From the Division of Cell and Molecular Biology,
Department of Biology, Boston University, Boston, Massachusetts
02215, § NIDDK, National Institutes of Health, Bethesda,
Maryland 20892, and ¶ AgResearch,
Ruakura, Hamilton, New Zealand
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ABSTRACT |
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Stat5b gene disruption leads to an
apparent growth hormone (GH) pulse insensitivity associated with loss
of male-characteristic body growth rates and male-specific liver gene
expression (Udy, G. B., Towers, R. P., Snell, R. G.,
Wilkins, R. J., Park, S. H., Ram, P. A., Waxman, D. J., and Davey, H. W. (1997) Proc. Natl. Acad. Sci.
U. S. A. 94, 7239-7244). In the present study, disruption of
the mouse Stat5a gene, whose coding sequence is ~90%
identical to the Stat5b gene, resulted in no loss of
expression in male mice of several sex-dependent,
GH-regulated liver cytochrome P450 (CYP) enzymes. By contrast, the loss
of STAT5b feminized the livers of males by decreasing expression of
male-specific CYPs (CYP2D9 and testosterone 16 The cytochrome P450s
(CYPs)1 are a superfamily of
heme proteins that hydroxylate steroid hormones and other endogenous
chemicals as well as numerous drugs and environmental carcinogens. CYPs are highly expressed in liver, where they are subject to complex hormonal regulation and sex-dependent expression.
Prototypic examples of sex-specific liver CYPs in the rat model are the
male-specific CYP2C11 (testosterone 16 Pulsatile GH, but not continuous GH, strongly activates the signal
transducer and transcriptional activator STAT5 in rat liver (12). STAT5
was originally identified in lactating mammary gland as a
prolactin-inducible transcription factor (13). Subsequently, two highly
conserved (~90% identical in coding sequence) STAT5 genes,
Stat5a and Stat5b, were identified and found to
be expressed in many tissues (14-17). Both STAT5 forms can be
activated in tissue culture by multiple cytokines and growth factors,
including GH, erythropoietin, epidermal growth factor, and various
interleukins (14, 18-21). STAT5 proteins thus have the potential to
contribute to multiple signaling pathways associated with cell growth
and differentiation. The proposed mediation by STAT5 of GH
pulse-regulated, sexually dimorphic liver gene expression (12) is
supported by the finding of a functional STAT5 response element in the
promoter region of several male-specific, GH pulse-regulated genes
(22).2 In addition, targeted
disruption of Stat5b leads to a major loss of multiple,
sexually differentiated responses associated with pulsatile GH
secretion (23), demonstrating that this GH pulse-activatable transcription factor (12) is essential for maintaining sexually dimorphic body growth rates and liver gene expression.
During mammary gland differentiation, STAT5a and STAT5b both undergo
prolactin-inducible tyrosine phosphorylation and bind as a
heterodimeric STAT5a-STAT5b complex at To address the role of STAT5a in the GH-regulated sexual dimorphism of
liver gene expression, we have examined the effects of
Stat5a gene disruption on hepatic CYP enzyme activities and protein expression. 129J congenic Stat5b Animals--
Generation of Stat5a and
Stat5b gene-disrupted mice has been described (23, 25). In
order to evaluate the effects of STAT5b deficiency in a congenic 129J
background, chimeric male mice (derived from 129J ES cells) were mated
with 129J females. The Stat5b+/ Preparation of Mouse Liver Homogenates, Cytosol, and
Microsomes--
Mouse liver tissues were snap-frozen in liquid
nitrogen and stored at Antibodies--
Rabbit polyclonal antibodies raised against
mouse STAT5a amino acids 774-793 and mouse STAT5b amino acids 776-786
were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)
(sc-1081 and sc-835, respectively). These STAT5 antibodies were shown
to be specific for STAT5a or STAT5b, respectively, under our Western blotting conditions. Some cross-reactivity was apparent in
electrophoretic mobility shift assay (EMSA) supershift analysis (see
Fig. 2B). Mouse monoclonal anti-STAT1 and anti-STAT3
antibodies purchased from Transduction Laboratories were raised against
STAT1 amino acids 591-731 (S21120) and STAT3 amino acids 1-178
(S21320), respectively. Anti-rat CYP3A (mouse monoclonal antibody
2-3-2) (29), rabbit polyclonal anti-rat CYP2B1 (30), and rabbit
polyclonal anti-mouse CYP2D9 antibodies (31) were used for Western blot analysis of microsomal CYP proteins. Anti-mouse CYP2D9
(anti-C-P45016 Western Blotting--
Liver cytosolic protein (40 µg) and
microsomal protein (20 µg) prepared from 8-9-week-old wild-type
Stat5a Microsomal Testosterone Hydroxylation Assay--
Microsomal
metabolism of testosterone was assayed as described previously (32)
using 25 µg of mouse liver microsomal protein incubated in 0.2 ml
containing 50 mM Tris buffer, pH 7.6, 3 mM MgCl2, and 14C-labeled testosterone (10 nmol,
~100,000 cpm). Reactions were initiated by the addition of 0.36 mM NADPH and terminated 20 min later by the addition of 1 ml of ethyl acetate. Testosterone and hydroxytestosterone metabolites
were extracted with ethyl acetate and then chromatographed on silica
gel TLC plates developed in solvent A (methylene chloride/acetone
(80:20, v/v)) and then solvent B (chloroform/ethyl acetate/ethyl
alcohol (70:17.5:12.5, v/v/v)). TLC plates were exposed overnight to
PhosphorImager plates followed by quantitation using a Molecular
Dynamics PhosphorImager instrument and ImageQuant software (Sunnyvale,
CA). In order to assess the homogeneity and identity of individual
radiolabeled testosterone metabolites, radioactive spots of interest
were cut from the aluminum-backed TLC plates, and the
14C-labeled monohydroxytestosterone metabolites were then
eluted with ethyl acetate. Unlabeled authentic hydroxytestosterone
metabolites were then individually cospotted with each of the unknown
14C-metabolites to verify co-migration in two independent
TLC solvent systems (32).
EMSA--
Total liver homogenate protein (15 µg) was
preincubated for 10 min at room temperature with 9 µl of gel mobility
shift buffer (12.5 mM Tris-HCl, pH 7.5 containing 10 fmol
of DNA probe, 2 µg of poly(dI-dC) (Boehringer Manheim), 5% glycerol,
1.25 mM MgCl2, 625 µM EDTA, and
625 µM dithiothreitol). Double-stranded oligonucleotide probe containing the STAT5 response element of the rat RNA Isolation and Reverse Transcription Polymerase Chain Reaction
(PCR)--
Total RNA was isolated from ~100 mg of mouse liver using
TRIZOL Reagent (Life Technologies, Inc.). First strand cDNA was
synthesized using random hexamer by adding 7 µl of a master mix
reaction buffer (1.5 mM MgCl2, 15 mM KCl, 10 mM Tris-HCl, pH 8.3, 10 mM dNTPs, 0.1 M dithiothreitol) to 2 µg of
RNA and 100 ng of random hexamer primer. Samples were incubated, first
for 50 min at 42 °C and then for 15 min at 70 °C. RNase H (1 µl, 2 units) was then added, followed by a 30-min incubation at
37 °C. 2 µl of each cDNA sample was amplified in 50 µl of
PCR reaction mixture (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2,, 0.01%
gelatin, 2 mM dNTPs, and 100 ng of each STAT5 PCR primer
(see below)). Amplification was conducted in a Stratagene thermal
cycler at 94 °C for 1 min, 55 °C for 1 min 10 s, and
72 °C for 1 min 30 s for 30 cycles. RNA that was not reverse
transcribed was included as a negative control to verify the absence of
genomic DNA or PCR product contamination. The efficiency of RNA
extraction and of reverse transcription was determined by including
Statistical Analysis--
Individual group comparisons were
performed using the two-tailed Student's t test
(p Stat5a Gene Disruption--
Liver cytosols prepared from
Stat5a
Our previous study of Stat5b Characterization of STAT5a and STAT5b DNA Binding Activity in Stat5
Gene-disrupted Mouse Liver--
We next used a STAT5-binding DNA probe
from the rat
The presence of STAT5 homo- or heterodimers in these liver samples was
further investigated by supershift analysis using anti-STAT5a and
anti-STAT5b antibodies (Fig. 2B). Anti-STAT5b antibody fully supershifted the STAT5b-containing DNA complex present in
Stat5a Expression of Sex-dependent CYPs in
Stat5a
In male 129J mice, the loss of STAT5b increased the female-dominant
testosterone 6
A similar conclusion can be drawn based on the effects of
Stat5a and Stat5b gene disruption on
sex-dependent CYP3A and CYP2B protein expression. Western
blot analysis indicated that the female-dominant liver CYP3A protein
band b was increased in Stat5b
We conclude that Stat5b gene disruption feminizes the livers
of male mice by increasing the expression of female-dominant CYP3A and
CYP2B proteins to the level of wild-type female mice, while partially
decreasing the expression of male-dominant CYP2D9 protein and its
associated testosterone 16 Selective Loss in Female Mice of Select CYP Enzyme Activities upon
Disruption of Stat5a or Stat5b--
We next investigated whether
either of the STAT5 proteins is required to maintain normal CYP enzyme
and protein expression in female mouse liver. Although disruption of
the Stat5a or Stat5b genes had no effect in males
on any of the sex-independent CYP activities examined (Fig.
7; Table
III), in females the loss of STAT5a or
STAT5b did result in a marked loss of some, but not all,
sex-independent testosterone hydroxylase activities. Thus, liver
microsomal testosterone 16 Loss of Female-specific CYP2B Protein in Female
Stat5a Relative Expression Levels of STAT5a and STAT5b mRNA in Mouse
Liver--
In view of the potential role of STAT5 heterodimerization
in regulating CYP enzyme expression suggested by the above experiments, we sought to determine the relative abundance of STAT5a and STAT5b in
mouse liver. STAT5 form-specific antibodies are available
(e.g. Fig. 1); however, the absence of purified STAT5
protein standards precluded a determination of the relative molar
abundance of STAT5a versus STAT5b protein using an
immunochemical approach. Consequently, we investigated the relative
levels of STAT5a and STAT5b mRNA in total mouse liver RNA using a
reverse transcription PCR method. Since the two STAT5 mRNAs are
~90% identical, we used an assay that incorporates an
NcoI restriction digestion step to distinguish a
PCR-amplified STAT5a cDNA fragment, which contains an
NcoI site, from the corresponding STAT5b cDNA, which
does not (see "Materials and Methods"). Results obtained from five
individual 129J × Black Swiss mouse livers are shown in Fig.
8A. NcoI digestion
of the PCR fragments revealed that the majority of the STAT5 cDNA
was derived from STAT5b mRNA in both male (Fig. 8A) and
female 129J × Black Swiss mice (data not shown), as indicated by
the minor fraction that was digested to yield the STAT5a-derived 310- and 240-bp NcoI fragments. In control experiments,
NcoI fully digested STAT5a cDNA amplified from a cloned
STAT5a plasmid using the same PCR primers, while there was no digestion
of a corresponding STAT5b cDNA fragment (data not shown).
Quantitation of the ratio of digested to undigested fragments revealed
that STAT5b corresponded to 95-96% of the total STAT5 mRNA in
both male and female 129J and 129J × Black Swiss mice and to
~90% of the total STAT5 mRNA in 129J × BALB/c mice (Fig.
8B and data not shown).
The present study demonstrates that STAT5a and STAT5b both play
important roles in the maintenance of sexually dimorphic liver CYP gene
expression in the mouse model. The two highly conserved (~90%
identical) STAT5 proteins, STAT5a and STAT5b, were shown to be
activated in mouse liver to form both homodimers (STAT5a-STAT5a; STAT5b-STAT5b) and heterodimers (STAT5a-STAT5b). STAT5b alone, most
likely in the form of a STAT5b-STAT5b homodimeric complex, was found to
be required to maintain the male-specific pattern of GH
pulse-stimulated liver Cyp expression. This was apparent from the loss of the male-specific CYP2D9 band b and the
increase in several female-predominant P450s in
Stat5b Strain-dependent Expression of Mouse Liver CYP
Testosterone Hydroxylases--
In contrast to the rat, where at least
24 hepatic CYP forms have been extensively characterized at a molecular
and regulatory level and CYP gene-specific catalytic and immunochemical
probes are widely available (39), the characterization of individual murine members of the CYP enzyme system is far less advanced, and in
many instances the relationship of specific mouse P450 proteins with
specific Cyp genes is uncertain. Moreover, unlike in the
rat, there are major strain differences in Cyp gene
expression patterns in the mouse (4, 34, 35) (Tables I and III). For example, whereas testosterone 15 Proposed Role of Hormone-induced Activation and Dimerization of
STAT5a and STAT5b--
In mammary gland, STAT5a and STAT5b are both
activated by prolactin (24). The generation of mice in which the genes
encoding prolactin receptor (43), STAT5a (25), and STAT5b (23) have been individually inactivated has demonstrated that in mammary gland
the heterodimeric STAT5a-STAT5b complex is the principal mediator of
mammopoietic and lactogenic signaling (44). Since STAT5a and STAT5b can
thus be activated by prolactin, in addition to GH, the decreased
expression of certain Cyp genes in both
Stat5a Implications for GH-regulated CYP Gene Expression--
In males,
but not females, Stat5b gene disruption increased
female-dominant testosterone hydroxylase enzyme activity and protein expression (Tables I and II). This elevation of normally
female-dominant liver enzyme levels suggests that STAT5b may negatively
regulate some GH-regulated liver-expressed genes in addition to its
demonstrated positive effects on the transcriptional activation of
certain male GH pulse-stimulated genes (22).2 This model is
in accord with other studies based on an analysis of CYP enzyme
patterns in GH-deficient lit/lit mice, where it is shown
that the low expression in male mouse liver of several female-specific,
GH-regulated CYP enzymes is at least in part due to the suppressive
effects of male GH pulses (2, 6). Precedent for an inhibitory effect of
activated STAT5b on gene expression is provided by the transcriptional
inhibition by prolactin-activated STAT5b of interferon-regulatory
factor-1 (51) and by the inhibitory effects of GH-activated STAT5b on
gene transcription stimulated by cross-talk with the nuclear receptor
PPAR
Distinct intracellular signaling pathways are activated by a nearly
continuous (female) compared with an intermittent (male) pattern of
plasma GH stimulation and have been implicated in the sex-dependent expression of certain Cyp genes in
rat liver (1). These include the JAK/STAT5b pathway in the case of
males (12) and pathway(s) that may involve a novel GH-regulated nuclear
factor, termed GHNF (53) and perhaps also phospholipase A2 signaling (54) in females. In addition, the present study demonstrates that in
female mouse liver STAT5a and STAT5b are both required for expression
and thus may be important regulators, of certain female-specific CYP
enzymes. The interactions of STAT5 with female-expressed Cyp
genes may be direct, e.g. could involve STAT5a-STAT5b
DNA-protein complexes, or may be indirect. Conceivably, Cyp
genes, even within the same gene subfamily, may respond to distinct
GH-dependent signaling pathways. Alternatively, a single
GH-activated signaling pathway may regulate different Cyp
genes, in some cases leading to activation and in other cases leading
to inhibition of Cyp expression. Further study will be
required to elucidate the multiple GH-activated signaling pathways that
activate members of this multigene family and to establish the precise
roles that STAT5a and STAT5b play in their expression.
After completion of the present study, Teglund et al. (55)
confirmed our earlier report (23) that Stat5b gene
disruption leads to loss of the GH pulse-regulated male pattern of
postpubertal body growth rate and liver gene expression. They also
reported the lack of an effect of Stat5a gene disruption on
male-specific CYP2D expression, in agreement with the present study.
-hydroxylase) while
increasing to female levels several female-predominant liver CYPs
(CYP3A, CYP2B, and testosterone 6
-hydroxylase). Since STAT5a is thus
nonessential for these male GH responses, STAT5b homodimers, but not
STAT5a-STAT5b heterodimers, probably mediate the sexually dimorphic
effects of male GH pulses on liver CYP expression. In female mice,
however, disruption of either Stat5a or Stat5b
led to striking decreases in several liver CYP-catalyzed testosterone
hydroxylase activities. Stat5a or Stat5b gene
disruption also led to the loss of a female-specific, GH-regulated hepatic CYP2B enzyme. STAT5a, which is much less abundant in liver than
STAT5b, and STAT5b are therefore both required for constitutive expression in female but not male mouse liver of certain GH-regulated CYP steroid hydroxylases, suggesting that STAT5 protein
heterodimerization is an important determinant of the
sex-dependent and gene-specific effects that GH has on the liver.
INTRODUCTION
Top
Abstract
Introduction
References
- and 2
-hydroxylase) and
the female-specific CYP2C12 (steroid sulfate 15
-hydroxylase) (1).
Marked sex-dependent differences in hepatic CYP profiles
are also seen in the mouse, where CYP2D9 (a testosterone
16
-hydroxylase) and CYP2A4 (a testosterone 15
-hydroxylase) are
expressed in males and females, respectively, in certain strains (2,
3). Sexually dimorphic expression of a mouse CYP2B testosterone
16
-hydroxylase has also been described (4-6). The expression of
these sexually dimorphic liver steroid hydroxylase CYPs is primarily
determined by the sexual dimorphism of plasma growth hormone (GH)
profiles (2, 7-9). Intermittent plasma GH pulses, a characteristic of
adult male rats, induce expression of male-specific CYP proteins and
their associated steroid hydroxylase activities, while the near
continuous presence of GH in the plasma of adult female rats induces
expression of female-specific and female-dominant liver CYP proteins
(1, 10). The plasma GH pattern in mice is pulsatile in both sexes; however, sex-specific responses of liver CYPs to plasma GH profiles can
be discerned by the distinct frequency of pulsation in males (interpulse interval of ~2.5 h) and females (interpulse interval <1
h) (11).
-interferon activation-like regulatory sites (TTCNNNGAA), such as that found in the mouse
-casein promoter (13, 24). Stat5a gene disruption has
demonstrated that STAT5a is critically required to activate and/or
repress yet unknown target genes that promote mammary gland
differentiation and lactogenesis (25). Mammary gland STAT5b can only
partially fill this function and then again only after repeated
hormonal stimulation through multiple pregnancies and episodes of
suckling (26). Although targeted disruption of Stat5b leads
to the loss of multiple sexually differentiated responses governed by a
pulsatile plasma GH profile (23), it is uncertain whether this loss
reflects a requirement for heterodimeric STAT5a-STAT5b complexes or
perhaps an absolute requirement for homodimeric STAT5b-STAT5b complexes for GH pulse-regulated liver gene expression. STAT5a and STAT5b exhibit
differences with respect to their tissue distribution (14, 15) and DNA
binding specificities (27) and in the sequences of their COOH-terminal
transcription activation domain (28). These two STATs could thus have
distinct functions with respect to their role in GH signaling and its
impact on the sexual dimorphism of liver gene expression.
/
mice were also examined to verify and extend using an inbred mouse
strain our earlier findings (23) on the effects of Stat5b gene disruption in 129J × BALB/c outbred mice. Evidence is
presented in support of the hypothesis that heterodimerized
(STAT5a-STAT5b) and homodimerized (STAT5b-STAT5b) STAT5 complexes play
distinct roles in the sexually differentiated responses of the liver to GH.
MATERIALS AND METHODS
progeny were
then bred to obtain the 129J congenic
Stat5b
/
and Stat5b+/+
mice used for these experiments. Experiments using
Stat5a
/
mice were carried out using
littermates obtained from 129J × Black Swiss outbred mice. In all
cases, comparisons of wild-type and STAT5-deficient mice were made
between littermates obtained from the crossing of heterozygote siblings
or of heterozygote females with null male siblings, all containing the
same 129J outcrossed background. Accordingly, strain-specific P450
allelic determinants are expected to segregate randomly in both
wild-type and Stat5 gene-disrupted mice. Efforts to obtain
liver tissue from 129J congenic Stat5a
/
mice
for direct comparison to the 129J congenic
Stat5b
/
mice were unsuccessful, since these
animals proved very difficult to breed. Although the possibility cannot
formally be excluded that a 129J-derived regulatory determinant of
liver CYP gene expression is tightly linked to the Stat5
locus and contributes to some of the liver CYP profile differences
between wild-type and Stat5a
/
129J × Black Swiss mice, this is considered unlikely.
80 °C until use. Liver (about 1 g) was
homogenized in 10 ml of homogenizing buffer (10 mM
Tris-HCl, pH 7.4, 1 mM EDTA, and 250 mM
sucrose) with the addition of phosphatase inhibitors (1 mM
sodium orthovanadate and 10 mM sodium fluoride) and a
protease inhibitor (100 µM phenylmethanesulfonyl
fluoride) and centrifuged at 9000 rpm for 15 min to obtain a total
tissue homogenate. Ultracentrifugation at 100,000 × g
for 1 h was carried out to separate microsomal pellets and the
cytosolic supernatant. Microsomal pellets were suspended in KPi buffer,
pH 7.4, containing 0.1 mM EDTA and 20% glycerol, and
stored at
80 °C until use. Microsomal protein concentrations were
determined using the Bradford assay kit (Sigma). Cytosolic and total
tissue homogenate protein concentrations were determined using the
Bio-Rad Dc detergent protein assay kit using bovine serum albumin as a standard.
) was generously provided by Dr. M. Negishi (NIEHS, National Institutes of Health, Research Triangle Park, NC).
/
or
Stat5b
/
mice were electrophoresed through
standard Laemmli SDS-polyacrylamide gels (10% gels for STAT protein
separation and 8% gels for mouse microsomal CYP protein separation),
transferred to nitrocellulose membranes, and then probed with anti-STAT
or anti-CYP antibodies. Membranes were blocked for 1 h at 37 °C
with 3% nonfat dry milk (Blotto) and 1% bovine serum albumin in a
high Tween buffer (0.3% Tween 20 in phosphate-buffered saline) for
anti-STAT1 and anti-STAT3 or with 2% Blotto and 2% bovine serum
albumin in TST buffer (10 mM Tris-HCl, pH 7.5, 0.1% Tween
20, 100 mM NaCl) for probing with anti-STAT5a and
anti-STAT5b. For microsomal CYP Western blotting, membranes were
blocked with 3% Blotto and 1% bovine serum albumin in TST buffer.
Incubations with primary antibody were carried out for 1 h at
37 °C at dilutions of 1:3000. Antibody binding was visualized on
x-ray film by enhanced chemiluminescence using the ECL kit from
Amersham Pharmacia Biotech. Nitrocellulose membranes were reprobed
after stripping in 62.5 mM Tris-HCl, pH 7.6, 2% SDS, 50 mM 2-mercaptoethanol for 20 min at 50 °C. Results are presented in figures prepared from grayscale scans of portions of the
x-ray films of each blot. Scans were obtained using a Cannon IX-4015 scanner outfitted with Ofoto scanning software.
-casein promoter (nucleotides
101 to
80; 5'-GGA-CTT-CTT-GGA-ATT-AAG-GGA-3') was 32P-end-labeled on one strand using T4 kinase and then
incubated with the protein sample for 20 min at room temperature and
then 10 min on ice to stabilize the STAT5-DNA gel shift complex (33). For supershift analysis, an additional 10-min incubation in the presence of STAT antibodies was carried out after the addition of the
labeled DNA probe. Samples were electrophoresed in a cold room through
a nondenaturing polyacrylamide gel (5.5% acrylamide, 0.07%
bisacrylamide) (National Diagnostics, Atlanta, GA) in 0.5 × TBE
buffer (44.5 mM Trizma-base, 44.5 mM boric
acid, 5 mM EDTA) following a 30-min preelectrophoresis
step. After electrophoresis of the samples into the gel for 20 min at
120 V, the gel apparatus was moved to room temperature to increase the
speed of protein migration. In some cases, the electrophoresis time was
increased and/or the percentages of acrylamide and bisacrylamide gel
were increased to 6.5% and 0.08%, respectively, to increase the
resolution of the STAT5-containing EMSA complexes.
-actin-specific primers as an internal control in each PCR reaction.
The STAT5 primers used in the experiment (forward primer, 5'-CAG GTG
AAG GCG ACC ATC AT-3'; reverse primer, 5'-TG CTG TTG TAG TCC TCG
AGG-3') amplify both STAT5a and STAT5b cDNA to produce a 550-base
pair PCR product using a protocol kindly provided by Dr. M. Negishi. To
determine the relative STAT5a and STAT5b mRNA levels in mouse
liver, the STAT5 PCR products were purified using a QIAquick PCR
purification kit (QIAGEN, Valencia, CA) and then digested with
NcoI restriction endonuclease (New England BioLabs, Beverly,
MA). PCR-amplified STAT5a cDNA obtained using the above primers
contains a single NcoI restriction site, which when cut
yields fragments 240 and 310 bp in length, while PCR-amplified STAT5b
cDNA does not contain the NcoI cutting site. Purified
PCR products were digested with NcoI (2 units) in New England BioLabs buffer 4 for 2 h at 37 °C. PCR products and
1-kilobase pair molecular marker were electrophoresed on a 1.5%
agarose gel and visualized by ethidium bromide staining, and their
relative levels were quantitated using ImageQuant software.
0.05).
RESULTS
/
mice were assayed for the expression
of individual STAT proteins by Western blotting. Wild-type mice showed
similar levels of liver cytosolic STAT5a protein in both males and
females, with some differences between individual animals apparent
(Fig. 1, lanes 1,
2, and 7-9). No STAT5a protein was detected in
the Stat5a
/
mice of either sex. Reprobing
with antibodies to other STAT proteins revealed that STAT5b, STAT1, and
STAT3 protein levels were not significantly changed by the
Stat5a gene disruption. This contrasts to the increase in
liver STAT1 in Stat5b
/
mice (23). STAT3 was
present in liver at a significantly higher level in females than in
males in the 129J × Black Swiss mice used in the present study
(Fig. 1, bottom panel, lanes
7-13 versus lanes 1-6).
No sex difference in hepatic STAT3 levels was seen, however, in 129J or
in 129J × BALB/c mice (data not shown).
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Fig. 1.
STAT proteins expressed in livers of
Stat5a /
mice. Liver cytosols (40 µg) were prepared from individual male (lanes
1-6) and female (lanes 7-13)
wild-type (lanes 1, 2, and
7-9) and Stat5a knock-out mice (lanes
3-6 and 10-13). Western blot analyses were
carried out by sequentially probing with each of the indicated
anti-STAT antibodies. Unidentified protein(s) cross-reactive with
anti-STAT1 and STAT3 antibodies are marked by an asterisk at
the left.
/
mice (23) was
carried out using 129J × BALB/c outbred mice. Since there are
significant strain differences in the patterns of CYP enzyme expression
in mouse liver (4, 34, 35), we generated
Stat5b
/
mice in the 129J inbred strain in
order to eliminate random genetic contributions from the 129J × BALB/c outcrossed background to individual animal variation.
Examination of the Stat5b
/
129J congenic
mice revealed effects of STAT5b disruption on liver STAT protein
expression similar to those seen earlier in outbred mice; STAT1 levels
were increased, while the expression of STAT3 and STAT5a was unchanged
(data not shown).
-casein gene in an EMSA of total liver homogenates to
examine the functional (DNA binding) activity of STAT5b protein in
Stat5a
/
mice. STAT5 protein present in
Stat5a
/
mouse liver was active in this DNA
binding assay (Fig. 2A,
lane 1). Given the absence of STAT5a protein in
these livers, we conclude that the DNA complex detected is composed of
STAT5b-STAT5b homodimers. These homodimers migrated distinctly faster
than STAT5a homodimers, which are present in liver homogenates prepared
from Stat5b
/
mice (lanes
4 and 5). A corresponding fast mobility complex
was formed by a GH pulse-activated rat liver homogenate
(lane 8), in agreement with earlier studies
indicating that STAT5b is the major STAT5 protein in this tissue (36,
37). The major complex observed in wild-type mice migrated at an
intermediate mobility compared with that present in
Stat5a
/
and
Stat5b
/
mice (lanes 2,
3, 6, and 7), indicating the presence
of STAT5a-STAT5b heterodimers.
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Fig. 2.
Gel shift analysis of liver STAT5 DNA binding
activity from wild-type, Stat5b /
and
Stat5a
/
mice (A) in
the absence or presence of supershifting anti-STAT5a and anti-STAT5b
antibodies (B). Liver extracts were prepared from
individual mice and then analyzed by EMSA for STAT5 DNA binding
activity as described under "Materials and Methods." A,
a distinct mobility difference between STAT5a-STAT5a homodimers and
STAT5a-STAT5b heterodimers is apparent from a comparison of
lanes 4 and 5 versus
lanes 6 and 7. A GH pulse-activated
male rat liver extract is shown in lane 8 for
comparison. B, supershift analysis using anti-STAT5a and
anti-STAT5b antibodies revealed some immune cross-reactivity between
the STAT5a and STAT5b antibodies (see text). Male rat total liver
homogenates were used as a positive control (lanes
1-3). Lane 14 shows that the
anti-STAT5a antibody can completely supershift the STAT5a-STAT5a
homodimer that is present in the Stat5b
/
male mouse liver. Arrows with asterisks at the
right mark supershifted protein-DNA complexes. Note distinct
mobilities of each of the nonsupershifted complexes shown in
lanes 4, 7, 10, and
13 as a function of the presence or absence of STAT5a or
STAT5b (cf. panel A).
/
mice (Fig. 2B,
lane 9). A supershift complex of similar mobility was obtained in wild-type mouse liver (lanes 6 and 12) and in male rat liver (lane
3), suggesting that these tissues contain homodimeric
STAT5b-STAT5b complexes in addition to the intermediate mobility
STAT5a-STAT5b heterodimeric complexes evident from Fig. 2A.
Some cross-reactivity of this anti-STAT5b antibody with STAT5a-STAT5a homodimers was apparent, however, as revealed by the more rapidly migrating supershift complex obtained with
Stat5b
/
mouse liver samples (lane
15). This latter complex was not present in significant
amounts in wild-type mouse liver (lanes 6 and
12) or male rat liver (lane 3),
indicating that the majority of activated STAT5a is complexed as a
heterodimer with STAT5b in wild-type liver. Anti-STAT5a antibody
yielded a nearly complete supershift of the STAT5a-STAT5a complexes
present in Stat5b
/
mouse liver
(lane 14) but only a partial supershift of the
STAT5-containing complexes present in wild-type mice (lanes
5 and 11) or in male rat liver (lane
2). The supershift pattern obtained with anti-STAT5a antibody is consistent with the presence of both STAT5b-STAT5b homodimer and STAT5a-STAT5b heterodimer, as demonstrated for
GH-activated STAT5a and STAT5b standards expressed in extracts of
transfected COS-1 cells; STAT5a antibody completely supershifted
STAT5a-STAT5a EMSA complexes, while it partially shifted STAT5a-STAT5b
and STAT5b-STAT5b complexes formed by the transfected COS-1 cell
extracts (data not shown). This cross-reactivity of anti-STAT5a with
STAT5b was also apparent from the disruption of the STAT5b-STAT5b
complex present in liver extracts from
Stat5a
/
mice (lane 8 versus lane 7). Since STAT5 DNA
binding activity is obligatorily dependent on tyrosine phosphorylation
of the STAT protein (38), we conclude based on these data that STAT5a
and STAT5b are both present in mouse liver in the activated form.
/
Male Mice--
Experiments were carried out to
ascertain whether STAT5a is required for expression of
sex-dependent liver CYP enzymes. To achieve this objective,
we first examined the patterns of CYP enzyme expression in male and
female Stat5a
/
and
Stat5b
/
mice, using the diagnostic CYP
substrate testosterone (32). Liver CYP enzyme patterns are known to
differ significantly between individual strains of mice (2, 34, 35). We
observed several differences in the sex-dependence of liver microsomal
testosterone hydroxylation in the two mouse strains used in this study,
129J × Black Swiss for Stat5a
/
and
129J inbred for Stat5b
/
. Three
female-dominant hydroxytestosterone metabolites were formed in
wild-type 129J × Black Swiss liver microsomal incubations
(2
-OH, 6
-OH (Fig. 3A)
and 7
-OH (Table I)), while only a
single female-dominant hydroxytestosterone metabolite (6
-OH; Fig.
3B) was formed by wild-type 129J mouse liver microsomes
(Table I). In the case of 129J × BALB/c outbred mice, two
female-dominant hydroxytestosterone metabolites (6
-OH and 6
-OH)
were formed (Table I). In addition, 129J inbred mouse liver microsomes
formed a male-specific 16
-OH-testosterone metabolite that was not
observed in the 129J × BALB/c outbred mice used in our earlier
Stat5b
/
studies (Fig. 3B; Table
I). This latter strain difference is the result of a repression of a
female-specific, CYP2B-dependent testosterone
16
-hydroxylase in 129J females (4).
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Fig. 3.
Influence of Stat5a and
Stat5b gene disruption on sex-dependent
liver microsomal testosterone hydroxylase activities. Testosterone
hydroxylase activities were determined in liver microsomes prepared
from wild-type mice (filled bars) and
Stat5a /
or
Stat5b
/
mice (speckled
bars). Hepatic microsomal proteins (25 µg) were incubated
with 14C-labeled testosterone in the presence of NADPH as
described under "Materials and Methods." Specific activities shown
are mean ± S.E. for n = 5 individual mice per
group. A single asterisk designates significant
differences from wild-type at p
0.05. The
double asterisks represent significant
differences from males at p
0.05.
Impact of Stat5a and Stat5b gene disruption on
sex-dependent liver microsomal testosterone hydroxylase
activities
-hydroxylase enzyme activity up to the level of
wild-type females (Fig. 3B). The same response to the loss
of STAT5b was seen for two CYP activities that have a female-dominant expression profile in 129J × BALB/c mice, testosterone
6
-hydroxylase and testosterone 6
-hydroxylase (Table I). By
contrast, the loss of STAT5a did not increase expression of any of the
female-dominant CYP enzyme activities assayed (testosterone 2
- and
6
-hydroxylase) in affected males (Fig. 3A; Table I). None
of the female-dominant testosterone hydroxylase activities in female
mouse liver was affected by Stat5a or Stat5b gene
disruption (Table I). We conclude that the loss of STAT5a in male mouse
liver does not lead to the increase in female-dominant CYP enzyme
activities that occurs in response to the loss of STAT5b.
/
males (Fig. 4B,
lanes 5-7 versus lanes
1-4), in agreement with the increase in CYP3A-diagnostic
testosterone 6
-hydroxylase activity (Fig. 3B). Two other
liver CYP proteins that are female-dominant in 129J × BALB/c
mice, CYP2B band a and CYP2B band b, were also increased in Stat5b
/
males to the much
higher female levels (Fig. 5A,
lanes 5-8 versus lanes
1-4; cf. lane 9). By
contrast, none of the female-dominant CYP proteins, i.e.
CYP3A band b and CYP2B band b, was increased in
Stat5a
/
male mice (Fig. 4A,
lanes 4-7 versus lanes
1-3; Fig. 5B, lanes 4-7
versus lanes
1-3).3
Stat5b
/
male mice also exhibited a
significant (albeit partial) loss of male-specific testosterone
16
-hydroxylase activity (Fig. 3B) and its associated
CYP2D band b (Fig.
6B, lanes
5-8 versus lanes 1-4). In
contrast, expression of the male-specific liver CYP2D band b
was unchanged in Stat5a
/
male mice (Fig.
6A, lanes 4-7 versus
lanes 1-3) (Table
II).
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Fig. 4.
CYP3A protein expression in
Stat5a /
and
Stat5b
/
mice. Shown are Western
blots of mouse liver microsomes (20 µg) prepared from wild-type and
Stat5a
/
(A) and
Stat5b
/
mice (B) using anti-CYP3A
antibodies. Of the three immune cross-reactive CYP3A bands, the
female-dominant band b was significantly elevated in
Stat5b
/
male mice (B,
lanes 5-7). No significant changes were observed
in either male or female Stat5a
/
mouse liver
microsomes. Band b is somewhat difficult to distinguish from
band a in panel B, in part due to the
fuzzy nature of the bands seen on this Western blot.
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Fig. 5.
Effect of Stat5a gene
disruption on hepatic microsomal CYP2B protein expression. Shown
are Western blots of male and female mouse liver microsomes from the
indicated strains, probed using anti-CYP2B polyclonal antibody. Three
immune cross-reactive CYP2B bands are seen (bands
a, b, and c, with band
c resolved into a doublet in A and C).
CYP2B band a is female-specific in wild-type 129 × BALB/c mice (A) but is expressed in both sexes in 129J × Black Swiss mice (B). CYP2B band b is
female-specific in both strains.
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Fig. 6.
Effect of
Stat5b /
on male-specific CYP2D9
protein expression in 129J mice. Shown are Western blots of mouse
liver microsomes probed using anti-mouse CYP2D9 antibody.
Band b shows the male predominance that is
characteristic of CYP2D9. This band is decreased near to female levels
in male Stat5b
/
mice but is unchanged in
male Stat5a
/
mice.
Impact of Stat5a and Stat5b gene disruption on
sex-dependent P450 protein levels
-hydroxylase activity to the lower level
characteristic of females. These data support our earlier proposal that
STAT5b is essential for maintaining the overall pattern of
male-specific liver CYP gene expression (23). By contrast, STAT5a is
apparently not required to maintain a male-specific pattern of
GH-regulated liver CYP protein expression in male mice. Together, these
studies indicate that STAT5b-STAT5b homodimers are likely to be
required, while STAT5b-STAT5a heterodimers are dispensable for the
male-specific pattern of liver CYP expression.
-hydroxylation was decreased in both
female Stat5a
/
mice and in female
Stat5b
/
mice (129J × BALB/c), while
testosterone 6
-hydroxylation was decreased in
Stat5b
/
females (129J strain) (Fig. 7).
Other sex-independent testosterone hydroxylases were unaffected
(e.g. 6
-hydroxylase and 2
-hydroxylase; left
panels of Fig. 7). Although testosterone 16
-hydroxylase is a male-specific enzyme activity in 129J inbred mice (Fig.
3B), this activity is high in both females and males in
wild-type 129J outcrossed mice (filled bars, Fig.
7). This latter finding is consistent with the characterization of the
female mouse liver testosterone 16
-hydroxylase enzyme as a
female-dominant P450 whose repression in 129J females is inherited as
an autosomal recessive trait (4). Moreover, the selective loss of
testosterone 16
-hydroxylase activity in female mouse liver in
response to the loss of either STAT5a or STAT5b (Table III, last
column; Fig. 7) indicates that both STAT5 forms are required to
maintain full expression of this constitutively expressed CYP gene
product in female but not male mice. Similarly, the selective loss in
female 129J Stat5b
/
mouse liver of
testosterone 6
-hydroxylase activity (Fig. 7B, right panel) indicates a requirement for both
STAT5 proteins to maintain expression of this P450 enzyme in
females.
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Fig. 7.
Effect of
Stat5a /
or
Stat5b
/
on sex-independent liver
testosterone hydroxylase activities. Testosterone hydroxylase
activities were determined in liver microsomes prepared from wild-type
mice (filled bars) and
Stat5a
/
or
Stat5b
/
mice (speckled
bars) as described under "Materials and Methods."
Specific activities shown are mean ± S.E. for n = 5 individual mice per group. A single asterisk
represents significant differences from wild type (p
0.05). Disruption of Stat5a or Stat5b is seen to
decrease expression in female but not male mouse liver of some but not
other sex-dependent, CYP-catalyzed testosterone hydroxylase
activities.
Effect of Stat5a and Stat5b gene disruption on sex-independent liver
microsomal CYP-catalyzed testosterone hydroxylase activities
/
Mice--
The mouse CYP2B subfamily contains
several liver-expressed proteins, including at least one whose
expression is female-predominant and growth hormone-regulated (6). In
view of the GH dependence of this CYP2B enzyme, we examined whether
Stat5a or Stat5b disruption impacts on its
expression. Western blot analysis revealed three CYP2B cross-reactive
proteins in 129J × Black Swiss mouse liver microsomes: band
a, which is sex-independent in this mouse strain,3
albeit variable in individual mice (Fig. 5B); band
b, which is female-specific; and band c, which in some
experiments could be resolved to give two bands (Figs. 5, A
and C). In male mice, Stat5a gene disruption did
not cause any notable change in the level of the sex-independent CYP2B
band a or any increase in the female-specific CYP2B
band b (Fig. 5B), as indicated above. By
contrast, a substantial loss of the female-specific band b
was seen in five of six female Stat5a
/
mice
(Fig. 5C, lanes 7-11
versus lanes 2-6, Table II, and data not shown). This response of CYP2B band b to
Stat5a gene disruption in females is in sharp contrast to
the lack of an effect of Stat5a gene disruption on other
female-specific or female-dominant liver CYP proteins or activities,
such as CYP3A band b (Fig. 4A) or testosterone
2
- and 6
-hydroxylase activity (Fig. 3A). CYP2B band b was not expressed in 129J male or female mouse liver,
precluding a determination of the impact of Stat5b gene
disruption on its expression in this strain. In 129J × BALB/c
outbred mice, CYP2B bands a and band b were both
expressed as female-dominant forms (Fig. 5A); band
a remained at the same level of expression in female
Stat5b
/
mice, while band b was
partially decreased in five of seven female Stat5b
/
mice (data not shown).
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Fig. 8.
Reverse transcription PCR analysis of liver
STAT5 mRNA levels. 2 µg of total RNA isolated from five
individual male 129J × Black Swiss mouse livers was
reverse-transcribed and then PCR-amplified with STAT5 primers that do
not distinguish between STAT5a and STAT5b, as described under
"Materials and Methods." A, the amplified PCR products
(550 bp), with or without NcoI digestion, as indicated, were
electrophoresed in an ethidium bromide-stained gel. The 550-bp STAT5b
cDNA fragment does not contain an NcoI site, whereas the
STAT5a cDNA is digested by NcoI to yield fragments of
240 and 310 bp. B, quantitation of the fractions of total
STAT5 cDNA that correspond to STAT5a (310- plus 240-bp fragments)
and STAT5b (550-bp fragment) after NcoI digestion, as
determined by integration of band intensities. In control experiments
not shown, the corresponding 550-bp cDNA amplified from cloned
STAT5 plasmid DNA was shown to be fully digested with NcoI
in the case of STAT5a and not at all digested with NcoI in
the case of STAT5b.
DISCUSSION
/
but not
Stat5a
/
male mouse liver (CYP3A band
b and CYP2B band b). By contrast, in female mouse
liver, STAT5a and STAT5b were both required for full expression of
several CYP proteins and activities, including a female-specific CYP2B
protein (band b; Fig. 5C), which apparently corresponds to the female-specific, GH-regulated mouse liver CYP2B protein with the same relative mobility described elsewhere (6). This
requirement of both STAT5a and STAT5b in female mouse liver suggests
that STAT5a-STAT5b heterodimers play a unique regulatory role in the
female. This contrasts with the role proposed for STAT5b homodimers in
male liver in mediating GH pulse regulation of sexually dimorphic liver
CYPs. Although a definitive cause-and-effect relationship is not
established by these findings, the loss of male-specific liver gene
expression in Stat5b
/
but not
Stat5a
/
mice could indicate a direct effect
of, and a specific requirement for, STAT5b-STAT5b homodimers to
regulate expression of male GH pulse-induced Cyp genes.
Alternatively, it is possible that other STAT5-containing complexes
(STAT5a-STAT5b and STAT5a-STAT5a) could be intrinsically capable of
regulating the male-expressed Cyp genes but might simply not
be present in sufficient amounts in Stat5b
/
mouse liver to satisfy the threshold requirements with respect to
transmission to the nucleus of a male, pulsatile plasma GH signal.
-hydroxylase activity has been associated with the female-predominant CYP2A4, and testosterone 16
-hydroxylase activity represents the male-specific CYP2D9 in some
mouse strains, these P450 enzyme activities are also associated with
other CYP gene products and consequently lose their sex-dependence in
other mouse strains (Table III) (4, 31). For example, testosterone 16
-hydroxylation is not only catalyzed by CYP2D9, but is also catalyzed by a female-expressed CYP2B enzyme that is expressed in
select mouse strains (4, 5, 40, 41). Accordingly, the high level of
liver microsomal testosterone 16
-hydroxylase activity seen in both
male and female wild-type 129J × Black Swiss and 129J × BALB/c mice (versus the male-specific expression of testosterone 16
-hydroxylase activity in 129J mice) (Fig. 7,
A and C, versus Fig. 3B)
probably results from the combined expression in the outcrossed strains
of the male-specific CYP2D9 band b (e.g. Fig. 6)
and the female-specific CYP2B, band b (Fig. 5). CYP2B band b is not expressed in 129J mice (data not shown), in
agreement with the repression via an autosomal recessive trait of the
female-predominant testosterone 16
-hydroxylase CYP enzyme in 129J
females (4). Consequently, the selective loss in female, but not male,
STAT5a and STAT5b knockout mice of certain sex-independent testosterone hydroxylase activities, namely 16
-hydroxylation (129J × BALB/c and 129J × Black Swiss) and 6
-hydroxylation (129J), is likely to reflect a loss of female-specific CYP gene products rather than a
loss in the female of sex-independent CYP gene products. Further
progress in linking the individual mouse liver microsomal testosterone
hydroxylase activities of each strain with their corresponding
Cyp genes will be necessary in order to further investigate
at the gene-regulatory level the differential effects of STAT5a-STAT5b
heterodimers versus STAT5b-STAT5b homodimers on liver CYP
gene expression that we hypothesize to occur on the basis of our
findings. Identification and further investigation of the specific
Cyp genes that may be regulated in this manner is likely to
be a formidable task, in view of the complexity of the mouse
Cyp gene superfamily (cf. three distinct classes
comprised of 16 closely related mouse Cyp2b genes, only two
of which have been identified (42)).
/
and
Stat5b
/
female mouse liver described in the
present study could conceivably reflect a loss of STAT5a- and
STAT5b-mediated liver prolactin signaling. However, while the long form
of prolactin receptor is expressed in liver tissue, the short form of
the receptor is much more abundant. Moreover, prolactin receptor short
form can exert a dominant-negative phenotype with respect to STAT
activation, such that prolactin-dependent STAT5 activation
is not achieved in liver tissue (12, 45, 46). Accordingly, female
Cyp gene expression in the liver is not likely to be
regulated by prolactin-induced STAT5a-STAT5b activation and
heterodimerization, but rather by GH or perhaps other cytokines that
can also activate STAT5a and STAT5b (47-49). The factors that regulate
the extent of dimerization between STAT5a and STAT5b have not been
identified, although the relative abundance of activated STAT5a and
STAT5b in the target tissue is likely to be a key factor. In liver
tissue, STAT5a appears to be the more minor expressed STAT5 form, both
in the rat (36, 37) and in the mouse (Fig. 8). Accordingly, the ratio
of activated STAT5b to STAT5a may be much greater than 1, such that
homodimeric STAT5b complexes dominate, particularly in males, where the
pulsatile plasma GH profile activates STAT5b much more efficiently than in females (12). It is conceivable, however, that in female liver
activated heterodimeric STAT5a-STAT5b complexes may be relatively abundant as a consequence of the down-regulation of STAT5b activation in response to the female-characteristic, nearly continuous plasma GH
profile (12, 33, 50). Thus, whereas the abundance of activated,
homodimeric STAT5b complexes may serve to maintain sexually dimorphic
GH responses in male liver, a heterodimeric STAT5a-STAT5b complex in
female liver could contribute to the expression of certain
female-specific Cyp genes, such as that which encodes CYP2B
band b.
(52). Thus, while GH pulse-induced expression of male-specific
Cyp genes such as rat CYP2C11 or mouse
Cyp2d9 may require direct STAT5b-DNA interactions, we
hypothesize that GH-dependent Cyp gene products
that are female-expressed (e.g. rat CYP2C12 or
mouse CYP2B, band b) may in part be regulated by
interactions between STAT5b and other factors (e.g. a
hypothetical repressor of the female-expressed Cyp genes).
These interactions could lead to inhibition of gene expression in the
male, giving rise to the observed female-specific pattern of gene expression.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. M. Negishi for provision of
antibody to CYP2D (anti-C-P45016) and for sharing a
reverse transcription PCR/restriction digest method for distinguishing
STAT5a and STAT5b mRNAs, and we thank Michael McLachlan for PCR
phenotyping of the Stat5b
/
and wild-type
mice used in this study.
![]() |
FOOTNOTES |
---|
* 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.
To whom correspondence should be addressed: Dept. of Biology,
Boston University, 5 Cummington St., Boston, MA 02215. Tel.: 617-353-7401; Fax: 617-353-7404; E-mail: djw{at}bio.bu.edu.
2 S. H. Park and D. J. Waxman, unpublished observations.
3 CYP2B band a is expressed in both males and females in 129J × Black Swiss mice, in contrast to its female-specific expression pattern in 129J × BALB/c mice (Fig. 5, B versus A).
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ABBREVIATIONS |
---|
The abbreviations used are: CYP, cytochrome P450; STAT, signal transducer and activator of transcription; EMSA, electrophoretic mobility shift assay; PCR, polymerase chain reaction; GH, growth hormone; bp, base pair(s).
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REFERENCES |
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