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Institute of Environmental Health Sciences, Wayne State University, Detroit, Michigan
(Received May 27, 2008; Accepted July 8, 2008)
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Abstract |
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). We previously reported that treatment of rats or primary cultured rat hepatocytes with squalestatin 1 (also known as zaragozic acid A), an inhibitor of squalene synthase, the first committed step in cholesterol biosynthesis, caused the selective induction of CYP2B (Kocarek et al., 1998), relative to other inducible P450s, and that this induction was attributable to squalene synthase inhibition and the accumulation of an endogenous isoprenoid(s) that is capable of activating CAR (Kocarek and Mercer-Haines, 2002) (see Fig. 1 for a scheme of the isoprenoid pathway).
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); GGPP synthase, which catalyzes the formation of GGPP (Kavanagh et al., 2006) that is also used in protein prenylation reactions (Basso et al., 2006); trans-prenyltransferase, which leads to the biosynthesis of ubiquinone (although in mammals, GPP is this enzyme's preferred substrate) (Dallner and Sindelar, 2000); cis-prenyltransferase, which leads to the formation of dolichol and dolichyl phosphate (Krag, 1998); and heme O synthase, which results in the conversion of heme B to heme O and heme A (Brown et al., 2002). In addition, FPP can be dephosphorylated to farnesol by one or more lipid phosphatases that have not yet been formally identified (Bansal and Vaidya, 1994), although recombinant human phosphatidic acid phosphatase 2a has been shown to be capable of catalyzing this reaction in vitro (Fukunaga et al., 2006). Farnesol can then be sequentially oxidized to farnesal, farnesoic acid, and a family of dioic acids that are excreted in large quantities by animals treated with squalestatin 1 (Bostedor et al., 1997; Vaidya et al., 1998).
Alendronate is an NBP that inhibits FDPS with nanomolar potency (Keller and Fliesler, 1999; Bergstrom et al., 2000). Alendronate and other drugs of its class, including risedronate and zoledronate, are used clinically for the prevention and treatment of osteoporosis, and this therapeutic efficacy is thought to be the result of FDPS inhibition, which decreases the availability of FPP and GGPP for protein prenylation reactions, in turn leading to activation of osteoclast apoptosis (Russell et al., 2007). Because FDPS catalyzes not only the formation of FPP but also the preceding metabolic reaction in which IPP and DMAPP condense to form GPP, alendronate treatment causes the accumulation of IPP and DMAPP (Keller and Fliesler, 1999; Bergstrom et al., 2000).
In this study, we used alendronate to test whether squalestatin 1-mediated CYP2B induction in primary cultured rat hepatocytes is dependent on the production of FPP. We report not only that squalestatin 1-mediated CYP2B induction is effectively abolished by alendronate treatment but also that phenobarbital-mediated CYP2B induction is partially suppressed, suggesting that one or more upstream isoprenoids (e.g., IPP or DMAPP) function as endogenous suppressors of CYP2B induction.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-32P]dATP were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Solvents for lipid extraction and chromatography (Burdick & Jackson) were purchased from Thermo Fisher Scientific (Hanover Park, IL). Other materials were purchased from the sources indicated below.
Primary Culture of Rat Hepatocytes. Hepatocytes were isolated from adult male Sprague-Dawley rats (250–350 g) (Harlan, Indianapolis, IN) as described previously (Kocarek and Reddy, 1996). Freshly isolated hepatocytes were suspended in Williams' E Medium supplemented with 0.25 U/ml insulin, 10–7 M triamcinolone acetonide, 100 U/ml penicillin, and 100 µg/ml streptomycin and plated into collagen (PureCol: 99.9% collagen; 
97% type I collagen with the remainder type III collagen)-coated plastic vessels. Three million viable hepatocytes were plated into 60-mm dishes (for FPP measurements and microsome preparation), 1.5 million cells were plated into the wells of 6-well plates (for RNA isolation), and 350,000 cells were plated into the wells of 12-well plates (for MTT assays). Twenty-four hours after plating, the medium was replaced with fresh medium containing 0.2 mg/ml Matrigel. The medium was replaced thereafter (without Matrigel) at 24-h intervals. Drug treatments were initiated 48 h after plating and were performed as described in the individual figure legends. Drugs were added to the culture medium as concentrated stock solutions in water.
Northern and Western Blot Analysis. Total RNA was isolated from hepatocytes cultured in six-well plates using TRIzol reagent (Invitrogen), and the RNA samples constituting each treatment group (three wells of hepatocytes per group) were pooled. Ten-microgram samples of RNA were resolved on denaturing agarose gels and analyzed by Northern blot hybridization, using the procedures and cDNA probes to CYP2B1, CYP3A23, HMG-CoA reductase, and 7S that have been described previously (Kocarek and Reddy, 1996). Because the CYP2B1 and CYP3A23 cDNA probes cross-react with multiple related transcripts, the bands detected with these probes are referred to as CYP2B and CYP3A. The CYP2B1 probe most probably hybridizes with CYP2B1 and CYP2B2 transcripts on Northern blots of primary cultured male rat hepatocyte RNA, whereas the CYP3A23 cDNA probe most probably detects CYP3A1, CYP3A2, and CYP3A23 transcripts on these Northern blots. CYP2B and 7S band intensities were measured using an Image Station 440CF with one-dimensional Image Analysis Software (version 3.6; Eastman Kodak Co., Rochester, NY), and the CYP2B values were normalized to the corresponding 7S values.
For microsome isolation, hepatocytes in 60-mm dishes were washed once with cold PBS and scraped into 1-ml of cold Cell Recovery Solution. The hepatocytes constituting each treatment group (two dishes of hepatocytes per group) were pooled and gently mixed by inversion for 15 min at 4°C to remove traces of Matrigel. The hepatocytes were then pelleted, washed once with cold PBS, and stored at –80°C until further processing. Cell pellets were suspended in 100 µl of 50 mM Tris-HCl, 1 mM EDTA, and 0.25 M sucrose, pH 7.4, and homogenized by sonication. Homogenates were centrifuged for 20 min at 20,000g at 4°C, and supernatants were centrifuged for 1 h at 105,000g at 4°C. The microsomal pellets were suspended in 40 µl of 50 mM Tris-HCl, 20% glycerol, and 1 mM EDTA, pH 7.4, and protein concentrations were measured using the Quickstart Bradford Protein Assay (Bio-Rad, Hercules, CA). SDS-polyacrylamide gel electrophoresis was performed using 10% Tris-HCl 12-well Ready-Gels (Bio-Rad) and 2 µg of microsomal protein per well, and resolved proteins were transferred onto Immunoblot polyvinylidene difluoride membranes (Bio-Rad). Immunoreactive CYP2B proteins were detected using a polyclonal antibody to CYP2B1 (Xenotech LLC, Lenexa, KS) and the ECL Plus Western Blotting Detection system (GE Healthcare, Piscataway, NJ).
PROD Activity. Reaction mixes were assembled in 96-well fluorescence plates and contained 20 µg of microsomal protein, 10 µM pentoxyresorufin, 5 mM MgCl2, 50 mM Tris-HCl, pH 7.5, and 1 mM NADPH (added last to initiate the reactions) in a total volume of 200 µl. Reactions were incubated in a SpectraMax Gemini fluorescence plate reader (Molecular Devices, Sunnyvale, CA) at 37°C, and fluorescence data (excitation wavelength = 522 nm; emission wavelength = 586 nm) were acquired every 45 s using SoftMax Pro software (Molecular Devices). Slopes were fit to the initial, linear portions of the fluorescence-time plots, and data were expressed as picomoles of resorufin formed per minute per milligram of microsomal protein (based on comparison to a standard curve generated by measuring the fluorescence of wells containing 0–5 pmol of resorufin in buffer). PROD activities were undetectable in samples incubated without NADPH.
MTT Activity. Hepatocytes cultured in 24-well plates were incubated with 0.3 ml of 2.4 mM MTT in PBS for 30 min at 37°C, washed once with PBS, and extracted with 0.3 ml of isopropanol with continuous shaking at room temperature for 30 min. Plates were centrifuged at 3000 rpm for 10 min, and 40-µl aliquots of the supernatants were added to 160 µl of isopropanol in the wells of a 96-well plate. Absorbance was measured at 560 nm using a SpectraMax Plus Absorbance Microplate Reader and SoftMax Pro software (Molecular Devices).
Measurement of FPP Levels. Cellular levels of FPP were measured according to the method described by Tong et al. (2005). After treatment, hepatocytes were recovered from 60-mm dishes using Cell Recovery Solution as described above. The hepatocytes constituting each treatment group (three dishes of hepatocytes per group) were pooled. Cellular protein contents were measured using the bicinchoninic acid assay (Smith et al., 1985). Cell pellets were extracted twice at 70°C with butanol-75 mM ammonium hydroxide-ethanol (1:1.25:2.75, v/v/v). Pooled supernatants were dried under nitrogen at 50°C, and residues were dissolved in 50 mM Tris-HCl, pH 7.5, containing 5 mM dithiothreitol, 5 mM MgCl2, 10 µM ZnCl2, and 1% octyl-β-D-glucopyranoside. Each farnesylation reaction contained extract equivalent to 200 µgof cellular protein, 500 pmol of N-dansylated carboxyl terminus of H-Ras peptide (dansyl-GCVLS; Sigma-Aldrich), and 136 units (250 ng) of recombinant rat protein farnesyl transferase (Calbiochem, La Jolla, CA) in a total volume of 50 µl. Additional reactions containing known quantities of FPP (7–42 pmol) were analyzed in parallel to the samples to permit construction of standard curves. Reactions were performed at 38°C for 120 min and terminated by addition of 50 µl of acetonitrile and 5 µl of 1.2 N hydrochloric acid. Denatured proteins were removed by centrifugation, and 20-µl aliquots of supernatants were analyzed by high performance liquid chromatography, using a Waters (Milford, MA) system (1525 binary HPLC pump, 2487 Dual 
Absorbance Detector, busSAT/IN Module, and Breeze software) and a Shimadzu RF-10AXL fluorescence detector. Separations were performed using a Symmetry C18 5.0 µm, 4.6 mm x 150 mm column protected by a guard column (Waters) and a binary mobile phase consisting of solvent A (25 mM ammonium acetate in 40% acetonitrile) and solvent B (20 mM ammonium acetate in 90% acetonitrile).
Chromatography was performed at room temperature with a mobile phase flow rate of 1 ml/min and was initiated upon sample injection with 38% B for 1 min, followed by linear transition over 7 min to 100% B and 100% B for 8 min, a return to initial conditions over 3 min, and re-equilibration. The free and farnesylated peptides were detected using an excitation wavelength of 335 nm and an emission wavelength of 528 nm. The retention time for the farnesylated peptide varied from 5.5 to 6.4 min, depending on the ambient temperature. Peak areas were determined, and data were calculated as picomoles of FPP per milligram of cellular protein.
Statistical Analysis. Numerical data (FPP levels, MTT activities, and normalized Northern blot data) from replicate experiments (i.e., from multiple independent hepatocyte preparations) were analyzed using one-way analysis of variance followed by the Newman-Keuls multiple comparison test, with p < 0.05 set as the level of significance. For this purpose, one treatment group was defined as the reference group and the data from the other treatment groups were expressed as percentages of the reference group value within each experiment. Therefore, the reference groups (defined as 100% in each experiment so there was no interexperiment variability) were excluded from the analyses, as were individual groups containing fewer than three samples. IC50 values were determined using the log(inhibitor) versus response-variable slope function of Prism 5.0 (GraphPad Software Inc., La Jolla, CA).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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40 µM), with average decreases of 75% and >90% occurring at alendronate concentrations of 60 and 100 µM, respectively (Fig. 2A). These effective alendronate concentrations were not toxic to the hepatocytes, as judged by the lack of effect on MTT activity (Fig. 2B). Alendronate was next evaluated for its ability to modify CYP2B, CYP3A, and HMG-CoA reductase expression, either when used alone or in combination with 0.1 µM squalestatin 1 or 100 µM phenobarbital (Fig. 3). Treatment with alendronate alone had no effect on CYP2B or CYP3A mRNA content (Fig. 3). However, cotreatment with alendronate effectively inhibited squalestatin 1-mediated CYP2B induction (IC50 30 µM), with complete suppression occurring at alendronate concentrations of 60 and 100 µM (Fig. 3). In addition, alendronate treatment produced a concentration-dependent suppression of phenobarbital-mediated CYP2B induction, which, although less pronounced than the absolute blockade that occurred for squalestatin 1-mediated induction, was nevertheless significant at an alendronate concentration of 100 µM (Fig. 3). Although phenobarbital is commonly regarded as an inducer of both CYP2B and CYP3A, this drug induces CYP2B and CYP3A with different concentration dependencies (Kocarek et al., 1990). Thus, although maximal CYP2B induction is achieved at 100 µM phenobarbital, this concentration produces only a slight elevation of CYP3A expression. Alendronate cotreatment suppressed the phenobarbital-mediated CYP3A induction that did occur in parallel with the above-described suppression of CYP2B induction (Fig. 3). As an additional observation, squalestatin 1 treatment produced the expected up-regulation of HMG-CoA reductase expression that signifies effective blockade of cholesterol biosynthesis (Fig. 3). By contrast, treatment with alendronate produced only very slight up-regulation of HMG-CoA reductase expression, even at a concentration (100 µM) that produced near complete inhibition of farnesyl diphosphate synthase (Figs. 2 and 3). Moreover, alendronate cotreatment produced a concentration-dependent suppression of the squalestatin 1-mediated HMG-CoA reductase up-regulation that was in close correspondence to the suppressive effects of alendronate on phenobarbital-inducible CYP2B and CYP3A expression (Fig. 3).
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The partial suppression of phenobarbital-mediated CYP2B induction that was produced by alendronate cotreatment prompted us to consider whether the effect was produced by alendronate itself or was a consequence of FDPS inhibition, as NBP treatments have been reported to cause accumulation of upstream isoprenoids, including isopentenyl pyrophosphate and dimethylallyl pyrophosphate (Keller and Fliesler, 1999; Bergstrom et al., 2000). To discriminate between these possibilities, we performed treatments with the HMG-CoA reductase inhibitor, pravastatin, to shut off the cholesterol biosynthetic pathway at the level of mevalonate and thereby prevent the ability of alendronate to cause accumulation of any isoprenoids. Pravastatin was specifically selected for use, as this drug produces effective upstream blockade of the cholesterol biosynthetic pathway but does not itself produce any effects on CYP2B expression (Kocarek and Reddy, 1996; Kocarek et al., 2002). The concentration-dependent ability of alendronate to suppress phenobarbital-mediated CYP2B induction was evaluated in the absence and presence of pravastatin (Fig. 4A). As previously seen, alendronate treatment produced concentration-dependent suppression of phenobarbital-mediated CYP2B induction. This suppression was attenuated when the hepatocytes were cotreated with pravastatin. In particular, the suppressive effect that was produced by 60 µM alendronate was largely prevented by pravastatin cotreatment, whereas the greater suppression that was produced by 100 µM alendronate treatment was partially reversed by pravastatin (Fig. 4A). Next, the effects of pravastatin-mediated HMG-CoA reductase blockade, combined with rescue by mevalonate treatment, were evaluated on alendronate (60 µM)-mediated suppression of phenobarbital-inducible CYP2B mRNA and immunoreactive protein levels (Fig. 4B) and CYP2B-mediated catalytic activity (Fig. 4C). Alendronate-mediated suppression of phenobarbital-inducible CYP2B expression was evident at all levels of measurement, as was pravastatin-mediated reversal of the suppression. Cotreatment of pravastatin-treated hepatocyte cultures with mevalonate restored the ability of alendronate to cause suppression of phenobarbital-mediated CYP2B induction (Fig. 4, B and C). Squalestatin 1-mediated induction of CYP2B mRNA, protein, and enzymatic activity was abolished by treatment with 60 µM alendronate (Fig. 4, B and C).
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Discussion |
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; Bergstrom et al., 2000), the alendronate concentrations (10–100 µM) that caused effective FPDS inhibition in cultured rat hepatocytes may seem high, these concentrations are comparable to those found to produce FPDS inhibition in cultured murine osteoclasts (Bergstrom et al., 2000). The complete blockade of squalestatin 1-mediated CYP2B induction that was produced by alendronate treatment is consistent with a requirement for ongoing FPP synthesis in the induction process, although a caveat is that FDPS inhibition prevents the production of both GPP and FPP. Nevertheless, these findings clearly demonstrate that squalestatin 1 is incapable of causing CYP2B induction unless isoprenoid biosynthesis is allowed to proceed at least to the level of GPP.
Phenobarbital was included in this study as a positive control CAR activator and CYP2B inducer, whose effect on CYP2B expression is unaffected by cotreatment with the upstream cholesterol biosynthesis inhibitor, pravastatin (Kocarek and Mercer-Haines, 2002). Our expectation was that phenobarbital-mediated CYP2B induction, in contrast with squalestatin 1-mediated induction, would be unaltered by alendronate cotreatment. However, alendronate treatment was found to produce a concentration-dependent suppression of phenobarbital-mediated CYP2B induction that, although less pronounced than the complete blockade of squalestatin 1-mediated induction that occurred, was nevertheless substantial. This finding prompted us to investigate whether the suppressive effect of alendronate on phenobarbital-mediated CYP2B induction could be attributed to a direct action of alendronate itself or rather whether the suppression was transduced as a consequence of FPDS blockade and accumulation of an upstream metabolite. Cotreatment of hepatocyte cultures with pravastatin to block mevalonate and therefore all isoprenoid biosynthesis attenuated the ability of alendronate to suppress phenobarbital-inducible CYP2B expression, and this attenuation was overridden by bypassing the pravastatin-mediated blockade through the addition of exogenous mevalonate. Pravastatin treatment effectively reversed the suppressive effect on phenobarbital-mediated CYP2B expression that was produced by 60 µM alendronate but only partially reversed the larger suppressive effect that was produced by 100 µM alendronate. These findings indicate that the suppressive effects of alendronate on phenobarbital-mediated CYP2B induction can be attributed both to FDPS inhibition with upstream metabolite accumulation (i.e., because some of the effect can be reversed by upstream blockade) and to direct drug action on the induction process (i.e., because some of the effect is not inhibited by pravastatin treatment).
The nature of the alendronate-generated endogenous bioactive suppressor of phenobarbital-mediated CYP2B induction can be surmised by the knowledge that alendronate treatment has been shown to cause accumulation of the upstream isoprenoids, IPP and DMAPP (Keller and Fliesler, 1999; Bergstrom et al., 2000). To date, IPP and/or DMAPP have been implicated as biologically active molecules in at least two ways. First, IPP and DMAPP have been identified as probable mediators of adverse inflammatory effects that are produced by NBP treatments. NBPs stimulate V
V
2 T cells to produce inflammatory cytokines in a manner that can be prevented by statin treatment and mimicked by IPP or DMAPP (Bürk et al., 1995; Tanaka et al., 1995; Kunzmann et al., 2000; Thompson and Rogers, 2004; Hewitt et al., 2005). It has been suggested that this action may underlie the rare occurrence of musculoskeletal adverse effects that occur in patients taking NBPs (Bock et al., 2007). IPP has also been identified as a tumor cell antigen that causes activation of VV2 T cells (Gober et al., 2003), which has implications for the mechanisms governing immunosurveillance in response to the metabolic derangements occurring in cancer cells. NBPs also cause necrotic and inflammatory effects in rats and mice, although these species lack VV2 T cells, and IPP also appears to underlie at least some of these effects (Deng et al., 2007). Such inflammatory effects could conceivably confound investigation of the ability of NBP treatment to modify hepatic P450 regulation in vivo.
Second, IPP is able to combine with adenosine monophosphate in a reaction catalyzed by aminoacyl-tRNA synthases to form a novel ATP analog, termed triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester (ApppI) (Mönkkönen et al., 2006). ApppI has been identified in extracts of cultured osteoclasts, macrophages, and glioma cells treated with an NBP (Mönkkönen et al., 2006), as well as in peritoneal macrophages isolated from zoledronate-treated mice (Mönkkönen et al., 2007). ApppI has been shown to induce apoptosis in osteoclast cells as a result of inhibition of the mitochondrial adenine nucleotide translocase (Mönkkönen et al., 2006). Therefore, NBP-mediated osteoclast apoptosis is probably the result of two mechanisms acting in combination; i.e., reduction of FPP and GGPP levels, which reduces the prenylation of critical proteins, and formation of ApppI, which causes disruption of adenosine nucleotide transport and mitochondrial function.
Whether or how either of the two above-described actions may contribute to the alendronate-mediated suppressive effects on CYP2B induction that we report in this study is a topic for further investigation. Because current evidence suggests that CAR can be activated by processes that cause activation of the energy-sensing AMP-activated protein kinase (Rencurel et al., 2005, 2006; Blättler et al., 2007; Shindo et al., 2007), it seems possible that ApppI formation followed by inhibition of adenine nucleotide translocase and alteration of hepatocellular ATP/AMP balance may contribute to the mechanism whereby alendronate treatment causes suppression of phenobarbital-inducible CYP2B expression. An additional possibility is that alendronate and/or one or more of the upstream isoprenoids are able to interact with CAR as inverse agonists. However, our initial experiments, using an in vitro human CAR interaction assay, have not supported the operation of such a suppressive mechanism.
It is clear that endogenous isoprenoids are capable of producing diverse effects on hepatic P450 expression. Achieving an understanding of the underlying mechanisms will both enhance our knowledge about the biological capacity of this complex series of endogenous molecules and improve our ability to predict the impact of isoprenoid-modifying treatments on xenobiotic metabolism.
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Footnotes |
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: CAR, constitutive androstane receptor (or NR1I3); FPP, farnesyl pyrophosphate (or farnesyl diphosphate); FDPS, farnesyl diphosphate synthase (or farnesyl pyrophosphate synthase); GPP, geranyl pyrophosphate; IPP, isopentenyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; NBP, nitrogen-containing bisphosphonate (or aminobisphosphonate); DMAPP, dimethylallyl pyrophosphate; MTT, 3-(4,5-di methylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; PBS, phosphate-buffered saline; PROD, pentoxyresorufin O-depentylase; ApppI, triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester.
Address correspondence to: Dr. Thomas A. Kocarek, Institute of Environmental Health Sciences, Wayne State University, 2727 Second Ave., Room 4000, Detroit, MI 48201. E-mail: t.kocarek{at}wayne.edu
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92 cells are stimulated in a cross-reactive fashion by a variety of phosphorylated metabolites. Eur J Immunol 25: 2052–2058.[Medline], macrophages, and T-cells. Int Immunopharmacol 7: 152–161.[CrossRef][Medline] cells recognize endogenous mevalonate metabolites in tumor cells. J Exp Med 197: 163–168. T cells in response to aminobisphosphonates is inhibited by statins. Clin Exp Immunol 139: 101–111.[CrossRef][Medline] T cells by aminobisphosphonates and induction of antiplasma cell activity in multiple myeloma. Blood 96: 384–392. T cells. Nature 375: 155–158.[CrossRef][Medline],-T-cell proliferation and activation in vitro. J Bone Miner Res 19: 278–288.[CrossRef][Medline]
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