Many P450 genes are induced by exogenous chemicals including
drugs, environmental toxins, and carcinogens. Elevated P450 expression
underlies many drug activation events and drug interactions that may
lead to their increased toxicity and carcinogenicity (Conney, 1967;
Gonzalez, 1989). Phenobarbital (PB) (
)is a prototype for a
group of structurally unrelated chemicals that still share the property
of activating many members of CYP subfamilies 2B, 2C, and 3A in animal
species (for reviews, see Nebert and Gonzalez (1987), Gonzalez(1989),
Porter and Coon(1991), Waxman and Azaroff (1992), and Denison and
Whitlock(1995)). The molecular details of this regulation in mammalian
cells are largely unknown, owing to the lack of PB-responsive cell
lines or other reliable in vitro assay systems.
An
extensive study of the barbiturate-regulated induction mechanism of
bacterial CYP102 gene led Fulco and associates to identify a 17-bp
sequence as the binding site for a barbiturate-regulated factor (He and
Fulco, 1991; Liang and Fulco, 1995). Based on sequence comparisons and in vitro protein binding studies (He and Fulco, 1991), it was
suggested that a similar sequence located in the CYP2B promoter
(-89/-73 bp) could be the PB-responsive element for this
and other mammalian P450 genes (Shaw and Fulco, 1993; Liang et
al., 1995). Some studies indicated that this so-called Barbie box
(consensus: 5`-ATCAAAAGCTGGAGG) may play a role in PB-induction of
mammalian genes. The element required for in vitro transcription of a CYP2B2 minigene (-88/-56 bp)
includes the Barbie box (Rangarajan and Padmanaban, 1989; Upadhya et al., 1992). The deletion or mutation of a Barbie box-like
sequence (-140/-124 bp) in rat 
-acid
glycoprotein (AGP) promoter suppressed both basal and dexamethasone- or
PB-induced CAT expression (Fournier et al., 1994). Other
studies argue against a PB-inducible role for the Barbie box. A
transgenic mouse line carrying up to -800 bp of CYP2B2
5`-flanking sequence expressed CYP2B2 mRNA only constitutively.
PB-dependent induction of 2B2 mRNA was evident when additional 5`
sequences were present (Ramsden et al., 1993). Furthermore, in
rat Qsj:SD strain CYP2B2 induction is defective even though CYP2B1 is
activated by PB. Basal expression from both genes can be detected, and
both CYP2B2 and CYP2B1 promoter sequences up to -800 bp matched
those of the parent strain, containing intact Barbie box sequences
(Hashimoto et al., 1988). As for AGP gene, the kinetics of PB
induction are much slower than for CYP2B (Fournier et al.,
1994), other studies do not demonstrate any protein binding to Barbie
box-like sequences in rat or mouse AGP promoter (Ratajczak et
al., 1992; Lee et al., 1993), and basal and
dexamethasone-dependent expression are governed by another elements at
-155/143 and -120/-105 bp (Ingrassia et al.,
1994). These conflicting data indicate that the role of Barbie box in
CYP2B regulation is not yet resolved.
The transcription of hepatic
P450 genes is rapidly activated by PB in vivo. While many
hepatoma cell lines have lost their ability to express P450s or respond
to PB, in some primary cultures CYP2B mRNAs can be increased (Schuetz et al., 1990; Waxman et al., 1990; Akrawi et
al., 1993; Aubrecht et al., 1993; Sidhu et al.,
1993). However, there is a paucity of data on CYP2B transcriptional
regulation or reporter gene assays in primary cells. Clearly, the
development of a faithful PB-inducible system would foster a better
understanding of induction processes involved.
In the present
report, we have now developed a PB-responsive primary hepatocyte
culture method suitable for reporter gene assays and mRNA analyses. We
have cloned and characterized the promoter element from the mouse Cyp2b10 gene, the major PB-inducible P450 (Honkakoski and
Lang, 1989; Aida and Negishi, 1991; Honkakoski et al., 1992a,
1992b). Our results suggest that Barbie box-like sequences do not have
a major transcriptional role, and that sequences important for
PB-induced transcription are located in the distal part of Cyp2b10 gene.
EXPERIMENTAL PROCEDURES
Reagents
Restriction enzymes were from Life
Technologies, Inc. or New England Biolabs. Random priming kit was from
Pharmacia Biotechn Inc. [
-
P]ATP (>5000
Ci/mmol), [
-
S]dATP (>1000 Ci/mmol),
[
-
P]dATP (>6000 Ci/mmol),
[
-
P]UTP (800 Ci/mmol), and
[
C]dichloroacetylchloramphenicol (56 mCi/mmol)
were purchased from Amersham Corp. Oligonucleotides were synthesized
using phosphoramidite chemistry on an Applied Biosystems DNA/RNA
synthesizer. Cell culture media, media supplements, and fetal bovine
serum were from Life Technologies, Inc., collagenase type I and
collagen I were from Sigma, and Matrigel and tissue culture dishes were
obtained from Becton Dickinson Labware (Bedford, MA). All other
chemicals were usually from Sigma or Boehringer Mannheim. TCPOBOP and
1,4-bis[2-(3-chloropyridyloxy)]benzene were synthesized by a
modification of the method of Kende et al.(1985), and
structures were verified using MS and
H NMR. The purities
exceeded 99% as judged by
H NMR.
Animals
Male, 8-10-week-old DBA/2J or
C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) were either
untreated or induced by a single intraperitoneal injection of PB (100
mg/kg, in saline) or TCPOBOP (3 mg/kg, in corn oil) (Honkakoski et
al., 1992a). Tissue samples were taken 16 h later, pooled, and
processed for total RNA (Chomczynski and Sacchi, 1987) and preparations
of liver nuclei (Legraverend et al., 1992) or nuclear extracts
(Gorski et al., 1986). The vehicles themselves had no effect
on CYP2B10 expression (Honkakoski et al., 1992a, 1992b).
Cloning of Cyp2b10 Genomic DNA
Standard molecular
biology protocols were followed (Maniatis et al., 1989).
DBA/2J genomic DNA was partially digested with MboI and
ligated into the BamHI site of EMBL3 vector. Approximately
10
phages were screened initially with 2B10 cDNA (pf3/46)
and subsequently with a EcoRI-StuI cDNA fragment
(+1/+158 bp) (Noshiro et al., 1988). Twenty-two
independent clones were isolated, and phage DNAs were further analyzed
by restriction mapping and hybridization with a CYP2B10 mRNA-specific
oligonucleotide (primer C, see below), subcloning into pUC vectors, and
dideoxy sequencing using universal and specific primers. Only one
clone, designated L4,was identified corresponding to the Cyp2b10 gene. Some fragments of the L4 phage DNA (Hind/Bam-4,
-1.4/+2.3 kb; Bgl/Pst-4, -4.3/-0.6 kb,
Hind/Pst-4, -1.4/-0.6 kb) were further subcloned into pUC
and M13mp18 vectors and sequenced.
Correspondence between Genomic DNA and mRNA
Sequence
We isolated total liver RNA from PB-induced male DBA/2J
mice to minimize the expression of related, female-specific CYP2B9 mRNA
(Noshiro et al., 1988) and used sequence information and
conserved exon-intron junctions of Cyp2b genes (Lakso et
al., 1991) for designing specific primers for 2B10. Ten micrograms
of RNA was reverse-transcribed using an oligo(dT) primer and Moloney
murine leukemia virus reverse transcriptase (Boehringer Mannheim). The
cDNA was amplified for 20 cycles using primers A
(5`-CGTGAATTCCTTGAAGGTTGGCTCAACGACAG, +314/+341 nucleotide of
pf3/46 cDNA) and B (5`-CGTGAATTCAACATTGGTTAGACCAGGACCATGG, 5` end of
CYP2B10 mRNA, deduced from primer extension) and Taq DNA
polymerase (Boehringer Mannheim). The amplified DNA was digested with EcoRI and ligated into the EcoRI site of M13mp18.
Single-stranded phage DNA from 10 independent colonies was isolated and
sequenced using M13 primers. Each sequence matched the deduced first
exon sequence of the genomic DNA, confirming that we had isolated the Cyp2b10 gene.
Determination of the Transcription Start Site
One
hundred micrograms of total liver RNA from untreated and PB-induced
male DBA/2J mice were reverse-transcribed at 42 °C for 1 h using
P-end-labeled primer C (5`-GAAGTTGCCACGGGACTTTGGG;
+78/+99 nucleotides of pf3/46 cDNA). The residual RNA was
destroyed by RNase A; resulting cDNAs, and G and G+A Maxam-Gilbert
reactions of the amplified genomic fragment (primers C and D
(5`-CAGCACACCCGCAGTCTCTTGT; nucleotides -306/-285) were
electrophoresed on 8% acrylamide, 6 M urea gels and
autoradiographed.
Cyp2b10 Gene Transcription in Liver
Nuclear
run-on assays were carried out essentially according to Legraverend et al. (1992) using identical amounts of nuclei (corresponding
to 400 µg of DNA) from untreated, PB- or TCPOBOP-induced male mouse
livers and 0.1 mCi of [
-
P]UTP. After
incubation at 30 °C for 20 min,
P-labeled RNA
transcripts were isolated and hybridized (20
10
cpm) to nitrocellulose-immobilized linear cDNAs (5 µg) for
p16
-14 recognizing both CYP2D9 and CYP2D10 mRNAs (Wong et
al., 1989), pf3/46 recognizing CYP2B10 mRNA,
-actin, and
pUC19 vector (Aida and Negishi, 1991). The washed (3
20 min at
68 °C, 0.1
SSC, 0.1% SDS) filters were then
autoradiographed.
Tissue Distribution and Induction of CYP2B10
mRNA
To avoid problems with cross-hybridization with the cDNA
probe due to large number of Cyp2b genes and low levels of
CYP2B10 mRNA in extrahepatic tissues, we chose RT-PCR method instead of
Northern blotting. The cDNAs were synthesized as above using 50 µg
of total tissue RNA from control and induced male mice. CYP2B10 cDNA
was amplified using Taq DNA polymerase and primers A and B (20
cycles for liver, 30 cycles for other tissues). The products were
electrophoresed through a 2% agarose gel and photographed. Control
experiments proved that RT-PCR was done under conditions where the
product yield was proportional to the cycle number and linearly
dependent on the amount of reverse-transcribed RNA. Southern blotting
and probing with EcoRI-StuI cDNA fragment and
sequencing of the 360-bp product were also done to verify the CYP2B10
message.
DNase I Protection and Gel Shift Assays
Nuclear
extracts from untreated and induced mouse livers were further purified
by heparin-agarose (Sigma) as described by Yoshioka et al. (1990). The eluted proteins were dialyzed against 10 mM Hepes, pH 7.6, 100 mM KCl, 10% glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride, and 1 µg/ml each of leupeptin,
aprotinin, and pepstatin, and concentrated to 4-7 mg/ml protein.
DNase I protection assays were carried out according to instructions in
the SureTrack kit (Pharmacia) using
P-end-labeled,
appropriately cut fragments from Hind/Bam-4 or Hind/Pst-4 plasmid DNA
or generated by amplification using a
P-end-labeled
primer. The DNA fragments were separated on standard sequencing gels,
which were then dried and autoradiographed. Gel shift assays were
performed using 1-5 µg of crude nuclear extract in
10-20 µl of 10 mM Hepes, pH 7.6, 15% glycerol, 2
µg of poly(dI-dC), 0.05% Nonidet P-40, 50 mM NaCl, and
about 30,000 cpm of
P-end-labeled oligonucleotide or DNA
fragment probe. The free and protein-bound probes were separated on
4.5% or 7% acrylamide gels in 0.5
TBE buffer prior to
autoradiography.
CAT Reporter Gene Construction
Based on the
detected transcription start sites, we chose HincII site
(+4) as the 3` end for the promoter construct. Other appropriate
restriction enzymes were used to cut fragments out of Hind/Bam-4 pDNA.
The fragments and SalI-digested pCAT-Basic vector (Promega,
Madison, WI) were treated with T4 or Klenow DNA polymerase (Life
Technologies, Inc.) and ligated using T4 DNA ligase (Stratagene, La
Jolla, CA). Following transformation into Escherichia coli HB101 cells (Life Technologies, Inc.), the recombinant plasmids
were purified twice on CsCl gradients, verified by restriction mapping
and sequencing over the junction sites, and checked for content of
supercoiled plasmid on agarose gels. The plasmids were named according
to distance of 5`-end of the insert from the transcription start site.
-4300CAT was generated by ligation of 3.7-kb PstI-PstI fragment from Bgl/Pst-4 plasmid into PstI-digested -1404-CAT plasmid. Other constructs were
made by amplification of specific regions of -1404-CAT using
primers into which HindIII (5`) or PstI (3`) were
incorporated. After digestion of DNAs with HindIII and PstI, the fragments were ligated into corresponding sites of
-1404-CAT plasmid. The purified plasmids were verified by
sequencing over the amplified region.
Transient Transfection
Electroporation was chosen
because DNA transfection takes place prior to plating, and equal cell
aliquots from same pool are taken for treatments, eliminating
differences in transfection efficiencies (see LeCam et
al.(1994), and references therein). It is also advantageous to be
able to perform transfection and start treatments quickly, limiting the
transcription taking place during transfection. Our preliminary
experiments with
-galactosidase and pCAT-Control plasmids
(Promega) proved that with the same cell preparation, transfection
efficiencies were similar regardless of DNA construct, even though
daily variations were observed. Primary hepatocytes were isolated from
male C57BL/6 mice using a two-step collagenase perfusion (0.4-0.5
mg/ml in HBSS supplemented with 10 mM Hepes, pH 7.4, and 1
µM porcine insulin; Sigma) and low speed centrifugations.
They were further purified by Percoll (Pharmacia) density
centrifugation to a viability of at least 90% (Nemoto and Sakurai,
1995). The cells were washed and suspended (20-30
10
/ml) in ice-cold PBS containing 20 mM Hepes, pH
7.4. The cells were electroporated at 960 microfarads and 150-170
V using 300 µg/ml sonicated herring sperm DNA as carrier and up to
100 µg/ml CAT construct. The electroporated cells were diluted into
prewarmed Williams' E medium supplemented with 7% fetal bovine
serum, ITS supplement (Sigma), and streptomycin-penicillin G (100
units/ml) and incubated for 5 min. From each pool, 3-ml aliquots were
dispensed on 60-mm cell culture dishes, and the cells were allowed to
attach for 30 min at 37 °C under 5% CO
. The unattached
cells were removed, and the dishes were washed with PBS. The cells
(2-3
10
/dish) then received fresh medium
without serum, containing 5 nM dexamethasone, and inducers or
vehicles (1 mM PB in saline; 50 nM TCPOBOP or
1,4-bis[2-(3-chloropyridyloxy)]benzene in dimethyl
sulfoxide). In some instances, cycloheximide (10 µM),
-amanitin (1 µM), actinomycin D (0.5
µM), GH (50 ng/ml), or EGF (20 ng/ml) were added 30 min
prior to the inducer. The transfection and CAT assay conditions were
optimized in preliminary experiments using pCAT-Control plasmid.
Culture conditions, treatment times, and doses were defined by
analyzing CYP2B10 mRNA and protein, and CAT expression from the
-1404-CAT and -64-CAT plasmids. Alternatively, cells were
plated without electroporation, allowed to attached, and washed with
PBS. Cells were transfected using 7.5 µg of DNA and 50 µg of
Lipofectin reagent (Life Technologies, Inc.) in 3 ml of medium (Shih
and Towle, 1995) without dexamethasone or serum. After an overnight
incubation, the medium was changed to additionally contain 5 nM dexamethasone and 0.5 mg/ml Matrigel, and inducers were added for
24 h.
Analysis of Cellular mRNAs and CAT Activity
The
cell medium was removed at 8 h, and cells were lysed using Trireagent
(Molecular Research Center, Inc., Cincinnati, OH). The CYP2B10 mRNA was
analyzed using the methods described above, and mouse albumin 180-bp
cDNA fragment was amplified using primers E (5`-AGACATCCTTATTTCTATGCCC)
and F (5`-CTGCATACTGGAGCACTTCATT). The cellular mRNA-derived PCR
products were cloned into pCRII vector (Invitrogen) and sequenced. The
verified cDNA fragments were used as probes in Northern blotting of
cellular RNA (Maniatis et al., 1989). For CAT assay, 22 h
after medium change, cells were washed with ice-cold PBS containing 1
mM EDTA, scraped off, and pelleted. The cell pellets were
lysed as described by Pothier et al.(1992) and assayed for
protein (Bradford, 1976) and CYP2B10 protein (Honkakoski et
al., 1992b). Equal amounts of cell extracts (150-200 µg
of protein) were then heat-treated and assayed for CAT activity (Gorman et al., 1982) with the exceptions that acetyl coenzyme A
concentration was 1.33 mM and incubation time was extended to
4 h.
RESULTS
Sequence Analysis of the Cyp2b10 Gene
We
isolated a genomic clone containing at least 13 kb of 5`-flanking
region, the first exon, and part of the first intron of the Cyp2b10 gene. The other 21 genomic clones did not contain any portions of
the Cyp2b10 gene, and they could be classified into seven
groups, confirming the large size of Cyp2b gene family (Lakso et al., 1991). The first exon 197-bp sequence (Fig. 1)
was 94%, 82%, and 79% similar to that of rat PB-inducible CYP2B2, and
those of non-inducible mouse Cyp2b9 and rat CYP2B3 (Jean et al., 1994), respectively. In 5`-flanking sequences, the
overall homology of Cyp2b10 to CYP2B2 was 83%. The Cyp2b10 promoter was 62% and 70% similar to that of CYP2B3 and Cyp2b9, respectively, within the proximal 400 bp; upstream of the (CA)
repeat, the similarity to Cyp2b9 dropped to only 36%. Only a
few differences from the CYP2B2 gene were noted; first, the (CA)
repeats (Suwa et al., 1985; Lakso et al., 1991) were
very short in Cyp2b10 (Fig. 1; -303/-300
bp). Second, the so-called Barbie box sequence (Shaw and Fulco, 1993)
was interrupted in Cyp2b10 by an insertion of 42 nucleotides,
the result being that sequence homology to the Barbie box consensus
decreased to 67% (Fig. 1, wavy lines). However, the
insertion was shared by mouse Cyp2b9 and rat CYP2B3 genes
(Jean et al., 1994), suggestive of loss of the 42-bp DNA
element from CYP2B1/2 genes after the rat-mouse divergence. An atypical
TATA box (CATAAAAG) and sequences resembling the binding sites for
transcription factors including Sp1, AP-1, C/EBP, Ets-1, HNF-5, and GR
were also found (Fig. 1).
Figure 1:
The nucleotide sequence of the Cyp2b10 promoter. The 5`-flanking region, the first exon, and
5`-end of the first intron of genes for Cyp2b10 (line
1), CYP2B2 (line 2), and Cyp2b-9 (line
3) are compared, with differences only shown. Deletions are shown
by dots. Putative nuclear protein binding sites and TATA box
are shown above the Cyp2b10 sequence. The Cyp2b10 transcription start sites are shown by asterisks, the
translation start codon is shown in boldface, and the intron
sequences in lowercase. The proximal promoter regions of Cyp2b10 and Barbie box consensus (5`-ATCAAAAGCTGGAGG) can be
aligned at two sites(-131, -88) on Cyp2b10 gene (wavy lines).
Transcription Start Site
We determined the
transcription start sites of Cyp2b10 gene by primer extension
analysis. Male mice were used to minimize the expression of CYP2B9 mRNA
and its possible cross-hybridization with the primer. Three bands
represent the start sites at adenines at 29, 30, and 34 bp downstream
of the putative TATA box, respectively (Fig. 2). Treatment by PB
did not change the location of this site, since the same fainter bands
were detected in control RNA. The observed start sites matched closely
to those of CYP2B1/2 in rat liver (Suwa et al., 1985).
Figure 2:
Determination of the Cyp2b10 transcription start site. Liver RNA (100 µg) from untreated (lane 2) and PB-treated (lane 3) DBA/2J male mice was
reverse-transcribed using 5`-end-labeled primer C as described under
``Experimental Procedures.'' The cDNAs and G (lane
1) and G+A (lane 4) reaction ladders were run on a
8% sequencing gel and autoradiographed.
Cyp2b10 Regulation in Mouse Tissues
Previous
Northern blot analysis showed that hepatic CYP2B10 mRNA was highly
induced within 3 h after PB or TCPOBOP injection (Aida and Negishi,
1991; Honkakoski et al., 1992a). To minimize detection and
cross-hybridization problems, CYP2B10 mRNA expression was measured by
RT-PCR. Although CYP2B10 mRNA was detected in lung and intestine after
longer amplification, PB-inducible expression was observed only in the
liver (Fig. 3). Sequencing of the amplified product yielded only
CYP2B10 mRNA in liver (not shown). The nuclear run-on assays showed
that both PB and TCPOBOP induced nascent
P-labeled CYP2B10
transcripts (detected by pf3/46 cDNA), while that of non-inducible
CYP2D9 plus 2D10 (p16
-14 cDNA) was relatively unchanged.
Furthermore, this induced transcription was largely unaffected by the
presence of GH, since the pattern was quite similar between intact and
hypophysectomized mice (Fig. 4). This confirms that pituitary
factors are not critical for Cyp2b10 expression in mice (Smith et al., 1993), as observed for CYP2B1/2 in rats (Yamazoe et al., 1987; Schuetz et al., 1990). The
transcription of
-actin was variable for unknown reasons. The
amount of CYP2D9 plus 2D10 transcripts was decreased by
hypophysectomy (Fig. 4), consistent with our previous reports
(Wong et al., 1989; Yoshioka et al., 1990).
Figure 3:
Liver-specific induction of Cyp2b10 gene. Fifty micrograms of total RNA from control (lanes
1), PB-treated (lanes 2), and TCPOBOP-treated (lanes
3) DBA/2J male mouse tissues were reverse-transcribed and
amplified for 20 cycles (liver) or 30 cycles (other tissues). One-fifth
of the amplification reaction was loaded on 2% agarose gel and
photographed.
Figure 4:
Transcriptional regulation of the Cyp2b10 gene. Hypophysectomized (Hypox) and
sham-operated (Sham) DBA/2J male mice were injected with corn
oil (lanes 1), PB (lanes 2), or TCPOBOP (lanes
3). Purified nuclei (400 µg of DNA) were incubated with
[
-
P]UTP for 20 min, and the isolated RNA
transcripts (20
10
cpm) were hybridized to
indicated immobilized linear plasmid DNAs (5 µg) detecting CYP2B10
(pf3/46), CYP2D9 plus CYP2D10 (p16
-14),
-actin mRNAs, or pUC
according to Legraverend et al.(1992). The filters were washed
(3
20 min) at 68 °C with 0.1
SSC, 0.1% SDS, and
autoradiographed.
Cyp2b10 Regulation in Mouse Primary Hepatocytes
We
then developed a primary cell culture system, in which endogenous
CYP2B10 mRNA could be rapidly induced. Northern hybridization (Fig. 5A) and RT-PCR assays (Fig. 5B)
displayed more than 10-fold increases of CYP2B10 mRNA in hepatocytes
within 8 h by treatment with PB, 100 nM dexamethasone, or
TCPOBOP, but not by 3-chloro derivative of TCPOBOP. Time course studies
indicated CYP2B10 mRNA was elevated already after 1 h, reaching a
maximum level at 8 h, as in vivo (data not shown). Most
importantly, the increase in CYP2B10 mRNA was blocked by pretreatment
with the transcriptional inhibitors
-amanitin and actinomycin D (Fig. 5, A and B, lanes 7 and 8) but not by cycloheximide, GH, or EGF (lane 6 and
data not shown). This indicates that Cyp2b10 gene
transcription is rapidly activated also in primary hepatocytes.
Intriguingly, Northern hybridization detected a non-inducible 2.7-kb
mRNA, in addition to 2.2-kb CYP2B10 mRNA (Fig. 5A).
Since CYP2B9 or CYP2B13 mRNAs could not be amplified from these cells
(not shown), the 2.7-kb mRNA may be a novel member of the Cyp2b family or an alternatively spliced variant. Nevertheless, it
served as an excellent internal control since both the 2.7-kb and
2.2-kb RNAs are newly transcribed and present in similar levels. The
abundant albumin mRNA was used to control RNA loading and quality, and
it was not changed by the treatments. We also found that low
concentrations (<10 nM) of dexamethasone in the medium are
essential for PB induction of CYP2B10 mRNA while higher levels (
100
nM) increase the basal level.
Figure 5:
Expression of endogenous Cyp2b10 gene in primary mouse hepatocytes. Panel A, Northern
blotting of 10 µg of cellular RNA from cells treated for 8 h with
vehicle (lane 1), 1 mM PB (lane 2), 50
nM TCPOBOP (lane 3), 50 nM 1,4-bis[2-(3-chloropyridyloxy)]benzene (lane
4), 100 nM dexamethasone (lane 5), 1 mM PB + 20 ng/µl EGF (lane 6), 1 mM PB
+ 1 µM
-amanitin (lane 7), and 1 mM PB + 0.5 µM actinomycin D (lane 8). The
probes used were a 158-bp first exon EcoRI-StuI
fragment for 2B10 mRNA (2.2 kb, upper part), and a 180-bp cDNA
fragment for mouse albumin mRNA (2.3 kb, lower part). Panel B, RT-PCR amplification of 10 µg of cellular RNA
from cells treated for 8 h as in panel A using specific
primers A and B for Cyp2b10 mRNA (24 cycles, 360-bp product) and panels
E and F for albumin mRNA (19 cycles, 180-bp product). One-fifth of the
amplification reactions were resolved on 2% agarose gels and
photographed.
Functional Analysis of Cyp2b10 Promoter
We
transfected primary hepatocytes by electroporation with various CAT
reporters containing successive 5`-deletions of the Cyp2b10 promoter. The promoters carrying 5`-flanking sequences up to
-775 bp had high basal transcriptional activities, mostly
contributed by sequences between -64 and -34 bp.
Elimination of the Barbie box-like sequences had no effect
(-376CAT versus -64CAT; Fig. 6A).
Sequences upstream of -775 bp decreased the basal activity
considerably, which suggested a negative regulatory element between
-971 bp and -775 bp of Cyp2b10 gene (Fig. 6A). Inducible transcription was observed only
with the two longest 5`-flanking sequences: PB or TCPOBOP treatments
resulted in 2.0-3.3-fold increases of CAT activity with the
-4300-CAT and -1404-CAT reporters, respectively (Fig. 6B, Table 1). This indicates that the
-1404/-971 bp region may contain DNA elements important for
PB induction, since further addition of 5` sequences did not enhance
induction of CAT. Importantly,
1,4-bis[2-(3-chloropyridyl-oxy)]benzene, which did not induce
2B10 mRNA (Fig. 5, A and B), also failed to
induce CAT activity from the -1404CAT reporter (Fig. 6C), whereas PB and TCPOBOP increased both 2B10
mRNA and CAT activity. The lipofection protocol gave qualitatively
similar results with 4-5-fold increase by TCPOBOP but somewhat
higher basal CAT activity (Fig. 6D).
Figure 6:
Basal and induced expression of Cyp2b10-driven CAT activity in transfected primary mouse
hepatocytes. Panel A, the indicated Cyp2b10 promoter/CAT constructs were electroporated into primary mouse
hepatocytes, cells were aliquoted and maintained in Williams' E
supplemented with ITS and 5 nM dexamethasone for 22 h. Panel B, the indicated Cyp2b10 promoter/CAT
constructs were electroporated into primary mouse hepatocytes, and
cells were aliquoted and maintained in Williams' E supplemented
with ITS and 5 nM dexamethasone without(-) or with
(+) 50 nM TCPOBOP for 22 h. Panel C, the
indicated Cyp2b10 promoter/CAT constructs were electroporated
into primary mouse hepatocytes, cells were aliquoted and maintained in
Williams' E supplemented with ITS and 5 nM dexamethasone
with the addition of vehicle (lane 1), 1 mM PB (lane 2), 50 nM 1,4-bis[2-(3-chloropyridyloxy)]benzene (lane
3), or 50 nM TCPOBOP (lane 4) for 22 h. Panel D. The indicated Cyp2b10 promoter/CAT
constructs were transfected using Lipofectin into primary mouse
hepatocytes for 16 h in Williams' E supplemented with ITS.
Thereafter, the cells were washed with PBS, and medium containing
additionally 5 nM dexamethasone and 0.5 mg/ml Matrigel was
added without(-) or with (+) 50 nM TCPOBOP for
another 24 h. CAT activities and protein were assayed as described
under ``Experimental
Procedures.''
Nuclear Protein Binding to Cyp2b10 Promoter
In the
light of the reported importance of the Barbie box to PB induction and
results of our functional studies, we performed DNase I protection with
heparin-agarose-enriched nuclear extracts. In the proximal promoter,
regions at -64/-45 and -235/-215 bp (Fig. 7A) appear to match the regions displaying
PB-enhanced binding within the CYP2B2 gene (Shephard et al.,
1994); however, no differences in protein binding was evident with Cyp2b10 gene as confirmed by gel shift assays (not shown).
Intriguingly, the regions resembling the Barbie box (Fig. 1)
were not protected in any of the samples (Fig. 7A).
This finding was confirmed by gel shift assays; furthermore, addition
of PB directly (He and Fulco, 1991) or together with cytosolic
fractions to nuclear extracts in vitro did not produce any
inducer-dependent complexes (not shown). This indicates that Barbie
box-like sequences of Cyp2b10 do not bind nuclear proteins. In
the -1404/971 bp region, we could not detect any reproducible
differences in the protection pattern between the samples (Fig. 7, B and C), further confirmed in gel
shift assays (not shown). The major distal protected segments included
-1354/1330 bp, which apparently corresponds to the GRE-like
element in CYP2B2 (Jaiswal et al., 1990), and
-1228/-1195 bp containing C/EBP and AGGTCA motifs.
Figure 7:
DNase I-protected elements of the proximal (panel A) and distal (panels B and C)
regions of Cyp2b10 gene. Heparin-agarose-enriched liver
nuclear proteins (2-20 µg) from untreated or PB-treated
DBA/2J male mice adjusted to 20 µg of total protein with bovine
serum albumin were incubated at room temperature with
P-end-labeled XbaI-HincII (panel
A), HindIII-FokI (panel B), or FokI-NcoI (panel C) DNA fragments for 20
min, and digested with 0.5-1 unit of DNase I for 30 s. Reaction
products were ethanol-precipitated after proteinase K digestion and
phenol-chloroform extraction, separated on a 8% sequencing gel with
G+A reaction ladders, and autoradiographed. Protected regions are
indicated with black bars and distances from the transcription
start site.
DISCUSSION
Cell Culture
The signaling mechanisms and DNA
elements involved in PB-induced transcription of CYP2B genes have not
been elucidated in detail. Hepatoma cell lines in general have lost
either their ability to express CYP2B genes or their responsiveness to
PB, reflecting the highly differentiated nature of the CYP expression.
Intensive efforts, therefore, have been made to develop primary
hepatocyte cultures responsive to PB (see, e.g., Schuetz et al.(1990), Waxman et al.(1990), Akrawi et
al.(1993), Aubrecht et al.(1993), Sidhu et
al.(1993), and Nemoto and Sakurai(1995)). In these systems, CYP2Bs
mRNAs and proteins are highly inducible, and a great deal has been
learned about inhibitory effects of serum, growth factors, cytokines,
and cyclic AMP (Waxman et al., 1990; Schuetz et al.,
1990; Aubrecht et al., 1993; Abdel-Razzak et al.,
1995; Clark et al., 1995; Sidhu and Omiecinski, 1995), as well
as the dexamethasone requirement for optimal CYP2B induction (Waxman et al., 1990; Kocarek et al., 1994; Nemoto et
al., 1995). However, the presence of feeder cells or Matrigel
underlay, or the formation of cell clusters, may compromise DNA
transfection into the cells (Pasco and Fagan, 1989). Similarly,
Matrigel overlay limits the transfection period to the first 4 h of
culture, whereas PB treatment is initiated usually 48 h later (Schuetz et al., 1990; Sidhu et al., 1993). This may result in
destabilization of transfected DNA, in loss of transcription factors,
and in uncontrolled transcription from the plasmid prior to treatment.
In some instances, induction kinetics seem to differ from the rapid
response in vivo. Given the long PB treatment (24-96 h),
some co-treatments may also affect CYP2B mRNA stability as well. With these concerns in mind, we have now developed a primary
hepatocyte culture in which endogenous Cyp2b10 gene is
maximally activated within 8 h. This activation was blocked by
pretreatment by transcriptional inhibitors but not by cycloheximide or
growth factors, and the activation kinetics mirrors the in vivo responses (Aida and Negishi, 1991; Honkakoski et al.,
1992a). Similar protocols were developed by
Salonpääet al. (1994)
and Lecam et al.(1994) for CYP2A5 and GH-regulated rat Spi 2.1
gene, respectively. Protocol differences include shorter cell
attachment time with serum, decreased dexamethasone levels, and plating
to higher density, all of which were found necessary for optimal
CYP2B10 mRNA expression.
Reporter Gene Studies
We then sought after DNA
transfection methods compatible with our cell system. We used
electroporation to avoid differences in transfection efficiency by
using the same pool of transfected cells for aliquoting for different
treatments (Paquereau and LeCam, 1992; LeCam et al., 1994).
Our observations with various Cyp2b10 deletion constructs
correlate well with the data from CYP2B2 transgenic mice. In both
cases, promoters containing about 800 bp of 5`-flanking sequence had
high basal expression but showed no PB-inducibility in hepatocytes.
Similarly, additional 5`-flanking sequences decreased the basal
activity but conferred PB-inducible transcription (Ramsden et
al., 1993). We found that the -1404/-971 bp region was
critical for Cyp2b10 inducibility. The importance of this
element was strengthened by our finding that another Cyp2b10 inducer TCPOBOP (but not its inactive 3-chloro derivative) also
increased CAT activity from -1404CAT reporter. Two other studies
have also indicated that a PB-responsive element (PBRE) might be
located in distal parts of PB-inducible genes. Hahn et
al.(1991) identified a PBRE between -5.9 and -1.1
kilobase pairs of the chicken CYP2H1 gene that exhibited 2.4-fold
increases. Trottier et al.(1995) located a PBRE of CYP2B2 gene
within -2318 and -2155 bp, showing 3.5- to 6.6-fold. The
163-bp CYP2B2 and 4800-bp CYP2H1 fragments could also confer PB
inducibility to heterologous promoters. Despite our attempts to link
-1404/-971 bp element and several overlapping fragments to
heterologous and proximal Cyp2b10 promoters, we could not
confer any PB inducibility to these constructs (not shown). This may be
due to disruption of interactions between the enhancer-binding
factor(s) and factors binding elsewhere on the Cyp2b10 gene,
such as the repressor element at -971/-775 bp. Apparently, these reporter genes are expressed differently, but so
are the endogenous PB-inducible CYP genes between species. For
instance, rat and mouse CYP2B forms differ in their response to
dexamethasone, TCPOBOP, and GH (Meehan et al., 1988; Poland et al., 1981; Honkakoski et al., 1992a; Smith et
al., 1993), and there are differences in tissue specificity, basal
liver expression, and sensitivity to cycloheximide within PB-inducible
CYP2B genes (Omiecinski, 1986; Ohmori et al., 1993; Dogra et al., 1993). It is therefore possible that some aspects of
the induction process may differ between species, and activation of Cyp2b10 gene may include a unique step in addition to the
principal mechanism common to PB-inducible P450 genes.
Protein-DNA Interactions
The well established
function of Barbie box in bacterial CYP102 gene transcription and its
homology to other PB-inducible gene promoters (He and Fulco, 1991; Shaw
and Fulco, 1993; Liang et al., 1995) has focused much
attention on the role of Barbie box-like sequences in mammalian gene
regulation (Upadhya et al., 1992; Fournier et al.,
1994). Intriguingly, a 42-bp insertion into Cyp2b10 sequence
not only splits the Barbie box, it also decreases its degree of
sequence identity. We did not detect any nuclear protein binding to Cyp2b10 Barbie box-like sequences. They did not mediate
PB-induction, nor did their deletion have any effect on basal promoter
activity, and importantly, the presence of 42-bp insertion had no
effect on the inducibility of the endogenous Cyp2b10 gene.
These findings imply that Barbie box may not be a common regulatory
element for PB-inducible expression. In support of this view,
PB-responsive mouse -1404/-971 bp and rat
-2318/-2155 bp fragments do not contain any Barbie box-like
sequences.In distal -1404/-971 bp region, none of the
protein binding sites displayed any major difference between control
and PB treatment, a finding identical to rat 2B2 163-bp region
(Trottier et al., 1995). There are several explanations for
this apparent difference between binding and functional studies. First,
a pre-existing factor might be modified by a signal derived from the
inducer, e.g. by phosphorylation, a mechanism known to
activate nuclear factors (Hunter and Karin, 1992). This would be quite
consistent with the rapid PB induction and its insensitivity to
cycloheximide. Other possibilities include that a factor is being
displaced by a similar, but functionally different factor (Isshiki et al., 1991), or that the induction is mediated by
protein-protein interactions (Konig et al., 1992) rather than
by a mechanism involving primarily DNA-protein recognition. Even though
it is unclear why a putative PBRE would be located in different regions
of the homologous rat and mouse CYP2B genes, we found similarities
between the rat 163-bp sequence and the mouse -1404/-971 bp
region. A 25-bp DNA fragment at -1.2 kilobase pairs of Cyp2b10 gene is 80% similar to a portion of the 163-bp
sequence, and an overlapping segment (-1228/-1195 bp) can
bind nuclear protein(s). Both rat and mouse 25-bp fragments carry core
binding sites (AGGTCA) for members of ligand-dependent transcription
factor superfamily. This is very interesting in view of reports that
some endocrine-sensitive factors, steroid hormones, and oxysterols
might modulate basal and/or PB-induced CYP2B expression (Larsen and
Jefcoate, 1995; Nemoto and Sakurai, 1995; Kocarek et al.,
1993). It has been suggested that PB-like inducers may act on CYP2B
genes indirectly by inhibition of endogenous sterol metabolism and
interfering with the natural sterol signaling of CYP2B expression
(Waxman and Azaroff, 1992). Our preliminary studies indicate that
several nuclear factors are able to bind the 25-bp segment, and we are
currently trying to purify and characterize these factors.