©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel cis-Acting Element in a Liver Cytochrome P450 3A Gene Confers Synergistic Induction by Glucocorticoids plus Antiglucocorticoids (*)

(Received for publication, June 6, 1995; and in revised form, August 18, 1995)

Linda C. Quattrochi (1) A. Scott Mills (2) Joyce L. Barwick (1) Charles B. Yockey (1) Philip S. Guzelian (1)(§)

From the  (1)Department of Medicine, Hepatobiliary Research Center, Section of Medical Toxicology, University of Colorado Health Sciences Center, Denver, Colorado 80262 and the (2)Department of Pathology, Medical College of Virginia, Richmond, Virginia 23298

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The induction by dexamethasone of rat liver CYP3A1 differs from classical glucocorticoid gene regulation in part because both glucocorticoids and antiglucocorticoids such as pregnenolone 16alpha-carbonitrile (PCN) induce CYP3A1 through transcriptional gene activation. In the present study, we transiently expressed in primary cultures of rat hepatocytes plasmids consisting of CYP3A1 5`-flanking sequences fused to a chloramphenicol acetyltransferase reporter plasmid. Deletional analysis identified a 78-base pair (bp) element located approximately 135 bp upstream of the transcriptional start site that was inducible by treatment of the cultures with dexamethasone or PCN and was induced synergistically by dexamethasone plus PCN. Nuclear extract from control rat liver protected two regions within the 78-bp sequence against digestion with DNase I. The same two regions were protected when nuclear extracts from dexamethasone-treated animals were used. Analysis of both of the ``footprints'' (FP1 and FP2) failed to reveal a classical sequence for the glucocorticoid-responsive element. A 33-bp element that includes FP1 sequences inserted into the chloramphenicol acetyltransferase reporter plasmid and transiently expressed in rat hepatocytes conferred a profile of dexamethasone and PCN induction similar to that of the 78-bp element. However, an Escherichia coli expressed glucocorticoid receptor protein failed to protect sequences within FP1 in DNase I footprinting experiments and failed to change its mobility in gel shift assays. Moreover, as judged by the gel shift assay, the specific protein binding to this fragment was the same whether nuclear extracts from the liver of untreated or dexamethasone-treated rats were used. We conclude that the activation of CYP3A1 gene transcription by glucocorticoids may involve proteins already bound to the controlling element in the CYP3A1 gene through a mechanism in which GR in the presence of hormone does not bind directly to CYP3A1 DNA.


INTRODUCTION

CYP3A1, a member of the cytochrome P450 supergene family, is prominently induced in liver microsomes of rats or in primary cultures of adult rat hepatocytes treated with the synthetic glucocorticoid, dexamethasone and, paradoxically, by such antiglucocorticoids as pregnenolone 16alpha-carbonitrile (PCN)^1(1, 2, 3, 4, 5) . Studies using isolated nuclei from rats treated with dexamethasone have demonstrated that transcriptional activation of the CYP3A1 gene is the primary mechanism underlying the induction process(6, 7, 8) . However, detailed pharmacologic analysis of these phenomena in culture disclosed that the time course of induction and the rank order and dose response of synthetic steroid inducers was different for CYP3A1 expression as compared to that for a typical glucocorticoid-responsive gene, tyrosine aminotransferase (TAT)(3, 4, 5) . Moreover, tests of agonist-antagonist relationships demonstrated that rates of de novo synthesis of immunoreactive CYP3A1 protein and accumulation of CYP3A1 mRNA were stimulated synergistically when hepatocyte cultures were incubated in the presence of dexamethasone plus PCN, even though the expression of TAT was inhibited by this same protocol(3, 4, 5) . Glucocorticoids up-regulate TAT by entering the hepatocyte and forming a complex with the glucocorticoid receptor (GR). This is followed by binding of the GR to two glucocorticoid-responsive elements (GRE) located in the 5`-flanking region of the TAT gene, an event that activates TAT gene transcription in a cooperative manner(9) . Based on our initial studies, we suggested that the regulation of CYP3A1 by glucocorticoids may proceed by a mechanism that differs from classical GR binding to a GRE that might be located upstream of the CYP3A1 gene(4) .

A possible explanation for our unusual findings was that glucocorticoids and antiglucocorticoids might affect induction of CYP3A1 at different steps in gene expression including post-transcriptional events such as translation or degradation of CYP3A1 mRNA. To investigate the role of CYP3A1 transcription specifically, we established a system for transient expression of 1.5 kilobases of DNA 5`-flanking the CYP3A1 gene^2 fused to a chloramphenicol acetyltransferase (CAT) reporter plasmid in primary cultures of adult rat hepatocytes that maintain synergistic inducibility of hepatocellular CYP3A1 mRNA by dexamethasone plus PCN(5) . We found that CAT expression from this transfected CYP3A1 chimeric plasmid was induced by treatment of the cultures with dexamethasone or with PCN and was induced synergistically by treatment with dexamethasone plus PCN while expression of a transfected control plasmid containing the classical glucocorticoid inducible MMTV gene was induced by dexamethasone, was not induced by PCN, and was only slightly induced by dexamethasone plus PCN(5) . These results establish that the unique features of CYP3A1 regulation by glucocorticoids largely involve effects on CYP3A1 gene transcription. Preliminary analysis of deletions of the 1.5 kilobases of CYP3A1 DNA defined a 165-base pair (bp) fragment approximately 200 bp upstream of the transcriptional start site that maintains dexamethasone and PCN inducibility and PCN synergy when transiently expressed in cultured hepatocytes(5) . In the present study, we have exploited the advantages of this culture system to closely define the functional glucocorticoid controlling DNA that flanks the CYP3A1 gene. Finding that the sequence lacks a classical GRE, fails to bind GR, but binds to a pattern of liver nuclear proteins from control or dexamethasone-treated rats that appears the same, we conclude that activation of CYP3A1 gene transcription by glucocorticoids may involve a novel indirect interaction of GR with CYP3A1.


MATERIALS AND METHODS

Animals and Materials

Adult male Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing 190-220 g were maintained with free access to animal chow and water for 1-2 weeks prior to use. Collagenase type I was purchased from Worthington Biochemical Corp. (Freehold, NJ), and all other tissue culture reagents were of the highest grade commercially available from Sigma. Matrigel was prepared from the Engelbreth-Holm-Swarm tumor(10) , kindly supplied by Dr. Hynda K. Kleinman, NIDR, NIH, Bethesda, MD. Other reagents were purchased from the following: Lipofectin (Life Technologies, Inc.), CAT-ELISA kit (Boehringer Mannheim), Nycodenz (Sigma), and enzymes (Life Technologies, Inc.). Dexamethasone was purchased from Sigma and PCN was a gift from John Babcock (Upjohn). pMMTVCAT (11) was provided by R. Miksicek and pBLCAT2 (12) was provided by G. Schutz (Institute for Cell and Tumor Biology, Heidelberg, Germany).

Preparation of Hepatocytes and DNA Transfections

Primary cultures of adult rat hepatocytes were prepared as described previously (13) . Hepatocytes freshly isolated by collagenase perfusion of the livers of untreated rats were incubated in a humidified atmosphere of 95% air, 5% CO(2) in our standard medium, a modification of Waymouth's MB-752 medium containing insulin (0.1 µM) as the only hormone. For DNA transfection experiments, 0.4 times 10^6 cells were plated in 35-mm plastic culture dishes (Lux) in 1 ml of medium supplemented with 5% fetal calf serum. The cultures were maintained from 24 to 66 h in serum-free Eagle's minimal essential medium (Life Technologies, Inc.) containing 0.1 µM insulin. At 66 h each dish of hepatocytes was washed with 1 ml of Opti-MEM (Life Technologies, Inc.) and then exposed to a mixture formed by combining 12 µg of Lipofectin with 3.5 µg of DNA (as specified by Life Technologies, Inc.). After 6 h of incubation with the cells, the Lipofectin/DNA mixture was removed and the cultures were incubated with 1 ml of Waymouth's medium containing 300 µg of Matrigel and inducers. Twenty-four h later the medium was replaced with Waymouth's medium containing inducers only. Inducers were added as 1000-fold stocks in dimethyl sulfoxide.

Assay of CAT Protein

After 48 h of treatment with inducers, cell lysates were harvested and assayed for CAT protein (CAT-ELISA assay, Boehringer Mannheim). Briefly, each dish was washed two times with phosphate buffered saline and then incubated 30 min at 4 °C in 1 ml of 40 mM Tris buffer, pH 7.4, containing 5 mM EDTA and 150 mM NaCl to solubilize the Matrigel. This buffer was replaced with 350 µl of lysis buffer (Boehringer Mannheim). Following a 30-min incubation at room temperature, the lysates were centrifuged at 12,000 times g for 10 min. CAT protein was measured according to instructions provided with the kit. Total cellular protein concentration was determined using a bicinchoninic acid microassay (Sigma). Transfection efficiency in several culture preparations was in the range of 5-10%, and CAT protein measured from duplicate plates varied by less than 10% .

Plasmid Constructions and Preparation of Double-stranded Oligonucleotides

The construction of the chimeric plasmid containing the CYP3A1 gene 5`-flanking sequences from -1525 to -56 bp upstream of the transcription start site has been described previously (5) . CYP3A1 sequences consisting of 5` and 3` deletions were generated by the polymerase chain reaction and sequenced (14) to confirm the accuracy of the amplification reaction. All CYP3A1 5`-flanking sequences were cloned in the 5` to 3` orientation in front of the thymidine kinase (TK) promoter of the pBLCAT2 expression vector(12) . Constructions containing these sequences inserted 5` to the CYP3A1 promoter appeared to support dexamethasone- and PCN-mediated CAT expression when transfected transiently into cultured hepatocytes, but the values were too low to give reliable results. Complimentary oligonucleotides spanning sequences protected against DNase I digestion (FP1, FP2), containing BamHI restriction sites at each end, were synthesized (Macromolecular Resources, Fort Collins, CO), annealed and the double-stranded (ds)-oligonucleotides purified by polyacrylamide gel electrophoresis. The ds-oligonucleotides were cloned into the BamHI site of pBLCAT2 as single and multiple copies and in both orientations. The FP1 oligonucleotide corresponds to the position -148 to -115 and FP2 oligonucleotide to -116 to -69. The sequence of the upper strands are as follows: FP1, 5`-GGATCCAATAGGCTGTAGATGAACTTCATGAACTGTCTAGGATCC-3`; FP2, 5`-GGATCCTAGGGGAAGAGAGTACCAAAGTCCACGTGAGGCAGAGGTGATCCATCTGGATCC-3`.

Treatment of Animals and Preparation of Nuclear Extract

Twenty-one-day-old male, Sprague-Dawley rats were given dexamethasone 21-acetate (Sigma) in phosphate-buffered saline plus 10% ethanol intraperitoneally at a concentration of 100 mg/kg for 3 h. After treatment, animals were sacrificed, livers removed, and nuclei prepared by centrifugation through Nycodenz(15) . Purified nuclei were extracted for proteins with high salt buffer containing 20 mM HEPES (pH 7.9), 12.5% glycerol, 1.5 mM MgCl(2), 0.1 mM EDTA, 0.5 mM dithiothreitol (DTT), 0.4 M KCl, and the protease inhibitors Pefabloc, leupeptin, and pepstatin A (Boehringer Mannheim). Extracts were dialyzed against 20 mM HEPES (pH 7.9), 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.2 mM Pefabloc, and 0.5 mM DTT, aliquoted, and stored at -80 °C until use. Protein concentration was determined using a bicinchoninic acid microassay.

DNase I Footprinting Assay

The 165-bp BamHI-HindIII fragment (position -220 to -56) was labeled with P at the BamHI site (upper strand). The labeled probe was incubated with 60 µg of crude nuclear extract. Incubations were performed as specified in the instruction manual for the HotFoot footprinting kit (Stratagene, La Jolla, CA). After incubation, a partial DNase I digest was carried out by adding 50 µl of DNase I buffer and concentrations of DNase I (Stratagene) from 0.02 to 0.1 unit. DNA was extracted with phenol-chloroform, recovered by ethanol precipitation, and analyzed on a 12% denaturing polyacrylamide gel. Sequencing of the DNA probe was performed by the Maxam-Gilbert method (16) using a sequencing kit from United States Biochemical Corp. An Escherichia coli expressed protein consisting of the DNA-binding domain of the glucocorticoid receptor (dbGR, apoT7X556) (17) was a gift from Dr. Keith Yamamoto, University of California, San Francisco. The footprinting assay using the dbGR was essentially as described for nuclear extracts, except that 600 ng of dbGR was preincubated for 30 min on ice with ZnCl (final concentration, 250 mM) prior to the binding reaction. The MMTV DNA fragment, from -222 to +104 relative to the transcriptional start site, in the 5` long terminal repeat of the mouse mammary tumor virus was labeled and used as a positive control for GR binding. This fragment contains three regions protected to varying degrees when used in nuclease protection assays employing partially purified receptor (18) .

Electrophoretic Mobility Shift Assay

The ds-oligonucleotide, FP1, corresponding to the position -148 to -115, was radiolabeled with [-P]ATP and polynucleotide kinase. For this assay, 15 µg of nuclear protein were incubated at room temperature for 20 min with 3 µg of poly(dI-dC) in a binding buffer consisting of 20 mM HEPES, pH 7.9, 60 mM KCl, 5 mM MgCl(2), 2 mM DTT, and 10% glycerol. The binding reaction was continued for 20 min after addition of labeled oligonucleotide to the reaction. Competitor DNA was added during the preincubation period, prior to addition of labeled oligonucleotide. Gel electrophoresis was conducted using 1 times TBE buffer (0.089 M Tris borate, 0.089 M boric acid, 0.002 M EDTA). The sequences of the oligonucleotides used in the electrophoretic mobility shift assay (upper strands) are as follows: FP1, 5`-GGATCCAATAGGCTGTAGATGAACTTCATGAACTGTCTAGGATCC-3`; GRE, 5`-CTAGGCTGTACAGGATGTTCTGCCTAG-3`; NF-kappaB, 5`-AGTTGAGGGGACTTTCCCAGG-3`.


RESULTS

Preliminary analysis of deletions of chimeric plasmid TKCAT-1525/-56 that contains CYP3A1 sequences from -56 to -1525 relative to the transcriptional start site, previously identified a region of the rat CYP3A1 gene between -220 and -56 that, when transiently expressed in primary cultures of adult rat hepatocytes, responded to treatment of the cells with dexamethasone or PCN and exhibited dexamethasone plus PCN synergy(5) . We noted that -220 to -56 contains neither a classical sequence for the 15-bp GRE (19) nor sequences identical to the consensus sequence, TGTTCT, for ``half-sites'' in the classical GRE(20) . We prepared additional deletion mutants of the chimeric CAT plasmid, TKCAT-220/-56 (Fig. 1), transiently expressed these in primary cultures of rat hepatocytes, and measured CAT expression following 48 h treatment of hepatocytes with 10 µM dexamethasone or PCN (Fig. 1). Deletion of sequences between -220 and -176 or between -71 and -56 resulted in no loss of dexamethasone or PCN induction of CAT expression, whereas a further deletion between -176 and -148 resulted in a 2-fold loss of dexamethasone induction. The deletion extending beyond position -56 to -37 bp appeared to reduce CAT expression by PCN treatment, although CAT expression by dexamethasone treatment was not altered. These data suggest the possibility that a negative element lies between -56 and -37. However, the induction by dexamethasone on both the TKCAT-176/-71 (20-fold) and TKCAT-148/-71 (10-fold) constructions was substantial. As shown previously for the TKCAT-220/-56 construction(5) , dexamethasone also produced dose-dependent increases in CAT expression for TKCAT-148/-71 (data not shown). From these results we concluded that the sequences between -148 and -71 are essential for the steroid inductions, although upstream sequences (-176 to -148) may potentiate hormone responsiveness.


Figure 1: Expression of chimeric CYP3A1-TKCAT plasmids in primary rat hepatocytes. Chimeric plasmids were generated by amplification of CYP3A1 sequences using polymerase chain reaction, and sequences cloned into the pBLCAT2 vector (TKCAT) as described under ``Materials and Methods.'' Negative numbers represent distances from the transcriptional start site. Chimeric plasmids were transiently expressed in primary cultures of rat hepatocytes. Cells were treated with dexamethasone (10 µM) or PCN (10 µM) for 48 h, followed by analysis of CAT expression. The -fold induction refers to the ratio of CAT protein of induced cells to uninduced control cells. Data are the average results (mean ± standard error of the mean) of at least three independent transfection experiments.



To examine whether trans-acting proteins bind to the identified CYP3A1 dexamethasone-responsive region, we performed in vitro DNase I footprinting assays. Radioactively labeled CYP3A1 DNA (-220/-56) was incubated with nuclear extracts from control rat liver, and then the mixture was digested with DNase I. The results of these ``footprinting'' studies showed two regions strongly protected and one region weakly protected against DNase I activity (Fig. 2, lane 3). The reaction was specific because addition of excess unlabeled DNA (-220/-56) was able to compete for binding to both protected regions (Fig. 2, lane 5), whereas the presence of excess nonspecific DNA in the reaction did not compete (Fig. 2, lane 6). Although dexamethasone induction of CYP3A1 in the liver might be expected to alter the pattern perhaps by creating additional footprints, use of nuclear extracts from dexamethasone-treated rat liver in the same experiment produced the identical footprints (Fig. 2, lane 4). Moreover, we found the same footprints when this experiment was carried out with the use of nuclear extracts isolated from untreated and dexamethasone-treated cultures of rat hepatocytes (data not shown).


Figure 2: DNase I footprint analysis of nuclear protein binding to the CYP3A1 dexamethasone-responsive element. The P-labeled CYP3A1 fragment from -220 to -56 (upper strand) was incubated with 60 µg of crude nuclear extract from untreated control and dexamethasone-treated animals. Lane 1, A+G sequence reaction; lane 2, CYP3A1 DNA, no nuclear extract; lane 3, control extract; lanes 4-6, induced extract. Specific binding was determined by competition reactions in which 100-fold molar excess of unlabeled CYP3A1 DNA (-220/-56) (lane 5, +) or nonspecific DNA (lane 6, +) was added prior to addition of labeled CYP3A1 DNA. Sequencing of CYP3A1 DNA (-220/-56) was performed by the Maxam-Gilbert method(16) . Footprints 1 and 2 (FP1, FP2) are marked by solid lines. The dashed line indicates a footprint possibly due to weak interactions. Base pairs protected are delineated by negative numbers representing distance from the start of transcription.



By alignment with the CYP3A1 DNA (-220/-56) sequence, we determined that the first protected region (FP1) was located from -135 to -117, while the second footprint (FP2) was from -108 to -85 ( Fig. 2and 3A). Thus, within the -220 to -56 DNA probe, both footprints, FP1 and FP2, were found within the 78-bp fragment identified from deletional analysis to be dexamethasone- and PCN-responsive (Fig. 1, TKCAT-148/-71). Close examination of the FP1 sequence (Fig. 3B) finds 8 bp in common with a 12-bp consensus GRE (19) and 4 of 5 nucleotides suggested as critical for GR binding(21) . The FP1 sequence also contains a palindromic sequence (underlined), ATGAACTTCAT, that overlaps one of two ``direct repeats'' of ATGAACT separated by 2 nucleotides (Fig. 3C). A 6-bp portion of these 7-bp repeated sequences, TGAACT, when examined on the lower strand (AGTTCA), is a DNA motif representing a high affinity binding site for several types of nuclear receptors including the peroxisome proliferator activator (22) and the retinoic acid receptors(23) . The CYP3A1 gene is not regulated by peroxisome proliferators or retinoic acids, thus emphasizing that a DNA binding site identified by sequence analysis is not necessarily functional. In addition, we identified a sequence at the 3` end of FP2 similar to that of an AP1 binding site (24) (Fig. 3A).


Figure 3: Summary of the DNA-protein interactions at the CYP3A1 dexamethasone-responsive element. A, regions protected against DNase I digestion, referred to as FP1 and FP2, of dexamethasone-responsive CYP3A1 (-220/-56) are indicated by solid lines. The dashed line indicates a footprint possibly due to weak interactions. The sequence similar to an AP1 binding site (24) is boxed. B, alignment of FP1 with the consensus GRE sequence(19) . The 5 bases critical for glucocorticoid receptor binding are shown in bold(21) . Lines between nucleotides show identity. C, the sequence of the ds-oligonucleotide containing the FP1 sequence (underlined) is shown. Arrows indicate its palindromic structure and a 7-bp direct repeat.



We prepared overlapping ds-oligonucleotides that span the 78-bp responsive region in CYP3A1 corresponding to -148 to -115 (contains FP1) and to -116 to -69 (contains FP2) to determine which if either of the sequences that were protected against DNase I retains responsiveness to inducers. When the ds-oligonucleotides were cloned into pBLCAT2 and transiently expressed in hepatocytes, only sequences spanning the area protected by FP1 (TKCAT-148/-115) were responsive to dexamethasone and PCN (6- and 3-fold, respectively) (Fig. 4A). The magnitude of the response of the FP1 containing construction to dexamethasone was comparable to the endogenous hepatic levels of CYP3A1 transcriptional activation by dexamethasone as demonstrated by nuclear run-on transcription assays(6, 7, 8) . When placed in the opposite orientation, i.e. 3` to 5`, this -148/-115 ds-oligonucleotide mediates a similar level of CAT induction; however, multiple copies of this sequence did not increase CAT induction over and above the results with a single copy (data not shown). Thus, this single 33-bp element is able to confer both dexamethasone and PCN induction on CYP3A1 gene expression in cultured hepatocytes, although sequences flanking this element may be important in the modulation of responsiveness, as indicated by an increase in CAT induction when sequences from -148 to -176 are included.


Figure 4: Transient expression in cultured hepatocytes of CYP3A1 5`-flanking sequences encompassing FP1 and FP2. A, ds-oligonucleotides corresponding to -148/-115 (contains FP1) and -116/-69 (contains FP2) were cloned 5` to TKCAT and transiently expressed in rat hepatocytes. Hepatocytes were treated with dexamethasone (10 µM) or PCN (10 µM). Footprint sequences are underlined. B, hepatocytes were transfected with either MMTVCAT or TKCAT-148/-115 and treated with either dexamethasone (0.1 µM), PCN (10 µM), or dexamethasone (0.1 µM) plus PCN (10 µM) for 48 h. CAT protein was determined as described under ``Materials and Methods.'' The -fold induction refers to the ratio of CAT protein of induced cells to uninduced control cells. Data are the average results (numbers above bars) of two to three independent experiments. Open bars, MMTVCAT; solid bars, TKCAT-148/-115. N.I., no induction; ratio of CAT protein of induced cells to uninduced control cells < 1.0.



We also demonstrated that the 33-bp element containing the FP1 sequence still confers (although at an attenuated level) the synergistic effect of PCN on CYP3A1 induction by dexamethasone demonstrated previously for the larger CYP3A1 5`-flanking DNA segments(5) . In cultured hepatocytes transfected with TKCAT-148/-115 (one copy of the 33-bp element) and treated with a low dose of dexamethasone (0.1 µM), with PCN (10 µM), or with their combination, the induction of CAT was <2-, 2-, and 10-fold, respectively (Fig. 4B). As a control, we transfected hepatocyte cultures with MMTVCAT, a vector containing a typical GR regulated gene promoter (11) and found, as expected from previous studies(5) , that dexamethasone treatment induced CAT expression and that PCN treatment induced no CAT expression and modestly antagonized dexamethasone induction of MMTVCAT (Fig. 4B).

Inasmuch as the 33-bp element is sufficient to confer dexamethasone responsiveness in cultured hepatocytes and contains sequences in its FP1 segment similar to a simple GRE (Fig. 3B), we performed in vitro DNase I footprinting to determine if the GR actually is capable of being bound by this CYP3A1 DNA fragment. When the radioactively labeled CYP3A1 DNA (-220/-56) containing the 33-bp element was incubated with saturating amounts (approximately 50 pmol) of dbGR, a bacterially expressed protein corresponding to the DNA-binding domain of the rat GR(17) , no protection of the CYP3A1 fragment could be detected (Fig. 5, lanes 12 and 13). In contrast, the MMTV promoter, a DNA control for GR binding that contains multiple GREs(18) , was bound by dbGR as shown by protected sequences within the MMTV promoter corresponding to the GRE showing partial dyad symmetry (-174/-185) (Fig. 5, lanes 4 and 5). These results suggest that if the GR is directly involved in the transcriptional activation of the CYP3A1 gene, it likely interacts with DNA-bound protein rather than with DNA itself.


Figure 5: Test for binding to the dexamethasone-responsive CYP3A1 element by dbGR (DNA binding domain of the glucocorticoid receptor protein) with the in vitro DNase I footprint assay. DNase I footprinting was carried out after binding the dbGR to the MMTV promoter and the CYP3A1-responsive element (-220/-56). Lane 1, A+G sequence for the MMTV promoter; lanes 2 and 3, MMTV promoter, no protein; lanes 4 and 5, MMTV promoter incubated with 600 ng of dbGR; lanes 6-9, CYP3A1 promoter (-220/-56) sequence; lanes 10 and 11, CYP3A1 promoter, no protein; lanes 12 and 13, CYP3A1 promoter incubated with 600 ng dbGR. - refers to absence of dbGR protein in the binding reaction. The GRE in the MMTV promoter protected against DNase I digestion is bracketed.



Further evidence against the possible direct binding of the GR with the CYP3A1 gene was developed with the use of the electrophoretic mobility shift assay. When we incubated bacterially expressed GR (dbGR) with the radiolabeled 33-bp ds-oligonucleotide-responsive element (containing FP1), no change in its mobility in gel electrophoresis was observed, whereas control incubations containing GR plus the TAT GRE were retarded in their migration as expected (data not shown). We did detect a protein-DNA complex when the radiolabeled 33-bp ds-oligonucleotide was incubated with liver nuclear extracts from untreated or from dexamethasone-treated rats (Fig. 6, lanes 2 and 3). As evidence of sequence specificity, the binding of nuclear proteins to the 33-bp oligonucleotide could be abolished in the presence of 50-fold molar excess of unlabeled 33-bp oligonucleotide, but not by 150-fold molar excess of an unrelated oligonucleotide containing a binding site for NF-kappaB (Fig. 6, lanes 4 and 7). The specific protein-DNA complex appeared to migrate with the same mobility in the gel regardless of whether nuclear extracts from untreated or dexamethasone-treated rat liver were used in the binding reaction (Fig. 6, upper arrow), even though the latter extract alone contained sufficient GR protein to be detected by Western blot analysis (data not shown). Also, addition of excess GRE oligonucleotide (50- and 150-fold) to the binding reaction did not result in competition for the complex, indicating that the GR was not part of the protein-DNA complex formed at the 33-bp element (Fig. 6, lanes 5 and 6). Failure of the GR-containing nuclear extracts from dexamethasone-treated rats to enhance retardation of the DNA probe's mobility does not exclude protein-protein interactions involving the GR because such complexes are well known to be subject to disruption under electrophoretic conditions in vitro(25, 26) .


Figure 6: Binding of rat liver nuclear proteins to the 33-bp responsive element. The 5`-end-labeled double-stranded oligonucleotide corresponding to the 33-bp responsive element (containing FP1 sequence) was incubated with liver nuclear extracts from untreated control (C) and dexamethasone-treated (DX) rats. For competition, 50-fold molar excess of unlabeled FP1 DNA, 50-fold (lane 5) and 150-fold (lane 6) excess of unlabeled GRE oligonucleotide, and 150-fold excess of nonspecific DNA (NS) was incubated prior to addition of probe. The GRE ds-oligonucleotide represents the human tyrosine aminotransferase gene response element, and the nonspecific DNA is a ds-oligonucleotide that contains the DNA binding site for NF-kappaB. The upper arrow indicates the position of the specific DNA-protein complex and the lower arrow the position of the free DNA probe.




DISCUSSION

CYP3A1 is one of a family of homologous liver cytochrome proteins inducible by glucocorticoids in many species(27) , including humans (28) , where the 3A cytochromes are the dominant forms accounting for the metabolism of numerous clinically important drugs including nifedipine and cyclosporin as well as such endogenous substrates as cortisol(29) . Isolation of the rat CYP3A1 gene (30) and the development of a system for primary culture of adult rat hepatocytes in which CYP3A1 expression and inducibility by dexamethasone can be maintained (31) has afforded an opportunity to investigate in detail, the unique mode of regulation of CYP3A1 by glucocorticoids. Inhibition of protein synthesis in hepatocyte cultures and in rat liver actually increases CYP3A1 mRNA, making this approach unhelpful in distinguishing induction of CYP3A1 by dexamethasone as a primary or secondary glucocorticoid response(32) . Favoring the former is a report that CYP3A1 is transcriptionally activated within 1 h of dexamethasone treatment to rats(8) . Attempting to penetrate this problem directly, we established previously that a 165-bp fragment contained within 1.5 kilobases of DNA 5`-flanking the CYP3A1 gene transiently expressed in hepatocyte cultures mirrored changes in accumulation of endogenous CYP3A1 mRNA in being transcriptionally activated by treatment of the cells with dexamethasone by a process that is slightly antagonized (RU486) or was actually enhanced synergistically by antiglucocorticoids (PCN)(5) . We now show that transcriptional responses to dexamethasone and PCN and to the synergistic effect of PCN are mediated by a single 33-bp element located at -135 in the CYP3A1 gene. Failure to find within these 33-bp a consensus GRE sequence (5`-AGAACAnnnTGTTCT-3`), multiple GRE half-sites (TGTTCT) (reviewed in (19) and (33) ), or sequences associated with a delayed, secondary glucocorticoid response element (34) is consistent with our proposal from pharmacologic studies that glucocorticoid induction of CYP3A1 proceeds by a non-classical pathway(3, 4) .

While it remains to be proven unequivocally that the GR is obligatory for dexamethasone induction of CYP3A1 in primary cultures of adult rat hepatocytes (or in rat liver), there is strong pharmacologic evidence in such cultures to support this idea (only glucocorticoids to the exclusion of androgens, estrogens and progestins induce CYP3A protein; (3) ). Sequence comparisons of the dexamethasone-responsive CYP3A1 33-bp segment (-148/-115) to mouse proliferin (25) and phosphoenolpyruvate carboxykinase (35) genes, previously demonstrated to bind the GR, suggested sufficient homology to a 12-bp consensus element (19) and critical GR binding nucleotides (21) to test whether the GR can bind to the functional CYP3A1 33-bp segment. However, assays of the binding of dbGR protein with the use of in vitro DNase I footprinting as well as electrophoretic mobility shift assay ruled out an interaction of GR with CYP3A1 at least under conditions in which binding occurs with the classical MMTV GRE (Fig. 5) or the TAT GRE (data not shown), respectively. Furthermore, neither of these techniques disclosed the appearance of an associated protein (such as the GR) that binds to the functional CYP3A1 33-bp segment when it undergoes dexamethasone induction, as would be expected from similar studies of classical GRE-controlled genes(9, 36) . It is a formal but unlikely possibility that proteins bound to the 3A1 enhancer sequence in the basal, steady state are ``switched'' with other dexamethasone induction-specific proteins that have the same characteristics in the two assay systems we employed. We conclude that the glucocorticoids and antiglucocorticoids activate expression of the CYP3A1 gene hormone-responsive element by enhancing transcription through a mechanism that may involve a protein or proteins already bound to the CYP3A1 enhancer in the basal, steady state.

Fresh insight into the control of CYP3A1 arose from DNase I footprinting studies, which disclosed the same pattern of factor(s) capable of binding to this gene in the basal, steady state and following dexamethasone treatment (Fig. 2). One of these footprint areas, FP1, was associated with a 33-bp segment that, by a mechanism that remains to be explained, was able to confer both dexamethasone and PCN induction and PCN synergy to a heterologous promoter when transiently expressed in rat hepatocytes. The possibility that dexamethasone and PCN exerted individual effects at separate sites on the CYP3A1 gene now seems less likely. However, inasmuch as DNA-binding factors, such as steroid receptors, bind as dimers to their cognate DNA binding sites, the presence of a palindrome and a 6-bp direct repeat, similar to synthetic and natural DNA response elements for some steroid receptors, within the 33-bp responsive element supports the conclusion that multiple DNA-binding proteins, possibly dimers, may contact sequences within this element. This direct repeat sequence is also found in the rat CYP3A2 gene and was recently reported to reside in a segment of the gene required for basal expression of a transfected CAT reporter gene and to bind proteins from rat liver nuclear extracts (37) . Wen and Locker (38) recently described a DNA binding site for a novel hepatocyte transcription factor within rat alpha-fetoprotein, a gene repressed in the developing liver by glucocorticoids(39) . This element, termed promoter-linked coupling element, that may compete for GR binding as the mechanism for glucocorticoid repression(38) , is similar to CYP3A1 FP1 in its sequence (8 of 12 bp in common), and in overlapping a GRE-like sequence. It remains to be determined whether it is this protein or members of the nuclear receptor family that recognize the CYP3A1 FP1 sequence and whether the GR, if it is involved at all, transcriptionally activates CYP3A1 gene expression through protein-protein interactions. New models associate the GR with DNA-bound constitutive factor without actual binding to DNA resulting in either down-regulation (40) or up-regulation of gene expression (41) .


FOOTNOTES

*
This work was supported by National Institutes of Health Grants ES05744 and ES04238 and University of Colorado Hepatobiliary Center Grant DK34914. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: UCHSC Medical Toxicology, B146, 4200 E. 9th Ave., Denver, CO 80262. Tel.: 303-270-3501; Fax: 303-270-7180.

(^1)
The abbreviations used are: PCN, pregnenolone 16alpha-carbonitrile; CAT, chloramphenicol acetyltransferase; TK, thymidine kinase; GR, glucocorticoid receptor; dbGR, DNA binding domain of the glucocorticoid receptor protein; GRE, glucocorticoid-responsive element; TAT, tyrosine aminotransferase; bp, base pair(s); ELISA, enzyme-linked immunosorbent assay; ds, double-stranded; DTT, dithiothreitol; MMTV, mouse mammary tumor virus.

(^2)
The sequence of the 5`-noncoding region of the genomic clone used in this study is completely identical with that of the recently isolated P450 RL33(42) .


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