In addition to suppressing cholesterol synthesis and uptake, oxysterols also activate glycerophospholipid and SM (sphingomyelin) synthesis, possibly to buffer cells from excess sterol accumulation. In the present study, we investigated the effects of oxysterols on the CDP-choline pathway for PtdCho (phosphatidylcholine) synthesis using wild-type and sterol-resistant CHO (Chinese-hamster ovary) cells expressing a mutant of SCAP [SREBP (sterol-regulatory-element-binding protein) cleavage-activating protein] (CHO-SCAP D443N). [3H]Choline-labelling experiments showed that 25OH (25-hydroxycholesterol), 22OH (22-hydroxycholesterol) and 27OH (27-hydroxycholesterol) increased PtdCho synthesis in CHO cells as a result of CCTα (CTP:phosphocholine cytidylyltransferase α) translocation and activation at the NE (nuclear envelope). These oxysterols also activate PtdCho synthesis in J774 macrophages. in vitro, CCTα activity was stimulated 2- to 2.5-fold by liposomes containing 5 mol% 25OH, 22OH or 27OH. Inclusion of up to 5 mol% cholesterol did not further activate CCTα. 25OH activated CCTα in CHO-SCAP D443N cells leading to a transient increase in PtdCho synthesis and accumulation of CDP-choline. CCTα translocation to the NE and intranuclear tubules in CHO-SCAP D443N cells was complete after 1 h exposure to 25OH compared with only partial translocation by 4–6 h in CHO-Mock cells. These enhanced responses in CHO-D443N cells were sterol-dependent since depletion with cyclodextrin or lovastatin resulted in reduced sensitivity to 25OH. However, the lack of effect of cholesterol on in vitro CCT activity indicates an indirect relationship or involvement of other sterols or oxysterol. We conclude that translocation and activation of CCTα at nuclear membranes by side-chain hydroxylated sterols are regulated by the cholesterol status of the cell.
The CDP-choline or Kennedy pathway is the primary route for de novo synthesis of PtdCho (phosphatidylcholine), the most abundant phospholipid in eukaryotic cells. This pathway supplies phospholipid that is essential for cell growth and division [1,2], as well as other metabolic processes such as the syntheses of SM (sphingomyelin) and phosphatidylserine, pulmonary surfactant, lipoproteins and bile . The rate-limiting step in the CDP-choline pathway is catalysed by CCT (CTP:phosphocholine cytidylyltransferase), an amphitropic enzyme that is active when membrane-associated and inactive in its soluble form [4,5]. CCTα and CCTβ isoforms are encoded by separate genes and have divergent tissue and subcellular distributions [6,7]. Unlike CCTβ, which is primarily in the cytosol and ER (endoplasmic reticulum), an N-terminal nuclear localization signal promotes CCTα translocation into the nucleus in many, but not all, cell types [8,9]. Nuclear CCTα undergoes translocation to the NE (nuclear envelope) and NR (nucleoplasmic reticulum) in response to two classes of lipid activators [10–12]. Electrostatic interaction between negatively charged lipids, such as fatty acids, and positive-charged residues in the α-helical domain M of CCTα promotes membrane association. Membranes enriched in type II lipids, such as DAG (diacylglycerol) and phosphatidylethanolamine, have increased curvature stress and discontinuities that enhance insertion of domain M [13,14]. This mechanism for CCTα activation allows for rapid modulation of the CDP-choline pathway in response to membrane content of PtdCho and its lipid precursors.
While it is apparent that CCTα and the CDP-choline pathway are subject to regulation by phospholipids and their catabolites, the effects of sterols and sterol precursors are poorly defined. Previous studies using cultured cells showed that CCTα and the CDP-choline pathway are co-regulated with cholesterol metabolism to ensure a constant ratio of phospholipid and cholesterol (reviewed in ). CCTα activity and PtdCho synthesis in macrophages were increased in response to cholesterol loading as a mechanism to maintain sterol/phospholipid ratios and prevent sterol toxicity [16,17]. Consistent with this interpretation, macrophages devoid of CCTα activity were more sensitive to cholesterol-induced cytotoxicity . Failure of PtdCho synthesis to keep pace with cholesterol accumulation eventually leads to activation of the ER unfolded protein response and CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein 10]-mediated apoptosis . The mechanism of increased PtdCho synthesis in this model involved enhanced dephosphorylation of membrane-associated CCTα . This appeared to be a macrophage-specific response since PtdCho synthesis was not increased in CHO (Chinese-hamster ovary) cells in response to a similar cholesterol-loading regime. CCTα and PtdCho synthesis in CHO cells is regulated by the SREBP (sterol-regulatory-element-binding protein)/SCAP (SREBP cleavage-activating protein) pathway [21,22], a sterol-regulated proteolytic cascade that processes SREBPs to mature transcription factors required for expression of cholesterol and fatty acid biosynthetic genes . CCTα activity and PtdCho synthesis were increased as a consequence of SREBP regulation of fatty acid synthesis [22,24] and by transcriptional induction of CCTα [25,26].
Oxysterols regulate cholesterol synthesis, esterification and efflux, but are also implicated in the control of PtdCho homoeostasis. 22OH (22-hydroxycholesterol) in combination with retinoic acid inhibit PtdCho synthesis in murine lung epithelial cells by promoting CCTα docking to MAPK (mitogen-activated protein kinase) and increased phosphorylation . Oxidized lipoproteins inhibited surfactant PtdCho synthesis by calpain-mediated proteolysis of CCTα . In contrast, 25OH (25-hydroxycholesterol) treatment of macrophages for 24 h increased PtdCho synthesis . While these studies provide insights into the long-term effects of cytotoxic concentrations of oxysterols on PtdCho, there is scant information on initial short-term response by the CDP-choline pathway, particularly with respect to direct effects of oxysterols on CCTα activity. In the present study, we investigated how oxysterols affect the PtdCho synthesis and CCTα using CHO cells and a mutant made sterol-resistant by expression of SCAP D443N (CHO-SCAP D443N). The D443N mutation in the SCAP sterol-sensing domain prevents the cholesterol-dependent interaction with Insig-1 and Insig-2 [29,30], resulting in increased cholesterol, cholesterol ester and PtdCho synthesis . Metabolic labelling, cell fractionation and immunofluorescence experiments showed that 22OH, 25OH and 27OH (27-hydroxycholesterol) activated CCTα by translocation to nuclear membranes in CHO cells, an effect that was enhanced in sterol-resistant CHO-SCAP D443N cells. Based on in vitro assays with PtdCho liposomes, oxysterols, but not cholesterol, behave as type II activators of CCTα.
25OH, 22OH and 27OH were purchased from Steraloids (Wilton, NH, U.S.A.). Complete™ protease inhibitor cocktail was from Boehringer Mannheim. [methyl-3H]Choline and [1-14C]acetate were from Mandel-NEN (Guelph, ON, Canada). [methyl-14C]Phosphocholine was purchased from Amersham Biosciences or American Radiochemicals. Tissue culture medium and reagents were purchased from Gibco BRL. LPDS [lipoprotein-deficient FCS (fetal calf serum)] was prepared as previously described .
CHO-SCAP D443N and CHO-Mock cells were derived from CHO7 cells (CHO cells adapted to growth in delipidated serum) by stable overexpression of pTK-HSV-SCAP (D443N)-T7 . CHO-SCAP D443N cells were maintained in DMEM (Dulbecco's modified Eagle's medium) with 5% LPDS and 0.3 μg of 25OH/ml. CHO-Mock cells were cultured in DMEM with 5% LPDS (medium A). For experiments, CHO-SCAP D443N and CHO-Mock cells were cultured in medium A. Wild-type CHO cells were maintained in DMEM with 5% (v/v) FCS (medium B). J774 macrophages were cultured in DMEM with 10% FCS.
Analysis of labelled PtdCho, choline metabolites and sterols
CHO cells were cultured in medium A or B for 24 h prior to the start of experiments. Cells then received oxysterol or solvent control and at different intervals were either (i) pulse-labelled with 2 μCi of [3H]choline/ml for 30 or 60 min or (ii) continuously labelled with 2 μCi of [3H]choline/ml (see the Figure legends for further details). After rinsing with 2 ml of cold PBS, cells were harvested by scraping in 1 ml of methanol/water (5:4, v/v) and transferred to screw-cap tubes. [3H]Choline-labelled PtdCho and water-soluble [3H]choline metabolites were extracted as previously described . Aliquots of the aqueous phase were resolved by TLC in ethanol/water/ammonia (48:95:6, by vol.), identified by spraying with phosphomolybdate, and radioactivity was quantified by scintillation counting. Radiolabelled PtdCho was measured by scintillation counting of an aliquot of the chloroform phase (>98% of radioactivity in PtdCho).
The synthesis of cholesterol and biosynthetic intermediates was measured by incubating CHO cells with 5 μCi of [14C]acetate/ml. Cells were harvested in 0.5 M NaOH, transferred to screw-cap tubes and saponified in 3 ml of ethanol and 0.5 ml of 50% KOH for 1 h at 60 °C. The sterol fraction was extracted with hexane and resolved by TLC in light petroleum/diethyl ether/acetic acid (60:40:1, by vol., boiling point, 35–60 °C). Thin-layer plates were exposed to a film at −70 °C and the bands corresponding to cholesterol and lanosterol were identified by co-migration with authentic standards.
CCTα assays, immunoblotting and immunofluorescence
CCTα activity was measured in soluble and particulate (total membrane) fractions of CHO cell homogenates as previously described [21,32]. Briefly, cells were homogenized in 20 mM Tris/HCl (pH 7.4), 5 mM DTT (dithiothreitol) and Complete™ protease inhibitor cocktail using a Dounce homogenizer and centrifuged for 30 min at 15000 g. The pellet was resuspended in the same buffer containing 0.25 M sucrose. CCT activity was assayed by measuring conversion of [methyl-14C]choline into CDP-[14C]choline in soluble and particulate fractions in the presence or absence of PtdCho/oleate (1:1, mol/mol) vesicles (prepared by sonication).
Recombinant rat CCTα (provided by Dr Rosemary Cornell, Simon Fraser University, Burnaby, BC, Canada) was assayed in the presence of sonicated PtdCho vesicles containing 25OH, 22OH or 27OH, with or without cholesterol. Briefly, stock solutions of oxysterols and cholesterol were combined with PtdCho, dried under nitrogen, resuspended in 50 mM Tris/HCl (pH 7.4), 100 mM NaCl and 10 mM MgCl2 and sonicated on ice for 15 s at a 60% power setting (repeated five times). Liposomes (100 μM) were pre-incubated with CCTα (0.4 ng) for 10 min on ice before initiation of the enzyme assay as described above.
Proteins in soluble and particulate fractions were separated by SDS/PAGE, transferred to nitrocellulose and incubated with a CCTα antibody raised against the entire domain P (provided by Dr Martin Post, University of Toronto Hospital for Sick Children, Toronto, ON, Canada) in Tris/HCl (pH 7.4), 150 mM NaCl and 5% (w/v) non-fat dried skimmed milk powder for 2–4 h at 20 °C. This was followed by a goat anti-rabbit horseradish peroxidase-coupled secondary antibody and visualization by the chemiluminescence method (Amersham Biosciences).
Endogenous CCTα in CHO cells was localized by immunofluorescence using a CCTα polyclonal antibody prepared against a domain P peptide (PSPSFRWPFSGKTSP) and a goat anti-rabbit Alexa Fluor® 488-conjugated secondary antibody in cells fixed with 4% (w/v) paraformaldehyde and permeabilized with 0.05% Triton X-100 . Images were acquired using either a Zeiss Axiovert 200M fluorescence microscope equipped with a ×100 oil immersion (1.4 NA) objective and axioCam HRm CCD camera (charge-coupled-device camera) or a Zeiss LSM510 confocal microscope with a ×100 oil immersion objective (1.4 NA).
Activation of CCTα and PtdCho synthesis by oxysterols
25OH is a potent suppressor of cholesterol synthesis and activates SM synthesis via oxysterol-binding-protein-mediated ceramide transport to the Golgi apparatus. To determine whether oxysterols also affected PtdCho, synthesis was quantified in CHO cells treated with 25OH for up to 10 h. PtdCho and levels of the CDP-choline pathway metabolites phosphocholine, CDP-choline and GPC (glycerophosphocholine) were determined by continuous labelling with [3H]choline (Figure 1). Dose–response experiments indicated that 2.5 μg of 25OH/ml (6.2 μM) provided maximal changes in PtdCho and CDP-choline but did not induce apoptosis based on lack of caspase cleavage of CCTα and PARP [poly(ADP-ribose) polymerase], and chromatin condensation (Figure 1A and results not shown). It was also noted that 25OH concentrations between 0.1 and 0.5 μg/ml increased [3H]choline incorporation into PtdCho and CDP-choline. Compared with solvent-treated controls, 25OH (2.5 μg/ml) promoted a significant accumulation of [3H]PtdCho at 8 and 10 h (Figure 1B). There was also a large increase in isotope incorporation into CDP-choline and slight decrease in [3H]phosphocholine indicative of CCTα activation. Increased PtdCho synthesis in 25OH-treated cells was also partially counterbalanced by degradation as indicated by increased [3H]GPC production at all time points.
25OH stimulates PtdCho synthesis in CHO cells
To determine whether other side-chain hydroxylated sterols activate PtdCho synthesis, CHO cells were treated with 22OH and 27OH (Figure 2). 27OH is the most abundant oxysterol in the bloodstream  and has been implicated in cholesterol regulation and transport . 22OH is a potent activator of the liver X receptor and of expression of genes involved in cholesterol disposal pathways . 25OH, 22OH and 27OH treatment of CHO cells for 8 h significantly increased [3H]choline incorporation into PtdCho and CDP-choline (Figures 2A–2D). For all three oxysterols, there was a trend towards increased incorporation into GPC and reduced phosphocholine, but this did not reach significance. PtdCho synthesis was also measured in J774 macrophages to determine whether this response to oxysterols was evident in other cell types (Figure 2E). 25OH, 22OH and 27OH treatment for 4 h produced a dose-dependent increase in [3H]choline incorporation into PtdCho that was greater in magnitude than in CHO cells.
Stimulation of PtdCho synthesis by 22OH, 25OH and 26OH
By virtue of an N-terminal nuclear localization signal, CCTα in CHO cells is in the nucleus and translocates to the NE in response to fatty acids or other lipophilic activators. Indirect immunofluorescence was used to determine whether oxysterol activation of PtdCho synthesis was a consequence of translocation of endogenous CCT to the NE (Figure 3). CCTα was diffusely distributed throughout the nucleoplasm in untreated cells (Figure 3A). Weak translocation of CCTα to the NE was evident after 4 h exposure to 25OH but by 4 and 8 h most of the cells (80%) displayed NE and localization. Translocation to the NE was also observed in CHO cells treated with 22OH and 27OH for 8 h (Figure 3B). Compared with oleate, which promotes rapid (10–20 min) and complete translocation to nuclear membranes , oxysterol caused incomplete CCTα translocation as indicated by residual diffuse nucleoplasmic staining.
Oxysterols promote CCTα translocation to the NE
Oxysterols stimulate CCTα activity in vitro
Similar to cholesterol , 25OH could behave as a type II amphiphilic CCTα activator. To test this, recombinant CCTα activity was assayed in the presence of PtdCho vesicles containing increasing mol% 25OH, 22OH or 27OH, and was compared with the known activator oleate (Figure 4A). PtdCho vesicles containing increasing oxysterols stimulated CCTα approx. 2-fold at 10 mol% compared with 5-fold activation by oleate. Based on this dose–response curve, it was decided to accurately measure activation of CCTα at <10 mol% oxysterol and compare this with cholesterol (Figures 4B–4D). Inclusion of 2.5 or 5 mol% 25OH, 27OH and 22OH resulted in significant 1.5–2.5-fold increase in CCTα activity compared with PtdCho liposomes without oxysterols (Figures 4B–4D). Interestingly, liposomes with 1–5 mol% cholesterol slightly inhibited CCTα activity even when 2.5 and 5 mol% oxysterol was included.
Activation of recombinant CCTα by oxysterols
Enhanced 25OH activation of CCTα in sterol-resistant CHO cells
CHO7 cells expressing a sterol-insensitive version of SCAP (D443N) have elevated cholesterol and fatty acid synthesis that is resistant to suppression by 25OH . Similar to other sterol-resistant CHO cell lines, CHO-SCAP D443N cells have increased PtdCho synthesis that is linked to post-transcriptional activation by fatty acids or fatty-acid-derived activator [22,24]. In the present context, these cells can be used to study whether unregulated SREBP–SCAP activity sensitizes PtdCho synthesis and CCTα to 25OH. Cells were treated with 25OH for the indicated times and, 1 h prior to harvest, were pulse-labelled with [3H]choline to monitor the CDP-choline pathway (Figure 5). Treatment of CHO-Mock cells with 25OH for up to 8 h did not significantly affect [3H]choline incorporation into PtdCho (Figure 5A). However, [3H]choline incorporation into [3H]CDP-choline increased 15–20-fold, while phosphocholine was transiently reduced by 2-fold at 4 h. The effect of 25OH on CHO-SCAP D443N cells was similar but the magnitude of the changes in the CDP-choline pathway was more dramatic (Figure 5A). PtdCho synthesis was transiently stimulated for up to 2 h before declining below baseline levels at 8 h, [3H]CDP-choline levels were increased 4-fold and [3H]phosphocholine was reduced by 60% after 8 h. [3H]GPC levels were increased similarly by 25OH in control and CHO-SCAP D443N cells. Because of parallel increases and decreases in CDP-choline pathway products, the stimulation of CCTα activity is more apparent when downstream (CDP-choline and PtdCho) and degradation (GPC) products are added together (Figure 5B). Total post-CCTα reaction products were increased in CHO-SCAP D443N cells compared with CHO-Mock cells, primarily as a result of the dramatic rise in [3H]CDP-choline levels. Thus, similar to results with CHO cells (Figure 1), 25OH stimulated CCTα and production of [3H]CDP-choline, effectively switching the rate-limiting step in the pathway to choline phosphotransferase.
PtdCho synthesis in 25OH-treated sterol-resistant CHO cells
To test whether the activation of CCTα by 25OH observed in [3H]choline-labelling experiments was accompanied by enzyme translocation to membranes, the distribution of CCTα between soluble and membrane fractions of 25OH-treated CHO-Mock and CHO-SCAP D443N cells was determined by enzyme assays and immunoblotting (Figure 6). A digitonin release assay used to measure soluble/membrane distribution of CCT [33,37] was not reliable under the conditions of the present study owing to differences in sterol content due to SCAP D443N expression and oxysterol treatment. Instead, cells were harvested after treatment with 25OH, homogenized, and soluble and particulate (total membranes) fractions were prepared by centrifugation . CCT activity was then assayed in these fractions in the presence and absence of PtdCho/oleate liposomes. CCT activity in untreated CHO-Mock cells was primarily in the soluble fraction (90%) and translocation to the particulate fraction was not evident after 25OH treatment for 4 h (Figure 6A). Untreated CHO-SCAP D443N cells had a similar initial distribution that was shifted towards the particulate fraction after 25OH treatment. Soluble and particulate fractions from control and 25OH-treated cells were then immunoblotted to confirm CCTα protein translocation (Figure 6B). Consistent with activity measurements, CCTα protein did not translocate to the particulate fraction in CHO-Mock cells, but a substantial shift in CCTα protein from the soluble to the particulate fraction was evident in 25OH-treated CHO-SCAP D443N cells.
Enhanced 25OH-mediated membrane translocation of CCTα in CHO-SCAP D443N cells
Since CCTα could dissociate from membranes during cell homogenization leading to an underestimation of the extent of membrane translocation, CCTα localization was also analysed by immunofluorescence and confocal microscopy in cells treated with 25OH for up to 6 h (Figure 7A). CCTα in untreated CHO-Mock and SCAP D443N cells was localized to the nucleoplasm with little evidence of localization to nuclear membranes, although some NE-associated CCTα was evident in CHO-SCAP D443N cells. 25OH treatment of CHO-Mock cells for 4–6 h resulted in translocation of CCTα to the NE in approx. 30–50% of cells. In 25OH-treated CHO-SCAP D443N, CCTα translocation to the NE was detected after 1 h and was evident in the entire cell population by 2 h. 25OH-treated CHO-SCAP D443N cells also displayed extensive localization to intranuclear membrane tubules (see 4 and 6 h panels) previously identified as the NR . Cytoplasmic CCTα was evident in approx. 10% of CHO-SCAP D443N cells after prolonged oxysterol treatment (indicated by arrows in 6 h panel) indicating nuclear export. To assess whether enhanced CCTα translocation in CHO-SCAP D443N cells involved altered cholesterol content or distribution, cells were depleted of cholesterol with cyclodextrin for 30 min, rinsed to remove cyclodextrin and 25OH was added for 2 h (Figure 7B). CHO-SCAP D443N cells treated with cyclodextrin had a marked reduction in 25OH-mediated CCTα localization to the NE, suggesting that cholesterol enhanced CCTα translocation.
25OH promotes translocation of CCTα to the NE in CHO cells
To test whether elevated cholesterol synthesis was involved in sensitizing CCTα and the CDP-choline pathway to 25OH in CHO-SCAP D443N cells, de novo synthesis was inhibited with lovastatin prior to 25OH addition (Figure 8). CHO-SCAP D443N cells were resistant to suppression of cholesterol synthesis by 25OH, as measured by [14C]acetate incorporation and TLC (Figure 8A), but lovastatin markedly reduced cholesterol synthesis to the level of untreated controls. If cholesterol or other products of the mevalonate pathway were required for CCTα activation by 25OH, then pretreatment of CHO-SCAP D443N cells with lovastatin should abrogate this effect. Similar to results shown in Figure 5, 25OH treatment of CHO-D443N cells for 6 h increased [3H]choline incorporation into CDP-choline, and reduced incorporation into phosphocholine and PtdCho, leading to a net increase in post-CCTα choline metabolites (Figure 8B). Treatment of either cell line with lovastatin alone had no effect on [3H]choline incorporation except to slightly reduce [3H]PtdCho synthesis in CHO-SCAP D443N cells so that it was no longer significant. Treatment of CHO-SCAP D443N cells with lovastatin prior to 25OH partially prevented [3H]CDP-choline accumulation, and restored [3H]choline incorporation into PtdCho, indicating that cholesterol or other mevalonate-derived metabolites sensitized CCTα to 25OH.
Lovastatin reverses the effect of 25OH on the CDP-choline pathway
Membrane composition is maintained by co-ordinating the synthesis, catabolism and efflux of phospholipids, sphingolipids and cholesterol. This ensures membrane integrity and also prevents inappropriate growth inhibition or proliferation related to production of bioactive lipids such as ceramide and DAG. PtdCho synthesis is co-regulated with sterol metabolism at several levels. For example, fatty acid production regulated by the SREBP/SCAP pathway [22,24], intermediates of the cholesterol biosynthetic pathway, such as farnesol and geranylgeraniol [33,38–40], and cholesterol [16,20], suppress or activate individual steps in the CDP-choline pathway. Here, we report that oxysterols, potent regulators of cholesterol homoeostasis, activate CCTα by promoting translocation to nuclear membranes.
Choline-labelling experiments in CHO and sterol-resistant CHO-SCAP D443N cells revealed that 25OH caused a sustained or transient increase in PtdCho, depletion of phosphocholine and accumulation of CDP-choline, indicative of CCTα activation. However, net PtdCho synthesis was markedly blunted by an apparent lack of conversion of CDP-choline into PtdCho and, to a lesser extent, degradation to GPC. As a result, PtdCho synthesis was largely unaffected in CHO-Mock cells but was transiently stimulated and then inhibited in CHO-SCAP D443N cells. Accumulation of CDP-choline was also observed in cells treated with other lipophilic alcohols, such as farnesol and oleyl alcohol, that promote apoptosis, inhibit CPT (cholinephosphotransferase) [40,41] and activate CCTα [10,33]. This accumulation has been attributed to inhibition of CPT under apoptotic conditions. Although 25OH and other oxysterols are reported to induce apoptosis in cultured cells [42,43], we found no evidence of apoptosis after treatment of CHO cells with 22OH, 25OH or 27OH for up to 10 h, probably as a consequence of using lower concentrations and shorter treatment times. A plausible explanation for the accumulation of CDP-choline is that activation of CCTα by oxysterols results in expansion of the CDP-choline pool, effectively shifting the rate-limiting step in the pathway due to substrate saturation of CPT. Fatty acid stimulation also promotes an accumulation of [3H]CDP-choline in CHO cells, indicating that physiological CCTα activators switch the rate-limiting step to CPT .
Treatment of CHO and CHO-SCAP D443N cells with 25OH caused CCTα translocation to membranes that were identified by immunofluorescence as the NE and the NR, a network of tubular invaginations of the NE that extends into the nucleoplasm [10,44,45]. Compared with CHO-SCAP D443N cells, translocation of CCTα was slow in CHO and CHO-Mock cells and incomplete even after 6 h. 25OH-mediated translocation of CCTα to the NR and NE was also accompanied by export from the nucleus in approx. 10% of CHO-SCAP D443N cells after 6 h (Figure 7A). During farnesol-induced apoptosis, CCTα was also translocated to the NE and subsequently exported to the cytoplasm by a mechanism that did not require caspase cleavage . We recently observed that CCTα was also exported from the nucleus during oleate stimulation (K. Gehrig, C. M. Morton and N. D. Ridgway, unpublished work), indicating that nuclear export is the result of extended CCTα activation and residency at the NE or NR.
Stimulation of recombinant CCTα activity by oxysterols in vitro supports the concept that increased activity in cells is the result of altered membrane structure that enhances CCTα translocation and activation. Lipophilic alcohols such as farnesol, oleyl alcohol and mono- and di-acylglycerol are type II lipids that activate CCTα [32,33,46] by increasing curvature stress and/or creation of discontinuities in lipid packing that promotes insertion of domain M [13,47]. Cholesterol is a weak type II activator of CCTα, affording only a 25% increase in activity at relatively high concentration (30–50 mol%) . 25OH has properties similar to cholesterol, such as formation of liquid-ordered and detergent-resistant membranes in model membranes [48,49], but promotes activation of CCTα at much lower concentrations (2.5–5 mol%). 25OH and other side-chain-substituted oxysterols differ from cholesterol by promoting membrane expansion at low mol% and, depending on the surface pressure, are oriented inversely (side chain at the membrane interface) or horizontal to the membrane interface . These physical properties of side-chain-substituted oxysterols could also promote surface packing defects or negative curvature stress that enhances association of CCTα domain M with membranes.
We previously showed that PtdCho synthesis was increased 2-fold in CHO-SCAP D443N cells as well as in two other sterol-resistant CHO cell lines due to elevated fatty acid synthesos [22,24]. Increased PtdCho synthesis in CHO-SCAP D443N was not accompanied by increased CCTα activity or membrane localization, yet these cells displayed increased sensitivity to 25OH. Thus expression of SCAP D443N produced an altered metabolic state that ‘primed’ CCTα for translocation in response to exogenous 25OH. When treated with oxysterols these cells displayed a transient increase in PtdCho synthesis and the intermediate CDP-choline, and increased CCTα translocation. Results showing that cyclodextrin and lovastatin prevented or blunted the effects of 25OH on CCTα translocation and PtdCho synthesis in CHO-SCAP D443N cells respectively suggest that this primed state is due to increased cholesterol, resulting from the sterol-resistant phenotype. The finding that lovastatin converted the [3H]choline metabolic profile in oxysterol-treated CHO-SCAP D443N cells into one resembling untreated cells suggests that oxysterol activation is dependent on cholesterol or other cholesterol derivatives. However, in vitro activity of CCTα was slightly decreased by inclusion of 5 mol% cholesterol in liposomes, indicating that the cholesterol is not directly involved or only activates in the context of a complex biological membrane. Since cyclodextrin potentially extracts other sterols, including oxysterols , and lovastatin reduces the level of all sterol and non-sterol intermediates, we cannot definitely identify the regulatory molecule involved.
Oxysterols produced as a consequence of an increased cellular cholesterol load could act directly on CCTα to increase PtdCho synthesis and prevent cholesterol toxicity. We argue that this could be a physiological mechanism to control PtdCho levels since (i) CCTα and PtdCho synthesis were activated in a dose-dependent manner by relatively low concentrations of oxysterols, (ii) endogenous sterol levels control sensitivity to exogenous oxysterols and (iii) macrophages had a robust response to oxysterols. While these findings have to be confirmed in other experimental settings, results show that direct activation of CCTα by oxysterols is a potential mechanism to maintain the sterol/phospholipid content of membranes within normal boundaries.
Robert Zwicker and Gladys Keddy are acknowledged for their excellent technical assistance.
Dulbecco's modified Eagle's medium
fetal calf serum
SREBP cleavage-activating protein
This work was supported by a Canadian Institutes of Health Research operating grant [grant number MOP 62916], a Cancer Care Nova Scotia trainee award (to T. A. L.) and an IWK trainee award (to K. G.).
Present address: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390-9046, U.S.A.