Activation of the cholesterol pathway and Ras maturation in response to stress

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Abstract

All cells depend on sterols and isoprenoids derived from mevalonate (MVA) for growth, differentiation, and maintenance of homeostatic functions. In plants, environmental insults like heat and sunlight trigger the synthesis of isoprene, also derived from MVA, and this phenomenon has been associated with enhanced tolerance to heat. Here, we show that in human prostate adenocarcinoma PC-3M cells heat shock leads to activation of the MVA pathway. This is characterized by a dose- and time-dependent elevation in 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) activity, enhanced sterol and isoprenoid synthesis, and increased protein prenylation. Furthermore, prenylation and subsequent membrane localization of Ras, a central player in cell signaling, was rapidly induced following heat stress. These effects were dose-dependent, augmented with repeated insults, and were prevented by culturing cells in the presence of lovastatin, a competitive inhibitor of HMGR. Enhanced Ras maturation by heat stress was also associated with a heightened activation of extracellular signal-regulated kinase (ERK), a key mediator of both mitogenic and stress signaling pathways, in response to subsequent growth factor stimulation. Thus, activation of the MVA pathway may constitute an important adaptive host response to stress, and have significant implications to carcinogenesis.

Introduction

The MVA pathway of sterol biosynthesis is highly conserved throughout the living world, from bacteria to humans (Casey, 1992; Grünler et al., 1994). The conversion of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) to MVA is catalyzed by HMG-CoA reductase (HMGR), the rate-limiting enzyme in the synthesis of sterols and isoprenoids (Goldstein and Brown, 1990). The use of HMGR inhibitors, like lovastatin (LOV) and its synthetic analogs, is the most effective clinical approach to abrogate hypercholesterolemia, the greatest risk factor for cardiovascular disease (Alberts et al., 1980). Lipid moieties derived from MVA are essential players in numerous events central to cell growth and differentiation, from electron transport to cell division. The maturation of Ras proteins, heterotrimeric G proteins (γ subunit), nuclear lamins (A and B), and rhodopsin kinase, among others, requires their covalent attachment to C15 (farnesyl) or C20 (geranylgeranyl) isoprenoids derived from MVA (Goldstein and Brown, 1990; Casey, 1992; Grünler et al., 1994). Isoprenylation, accomplished by prenyltransferases, is the first of four possible post-translational modifications that render these proteins more lipophilic and allow for their anchorage within the lipid bilayer of membranes. Isoprenylation-dependent membrane anchorage and subcellular localization of these proteins is often required for their maturation and function (Casey, 1992; Marshall, 1993; Grünler et al., 1994; Tilbrook et al., 1995). Because of the marked dependency of cancer cells on isoprenylated molecules for cell growth, inhibitors targeting prenyltransferases or HMGR have been proposed as candidates for cancer intervention (Gibbs et al., 1994; Larner et al., 1998).

Many components of the MVA pathway in animal cells are structurally and functionally similar to those of plants cells (Bach, 1995). In plants, environmental insults like heat, sunlight and water stress lead to the synthesis of MVA-derived lipids such as isoprene. The simplest member of the isoprenoid family, isoprene can be synthesized in large amounts by a variety of organisms ranging from bacteria to humans, and is easily measured in human breath (Sharkey, 1996). In plants, its increased synthesis upon exposure to environmental insults, particularly heat stress, has been associated with enhanced thermotolerance (Sharkey and Singsaas, 1995). A current working hypothesis for the mechanism of thermal protection by isoprene is that it `alters photosynthetic membrane properties in order to allow plants to cope with large fluctuations in temperature that occur in sunlit leaves' (Sharkey, 1996). Since the MVA pathway is highly conserved in all living cells, we hypothesized that the ability to activate the synthesis of some isoprenoids and/or sterols in order to favorably respond to adverse stimuli may also exist in mammalian cells. Indeed, there is limited evidence to support this view. For example, early studies reported elevations of plasma cholesterol following a variety of stresses in vivo (Bucher et al., 1958; Kritchevsky, 1958; Servatius et al., 1993). More recently, however, it was reported that, in cultured rat hepatocytes, acute stress is associated with an inhibition of both the cholesterol and fatty acid biosynthetic pathways (Corton et al., 1994; Hardie and Carling, 1997).

In our present study, we have examined the effects of heat shock on the de novo isoprenoid and sterol synthesis in the human prostate adenocarcinoma cell line PC-3M. We present evidence that exposure of these cells to heat shock results in the dose- and time-dependent activation of HMGR, leading to enhanced synthesis of isoprenoids and sterols. Additional results are presented which suggest that stress-induced biosynthesis of isoprenoids is associated with increased protein prenylation, and specifically with the elevation in membrane-bound v-Ha-Ras levels, which may in turn allow for enhanced activation of ERK, a central player in the stress response. Taken together, our results suggest that the activation of the MVA pathway by heat stress does occur in mammalian cells, and may provide novel insight into the cellular mechanisms utilized to favorably respond and adapt to environmental insults.

Results

Stress-induced synthesis of sterols and isoprenoids through the activation of HMGR

To study the effects of cellular stress on the MVA pathway, we measured the syntheses of sterol and non-sterol products of MVA following exposure of PC-3M cells to 42 or 44°C. A dose-dependent increase in the incorporation of [14C]acetate into squalene, lanosterol, free cholesterol and cholesteryl esters was observed in cells subjected to 42°C heat treatment for 1, 2 or 4 h (Figure 1). Exposure of cells to 44°C caused a significantly greater elevation in the de novo synthesis of these lipid moieties, including cholesterol esters, while the accumulation of free cholesterol declined with these higher doses of heat. The latter might be the consequence of a block in the conversion of lanosterol into cholesterol and/or the more rapid esterification of free cholesterol into cholesteryl esters. In all cases, the presence of LOV (30 μM), a competitive inhibitor of HMGR, the rate-limiting enzyme in the pathway, prevented the enhanced sterol synthesis with heat stress while fatty acid levels were unaffected (not shown). Importantly, the stress-dependent activation of sterol synthesis was not limited to PC-3M cells, but occurred similarly in other human cancer cells from diverse lineages, such as melanoma 1011 cells and the Jurkat T-cell line (our unpublished results).

Figure 1
figure1

Increased synthesis of sterols by heat stress. Chromatogram of non-saponifiable lipids from cells radiolabeled during the 14-h period following stress, in the presence or absence of LOV (30 μM). The numeric values shown below indicate the fold induction in [14C]acetate incorporation into the non-saponifiable lipid of interest with respect to control (C) levels. The chromatogram and values shown here are representative of more than four independent experiments. The positions of standards are indicated

Next, we examined whether the synthesis of smaller derivatives of MVA like the free (not protein-bound) isoprenoids, farnesol (FOH) and geranylgeraniol (GGOH), was likewise altered by these treatments (Figure 2). Consistent with the results presented above, exposure of cells to 42 and 44°C triggered the de novo synthesis of FOH and GGOH in a dose-dependent manner, with a greater effect seen with 44°C treatment (in a representative TLC, the [14C]acetate incorporated into FOH or GGOH ranged from about 4000 c.p.m. in controls to 7000 – 14 000 c.p.m. in treated cells, approximately 1 – 4% of the total [14C]acetate incorporated). Simultaneous LOV treatment again inhibited the heat-induced incorporation of [14C]acetate into isoprenoids.

Figure 2
figure2

Increased synthesis of isoprenoids by heat shock. (Left), Fold induction in [14C]acetate incorporation with respect to control of hexane-extractable and dephosphorylated isoprenoids, FOH and GGOH, from cells treated in the presence or absence of LOV as indicated. (Right), A similar analysis from cells treated for one to three consecutive days is shown. The Rfs for FOH and GGOH were 0.47±0.03 and 0.29±0.02, respectively. Values represent mean±s.e.m. of three independent experiments, a44°C, 2 h (+LOV)

We then evaluated if this response could be enhanced if cells were repeatedly exposed to stressful stimuli, as may occur in natural settings or therapeutic regimens. When cells were treated with relatively low doses of heat shock (42°C) over 2 or 3 consecutive days, the synthesis of isoprenoids (Figure 2) and sterols (not shown) was further activated above levels seen with a single exposure. This phenomenon might represent either a cumulative effect, or an adaptive response to subsequent insults after a `priming' exposure to the stressful stimulus, or both. In all instances, treatment with LOV (30 μM) effectively inhibited the stress-induced [14C]acetate incorporation into isoprenoids and sterols.

Since HMGR constitutes the key regulatory enzyme of the MVA pathway and has been shown to be modulated by stress in plants (Stermer et al., 1994), we hypothesized that the stress-induced increase in synthesis of MVA products described above might result, at least in part, through an elevation in HMGR activity. Indeed, treatment of cells with 42°C (4 h) or 44°C (1 h) resulted in a time- and dose-dependent increase in microsomal HMGR activity that reached a maximum 4 – 5-fold induction 3 h after cells had been heated at 44°C for 1 h (Figure 3). The kinetics of induction and subsequent decline in enzymatic activity observed with these heat treatments are consistent with the fact that HMGR activity is highly regulated by its products at multiple levels, including transcription, translation and post-translational events (Goldstein and Brown, 1990; Moriyama et al., 1998). Thus, it is also possible that this significant elevation in HMGR activity may result from its modulation at more than one level: enhanced HMGR expression, reduced degradation of the enzyme (likely by dephosphorylation), and other post-translational modifications affecting enzyme activity. Particularly upon exposure to 44°C, the gradual decrease in activity observed following the 3-h time point is likely the result of a negative feed-back inhibition by the accumulating mevalonate and sterols. As expected, in cells treated with 42°C this phenomenon occurred to a much lesser extent, and maximal HMGR activity was observed only 6 h after heat stress. In all instances, enzyme activity was completely abolished when the reaction was performed in the presence of 30 μM LOV (not shown). Importantly, repetitive stresses did not significantly augment HMGR activity (our unpublished results). Therefore, it is likely that additional enzymes downstream from MVA synthesis are also modulated by heat stress, and these enzymes may be further activated with repeated insults.

Figure 3
figure3

Activation of HMGR by heat. (a) A representative chromatogram depicting the kinetics of enzyme activation ([14C]HMG-CoA conversion to [14C]MVA) following a single treatment at 44°C for 1-h. (b) Fold increase in microsomal HMGR activity at the times indicated after a single heat shock (42°C, 4 h or 44°C, 1 h) relative to that of untreated controls. Enzymatic activity is also expressed in nmol of MVA formed per min per mg of microsomal protein. Values represent mean±s.e.m. of five independent experiments

Increased protein prenylation and Ras maturation by heat stress

Protein prenylation, or the covalent attachment of an isoprenoid group to proteins, is a mandatory step in the maturation and membrane localization of certain proteins, such as Ras, that are essential for cell growth and differentiation. Considering the elevation of isoprenoid synthesis described above (Figure 2), we examined whether heat stress would ultimately have an effect on protein prenylation in our model system. To address this question, we first evaluated the synthesis and utilization of endogenous MVA, i.e. its incorporation into proteins, in a standard competition assay in which cells are first pretreated with LOV to deplete the preexisting pools of endogenous MVA, then subsequently cultured in the presence of exogenous [14C]MVA as previously described (Mumby et al., 1990; Siddiqui et al., 1998). Thus, cells were pretreated with LOV (10 μM) for 16 h. After the removal of LOV, cells were subjected to heat stress. Incorporation of [14C]MVA (50 μCi/ml) into proteins was assessed over the 7-h period following heat shock. As shown in Figure 4, radiolabeling of proteins with [14C]MVA was significantly lower in cells exposed to 42 or 44°C with respect to control cells, suggesting that endogenous relative to radiolabeled MVA levels were elevated by heat stress. This effect was particularly pronounced for proteins within the 20 – 25-kDa molecular weight range, a size interval encompassing the Ras superfamily of proteins. The heat-induced, dose-dependent increase in the MVA incorporation is in agreement with the rapid activation of HMGR, the enzyme catalyzing MVA synthesis. In addition, the pattern of [14C]MVA incorporation into proteins is in keeping with the induction of isoprenoid synthesis shown in Figure 2, since it was also potentiated with repeated exposures to heat (42°C) over consecutive days, and was again more pronounced with 44 than with 42°C. Furthermore, the differences in [14C]MVA utilization between control and heat stressed cells were abolished when heat treated cells were radiolabeled in the presence of LOV (30 μM), where the pattern of labeling resembled that of cells treated with LOV alone. This observation strongly suggests that our results do not represent a toxicity-related reduction of total MVA utilization caused by heat. Instead, it further validates our hypothesis that the decline in [14C]MVA incorporation into proteins following heat stress is likely due to an elevated ratio of MVA/[14C]MVA by the increased de novo synthesis of endogenous MVA, and resulting in accumulating pools of `cold' isoprenoids available for protein prenylation.

Figure 4
figure4

Changes in [14C]MVA incorporation into proteins following heat stress. After a 16-h LOV pretreatment (10 μM) in order to maximize exogenous [14C]MVA incorporation, LOV was removed and cells were either treated with heat of left untreated (C); cells continuously exposed to 30 μM LOV served as negative controls. Following incubation with [14C]MVA (50 μCi/ml), radiolabeled proteins were resolved by SDS – PAGE, transferred onto membranes and analysed with a PhosphorImager (left). Membranes were stained with fast green to visualize protein loading (right)

Membrane-bound Ras proteins are pivotal components of signal transduction pathways triggered by proliferative as well as stressful stimuli (Kyriakis and Avruch, 1996). Considering the absolute requirement of isoprenylation for the maturation, membrane targeting and activation of Ras (Hancock et al., 1989), we examined the extent to which heat stress-triggered induction of isoprenoid synthesis would ultimately influence Ras (v-Ha-Ras) membrane localization in our model system. To this end, lysates of heat-shocked PC-3M cells (3 h post-stress) were separated into membrane and soluble fractions, and subjected to Western blot analysis. Although levels of cytoplasmic Ras were expectedly low, membrane-bound, and therefore farnesylated Ras, increased in a dose-dependent fashion, a phenomenon that was further enhanced with additional treatments (Figure 5a). Next, we evaluated if increased de novo Ras expression by heat was contributing to the accumulation of membrane-bound Ras. First, the levels of Ras mRNA were not significantly altered in response to these stresses (not shown). Labeling of cellular proteins with [35S]methionine/cysteine during the 7-h period after heat shock, followed by Ras immunoprecipitation, showed little or no increase in Ras protein synthesis (Figure 5b) after one or more 42°C treatments. Hence, the observed changes in the levels of membrane-bound Ras seen in Figure 5a are likely due to post-translational events, specifically Ras farnesylation, possibly as a result of increased isoprenoid availability. However, subtle changes in Ras expression might also contribute to this effect.

Figure 5
figure5

Changes in Ras prenylation and sub-cellular localization after heat shock. (a) Western analysis of Ras expression in membrane and soluble (cytoplasmic) fractions from PC-3M cultures 3 h after the last heat treatment. (b) De novo Ras synthesis following stress. [35S]met/cys-radiolabeled Ras immunocomplexes were resolved by SDS – PAGE, blotted onto membranes and analysed with a PhosphorImager. (c) Immunoprecipitation of 14C-MVA-labeled, membrane-bound Ras protein ([14C]M-Ras). Following a 16-h pretreatment with LOV (10 μM), cells were heat-stressed of left untreated. All cells were then labeled with 14C-MVA (50 μCi/ml) as described in Figure 4, and Ha-Ras immunocomplexes were precipitated, resolved by SDS – PAGE, transferred onto membranes and analysed with a PhosphorImager. (d) Western analysis of the kinetics of Ras membrane localization following heat treatment. After a 16-h LOV pretreatment (in order to accumulate Ras in the cytoplasm), LOV was removed, cells were either heat shocked or untreated, and harvested at the times indicated. In the case of treatment with 44°C in the presence of LOV 30 μM (+LOV), only the 6-h time point is shown. `0 h' represents the time point immediately after heat treatment. M-Ras, membrane-bound Ras; C-Ras, cytoplasmic Ras

In order to directly examine the changes in Ras isoprenylation, a competition study using [14C]MVA was performed as described above, pre-treating cells with LOV, after which cells were heat-stressed, and radiolabeled. 14C-MVA-labeled v-Ha-Ras immunocomplexes were then resolved by SDS – PAGE. In keeping with the changes observed in total protein prenylation shown in Figure 4, Ras labeling with exogenous 14C-MVA was also reduced slightly after a single exposure to 42°C, and significantly more with subsequent treatments or with a single exposure to 44°C (Figure 5c). These results suggest that the heat-triggered elevation in endogenous MVA was sufficient to allow higher incorporation of `cold' relative to radiolabeled MVA into FOH required for Ras prenylation. Next, we evaluated the kinetics of the sub-cellular localization and maturation of Ras following exposure to heat. Since Ras is predominantly found prenylated and membrane-bound in cancer cells, we again pretreated cells with 10 μM LOV, which led to the accumulation of unprenylated Ras in the cytoplasm. LOV was then removed and cells were either left untreated (control) or were heated to 44°C. At various times thereafter, cells were harvested and protein extracts evaluated for Ras expression by Western blot analysis, allowing for the identification of two main bands corresponding to the cytoplasmic (C-Ras) and membrane (M-Ras) associated protein (confirmed by Western blot analysis of membrane-enriched and soluble fractions). As shown in Figure 5d, exposure of PC-3M cells to 44°C for 30 min resulted in a rapid shift (significant after 3 h) of unprenylated Ras (C-Ras) to the prenylated, membrane-bound Ras (M-Ras) form. Moreover, the shift from the cytoplasmic to the membrane-associated mature form of Ras was almost completed by 6 h after heat treatment. When cells were exposed to 44°C in the presence of LOV, prenylation was prevented, thereby limiting Ras to the cytoplasm. In control cells, however, the recovery from LOV treatment occurred much more slowly, and C-Ras was still readily detectable at the 20-h time-point. Taken together, our findings support the view that the accumulation of MVA-derived moieties following heat stress is associated with an increased maturation and membrane localization of Ras.

Isoprenylation and subcellular localization of Ras are critical steps for its function as a key intermediate within the extracellular signal-regulated kinase (ERK) signaling pathways (de Vries-Smits, 1992). One of several mitogen-activated protein kinase pathways involved in stress signaling, ERK activation has been often associated with enhanced cell survival and transformation (Cowley et al., 1994; Mansour et al., 1994; Guyton et al., 1996). Thus, we evaluated the possibility that prior heat stress treatment would influence subsequent activation of ERK by serum stimulation. Accordingly, ERK activity was assessed in PC-3M cells that were either stimulated with serum alone or were `primed' earlier with a single mild heat shock treatment (42°C, 4 h). As shown in Figure 6, ERK activity was slightly elevated by heat stress alone, but had returned to basal levels 3 h later, prior to subsequent stimulation with serum. Cells primed in this fashion elicited a significantly more pronounced and sustained activation of ERK by serum than did unheated cells. ERK activation was elevated to even higher levels in cells subjected to this mild heat stress on two consecutive days (not shown), in keeping with the augmented membrane-bound Ras levels after 2 days of heat stress shown in Figure 5. Interestingly, a similar study examining the activation of JNK (c-jun-N-terminal kinase), a stress signaling pathway often associated with cell death, showed no changes after heat stress (not shown). Taken together, these results support our hypothesis depicted in Figure 7 that the stress-induced activation of the MVA pathway may constitute part of an adaptive mechanism to favorably respond to stressful stimuli, possibly by enhancing the activation capacity of the ERK signaling pathway.

Figure 6
figure6

Increased activation of ERK in PC-3M cells primed with heat shock. Following a 16-h serum starvation period, cells were subjected to one of the following three treatments: single exposure to 42°C, treatment with 30% fetal bovine serum (FBS) alone, or treatment with 42°C as a priming stress followed by FBS stimulation 3 h later. ERK activity was then assessed at the indicated time points

Figure 7
figure7

Model depicting the activation of the MVA pathway as an adaptive response to stress and its potential impact on Ras protein localization and the MAP kinase cascade

Discussion

Although it has been demonstrated in plants that HMGR activity is modulated by light, wounding or infection (Stermer et al., 1994), and that stress-tolerizing MVA derivatives are generated in response to environmental insults, particularly heat (Sharkey and Singsaas, 1995; Sharkey, 1996), the existence of similar mechanisms in animal cells has not previously been described. Our study is the first to show that some mammalian cells also respond to heat stress by stimulation of the MVA biosynthetic pathway, at least in part through HMGR activation. Importantly, some of these effects were also observed in other cell lines and upon stimulation with other stresses, such as ultraviolet radiation (UV)-C or arsenite treatment (our unpublished results). In addition, repeated stresses induced higher isoprenoid and sterol synthesis, and increased protein prenylation, particularly that of the v-Ha-Ras oncoprotein, suggesting that MVA activation may constitute an important adaptive response to stress with broad consequences in cancer biology.

In contrast to our findings, Corton et al. (1994) have reported that in primary rat hepatocytes, the synthesis of both fatty acids and cholesterol are inhibited by stresses such as heat shock (45°C) and arsenite treatment (500 μM) that lead to the depletion of ATP and elevate AMP levels. High AMP then leads to the stimulation of AMP-activated protein kinase, which serves to phosphorylate and thus down-regulate the activities of the rate-limiting enzymes in these pathways, acetyl-CoA carboxylase and HMGR. The authors have hypothesized that this response would serve to preserve the pools of ATP that are required for vital cellular processes (Corton et al., 1994; Hardie and Carling, 1997). The reason(s) for the differences seen in our study and those of Corton's group are not clear, but could reflect cell type differences (hepatocytes versus prostate, or transformed versus non transformed cells) or the severity of the stress for the particular cell type examined. In this regard, it is worth noting that in their studies, Corton et al. (1994) did, like us, observe a small enhancement in cholesterol synthesis with 42°C heat stress. Furthermore, our findings are in agreement with other reports, some dating back to the 1930's, which describe changes in cholesterol levels in rats after tail-shock (Servatius et al., 1993), Triton-X treatment, γ-radiation (Bucher et al., 1958), and UV-radiation (Kritchevsky, 1958), and in humans with agents such as UV- or X-radiation (Kritchevsky, 1958). Our results regarding the enhanced Ras maturation following stress are consistent with another recent report indicating an important link between the activation of the MVA pathway and subsequent Ras processing. By culturing chick heart cells in medium supplemented with lipoprotein-depleted serum, Gadbut et al. (1997) observed a 40% increase in total cellular cholesterol. This considerable stimulation of the MVA pathway resulted in a significant increase in farnesylated and membrane-associated Ras, providing the first direct evidence that the prenylation of proteins, and in particular that of Ras, is a regulated event that could be subject to modulations in the MVA pathway of cholesterol synthesis. This finding strongly supports our conclusions regarding the changes in protein prenylation in our mammalian stress model. Indeed, increased Ras expression and its prenylation has been reported for UV-C-induced skin tumors in mice (Khan et al., 1994). It is also important to note that, while our study has focused on Ras, it is only one of many important regulatory molecules (within and outside of the Ras superfamily) that are subject to regulation via farnesylation or geranylgeranylation. Hence, the enhanced isoprenoid synthesis during stress is likely to impact on these activities as well.

The protective role of these isoprenoids and sterols against cellular damage is supported by studies demonstrating that inhibitors of the MVA pathway and prenyltransferases act as radiosensitizers (Miller et al., 1993; Bernhard et al., 1998). Moreover, the onset of abnormal Ras expression and maturation that may occur during the early stages of neoplastic transformation have been associated with resistance to chemotherapy and radiation (Sklar, 1988; Samid et al., 1991; Miller et al., 1993; Miller and Samid, 1995). Furthermore, our findings could have direct clinical relevance, as localized hyperthermia is used for the treatment of benign prostate hyperplasia (Larsson and Collins, 1995) and prostate cancer (Servadio and Leib, 1991; Madersbacher et al., 1998). Indeed, the heat shock conditions in our study are well within the range used clinically, since hyperthermia sessions are often implemented once or twice a week, reaching tissue temperatures of 40 – 60°C for 1 h. The possibility of inducing Ras maturation with heat could raise concerns regarding such treatment protocols. Stress-associated increases in isoprenoids could ultimately favor the survival and selection of (pre)malignant cells that acquire elevated mature Ras proteins and possibly increased ERK activity (see Figures 6 and 7), two parameters linked with mitogenesis and neoplastic transformation (Tilbrook et al., 1995; Cowley et al., 1994; Mansour et al., 1994; Khosravi-Far et al., 1995; Okazaki and Sagata, 1995; Guyton et al., 1996). Importantly, our findings could provide new insight into the etiology and/or significance of the high levels of isoprenoids or sterols found in tumor cells (Yasuda and Bloor, 1932; Siperstein, 1995; Bernhard et al., 1998).

Materials and methods

Cell culture and reagents

PC-3M cells were maintained in RPMI 1640 medium (Life Technologies, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), HEPES buffer and antibiotics. In order to better monitor the parameters under investigation, we adopted the standard treatment protocol of a 10 – 16-h serum starvation period prior to any stressful treatments. This pre-treatment step was performed to lower the base level activities of enzymes such as HMGR or ERK (and thus better detect subsequent enzyme activations), and also to avoid the presence of exogenous lipids during lipid radiolabeling.

Sterol and isoprenoid analysis

Actively growing, subconfluent PC-3M cells were exposed to heat shock (42 or 44°C) on 1 – 3 consecutive days. Cells were then serum-starved for 10 h prior to the last stress and radiolabeling. Cells receiving LOV (30 μM) were also treated at this time. Immediately after the last heat shock, 7 μCi/ml of [14C]acetate (American Radiolabeled Chemicals, St. Louis, MO, USA) was added to the cultures. Cells were labeled for 14 h, then washed and harvested in cold PBS, pelleted, and resuspended in 75% ethanol. Non-saponifiable lipids were extracted as previously described (Horie et al., 1993). Extract volumes normalized to cellular protein were resolved by thin-layer chromatography (TLC) in silica gel plates, in the presence of cold standards as carriers as well as radioactive standards, using hexane:diethyl ether:acetic acid (65 : 35 : 1) as the first solvent system. In order to resolve squalene (SQ) and cholesteryl esters (CE) that had accumulated near the solvent front, the same TLC plates were turned 180°, and a second solvent system was used (petroleum ether:ethyl ether:acetic acid, 95 : 5 : 0.5), allowing the front to move up to 1/3 of the TLC plate (about 3 in). The persistent cholesteryl esters band observed even after LOV treatment is likely the product of preexisting `cold' cholesterol esterified with de novo radiolabeled fatty acids, which are unaffected by LOV treatment. For isoprenoid analysis, ethanol-extracted (total) lipids were dephosphorylated (Fujii et al., 1982) and resolved with cold and radioactive standards (American Radiolabeled Chemicals) by reverse-phase TLC (LKC18, Whatman, Hillsboro, OR, USA) in acetone:water (8 : 2). In all cases, positions of radiolabeled lipids and standards were determined by exposure of TLC plates to I2 vapors. Analysis and quantitation was performed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA). LOV was kindly provided by Dr Yu-shen Chao (Merck, Rahway, NJ, USA).

HMGR assay

Actively growing, subconfluent PC-3M cells were placed in serum-free medium 16 h prior to heat shock (42°C, 4 h or 44°C, 1 h). At the times indicated, cells were harvested and microsomes were isolated in a buffer containing 100 mM NaCl, 20 mM potassium phosphate pH 7.4, 30 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM NaF, 5 mM DTT, 2 μM leupeptin, and 2 μM aprotinin, and stored frozen in 30% glycerol. HMGR activity was assayed, using [14C]HMG-CoA (American Radiolabeled Chemicals) as a substrate, as previously described (Shapiro et al., 1974). [14C]MVA formed was resolved by silica gel TLC in benzene:acetone (1 : 1), quantitated with a PhosphorImager, and normalized to microsomal protein concentrations. Enzyme activity was expressed as nmol of MVA formed per min per mg of microsomal protein.

Analysis of protein prenylation

Actively growing cells were serum-starved and pretreated with 10 μM LOV for 16 h prior to the last heat treatment in order to prevent the dilution of (R,S)-[2-14C]MVA (50 – 60 Ci/mmol, ARC) with endogenous MVA. Following stress, cells were labeled with 50 μCi/ml 14C-MVA (equivalent to 130 μg cold MVA/ml) for 7 h. Cells were harvested in RIPA buffer (10 mM phosphate buffer pH 7.4, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, 100 mM NaCl, 0.1% sodium azide) supplemented with protease inhibitors, vortexed extensively and centrifuged at 25 000 g for 15 min. Protein concentrations were determined with the BioRad DC reagent. Fifty-μg aliquots were resolved by SDS – PAGE (12% acrylamide), and transferred onto PVDF membranes (Millipore Corporation, Bedford, MA, USA). Protein labeling was assessed with a PhosphorImager and membranes were also stained with fast green to visualize protein loading.

Cellular fractionation and Ras membrane localization

Actively growing, subconfluent cells were serum-starved, and cells receiving LOV were treated 16 h prior to the last heat treatment. Three hours after the last stress, cells were scraped into cold PBS containing protease inhibitors. After three cycles of freezing/thawing in 50 mM Tris pH 7.4, 0.5 mM EDTA, insoluble (membrane) fractions were separated from soluble fractions by centrifugation at 14 000 r.p.m. for 20 min, and washed three times. Soluble and membrane fractions were resolved by SDS – PAGE (14% acrylamide), and transferred onto PVDF membranes. In order to evaluate the kinetics of Ras sub-cellular localization, cells were pre-treated with 10 μM LOV as described above. After the removal of LOV, cells were exposed to heat or left untreated. Lysates were collected at the times indicated by scraping in RIPA buffer supplemented with protease inhibitors, vortexed, centrifuged and 40-μg aliquots were resolved by SDS – PAGE. In all cases, PVDF membranes were incubated with anti-Ha-Ras (Transduction Laboratories, Lexington, KY, USA) and visualized by Enhanced Chemiluminescence (ECL, Amersham, Arlington Heights, IL, USA).

Ras immunoprecipitation

Cells were treated, radiolabeled with either 14C-MVA as described above or with [35S]methionine/cysteine (200 μCi/ml) for 7 h, and harvested in RIPA buffer with protease inhibitors. Immunocomplexes were precipitated overnight using 400 μg of lysates by incubation with monoclonal anti-v-Ha-Ras antibodies (Y13-259, Oncogene Science, Cambridge, MA, USA) and protein G Sepharose (Sigma, St. Louis, MO, USA), washed three times using RIPA buffer, pelleted, resolved by SDS – PAGE, and transferred onto PVDF membranes. Protein labeling was analysed with a PhosphorImager.

ERK assay

Actively growing, subconfluent PC-3M cells were serum starved for 16 h prior to heat stress (42°C, 4 h). Cells were either heat-shocked in order to activate the MVA pathway or were left untreated. Three hours later, cells were stimulated with FBS (30%) or left unstimulated. At the times indicated cells were harvested, and ERK activity was analysed by an immunocomplex assay using myelin basic protein as a substrate, as previously described (Guyton et al., 1996).

Statistical analysis

An unpaired Student's t-test was used to assess differences between two groups. A value of P<0.05 was considered significant.

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Acknowledgements

We are grateful to Drs Tak-Yee Aw and Xiantao Wang for their valuable assistance, Dr Steven Branch for helpful discussions regarding the HMGR assay, Jennifer Martindale for critical reading of the manuscript, and Dr Dan L Longo for his advice and constant support.

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Correspondence to Nikki J Holbrook.

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Shack, S., Gorospe, M., Fawcett, T. et al. Activation of the cholesterol pathway and Ras maturation in response to stress. Oncogene 18, 6021–6028 (1999) doi:10.1038/sj.onc.1203002

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Keywords

  • cholesterol
  • 3-hydroxy-3-methylglutaryl-CoA reductase
  • ras
  • stress

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