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NEUROPHARMACOLOGY
Pathologisches Institut, Abteilung Neuropathologie, Neurozentrum, Universitätsklinik Freiburg, Freiburg, Germany
Received January 22, 2007; accepted May 14, 2007.
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; Nelson et al., 2004). Although the main site for P450-dependent drug metabolism is the liver, P450 isoforms also occur in defined neuronal or glial cell populations of the brain (Volk et al., 1995; Strobel et al., 2001). The function of P450 in brain is poorly understood and the object of intense discussion. The need for understanding brain P450 function is gaining more and more in importance, as by far the most of the neuroactive drugs used in therapy of neurological diseases such as epilepsy, anxiety disorders, or depression interact with specific P450 isoforms either as substrate or inducer (Flockhart DA, Cytochrome P450-Drug Interaction Table: http://medicine.iupui.edu/flockhart). These compounds have a lipophilic structure that facilitates passing the blood-brain barrier and penetration into the central nervous system (Meyer et al., 2004). The cerebral isoforms CYP2B1, CYP2B10, CYP2C29, CYP3A11, and CYP3A13 have been found to be inducible by antiepileptic drugs (AEDs) such as phenytoin or phenobarbital in rats and mice (Volk et al., 1995; Thuerl et al., 1997; Meyer et al., 2001; Hagemeyer et al., 2003). Therefore, a major endeavor in clinic and research is to understand the underlying molecular mechanisms of drug therapy within the central nervous system to improve present therapeutic strategies.
Several recent reports indicate that function and regulation of brain P450 is different from those processes in liver. It has been shown that enzymatic activity of brain P450 is dependent on the availability of the prosthetic group heme (Meyer et al., 2002, 2005) and that glial and neuronal P450 have different function (Meyer et al., 2004). Some studies indicate that brain P450 may act in mediation of steroid hormone signaling (Wang et al., 2000; Hagemeyer et al., 2003; Meyer et al., 2006). We hypothesize that brain P450 has functions beyond drug metabolism. In a previous study, we demonstrated concomitant up-regulation of CYP3A11 and androgen receptor (AR) as well as enhanced testosterone metabolism and up-regulation of CYP19 (aromatase) in the hippocampus of phenytoin-treated mice (Meyer et al., 2006). These observations indicate a connection between P450-inducing AEDs, decreased sex hormone levels, and altered androgen signaling and are further supported by clinical data from epileptic patients. Treatment of epilepsy with P450-inducing AEDs is frequently associated with endocrine and reproductive disorders in both men and women. These side effects include bad mood states, menstrual disorders, polycystic ovaries, infertility, impotence, and obesity. They are thought to originate from reduced testosterone levels, most probably by its enhanced catabolism within the brain (Isojarvi et al., 2005). Taken together, both the clinical observations and the data from mouse hippocampus imply a crucial function of P450 in AR regulation within the central nervous system.
To clarify the interplay between drug application, subsequent P450 induction, and steroid hormone signaling in brain, we established an in vitro assay to obtain more specific and detailed information about the function and regulation of P450. Initially, we used mouse hepatic cell line Hepa1c1c7 to investigate common principles of P450 regulation and P450 dependent effects on steroid hormone receptors. As a neuronal-type system, we selected rat pheochromocytoma PC-12 cells. This cell line is well established as a neuronal model, because a neuronal phenotype can be induced by application of nerve growth factor (NGF) (Khvotchev et al., 2003).
In the present study, we demonstrate that in both the hepatic and the neuronal-type cell line, androgen receptor up-regulation as well as enhanced AR-dependent signal transduction observed due to AED application are mediated by CYP3A2 or CYP3A11. This observation was verified by the use of CYP3A11-specific short interfering RNA (siRNA) molecules and the CYP3A inhibitor ketoconazole. We conclude that CYP3A isoforms themselves are main regulators of AR expression and downstream signaling. This is a novel function for CYP3A that demonstrates the relevance of understanding the action of P450 in steroid hormone-sensitive areas, especially in the brain.
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The pcDNA1.1Amp expression vector was purchased from Invitrogen BV (Groningen, The Netherlands), the MMTV-ARE-CAT and ARE-TK-CAT reporter constructs were a generous gift from R. Schüle (University Hospital Freiburg, Freiburg, Germany) and have been described previously (Schüle et al., 1990). All other reagents were from commercial sources at the highest purity available.
Cloning of CYP3A11 Full-Length and CYP3A11-GFP-Fusion Constructs. Specific primers for generation of mouse CYP3A11 full-length cDNA were designed using the DNA-Star software package (GATC Biotech, Konstanz, Germany), based on National Center for Biotechnology Information nucleotide database entry NM_007818 as a template. The CYP311 forward primer (sense, including the NotI site) corresponded to nucleotides 1 to 21 (5'-CAGCGGCCGCGAGGGAAGCATTGAGGAGGAT-3'), the reverse primer (antisense, including the XbaI site) corresponded to nucleotides 1697 to 1720 (5'-CATCTAGAACCAGGCATCAAAACCAATCTATT-3'). In addition, for nested PCR, the forward primer corresponded to nucleotides 291 to 310 (5'-ATGGGGGTTGTTTGATGG TC-3') and the reverse primer to nucleotides 841 to 859 (5'-GGCGGCTTTCCTTCATTCT-3'). Total RNA from mouse hippocampus was isolated using an RNeasy Kit. One microgram of total RNA was reverse transcribed at 42°C for 1 h. The full-length cDNA was amplified by long-template PCR. Separated PCR products were extracted from the agarose gel and purified with a Qiaquick Gel Extraction Kit (QIAGEN) according to the manufacturer's protocol. A 1730-bp NotI-XbaI fragment of the full-length CYP3A11 cDNA was cloned in sense orientation into pcDNA1.1_Amp expression vector (Invitrogen BV) using a Rapid Ligation Kit 1 (Roche Diagnostics).
The CYP3A11-GFP fusion construct was used as a positive control for the RNA interference experiments. A full-length CYP3A11 cDNA, missing the stop codon, was generated from mouse brain cDNA and ligated in-frame into the pEGFP-N1 vector (BD Biosciences, Heidelberg, Germany). Hence, we obtained a CYP3A11-GFP fusion protein with the P450 fused at the N terminus of GFP. The CYP3A11 forward primer (sense strand), containing an additional XhoI cutting site, corresponded to nucleotides 4 to 21 (5'-CAC CTCGAG GGAAGCATTGAGGAGGAT-3'), and the CYP3A11 reverse primer, with a SacII cutting site, to nucleotides 1572 to 1590 (5'-CAC CCGCGG TGCAGTCATAACTGGAGCA-3') (based on CYP3A11 GenBank Accession Number nm_007818). Long-template PCR was performed using Pyrococcus woesei proofreading polymerase. The full-length CYP3A11 and CYP3A11-GFP plasmids were transformed into Escherichia coli strain XL-1 blue and prepared using Plasmid Midi Kit (QIAGEN). Finally, both plasmids were checked by nested PCR and cycle sequencing using the MEGABACE 500 system (GE Healthcare, Little Chalfont, Buckinghamshire, UK) with ABI BigDye 2.0 Mix and documented accurate sequences.
Cell Culturing Conditions. Mouse hepatoma Hepa1c1c7 cells were grown as a monolayer in Dulbecco's modified Eagle's medium (with GlutaMAX, supplemented with 4500 mg/ml glucose) containing 10% (v/v) fetal bovine serum and 1% penicillin-streptomycin solution (Invitrogen, Karlsruhe, Germany). Rat pheochromocytoma cells PC-12 (DSZM, Braunschweig, Germany) were grown in RPMI 1640 Glutamax (Invitrogen) supplemented with 10% horse serum (Invitrogen)/5% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin (Invitrogen) on collagen type IV-coated (25 µg/ml) cell culture flasks (Falcon; BD Biosciences Discovery Labware, Bedford, MA). Cultures were maintained in a humidified incubator at 37°C with 5% CO2. Cells were subcultured every 3 to 5 days with Accutase (PAA Laboratories, Coelbe, Germany). Before experiments, PC-12 cells were supplemented with 0.1 µg/ml NGF (Sigma-Aldrich) for neuronal differentiation.
Treatment of Cell Lines with Antiepileptic Drugs. The antiepileptic drugs phenytoin and phenobarbital were dissolved in dimethyl sulfoxide and added diluted to a final concentration of 10 or 100 µM for 24 or 48 h. Ketoconazole was used at 10 µM concentration. These drug concentrations were proved to be nontoxic for the cells by a cytotoxicity assay (Cytotoxicity Detection Kit, Roche Diagnostics), on the basis of lactate dehydrogenase release, as follows: 2.5 x 104 cells/well in a 96-well plate were treated with 100 nM, 1 µM, 10 µM, 100 µM, and 1 mM of the respective drug. After 48 h of treatment, cell viability was measured spectrophotometrically at 490 nm by examining the formation of formazan resulting from 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride bioreduction (data not shown). The steroid hormone testosterone, dissolved in dimethyl sulfoxide, was added diluted to a final concentration of 100 nM to provide sufficient concentrations of P450 substrate. By omitting testosterone, its influence on AR transcription and activation was illustrated. All drug and hormone concentrations used have been validated previously to resemble therapeutic conditions (Thuerl et al., 1997; Tauer et al., 1998).
Immunoblot against CYP3A11 and AR. Quantities of 106 Hepa1c1c7 or PC-12 cells were homogenized by sonication (homogenate). Subfractionation was performed by differential centrifugation as described earlier (Meyer et al., 2002). To achieve microsomes the cell homogenates were centrifuged at 9000g. The 9000g supernatant (9S) was directly applied to ultracentrifugation at 150,000g for 30 min at 4°C in a Beckman benchtop ultracentrifuge (Beckman, Krefeld, Germany). The resulting pellet was designated as microsomes (mc). The 9000g pellet (9P) contains the cell nuclei and was used to investigate AR localization in Hepa1c1c7 cells. Denatured protein was resolved on 9% sodium dodecylsulfate-polyacrylamide gels. As a reference, mouse liver microsomes were used. Proteins were transferred to polyvinylidene difluoride membranes (Immobilon P; Millipore, Schwalbach, Germany) in buffer (125 mM Tris and 960 mM glycine). Incubations were performed with a CYP3A polyclonal antibody (dilution 1:2000) followed by exposure to horseradish peroxidase-conjugated IgG goat anti-rabbit (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA) at a dilution of 1:10,000. The specificity of the antibodies for the antigen was proved previously (Hagemeyer et al., 2003). AR detection was performed with an AR polyclonal antibody (dilution 1:500) followed by exposure to the horseradish peroxidase-conjugated IgG at a dilution of 1:5000. The immunopositive bands were visualized with enhanced chemoluminescence (GE Healthcare). The intensity of the immunostained bands was evaluated by scanning densitometry using Tina 2.10 software (Raytest, Straubenhardt, Germany).
Fluorescence Double-Labeling Immunocytochemistry. Cells were grown on Thermanox slides (Nunc, Wiesbaden, Germany) and fixed in paraformaldehyde (4% v/v) for 30 min at room temperature. Unspecific binding sites were blocked by phosphate-buffered saline/0.3% Triton X-100 (v/v)/1% bovine serum albumin. The cells were incubated in a cocktail of primary antibodies (rabbit anti-AR, dilution 1:200 and mouse anti-NFpan, dilution 1:6) in phosphate-buffered saline/0.3% Triton X-100 (v/v)/1% bovine serum albumin at 4°C overnight. Afterwards, the binding sites of the primary antibodies were labeled by fluorescence-coupled secondary antibodies applied for 2 h at 4°C (GAR-RRX; Jackson ImmunoResearch Laboratories, Inc., and GAM-Alexa 488; Invitrogen). Samples were washed again and cover-slipped in Elvanol [20% polyvinyl alcohol (Calbiochem, Darmstadt, Germany) and 1% 1,4-diazabicyclo[2.2.2]octane (Aldrich, Steinheim, Germany) in 0.2 M Tris-HCl, pH 8.5]. Controls were performed by omitting one or both primary antibodies. Thus, there was specific staining for only one of the antigens, or, in the latter case, there was no specific staining at all. Fluorescent signals of 2-µm-thick optical sections were examined by using a confocal laser scanning microscope (TCS NT; Leica, Heidelberg, Germany).
Immunocytochemistry. Cells used for immunocytochemistry (2.5 x 105) were grown on Thermanox coverslips, and 48 h after drug application, cells were fixed for 30 min in 4% paraformaldehyde in sodium phosphate buffer (pH 7.4). For immunostaining, we followed the instructions for the Zymed kit (Invitrogen, Heidelberg, Germany). The antibody against the AR was used at a dilution of 1:400. All samples were incubated overnight at 4°C. The specificity of the antibody has been demonstrated previously (Meyer et al., 2006). Negative controls were treated with only the secondary antibodies and proved to exert no immunosignal. Chromogen staining using 3,3'-diaminobenzidine was performed for 15 min at room temperature. Samples were washed again and coverslipped in Elvanol as described previously.
Transfection of Cell Lines with ARE-CAT Constructs and Full-Length CYP3A11. An ARE-CAT reporter construct was used to report androgen receptor activation. For Hepa1c1c7, 106 cells were pretreated for 48 h with a 10 µM concentration of the designated antiepileptic drug and 100 nM testosterone on six-well dishes. Afterwards, 2 µg of MMTV-ARE-CAT construct was transfected into the cells by electroporation using a specific Nucleofector solution (Cell Line Nucleofector Kit R, VCA-1001) as described below. The transfection efficiency in Hepa1c1c7 cells achieved was 75 to 80%.
PC-12 cells were transfected either with the full-length CYP3A11 plasmid (FL3a11) alone for quantitative real-time RT-PCR investigations or in combination with an ARE-CAT construct for the CAT reporter gene assay (CAT ELISA). Each transfection was performed using 106 cells, a specific Nucleofector solution (Cell Line Nucleofector Kit V, VCA-1003), 0.5 to 1 µg of FL3a11, or 1 µg of empty pcDNA1.1Amp vector as a negative control. For the transactivation assay, we added 2 µg of ARE-TK-CAT reporter construct. Transfection efficiency for PC-12 was approximately 35 to 40%, which is in accordance with the manufacturer's cell line-specific protocol (Amaxa Biosystems). After electroporation, PC-12 cells were plated onto collagen-coated six-well dishes supplemented with fresh media and analyzed after 5 to 8 h by real time RT-PCR or after 24 to 48 h by the CAT ELISA, respectively.
RNA Interference. Three different duplex siRNAs with TT ends, located on different exons, against both CYP3A11 and GAPDH were designed using a commercial siRNA design tool algorithm (Ambion, Austin, TX). GAPDH siRNA was used as a positive control. The selected siRNA duplexes were cyp3a11_01 (5'-AAGGGCAGCATTGATCCTTAT-3') (exon 7), cyp3a11_02 (5'-AATTCGACATGGAGTGCTATA-3') (exon 2), and cyp3a11_03 (5'-AACAACCCAGAGGATCCTTTT-3') (exon 3) for CYP3A11, GAPDH_01 (5'-AACTACATGGTCTACATGTTC-3'), GAPDH_02 (5'-AACCACGAGAAATATGACAAC-3'), and GAPDH_03 (5'-AAGTATGATGACATCAAGAAG-3') for GAPDH and as a nonsilencing control a scrambled antisense siRNA (5'-AATTCTCCGAACGTGTCACGT-3'). All siRNA nucleotides were compared with mouse and rat genome databases (NCBI nBLAST) and demonstrated exclusive specificity against the intended target genes. Each siRNA was used in a final concentration of 50 nM. Initially, CYP3A11 siRNA efficiency was tested by cotransfection with 1 µg of CYP3A11-GFP fusion plasmid in Hepa1c1c7. As expected, the silencing targeted against CYP3A11 resulted in a nearly complete loss of fluorescence of the CYP3A11-GFP fusion protein (data not shown).
For Hepa1c1c7, 106 cells were treated for 48 h with 100 µM concentrations of the designated antiepileptic drug before being transfected with 50 nM CYP3A11, GAPDH, or nonsilencing siRNA by the electroporation technique using the Nucleofector device (Nucleofector program T-20). After an additional 48 h, mRNA expressions of AR, CYP3A11, and GAPDH were analyzed by quantitative real time RT-PCR, and the resulting AR-dependent signaling was checked by the CAT reporter gene assay (see below). For PC-12, the CYP3A11 siRNA nucleotides were cotransfected with those vectors used in either the CYP3A11 overexpression or the transactivation assays.
RNA Isolation and Real-Time PCR. Total RNA was isolated using an RNeasy Kit. Two micrograms of total RNA was reverse-transcribed as described above. Real-time PCR was performed on an ABI PRISM 7700 system (TaqMan) using sequence detector software, version 1.9 (Applied Biosystems, Darmstadt, Germany). TaqMan primers and probes for cyp3a11 (NM 007818), cyp3a2 (U 09742), mouse AR (GenBank Accession Number X53779), rat AR (NM 012052.1), and GAPDH (NM 008084) were designed with the help of Primer Express software (version 1.5; Applied Biosystems). TaqMan probes coupled to a 5'-fluorophore (FAM) and a 3'-quencher (TAMRA) were manufactured by Applied Biosystems. A 15-min initial denaturation at 95°C was followed by a 40-cycle two-step PCR with each 30-s annealing/elongation at 60°C and 30-s denaturation at 95°C. GAPDH was chosen as the housekeeping gene, because neither testosterone nor the antiepileptic drugs used are known to regulate GAPDH in brain (Joe and Ramirez, 2001). Primer and probe concentrations were optimized as follows: mAR forward, 5'-TTAACGTCCTGGAAGCCATTG-3' (900 nM); mAR reverse, 5'-CAAAGGAATCTGGTTGGTTGTTG-3' (900 nM); rAR forward, 5'-CTTTCTTAATGT-CCTGGAAGCCAT-3'(900 nM); rAR reverse, 5'-AAGGAATCAGGCTGGTTGT TGT-3' (900 nM); and AR probe, 5'-FAM-CCAGGAGTGGTGTGTGCCGGACAT-TAMRA-3' (100 nM) (located on r+mAR gene, exon 4); mCyp3a11 forward, 5'-CCCAAAGGGTCAACAGTGATG-3' (900 nM); mCyp3a11 reverse, 5'-CTTCAGGCTCTGACCAGTGCT-3' (900 nM), and mCyp3a11 probe, 5'-FAM-TTCCATCTTATGCTCTTCACCATGACCA-TAMRA-3' (100 nM) (located on cyp3a11 gene, exon 6); rCyp3a2 forward, 5'-AAACCACCAGCAGCACACTCT-3' (900 nM); rCyp3a2 reverse, 5'-TCCTCCTGCAGTTTCTTCTGAAT-3' (900 nM); and rCyp3a2 probe, 5'-TCTTGTATTTC-CTGGCCACTCACCCTGA-3' (100 nM) (located on cyp3a2 gene, exon 6). Levels of the GAPDH housekeeping gene were determined for internal normalization using GAPDH forward, 5'-CCAGAACATCATCCCTGCATC-3'; GAPDH reverse, 5'-GGTCCTCAGTGTAGCCCAAGAT-3'; and GAPDH probe, 5'-FAM-CCGCCTGGAGAAACCTGCCAAGTATG-TAMRA-3' (located on GAPDH gene, exons 5–6). Results were demonstrated as the ratio of the quantity of the target gene versus expression of the GAPDH housekeeping gene and finally normalized against the values of vehicle or untreated controls. All primers and probes were compared with the mouse and rat genome database (NCBI nBLAST) and demonstrated exclusive specificity against the intended target genes. In addition, all primers were checked in a standard RT-PCR reaction with cDNA of both cell lines before TaqMan real-time PCR and demonstrated single bands at the expected nucleotide size in ethidium bromide staining (data not shown). This allowed further exclusion of genomic DNA contamination, as the GAPDH primer set enables exon spanning.
Endogenous AR expression in both cell lines was evidenced by nonquantitative RT-PCR using mouse AR- and rat AR-selective TaqMan primer pairs. The PCR products obtained had the expected size at 71-bp/74-bp length and were separated by electrophoresis using 3.5% MetaPhor high-resolution agarose (Cambrex Bio Science, Rockland, Inc., Rockland, ME). GeneRuler 100-bp DNA ladder (Fermentas, St-Leon-Rot, Germany), and DNA Molecular Marker X (Roche Diagnostics) as markers.
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Statistical Analysis. Each experiment was confirmed two times at least and measured in triplicate. The interassay coefficients of variation are indicated in the respective figure legends and were generally less than 10%. Data are presented as means ± S.D. of a representative experiment for each section. For statistical analysis, we used unpaired two-group t tests (two-tailed t test) or, for multiple comparisons, an analysis of variance with a post hoc correction (Bonferroni). Data processing was performed using MS Excel 2003 and SPSS 11.0.1 software (SAS Institute, Cary NC). For transient transfections, data were normalized against the transfection efficiencies. The accepted significance level was p < 0.05, at least.
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), validated by expression of NFpan. As displayed by fluorescence double-labeling immunocytochemistry, NFpan is clearly coexpressed with endogenous AR in NGF-treated PC-12 cells (Fig. 2b).
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Induction Model: Treatment of Hepatic and Neuronal Cells with P450-Inducing Drugs
We applied the commonly used CYP3A-inducers phenytoin and phenobarbital as equivalents in both cell lines (Volk et al., 1995).
Regulation of CYP3A11 and AR mRNA Expression in Hepa1c1c7 Cells. Hepa1c1c7 cells were generally used as a model for P450 investigations because these cells are known to be very easily induced by AEDs (Karenlampi et al., 1989). We therefore used this cell line as a model to investigate general relations between AR, P450, and AED treatment.
Application of phenytoin to Hepa1c1c7 cells resulted in an orchestrated, congruent up-regulation of CYP3A11 and AR mRNA, as analyzed by quantitative real-time PCR (Fig. 3, a and b). We found 7-fold induction of CYP3A11 and 12-fold induction of AR. According to this observation, we analyzed whether specific silencing of CYP3A11 also affects AR mRNA regulation. After application of specific CYP3A11 siRNA nucleotides, both target genes were down-regulated (Fig. 3, a and b). We found effective silencing of CYP3A11 (80%) as well as AR down-regulation (75%) in phenytoin-treated cells using specific CYP3A11 siRNA.
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After treating the cells with 100 µM phenytoin in presence of nonsilencing siRNA, we found CAT expression to be up-regulated to approximately 40% compared with that in untreated Hepa1c1c7 cells, indicating enhanced receptor activation (Fig. 3c). Transfection of the cells with CYP3A11 siRNA reversed this activation completely. We found CAT expression of the phenytoin-treated cells to be in the same range as that in the untreated nonsilencing controls. Untreated cells also revealed significant reduction of receptor activation to approximately 40% as a result of CYP3A11 silencing (Fig. 3c).
Regulation of AR Protein Expression in Hepa1c1c7-cells after Treatment with the CYP3A Inhibitor Ketoconazole. AR protein expression was found to be increased approximately 3.5-fold after treatment with phenobarbital compared with the untreated cells (Fig. 4a). In combination with the CYP3A inhibitor ketoconazole, this AED-dependent AR protein up-regulation was completely reversed to basal levels, as evaluated by immunoblot (Fig. 4a). These findings were corroborated by immunocytochemistry, as the cells treated with phenytoin and phenobarbital clearly showed higher AR-staining intensity (Fig. 4b).
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Application of phenytoin and phenobarbital to PC-12 cells resulted in a significant induction of endogenous CYP3A2, which was initially shown to be the most prominent CYP3A isoform in PC-12 cells. Phenobarbital increased CYP3A2 mRNA expression approximately 25-fold and phenytoin 12-fold, as analyzed by quantitative real-time PCR (Fig. 5a). Furthermore, phenobarbital treatment doubled AR mRNA expression compared with that in untreated PC-12 cells. This effect was completely reversed in the case of a combination of inducer with the inhibitor ketoconazole (Fig. 5b). Finally, similar effects were observed for AR protein expression. Phenytoin treatment increased AR protein levels 2.5-fold and phenobarbital 3.5-fold compared with levels in untreated PC-12 cells. In both cases, AR protein expression decreased to basal levels, if inducers were used in combination with ketoconazole (Fig. 5c).
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We observed up-regulation of both CYP3A11 and AR mRNA (Fig. 6, a and b). Overexpression of 1 µg of FL3a11 resulted in approximately 4-fold enhanced AR mRNA expression compared with the vector controls in the presence of the nonsilencing siRNA (Fig. 6b). Next, we analyzed whether specific silencing of CYP3A11 also affects AR mRNA regulation in PC-12. Cotransfection of 1 µg of FL3a11 with the specific CYP3A11 siRNA resulted in down-regulation of both target genes. CYP3A11 mRNA expression was inhibited to approximately 75% compared with the nonsilencing control samples (Fig. 6a). The expression of AR mRNA was found to be decreased almost to the same level as that in the vector controls after application of CYP3A11 siRNA (Fig. 6b).
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Regulation of AR Signaling by CYP3A11. We investigated whether CYP3A11 overexpression leads to increased receptor activation and signaling. Hence, we established a transactivation assay including both the FL3a11 overexpression vector and an ARE-CAT reporter. We found approximately 3.5-fold enhanced androgen receptor activity due to CYP3A11 overexpression in the presence of nonsilencing siRNA compared with that in the vector-only samples. Addition of specific CYP3A11 siRNA to this system resulted in decreased expression of CAT to levels of the vector control (Fig. 6c). These results of the CAT-ELISA assay are concordant with our findings achieved by quantitative real-time PCR.
Influence of Testosterone Withdrawal on AR Expression and Activation
To clarify the role of testosterone itself on AR expression and activation, both methodical procedures, quantitative real-time PCR and the reporter gene assay, were performed in Hepa1c1c7 cells as mentioned before but by omitting the steroid hormone. In absence of testosterone, application of 100 µM phenytoin again resulted in congruent up-regulation of CYP3A11 (80%) and AR mRNA (50%) (Fig. 7a) but failed to enhance AR activation as measured by CAT ELISA (Fig. 7b).
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The use of cell line models facilitates investigation of the coherence between drug application, P450 function, and AR regulation at the molecular level. We initially started with drug-dependent induction of CYP3A11 in Hepa1c1c7 cells. This cell line is known to be very easily induced by AEDs (Karenlampi et al., 1989) and expresses CYP3A11 as well as AR constitutively. We decided to use this cell line to investigate the common principles of P450 regulation and its effects on steroid hormone receptor regulation. Using this so called "induction model," we demonstrated that CYP3A11, after induction by AEDs, modulates AR expression and signaling in a congruent manner. If CYP3A11 expression is silenced or if it is chemically inhibited by ketoconazole, AR expression and signaling decrease. These findings point to a general function of P450 in regulation of steroid hormones and their receptors in endocrine tissues.
Brain P450 is regulated differently from hepatic isoforms. This fact has to be taken into consideration when this model is applied to brain. Both in vitro and mouse in vivo models have revealed that the availability of heme is a limiting step in brain P450 function (Meyer et al., 2002, 2005). This observation indicates a fundamental difference between brain P450 and that in liver, in which a constitutive heme pool is provided (Giger and Meyer, 1983). Nevertheless, previous studies in rodents encouraged the concept that brain P450s have a prominent function in steroid hormone regulation (Rosenbrock et al., 1999; Wang et al., 2000; Meyer et al., 2006).
To validate this concept on molecular levels, we established a neuronal cell line model using PC-12 cells. These cells exert a neuronal phenotype by application of NGF and are therefore commonly used as a neuronal model (Khvotchev et al., 2003). This observation was corroborated by detection of the neurofilament subtypes (60, 160, and 200 kDa, called NFpan) in our cell line system. Furthermore, we could demonstrate constitutive expression of AR mRNA and protein in PC-12 cells by RT-PCR using specific rAR primers and by coexpression of AR protein with NFpan. According to several other reports, constitutive AR expression in PC-12 cells is very weak and hardly able to be detected (Lustig et al., 1994; Nguyen et al., 2005). Nevertheless, if P450 was either induced by AEDs (CYP3A2) or expressed heterologously (CYP3A11), AR was expressed at evident levels. We conclude that significant AR expression in PC-12 cells is dependent on sufficient P450 presence. However, compared with Hepa1c1c7 cells, higher expression levels of CYP3A2 or CYP3A11 are obviously necessary in PC-12 cells to activate the AR. This observation is corroborated by the findings that chemical inhibition of CYP3A2 by ketoconazole or silencing of recombinant CYP3A11 both led to down-regulation of AR expression and signaling in PC-12 cells. Ketoconazole was originally known as a potent inhibitor of CYP3A activity, and recent reports also claim that it has transcriptional inhibition potency (Huang et al., 2007).
The PC-12 cell assay has a further advantage. It allows heterologous expression of murine CYP3A11 on a rat background. Thereby, we can mimic P450 induction in a drug-independent model excluding direct drug-mediated effects on AR regulation. This approach clearly demonstrates that AR expression and signaling are regulated in the presence of CYP3A11 and not by a possible interaction of the drug with the receptor. These findings are substantiated by other studies investigating AED action on reproductive function. An in vivo study observed phenobarbital-dependent effects on reproductive function of rats accompanied by altered sex hormone levels (Gupta et al., 1980). Other studies investigating direct interactions of AEDs with AR revealed that the P450 inducer carbamazepine and the noninducer valproate did not show any direct AR agonist activity (Death et al., 2005). Actually, valproate exerted an antagonistic activity on AR that is consistent with the endocrine side effects of valproate observed by others (Nelson-DeGrave et al., 2004).
We hypothesize that the signal transduction between CYP3A and the AR after drug treatment proceeds by a mechanism leading to a compensatory activation of ARs with enhanced P450-dependent androgen metabolism. We could demonstrate that withdrawal of the CYP3A11 substrate testosterone in phenytoin-treated cells prevents AR activation, whereas AR expression remains up-regulated, although less prominent than in the presence of testosterone. Studies from brain and thyroid gland demonstrate that testosterone is important for AR autoregulation (Lu et al., 1999; Banu et al., 2002). We assume that testosterone has to be present as a substrate or ligand to enhance and activate the P450 AR system. This assumption allows deeper mechanistic insight into the role of CYP3A11 in AR activation. It seems that CYP3A11 itself has influence on AR transcription, whereas AR activation was shown to be dependent on the availability of both CYP3A11 and its substrate testosterone. Previous studies demonstrate that conversion of testosterone to its hydroxylated metabolites is substantially increased in brain as a consequence of phenytoin administration (Rosenbrock et al., 1999; Meyer et al., 2006). In addition, this CYP3A11-dependent depletion of testosterone in mouse hippocampus is accompanied by increased AR expression (Meyer et al., 2006). The hormone-sensitive cells might get a signal to compensate for reduced hormone levels by receptor up-regulation to maintain overall hormonal influence on cellular procedures. This ligand metabolism-based hypothesis is corroborated by several in vitro and in vivo studies that have revealed similar effects about androgen-dependent AR regulation (Quarmby et al., 1990; Krongrad et al., 1991). In addition, estrogen receptor 
was up-regulated in a compensatory manner due to estrogen depletion (Agarwal et al., 2000; Kim et al., 2004).
A putative enzyme-receptor cross-talk seems plausible to link drug-mediated CYP3A11 induction with AR signaling. It is known from drug-inducible liver CYP3A isoforms that enzyme-receptor cross-talk leads to diversified signaling (Handschin and Meyer, 2003). So, after phenobarbital induction, the CYP3A-regulating nuclear receptor, constitutive androstane receptor, was shown to be related to AMP-activated protein kinase activation (Rencurel et al., 2006). AMP-activated protein kinase, as the main enzyme regulating lipolysis, also exerts cross-talk with peroxisome proliferator-activated receptor , another nuclear receptor active in lipid and energy homeostasis. Therefore, several CYP3A-inducing drugs are effective in the regulation of cellular energy turnover. We could demonstrate that CYP3A2 and CYP3A11 are crucial and effective mediators of AR signaling. This finding highlights a novel function for P450s, and it expands our knowledge of P450 function, especially in steroid hormone-sensitive areas. The role of CYP3A11 in steroid hormone receptor regulation after drug induction needs further investigation, which would go far beyond the scope of the present study.
Our findings may have a strong impact in the pathology of neurological diseases. Clinical and animal studies imply that P450s regulating steroid metabolism can influence neurological functions, as neuroactive steroids are associated with memory, behavior, mood, neuroprotection, aging, and neurotransmission. In addition, steroid hormones were credited with playing an important role in brain development, function, and plasticity (for review, see Melcangi and Panzica, 2006). Therefore, it seems conclusive that changes in hormonal signal transduction lead to severe reproductive and endocrine as well as mental disorders. This conclusion is substantiated by recent clinical data implying complex interactions between P450-inducing AEDs and the occurrence of reproductive and endocrine disorders (Herzog and Fowler, 2005; Isojarvi et al., 2005). Epileptic patients receiving the classic P450 inducers demonstrate an unusually high occurrence of these disorders compared with those treated with noninducing AEDs.
The relation between P450 induction, altered steroid hormone metabolism, and AR-signaling may be of particular interest for research, clinical use, and drug design, because 70 to 80% of all clinically used drugs interact with P450 (Ingelman-Sundberg, 2004). This may influence drug design or drug use, as such relations can lead to adverse drug effects with dramatic costs ($100 billion in the United States) (Ingelman-Sundberg, 2004) or to neuroprotective effects (Nguyen et al., 2005). The present study clearly demonstrates that the role of P450 in brain or, in general, is not restricted to hormonal clearance and degradation. The enhanced expression of P450s after drug treatment obviously leads to activation of AR and to altered steroid hormone signaling with more or less severe consequences for the patient.
In conclusion, our study demonstrates an essential role of P450s as endocrine modulators due to drug-dependent induction. Our data contribute to understanding of the overall action of AEDs and the role of P450 in brain, thereby helping to improve drug design.
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Footnotes |
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Parts of this study were presented as follows: Gehlhaus M, Schmitt N, Hagemeyer CE, Knoth R, Volk B, and Meyer RP (2004) Anticonvulsive drugs modulate steroid hormone signalling in hippocampus of mouse brain, in Proceedings of the 15th International Symposium on Microsomes and Drug Oxidations, 15th International Symposium on Microsomes and Drug Oxidations; 2004 July 4–9; Mainz, Germany, and Gehlhaus M, Schmitt N, Hock M, Knoth R, Volk B, and Meyer RP (2006) Cytochrome P450 CYP3A11 links neuroactive drugs to androgen signalling in hepatic and neural cell lines, Proceedings of the 16th International Symposium on Microsomes and Drug Oxidations, 16th International Symposium on Microsomes and Drug Oxidations; 2006 September 3–7; Budapest, Hungary.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: P450, cytochrome P450; AED, antiepileptic drug; AR, androgen receptor; NGF, nerve growth factor; siRNA, short interfering RNA; NFpan, neurofilament pan; PCR, polymerase chain reaction; Q-PCR, quantitative PCR; CAT, chloramphenicol-acetyltransferase; ELISA, enzyme-linked immunosorbent assay; MMTV, mouse mammary tumor virus; ARE, androgen-responsive element; GFP, green fluorescent protein; bp, base pair; RT, reverse transcriptase; FL3a11, full-length cytochrome P450 3a11; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mAR, mouse AR; rAR, rat AR.
Address correspondence to: Dr. Ralf P. Meyer, Pathologisches Institut, Abt. Neuropathologie, Neurozentrum, Universität Freiburg, Breisacherstraße 64, D-79106 Freiburg, Germany. E-Mail: ralf.meyer{at}uniklinik-freiburg.de
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