The intracellular concentration of cholesterol is maintained within a narrow range by several feedback mechanisms that halt intracellular sterol synthesis and uptake via the LDL ()receptor(1) . Elevation of cellular cholesterol results in suppression of transcription of genes for several cholesterol biosynthetic enzymes and the LDL receptor. Transcription and translation of HMG-CoA reductase mRNA are reduced, and degradation of the enzyme is increased by sterol and nonsterol factors(2) . Excess cholesterol within the cell is rapidly converted to its ester by the action of acyl-CoA:cholesterol acyltransferase and stored in cytoplasmic droplets(3) . The net result is decreased cholesterol synthesis and storage of cholesterol until such time that it is required for synthesis of membranes, lipoproteins, bile acids, or hormones.

The cellular metabolite responsible for feedback repression of cholesterol synthesis has not been identified. Presumably, when sterol concentrations reach a critical level, cholesterol or a related metabolite triggers a regulatory cascade that culminates in transcriptional suppression and the other sterol-regulated events described above. A class of potential regulatory molecules is oxygenated sterols. These cholesterol derivatives contain, in addition to the 3-hydroxyl group, a hydroxyl, keto, or epoxide moiety(4, 5) . Oxysterols, such as 25-hydroxycholesterol, produce regulatory responses in cultured cells similar to those of LDL, and the most potent are effective at nanomolar concentrations. The mechanism of oxysterol suppression of cholesterol synthesis is unclear. A high affinity binding protein for oxysterols, oxysterol-binding protein (OSBP), has been identified (6, 8, 9) that displays affinity for various oxysterols proportional to suppression of HMG-CoA reductase and cholesterol synthesis in cultured cells(4, 7) . A clue to OSBP function came from immunofluorescence studies on CHO cells overexpressing the rabbit OSBP cDNA. OSBP was found to undergo translocation from a cytoplasmic/vesicular compartment to the Golgi apparatus in the presence of ligand(10) . This suggested that the Golgi apparatus could be a target for oxysterol action, perhaps by modifying sterol trafficking between cholesterol-rich plasma membrane and cholesterol-poor ER(11) .

Since localization of OSBP to the Golgi apparatus was disrupted by BFA (10) , we further tested whether BFA would disrupt the regulatory actions of OSBP or other oxysterol signaling pathways requiring an intact Golgi apparatus. Here, BFA and monensin was used to assess the involvement of the Golgi apparatus in mediating the effects of 25-hydroxycholesterol on suppression of mRNAs for several sterol-regulated genes in CHO and BFA-resistant PtK1 cells. The effect of BFA on stimulation of cholesterol esterification by 25-hydroxycholesterol was also investigated.

EXPERIMENTAL PROCEDURES

Materials

Cell Culture

mRNA Analysis

Fluorescence Staining of the Golgi Apparatus

Acyl-CoA:Cholesterol Acyltransferase Assays

Protein Synthesis

RESULTS

Dose-dependent Reversal by Brefeldin A of 25-Hydroxycholesterol Suppression of mRNAs

Figure 1: 25-Hydroxycholesterol-mediated suppression of mRNA in BFA-treated CHO cells. CHO cells were cultured in medium B for 18-24 h prior to the start of experiments. Cells then received 6 ml of medium B containing the indicated concentrations of BFA (0-2 µg/ml). Following a 15-min incubation at 37 °C, new medium was added containing 25-hydroxycholesterol (2.5 µg/ml) and the indicated concentrations of BFA. Cells were incubated for 4 h and harvested, and mRNA was quantitated. The results are expressed relative to values for cells grown in medium B without additions and are the average of two separate experiments normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA.

Results of experiments in which cells were pretreated with 25-hydroxycholesterol (2.5 µg/ml) for 4 h prior to the addition of BFA and oxysterol are shown in Fig. 2. BFA increased HMG-CoA reductase and LDL receptor mRNA levels in a dose-dependent fashion by 30-40% relative to control, but did not return mRNA to fully induced levels. HMG-CoA synthase mRNA was suppressed more completely than the other three mRNAs, but the degree of reversal by BFA (30% increase relative to control) was similar when compared with the LDL receptor and HMG-CoA reductase. FPP synthase mRNA was also measured in these experiments and was found not to be suppressed as efficiently by 25-hydroxycholesterol compared with the other three mRNAs or induced by BFA to the same degree.

Figure 2: Reversal of mRNA suppression by BFA in CHO cells pretreated with 25-hydroxycholesterol. Cells were cultured for 18-24 h in medium B prior to the addition of 6 ml of medium B containing 25-hydroxycholesterol (2.5 µg/ml). After a 4-h incubation at 37 °C, cells received fresh medium B containing 25-hydroxycholesterol (2.5 µg/ml) and the indicated concentrations of BFA. After 3 h, cells were harvested, total RNA was isolated, and mRNA was quantitated as described under ``Experimental Procedures.'' Results are expressed relative to control cells grown in medium B without oxysterol or BFA and are the average of two separate experiments normalized to expression of glyceraldehyde-3-phosphate dehydrogenase.

The morphology of the Golgi apparatus in cells treated with increasing concentrations of BFA was monitored using FITC-labeled lentil lectin (Fig. 3). FITC-labeled lentil lectin staining of untreated CHO cells revealed brightly fluorescent structures clustered at one pole of the nucleus, indicative of the Golgi apparatus (Fig. 3A). Treatment with 0.1 µg/ml BFA (Fig. 3B) for 4 h did not alter this pattern appreciably; however, treatment with 0.2 µg/ml BFA (Fig. 3C) produced some fragmenta-tion of the Golgi apparatus and increased diffuse staining. This latter BFA concentration produced a mixed population of cells with intact, partially disrupted, or absent Golgi staining. Higher concentrations of BFA (0.5 and 1 µg/ml; Fig. 3, D and E, respectively) resulted in uniform disruption of the Golgi apparatus and diffuse staining around the nucleus, indicating absorption of lentil lectin-binding oligosaccharides into the ER. BFA concentrations that produced partial or complete disruption of the Golgi apparatus correlated with reversal of 25-hydroxycholesterol suppression of mRNAs as shown in Fig. 1and 2. FITC-labeled lentil lectin staining of the Golgi apparatus in the presence of 25-hydroxycholesterol (2.5 µg/ml) and increasing BFA concentrations showed patterns of staining indistinguishable from results shown in Fig. 3(data not shown).

Figure 3: Fluorescence localization of the Golgi apparatus in BFA-treated CHO cells. Cells were cultured in medium B for 18 h prior to the addition of fresh medium B containing 0 (A), 0.1 (B), 0.2 (C), 0.5 (D), or 1.0 (E) µg/ml BFA. After treatment with BFA for 4 h, cells were fixed and stained with FITC-labeled lentil lectin as described under ``Experimental Procedures.'' Control cells received ethanol. Bar, 10 µm.

The concentration of BFA required to disrupt the Golgi apparatus in these studies was found to be higher then previously reported: 0.1 µg/ml BFA is usually sufficient to collapse the Golgi apparatus into the ER(24) . This can be explained by the relatively long incubation times required to achieve suppression of sterol-regulated transcription and active degradation of BFA in CHO cells(25) . However, extended incubations with relatively high concentrations of BFA did not adversely affect cell viability. Incubation of CHO cells for 4 h with 0.1 and 1.0 µg/ml BFA inhibited protein synthesis (as measured by [S]methionine/cysteine incorporation) by 0 and 15%, respectively, relative to untreated controls.

Effect of BFA on mRNA Levels in Control and Oxysterol-treated Cells

Figure 4: Suppression of mRNA by 25-hydroxycholesterol in control and BFA-treated CHO cells. A, CHO cells were cultured in medium B for 18-24 h prior to the start of experiments. Cells were pretreated with 6 ml of medium B containing either 1 µg/ml BFA () or ethanol solvent () for 15 min before the addition of 2.5 µg/ml 25-hydroxycholesterol. Cells were incubated for the indicated times, RNA was harvested, and mRNAs for sterol-regulated genes were assayed as described under ``Experimental Procedures.'' Results are the means ± S.D. of four separate experiments. Values are relative to expression in cells cultured for 4 h in medium B with no additions. B, shown are representative autoradiograms of S1 nuclease protection and primer extension assays. The band above the regulated HMG-CoA synthase S1 nuclease product is residual undigested probe. Films were developed after 2-12 h of exposure to dried gels at -70 °C. 25-OH, 25-hydroxycholesterol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Disruption of the Golgi Apparatus Is Necessary to Reverse Suppression of mRNA by 25-Hydroxycholesterol

Figure 5: BFA does not reverse suppression of HMG-CoA reductase mRNA by 25-hydroxycholesterol in PtK1 cells. CHO and PtK1 cells were grown in medium B (supplemented with 1 mM pyruvate for PtK1 cells) for 24-36 h prior to treatment for 6 h with medium B containing ethanol solvent (no additions (NA)), 2.5 µg/ml 25-hydroxycholesterol (25-OH), 1 µg/ml BFA, or both (BFA/25-OH). Cells received BFA 15 min prior to oxysterol addition. RNA was harvested, and HMG-CoA reductase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were quantitated as described under ``Experimental Procedures.'' Results are expressed relative to cells that received no additions and are the means ± S.D. of five separate experiments.

We also investigated the effect of forskolin on Golgi disruption by BFA. Forskolin was previously shown to prevent disruption of the Golgi apparatus by BFA via a cAMP-independent mechanism(24) . This was not the case for mRNA suppression, as illustrated by the lack of effect of forskolin on BFA reversal of oxysterol suppression (Fig. 6). Treatment of cells with 100 µM forskolin had no effect on base-line mRNA levels of HMG-CoA reductase or HMG-CoA synthase. Similarly, forskolin in combination with BFA and 25-hydroxycholesterol did not suppress mRNA levels to those of 25-hydroxycholesterol-treated cells during a 4-h incubation period. Forskolin (100 µM) prevented disruption of the Golgi apparatus by a 30-min treatment with 0.1 µg/ml BFA, as determined by FITC-labeled lentil lectin fluorescence staining, but was ineffective when cells were treated for 4 or 6 h with 1 µg/ml BFA (data not shown).

Figure 6: Forskolin does not reverse BFA suppression of HMG-CoA reductase and HMG-CoA synthase mRNAs by 25-hydroxycholesterol. CHO cells were cultured in medium B for 18-24 h prior to the start of experiments. Cells were pretreated with forskolin (FK; 100 µM) and BFA (1 µg/ml) for 60 and 30 min, respectively, prior to the addition of 25-hydroxycholesterol (25-OH; 2.5 µg/ml) for 4 h (cells treated with BFA/25-hydroxycholesterol/forskolin received forskolin first). Cells not receiving these additions (no additions (N/A)) were mock-treated with ethanol solvent. Results are expressed relative to values for cells grown in medium B and are the average of two separate experiments normalized to expression of glyceraldehyde-3-phosphate dehydrogenase.

Effect of Monensin on Oxysterol-regulated mRNA

Figure 7: 25-Hydroxycholesterol suppression of mRNA in CHO cells treated with monensin. CHO cells were grown in medium B for 18 h prior to the start of experiments. A, fresh medium B containing 2.5 µg/ml 25-hydroxycholesterol (25-OH); 10 µM monensin (Mon) and 2.5 µg/ml 25-hydroxycholesterol; 100 µM monensin and 2.5 µg/ml 25-hydroxycholesterol; or 100 µM monensin was added to cells for 4 h. Cells were pretreated with the indicated concentration of monensin for 30 min prior to oxysterol addition. Results are the means ± S.D. of four experiments. B, cells received medium B with 25-hydroxycholesterol (2.5 µg/ml) for 4 h. Fresh medium was added containing the indicated concentrations of monensin and 2.5 µg/ml 25-hydroxycholesterol for an additional 3 h. Total RNA was isolated, and mRNAs for the LDL receptor (), HMG-CoA reductase (), HMG-CoA synthase (), and FPP synthase () were assayed as described under ``Experimental Procedures.'' Results are the average of two experiments and are expressed relative to mRNA from cells grown in medium B with no additions.

Similar to the results for BFA shown in Fig. 2, monensin (50-100 µM) reversed the suppressive effects of 25-hydroxycholesterol on HMG-CoA reductase after CHO cells had been exposed to oxysterol for 4 h (Fig. 7B). However, the ionophore did not reverse suppression of mRNA for the LDL receptor, HMG-CoA synthase, or FPP synthase. The effect of monensin on Golgi apparatus structure was assessed by fluorescence staining with FITC-labeled lentil lectin (Fig. 8). Monensin caused the Golgi apparatus to lose its compact perinuclear staining pattern and to assume a more disaggregated and dilated structure. However, staining did not appear to become diffuse and associated with other organelles as observed with BFA. Protein synthesis in CHO cells (as measured by [S]methionine/cysteine labeling) was inhibited by 40 and 60% during a 4-h incubation with 10 and 100 µM monensin, respectively.

Figure 8: Fluorescence localization of the Golgi apparatus in monensin-treated CHO cells. Cells were cultured in medium B for 18 h prior to the addition of fresh medium B containing 0 (A), 10 (B), or 100 (C) µM monensin. After treatment with monensin for 4 h, cells were fixed and stained with FITC-labeled lentil lectin as described under ``Experimental Procedures.'' Control cells received ethanol. Bar, 10 µm.

Prevention of oxysterol suppression of mRNAs by BFA or monensin could be due to message stabilization. To test this possibility, we treated CHO cells with actinomycin D (10 µg/ml) in the presence of 1 µg/ml BFA, 100 µM monensin, or no additions and determined transcript levels for HMG-CoA reductase, HMG-CoA synthase, and glyceraldehyde-3-phosphate dehydrogenase after 2 and 4 h (Fig. 9). The half-life of HMG-CoA reductase was 4.4 h in control cells, compared with 5.2 h in BFA-treated cells and 9.2 h in monensin-treated cells. Glyceraldehyde-3-phosphate dehydrogenase mRNA was considerably more stable, and half-lives ranged from 20 h in control and BFA-treated cells to 45 h in monensin-treated cells (glyceraldehyde-3-phosphate dehydrogenase mRNA levels decreased by <10% after 4 h). HMG-CoA synthase mRNA stability was not appreciably altered by BFA or monensin treatment (half-lives ranged from 7 to 5.5 h). Similarly, LDL receptor mRNA stability was unaltered by BFA or monensin (data not shown).

Figure 9: Effect of BFA and monensin on stability of mRNAs for HMG-CoA reductase, HMG-CoA synthase, and glyceraldehyde-3-phosphate dehydrogenase. CHO cells were cultured in medium B for 18 h prior to the addition of medium B containing 10 µg/ml actinomycin D and either 100 µM monensin (, ), 1 µg/ml BFA (, ), or ethanol solvent alone (, ). Cells were harvested for RNA immediately following medium addition (0 h) and at 2 and 4 h. Solidsymbols in the upperpanel indicate glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, and opensymbols indicate HMG-CoA reductase mRNA. Results are representative of two separate experiments.

BFA- and Oxysterol-activated Cholesterol Esterification

DISCUSSION

In this study, we sought to identify the Golgi apparatus as a target for the action of oxysterols in suppressing transcription of sterol-regulated genes. The recent identification of a membrane-bound transcription factor in the ER that undergoes sterol-regulated proteolysis to a soluble nuclear form is the first clear evidence that membrane sterol content regulates transcription(30, 31) . However, it is still unclear how ER cholesterol content is regulated and the immediate target for this pool of regulatory sterol. Results from this study indicate that disruption of the Golgi apparatus by BFA makes the aforementioned pathway for sterol-regulated transcription insensitive to oxysterol.

Measurement of steady-state mRNA levels of four sterol-regulated genes revealed that BFA consistently prevented suppression by 25-hydroxycholesterol in CHO cells. BFA is a fungal toxin that selectively disrupts the cis/medial/trans-elements of the Golgi apparatus(32) , with limited effects on the trans-Golgi network, endosomal system, and lysosomes(33, 34) . BFA blocks anterograde transport from the ER to the Golgi apparatus in the presence of sustained retrograde movement, causing absorption of Golgi membranes and contents into the ER(32) . This is achieved by inhibiting GTP exchange on the ADP-ribosylation factor, thus preventing coatomer assembly and vesicle formation(35, 36) . The degree to which BFA prevented oxysterol suppression of transcription varied, as did the extent of suppression by 25-hydroxycholesterol of the four mRNAs. The reasons for these differences are unclear, but suggest that the promoters of the four genes respond differently to oxysterol and disruption of the Golgi apparatus by BFA. This was most evident for HMG-CoA synthase, where BFA pretreatment ( Fig. 1and Fig. 4) was only partially effective in preventing suppression by 25-hydroxycholesterol. This suggests that there is more than one mechanism for suppression of HMG-CoA synthase mRNA and that BFA is only effective in blocking one component of transcriptional suppression. There is some evidence that sterols act via different response elements and transcription factors depending on the promoter. HMG-CoA reductase is suppressed by sterols, but suppression is not mediated by the same sterol regulatory element (37) or sterol regulatory element-binding proteins (30, 38, 39) responsible for sterol-regulated transcription of the LDL receptor or HMG-CoA synthase. Similarly, sterol-mediated suppression of FPP synthase transcription does not seem to involve sterol regulatory element-like promoter sequences(23) . However, there must be a common sterol-dependent mechanism that initiates transcriptional suppression of these genes that is in turn inhibited by BFA.

Several findings from this study suggest that an intact functional Golgi apparatus is required for oxysterol-mediated transcriptional suppression. First, disruption by BFA of the fluorescence staining pattern of a Golgi apparatus-specific lectin was found to correlate with inhibition by 25-hydroxycholesterol of sterol-regulated mRNA. Second, PtK1 cells were found to be resistant to BFA effects on 25-hydroxycholesterol-mediated suppression of HMG-CoA reductase mRNA. This rules out the possibility that BFA antagonizes 25-hydroxycholesterol by acting on a target outside the Golgi apparatus. Results with PtK1 cells were also important since forskolin did not antagonize BFA in our system. In other studies, forskolin was only partially effective in reversing stimulation of sphingolipid synthesis (28, 40, 41) or cholesterol esterification (28, 29) by BFA. Third, BFA partially restored mRNA levels in CHO cells pretreated with 25-hydroxycholesterol for 4 h. This finding is particularly important since it suggests that an intact functional Golgi apparatus is necessary for suppression by oxysterol to be maintained after an initial regulatory signal has been received.

Findings similar to those described above for BFA were also seen with another Golgi apparatus-specific agent, monensin. However, in the case of monensin, reversal of 25-hydroxycholesterol suppression was only observed for HMG-CoA reductase mRNA; HMG-CoA reductase mRNA degradation was reduced; and mRNAs for the other three genes were decreased. Differences in the mechanism of action of BFA and monensin could account for some of these results. Unlike BFA, monensin did not completely fragment the Golgi apparatus, which appeared to vacuolate as previously reported(27) , and is specific for the trans-elements of the organelle. It should also be noted that a concentration of monensin 10-fold greater than that required to elicit effects in other systems (27) was necessary to reverse HMG-CoA reductase mRNA suppression.

BFA is known to stimulate cholesterol esterification and to modify lipid metabolism in several cell types(28, 29, 40, 41) , a result that is confirmed here using CHO cells. In this study, BFA and 25-hydroxycholesterol stimulated cholesterol esterification to a similar extent, but when added to cells simultaneously, 70% of the individual activity was reached. There are two possible explanations. First, acyl-CoA:cholesterol acyltransferase is not maximally activated by either 25-hydroxycholesterol and BFA, and adding both compounds to cells is not 100% additive due to saturation of enzyme activity. Second, BFA could have a minor inhibitory effect on 25-hydroxycholesterol-stimulated acyl-CoA:cholesterol acyltransferase activity, but this is masked by the stimulation BFA itself causes. Both of these scenarios imply that BFA has little or no effect on 25-hydroxycholesterol-stimulated acyl-CoA:cholesterol acyltransferase activity. BFA has been shown to partially block increased cholesterol esterification caused by sphingomyelinase treatment of intact cells, suggesting that it will modify cholesterol influx to the ER and acyl-CoA:cholesterol acyltransferase activity under some conditions (42) .

The observation that BFA and 25-hydroxycholesterol stimulate acyl-CoA:cholesterol acyltransferase activity suggests that both compounds cause sequestration of cholesterol within the ER, where it is available for esterification. This could be accomplished by stimulating influx of cholesterol from another membrane (i.e. Golgi apparatus) or inhibiting movement of cholesterol out of the ER. Since BFA does not inhibit cholesterol transport from the ER (43) and based on the mechanism of action of BFA and results with PtK1 cells, the latter scenario involving redistribution of Golgi apparatus cholesterol seems more probable. If absorption of Golgi apparatus cholesterol into the ER is responsible for BFA-mediated acyl-CoA:cholesterol acyltransferase activation, then the Golgi apparatus must be a relatively rich source of cholesterol. The cholesterol content of the Golgi apparatus can be altered by LDL deprivation or increased cholesterol synthesis, as measured indirectly by the fluorescence properties of a ceramide analogue that localizes to the Golgi apparatus (44) . Cisternae of the Golgi apparatus have a distinct polarity in cholesterol content, with the trans-elements enriched in sterol relative to the cis-elements(45, 46) . Changes in cholesterol content of the Golgi apparatus might indicate that flow of sterol into or out of the organelle was perturbed, perhaps as the result of increased cholesterol synthesis or uptake of lipoproteins.

Little is known of the effects of BFA on other aspects of cholesterol regulation. BFA did not influence degradation of a HMG-CoA reductase/-galactosidase fusion protein in response to mevalonate, but did increase the protein half-life 2-fold in the absence of this degradative signal(47) . The lack of effect of BFA on mevalonate-mediated degradation precludes the Golgi apparatus in reductase degradation. However, stabilization in the absence of mevalonate is difficult to reconcile given that increased ER cholesterol levels and cholesterol esterification result from BFA treatment.

OSBP is a potential candidate for transducing the effects of oxysterols either by interaction with the Golgi apparatus or by release from a vesicular or cytoplasmic pool(7) . If OSBP translocation/attachment to the Golgi apparatus is required for transducing the effects of oxysterols, then disruption of the Golgi apparatus would interrupt this process. This is precisely the result when BFA is used to antagonize 25-hydroxycholesterol suppression of sterol-regulated mRNAs. There is no direct evidence that OSBP has signal transduction properties, but recent reports have shown that OSBP and a number of other proteins with known or suspected roles in signal transduction have a domain with homology to pleckstrin, a protein kinase C substrate in platelets(48) . The pleckstrin homology domain is thought to mediate protein-protein or protein-lipid interactions(49, 50) .

If, as this study suggests, the Golgi apparatus is involved in transducing the signal for transcriptional suppression by 25-hydroxycholesterol, then it follows that lipid or protein sorting and trafficking in the Golgi network are regulated by oxysterols. Alternatively, absorption of Golgi proteins or lipids into the ER makes transcription insensitive to 25-hydroxycholesterol by a mechanism that has yet to be identified.

FOOTNOTES

*

This work was supported by a grant and a scholarship (to N. D. R.) from the Medical Research Council of Canada and by an establishment grant from Izaak Walton Killam Children's Hospital. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Dept. of Pediatrics and Biochemistry and the Atlantic Research Center, Dalhousie University, 5849 University Ave., Halifax, Nova Scotia B3H 4H7, Canada. Tel.: 902-494-7133; Fax: 902-494-1394.

()

The abbreviations used are: LDL, low density lipoprotein; HMG, 3-hydroxy-3-methylglutaryl; OSBP, oxysterol-binding protein; CHO, Chinese hamster ovary; ER, endoplasmic reticulum; BFA, brefeldin A; FITC, fluorescein isothiocyanate; FPP synthase, farnesyl-diphosphate synthase.

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