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Vol. 28, Issue 3, 268-278, March 2000
Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee (E.G.S., C.B., K.Y.); Deutsches Krebsforschungszentrum, Heidelberg, Federal Republic of Germany (W.S., G.S.); Division of Analytical Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Kitaku, Sapporo, Hokkaido, Japan (T.K.); Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California (L.B.); and Baker Medical Research Institute, Melbourne, Australia (K.M., T.J.C.)
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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Cytochrome P-4503A, CYP2B, and P-450 reductase are induced
by glucocorticoids, antiglucocorticoids such as pregnenolone
16
-carbonitrile, and drugs such as rifampin and phenobarbital.
Although the pregnane X receptor is reported to mediate steroid and
drug activation of CYP3A via a conserved cis-element in
CYP3A genes, discrepancies exist between the induction of the
endogenous CYP3A genes and the activation of the pregnane X
receptor. It is a formal possibility that the glucocorticoid
receptor may account for some of these discrepancies. To determine the
requirement in vivo of the glucocorticoid receptor in expression of
CYP3A and CYP2B, we compared the induction of these proteins in the
livers of normal mice and mice with a targeted mutation in the
glucocorticoid receptor. Mice lacking the glucocorticoid receptor show
no difference in constitutive hepatic expression of CYP3A but show a
decrease in the level of CYP2B. Glucocorticoid receptor-deficient mice
challenged with either dexamethasone or pregnenolone 16-carbonitrile
failed to induce CYP2B proteins, whereas CYP2B was readily induced in
(+/+) mice. In contrast, CYP3A and P-450 reductase proteins were
induced by either inducer in wild-type and glucocorticoid receptor-null mice. Similarly, rifampin induced CYP3A in either wild-type or glucocorticoid receptor-null mice. Despite reports that rifampin is a
nonsteroidal ligand for the human glucocorticoid receptor, rifampin
failed to induce tyrosine aminotransferase in mice regardless of
glucocorticoid receptor genotype, and rifampin did not compete for
ligand binding to either mouse or human glucocorticoid receptor. Phenobarbital induced CYP3A, CYP2B, and P-450 reductase in all mice,
but the amplitude of induction was diminished 37% in glucocorticoid receptor-null mice. Thus, there are distinctly different essential requirements of CYP3A, CYP2B, and P-450
reductase genes for the glucocorticoid receptor in their
induction by steroids and drugs.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Glucocorticoids (both agonists
and antagonists), rifampin, and phenobarbital induce cytochromes P-450,
particularly CYP3A11 and 3A13 in mice and CYP3A4 in humans in vivo
(Watkins et al., 1985
; Wrighton et al., 1985b; Yanagimoto et al., 1997)
and in their cultured hepatocytes (Schuetz and Guzelian, 1984; Strom et
al., 1996). Rat CYP3A1 is unresponsive to rifampin (Wrighton et al.,
1985b). Many of these same compounds induce rat, mouse, and human CYP2B
proteins (Strom et al., 1996; Honkakoski and Negishi, 1998). In humans,
these inductions lead to clinically important drug-drug interactions
when glucocorticoids, phenobarbital, or rifampin is administered
concurrently with medications (e.g., cyclosporin A, contraceptives, and
so on) that are normally metabolized by these CYPs. Understanding the
molecular mechanisms leading to drug and steroid induction of CYP3A and
CYP2B, and particularly whether these cytochromes share identical
regulatory mechanisms, ultimately leads to better models for screening
and predicting drug interactions.
In probing the mechanism of glucocorticoid induction of these
cytochromes, an unresolved issue has been the possible direct or
indirect involvement of the glucocorticoid receptor in CYP3A induction.
Although glucocorticoids induce the expression of many genes by
ligand-mediated activation of the glucocorticoid receptor, we
determined that glucocorticoid and, paradoxically, antiglucocorticoid induction of CYP3A reflected a nonclassic glucocorticoid-receptor induction process (Schuetz and Guzelian, 1984). Several recent reports
have identified a unique nuclear hormone receptor, the pregnane X
receptor (PXR)1
(Kliewer et al., 1998; Lehmann et al., 1998; also referred to as hSXR,
Blumberg et al., 1998), which appears to mediate steroid and drug
induction of CYP3A. Mouse and human PXRs are ligands activated by high
concentrations of glucocorticoid agonists (dexamethasone) and
phenobarbital and in a species-specific manner by the glucocorticoid antagonist (pregnenolone 16-carbonitrile; PCN) and by rifampin (Kliewer et al., 1998; Lehmann et al., 1998). Ligand-stimulated PXR
transcriptionally activates hormone response elements found in the rat
CYP3A1 (Kliewer et al., 1998) and human CYP3A4
genes (Blumberg et al., 1998; Lehmann et al., 1998), elements that are conserved in the mouse CYP3A11 gene (Toide et al., 1997).
However, closer inspection of these and other reports suggests that the
PXR may not totally account for drug and steroid induction of CYP3A,
and therefore, the possibility exists that the glucocorticoid receptor
is also required for the induction of this cytochrome. First,
dexamethasone, which is an efficacious inducer of rat, mouse (Wrighton
et al., 1985b; Yanagimoto et al., 1997), and human CYP3A (Watkins et
al., 1989), was only a weak activator of mouse (Kliewer et al., 1998)
and human (Bertilsson et al., 1998; Blumberg et al., 1998) PXRs.
Indeed, Bertilsson et al. (1998) found no activation of human PXR by
dexamethasone, concluding that there must be additional mechanisms for
its induction of CYP3A4. Second, none of the four reports on PXR have
used the authentic CYP3A4 5'-flanking sequences (which contain putative
glucocorticoid receptor binding elements; Ogg et al., 1999); instead,
only reporters with multiple copies of the PXR binding motif were
used. Therefore, it cannot be concluded with certainty that PXR
is the singular transcription factor required for steroid and drug
induction of CYP3A. Indeed, it was recently reported (Pereira et al.,
1998) that a functional glucocorticoid response element (GRE) bound glucocorticoid receptor in the rat CYP3A1 gene, and it was suggested that cooperation of the upstream GRE and downstream elements (e.g., PXR
element) may be required for the maximal response of CYP3A to
glucocorticoids. Similarly, cotransfection of glucocorticoid receptor
with the CYP3A4 promoter in HepG2 cells demonstrated a requirement of
glucocorticoid receptor for CYP3A induction by steroids and drugs (Ogg
et al., 1999). Third, the antiglucocorticoid RU486 [a mouse (Kliewer
et al., 1998) and human (Lehmann et al., 1998) PXR activator]
paradoxically blocks dexamethasone transcriptional activation of the
rat CYP3A1 and human CYP3A4 promoters (Burger et al., 1992; Ogg et al.,
1999), again suggesting a role for the glucocorticoid receptor in
dexamethasone induction of CYP3A. Finally, rifampin is a good inducer
of mouse CYP3A (Wrighton et al., 1985b; Yanagimoto et al., 1997), yet
rifampin is reported to be a weak activator of mouse PXR, suggesting
there may be an alternate mechanism for rifampin induction of CYP3A in
the mouse. However, rifampin was recently demonstrated to be a
nonsteroidal ligand for the human glucocorticoid receptor (Calleja et
al., 1998). Therefore, the possibility existed that the glucocorticoid
receptor played a role in steroid and rifampin induction of CYP3A.
There also is evidence that the classic glucocorticoid receptor may
participate in regulation of the CYP2B1/2 genes. For example, the mouse
CYP2B10 and rat CYP2B1/2 (Stoltz et al., 1998) genes contain GREs, and
the rat CYP2B2 GRE is functionally activated by glucocorticoids
(Jaiswal et al., 1990). There are indications as well that the
glucocorticoid receptor is required for maximal phenobarbital induction
of CYP2B10 in mice and of CYP2B2 in rats (Shaw et al., 1993; Honkakoski
and Negishi, 1998), and a putative GRE has been identified as part of a
phenobarbital response unit in the 5'-flanking sequences of the rat
CYP2B2 gene (Stoltz et al., 1998).
To determine whether the induction of these structurally dissimilar
gene products by steroids and drugs requires the glucocorticoid receptor, we analyzed the induction of these CYP genes in
glucocorticoid receptor-null mice. The majority of mutant mice carrying
disruption of the glucocorticoid receptor gene die within minutes of
birth due to atalectasis of the lungs (Cole et al., 1995), with
12% of the homozygous mutants surviving. The glucocorticoid
receptor-null mice have impaired activation of many hepatic enzymes,
including gluconeogenic enzymes and tyrosine aminotransferase (TAT),
which require the glucocorticoid receptor for glucocorticoid activation (Cole et al., 1995). By using adult glucocorticoid receptor-null mice,
we have found that there is an absolute requirement of the glucocorticoid receptor for steroid induction of CYP2B, but the receptor is not obligatory for glucocorticoid, antiglucocorticoid, or
rifampin induction of CYP3A or P-450 reductase.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Materials.
Mouse 3T3-L1 cells were obtained from American Type Culture Collection
(Rockville, MD). Dulbecco's modified Eagle's medium with no
phenol red (DMEM) and the nonradioactive steroids aldosterone, dexamethasone, and rifampin were obtained from Sigma Chemical Co. (St.
Louis, MO). FBS was obtained from CSL (Melbourne, Australia). The
highly specific synthetic glucocorticoid and progesterone receptor
antagonist RU38486
(17
-hydroxy-11-4-dimethylamino-phenyl-17-1-propynl-estra-4,9-dien-3-one) was a generous gift from Roussel-UCLAF (Paris, France).
[3H]Aldosterone (60-80 Ci/mmol) was obtained
from New England Nuclear (Boston, MA).
[3H]Dexamethasone (60-80 Ci/mmol) was obtained
from Amersham (Buckinghamshire, UK).
Mice.
All experiments with glucocorticoid receptor (+/+) and (
/) mice
were carried out using littermates of C57BL6-129/J (+/) heterozygote
intercrosses (Cole et al., 1995) and represented the 12% survivors of
the (/) offspring. Glucocorticoid receptor genotype of all
littermates was determined by Southern blotting of tail DNAs. Mice
ranged in age from 7 weeks to ~1 year. Three mice (representing males
and females) of (+/+) genotype and three mice of (/) genotype were
used for each drug treatment. For drug induction studies, mice were age
matched as closely as possible. Mice received daily i.p. injections of
50 mg/kg dexamethasone or PCN (Upjohn Co.) or rifampin for 2 days and
were sacrificed on day 3. PCN was administered in water containing 2%
Tween 80. Dexamethasone was administered in corn oil. Rifampin was
administered in water containing 10% DMSO. Sodium phenobarbital in
water was administered daily i.p. (75 mg/kg) for 3 days, and the mice
were sacrificed on day 4.
RNA Isolation and Northern Blot Analysis.
Total RNA was isolated from mouse livers and analyzed by Northern blot
with CYP3A11 cDNA (Yanagimoto et al., 1997). Blots were analyzed using
a PhosphorImager or autoradiographed, and band intensities were
quantified with Image Quant software or densitometry.
Immunoblot Analysis.
Mouse liver microsomes were prepared (Wrighton et al., 1985b), and 10 or 20 µg of protein was analyzed on 10% slab polyacrylamide gels,
electrophoresed, and immunoblotted with monoclonal anti-rat CYP3A1
Ig8 (Hostetler et al., 1987), polyclonal goat anti-rat CYP3A1
antibodies (Hostetler et al., 1987), or polyclonal goat anti-rat CYP2B
antibodies (raised against purified rat CYP3A and CYP2B proteins)
previously developed by Dr. S. Wrighton (Eli Lilly & Co., Indianapolis,
IN) and described (Wrighton et al., 1985a; Hostetler et al., 1989).
Monoclonal antibodies against rat CYP1A (CD2, 3, and 5) and rat
CYP2B1 (monoclonal antibodies BE28.2 and BE26.2) were previously
obtained from Dr. Paul Thomas (Rutgers University, Piscataway, NJ).
Monoclonal H-8 antibody against rat CYP2B (from Dr. Milton
Adesnik, Albert Einstein University, NY) was used previously in our
laboratory (Schuetz et al., 1986). Sheep anti-TAT was obtained from Dr.
Robert Ivarie (University of Georgia, Athens, GA) (Schuetz and
Guzelian, 1984). Rabbit anti-rat CYP4A (from Dr. Richard Okita,
Washington State University, Pullman, WA) and anti-rat P-450 reductase
(from Dr. Ken Thummel, University of Washington, Seattle, WA) were
generously provided. All primary antibodies were followed by
appropriate secondary antibodies coupled with peroxidase and developed
with the enhanced chemiluminescence detection system (Amersham).
Competition Binding Studies. Steroid binding to receptors was performed with a whole-cell binding assay. 3T3-L1 cells were plated at a density of 1 to 2 × 105 cells/35-mm well and grown to confluence with DMEM supplemented with 10% FBS. At confluence, the cells were washed twice with DMEM without serum to remove endogenous steroids and serum proteins. Then, 2.5 ml of fresh medium without serum was added, and the cells were incubated for 30 min at 37°C. Cells were then incubated in a total volume of 2.5 ml with either: 1) 2 nM [3H]aldosterone plus 1 µM RU38486 and increasing concentrations (0-1000 nM) of nonradioactive aldosterone or rifampin, or 2) 2 nM [3H]dexamethasone and increasing concentrations (0-1000 nM) of nonradioactive dexamethasone or rifampin. After incubation for 60 min at 37°C, the cells were washed twice with isotonic saline, scraped, and counted in a scintillation counter.
Human brain tumor tissue (100 mg) was homogenized in ice-cold buffer consisting of 8.5 mM Na2HPO4 · 7H2O, 1.5 mM KH2PO4, 10 mM Na2MoO4 · 2H2O, 20% (v/v) glycerol, and 2 mM monothiolglycerol, pH 7.4, and homogenates were centrifuged at 150,000g for 60 min at 4°C to yield cytosol. Cytosol protein levels (2-4 mg/ml) were determined with the Bradford assay. For competition studies, cytosols (100 µl) were incubated for 16 to 18 h at 4°C with 2 mM tracer, [3H]dexamethasone (100 µl), and increasing concentrations (0-10,000 nM) of nonradioactive dexamethasone or rifampicin (100 µl). The total incubation volume was 300 µl. Bound and free steroids were separated by the addition of 300 µl of an ice-cold suspension of hydroxyapatite (15%, w/v) in 50 mM tris(hydroxymethylaminomethane) and 10 mM KH2PO4, pH 7.2. After incubation for 20 min at 4°C with intermittent shaking, tubes were centrifuged (1000g, 5 min), the supernatant was aspirated, and the pellet was washed three times with ice-cold buffer (8.5 mM Na2HPO4 · 7H2O, 1.5 mM KH2PO4, and 10 mM Na2MoO4 · 2H2O, pH 7.2). Washed hydroxyapatite pellets were resuspended in 2 ml of ethanol at room temperature for 15 min and centrifuged, and the supernatant was taken for liquid scintillation spectrometry.| |
Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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We first examined the expression of CYP3A mRNA in livers of
untreated male and female mice. Northern blot analysis of total RNA
from male or female glucocorticoid receptor (+/+) and (/) mice
probed with a CYP3A cDNA showed they expressed similar levels of CYP3A
mRNA regardless of glucocorticoid receptor genotype (Fig. 1). Analysis of
poly(A)+ RNA from female mice confirmed that
there was no effect of glucocorticoid receptor genotype on constitutive
expression of CYP3A mRNA (not shown).
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We next investigated the expression of hepatic cytochromes P-450 using
antibodies that cross-react with CYP1A1 and CYP1A2. Only CYP1A2 is
constitutively expressed in mouse liver, and its expression was similar
in glucocorticoid receptor (+/+) and (/) mice regardless of gender
(Fig. 2, A and B). Expression of the multiple hepatic proteins immunoreactive with monoclonal anti-CYP3A1 (Fig. 2, A and B) or with polyclonal anti-CYP3A1 (not shown) was unaffected by the presence of the glucocorticoid receptor in either male or female mice. Expression of CYP3A proteins was variable among
both glucocorticoid receptor (+/+) and (/) females and could be due
to age and mixed genetic background differences among the mice.
However, because there was no uniform effect of glucocorticoid genotype
on CYP3A protein, we conclude that the glucocorticoid receptor does not
regulate constitutive CYP3A expression. Figure 2C illustrates the
dramatic gender difference in the hepatic expression of CYP3A proteins
in untreated male and female mice used in this study, consistent with
the sexually dimorphic expression of many CYPs in certain strains of
mice (Teglund et al., 1998; Park et al., 1999).
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We next attempted to identify which of the mouse liver proteins
immunoreactive with anti-CYP3A antibodies was CYP3A11. To date, two
highly related mouse CYP3A cDNAs expressed in adult mouse liver have
been cloned, CYP3A11 (Yanagimoto et al., 1992) and
CYP3A13 (Yanagimoto et al., 1994) (accession nos. X60452 and
X63023, respectively), but only CYP3A11 has been purified (Bornheim and
Correia, 1990). We previously determined that there are four proteins
in mouse liver that immunoreact with CYP3A antibodies (Schuetz et al.,
1996). On SDS-polyacrylamide gel electrophoresis, one CYP3A doublet
selectively immunoreacts with monoclonal anti-CYP3A1, and the other
doublet (faster migrating) immunoreacts with monoclonal anti-CYP3A4
(Schuetz et al., 1996). We observed these same four proteins in more
than 14 inbred strains of female mice (E.G.S., unpublished
observation). Monoclonal anti-CYP3A1 (Fig. 2D) and polyclonal
anti-CYP3A1 antibody (not shown) each recognized purified CYP3A11 as
the same single immunoreactive protein on immunoblots. Immunoreactive
CYP3A11 comigrated with the immunoreactive protein in male liver
microsomes and with the higher-molecular-weight protein in female mouse
liver microsomes (Fig. 2D). Mixing experiments of CYP3A11 together with
the mouse liver microsomes revealed comigration of CYP3A11 with the
upper band in females (not shown). It is possible CYP3A13 is the
lower-molecular-weight anti-CYP3A1 protein in mouse liver (Yanagimoto
et al., 1997).
Dexamethasone, PCN, and rifampin are known inducers of mouse liver
CYP3A proteins and mRNAs (Wrighton et al., 1985b; Yanagimoto et al.,
1997). The treatment of male mice with dexamethasone, PCN, or rifampin
induced CYP3A proteins in both glucocorticoid receptor (+/+) and
(/) genotypes (Figs. 3 and
4). Although the total amount of induced
CYP3A protein within treatment groups was similar, regardless of
glucocorticoid receptor genotype [e.g., PCN (+/+) and (/) had
similar amounts of immunoreactive CYP3A protein], given the
heterogeneity in the control levels of CYP3A and without full
dose-response analysis, we cannot say with certainty that the proteins
are induced to exactly the same extent. However, it is clear that the
glucocorticoid receptor is not essential for these agents to induce
CYP3A. Because dexamethasone and PCN are inducers of mouse and rat
liver CYP2B (Hardwick et al., 1983; Honkakoski and Negishi, 1998), we
next determined whether the glucocorticoid receptor had a role in mouse
CYP2B induction. Although some investigators find CYP2B10 is the
higher-molecular-weight species on immunoblots (Jarukamjorn et al.,
1999), we lack authentic purified proteins to confirm the identity of
the actual immunoreactive CYP2B proteins and refer hereafter to
the upper immunoreactive band as CYP2B because in male mice only
the higher-molecular-weight species was significantly induced by
dexamethasone and PCN (Figs. 3 and
5) and phenobarbital (Figs. 5 and
6), which is consistent with this being a
CYP2B protein. Surprisingly, dexamethasone and PCN induced CYP2B only
in glucocorticoid receptor (+/+) mice (Figs. 3 and 5). Moreover, basal
expression of CYP2B (higher-molecular-weight protein) was significantly
decreased in the glucocorticoid receptor (/) compared with (+/+)
mice (Figs. 3 and 6). There was no induction by rifampin of CYP2B
proteins in any of the mice. No attempt was made to measure enzyme
activities for CYP2B because there have been no definitive studies to
date correlating distinct CYP2B mouse liver proteins with microsomal
testosterone hydroxylase activities in C57BL/6J and 129SvJ mice.
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The researchers at numerous laboratories are routinely using antibodies
raised against rat liver CYPs or other rat liver proteins to study
regulation of the orthologous genes in mice (e.g., Park et al., 1999).
Several lines of evidence together support the specificity of
antibodies raised against rat CYPs recognizing the corresponding
orthologous mouse gene products in this study. First, purified CYP3A11
immunoreacted with the CYP3A1 monoclonal and polyclonal IgGs (Fig. 2D),
and these CYP3A antibodies have been previously shown to be specific
for CYP3A proteins (Hostetler et al., 1987). Second, the deduced amino
acid sequence of CYP3A11 is 87.3 and 84.9% identical with rat
CYP3A1/2, respectively (Yanagimoto et al., 1992), and CYP3A13 shows
71% amino acid identity with CYP3A11 (Yanagimoto et al., 1994). By
definition, the amino acid identity between P-450 gene families is
40%, making it most likely that the antibodies recognize mouse CYP3A
(or CYP2B). Third, we used a panel of anti-CYP3A and anti-CYP2B
monoclonal and polyclonal antibodies and compared the immunoblots of
liver microsomes from the treated mice (Figs. 4 and 5). Two separate
preparations of polyclonal anti-CYP3A1 IgG and a CYP3A1 monoclonal IgG
gave the same pattern of immunoreactive proteins. Similarly, three
unique monoclonal and one polyclonal IgG raised against rat CYP2B gave identical results, making it exceedingly likely that the
higher-molecular-weight protein recognized by these four different
anti-CYP2B antibodies is indeed a mouse CYP2B protein (tentatively
identified as CYP2B10). The specific CYP2 family lower-molecular-weight
immunoreactive proteins remain undefined. Fourth, the monoclonal IgGs
had previously been raised against purified rat liver CYP2B and CYP3A
proteins, and all of the polyclonal IgGs have previously been
backabsorbed against microsomes from a 3-methylcholanthrene-treated
male rat (Hostetler et al., 1987) to prevent promiscuous
immunoreactivity with other CYP family members. In addition, other
investigators (Park et al., 1999) who examined CYP proteins in mouse
liver microsomes found similar patterns of bands immunoreacting with
anti-rat CYP2B antibodies (Jarukamjorn et al., 1999; Park et al., 1999)
and have identified CYP2B10 and CYP2B9 as the slower and faster
migrating immunoreactive proteins, respectively. Finally, the
regulatory patterns of the CYP3A and the higher-molecular-weight CYP2B
protein in these mice conform to their patterns of induction by
dexamethasone, rifampin, PCN, and phenobarbital that have been reported
previously (Wrighton et al., 1985b; Yanagimoto et al., 1997; Honkakoski
and Negishi, 1998; Jarukamjorn et al., 1999). In total, the antibodies used in this study identify CYP3A and CYP2B proteins.
Rat liver P-450 reductase is also induced by drug and steroid
treatments (Simmons et al., 1987). We examined regulation of the
orthologous mouse liver P-450 reductase in microsomes from these same
mice. P-450 reductase was induced by dexamethasone, PCN, and rifampin
regardless of glucocorticoid receptor genotype (Fig. 3). Further
analysis of these same microsomes with antibodies against rat CYP4A
proteins revealed multiple immunoreactive bands whose expression was
unaffected by glucocorticoid receptor genotype. These results
demonstrate that the glucocorticoid receptor is required for agonist
and antagonist induction of CYP2B but not for steroidal and
nonsteroidal induction of CYP3A and P-450 reductase proteins.
Because the constitutive hepatic expression of CYP3A and CYP2B was
sexually dimorphic in these mice (i.e., females expressed significantly
greater amounts of these cytochromes than males; Fig. 2C and not shown
for CYP2B), it was impossible to compare CYP induction in microsomes
from male and female mice on the same blots. Therefore, we next
examined the induction of hepatic CYP3A and CYP2B in female
glucocorticoid receptor (+/+) and (/) mice. The magnitude of
rifampin induction of both CYP3A proteins in females was less robust
than that in males due to the higher basal level of CYP3A in female
mice (not shown) but supported the results in male mice that the
glucocorticoid receptor is not required for induction of CYP3A. As in
the male mice, CYP2B was not induced by rifampin in females regardless
of glucocorticoid receptor genotype (not shown).
To determine the influence of glucocorticoid receptor genotype on CYP3A
and CYP2B mRNAs, we examined hepatic mRNAs from male mice on Northern
blots (Fig. 7). Hybridization with a
CYP3A11 cDNA revealed induction of CYP3A mRNA by dexamethasone, PCN,
and rifampin in glucocorticoid receptor (+/+) and (/) mice. The mRNA supported the protein results that the glucocorticoid receptor is
not required for CYP3A to be induced by either dexamethasone, PCN, or
rifampin. Using a CYP2B10 cDNA, we were unable to detect good
hybridizable CYP2B mRNA in male livers (not shown), a finding consistent with observations by others of the low expression of CYP2B
mRNAs in control and steroid-treated male mouse liver in vivo (Nemoto
and Sakurai, 1995).
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Analysis of TAT protein in cytosols from these same livers showed that
dexamethasone induced TAT only in glucocorticoid receptor (+/+) mice
(Fig. 8), demonstrating the requirement
of the glucocorticoid receptor for this induction (as has been seen in
other glucocorticoid receptor-null mice; Reichardt et al., 1998) and
confirming the absence of functional glucocorticoid receptor in the
null mice (Fig. 8). The antiglucocorticoid PCN suppressed TAT protein
but only in the livers of mice lacking the glucocorticoid receptor (Fig. 8). However, there was no apparent induction of TAT protein by
rifampin in any of the mice (Fig. 8) regardless of glucocorticoid receptor genotype.
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This result was surprising because a recent report suggested that
rifampin is a high-affinity nonsteroidal ligand for the human
glucocorticoid receptor in liver (Calleja et al., 1998). We therefore
tested rifampin in a dose-dependent manner for its ability to compete 2 nM [3H]dexamethasone from glucocorticoid
receptor in extracts from mouse 3T3-L1 cells (Fig.
9A). Rifampin did not compete with
[3H]dexamethasone for binding to GR even at a
concentration of 1 µM. The IC50 value for
dexamethasone binding was determined to be 8.5 nM. Similar results were
found when we tested rifampin for competition of
[3H]aldosterone binding to the mouse
mineralocorticoid receptor in mouse 3T3-L1 cells and also found no
ability to compete at concentrations of up to 1 µM, whereas the
IC50 value for aldosterone binding to the mouse
mineralocorticoid receptor was 2.4 nM (not shown). We also tested
rifampin for competition of [3H]dexamethasone
binding to the human glucocorticoid receptor (Fig. 9B) and also found
no ability to compete at concentrations up to 10 µM. The
IC50 value for dexamethasone binding to the human glucocorticoid receptor was 4.6 nM. This indicates that rifampin showed
no physiological binding affinity for glucocorticoid receptor in either
species tested.
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Because we and others have found that glucocorticoid treatment can
affect the magnitude of phenobarbital induction of CYP (Kocarek et al.,
1994; Sidhu and Omicinski, 1995), we used glucocorticoid receptor-null
mice to determine whether the glucocorticoid receptor had a role in
phenobarbital induction of CYPs. The glucocorticoid receptor was not
obligatory for phenobarbital to induce CYP2B or CYP3A proteins (see
Fig. 6). However, the magnitude of CYP2B and CYP3A induction by
phenobarbital was attenuated in glucocorticoid receptor-null male mice,
although it was not obvious in these immunoblots developed to detect
CYP levels in untreated control mice (Fig. 6A). Quantitative
immunoblots (Fig. 6B) revealed that CYP2B and CYP3A were induced in
receptor (/) mice to only 63% of their induction in (+/+) mice
(100%). Female mice contained an additional CYP2B protein (migrating
between the two immunoreactive proteins in male microsomes) that is
constitutively expressed and inducible by phenobarbital in both
glucocorticoid receptor (+/+) and (/) female mice (Fig. 6). It is
possible that this additional protein is CYP2B9 because compared with
males, female mice express greater amounts of CYP2B9 (Nemoto and
Sakurai, 1995; Jarukamjorn et al., 1999).
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Although recent reports identify PXR as a mediator of steroid and
drug induction of CYP3A, several lines of evidence suggest that the
glucocorticoid receptor may also be required for induction of this
protein: 1) the weak activation of mouse or human PXR (Blumberg et al.,
1998; Kliewer et al., 1998; Lehmann et al., 1998) or even no activation
(Bertilsson et al., 1998) of human PXR by dexamethasone, 2) synergistic
interaction between PCN and dexamethasone for induction of the
endogenous rat CYP3A genes (Schuetz and Guzelian, 1984; Burger et al.,
1992), 3) the glucocorticoid receptor and traditional GREs in the CYP3A
gene were reported to mediate dexamethasone induction of the rat CYP3A1
promoter (Pereira et al., 1998), 4) the antiglucocorticoid (PXR ligand) RU486 blocks dexamethasone transcriptional activation of the CYP3A1 (Burger et al., 1992) and CYP3A4 (Ogg et al., 1999) promoters, and 5)
rifampin is an efficacious inducer of mouse CYP3A (Wrighton et al.,
1985b; Yanagimoto et al., 1997) but a weak activator of mouse PXR
(Lehmann et al., 1998). These results suggested that there may be
additional transcription factors (e.g., the glucocorticoid receptor)
required for dexamethasone and rifampin induction of CYP3A. Using the
glucocorticoid receptor (+/+) and (/) mice, we demonstrate that
CYP3A induction by glucocorticoids, antiglucocorticoids, and rifampin
proceeds via a mechanism that does not require the glucocorticoid
receptor. Moreover, there appears to be no influence of the
glucocorticoid receptor on basal expression of the CYP3As. In addition,
the inability of rifampin to induce TAT or to displace dexamethasone
from the mouse or human glucocorticoid receptor suggests that rifampin
cannot be a ligand for this receptor.
We compared the induction of CYP3A with that of CYP2B in the
glucocorticoid receptor (+/+) and (/) mice to determine whether the
glucocorticoid receptor was a shared regulatory characteristic. In
striking contrast to CYP3A, our results demonstrate that the glucocorticoid receptor is obligatory for both maximal constitutive expression of CYP2B and for either dexamethasone or PCN to induce CYP2B. This result seems paradoxical at first because PCN is an antiglucocorticoid and cannot activate the glucocorticoid receptor (Schuetz and Guzelian, 1984). Although questions regarding the precise
molecular mechanisms of CYP2B induction by PCN and dexamethasone remain
to be determined (i.e., is PXR mediating steroid induction of CYP2B?),
based on our results, we propose a model in which dexamethasone and PCN
induction of CYP2B requires both PXR and the glucocorticoid receptor.
In support of this hypothesis, PXR is the only transcription factor
identified that can mediate PCN signaling (Kliewer et al., 1998). In
addition, we previously demonstrated that the mechanism by which
dexamethasone induces CYP2B shares similar characteristics with
nonclassic glucocorticoid induction of CYP3A (Kocarek et al., 1994), a
mechanism now known to be mediated by PXR (Kliewer et al., 1998;
Lehmann et al., 1998). Intriguingly, a xenochemical response module in
the mouse Cyp2b10 and rat CYP2B1 and
2B2 genes (Park et al., 1996; Stoltz et al., 1998) contains nuclear hormone response elements (including a DR4, a potential target
sequence for PXR; Blumberg et al., 1998) and a putative GRE (Stoltz et
al., 1998).
There are conflicting data in the literature regarding the contribution
of glucocorticoids to phenobarbital induction of CYPs. Although most
reports found that phenobarbital induction of rat or mouse CYP2B either
required or was potentiated by glucocorticoids, high concentrations of
dexamethasone can suppress phenobarbital induction of CYP2B in rats
(Kocarek et al., 1994; Sidhu and Omicinski, 1995; Honkakoski and
Negishi, 1998). Mice with targeted gene disruption of the
glucocorticoid receptor offered a way to determine its contribution to
phenobarbital induction in mice in vivo. Our results demonstrate that
the glucocorticoid receptor is not essential for phenobarbital to
induce CYP2B but does contribute to maximum phenobarbital induction of
CYP2B in mouse liver. Surprisingly, a parallel effect of glucocorticoid
receptor abrogation on the magnitude of phenobarbital induction of
CYP3A was also found, suggesting the receptor may affect a common
phenobarbital induction mechanism (Sueyoshi et al., 1999).
Our result that rifampin was not capable of binding with either the
human or mouse glucocorticoid receptor is in stark contrast to an
original report (Calleja et al., 1998) that rifampin is a nonsteroidal
ligand for the human glucocorticoid receptor in HepG2 human
hepatoblastoma cells. However, similar to our results, two other
groups, using A549 human alveolar cells (Jaffuel et al., 1999) or mouse
AtT20 pituitary corticotroph cells and COS-7 monkey kidney cells
cotransfected with human glucocorticoid receptor (Ray et al., 1998),
have failed to find that rifampin interacts with the glucocorticoid
receptor. One possible explanation for the discrepancy between the
original (Calleja et al., 1998) versus all subsequent reports is that
in HepG2 cells, the authors may have been looking at PXR-mediated
activation of the MMTV (mouse mammary tumor virus) promoter.
Given that some HepG2 cells express a low level of PXR (E.G.S.,
unpublished observation) and that the MMTV enhancer has a
potential nuclear receptor binding motif previously shown to bind
LXR/RXR heterodimers (Willy et al., 1995) it is possible that rifampin
activated PXR/RXR bound and activated MMTV in their experiments.
Because AtT20, A549, and COS-7 cells presumably lack PXR, these cell
lines would be incapable of rifampin activation of MMTV. In addition,
although our data demonstrate that rifampin is not binding to the
ligand binding pocket of the receptor in the classic sense, we cannot
rule out rifampin activating GCR via another part of the receptor or
even via another associated protein.
A recent report found that DMSO, the vehicle used in the rifampin
treatments, can increase rat CYP3A by stabilizing the CYP3A protein in
primary rat hepatocytes (Zangar and Novak, 1998). To confirm that
rifampin, and not DMSO, was inducing CYP3A, we compared rifampin
induction of CYP3A in ethanol or DMSO and found that CYP3A was
equivalently induced when administered in either vehicle (not shown).
Similarly, DMSO had no effect on either basal CYP2B or on the extent of
CYP2B induction by phenobarbital (not shown), consistent with
the lack of effect of DMSO on induction of CYP2B in primary mouse
hepatocytes (Honkakoski and Negishi, 1998) and demonstrating that the
lack of CYP2B induction by rifampin was not due to the DMSO vehicle.
Thus, the effects of rifampin on these cytochromes are specific to
rifampin and not a side effect of the DMSO.
Our results now reveal what may be an important species difference in
rifampin induction of CYP2B. At the dose and duration administered,
rifampin clearly did not induce mouse CYP2B, whereas we previously
identified rifampin as an efficacious inducer of CYP2B6 in primary
cultures of human hepatocytes (Strom et al., 1996). Because rifampin
induced hepatic CYP3A, it clearly was biologically active in the
hepatocyte. However, in the livers of these same mice, CYP2B appeared
to be refractory to rifampin treatment and even appeared to be somewhat
decreased. These results are similar to previous reports that even
within the same species, rifampin can have both inductive and
repressive effects on hepatic CYPs (Lange et al., 1984). Moreover,
within a single CYP gene family (the CYP3As), there are noted species
differences in rifampin activation of the rat (unresponsive) versus
mouse and human (responsive) CYP3A proteins (Kocarek et al., 1995).
Could differences in genetic backgrounds (i.e., progeny are 129 × C57BL/6 heterozygotes) contribute to differences in CYP3A or
CYP2B induction between the animals under study? The progeny would be
expected to have random segregation of 129 or C57BL/6 P-450 allelic
determinants. Unfortunately, 100% of isogenic glucocorticoid receptor
(/) animals die at birth (Cole et al., 1995), necessitating the use
of heterozygote progeny. Although there are well documented strain
differences in the induction of some P-450s in mice (e.g., the CYP1A
family), in a survey of 14 different strains of mice (including
C57BL/6) we failed to identify any strain in which CYP3A was not
induced by either dexamethasone or PCN (E.G.S., unpublished
observation). Moreover, in this study, the induction of CYP3A proteins
in male mice by dexamethasone, PCN, or rifampin was the same in all
mice tested regardless of glucocorticoid receptor genotype and
regardless of the admixture of genetic background. Finally, because
CYP2B and CYP3A are induced in both parental strains (C57BL/6J and
129J) mice by dexamethasone (not shown), the complete loss of CYP2B
induction by this agent in glucocorticoid receptor (/) mice can
only be attributed to disruption of the glucocorticoid receptor locus.
Understanding the way in which drugs induce the CYPs (particularly
whether they share identical regulatory mechanisms) is biologically
relevant because it ultimately leads to better models for screening and
predicting drug interactions that occur due to the induction of
individual CYPs. Can we extrapolate these results to the regulation of
human CYPs? We propose that analogous to mouse CYP3A, the
glucocorticoid receptor will not be required for the induction of human
CYP3A4 by glucocorticoids or rifampin because human PXR is activated by
these agents and binds to a conserved hormone response element in the
CYP3A4 gene (Lehmann et al., 1998). Moreover, we were unable to find an
interaction of rifampin with human glucocorticoid receptor. However, if
there are functional GREs in the human CYP3A genes, it is still
possible that the glucocorticoid receptor could contribute to maximum
glucocorticoid induction of human CYP3A. Importantly, rifampin and
glucocorticoids are not selective inducers of human CYP3A but induce
other isozymes of CYP, including CYP2B6 (Strom et al., 1996) and
activities associated with CYP2C19 (Zhou et al., 1990) in human liver.
It remains to be determined whether glucocorticoid induction of human
CYP2B6 parallels our results with mouse CYP2B and absolutely requires the glucocorticoid receptor.
Because CYP3A and CYP2B proteins are up-regulated by many of the same
drugs and xenobiotics, it was originally assumed that they were induced
by a common mechanism, analogous to the paradigm of the aryl
hydrocarbon receptor mechanism and pleiotropic induction of multiple
detoxification proteins. However, we and others have demonstrated that
induction of CYP3A and CYP2B differs in several respects. For example:
1) the time course of induction of these two cytochromes differs
dramatically after treatment with PCN or phenobarbital (Hardwick et
al., 1983), 2) protein synthesis inhibitors have opposite effects on
the induction of these cytochromes by steroids and phenobarbital
(Burger et al., 1990), 3) induction of these cytochromes can be
differentiated by dose of inducer (Kocarek et al., 1990), and 4)
phenobarbital induction of these two genes may proceed by different
nuclear receptors (Honkakoski et al., 1998; Lehmann et al., 1998). We
now add that glucocorticoid and antiglucocorticoid inductions of CYP2B
and CYP3A have distinctly different requirements for the glucocorticoid receptor.
| |
Footnotes |
|---|
Received August 11, 1999; accepted November 9, 1999.
This work was supported by National Institutes of Health Research Grants ES08658, ES05851, P30-CA21765, and DA04265 (L.M.B.); a block grant from the National Health and Medical Research Council of Australia; and the American Lebanese Syrian Associated Charities (ALSAC).
Send reprint requests to: Dr. Erin Schuetz, Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105. E-mail: erin.schuetz{at}stjude.org
| |
Abbreviations |
|---|
Abbreviations used are:
PXR, pregnane X
receptor;
DMEM, Dulbecco's modified Eagle's medium;
TAT, tyrosine
aminotransferase;
GRE, glucocorticoid response element;
PCN, pregnenolone 16-carbonitrile.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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) and
rifampin (RIF, 
). The IC50 value, the concentration of
cold competitor at which 50% of [3H]dexamethasone is
competed off, is 8.5 nM (dexamethasone binding set at 100%).
The IC50 value for rifampin was 
1000 nM. B,
specificity of 2 nM [3H]dexamethasone binding in human
brain tumor tissue: competition by dexamethasone (DEX,