(Received for publication, December 22, 1995)
From the
We have previously reported that degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-limiting enzyme in the isoprenoid pathway leading to cholesterol production, can be accelerated in cultured cells by the addition of farnesyl compounds, which are thought to mimic a natural, nonsterol mevalonate metabolite(s). In this paper we report accelerated reductase degradation by the addition of farnesol, a natural product of mevalonate metabolism, to intact cells. We demonstrate that this regulation is physiologically meaningful, shown by its blockage by several inhibitory conditions that are known to block the degradation induced by mevalonate addition. We further show that intracellular farnesol levels increase significantly after mevalonate addition. Based on these results, we conclude that farnesol is a nonsterol, mevalonate-derived product that plays a role in accelerated reductase degradation. Our conclusion is in agreement with a previous report (Correll, C. C., Ng, L., and Edwards, P. A.(1994) J. Biol. Chem. 269, 17390-17393), in which an in vitro system was used to study the effect of farnesol on reductase degradation. However, the apparent stimulation of degradation in vitro appears to be due to nonphysiological processes. Our findings demonstrate that in vitro, farnesol causes reductase to become detergent insoluble and thus lost from immunoprecipitation experiments, yielding apparent degradation. We further show that another resident endoplasmic reticulum protein, calnexin, similarly gives the appearance of protein degradation after farnesol addition in vitro. However, after the addition of farnesol to cells in vivo, calnexin remains stable, whereas reductase is degraded, providing further evidence that the in vivo effects of farnesol are physiologically meaningful and specific for reductase, whereas the in vitro effects are not.
The isoprenoid metabolic pathway, which leads to the production
of cholesterol among other essential cellular products, is tightly
regulated at the conversion of 3-hydroxy-3-methylglutaryl-coenzyme A
(HMG-CoA) ()to mevalonate, catalyzed by the enzyme HMG-CoA
reductase, a 97-kDa glycoprotein of the endoplasmic reticulum
membrane(1, 2) . The levels of this enzyme are
governed by regulation of
transcription(3, 4, 5) , mRNA
translation(6, 7, 8, 9) , and enzyme
degradation(10, 11, 12, 13) .
We have studied the regulation of the degradation rate of reductase. Although production of sterols has a role in triggering accelerated reductase degradation, it has become clear that an unidentified nonsterol product is necessary for this acceleration to occur. This has been shown by treating cells with a potent competitive inhibitor of reductase, compactin, or by using a cell line, UT2, that lacks HMG-CoA reductase. Under these conditions exogenous cholesterol does not trigger accelerated degradation of reductase, unless the metabolic block is bypassed with exogenous mevalonate(14, 15) . The reciprocal relationship also appears to exist that the nonsterol component requires the presence of the sterol regulatory component to cause accelerated degradation. This has been demonstrated using inhibitors of enzymes in the squalene branch of the isoprenoid pathway(16) , and in our current study using a cell line deficient in squalene synthase. In both of these instances endogenous production of sterols, but not nonsterol products, was effectively blocked, and in both instances accelerated reductase degradation did not occur. The identity of the nonsterol mevalonate-derived regulatory component, however, has remained unknown. Recently Bradfute and Simoni (17) and others (18) reported that reductase degradation can be accelerated in intact cultured cells by the addition of farnesyl derivatives, which appear to act by mimicking the elusive mevalonate-derived metabolite.
Here we report acceleration of reductase turnover in intact cells by the addition of farnesol. We demonstrate that this regulation is physiologically meaningful by showing that the effect of farnesol is sensitive to inhibitory agents and a mutational condition that are known to stunt the regulatory effect of exogenously added mevalonate. Furthermore we report that intracellular levels of farnesol increase after mevalonate addition, a treatment known to accelerate reductase degradation, and decrease after compactin addition, a treatment that blocks mevalonate production and is known to increase reductase stability.
The possible role of farnesol in regulation of reductase degradation has been recently suggested (19) based on findings in an in vitro system. However, several findings of ours suggest that reductase protein loss in this system is largely due to nonphysiological causes. We show that a significant fraction of reductase protein becomes detergent-insoluble during incubation with farnesol in vitro, causing a depletion of immunoprecipitable reductase and thus the appearance of degradation. Also, our studies of another endoplasmic reticulum resident protein, calnexin, reveal that this protein likewise appears to be rapidly lost in permeabilized cells in response to farnesol but actually is largely rendered detergent-insoluble. Neither of these apparently nonphysiological effects occur in intact cells treated with farnesol. Based on our findings, we conclude that farnesol is a likely physiological, nonsterol regulatory molecule with a critical role in accelerated reductase degradation.
For studies of calnexin degradation, the above procedure was
followed except for the following changes. After the pulse and chase
periods, cells were solubilized in 1.0 ml of HBS buffer (50 mM HEPES, pH 7.5, 200 mM NaCl) supplemented with 1% SDS and
boiled for 5 min. Lysates were centrifuged 30 min at 16,000 g, and then supernatants were removed and diluted 10-fold with
HBS containing 1% Triton X-100. Samples were precleared with protein
A-Sepharose and then incubated overnight with 10 µl of
anti-calnexin antibody at 4 °C. Adsorbance of immunoprecipitated
protein was done as in the above procedure, except that washes were
done with HBS containing 1% Triton X-100 and 0.1% SDS.
Figure 1: Farnesol causes accelerated degradation of HMG-CoA reductase in vivo. As described under ``Experimental Procedures,'' CHO cells were pulse-labeled, then chase was done in the presence (closed symbols) or the absence (open symbols) of 30 µM farnesol for 0, 4, 6, 8, or 10 h, and then cells were solubilized. Reductase was immunoprecipitated and subjected to SDS-PAGE, and then bands were quantified by Bio-Rad Molecular Imager. The image presented is from one representative experiment of six, and the graph is a compilation of these experiments.
Additionally, we found that 30 µM farnesol causes a decrease in the rate of reductase synthesis (data not shown). This also has been demonstrated to occur due to the addition of mevalonate (14) or the addition of farnesyl acetate (17) in vivo.
Figure 2: Farnesol-induced accelerated degradation of HMG-CoA reductase is blocked by ALLN and by thapsigargin. CHO cells were pulse-labeled, and then chase was done in the presence of 30 µM farnesol, with or without either 20 µg/ml ALLN (upper gel) or 1 µM thapsigargin (THAP, lower gel) for 2, 4, 6, or 8 h. Solubilization, immunoprecipitation, and electrophoresis were performed as described under ``Experimental Procedures.'' Bands were quantified by densitometer, relative to a 100% value at time zero (not shown). A graph of reductase degradation is presented, showing farnesol alone (open circles), farnesol + ALLN (closed circles), or farnesol + thapsigargin (closed triangles).
Figure 3: SSD cells exhibit a stunted regulatory effect of farnesol. CHO (upper gel) and SSD (lower gel) cells were pulse-labeled, and then chase was done in the presence of either 20 mM mevalonate (MVA) or 30 µM farnesol (FARN) for 0, 2, 4, or 8 h. For basal degradation, chase was done with no addition for 0, 4, or 8 h. Solubilization, immunoprecipitation, and electrophoresis were done as described under ``Experimental Procedures.'' Band intensities were quantified by densitometer. Graphs showing reductase degradation in CHO cells and SSD cells are presented, with no addition (open circles), mevalonate (closed circles), or farnesol (closed triangles) shown.
Figure 4: Farnesol regulation requires exogenous sterols in SSD cells. A pulse-chase study of reductase degradation was done as described under ``Experimental Procedures.'' The chase period was for 8 h with a range of 25-hydroxycholesterol concentrations from 2.5 to 0.025 µM, each with no farnesol (open columns) or with 30 µM farnesol (shaded columns) present. Band intensities were quantified by Molecular Imager and are presented relative to a 100% value at time zero of the chase period.
Figure 5: Intracellular farnesol levels rise following mevalonate addition. CHO and SSD cells were grown in ten 150-mm plates per sample and then were left untreated (open columns) or treated with 20 mM mevalonate (shaded columns) for 3 h. Cells were then subjected to lipid saponification and extraction as described under ``Experimental Procedures.'' Farnesol levels were determined by HPLC as described under ``Experimental Procedures'' using a set of known quantities of farnesol as standards.
Of further interest is a comparison between intracellular farnesol levels in SSD cells and wild type cells. The initial studies of SSD cells (26) revealed that these cells secrete a considerable amount of farnesol into the culture medium. Consistent with this observation, we found intracellular farnesol levels to be approximately twice as high in SSD cells compared with wild type cells, with or without mevalonate addition (Fig. 5).
To further examine this matter, we tested another protein as an internal standard to determine whether the effect caused by farnesol in vitro is specific for the loss of reductase. Calnexin, a chaperone protein implicated in protein folding and retention in the endoplasmic reticulum, is a good control based on the fact that, like reductase, it is an integral membrane-spanning resident protein of the endoplasmic reticulum(31) . The effect of farnesol on the loss of calnexin was measured both in permeabilized cells and in intact cells. As shown in Fig. 6A, the addition of farnesol to permeabilized cells results in very rapid loss of calnexin protein in a dose-dependent manner. However, following farnesol addition to intact cells, calnexin remains stable, whereas the degradation of reductase is accelerated (Fig. 6B). These results suggest that in vitro, the loss of reductase caused by farnesol is nonspecific and nonphysiological, whereas in vivo the effect is specific for reductase and physiologically meaningful.
Figure 6: Farnesol causes calnexin loss in permeabilized cells but not in intact cells. A, protein loss in vitro is shown. Pulse-labeled CHO cells were permeabilized as described under ``Experimental Procedures'' and then incubated 4.5 h with 0, 25, 50, or 100 µM farnesol. Calnexin was immunoprecipitated, and samples were subjected to SDS-PAGE. Bands were quantified by Molecular Imager, and values are shown relative to a 100% value at time zero of the chase period. B, protein loss in vivo is shown. Pulse-labeled CHO cells were chased in the absence (open symbols) or the presence (closed symbols) of 30 µM farnesol and then solubilized. Samples were immunoprecipated with antibodies either to reductase (HMGR, circles) or calnexin (CNX, triangles) and then subjected to SDS-PAGE, and the bands were quantified by Molecular Imager. Reductase and calnexin bands shown are from two different exposure times of the same gel. The antibodies used to immunoprecipitate calnexin were successfully characterized in an immunoinhibition experiment using a peptide corresponding to the calnexin epitope (data not shown).
Figure 7:
Farnesol causes HMGal protein to become
detergent insoluble in permeabilized cells. A, results of a
pulse-chase study in permeabilized cells are shown. CHO-HMGal cells
were metabolically labeled and permeabilized (see ``Experimental
Procedures''), then incubated ± 50 µM farnesol
for the indicated times, and then subjected to immunoprecipitation and
electrophoresis. Intensities of HMGal protein bands were quantified by
densitometry. B, results of HMGal activity determination for
samples at times and conditions in A are shown. Solubilized
cells were assayed for -galactosidase activity by addition of 1
mg/ml o-nitrophenyl-
-galactopyranoside, followed by
colorimetric assay at 420 nm using a Beckman DU-64 spectrophotometer. C, results of cell lysate fractionations from samples at above
times and conditions are shown. Solubilized cell samples (see
``Experimental Procedures'') were centrifuged at 16,000
g for 30 min, and supernatants and pellets were
separated. For
-galactosidase activity determination, pellets were
resuspended in solubilization buffer to a volume equal to supernatants,
and each was assayed for HMGal activity as described in B. FARN, farnesol.
Figure 8:
Immunoblot analysis shows that both
reductase (HMGR) and calnexin (CNX) are rendered
detergent insoluble by farnesol in permeabilized cells. CHO cells were
permeabilized and treated for 6 h with or without 50 µM farnesol and then solubilized and centrifuged at 16,000 g for 30 min, and then pellets and supernatants were
separated. For immunoblotting, pellets were directly treated with
loading buffer, whereas supernatants were treated with 70% acetone to
precipitate proteins, and then this pellet was dissolved in loading
buffer. Gel electrophoresis, blot transfer, and antibody staining (with
either reductase or calnexin primary antibodies) were performed as
described under ``Experimental
Procedures.''
Studies aimed toward determining the identity of the regulatory nonsterol, mevalonate-derived metabolite involved in HMG-CoA reductase degradation have been subject to certain limitations when performed in living cells. This is because many of these candidate pathway metabolites, such as isopentenyl pyrophosphate, geranyl pyrophosphate, and farnesyl pyrophosphate, are polar molecules and are thus not permeant to cells. This has led to the employment of other strategies, one of which has involved the use of nonpolar artificial isoprenoid analogues, which were added to intact cells to determine whether reductase degradation could be accelerated, presumably by these artificial products mimicking some natural product. This approach showed some success, as farnesylated tocopherol analogs (18) and farnesyl acetate and farnesyl ethyl ether (17) were shown to accelerate reductase degradation in vivo. These results suggested that some farnesyl metabolite(s) in the isoprenoid pathway is the regulatory molecule.
Another strategy has involved the use of permeabilized cells, which do not present a permeability barrier. We showed previously (13) that mevalonate-induced accelerated reductase degradation persisted in cells after permeabilization with digitonin, but only if cells were pretreated with mevalonate before permeabilization. Presumably this period was necessary for production/accumulation of adequate levels of the mevalonate-derived regulator(s) so that regulated degradation could be underway at the time of permeabilization. Using a modification of our permeabilized cell system, Correll et al.(19) were able to demonstrate apparent accelerated degradation of reductase by addition of farnesol, without the pretreatment period required for mevalonate.
Farnesol is produced from farnesyl pyrophosphate in cells, in a reaction catalyzed by an allyl pyrophosphatase(28, 29, 30) , which diverts some farnesyl pyrophosphate from its primary metabolic route toward cholesterol. We had not tested farnesol in our permeabilized cell system(13) , and in the studies of farnesyl acetate in vivo(17) , farnesol had been tested but at a very limited set of concentrations, which in hindsight were either too low to elicit a regulatory response or so high that toxicity resulted. In the current study we have more rigorously tested farnesol in vivo, using concentrations low enough to be nontoxic to cells but higher than those previously found to be ineffectual and have found that farnesol indeed causes accelerated degradation of reductase. This is an important finding, because it is the first evidence of a natural nonsterol mevalonate-derived metabolite causing this effect when added to cells in vivo. Also of importance are our findings that this effect of exogenous farnesol in accelerating reductase degradation can be blocked by ALLN, by thapsigargin, and in a CHO cell line missing the enzyme squalene synthase (see Fig. 2and Fig. 3). Accelerated degradation by exogenous mevalonate has been shown to be sensitive to all three of these conditions, so our hypothesis regarding the role of farnesol is supported by these findings.
Although the in vitro results of Correll et al.(19) were controlled for degradation of total cell protein in their study, for the following reasons we feel that their reported loss of reductase in vitro is not due to physiologically meaningful causes. One, the farnesol-induced degradation of reductase in permeabilized cells is extremely rapid, considerably more so than observed in mevalonate-pretreated permeabilized cells (13) or even intact cells treated with mevalonate or farnesol. Two, in order to achieve significant acceleration of reductase loss in vitro, farnesol must be used at concentrations of 50 µM and higher, which are levels we have found to be highly toxic when added to intact cells. Three, our studies show that farnesol causes a second resident endoplasmic reticulum membrane protein, calnexin, to be rapidly lost in permeabilized cells. This loss of calnexin is not observed in farnesol-treated intact cells. Four, our studies show that farnesol, when added to permeabilized cells, causes a significant fraction of reductase (and calnexin) to become detergent insoluble, and therefore not recoverable in the usual immunoprecipitation from cell lysates. Although the amount of reductase and calnexin protein that is rendered detergent insoluble by farnesol is significant, it does not account for the total amount of each protein that is lost (compare Fig. 8with Fig. 7A and Fig. 6A). We suggest that a certain degree of nonspecific proteolysis occurs in vitro, perhaps brought about by farnesol disrupting the endoplasmic reticulum.
We believe that our findings, which demonstrate physiologically meaningful reductase degradation in vivo by a natural mevalonate product, serve as an important extension of previous in vivo studies utilizing farnesyl analogs and show that farnesol is a nonsterol mevalonate product with a key role in the regulated degradation of reductase.