Dual roles for cholesterol in mammalian cells

Abstract

The structural features of sterols required to support mammalian cell growth have not been fully defined. Here, we use mutant CHO cells that synthesize only small amounts of cholesterol to test the capacity of various sterols to support growth. Sterols with minor modifications of the side chain (e.g., campesterol, β-sitosterol, and desmosterol) supported long-term growth of mutant cells, but sterols with more complex modifications of the side chain, the sterol nucleus, or the 3-hydroxy group did not. After 60 days in culture, the exogenous sterol comprised >90% of cellular sterols. Inactivation of residual endogenous synthesis with the squalene epoxidase inhibitor NB-598 prevented growth in β-sitosterol and greatly reduced growth in campesterol. Growth of cells cultured in β-sitosterol and NB-598 was restored by adding small amounts of cholesterol to the medium. Surprisingly, enantiomeric cholesterol also supported cell growth, even in the presence of NB-598. Thus, sterols fulfill two roles in mammalian cells: (i) a bulk membrane requirement in which phytosterols can substitute for cholesterol and (ii) other processes that specifically require small amounts of cholesterol but are not enantioselective.

Sterols are essential components of eukaryote membranes. Their incorporation enhances the packing of the acyl chains of phospholipids in the hydrophobic phase of the bilayer, increases its mechanical strength, and reduces its permeability (1). Despite this crucial role, most animal species (e.g., nematodes and arthropods) cannot make sterols and so must get them from the diet. Vertebrates, by contrast, make sterols de novo from acetyl-coenzyme A and so do not require exogenous sterols. Accordingly, studies to probe sterol requirements of eukaryotes have used in invertebrates (2, 3), protazoans (4, 5), or yeast strains defective in sterol synthesis (6–8), so that the sterol composition of the organism can be controlled exogenously.

Although phytosterols can account for a substantial portion of total dietary sterols (approximately one-third in humans), vertebrates systematically exclude them from the body. Cholesterol predominates in the membranes of most animals and is virtually the exclusive sterol of vertebrates. Invertebrates such as insects typically convert phytosterols to cholesterol by dealkylating C24 in the side chain, thereby furnishing cholesterol needed by membranes and preventing accumulation of noncholesterol sterols. Mammals and other vertebrates can either make sterols de novo or get cholesterol from the diet. Accumulation of other dietary sterols in these animals is prevented by the action of two ATP-binding cassette transporters, ABCG5 and ABCG8 (9), which function as a heterodimer to limit intestinal absorption and facilitate biliary excretion of noncholesterol sterols (10, 11). Thus, cholesterol comprises the great majority of vertebrate sterols, even in animals ingesting large quantities of phytosterols.

The biological basis for selection of cholesterol as the major sterol in animal membranes is unclear. Bloch (12) proposed that cholesterol is the most effective sterol at modulating membrane properties and that sterols with bulkier side chains owing to alkylation at C24 interfered with sterol packing of the acyl chains of membrane phospholipids (1). Silbert and colleagues (13) examined the ability of noncholesterol sterols to support growth of a mouse fibroblast cell line (S₂) that fails to demethylate lanosterol, a late step in the cholesterol synthesis, and is thus a cholesterol auxotroph. These cells grew at reduced rates when the plant sterol campesterol (24α-methylcholesterol) was substituted for cholesterol in the medium and failed to grow when provided the other major plant sterol, β-sitosterol (24α-ethylcholesterol). These data suggest that cholesterol is specifically required for growth of mammalian cells. Because only a single mutant cell line was examined, it remains possible that other mutations acquired during mutagenesis could be responsible for their specific requirement for cholesterol.

To determine the structural features that sterols need to support growth of mammalian cells, we used three different mutant CHO cell lines that make only small amounts of cholesterol. They fail to activate sterol regulatory element-binding protein (SREBP) 1 and SREBP-2, membrane-bound transcription factors that target multiple genes involved in cholesterol synthesis and lipoprotein uptake. SREBPs are made as ≈120-kDa precursors located in the membranes of the endoplasmic reticulum (ER) and nuclear envelope. When cellular cholesterol falls, SREBP cleavage-activating protein (SCAP) escorts SREBP from the ER to the Golgi, where it is sequentially cleaved by two different enzymes, site-1 protease (S1P) and site-2 protease (S2P), to release the transcriptionally active N-terminal domain, which then goes to the nucleus, where it activates genes needed for lipid synthesis (14).

We studied three independent cell lines harboring mutations in SCAP (SRD-13A cells), S1P (SRD-12B cells), or S2P (M19 cells) (15–17). These mutations block SREBP processing and reduce cholesterol synthesis by >95%. Because the mutant cells do not survive in media lacking cholesterol and unsaturated fatty acids, they offer an excellent system for determining the ability of various sterols, alone or in combination, to support growth of mammalian cells.

Here, we demonstrate sterol synergism in mammalian cells, indicating that sterols also fulfill multiple functions in mammals. A bulk requirement can be met by various closely related sterols, including cholesterol, β-sitosterol, campesterol, and ent-cholesterol. The finding that ent-cholesterol (see below) supports growth of mammalian cells suggests that the bulk of cellular sterols serve a membrane function involving interaction with nonenantioselective lipids rather than with proteins, which usually are stereospecific.

Materials and Methods

Materials. Lipoprotein-deficient FBS (LPDS) (ρ > 1.215 g/ml containing >2.8 μg/ml cholesterol), sodium mevalonate, sodium oleate, and sodium compactin were prepared as described in refs. 18 and 19. β-Sitosterol was purchased from Sigma. Enantiomeric cholesterol, a stereoisomer of natural cholesterol, has the opposite configuration at every stereogenic (“chiral”) center (natural cholesterol is 3R, 8S, 9S, 10R, 13R, 14S, 17R, 21R; ent-cholesterol is 3S, 8R, 9R, 10S, 13S, 14R, 17S, 21S). It was made as described in ref. 20. All other sterols were from Steraloids (Newport, RI). Purity of the sterols was determined by GC/MS. The squalene epoxidase inhibitor NB-598 was a gift from Banyu Pharmaceutical (Tokyo) or was purchased from Sigma. Each lot was prepared as a nominally 10 mM stock in DMSO and titered to determine the lowest concentration that consistently killed all WT cells in the absence of added sterols under our assay conditions (ranging from 0.5 to 3.0 μM).

Cultured Cells. Cells were maintained in monolayer culture at 37°C in a 9% CO₂ incubator. CHO-7 cells are a subline of CHO-K1 cells selected for growth in LPDS (21). They are maintained in medium A (a 1:1 mixture of Ham's F12 medium and DMEM containing 100 units/ml penicillin and 100 μg/ml streptomycin sulfate) supplemented with 5% (vol/vol) LPDS (18, 19). M19 cells (22), SRD-12B cells (23), and SRD-13A cells (17) are previously described mutant CHO cells auxotrophic for cholesterol, mevalonate, and unsaturated fatty acid due to a deficiency of S2P (15), S1P (16), or SCAP (17), respectively. They were maintained in medium B (medium A supplemented with 5% FBS/1 mM sodium mevalonate/20 μM sodium oleate/5 μg/ml cholesterol). The stock cultures of mutant cells were selected with amphotericin B weekly to maintain the mutant phenotype (23).

Growth Assays. For assays, cells were plated on day 0 at a density of 30,000 cells per well in six-well plates in medium A (for WT) or medium B (for mutants). On day 1, cells were washed with PBS and refed with medium A supplemented with LPDS, 1 mM sodium mevalonate, 20 μM sodium oleate, and various sterols as indicated in the figure legends. Cells were refed every 1–2 days. On day 10, cells were washed, fixed in 95% ethanol, and stained with crystal violet or analyzed for sterol content as described below.

For long-term growth assays (>60 days), cells were plated at 500,000 per 100-mm plate and fed every 2–3 days. Once each week, sets of cells were split 1:5 and 1:10 into fresh plates.

To quantify growth, stained plates were scanned on a UMAX Astra 4000U scanner (UMAX Technologies, Dallas) in transmitted light mode at a resolution of 300 dots per inch. The images were analyzed by using nih imagej software to determine the average pixel value from a circular area of 64,744 pixels, corresponding to one-half the area of the well, centered on the well. The resultant values were subtracted from 255 to yield arbitrary units corresponding to the degree of growth at each condition tested. This method yielded results comparable (r² = 0.933) to quantitation of total cellular protein by standard solution assay (BCA Protein Assay kit, Pierce).

Determination of Cellular Sterol Composition. On day 10, cells were washed once with PBS, harvested by scraping in PBS, and transferred to a 15-ml conical tube. After centrifugation, the cell pellet was washed once with PBS. After removal of the supernatant, the pellet was frozen in liquid nitrogen and stored at -80°C until analyzed.

For analysis, we resuspended cell pellets in 1 ml of 0.1 M NaOH and vortexed them for 30 min. We determined protein concentration as above. An aliquot of ethanol containing the internal standards 5α-cholestane (50 μg) and epicoprostanol (2.5 μg) was added to 200 μl of cell lysate, and sterols were hydrolyzed by heating (to 100°C) in ethanolic KOH (100 mM) for 2 h. Lipids were extracted in petroleum ether, dried under nitrogen, and derivatized with hexamethyldisilazane-trimethylchlorosilane. GC/MS analysis was performed by using a 6890N gas chromatograph coupled to a 5973 mass selective detector (both from Agilent Technologies, Palo Alto, CA). The trimethylsilyl-derived sterols were separated on an HP-5MS 5%-phenyl methyl polysiloxane capillary column (30-m × 0.25-mm inside diameter × 0.25-μm film) with carrier gas helium at the rate of 1 ml/min. The temperature program was 150°C for 2 min, followed by increasing the temperature by 20°C per min up to 280°C and holding it for 13 min. The injector was operated in the splitless mode and was kept at 280°C. The mass spectrometer was operated in selective ion monitoring mode. The extracted ions were 458.4 (cholesterol), 343.3 (desmosterol), 458.4 (lathosterol), 456.4 (zymosterol), 382.4 (campesterol), 393.4 (lanosterol), and 396.4 (β-sitosterol).

Results

Noncholesterol Sterols Support Growth of Mammalian Cells. The mutant cell lines used in this study are maintained in medium containing 5 μg/ml cholesterol to support optimum growth (17, 22, 23). To determine the minimum dose of exogenous cholesterol required to support growth of these cells, cells were grown in media containing between 0 and 5 μg/ml cholesterol. All three cell lines showed substantial growth at 2 or 3 μg/ml cholesterol (see the supporting information, which is published on the PNAS web site); thus, these concentrations were used for subsequent studies. Next, we tested a panel of different sterols for the ability to support growth of the three cell lines. Specific sterols were selected to examine the features of the side chain, the sterol nucleus, and the 3-hydroxy group that are required for cell growth (Fig. 1). WT cells grew well in the absence of sterols and in the presence of all sterols tested except cholestenone, a cholesterol metabolite with a hydroxy group at C3 and a Δ4 double bond, and lanosterol. This biosynthetic precursor to cholesterol contains three methyl groups (4, 4′, and 14) and a 5β-H. Thus, the conformation of its A/B ring system is different from that of cholesterol.

Fig. 1.

Growth of cholesterol auxotrophs in noncholesterol sterols. On day 1, cells (WT and auxotrophs) were fed with medium A containing 5% (vol/vol) LPDS, 1 mM mevalonate, 20 μM oleate, and the indicated sterol at 2 μg/ml in ethanol (final concentration of 0.2% ethanol for all media). Cells were refed every 2 days thereafter until day 10, when cells were fixed and stained.

Absent added sterols, auxotrophic cell lines failed to grow (Fig. 1). Cholesterol and its immediate precursor, desmosterol, supported growth equally well. Sterols with minor modifications of the side chain, including unsaturation (desmosterol) or methylation or ethylation of C24 (campesterol and β-sitosterol), supported robust growth. Cells that were fed sterols with more complex side-chain modifications (stigmasterol) or lacking the side chain (androsterol) showed little or no growth (Fig. 1). Under our assay conditions, cholesterol and β-sitosterol supported similar growth rates in mutant and WT cells (see the supporting information).

Lathosterol, the Δ7 isomer of cholesterol, supported cell growth (Fig. 1), but the cells efficiently metabolized it to cholesterol (see below). Although the three auxotrophic cell lines respond similarly to most sterols tested, for some, the S1P^- cells respond differently than do S2P^- or SCAP^- cells (e.g., poor growth in desmosterol and no growth in lathosterol). We do not currently understand the basis of these differences among the auxotrophic cell lines.

Modification of the sterol nucleus by 5α saturation (dihydrocholesterol) or isomerization of the double bond from Δ5 to Δ4 (4-cholesten-3-β-ol) resulted in very poor cell growth (Fig. 1). An intact 3-hydroxy group proved essential for sterols to support growth. When this group was oxidized (5-cholesten-3-one), deleted (5-cholestene), or epimerized (5-cholesten-3α-ol), the auxotrophs failed to grow (Fig. 1). Therefore, a planar nucleus and an unblocked equatorial hydroxyl group are essential to the function of sterols in mammalian cells.

Added Sterols Are Major Sterols of Auxotrophic Cells. To determine whether the mutant cells converted noncholesterol sterols to cholesterol, we analyzed cellular sterol content by GC/MS (see the supporting information). In WT cells, cholesterol was the predominant sterol under all conditions tested. Exogenous sterols comprised <38% of total cellular sterols after 10 days of growth, presumably reflecting endogenous synthesis of cholesterol in WT cells. The accumulation of desmosterol when these cells are fed stigmasterol likely reflects its competitive inhibition of sterol Δ24-reductase, the enzyme that converts desmosterol to cholesterol (24). In the mutant cell lines, the exogenous sterols comprised 64–96% of total cellular sterols after 10 days. Thus, exogenous sterols were not converted into cholesterol, except in cells fed lathosterol, where cholesterol comprised >60% of total sterol. Basal activity of lathosterol 5-desaturase in these cells may be sufficient to catalyze the conversion of lathosterol to cholesterol even in the absence of SREBP-mediated transcription. Alternatively, the reaction may be catalyzed by other enzymes in hamster cells.

To further assess the responses of the auxotrophic cell lines to noncholesterol sterols, we performed dose–response experiments with the sterols that supported growth: cholesterol, β-sitosterol, campesterol, and stigmasterol (Fig. 2). Desmosterol had previously been shown to support growth of rodent cells in the absence of cholesterol (25, 26) and was not tested further. Although significant variation in growth was observed among the auxotrophic cells at lower doses of sterol, growth was similar to that of WT cells when β-sitosterol or campesterol was added at a concentration of 3 μg/ml (Fig. 2). Stigmasterol supported growth of auxotrophs less well than did the other phytosterols and showed a weak inhibitory effect on WT cells.

Fig. 2.

Effect of sterol concentration on cell growth. On day 1, cells (WT and auxotrophs) were fed with medium containing 5% (vol/vol) LPDS, 1 mM mevalonate, 20 μM oleate, and the sterol to be tested at the indicated concentration (final concentration of 0.3% ethanol for all media). Cells were refed every 2 days thereafter until day 10, when cells were fixed and stained. Growth was quantitated as described and plotted as arbitrary units.

Toxic Sterols. Lanosterol and cholestenone not only failed to support growth of the cholesterol auxotrophs but also killed WT cells (Fig. 1). Previous studies showed that 25-hydroxycholesterol (25HC), which also kills WT cells, suppresses the cleavage of SREBPs, resulting in a transcriptional deficit of cholesterol biosynthetic genes (27). The toxic effects of 25HC (1 μg/ml) can be overcome by providing exogenous cholesterol to the cells (21). We thus tested the ability of cholesterol to rescue cells from the toxic effects of lanosterol and cholestenone. WT cells grew well in medium supplemented with LPDS both in the absence of exogenous sterols and in the presence of 7-dehydrocholesterol (cholesta-5,7-diene-3β-ol). Addition of cholestenone or lanosterol at 2 μg/ml killed the cells (Fig. 3A). Cholesterol supplied as free cholesterol (5 μg/ml in ethanolic solution) or as LDL from complete FBS, rescued cell growth in the presence cholestenone (Fig. 3A) and lanosterol (Fig. 3B). In contrast with 1 μg/ml 25HC, at 2 μg/ml, 25HC was toxic under the conditions tested, even when cholesterol was added to the media, which may reflect actions of this compound on processes other than SREBP cleavage.

Fig. 3.

Effects of sterols on SREBP-2 cleavage. On day 1, WT cells were fed with medium A containing 5% (vol/vol) LPDS, 1 mM mevalonate, 20 μM oleate, and the indicated sterols at 3 μg/ml (final concentration of 0.3% ethanol for all media). Cells were refed every 2 days thereafter. (A) Cells were fixed and stained on day 5. (B) Cells were fixed and stained on day 10. (C)On day 0, WT cells were set up in medium A at 500,000 per 10-cm dish. On day 2, cells were refed inducing medium [medium A containing 5% (vol/vol) LPDS, 50 μM compactin, and 50 μM mevalonate (17)] or the same medium further supplemented with 10 μg/ml cholesterol and a 2 μg/ml concentration of the indicated sterol. All media were adjusted to 0.3% ethanol. After 4.5 h, N-acetyl-leucinyl-leucinyl norleucinal (ALLN) was added to a final concentration of 50 μg/ml. Cells were harvested 90 min later and lysed in SDS buffer [10 mM Tris·HCL, pH 7.6/100 mM NaCl/1% SDS containing protease inhibitors pepstatin A (5 μg/ml), lupeptin (10 μg/ml), ALLN (25 μg/ml), aprotinin (1,000 units/ml), and phenymethylsulfonyl fluoride (0.5 mM)]. Aliquots (50 μg of protein) were analyzed by SDS/PAGE and Western blotting with 5 μg/ml monoclonal antibody 7D4 against hamster SREBP-2 (39). P, membrane-bound SREBP-2 precursor; N, cleaved, nuclear SREBP-2.

To determine whether the toxicity of cholestenone was mediated by a mechanism similar to that observed for 25HC, we tested its ability to repress cleavage of SREBP-2. In this experiment, cells were cultured overnight in the presence of compactin (an inhibitor of 3-hydroxy-3-methyglutaryl-coenzyme A reductase that induces SREBP cleavage owing to cholesterol depletion) and mevalonate and then treated with the indicated sterol at 2 μg/ml for 5 h before harvest. Cells not treated with sterol (Fig. 3C, lanes 1 and 5) or treated with 7-dehydrocholesterol (lane 2) or cholestenone (lane 3) showed no suppression of SREBP-2 cleavage. Cholesterol delivered in ethanolic solution also failed to suppress cleavage of SREBPs, most likely because of its very low solubility. It does suppress cleavage when delivered as a complex with methyl-β-cyclodextrin (28). 25HC delivered in ethanol abolished cleavage (lane 4). These results suggest that the mechanism by which cholestenone kills WT cells is distinct from that of 25HC and that it does not involve acute suppression of SREBP cleavage. Lanosterol also fails to suppress cleavage of SREBPs under these conditions, and its cytotoxic effects are not due to acute suppression of SREBP cleavage (29).

Small Amounts of Cholesterol Are Essential for Cell Growth. Even after 60 days in cholesterol-free medium containing 2 μg/ml campesterol, cholesterol comprised 3% of total sterols in the auxotrophic cells. These trace levels of cholesterol presumably reflect residual endogenous synthesis and are not sufficient to support cell growth (Figs. 1 and 2). To determine whether β-sitosterol and campesterol can support cell growth in the absence of cholesterol, WT and SCAP^- cells were grown in the presence of a squalene epoxidase inhibitor, NB-598 (30). NB-598 blocks cholesterol synthesis but does not block formation of early intermediates in the cholesterol synthetic pathway (e.g., isoprenoids) that are required for cell growth. Cholesterol supported growth of both WT and SCAP^- cells grown in NB-598 (Fig. 4); however, growth was markedly reduced when cells were grown in campesterol plus NB-598. β-Sitosterol alone did not support growth of either cell line in the presence of NB-598 (Fig. 4A). This finding suggests that small amounts of cholesterol are essential for cell growth, even when cells are provided with permissive doses of noncholesterol sterols such as β-sitosterol.

Fig. 4.

Growth in the presence of the squalene epoxidase inhibitor NB-598. (A) Cholesterol content of WT and auxotrophic cells. On day 0, cells were set up as described in Materials and Methods. On day 1, cells were washed and refed medium A supplemented with 3% (vol/vol) LPDS/1 mM sodium mevalonate/20 μM sodium oleate/3 μg/ml of the indicated sterol. Cells received vehicle alone (-) or 3 μM NB-598 (+). Each condition was set up in quadruplicate. On day 10, two wells were harvested for sterol analysis, and the remaining wells were fixed and stained as described. Sterol content could not be determined for samples having no surviving cells. The graph shows total cholesterol content for each sample. Error bars indicate the range of values. Below each sample is a fixed and stained dish showing growth. (B) Cholesterol stimulation of growth. Cells were set up as in A, but the medium contained 0.5 μM NB-598 and the indicated concentrations of cholesterol in the presence or absence of 3 μg/ml β-sitosterol. Each condition was set up in triplicate. Cells were refed every 1–2 days until day 10, when cells were fixed and stained. Growth was quantified as described. The standard error of the mean for all samples ranged between 0.4 and 7.0. (C) Cellular cholesterol content (percentage of total sterols) versus the cholesterol composition of growth medium (percentage of total sterols) for the cells that received β-sitosterol. Cellular sterols were measured by GC/MS as described. Total sterols ranged from 0.442 to 5.533 μg/well. r² = 0.985 by linear regression analysis.

To test this hypothesis, we added increasing concentrations of cholesterol to the media of cells grown in the presence of NB-598 and in the presence or absence of 3 μg/ml β-sitosterol (Fig. 4B). Addition of cholesterol at levels that are themselves too low to support growth markedly enhanced growth of cells provided with β-sitosterol. Similar results were observed for SCAP^- cells (not shown). The cellular sterol composition in these experiments closely reflected the composition of sterols added to the media (r² = 0.99; Fig. 4C). Thus, mammalian cells have a specific requirement for cholesterol that cannot be fulfilled by other sterols.

Requirement for Cholesterol Is Not Enantiospecific. Interactions between sterols and proteins tend to be enantioselective, whereas those between sterols and membrane lipids show little or no enantioselectivity. To define the stereospecificity of the cellular sterol requirement, we grew cells in the presence of ent-cholesterol that has the opposite configuration at every chiral center as compared with native cholesterol (20). Auxotrophic cells grew equally well when either cholesterol or ent-cholesterol was added to the medium (Fig. 5B). In the presence of NB-598, WT and SCAP^- cells grew equally well in both enantiomers (Fig. 5C). Ent-cholesterol can satisfy the bulk requirement that can also be met by various modified sterols (e.g., campesterol and β-sitosterol) and meet the specific requirement for small amounts of cholesterol itself (see the supporting information). Therefore, in mammalian cells, the essential functions of cholesterol do not depend on its absolute configuration.

Fig. 5.

Requirement for cholesterol is not enantiospecific. (A) Cholesterol (Upper) and ent-cholesterol (Lower), its mirror image. (B) Ent-cholesterol supports growth of auxotrophic cells. The experiment was conducted as described in the legend of Fig. 1. (C) Ent-cholesterol supports growth in the presence of NB-598. The experiment was conducted as described in the legend of Fig. 4. Error bars indicate the range of values. Below each sample is a fixed and stained dish showing growth.

Discussion

Our major finding is that the cellular requirement for sterols is highly specific with respect to structure but is not enantioselective. Sterols with complex side chains or with modifications of the nucleus or the 3-hydroxy group did not support growth in three cell lines that require exogenous sterols. The major plant sterols, β-sitosterol and campesterol, supported growth similarly to cholesterol (Figs. 1 and 2) without being converted to cholesterol. Thus, these phytosterols can substitute for cholesterol as the primary sterol in the plasma membrane. However, the cholesterol auxotrophs invariably contained small amounts of cholesterol (<10% of total sterol), even after 60 days in culture in the presence of noncholesterol sterols, owing to residual cholesterol synthesis.

We therefore examined the effect of inhibition of cholesterol synthesis with the squalene epoxidase inhibitor NB-598. This treatment completely blocked growth of cells supplemented with β-sitosterol and markedly reduced growth of cells cultured in campesterol. Growth of cells cultured in the presence of the inhibitor NB-598 was restored when the media were supplemented with cholesterol or ent-cholesterol. These data indicate that mammalian cells can use several naturally occurring sterols in their plasma membranes but that small amounts of cholesterol or ent-cholesterol are required for an unidentified, essential process. We cannot exclude the possibility that the small amounts of cholesterol remaining in auxotrophs and cells treated with NB-598 (Fig. 5A) may be essential to yet other processes in which neither phytosterols nor ent-cholesterol can function.

All naturally occurring sterols include a planar ring system, an aliphatic side chain, and a hydroxyl group at the C-3 position (1). The 3β-OH group confers an amphiphilic character on the otherwise hydrophobic cholesterol molecule and thereby orients it in membranes. Whereas 3β-OH sterols are ubiquitous in nature, Bloch and his colleagues (32, 33) found that methyl cholesterol ethers supported growth of the sterol requiring prokaryote Mycoplasma capricolum and of a sterol auxotrophic mutant yeast strain (6, 33). Thus, Bloch questioned whether the 3β-OH is mandatory for sterol function or simply arises as a consequence of sterol synthesis. 5-Cholestene, cholestenone, and 3α-epicholesterol did not support growth of auxotrophic cells, which agrees with previous work in which cholesteryl methyl-ether and 3α-epicholesterol failed to support growth of a macrophage-like cell line in lipid-depleted medium (34). These findings suggest that an equatorial hydroxyl group at C-3 is essential for mammalian cells.

A planar ring system is also a common feature of sterols that support mammalian cell growth. Cholesterol has a tetracyclic ring structure consisting of three six-membered rings, denoted A, B, and C, and one five-membered ring denoted D (Fig. 1). The B–C and C–D ring fusion occurs at single bonds whose substituents are trans, allowing the ring system to adopt a planar conformation. The double bond at the AB ring junction prevents the A and B rings from adopting full chair conformations. This Δ5 double bond is essential for cell growth; dihydrocholesterol, the 5α-reduced derivative of cholesterol, did not support growth of the auxotrophs in this study, even though it retains the coplanar conformation of the A and B cyclohexyl rings. A mutant mouse fibroblast line (LM) in which sterol synthesis is blocked at desmosterol grew at reduced rates when supplemented with dihydrocholesterol. In that study, the mutant LM cells adapted to growth in dihydrocholesterol by desaturating and elongating the fatty acids in their membrane phospholipids (13). The enzymes that thus modify fatty acids are SREBP targets. The auxotrophs lack active SREBP, so the failure of dihydrocholesterol to support even modest growth may result from an inability of the auxotrophs to alter the fatty acid composition of their phospholipids.

Cyclization of squalene epoxide to lanosterol generates a rigid tetracyclic ring system with a flexible isooctyl side chain that is retained in all naturally occurring sterols. Our results show that the side chain is essential for growth of mammalian cells. Androsterol, which has no side chain, did not support growth. Modification of the side chain had varying effects; C-24-alkylated sterols with saturated side chains (campesterol and β-sitosterol) supported robust growth. Stigmasterol (unsaturated at C-22) supported growth only poorly.

An unanticipated finding of this study was that the precursor of sterols in fungi and animals, lanosterol, was toxic to WT cells. Toxicity was not observed with later stage precursors such as lathosterol or desmosterol. Cholesterol rescued lanosterol toxicity, indicating that lanosterol inhibited cholesterol synthesis. Similar findings were observed with the synthetic sterol cholestenone. The toxic sterol 25HC kills cells by inhibiting SREBP cleavage, thereby indirectly suppressing cholesterol synthesis. However, neither lanosterol (not shown) nor cholestenone suppressed cleavage of SREBP-2 (Fig. 5C). These data suggest that lanosterol and cholestenone inhibit cholesterol synthesis by an SREBP-independent pathway. Recently, Song et al. (29) demonstrated that lanosterol accelerates degradation of 3-hydroxy-3-methyglutaryl (HMG)-coenzyme A (CoA) reductase, an essential enzyme in cholesterol biosynthesis. This effect is independent of SREBP cleavage. In contrast to lanosterol, cholestenone does not promote degradation of HMG-CoA reductase (29). Deciphering how cholestenone inhibits cholesterol synthesis may thus provide insight into the mechanisms regulating cholesterol homeostasis.

The C24-alkylated sterols campesterol and β-sitosterol are abundant in nature and are the major dietary sterols of herbivores. Because these sterols meet the bulk membrane requirement of mammalian cells, why is this class of sterols so assiduously excluded from accumulating in vertebrates? Our data strongly suggest that the adverse effects of plant sterols are not a consequence of disruption of membrane integrity. Rather, certain noncholesterol sterols may disrupt cholesterol homeostasis. The accumulation of noncholesterol sterols in the adrenal glands of mice lacking ABCG5 and ABCG8 causes profound alterations in cholesterol metabolism. The cholesterol content of the adrenal glands in these animals is reduced 90%. Despite this pronounced reduction, these animals fail to up-regulate the cholesterol homeostatic machinery (38). When these animals were treated with ezetimibe, a drug that blocks sterol absorption, the content of noncholesterol sterols in the adrenal gland decreased sharply, and cholesterol homeostasis approached normal. Thus, the active exclusion of noncholesterol sterols from the body may be needed to maintain normal cholesterol homeostasis.

Studies of sterol requirements in the prokaryotic sterol auxotroph M. capricolum showed that these cells grew better when supplied with both cholesterol and lanosterol than with either sterol alone (35). These findings were taken to indicate that sterols play more than one role in membranes. Later, sterol synergism was demonstrated in unicellular eukaryotes including yeast (36) and Paramecium (37). Yeast mutants that require exogenous sterols can grow in a variety of sterols if trace amounts of ergosterol are present. Analysis of cellular free sterol levels and plasma membrane properties suggested that sterols such as cholesterol and dihydrocholesterol satisfy a bulk membrane requirement, whereas ergosterol serves a highly specific function (35). A similar phenomenon was observed in the natural auxotroph Paramecium tetraurelia, which has an absolute requirement for one of a small group of structurally related phytosterols including stigmasterol, β-sitosterol, and poriferasterol (37). Stigmasterol at levels as low as 20 ng/ml supports growth if a second relatively nonspecific sterol is provided at higher concentrations (1 μg/ml).

The function of the small quantities of cholesterol required by auxotrophs to sustain growth remains elusive. It may be that cholesterol plays an essential structural role that cannot be met by C24-alkylated sterols. Alternatively, cholesterol may be a precursor of an essential metabolite that cannot be made from C24-alkylated sterols. Ent-cholesterol rescued growth of auxotrophs in the presence of NB-598, suggesting that the essential function of cholesterol is not mediated by an enantiospecific protein. Further studies will be required to elucidate the cellular process or processes that specifically require cholesterol to the exclusion of other sterols.