incubation with HDL3 led to stimulation of 3H-labeled sterol translocation from intracellular sites to the cell surface which preceeded maximum 3H-labeled sterol efflux. A similar pattern was demonstrated for macrophages that were preloaded with cholesterol derived from either low density lipoprotein (LDL), acetyl-LDL, or phospholipase C-modified LDL. However, in macrophages that were not loaded with cholesterol, HDL3 stimulated net movement of 3H-labeled sterol from the plasma membrane into intracellular compartments, the opposite direction from that seen for cholesterol-loaded cells. A similar influx pattern was found in nonloaded macrophages and fibroblasts that were labeled with trace amounts of exogenous [3H]cholesterol. Cholesterol translocation from intracellular pools to the cell surface of cholesterol-loaded macrophages appeared to be stimulated by receptor binding of HDL, since chemical modification of HDL with tetranitromethane (TNM), which abolishes its receptor binding, reduced its ability to stimulate 3H-labeled sterol translocation and efflux. In nonloaded cells, however, the ability of HDL3 to stimulate sterol efflux and movement of sterol from the plasma membrane into intracellular pools was unaffected by TNM modification. Thus, binding of HDL to its receptor on cholesterol-loaded macrophages appears to promote translocation of intracellular cholesterol to the plasma membrane followed by cholesterol efflux into the medium. However, in nonloaded macrophages, HDL stimulates sterol movement from the plasma membrane into intracellular pools by a receptor-independent process.