Materials and Reagents
Phenobarbital (sodium salt), phenytoin (5,5-diphenylhydantoin, sodium salt), 5α-androstenol, 5-pregnen-3β-ol-20-one-16α-carbonitrile (PCN), and 1, 4-bis-[2-(3, 5,-dichloropyridyloxy)] benzene (TCPOBOP) were purchased from Sigma-Aldrich (St. Louis, MO). DMSO and all other common reagents not listed were purchased from Sigma-Aldrich or other common vendors. Cell culture media, fetal bovine serum, 100X Penicillin-streptomycin-glutamine solution and trypsin/EDTA were purchased from Invitrogen (Carlsbad, CA). All oligonucleotides were purchased from Sigma-Genosys (The Woodlands, TX) at 50 nmol scale and desalted. HepG2 cells were purchased from the American Type Culture Collection (Manassas, VA).
C3H/HeNCrlBR(C3H) mice were purchased from Charles Rivers Laboratory (Wilmington, MA). A CAR-null mouse (Ueda et al., 2002
) was first crossbred with C3H to generate CAR heterozygous offspring. Subsequently, CAR heterozygous offspring were repeatedly backcrossed with C3H mice until the genetic background became over 95% C3H. The obtained heterozygous mice were bred to produce the wild-type and CAR-null C3H mice. PXR-null 129S1/Sv*129x1/SvJ*C57BL/6 and congenic wild-type mice were obtained from Jeff Staundinger (Staudinger et al., 2001b
) and maintained at NIEHS. Mice were fed with a standard solid diet and tap water ad libitum for 5 days. Eight to fifteen week old male mice received corn oil (vehicle), phenobarbital (80 mg/kg), or phenytoin (80 mg/kg) once daily via gavage at a volume of 10 ml/kg for 4 consecutive days. Mice treated with PCN (80 mg/kg) were orally dosed once daily for 3 consecutive days using corn oil as vehicle. Animals were sacrificed 24 hrs after the last dose. The livers were removed for total RNA isolation and microsome isolation. The NIEHS Animal Care and Use Committee approved all animal procedures.
Total RNA Isolation & Quantitative RT-PCR
Total RNA was extracted using an ABI 6100 Nucleic Acid PrepStation. All chemicals for the ABI 6100 were purchased from Applied Biosystems (Foster City, CA). Total RNA from individual mice was isolated and stored at −80°C. Prior to reverse transcription, equal amounts of RNA from each individual RNA sample was pooled within each experimental group consisting of 3–5 mice according to treatment and genotype. Quantitative RT-PCR analysis was performed using a two step process. An initial reaction with MuLV Reverse Transcriptase (Applied Biosystems) followed by a subsequent quantitative PCR reaction using 2X SYBR Green Master Mix (Applied Biosystems). Reverse transcription was performed with 100 ng of total RNA combined with 1X PCR Buffer II, 0.4 μl (8 units) of Rnase inhibitor (Applied Biosystems), 5.5 mM MgCl2, 0.5 mM dATP, dCTP, dTTP, and dGTP (each), 2.5 μM random hexamers (Applied Biosystems), and 0.5 μl (25 units) of MuLV Reverse Transcriptase in a final volume of 20 μl. Reverse transcription reactions were incubated in a PCR System 9700 Thermocycler (Applied Biosystems) using the following cycling parameters: 25°C for 10 min, 42°C for 60 min, 95°C for 5 min (inactivation), and 4°C hold. The subsequent quantitative PCR reaction was performed on an ABI Prism 7900HT Sequence Detection System (Applied Biosystems). PCR reactions contained 0.5 μl of cDNA template, 1X SYBR Green Buffer Master Mix and 2.5 pmol of forward and reverse primer in a final volume of 10 μl. Gene specific primer set sequences are as follows: Cyp2b10 (Forward, 5′ACCCCACGTTCCTCTTCA3′; Reverse, 5′-CAGCAGGCGCAAGAACTGA-3′); Cyp2c29 (Forward, 5′-GTATTTGGGCTCAAAGCCTACTGTCA-3′; Reverse, 5′-CAGGGTCATGAGTGTAAATCGTCTCA-3′), Cyp2c37 (Forward, 5′-GATGGCAATCAACCATTGC-3′; Reverse, 5′-GCCGATCACATGCTCAATT-3′), Cyp3a11 (Forward, 5′-GTCAAACGCCTCTCCTTGCTG-3′; Reverse 5′-GGCTTGCCTTTCTTTGCCTTC-3′), and β-actin (Forward, 5′-CCTAGAAGCATTTGCGGTGCACGATG-3; Reverse, 5′-TCATGAAGTGTGACGTTGACATCCGT-3′). PCR cycling parameter were as follows: 50°C for 2 min hold, 95°C for 10 min hold, 94°C for 30 sec (denaturation), 60°C for 30 sec (annealing), and 72°C for 30 sec (extension). Denaturation, annealing, and extension temperatures and times were repeated for 42 cycles. PCR products were analyzed using gel electrophoresis, dissociation curve analysis, and by dye terminator DNA sequencing (Applied Biosystems) to determine single product formation and gene specificity. Standard curves (log of template dilution vs Ct value) for each gene specific primer set were used to determine relative mRNA content for each target gene. Each gene specific PCR reaction was performed in triplicate within each pooled experimental group. The triplicate values obtained from each gene specific PCR reaction was used to determine a relative starting template amount mean and standard error for each of the experimental groups.
Electrophoretic mobility Shift Assay (EMSA)
EMSA was performed on a 5% polyacrylamide gel using 0.5X TBE running buffer. Oligonucleotides were labeled with [α-32P]dCTP and probe was purified by Microspin G-25 columns (Amersham Biosciences) to remove unincorporated dNTP’s. Oligonucleotide sequences used in EMSA were as follows: CYP2C9 -1839 CAR-RE (Forward, 5′-CTAGACCAAACTCTTCTGACCTCT-3′; Reverse, 5′-CTAGAGAGGTCAGAAGAGTTTGGT-3′); Cyp2c37 -1890 CAR-RE (Forward, 5′-CTAGAGTTCTCTCCTGGATGAATTTGGGT-3′; Reverse, 5′-CTAGACCCAAATTCATCCAGGAGAGAACT-3′); Cyp2c37 -2065 CAR-RE (Forward, 5′-CTAGGTTACTGTGCTGGGTGAACTGTGTT-3′; Reverse, 5′-CTAGAACACAGTTCACCCAGCACAGTAAC-3′); Cyp2c37 -2791 CAR-RE (Forward, 5′-CTAGAAAAGCAAACTTTTCTGAACTCCATG; Reverse, 5′-CTAGCATGGAGTTCAGAAAAGTTTGCTTTT-3′); Cyp2c37 -2820 CAR-RE (Forward, 5′-CTAGAGCCCGTATCACAAAGTTCAACAAG-3′; Reverse, 5′-CTAGCTTGTTGAACTTTGTGATACGGGCT-3′); Cyp2c37 -3119 CAR-RE (Forward, 5′-CTAGATTAGTGAAATCAAAATGTGATGTATGAAATTCAAG-3′; Reverse, 5′-CTAGCTTGAATTTCATACATCACATTTTGATTTCACTAAT-3′); Cyp2c37 -3205 CAR-RE (Forward, 5′-CTAGCAAATAGAACAACATAAACTGAGAC; Reverse, 5′-CTAGGTCTCAGTTTATGTTGTTCTATTTG3′). Labeled probe (~100,000 cpm per rxn) was applied to each binding reaction in 2 μl of 5X binding buffer, 0.5 μg/ul poly(dI-dC), and 1 μl of each in vitro transcribed/translated protein (mCAR and hRXR) in a final volume of 10 μl. 5X binding buffer was composed of 20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, and 50 mM Tris-HCl (pH 7.5). After addition of probe, the reactions were peformed at room temperature for 20 minutes before being loaded on a polyacrylamide gel and electrophoresed. Gels were then dried and exposed to film for 16 to 24 hours at −80°C.
Expression Vectors and Cloning of the Cyp2c37 5′-Flanking Region
The expression construct pCR3-mCAR was previously described (Sueyoshi et al., 1999
) and pGEMT-hRXR was a kind gift by Ronald Evans. PCR was performed to isolate a 1.97 kb fragment of the Cyp2c37
5′-flanking sequence using the following primers: Forward, 5′-TGCTGTTAGCTGGTCTTGTTCTCTC-3′; Reverse, 5′-CCATGGAGATTCTTCTTACTGACACA-3′. The reverse primer introduced an Nco I restriction site at the 3′ end of the PCR product. The PCR fragment was generated using mouse genomic DNA from Promega (Madison, WI). This fragment was then subcloned into pCR2.1 Topo TA vector (Invitrogen) following manufacturer’s protocol. The endonucleases Bgl II and Nco I were used to remove a 1.8 kb fragment of the Cyp2c37
5′-flanking region from the pCR2.1 Topo TA subclone. This 1.8 kb fragment was then ligated into a Bgl II and Nco I digested pGL3Basic luciferase reporter vector (Promega) producing the Cyp2c37
-1.8 kb luciferase reporter. The Cyp2c37
5′-flanking sequence from -1842 bp to -2887 bp was amplified from murine genomic DNA (Promega) by PCR using forward primer 5′-ACGCGTGGGAAATATAGGGCAATGTGCATC-3′ and reverse primer 5′-GAAGATCTATAGCACAGGGTCAGCTC-3′. The forward primer introduced a Mlu I site in the 5′ end of the PCR product for Cyp2c37
5′-flanking sequence. This ~1Kb fragment was then subcloned into pCR2.1 Topo TA vector (Invitrogen), then digested by Bgl II to excise an ~876 bp fragment using interior Bgl II sites, and finally ligated into a Bgl II digested Cyp2c37
-1.8 kb luciferase reporter to create the Cyp2c37
-2.7 kb luciferase reporter. The subclone for the -1842 bp to -2887 bp Cyp2c37
5′-flanking sequence was digested by Stu I and Mlu I to remove an ~273 bp fragment (-2887 bp to -2614 bp). Subsequent ligation of this fragment into a Stu I and Mlu I digested Cyp2c37
-2.7 kb luciferase reporter created the Cyp2c37
-2.9 kb luciferase reporter. To create the Cyp2c37
-3.6 kb luciferase reporter, murine genomic DNA was again used as a template to amplify the Cyp2c37
5′-flanking sequence from -2540 bp to -3598 bp using a forward primer 5′-CGCGTTTGTAGAGTTAGAAAACACAATTTGC-3′ which also introduced a Mlu I site and the reverse primer 5′-CCCAGTTTCTCATGCTCAAATGGAA-3′. The PCR product -2540 bp to -3598 bp of the Cyp2c37
5′-flanking sequence was subcloned into pCR2.1 Topo TA vector (Invitrogen) and digested with Mlu I and Stu I to excise an ~1050 bp fragment of Cyp2c37
5′-flanking sequence. This fragment was then ligated into a Stu I and Mlu I digested Cyp2c37 -2.9 kb luciferase reporter, producing the Cyp2c37
-3.6 kb luciferase reporter. Mutations of putative binding sites in Cyp2c37
luciferase reporters were produced by site-directed mutagenesis using QuikChange (Stratagene, La Jolla, CA) on the Cyp2c37
-2.9 kb luciferase reporter background. Mutant oligonucleotides used in the site-directed mutagenesis reactions are as follows: -2791Bmut forward, 5′-ATAAAAGCAAACTTTTCCCCC
CTCCATGCAATAAAAACAGTG-3′; -2791Bmut reverse, 5′-CACTGTTTTTATTGCATGGAGCCCC
GAAAAGTTTGCTTTTAT-3′; -2791Amut forward, 5′-CAAGTTATAAAAGCCCC
CTCCATGC-3′; -2791Amut reverse, 5′-GCATGGAGGGGG
GCTTTTATAACTTG-3′. (mutations underlined) Dye terminator DNA sequencing (Applied Biosystems) was used to verify all sequences.
Transcriptional Activation Assays
HepG2 cells were maintained in Eagle’s minimum essential media (Invitrogen) supplemented with 10% fetal bovine serum and 1X penicillin-streptomycin-glutamine solution (Invitrogen). Cells were plated into 24 well plates at a density of ~100,000 cells/well. Transfections included 100 ng of nuclear receptor (pCR3-mCAR), 100 ng of each luciferase promoter construct, and 1 ng of pRL-tk transfection control. Twenty-four hours after transfection, cells were treated with drugs or vehicle and incubated for an additional 24 hours. Cells were subsequently lysed with 100 μl of 1X passive lysis buffer (Promega) for 30 min at room temperature with gentle rocking, and dual luciferase assays (Promega) were then performed on cell lysates per manufacturer’s procedures.
Results from luciferase assays and quantitative RT-PCR are expressed as mean +/− standard error of triplicate determinations. Single statistical comparisons were made using Dunnett’s Method; however, Tukey-Kramer HSD Test was utilized for multiple statistical comparisons when appropriate. The statistical tests were performed using JMP version 5.1 software (SAS Institute, Inc., Cary, NC) and the criterion of significance was set at p = 0.05.