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J Biol Chem, Vol. 274, Issue 52, 37117-37124, December 24, 1999
From the Laboratory of Drug Metabolism, Division of
Pharmacobiodynamics, Graduate School of Pharmaceutical Sciences,
Hokkaido University, N12W6, Kita-ku,
Sapporo, Hokkaido 060-0812, Japan
The purpose of this study was to clarify the
mechanism(s) responsible for the growth hormone (GH)-induced expression
of the CYP2C12 gene. To identify a functional GH-responsive
element (GHRE) in vivo, we performed the direct injection
of promoter-luciferase chimeric genes into female rat livers. The
results showed that the luciferase activity was decreased to
approximately 20% by the deletion of the sequence between nucleotides
CYP2C12 is known to be one of the steroid hydroxylases and is
constitutively expressed in female rats but not in male rats (1-4).
The sex-specific expression of the CYP2C12 gene is regulated by the plasma growth hormone
(GH)1 pattern (5-12).
Although the pattern of GH secretion in male rats is characterized by
the peaks of large amplitude every 3-4 h with undetectable levels in
interpulse periods, the pattern of GH secretion in female rats shows
more frequent oscillation of small amplitude (13, 14). Thus, the
transcription of the CYP2C12 gene is thought to be activated
by the constant level of GH seen in female rats (9, 10, 12).
The effects of GH are mediated by a GH receptor, which belongs to a
cytokine/hematopoietin receptor superfamily (15). On the basis of a
proposed pathway for the induction of gene expression by GH, the
binding of GH to the GH receptor promotes the association of the GH
receptor with Janus kinase 2 and the tyrosyl phosphorylation of the
Janus kinase 2. (16). Subsequently, the activated Janus kinase 2 phosphorylates the tyrosine residues of the STAT protein. After the
formation of the homodimer of the STAT protein or the heterodimer of
the STAT protein with other factor(s) in the cytoplasm, the complex
translocates to the nucleus and then binds to target sequences (17).
Among the STAT family, STAT1, STAT3, and STAT5 have been identified as
GH-stimulated proteins (15, 18). Particularly, STAT5 (19) has been
reported to participate in the GH-related expression of some genes
including c-fos (20), serine protease inhibitor 2.1 (21),
and the acid-labile subunit gene (22). Pulsatile plasma GH secretion
seen in male rats but not in female rats has been shown to activate
STAT5 in the liver (23, 24). Based on these lines of evidence, STAT5
has been regarded as the male-specific regulator for the expression of
genes including the sex-limited protein gene (25) and
CYP3A10 (26).
To date, the mechanism responsible for the GH-dependent
activation of the CYP2C12 gene in females has been studied
in vitro. HNF-6 has been reported to be involved in the
GH-dependent transcription of the CYP2C12 gene
(27). Additionally, it was found that the female-enriched
GH-dependent complex, termed GHNF, bound to five distinct
regions within the CYP2C12 promoter region between
nucleotides To identify a functional factor(s) responsible for the modulation of
the GH-dependent transcription of the CYP2C12
gene, we attempted to search the cultured cells to show the
inducibility of CYP2C12 by GH. However, no suitable cultured cells were
found. Recently, it has been reported that the direct injection of
plasmid DNA into the liver is a useful method to assay the in
vivo activity of the CYP2B and CYP2C
promoters (29). Thus, we employed the direct injection method to search
for a critical factor(s) responsible for the GH-dependent
expression and to determine the relative contribution of each
transcription factor to the GH-dependent expression of the
CYP2C12 gene.
In the present study, we provide evidence that the
GH-dependent and liver-specific expression of the
CYP2C12 gene in female rats results from a cooperative
regulation with STAT5, HNF-4, HNF-6, and factors that bind to elements
2C12-I and 2C12-II in the upstream region of the CYP2C12 gene.
Animal Treatments--
Adult female Harlan Sprague Dawley rats
(8 weeks old; Sankyo Experimental Animals, Tokyo, Japan) were used.
When necessary, female rats were hypophysectomized at 6 weeks of age.
Recombinant human methionylated GH (Somatonorm, Kabi Vitrum, Stockholm,
Sweden) was kindly supplied by Sumitomo Pharmaceutical Co. (Osaka,
Japan). Human GH was administered by a continuous infusion (0.94 IU/kg·day), which mimics female-type GH secretion, with an osmotic
minipump (model 2001, Alza, Palo Alto, CA) or by a pulsatile injection (0.94 IU/kg·day), which mimics male-type GH secretion (10), for 6 days.
Isolation of the 5'-Flanking Region of the CYP2C12 Gene--
To
isolate the 5'-flanking region of the CYP2C12 gene (30), a
gene library prepared from the rat genomic DNA, which had been cleaved
by Sau3A I and cloned into Construction of 5'-Deletion Mutants for Luciferase
Assay--
Luc5132 reporter plasmid was constructed as follows. A
HindIII site was introduced downstream of the nucleotide +16
of pUC5.2 by site-directed mutagenesis (pUC5.2(+16)). pUC5.2(+16) was
then cleaved with XhoI and HindIII. The fragment
corresponding to nucleotides from Direct DNA Injection--
Direct DNA injection was performed
with minor modifications of the method previously described (34, 29).
The CYP2C12-Luc chimeric genes (600 µg of plasmid DNA
diluted with 1.5 ml of Dulbecco's modified Eagle's medium (Nisssui
Pharmaceutical, Tokyo, Japan)) were injected into the peripheral site
of single liver lobes. Rats were sacrificed 7 days later to obtain the
injected liver weighing from 0.1 to 0.2 g. Human GH was
administered 1 day after the DNA injection by a continuous infusion for
6 days (30). When a reporter plasmid with TK promoter was injected into
the livers of hypophysectomized rats, GH was administered 5 days prior to the DNA injection. Rats were sacrificed 1 day after the DNA injection, when the highest luciferase activity was seen.
Luciferase Assay--
Three ml of LC Preparation of Rat Liver Nuclear Extracts--
Nuclear extracts
were prepared from the livers from hypophysectomized rats and from the
hypophysectomizd rats treated with GH by the constant infusion as
described elsewhere (35) except for 1% aprotinin (Sigma) in an initial
homogenization buffer. Subsequently, the extracts were snap-frozen in
0.01-ml aliquots at 5-10 mg of protein/ml and immediately frozen in
liquid nitrogen for storage.
Gel Shift Assay--
The gel shift assay was performed with 20 µl of a reaction mixture containing 25 mM Hepes (pH 7.8),
100 mM KCl, 5 mM MgCl2, 0.3 mM EDTA, 0.5 mM dithiothreitol, 2.5% glycerol,
2% Ficoll 400, 10 µg of liver nuclear extracts, and a
32P-labeled probe (10 fmol). The mixture was incubated at
0 °C for 90 min. Resulting DNA-protein complex was subjected to a
6% polyacrylamide gel in a buffer containing 25 mM Tris
borate and 0.5 mM EDTA. For a competition assay, liver
nuclear extracts were preincubated at 0 °C for 30 min with a 20-fold
molar excess of an unlabeled oligonucleotide. CRE and mutated CRE used
for a competition assay were obtained from Santa Cruz Biotechnology,
Inc. (Santa Cruz, CA). For a supershift assay, liver nuclear extracts
were preincubated at 0 °C for 30 min with 1 µl of antibodies to
STAT1 Southwestern Blot Analysis--
Southwestern blot analysis was
performed according to the method of Miskimins et al.
(43).
Direct Injection of the 5'-Deletion Mutants of the CYP2C12 Gene
into Female Rat Livers--
To examine whether or not the Luc5132
reporter gene was activated in vivo by GH secreted in a
female-type pattern, we injected the DNA of Luc5132 into the liver of
female rats (Fig. 1A). As expected, the luciferase activity was detected in the liver transfected with Luc5132. Hypophysectomy of female rats resulted in the abolishment of the luciferase activity. However, the activity was restored to the
level seen in intact rats by the continuous infusion of GH to the
livers of hypophysectomized female rats. These results indicate that
the direct DNA injection is a useful method to clarify the mechanism
responsible for the GH-dependent transcriptional regulation
of the CYP2C12 gene.
Identification and Characterization of GHREs in the Upstream Region
of the CYP2C12 Gene--
To identify possible regulatory element(s)
involved in the GH-induced expression of the CYP2C12 gene,
the 5'-deletion mutants of the gene were constructed as shown in Fig.
1B. When the mutants were injected into livers of female
rats, the maximal activity of luciferase was seen in the liver that
received Luc4213. The activity was decreased to approximately 20% by
the deletion of the nucleotide sequences between
The sequence of the 5'-flanking region of the CYP2C12 gene
was found to contain 9-bp palindrome sequences (5'-TTCCTAGAA-3'), designated as GHRE1 and GHRE2, in a region between nucleotides Binding of STAT5 to the GHRE of the CYP2C12 Gene--
Searching
the sequence similar to the GHRE, we found that the sequence of the
GHRE overlapped with those of STAT-binding sites (Fig.
4). Particularly, the sequence of the
GHRE was completely identical to the STAT5 binding sequence found in
the acid-labile subunit gene (22) and interleukin-2 receptor Cooperative Regulation of the CYP2C12 Gene by STAT5 and Other
Transcriptional Factors--
The liver-specific expression of CYP2C12
may not be accounted for solely by STAT5, since this protein is also
expressed in extrahepatic tissues. Thus, we postulated that
liver-specific factors other than STAT5 were required for the
expression of CYP2C12 in the livers of female rats. To identify the
regulatory regions necessary for the liver-specific expression of the
CYP2C12 gene, we performed a luciferase assay with reporter
plasmids, Luc4200
To examine whether 2C12-I and 2C12-II were responsible for the
regulation of the CYP2C12 promoter by GH, we constructed
deletion mutants with GHRE and 2C12-I or/and 2C12-II (Fig.
7E). Insertion of both 2C12-I and 2C12-II restored the
transcriptional activity to the level seen in the liver transfected
with Luc4200, although the injection of a reporter plasmid carrying
2C12-I or 2C12-II alone did not induce the activity. Taking these
results together, we conclude that the GH-dependent
expression of the CYP2C12 gene is regulated cooperatively by
STAT5, HNF-4, HNF-6, and an unknown factor(s), which binds to 2C12-I
and 2C12-II.
Activation of CYP2C12 Promoter by GH Secreted in Male Rats--
To
examine whether or not the luciferase activity derived from the Luc5132
reporter plasmid was activated by GH secreted in male rats, we injected
Luc5132 into the livers of male rats (Fig. 8). Interestingly, it was found that the
luciferase activity was detected not only in female rats but also in
male rats. The hypophysectomy of rats abolished the luciferase
activity. As expected, the pulsatile as well as constant infusion of GH
to hypophysectomized rats increased the activity. These results
indicate that the pulsatile GH secretion also induces transcription
factors responsible for the induction of CYP2C12.
In an attempt to study the mechanism responsible for the
transcriptional regulation of a certain gene, cultured cells in which the gene expression is seen are generally used, since the cultured cells possess a transcription factor(s) necessary for the gene expression. However, to our knowledge, suitable cultured cells that
show the inducibility of CYP2C12 by GH have not been found. In
addition, it is hard to reproduce the female- or male-type GH
stimulation with cells in culture. Therefore, we employed the direct
DNA injection system developed by Kemper et al. (29). As
expected, a luciferase activity in the liver transfected with Luc5132
was clearly detected. Thus, the direct DNA injection was evaluated to
be a useful tool to explore the mechanism of GH-dependent transcriptional regulation.
The binding sites of HNF-4 (33), HNF-6 (27), C/EBP The sequence of GHRE, TTCCTAGAA, was identical to that of interferon
CYP2C12 is unique in that its expression is female-specific. On the
other hand, STAT5 is considered to be a male-enriched transcriptional
factor, although we have shown that STAT5 serves as the modulator of
CYP2C12 gene expression in female as well as male rats.
Actually, the activation of the Luc5132 reporter gene was also seen in
male rats (Fig. 8). An unknown suppressor protein(s) involved in the
gender-related difference in the expression of the CYP2C12 may be
present in male rat livers. Additionally, it is also possible to assume
that the CYP2C12 gene is inactivated at a chromatin level in
male rats. Since a naked plasmid was transfected into the livers, sex
difference in chromatin structure cannot be seen in the present
experiments. These possibilities remain to be examined.
HNF-6 has also been reported to show a sex difference in the expression
level (27). However, HNF-6 functioned as the activator of the Luc5132
reporter gene in male rats (Fig. 8). Thus, HNF-6 may not be a critical
factor determining the sex-related difference.
We demonstrated that HNF-4 was a major factor to regulate the
expression of the CYP2C12 gene. Our result does not agree
with a previous work (33). Although the transactivation of the
CYP2C12 gene by HNF-4 alone was weak (33), HNF-4 was needed
for the expression of CYP2C12 when it cooperated with STAT5 and the
factors binding to 2C12-I and 2C12-II. In addition, the contribution of C/EBP To date, it has been reported that a SRY-like protein inhibits the
binding of C/EBP We identified novel regulatory regions, 2C12-I and 2C12-II. CREB is
known to be a transcription factor bound to CRE (52) and known to
interact with p300/CBP (53), which functions as histone
acetyltransferase. In addition to CREB, STAT5 also interacts directly
with p300/CBP (54). Therefore, STAT5 may function synergistically with
CREB through p300/CBP.
It has been reported that Ca2+ channel blocker inhibits the
expression of the CYP2C12 gene in rat hepatocytes (55). The
activation of CREB occurs in response to elevated intracellular
Ca2+ through
Ca2+/calmodulin-dependent protein kinase (56).
Thus, the Ca2+-dependent expression of the
CYP2C12 gene can be explained by the activation of CREB.
In addition to 2C12-I and 2C12-II, negative regulatory region ( It has been proven that STAT5 interacts directly with a glucocorticoid
receptor (48) or a p300/CBP (54). These complexes regulate
cooperatively the prolactin-dependent expression of the We thank Sumitomo Pharmaceutical Co. (Osaka,
Japan) for kindly supplying human recombinant GH.
*
This work was supported in part by a Grant-in-Aid from the
Ministry of Education, Science, Sports and Culture of Japan, the Core
Research for Evolutional Science and Technology, and the Program for
Promotion of Fundamental Studies in Health Sciences of the Organization
for Drug ADR Relief, R & D Promotion and Product Review of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviations used are:
GH, growth hormone;
GHNF, GH nuclear factor;
GHRE, GH-responsive element;
CRE, cyclic
AMP-response element;
CREB, CRE-binding protein;
CBP, CREB-binding
protein;
C/EBP, CCAAT/enhancer-binding protein;
HNF, hepatocyte nuclear
factor;
STAT, signal transducer and activator of transcription;
TAT, tyrosine aminotransferase;
TK, thymidine kinase;
TTR, transthyretin;
bp, base pair(s).
Cooperative Regulation of CYP2C12 Gene Expression
by STAT5 and Liver-specific Factors in Female Rats*
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ABSTRACT
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4213 and 4161. Within this region, two copies of a possible GHRE
were present. The sequence of the GHRE was overlapped with that of an
interferon-
-activated sequence, known to be recognized by the signal
transducer and activator of transcription (STAT) proteins. In fact, a
supershift assay showed that STAT5 was capable of binding to the core
sequence of the GHRE. Furthermore, a luciferase assay with reporter
plasmids, 
4161/3781, mutated hepatocyte nuclear factor-4
(HNF-4), and mutated HNF-6, revealed that the GH-stimulated expression
of the CYP2C12 gene was regulated cooperatively by STAT5,
HNF-4, HNF-6, and the factor(s) that binds to the elements, 2C12-I
(4095 to 4074) and 2C12-II (4072 to 4045). The cooperative
regulation by STAT5 and the liver-enriched transcription factors
account for the GH-dependent and the liver-specific
expression of the CYP2C12 gene in female rats.
INTRODUCTION
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ABSTRACT
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MATERIALS AND METHODS
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DISCUSSION
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1560 and +60 (28). However, it has not been clarified as
yet whether or not the above factors play a key role in the in
vivo expression of the CYP2C12 gene. In addition to the
uncertainty of the in vivo role of these factors, a
functional GH-responsive element(s) has not yet been clarified in the
regulatory region of the CYP2C12 gene.
MATERIALS AND METHODS
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FIXII vector (Stratagene, La
Jolla, CA), was screened with the fragment of a CYP2C12
promoter as a probe. Consequently, a positive clone containing the
9-10-kilobase fragment of the CYP2C12 gene was obtained.
The clone was digested with XhoI and HindIII to
obtain a 5'-flanking region between nucleotides 5132 and +113 from
the transcription start site (30). The resultant fragment was subcloned
to the XhoI/HindIII site of pUC19 (pUC5.2).
5132 to +16 was ligated into the
unique XhoI-HindIII site of basic vector 2 (Toyoinki, Tokyo, Japan). Reporter plasmids, Luc4213, Luc4118, and
Luc1944, were constructed by the digestion of Luc5132 with restriction
enzymes, HincII, BstXI, and BamHI, respectively. Luc4200, Luc4187, and Luc4161 were generated by polymerase chain reaction using S-4200, S-4187, or S-4161 as a 5'-primer and AS-3614 as a 3'-primer. Synthesized fragments were digested with BglII at position 3780 in the
CYP2C12 gene. Resultant fragments were inserted into the
SmaI/BglII site of Luc3780. To construct
Luc4200GHRE2 without the region between nucleotides 4174 and
4162, the region corresponding to nucleotides from 4200 to 4175
was inserted into the KpnI site of Luc4161. To confirm the
contribution of GHRE to GH-dependent transcriptional activity, reporter plasmids, 1×GHRE-LucTK, 2×GHRE-LucTK,
4×GHRE-LucTK, and GHRE-Luc1944 were constructed. TK promoter was
inserted into the BglII/HindIII site of basic
vector 2 (LucTK). 1×GHRE, 2×GHRE, or 4×GHRE was then introduced into
the XhoI site of LucTK. The copy number and the direction of
the oligonucleotides inserted into the reporter plasmids were confirmed
by a sequence analysis (31). To construct Luc4200(4161/3781) or
Luc4200 (4161/4118), the region corresponding to nucleotides
from 4200 to 4162 was inserted into the
KpnI/NheI site of Luc3780 or Luc4118. To
construct Luc4200(3776/3138), Luc4200(3137/1944),
Luc4200(1939/536), Luc4200(535/81),
Luc4200(4122/4036), or Luc4200(4030/3781), Luc4200 was
digested with BglII/EcoRV,
EcoRV/BamHI, BamHI/StuI, StuI/MscI, BstXI/HgaI, or
HgaI/BglII, respectively. Resultant fragments
were blunt-ended and self-ligated. To construct GHRE-Luc3780, the
region corresponding to nucleotides from 4200 to 4162 was inserted
into the KpnI/NheI site of Luc3780. To construct
GHRE-2C12-I-Luc3780 and GHRE-2C12-II-Luc3780, the regions corresponding
to nucleotides from 4095 to 4074 (2C12-I) and from 4072 to 4045
(2C12-II) were cloned into the GHRE-Luc3780. To construct
GHRE-2C12-I/II-Luc3780, the region corresponding to nucleotides from
4072 to 4045 (2C12-II) was cloned into the GHRE-2C12-I-Luc3780.
Luc4200mtC/EBP, Luc4200mtHNF-4, and Luc4200mtHNF-6 were generated
by site-directed mutagenesis. The oligonucleotide primers used
for the synthesis of DNA fragment or site-directed mutagenesis are as
follows: S-4200, 5'-AAATTTCCTAGAAGTGAAATTG-3'; S-4187,
5'-GTGAAATTGTGGTAAATTCC-3'; S-4161, 5'-TCATTGCCAGAGGAGACA-3'; AS-3614,
5'-TGATGAGTGAGAACATTCTA-3'; 4200/4175,
5'-CAAATTTCCTAGAAGTGAAATTGTGGTGGTAC-3' (sense strand) and
3'-CATGGTTTAAAGGATCTTCACTTTAACACCAC-5' (antisense strand);
4200/4162, 5'-CAAATTTCCTAGAAGTGAAATTGTGGTAAATTCCTAGAACG-3' (sense
strand) and 3'-CATGGTTTAAAGGATCTTCACTTTAACACCATTTAAGGATCTTGCGATC-5' (antisense strand); GHRE, 5'-TCGAGTTTCCTAGAAGCTCGAGTTTCCTAGAAGC-3' (sense strand) and 3'-CAAAGGATCTTCGAGCTCAAAGGATCTTCGAGCT-5' (antisense strand); mutated 2C12-C/EBP (228/205) (32),
5'-TGAGTGTAGATATCGGTGTTACAT-3' and 3'-ACTCACATCTATAGCCACAATGTA-5';
mutated 2C12-HNF-4 (122/96) (33), 5'-ATCATTGAGTCTGTCTTCATTTGAAAG-3'
and 3'-TAGTAACTCAGACAGAAGTAAACTTTC-5'; mutated 2C12-HNF-6 (52/30)
(27), 5'-GCAAAAGATGGTTTTTTATGGTG-3' and
3'-CGTTTTCTACCAAAAAATACCAC-5'.
/PGC-51 lysis buffer
(Toyoinki, Tokyo, Japan) per 1 g of liver tissue was added to
prepare liver cell extracts. After homogenization with a Dounce
homogenizer, insoluble materials were removed by centrifugation. The
supernatants were kept frozen at 80 °C until use. Luciferase
activity was measured using a Berthold Lumat LB 9501 luminometer with
photon detection integrated over 10-s intervals. Statistical
significance was judged by a Mann-Whitney U test.
, STAT3, STAT5, STAT5a, CREB1, or CREB2. These antibodies to
STAT proteins were purchased from Santa Cruz Biotechnology. After a
labeled probe was added, the mixture was incubated at 0 °C for 90 min. The oligonucleotide primers used as a probe are as follows: GHRE1,
5'-AAAATTTCCTAGAAGTG-3' and 3'-TTAAAGGATCTTCACTT-5'; GHRE2,
5'-GAAATTGTGGTAAATTCCTAGAACTC-3' and 3'-TTAACACCATTTAAGGATCTTGAGTA-5';
Ly6E interferon -activated sequence core site (36),
5'-GATCATATTCCTGTAAGTGAT-3' and 3'-TATAAGGACATTCACTACTAG-5'; M67
sis-inducible element from c-fos (37),
5'-GATCCATTTCCCGTAAATCAT-3' and 3'-GTAAAGGGCATTTAGTACTAG-5'; rat
2-macroglobulin acute-phase response element (38),
5'-GATCGGAATTCCCAGAAGGAT-3' and 3'-CCTTAAGGGTCTTCCTACTAG-5'; rat
-casein gene STAT5/mammary gland factor response element (39),
5'-GATCGGACTTCTTGGAATTAAGGGA-3 and 3'-CCTGAAGAACCTTAATTCCCTCTAG-5'; mutated -casein, 5'-GATCGGACTTAGTTTAATTAAGGGA-3' and
3'-CCTGAATCAAATTAATTCCCTCTAG-5'; mouse -globin GATA-1 binding site
(40), 5'-TCCGGCAACTGATAAGGATTCCCT-3' and
3'-AGGCCGTTGACTATTCCTAAGGGA-5'; TTR (41), 5'-TCTGATTATTGACTTAGTCAAG-3' and 3'-AGACTAATAACTGAATCAGTTC -5'; TAT (42),
5'-GACGTTTCTCAATATTTGCTCTGGCAG-3' and
3'-TGCAAAGAGTTATAAACGAGACCGTCT-5'.
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ABSTRACT
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MATERIALS AND METHODS
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View larger version (17K):
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Fig. 1.
Identification of GH-responsive regions in
the CYP2C12 gene. A, effect of
hypophysectomy or GH administration on luciferase activity in the liver
transfected with Luc5132. A Luc5132 deletion mutant was injected into
the livers of nontreated (NT) and hypophysectomized
(Hypox) female rats and female rats that were
hypophysectomized and received the infusion of human GH for 6 days
(Hypox + GH). B, luciferase assay with the
5'-deletion mutants of the 5'-flanking region of the CYP2C12
gene. The construction of deletion mutants is described under
"Materials and Methods." The numbers given to the
deletion mutants indicate the 5'-ends of the 5'-flanking sequence of
the CYP2C12 gene counted negatively from a transcriptional
start site. These mutants were injected into the liver of adult female
rats. All values represent the mean ± S.D. from independent
experiments shown in parentheses. The data are expressed as
the ratio of the luciferase activity of each deletion mutant to the
basal activity obtained with Luc5132. *, p < 0.05, significantly different between two groups (Mann-Whitney U
test).
4213 and 4161.
Further deletion between 4118 and 1944 also resulted in a
significant decrease to a basal activity. These results suggest that at
least two regions are necessary for the GH-dependent
expression of the CYP2C12 gene in female rats.
4213
and 4161 (Fig. 2A). Compared
with elements reported so far, the sequence of the GHREs overlapped
with those of the interferon -activated sequence and acute-phase
response element (5'-TTCCNNNAA-3'), known to be the binding sites of
STAT proteins. As can be seen in Fig. 2B, the deletion of
either the GHRE1 or the GHRE2 caused an 80-90% decrease in the
luciferase activity. This result indicates that both GHREs contribute
to the GH-dependent activation of the CYP2C12
gene in female rats. Furthermore, a gel shift assay using the GHRE1 or
the GHRE2 as a probe showed that a factor(s) in nuclear extracts from
female livers bound to each GHRE (Fig. 2C). Interestingly, the binding of the constitutive factor(s) to the GHREs disappeared with
the hypophysectomy of female rats. The binding of the factor(s) to the
GHREs was, however, restored by the continuous infusion of GH, which
mimics female-type GH secretion, to hypophysectomized female rats. To
further confirm the contribution of the GHREs to the
GH-dependent expression of the CYP2C12 gene,
GHRE-Luc1944, which contains 10 copies of the GHRE, was injected into
the liver of female rats (Fig.
3A). The results indicate that
the luciferase activity in the liver transfected with GHRE-Luc1944 is
much higher than that in the liver transfected with Luc1944. In the
livers from hypophysectomized female rats, the activation of the
GHRE-Luc1944 was not seen. The continuous infusion of GH to
hypophysectomized rats elevated the luciferase activity of GHRE-Luc1944
by approximately 10-fold. To further characterize GHRE, 1×GHRE-,
2×GHRE-, and 4×GHRE-LucTK were used for a luciferase assay. The
luciferase activity was significantly increased with the copy number of
GHRE (Fig. 3B). Additionally, the luciferase activity with
4×GHRE-LucTK was elevated by the infusion of GH to the livers of
hypophysectomized rats (Fig. 3C).
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Fig. 2.
Characterization of two GHREs found in the
5'-flanking region of the CYP2C12 gene.
A, nucleotide sequence of the CYP2C12 gene
between nucleotides 4213 and 4161. Possible STAT-binding elements,
GHRE1 and GHRE2, are shown in boldface type. B,
effects of the deletion of GHRE1 and/or GHRE2 on transcriptional
activity. The construction of deletion mutants is described under
"Materials and Methods." All values represent the mean ± S.D.
from four independent experiments. The data are expressed as the ratio
of the luciferase activity of each deletion mutant to the basal
activity obtained with Luc4200. *, p < 0.05, significantly different from the activity in the liver transfected with
Luc4200. C, gel shift assay with two GHREs. Gel shift assay
was performed as described under "Materials and Methods." Synthetic
GHRE1 and GHRE2 were used as labeled probes. Nuclear extracts were
prepared from nontreated females (NT), hypophysectomized
females (Hypox), and hypophysectomized females infused with
human GH for 6 days (Hypox + GH).
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Fig. 3.
GH-dependent activation of the
CYP2C12 or TK promoter through the GHRE.
A, GHRE-Luc1944 was constructed as described under
"Materials and Methods." The reporter plasmid was injected into the
livers of nontreated females (NT), hypophysectomized females
(Hypox), and hypophysectomized females infused with human GH
for 6 days (Hypox + GH). B, effects of the copy
number of GHRE on the transcriptional activity of TK promoter.
C, GH-dependent activation of 4×GHRE-LucTK.
4×GHRE-LucTK was injected into the livers of hypophysectomized females
(Hypox) or hypophysectomized females infused with human GH
for 5 days (Hypox + GH). All values represent the mean ± S.D. from independent experiments shown in parentheses.
The data are expressed as the ratio of the luciferase activity of each
deletion mutant to the basal activity obtained with GHRE-Luc1944 or
4×GHRE-LucTK in female rats. * and **, significantly different between
two groups at p < 0.05 and p < 0.01, respectively.
gene
(44). The STAT3 binding sites of the 2-macrogloblin gene
(38) and the STAT5 binding sites of the -casein gene (39) possessed only one base change as compared with the GHRE sequence. To confirm the
binding of STAT protein to the GHRE2, we performed a competition assay
with STAT-binding sequences seen in the rat -casein, rat 2-macroglobulin, and human Ly6E and M67 genes
(Fig. 5A). We found that the
binding of the GH-stimulated factor(s) to the GHRE2 disappeared with
the presence of a 20-fold molar excess of the STAT-binding sequences of
the -casein and 2-macroglobulin genes. The competitor of Ly6E and M67 partially inhibited the formation of the
complex of the GH-stimulated factor(s) with the GHRE2. Unlike the
competitors, the -casein, 2-macroglobulin, Ly6E, and
M67 genes, mutated -casein, and nonspecific competitor,
GATA-1, did not affect the binding of the complex to the GHRE2. To
identify a STAT protein to bind to the GHRE, a supershift assay using
antibodies to STAT1, STAT3, STAT5, and STAT5a was performed (Fig. 5,
B and C). The results showed that a supershifted
band appeared in the presence of antibodies against STAT5 and STAT5a.
Thus, it appeared that STAT5a is one of the modulators for the
expression of CYP2C12 in the livers of female rats.
View larger version (28K):
[in a new window]
Fig. 4.
Alignment of the binding Sites of STATs.
The sequences are aligned with 9-bp nucleotides, TTCCNNNAA, known to be
a consensus binding motif for STAT proteins.
View larger version (49K):
[in a new window]
Fig. 5.
Binding of STAT5 to the GHRE.
A, effects of competitors on the binding of STAT proteins to
GHRE2. A 32P-labeled double strand GHRE2 was incubated with
10 µg of nuclear extracts prepared from the liver of female rats in
the presence of a 20-fold molar excess amount of a competitor at
0 °C for 90 min. mt, mutated; 2-MG,
2-macroglobulin. B and C, supershift assay
using antibodies to STAT1, STAT3, STAT5, and STAT5a. A
32P-labeled double strand GHRE2 was incubated with nuclear
extracts prepared from the liver of female rats in the presence of
antibodies to STAT1, STAT3, STAT5, or STAT5a as described under
"Materials and Methods."
(4161/3781), Luc4200(3776/3138),
Luc4200(3137/1944), Luc4200(1939/536), or
Luc4200(535/81) (Fig.
6A). The deletion of the
nucleotides from 4161 to 3781 or from 535 to 81 lowered
the transcriptional activity, although the deletion from 3776 to
3138 increased the luciferase activity. The region from 3776 to
3138 may be a negative regulatory region. Within the region between
535 and 81, the putative binding sites of C/EBP and HNF-4, which
is known to be a liver-enriched factor, were present. The mutation within the C/EBP-binding sequence did not significantly affect the
transcriptional activity (Fig. 6B). However, the mutation of
the consensus sequence of HNF-4 decreased the activity to approximately one-twentieth of the level seen with Luc4200. Similarly, it was found
that the mutation of the sequence of HNF-6 caused the loss of the
transcriptional activity. In addition to these results, we identified
that a novel regulatory region was located in the region between
nucleotides 4161 and 3781 (Fig. 6A). Further study using
Luc4200(4161/4118), Luc4200(4122/4036), and Luc4200(4030/3781) demonstrated that enhancer elements existed between nucleotides 4122 and 4036 (Fig. 6C). A computer
search revealed that this region contained putative CRE (4095 to
4074) and HNF-3 (4072 to 4045) sequences. We designated these
elements as 2C12-I (4095 to 4074) and 2C12-II (4072 to 4045),
respectively (Fig. 7A). No
factors bound to any region other than 2C12-I and 2C12-II (data not
shown). To characterize a factor(s) binding to 2C12-I, a gel shift
assay using a mutated probe or competitors was performed (Fig.
7B). The result showed that five factors (I to V) bound to
2C12-I. These factors did not recognize mt2C12-I. Two shifted bands (I
and II) disappeared by the addition of a 20-fold molar excess of the
consensus CRE sequence. Unlike the CRE, mutated CRE did not affect the
binding of the factors to 2C12-I. Furthermore, two shifted bands (II
and III) were supershifted by antibodies against CREB1 (Fig.
7B). This result indicates that CREB1 but not CREB2
recognizes 2C12-I. Unknown factors (I, IV, and V) are currently under
examination. 2C12-II contained a sequence similar to the binding
sequence of HNF-3 (Fig. 7A). To examine whether or not HNF-3
bound to 2C12-II, a gel shift assay with mutant probes or competitors
was carried out (Fig. 7C). The binding of factors to 2C12-II
was diminished by the introduction of mutations (mt2) in the core
sequence of HNF-3 binding sequence. Neither mt1 nor mt2 was effective.
The sequences of TTR (5'-TCTGATTATTGACTTAGTCAAG-3') and TAT
(5'-GACGTTTCTCAATATTTGCTCTGGCAG-3') were reported to be the typical
binding sites of HNF-3 (41, 42). The binding of HNF-3 to TTR was not
inhibited even in the presence of a 250-fold molar excess of the
2C12-II sequence, although the band decreased in the presence of TAT.
Thus, it appeared that the factors binding to 2C12-II were not
identical to HNF-3. Southwestern blot analysis using 2C12-II as a probe
demonstrated that at least five nuclear factors specifically recognized
the sequence of 2C12-II (Fig. 7D). The molecular masses of
these binding factors were estimated to be 63, 47.5, 40, 36, or 34.5 kDa. The molecular weights of HNF-3, -3, and -3 have been
reported to be 50, 47, and 42 kDa (45), respectively. Although the
molecular mass of a factor (47.5 kDa) was similar to that of HNF-3,
other four factors were different from HNF-3. These results indicate
that the factors distinct from HNF-3 recognize 2C12-II.
View larger version (19K):
[in a new window]
Fig. 6.
Identification of regulatory regions coupled
with the GHREs. A, luciferase assay with the
5'-deletion mutants, Luc4200 (4161/3781),
Luc4200(3776/3138), Luc4200(3137/1944),
Luc4200(1939/536), or Luc4200(535/81). B,
effects of mutations into C/EBP, HNF-4, or HNF-6 binding sites on
transcriptional activity. C, luciferase assay with the
5'-deletion mutants, Luc4200(4161/3781),
Luc4200(4161/4118), Luc4200(4122/4036), or
Luc4200(4030/3781). These mutants were injected into the liver
of adult female rats. All values represent the mean ± S.D. from
independent experiments shown in parentheses. The data are
expressed as the ratio of the luciferase activity of each deletion
mutant to the basal activity obtained with Luc4200. ** and ***,
significantly different from the transcriptional activity in the liver
transfected with Luc4200 at p < 0.01 and
p < 0.001, respectively.
View larger version (21K):
[in a new window]
Fig. 7.
Characterization of 2C12-I and 2C12-II found
in the 5'-flanking region of the CYP2C12 gene.
A, nucleotide sequences of 2C12-I and 2C12-II. CRE and HNF-3
binding elements are in boldface type. The sites of mutated
(mt) sequences are underlined. B, gel shift assay
using 2C12-I and mt2C12-I. C, gel shift assay with 2C12-II
or mutant probes. To examine the effects of competitors on the binding
of HNF-3 to a TTR probe, a competitor DNA, TTR, 2C12-II, or TAT was
added at a 25-, 100-, or 250-fold molar excess of the probe TTR.
D, Southwestern blot analysis. Nuclear extracts (60 µg)
from the liver of female rats were incubated with
32P-labeled 2C12-II or mt2. E, function of 2C12-I or/and 2C12-II in GH-dependent activation.
The construction of deletion mutants is described under "Materials
and Methods." These mutants were injected into the liver of adult
female rats. All values represent the mean ± S.D. from
independent experiments shown in parentheses. The data are
expressed as the ratio of the luciferase activity of each deletion
mutant to the basal activity obtained with GHRE-2C12-I/II-Luc3780. **,
significantly different from the transcriptional activity in the liver
transfected with GHRE-2C12-I/II-Luc3780 at p < 0.01.
View larger version (41K):
[in a new window]
Fig. 8.
Effect of pulsatile GH secretion on
luciferase activity in the liver transfected with Luc5132. Luc5132
was injected into livers of nontreated rats (NT),
hypophysectomized (Hypox) rats, and rats hypophysectomized
and receiving the infusion of human GH by a continuous infusion
(GH(c)) or a pulsatile infusion (GH(p)) (10) for
6 days. All values represent the mean ± S.D. from independent
experiments shown in parentheses. The data are expressed as
the ratio of the luciferase activity of each deletion mutant to the
basal activity obtained with Luc5132 in nontreated female rats.
M, male rats; F, female rats.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(32), insulin
response element-A-binding protein (46), and GHNF (28) have been
reported as the transcriptional regulator of the CYP2C12
gene. These elements were located between nucleotides 1560 and +60 in
the CYP2C12 gene. On the other hand, the GHREs were located
in the upstream region between nucleotides 4200 and 4161 and
possessed functional STAT5-binding sites. The GHREs identified in the
present study are expected to be distinct from the elements reported so
far. In the CYP2C12 gene, two GHREs, GHRE1 and GHRE2, were
present in tandem. We showed in this paper that both GHRE1 and GHRE2
were essential for the transcriptional activation of the
CYP2C12 gene (Fig. 2B). This finding suggests that the two GHREs seem to function synergistically. Supporting this
phenomenon, it has been reported that two copies of the STAT5 binding
site are required for the maximal induction of serine protease
inhibitor 2.1 by GH (21).
-activated sequence seen in the bovine -casein (47) and mouse
acid-labile subunit genes (22), known to be recognized by both STAT5a
and STAT5b. However, STAT5b was reported to be suppressed by GH
secreted in female rats but not male rats. In fact, the amount of
STAT5b in the female rat livers was estimated to be low (24, 49, 50).
Our result of a supershift assay demonstrated that STAT5a is a major
regulator of the CYP2C12 gene in females. It has been
reported that STAT5a-STAT5b heterodimer was necessary for the
expression of female-specific CYP2B enzyme (51). Thus, the
heterodimerization of STAT5 proteins may be important for the
expression of CYP2C12 in rats and mice.
to the CYP2C12 expression was small as compared with the results of the previous work (32). This disagreement may be explained
as follows. The methodology of direct DNA injection was employed in the
present study, while primary hepatocytes and cultured cells such as
HepG2 were used in the previous work. The C/EBP expression plasmid
was transfected into the cultured cells. Cultured cells transfected
with C/EBP may not reflect the in vivo expression of the
CYP2C12 gene.
in cultured cells (46). Since the in vivo function of a SRY-like protein is not analyzed in the present study, we cannot exclude the possibility that a SRY-like protein is
related to the in vivo expression of CYP2C12. The regulation of the CYP2C12 gene by a SRY-like protein is currently under examination.
3776
to 3138) was present in the upstream region of the CYP2C12 gene. However, we could not find a typical negative regulatory region
reported so far. Further study is needed for the identification of a
factor(s) involved in the negative regulation.
-casein gene in the mammary gland. We demonstrated in this study that CREB and liver-specific factors together with STAT5 were required
for the transcriptional activation of the CYP2C12 promoter by GH. The GH-stimulated and the liver-specific expressions of CYP2C12
in female rats were confirmed as explicable by the cooperative regulation by STAT5, the liver-specific factors, and other factors such
a CREB.
ACKNOWLEDGEMENT
FOOTNOTES
To whom all correspondence should be addressed: Laboratory of Drug
Metabolism, Division of Pharmacobiodynamics, Graduate School of
Pharmaceutical Sciences, Hokkaido University, N12W6, Kita-ku, Sapporo,
Hokkaido 060-0812, Japan. Tel./Fax: 81-11-706-4978; E-mail: kamataki@pharm.hokudai.ac.jp.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1.
Kamataki, T.,
Maeda, K.,
Yamazoe, Y.,
Nagai, T.,
and Kato, R.
(1983)
Arch. Biochem. Biophys.
225,
758-770[CrossRef][Medline]
[Order article via Infotrieve]
2.
MacGeoch, C.,
Morgan, E. T.,
Halpert, J.,
and Gustafsson, J.-Å.
(1984)
J. Biol. Chem.
259,
15433-15439 3.
Kamataki, T.,
Maeda, K.,
Shimada, M.,
Kitani, K.,
Nagai, T.,
and Kato, R.
(1985)
J. Pharmcol. Exp. Ther.
233,
222-228 4.
Zaphiropoulos, P. G.,
Mode, A.,
Ström, A.,
Möller, C.,
Fernandez, C.,
and Gustafsson, J.-Å.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
4214-4217 5.
Kamataki, T.,
Shimada, M.,
Maeda, K.,
and Kato, R.
(1985)
Biochem. Biophys. Res. Commun.
130,
1247-1253[CrossRef][Medline]
[Order article via Infotrieve]
6.
MacGeoch, C.,
Morgan, E. T.,
and Gustafsson, J.-Å.
(1985)
Endocrinology
117,
2085-2092 7.
Kato, R.,
Yamazoe, Y.,
Shimada, M.,
Murayama, N.,
and Kamataki, T.
(1986)
J. Biochem. (Tokyo)
100,
895-902 8.
Yamazoe, Y.,
Shimada, M.,
Kamataki, T.,
and Kato, R.
(1986)
Jpn. J. Pharmacol.
42,
371-382[Medline]
[Order article via Infotrieve]
9.
MacGeoch, C.,
Morgan, E. T.,
Cordell, B.,
and Gustafsson, J.-Å.
(1987)
Biochem. Biophys. Res. Commun.
143,
782-788[CrossRef][Medline]
[Order article via Infotrieve]
10.
Legraverend, C.,
Mode, A.,
Westin, S.,
Ström, A.,
Eguchi, H.,
Zaphiropoulos, P. G.,
and Gustafsson, J.-Å.
(1992)
Mol. Endocrinol.
6,
259-266 11.
Mode, A.
(1993)
J. Reprod. Fertil.
46 (suppl.),
77-86[CrossRef]
12.
Sundseth, S. S.,
Alberta, J. A.,
and Waxman, D. J.
(1992)
J. Biol. Chem.
267,
3907-3914 13.
Tannenbaum, G. S.,
and Martin, J. B.
(1976)
Endocrinology
98,
562-570 14.
Edén, S.
(1979)
Endocrinology
105,
555-560 15.
Argetsinger, L. S.,
and Carter-Su, C.
(1996)
Physiol. Rev.
76,
1089-1107 16.
Argetsinger, L. S.,
Campbell, G. S.,
Yang, X.,
Witthuhn, B. A.,
Silvennoinen, O.,
Ihle, J. N.,
and Carter-Su, C.
(1993)
Cell
74,
237-244[CrossRef][Medline]
[Order article via Infotrieve]
17.
Darnell, J. E.,
Kerr, I. M.,
and Stark, G. R.
(1994)
Science
264,
1415-1421 18.
Ram, P. A.,
Park, S.-H.,
Choi, H. K.,
and Waxman, D. J.
(1996)
J. Biol. Chem.
271,
5929-5940 19.
Wood, T. J. J.,
Sliva, D.,
Lobie, P. E.,
Pircher, T. J.,
Gouilleux, F.,
Wakao, H.,
Gustafsson, J.-Å.,
Groner, B.,
Norstedt, G.,
and Haldosén, L.-A.
(1995)
J. Biol. Chem.
270,
9448-9453 20.
Gronowski, A. M.,
Zhong, Z.,
Wen, Z.,
Thomas, M. J.,
Darnell, J. E.,
and Rotwein, P.
(1995)
Mol. Endocrinol.
9,
171-177 21.
Sliva, D.,
Wood, T. J. J.,
Schindler, C.,
Lobie, P. E.,
and Norstedt, G.
(1994)
J. Biol. Chem.
269,
26208-26214 22.
Ooi, G. T.,
Hurst, K. R.,
Poy, M. N.,
Rechler, M. M.,
and Boisclair, Y. R.
(1998)
Mol. Endocrinol.
12,
675-687 23.
Waxman, D. J.,
Ram, P. A.,
Park, S.-H.,
and Choi, H. K.
(1995)
J. Biol. Chem.
270,
13262-13270 24.
Gebert, C. A.,
Park, S.-H.,
and Waxman, D. J.
(1997)
Mol. Endocrinol.
11,
400-414 25.
Varin-Blank, N.,
Dondi, E.,
Tosi, M.,
Hernandez, C.,
Boucontet, L.,
Gotoh, H.,
Shiroishi, T.,
Moriwaki, K.,
and Meo, T.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
8750-8755 26.
Subramanian, A.,
Teixeira, J.,
Wang, J.,
and Gil, G.
(1995)
Mol. Cell. Biol.
15,
4672-4682[Abstract]
27.
Lahuna, O.,
Fernandez, L.,
Karlsson, H.,
Maiter, D.,
Lemaigre, F. P.,
Rousseau, G. G.,
Gustafsson, J.-Å.,
and Mode, A.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
12309-12313 28.
Waxman, D. J.,
Zhao, S.,
and Choi, H. K.
(1996)
J. Biol. Chem.
271,
29978-29987 29.
Park, Y.,
Li, H.,
and Kemper, B.
(1996)
J. Biol. Chem.
271,
23725-23728 30.
Zaphilopous, P. G.,
Westin, S.,
Ström, A.,
Mode, A.,
and Gustafsson, J.-Å.
(1990)
DNA Cell Biol.
9,
49-56[Medline]
[Order article via Infotrieve]
31.
Sanger, F.,
Nicklen, S.,
and Coulson, A. R.
(1977)
Proc. Natl. Acad. Sci. U. S. A.
74,
5463-5467 32.
Tollet, P.,
Lahuna, O.,
Ahlgren, R.,
Mode, A.,
and Gustafsson, J.-Å.
(1995)
Mol. Endocrinol.
9,
1771-1781 33.
Ström, A.,
Westin, S.,
Eguchi, H.,
Gustafsson, J.-Å.,
and Mode, A.
(1995)
J. Biol. Chem.
270,
11276-11281 34.
Malone, R. W.,
Hickman, M. A.,
Lehmann-Bruinsma, K.,
Sih, T. R.,
Walzem, R.,
Carlson, D. M.,
and Powell, J. S.
(1994)
J. Biol. Chem.
269,
29903-29907 35.
Gorski, K.,
Carneiro, M.,
and Schibler, U.
(1986)
Cell
47,
767-776[CrossRef][Medline]
[Order article via Infotrieve]
36.
Khan, K. D.,
Shuai, K.,
Lindwall, G.,
Maher, S. E.,
Darnell, J. E.,
and Bothwell, A. L. M.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
6806-6810 37.
Wagner, B. J.,
Hayes, T. E.,
Hoban, C. J.,
and Cochran, B. H.
(1990)
EMBO J.
9,
4477-4484[Medline]
[Order article via Infotrieve]
38.
Wegenka, U. M.,
Buschmann, J.,
Lütticken, C.,
Heinrich, P. C.,
and Horn, F.
(1993)
Mol. Cell. Biol.
13,
276-288 39.
Wakao, H.,
Gouilleux, F.,
and Groner, B.
(1994)
EMBO J.
13,
2182-2191[Medline]
[Order article via Infotrieve]
40.
Tsai, S.-F.,
Martin, D. I. K.,
Zon, L. I.,
D'Andrea, A. D.,
Wong, G. G.,
and Orkin, S. H.
(1989)
Nature
339,
446-451[CrossRef][Medline]
[Order article via Infotrieve]
41.
Costa, R. H.,
Grayson, D. R.,
and Darnell, J. E.
(1989)
Mol. Cell. Biol.
9,
1415-1425 42.
Nitsch, D.,
and Schütz, G.
(1993)
Mol. Cell. Biol.
13,
4494-4504 43.
Miskimins, W. K.,
Roberts, M. P.,
McClelland, A.,
and Ruddle, F. H.
(1985)
Proc. Natl. Acad. Sci. U. S. A.
82,
6741-6744 44.
Lécine, P.,
Algarté, M.,
Rameil, P.,
Beadling, C.,
Bucher, P.,
Nabholz, M.,
and Imbert, J.
(1996)
Mol. Cell. Biol.
16,
6829-6840[Abstract]
45.
Lai, E.,
Prezioso, V. R.,
Tao, W.,
Chen, W. S.,
and Darnell, J. E.
(1991)
Genes Dev.
5,
416-427 46.
Buggs, C.,
Nasrin, N.,
Mode, A.,
Tollet, P.,
Zhao, H.-F.,
Gustafsson, J.-Å.,
and Alexander-Bridges, M.
(1998)
Mol. Endocrinol.
12,
1294-1309 47.
Cella, N.,
Groner, B.,
and Hynes, N. E.
(1998)
Mol. Cell. Biol.
18,
1783-1792 48.
Stöcklin, E.,
Wissler, M.,
Gouilleux, F.,
and Groner, B.
(1996)
Nature
383,
726-728[CrossRef][Medline]
[Order article via Infotrieve]
49.
Fernández, L.,
Flores-Morales, A.,
Lahuna, O.,
Sliva, D.,
Norstedt, G.,
Haldosén, L.-A.,
Mode, A.,
and Gustafsson, J.-Å.
(1998)
Endocrinology
139,
1815-1824 50.
Gebert, C. A.,
Park, S.-H.,
and Waxman, D. J.
(1999)
Mol. Endocrinol.
13,
213-227 51.
Park, S.-H.,
Liu, X.,
Hennighausen, L.,
Davey, H. W.,
and Waxman, D. J.
(1999)
J. Biol. Chem.
274,
7421-7430 52.
Comb, M.,
Birnberg, N. C.,
Seasholtz, A.,
Herbert, E.,
and Goodman, H. M.
(1986)
Nature
323,
353-356[CrossRef][Medline]
[Order article via Infotrieve]
53.
Chrivia, J. C.,
Kwok, R. P. S.,
Lamb, N.,
Hagiwara, M.,
Montminy, M. R.,
and Goodman, R. H.
(1993)
Nature
365,
855-859[CrossRef][Medline]
[Order article via Infotrieve]
54.
Pfitzner, E.,
Jähne, R.,
Wissler, M.,
Stoecklin, E.,
and Groner, B.
(1998)
Mol. Endocrinol.
12,
1582-1593 55.
Tollet, P.,
Hamberg, M.,
Gustafsson, J.-Å.,
and Mode, A.
(1995)
J. Biol. Chem.
270,
12569-12577 56.
Matthews, R. P.,
Guthrie, C. R.,
Wailes, L. M.,
Zhao, X.,
Means, A. R.,
and McKnight, G. S.
(1994)
Mol. Cell. Biol.
14,
6107-6116
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M. Endo, Y. Takahashi, Y. Sasaki, T. Saito, and T. Kamataki Novel Gender-Related Regulation of CYP2C12 Gene Expression in Rats Mol. Endocrinol., May 1, 2005; 19(5): 1181 - 1190. [Abstract] [Full Text] [PDF]
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 S Eleswarapu and H Jiang Growth hormone regulates the expression of hepatocyte nuclear factor-3 gamma and other liver-enriched transcription factors in the bovine liver J. Endocrinol., January 1, 2005; 184(1): 95 - 105. [Abstract] [Full Text] [PDF]
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P. Kotokorpi, C. Gardmo, C. S. Nystrom, and A. Mode Activation of the Glucocorticoid Receptor or Liver X Receptors Interferes with Growth Hormone-Induced akr1b7 Gene Expression in Rat Hepatocytes Endocrinology, December 1, 2004; 145(12): 5704 - 5713. [Abstract] [Full Text] [PDF]
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H. Helander, J.-A. Gustafsson, and A. Mode Possible Involvement of Truncated Signal Transducer and Activator of Transcription-5 in the GH Pattern-Dependent Regulation of CYP2C12 Gene Expression in Rat Liver Mol. Endocrinol., July 1, 2002; 16(7): 1598 - 1611. [Abstract] [Full Text] [PDF]
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 S.-H. Park and D. J. Waxman Inhibitory Cross-talk between STAT5b and Liver Nuclear Factor HNF3beta . IMPACT ON THE REGULATION OF GROWTH HORMONE PULSE-STIMULATED, MALE-SPECIFIC LIVER CYTOCHROME P-450 GENE EXPRESSION J. Biol. Chem., November 9, 2001; 276(46): 43031 - 43039. [Abstract] [Full Text] [PDF]
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M. C. Garcia, C. Thangavel, and B. H. Shapiro Epidermal Growth Factor Regulation of Female-Dependent CYP2A1 and CYP2C12 in Primary Rat Hepatocyte Culture Drug Metab. Dispos., February 1, 2001; 29(2): 111 - 120. [Abstract] [Full Text]
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G. B. Ehret, P. Reichenbach, U. Schindler, C. M. Horvath, S. Fritz, M. Nabholz, and P. Bucher DNA Binding Specificity of Different STAT Proteins. COMPARISON OF IN VITRO SPECIFICITY WITH NATURAL TARGET SITES J. Biol. Chem., February 23, 2001; 276(9): 6675 - 6688. [Abstract] [Full Text] [PDF]
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N. Delesque-Touchard, S.-H. Park, and D. J. Waxman Synergistic Action of Hepatocyte Nuclear Factors 3 and 6 on CYP2C12 Gene Expression and Suppression by Growth Hormone-activated STAT5b. PROPOSED MODEL FOR FEMALE-SPECIFIC EXPRESSION OF CYP2C12 IN ADULT RAT LIVER J. Biol. Chem., October 27, 2000; 275(44): 34173 - 34182. [Abstract] [Full Text] [PDF]
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