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Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston, Massachusetts 02215
Address all correspondence and requests for reprints to: Dr. David J. Waxman, Department of Biology, Boston University, 5 Cummington Street, Boston, Massachusetts 02215. E-mail: djw{at}bio.bu.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 ng/ml). In adult female rats,
GH release from the pituitary is more frequent, resulting in a more
persistent presence of circulating GH at an average plasma level of
approximately 40 ng/ml (2, 6, 7, 8). Recent investigations focusing on the mechanism by which hepatocytes discriminate between circulating plasma GH patterns have implicated the transcription factor STAT5b as an important intracellular mediator of GH pulse-activated, male-specific liver gene expression (9, 10, 11). STAT5b and the closely related STAT5a (>90% identical) (12, 13) belong to a family of Signal Transducers and Activators of Transcription that mediate the effects of a broad range of cytokines, growth factors, and hormones on various target tissues, including the liver (14). Exogenous administration of GH to hypophysectomized (GH-depleted) rats leads to the rapid activation of cytoplasmic liver STAT52 to yield a tyrosine phosphorylated nuclear dimer of STAT5 that has DNA-binding and transcriptional activity (11, 15). By contrast, treatment of hypophysectomized rats with GH administered continuously, i.e. in a female-like manner, effects a dramatic decrease in liver STAT5 activity (11, 16). STAT5a and STAT5b are both activated by male plasma GH pulses (16), although STAT5b, but not STAT5a, is obligatory for maintenance of the male-pattern of hepatic gene expression, as demonstrated in gene knockout studies (10, 17, 18).
The kinetics of STAT5b activation following GH pulse stimulation have been elucidated using the cell line CWSV-1, an SV40-immortalized rat hepatocyte-derived cell line that is responsive to GH (19). Application of intermittent GH pulses, but not continuous GH treatment, strongly activates STAT5b, which is the major STAT5 form present in these cells (20) and in liver (17). Repeated application of GH pulses to the cells stimulates repeated cycles of STAT5b activation via tyrosine phosphorylation and nuclear translocation, followed by deactivation via tyrosine dephosphorylation and return to the cytosol (21). Full STAT5b responsiveness to a second GH pulse requires a minimum off-period of 2.5 h (20), similar to the off-time between successive GH pulses seen in adult male rats in vivo (6, 7). This responsiveness of STAT5b to GH pulses applied in cell culture or given to hypophysectomized rats supports the proposal (11) that the substantially higher STAT5 activity in male compared with female rat liver is a direct reflection of the activation of STAT5b by physiological male GH pulses. A more direct evaluation of this hypothesis requires the examination of the temporal relationship in intact male rats between the occurrence of a plasma GH pulse and the presence of liver STAT5 in its active form. This would help establish whether STAT5b is repeatedly activated by the endogenous male-specific plasma GH pattern per se, or alternatively, whether the high STAT5b activity seen in male liver is due to other endogenous male-specific factors.
The onset of the sexual dimorphism of pituitary GH secretion during development is well characterized (22, 23). Before puberty, GH is present at low levels in the plasma of both male and female rats, and consequently, GH-responsive, sexually dimorphic hepatic genes are expressed at a low level (CYP2C12) or not at all (CYP2C11). Beginning at puberty (approximately 5 weeks postnatal in the rat), male rats exhibit their characteristic pulsatile plasma GH pattern leading to expression of CYP2C11 and loss of the low prepubertal levels of CYP2C12 (24, 25, 26). STAT5b is proposed to contribute to the GH-regulated expression of these and other sexually dimorphic liver genes (27); however, the expression of STAT5b and its activation during the course of male postnatal development have not been investigated. If STAT5b is indeed an intracellular mediator of the effects of plasma GH pulses on male-specific liver genes, then changes in liver STAT5b activity would be expected to accompany changes in circulating GH during postnatal development. To investigate these issues, we presently address the following questions. Does liver STAT5 activity vary in direct response to the occurrence of a plasma GH pulse? Do changes in liver STAT5 activity correlate with the developmental onset of CYP2C11 expression? Are the factors required for STAT5 signal transduction, including STAT5a, STAT5b, and the tyrosine kinase JAK2, expressed before puberty, or is the expression of these factors itself dependent on pubertal GH stimulation? Finally, is the activation of hepatic STAT5 by plasma GH pulses sufficient to activate CYP2C11 and confer a male pattern of hepatic gene expression? Our findings lead us to conclude that liver STAT5 is temporally activated in response to successive plasma GH pulses and is developmentally activated in parallel to CYP2C11 gene expression. However, STAT5 activation alone, although necessary, is not itself sufficient to induce an adult male pattern of liver gene expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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STAT5 activity analysis in rat liver homogenates
Rat liver homogenates were prepared from frozen liver tissue and
electrophoretic mobility shift analysis (EMSA) assays were performed
using a STAT5-specific DNA probe derived from the promoter of the rat
ß-casein gene, as previously described (16, 20). Approximately
200–400 mg of frozen rat liver tissue was homogenized at 4 C in 2 ml
of ice-cold homogenization buffer (10 mM Tris pH 7.6, 1
mM EDTA, 250 mM sucrose) containing a mixture
of protease inhibitors and phosphatase inhibitors. Samples were
centrifuged at 9,000 rpm for 20 min at 4 C. Supernatants were
aliquoted, snap-frozen in liquid nitrogen, and stored at -80 C. The
validity of using liver homogenates prepared in this manner to assay
liver nuclear STAT5 activity has been previously established (16). This
assay measures both STAT5a and STAT5b, although STAT5b is the more
abundant contributor to total hepatic STAT5 EMSA activity. EMSA gels
were dried and exposed to phosphorimager plates for 1–3 days. Analyses
were done on a Molecular Dynamics, Inc. PhosphorImager
(Sunnyvale, CA) with quantitation using ImageQuant software (16).
Background values (typically corresponding to 2–5% of a maximal male
liver STAT5 signal) were determined based on the average of 2–4 blank
regions from each gel and were subtracted from all samples on the gel
to yield net activity values. These values were then expressed as a
percentage of a standard high STAT5 activity male rat liver sample or
the average of several such male rat liver samples.
Statistical analyses were performed using Prism GraphPad Software, Inc. Linear and nonlinear (rectangular hyperbola)
regressions were performed on the same data set. The saturation curve
shown (see Fig. 2C
) was drawn by the computer from the nonlinear
regression analysis performed.
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Microsomal testosterone hydroxylation assay
Cytochrome P450-dependent microsomal metabolism of testosterone
was assayed at 37 C with shaking (29). Incubations contained 20 µg of
rat liver microsomal protein in 0.2 ml containing 100 mM
Tris buffer, pH 7.6, 0.5 mM MgCl2,
and 14C-labeled testosterone (10 nmol, 
100,000
cpm; Amersham Pharmacia Biotech, Arlington Heights, IL).
Reactions were initiated by the addition of 0.98 mM NADPH
and terminated 10 min later by the addition of 1 ml ethyl acetate.
Testosterone and hydroxytestosterone metabolites were extracted with
ethyl acetate and then chromatographed on silica gel TLC plates
developed sequentially in solvent A [methylene chloride/acetone
(80:20, vol/vol)] and then solvent B [chloroform/ethyl acetate/ethyl
alcohol (70:17.5:12.5, vol/vol/v)] (29). TLC places were
exposed to Molecular Dynamics, Inc. Phosphoimager plates
for 48 h and the radioactivity content and molar abundance of each
individual testosterone metabolite then quantitated using ImageQuant
software.
Western blotting
Liver microsomes (40 µg) or whole liver homogenates (40 µg)
were electrophoresed through Laemmli SDS gels (7.5% gels) run at
constant current and a starting voltage of 75 V, with cross-over to a
constant voltage of 170 V. Gels were electrotransferred to
nitrocellulose and probed with the following antibodies: anti-STAT5a,
anti-STAT5b and anti-JAK2 (antibodies sc-1081, sc-835, sc-294,
respectively, Santa Cruz Biotechnology, Inc., Santa Cruz,
CA), or anti-CYP2C11 (generously provided by Dr. J. Capdevilla,
Vanderbilt University, Nashville, TN). Blocking and antibody probing
conditions were as previously described (11). Detection on x-ray film
was accomplished by enhanced chemiluminescence using ECL reagents
(Amersham Pharmacia Biotech).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, the
individual male rats exhibited large differences in hepatic STAT5
activity (panels A and B). Furthermore, GH RIA analysis of plasma
obtained from the same rats revealed a direct relationship between the
presence of significant amounts of GH (e.g. rats 1, 3, 4, 7,
and 8; Fig. 1C) and the occurrence of a strong liver STAT5 EMSA signal
(c.f., Fig. 1B)
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. STAT5 activity values were calculated
relative to the average value of 4
livers1 showing a typical
high STAT5 activity profile (livers 19, 22, 25, 27; Fig. 2A). A
correlation between liver STAT5 activity (panel A) and plasma GH at the
time of liver excision (panel B) was apparent, both when analyzed by
linear regression analysis (r = 0.84; data not shown), and when a
nonlinear regression analysis (rectangular hyperbola) was performed
(r = 0.85) (Fig. 2C). STAT5 activation is shown to plateau with
increasing plasma GH concentration (Bmax =
163.2% relative STAT5 activity). A similar correlation (r = 0.84)
was seen when the high outlier data point (liver 29) was removed
(Bmax = 104%). These results indicate that liver
STAT5 activity reaches a maximal level beyond which there is diminished
response to further increases in plasma GH levels. This maximal STAT5
activity level may reflect saturation of GH receptors present on the
cell surface and/or depletion of the unphosphorylated STAT5 pool
present in the cytosol.
To distinguish basal liver STAT5 activity from the GH pulse-inducible
liver STAT5 activity, individual male rats were designated plasma GH
positive where plasma GH levels at the time of liver excision were
>3.7 ng/ml, corresponding to 3-fold above the least detectable GH
concentration under the conditions of the RIA (28). Rats having plasma
GH values below this level were designated GH pulse-negative, and
presumably correspond to animals killed between plasma GH pulses.
Analysis of the 29 adult male samples using these criteria revealed
(Table 1) that 28 individuals exhibited a
direct correlation between liver STAT5 activity and the occurrence of a
GH pulse: 17 plasma GH-positive rats all showed substantial liver STAT5
activity [i.e. activity 
the average STAT5 activity
level of 9.6 ± 1.7% seen in adult female rats (16)]; and 11
rats showed low STAT5 activity and low GH levels. The one rat that did
not fit this general pattern (rat 20; Fig. 2C, circled data point)
showed a high plasma GH level (34 ng/ml) but very low STAT5 activity
(1.4%).
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; Table 1). Furthermore,
no correlation (r = 0.02) between liver STAT5 activity and plasma
GH level was seen among the 11 samples in the low STAT5 and low plasma
GH level group. Also, the STAT5 activity level of the low plasma GH
group (2.8 ± 0.8%) is significantly lower than that of adult
females determined earlier using the same methodology (9.6 ±
1.7%) (16). The absence of a correlation between STAT5 activity and
plasma GH level in the low plasma GH group suggests that a threshold
level of circulating GH is required for activation of STAT5. The
apparent threshold GH level, 3.7 ng/ml, compares favorably with the
reported Kd of the GH-GH receptor complex [2.2
ng/ml; (30)]. The positive correlation in individual adult male rats
between liver STAT5 activity and the presence of significant levels of
GH (>3.7 ng/ml) at the time of liver excision provides strong support
for the earlier proposal, based on hypophysectomized rat studies (11),
that liver STAT5 directly responds to each successive plasma GH pulse,
and thus undergoes repeated cycles of activation and nuclear
translocation in intact adult male rats.
Activation of STAT5 during postnatal development
Expression of the male-specific, GH pulse-activated
CYP2C11 is not detected in rat liver until 4.5–5
weeks of age (25), i.e. the time of onset of the plasma GH
pulses that characterize pubertal and adult male rats. Prepubertal rats
are characterized by a more continuous presence of low-levels of plasma
GH (22, 23), which supports expression at a low level of the adult
female rat P450 form CYP2C12 in both male and female rats at 3–4 wk of
age (25). At puberty, when pulsatile pituitary GH secretion begins,
CYP2C11 gene expression begins. To ascertain whether there
is a correlation between the developmental onset of sex-specific GH
profiles (and consequently, CYP2C11 expression) and liver
STAT5 activation, liver homogenates prepared from individual male rats
killed at different time points after birth were assayed for STAT5 EMSA
activity (Fig. 3). Low liver STAT5
activity was seen in rats aged 4 days or 2 weeks. Liver STAT5 activity
in 4-week-old rats was somewhat higher, but still low compared with
that of pubertal and adult rats, and with no apparent dependence
on the plasma GH concentration (Table 2).
The 4-week-old rats exhibited low GH levels (ranging from 1.5–12
ng/ml) except for one sample which had an unusually high level of
plasma GH (359 ng/ml) but little STAT5 activity (Fig. 3A, lane 17).
Beginning at week 5, the differential activation of liver STAT5 in
individual male rats was seen, and this activation correlated with the
presence of GH in plasma at the time of liver excision (Fig. 3B, lanes
2–9). This plasma GH-dependent activation of STAT5 was also observed
in rats aged 8 and 12 weeks (Fig. 3C; Table 2), as seen earlier for the
larger group of adult males (Fig. 2). Western blot analysis of these
same liver samples revealed a striking increase in CYP2C11 protein
beginning at 5 wk (Fig. 4), which
paralleled the onset of the pulsatile STAT5 activation profile (Fig. 3).
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).
Prepubertal expression of these proteins is thus independent of the
plasma GH pulses that occur in pubertal and adult male rats.
Accordingly, given that GH receptor is also expressed in prepubertal
rat liver (33), the low-level liver STAT5 activity seen in rats aged 4
weeks and younger is suggested to result from the absence of a
sufficiently strong stimulatory plasma GH pulse in these animals,
rather than the absence of the protein factors required for activation
of the STAT5 signaling cascade.
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). This response confirms the presence
of functional GH receptor, JAK2 kinase and STAT5 in prepubertal rat
liver, and indicates that the activation of STAT5 by GH does not
require additional factors that are expressed following puberty.
Treatment of the prepubertal rats with GH at 50 µg/100 g BW
stimulated activation of liver STAT5 (55 ± 8% of adult male
control, mean ± SEM, n = 5; Fig. 6, lanes 14–18
vs. 4 ± 1.3% STAT5 activity in sham-injected
controls, mean ± SEM, n = 5, Fig. 6, lanes 5–9) (P < 0.05). Administration of a
physiologic GH dose of 3 µg/100 g BW also led to a significant,
albeit less-pronounced, activation of STAT5 (16 ± 2.2% of adult
male control, mean ± SEM, n = 4, Fig. 6, lanes 10–13; vs. 4 ± 1.3% in sham-injected
controls, mean ± SEM, n = 5, lanes
5–9) (P < 0.05). This finding is in contrast to our
earlier investigations in hypophysectomized rats (11) where GH at a
replacement dose of 3 µg/100 g BW stimulated maximal STAT5
activation. We conclude that the STAT5 signaling cascade is
intrinsically functional and responsive to GH in prepubertal rat liver,
albeit with a requirement for higher GH concentrations to achieve
maximal STAT5 activation compared with hypophysectomized adult
rats.
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Two-week-old pups were administered GH by sc injection
twice-daily (0800 h and 2000 h) for periods ranging from 1 to 7
days at a dose of 50 µg GH/100 g BW. Pups were killed 12 h after
the last GH injection. This hormone injection regimen is effective in
restoring normal adult male levels of liver CYP2C11 mRNA and activity
in hypophysectomized adult rats (36). Moreover, this dose of GH
activates STAT5 in prepubertal rats to a level (Fig. 6) that is more
than sufficient to restore CYP2C11 expression in
hypophysectomized adults (see Discussion). Western blot
analysis of liver microsomes prepared from the GH pulse-treated pups
revealed no induced expression of CYP2C11 protein (Fig. 7A). This finding was confirmed by
enzymatic analysis of liver microsomal, P450-dependent testosterone
hydroxylase activities. Whereas adult male rats exhibited high
CYP2C11-dependent liver microsomal testosterone 2
- and
16-hydroxylase activity, GH pulse-treatment of prepubertal pups did
not increase these activities above that of the very low levels seen in
sham-treated pups (Table 3). Thus, in
contrast to the GH pulse responsiveness of hypophysectomized adult
rats, hepatic enzyme profiles were not masculinized by GH pulse
injection into prepubertal rats. Analysis of female-dependent,
GH-regulated hepatic microsomal activities revealed no significant
changes in testosterone 7-hydroxylase (CYP2A1-dependent) and
steroid 5-reductase activities (Table 3).
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, B and C, pups treated with GH for either 2 or 7
days and killed 12 h after final injection showed little residual
STAT5 activity, which is a consequence of STAT5 being dephosphorylated
and returned to the cytosol (20, 21) during the intervening 12 h.
However, pups given one additional injection of GH and killed 30 min
later clearly showed high levels of liver STAT5 activity, indicating
that STAT5 is repeatedly activated by the exogenous GH pulses over the
course of the 7-day study. Analysis of body growth revealed no significant differences in absolute weights or growth rates between sham and GH-treated pups over the 7-day hormone treatment period. GH-treated pups showed an average daily weight gain of 2.04 ± 0.12 g (mean ± SEM, n = 9) compared with 1.72 ± 0.14 g for sham-injected rats (n = 6) (P < 0.05). This supports a previous report that the rapid body growth of rats from 2 to 3 weeks of age is not further stimulated by twice-daily injection of recombinant human GH (300 µg/100 g BW) (37).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Of the 29 adult male rats examined in the present study, one individual
did not exhibit the correlation between liver STAT5 activity and
circulating GH shown by all of the other rats. The high plasma GH and
low STAT5 activity seen in this rat (Fig. 2 and Table 1) could
correspond to a situation where the liver is excised very early during
a GH pulse, i.e. before there has been time for efficient
assembly of the GH receptor/JAK2 kinase complex and STAT5 tyrosine
phosphorylation. Indeed, in hypophysectomized rats given GH by
ip injection, liver STAT5 tyrosine phosphorylation does not occur until
5–10 min after GH administration (38). The small number of rats that
did not fit the correlation between liver STAT5 activity and the
presence of significant GH in plasma (1 out of 18 GH positive
individuals) (Table 1) is consistent with the activation of liver STAT5
being a relatively rapid event. All of the rats killed at the time when
plasma GH was low (3.7 ng/ml) displayed low STAT5 activity,
suggesting this group corresponds to animals killed during a plasma GH
interpulse interval ("trough period"), at which time STAT5
molecules activated by the prior plasma GH pulse have already been
dephosphorylated and returned back to the cytosol. Furthermore, the
average liver STAT5 activity in females (9.6 ± 1.7%) (16) is
substantially higher than that of GH-negative males (2.8 ±
0.8%). This indicates that the STAT5 activity of female rats is
significant, albeit much lower than the peak level of STAT5 activity
obtained in male rats stimulated by a plasma GH pulse. Accordingly,
STAT5, when activated by GH in adult female liver, could contribute to
the regulation of liver gene expression. Examples of this regulation
may include a female-specific, GH-regulated hepatic CYP2B enzyme, which
requires both STAT5a and STAT5b for full expression in adult female
mice (17), and the liver enriched transcription factor HNF6, which is
transcriptionally activated by GH in rats in a STAT5-dependent
manner (39).
The absence of a correlation in the low STAT5 activity group between
plasma GH level and liver STAT5 activity (r = 0.2) suggests that
the low basal liver STAT5 activity in male rat liver may not be due to
pulsatile plasma GH stimulation but may result from stimulation of
STAT5 signaling by cytokines or other endogenous factors. Together,
these findings suggest that a threshold plasma GH pulse level, which
is 3.7 ng/ml, is required for efficient STAT5 activation.
Limitations of these correlative observations include the fact that the
precise threshold for male-specific, GH pulse-dependent STAT5
activation cannot be determined and the uncertainty of whether the
plasma GH values assayed for individual rats correspond to samplings
taken during the "upswing" or "downswing" phase of the GH
pulse. Further investigations, including direct monitoring of the
temporal relationship between plasma GH profiles and liver STAT5
activation patterns in individual rats, will be necessary to address
these points.
The repeated activation and deactivation of STAT5 in the liver raises the possibility that GH pulse-activated, male expressed genes, such as CYP2C11, may be transcribed in an intermittent, or pulsatile, manner in direct relation to the intermittent presence of STAT5 transcription factor in its active form in the nucleus. In an alternative model, suggested by the observed transcriptional inhibitory potential of STAT5b in some systems (40, 41, 42, 43), STAT5 could act to repress CYP2C11 transcription, such that the inactivation of nuclear STAT5 at the conclusion of a plasma GH pulse serves as the stimulus that leads to CYP2C11 derepression and transcriptional activation. If this latter model is correct, then the temporal profile of CYP2C11 transcription initiation would correlate negatively with the liver’s STAT5 activation status and plasma GH levels. However, given the role of STAT5b as a positive regulator of male-specific liver CYP gene expression evident from STAT5 knockout mouse studies (10), the latter model seems unlikely. Other models are possible, however, including the indirect involvement of STAT5 in transcription of male-specific GH-regulated liver genes. The potential role of STAT5 as an indirect mediator of CYP2C11 expression is supported by the fact that a minimum of 2–3 days of GH pulse-treatment of hypophysectomized rats is required to restore CYP2C11 expression (4, 44). Because GH pulses activate liver STAT5 rapidly, within 15 min (11), this finding indicates that additional GH-dependent liver factors must be expressed before the transcriptional activation of CYP2C11 can occur. In agreement with this model, GH-activated STAT5 appears to be required, either directly or indirectly, for the expression of the liver-enriched transcription factors HNF6, HNF4, and HNF3ß (39, 45). These liver factors, in turn, may contribute to the expression of male-specific, liver expressed P450 genes, such as CYP2C11, perhaps acting in concert with STAT5. According to this model, transcription of genes such as CYP2C11 would be dependent on, but not necessarily temporally related to the plasma GH profile and nuclear STAT5 status of the liver. Further studies, including transcription initiation analysis in individual male livers that differ in STAT5 activation status, will be required to distinguish between these and other potential regulatory mechanisms.
The presence in prepubertal rats of the key protein factors required
for GH-stimulated STAT5 signaling, namely GH receptor (33), JAK2,
STAT5a, and STAT5b (Fig. 5) provided a unique opportunity to
investigate the sufficiency of STAT5 DNA-binding activity for
CYP2C11 expression in male rats. The precocious activation
of liver STAT5 in prepubertal rats required supraphysiological GH
doses, suggesting that mechanism(s) exist to moderate the
responsiveness of prepubertal rats to GH and thereby maintain hepatic
STAT5 activity at a low level. These mechanisms could include: more
efficient sequestration by plasma GH binding protein or enhanced plasma
GH clearance; a lower abundance of liver GH receptors (33); and less
efficient STAT5 activation or enhanced STAT5 dephosphorylation in
prepubertal compared with adolescent and adult rats. Although
twice-daily GH pulse treatment of 2-week-old rats for 7 days resulted
in the repeated activation of STAT5, it did not lead to an induction of
CYP2C11 gene expression. This finding was further confirmed
by the lack of masculinization of hepatic enzyme profiles, evaluated by
microsomal testosterone hydroxylase activity, and by the lack of
significant additional weight gain in GH-treated compared with
sham-injected immature rats. The ineffectiveness of exogenous GH pulses
with respect to prepubertal CYP2C11 activation cannot be
explained by the somewhat lower than maximal liver STAT5 activity that
we obtained (55% of adult male level), insofar as even a low GH dose
(e.g. 1 µg GH/100 g BW, corresponding to 25% of a normal,
physiological GH peak) induces full expression of CYP2C11 in
hypophysectomized adult rats (34), even though liver STAT5 is only
partially activated at this GH dose in the same hypophysectomized rat
model (38). Rather, the absence of CYP2C11 expression under
conditions where liver STAT5 is repeatedly activated over a 7-day
period (Fig. 7) suggests that prepubertal rat liver may be
intrinsically unresponsive to STAT5-stimulated gene expression.
Additionally, postpubertal liver factors other than STAT5 alone may be
required for efficient gene induction in the case of CYP2C11
and other male-expressed genes.
The liver-enriched transcription factors that presumably cooperate with STAT5 to achieve the male-specific pattern of liver gene expression which characterizes CYP2C11 and other sexually dimorphic, GH-regulated P450 genes are not known. Potential candidates include the liver-enriched transcription factors HNF1ß, HNF3ß and DBP, whose mRNA levels are very low during early postnatal periods compared with adults (46) and whose absence could conceivably be a determinant of the unresponsiveness of CYP2C11 to precocious activation by liver STAT5. Furthermore, other developmentally regulated factors may be required. For example, circulating androgen is required for full expression of CYP2C11. Birth-castrated rats do not express CYP2C11 protein or activity at adulthood (25, 26, 47), and full expression requires androgen replacement during both prepubertal and postpubertal periods (47). However, this androgen requirement is generally presumed to be a consequence of the effects of sex-steroids on GH-releasing hormone and somatostatin in the hypothalamus, leading to regulation of the circulating GH pattern, rather than a direct consequence of sex-steroid action on the liver (48, 49, 50). Nevertheless, given our findings regarding the insufficiency of GH pulse-activated STAT5 for stimulating male CYP expression, one cannot rule out the possibility that androgen-dependent factors other than pulsatile GH act in concert with STAT5 to stimulate the male-specific pattern of liver gene transcription.
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Acknowledgments |
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Footnotes |
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2 Liver STAT5 is predominantly comprised of
STAT5b, but includes the less abundant STAT5a. Both STAT forms
contribute to the EMSA activities measured in this study (16 17 ).
Received March 29, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M. G. Holloway, Y. Cui, E. V. Laz, A. Hosui, L. Hennighausen, and D. J. Waxman Loss of Sexually Dimorphic Liver Gene Expression upon Hepatocyte-Specific Deletion of Stat5a-Stat5b Locus Endocrinology, May 1, 2007; 148(5): 1977 - 1986. [Abstract] [Full Text] [PDF]
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D. J. Waxman and C. O'Connor Growth Hormone Regulation of Sex-Dependent Liver Gene Expression Mol. Endocrinol., November 1, 2006; 20(11): 2613 - 2629. [Abstract] [Full Text] [PDF]
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K. H. Clodfelter, M. G. Holloway, P. Hodor, S.-H. Park, W. J. Ray, and D. J. Waxman Sex-Dependent Liver Gene Expression Is Extensive and Largely Dependent upon Signal Transducer and Activator of Transcription 5b (STAT5b): STAT5b-Dependent Activation of Male Genes and Repression of Female Genes Revealed by Microarray Analysis Mol. Endocrinol., June 1, 2006; 20(6): 1333 - 1351. [Abstract] [Full Text] [PDF]
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M. G. Holloway, E. V. Laz, and D. J. Waxman Codependence of Growth Hormone-Responsive, Sexually Dimorphic Hepatic Gene Expression on Signal Transducer and Activator of Transcription 5b and Hepatic Nuclear Factor 4{alpha} Mol. Endocrinol., March 1, 2006; 20(3): 647 - 660. [Abstract] [Full Text] [PDF]
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 T. Landsman and D. J. Waxman Role of the Cytokine-induced SH2 Domain-containing Protein CIS in Growth Hormone Receptor Internalization J. Biol. Chem., November 11, 2005; 280(45): 37471 - 37480. [Abstract] [Full Text] [PDF]
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 M. Rahmaniyan, K. Patrick, and N. H. Bell Characterization of recombinant CYP2C11: a vitamin D 25-hydroxylase and 24-hydroxylase Am J Physiol Endocrinol Metab, April 1, 2005; 288(4): E753 - E760. [Abstract] [Full Text] [PDF]
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A. S. Verma, R. N. Dhir, and B. H. Shapiro Inadequacy of the Janus Kinase 2/Signal Transducer and Activator of Transcription Signal Transduction Pathway to Mediate Episodic Growth Hormone-Dependent Regulation of Hepatic CYP2C11 Mol. Pharmacol., March 1, 2005; 67(3): 891 - 901. [Abstract] [Full Text] [PDF]
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C. A. Wiwi and D. J. Waxman Role of Hepatocyte Nuclear Factors in Transcriptional Regulation of Male-specific CYP2A2 J. Biol. Chem., February 4, 2005; 280(5): 3259 - 3268. [Abstract] [Full Text] [PDF]
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 J. E. Rowland, A. M. Lichanska, L. M. Kerr, M. White, E. M. d'Aniello, S. L. Maher, R. Brown, R. D. Teasdale, P. G. Noakes, and M. J. Waters In Vivo Analysis of Growth Hormone Receptor Signaling Domains and Their Associated Transcripts Mol. Cell. Biol., January 1, 2005; 25(1): 66 - 77. [Abstract] [Full Text] [PDF]
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 E. V. Laz, C. A. Wiwi, and D. J. Waxman Sexual Dimorphism of Rat Liver Nuclear Proteins: Regulatory Role Of Growth Hormone Mol. Cell. Proteomics, December 1, 2004; 3(12): 1170 - 1180. [Abstract] [Full Text] [PDF]
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T. C. Brelje, L. E. Stout, N. V. Bhagroo, and R. L. Sorenson Distinctive Roles for Prolactin and Growth Hormone in the Activation of Signal Transducer and Activator of Transcription 5 in Pancreatic Islets of Langerhans Endocrinology, September 1, 2004; 145(9): 4162 - 4175. [Abstract] [Full Text] [PDF]
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C. A. Wiwi, M. Gupte, and D. J. Waxman Sexually Dimorphic P450 Gene Expression in Liver-Specific Hepatocyte Nuclear Factor 4{alpha}-Deficient Mice Mol. Endocrinol., August 1, 2004; 18(8): 1975 - 1987. [Abstract] [Full Text] [PDF]
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 D. J. Waxman and J. L. Celenza Sexual dimorphism of hepatic gene expression: novel biological role of KRAB zinc finger repressors revealed Genes & Dev., November 1, 2003; 17(21): 2607 - 2613. [Full Text] [PDF]
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Y. Kasukawa, D. J. Baylink, R. Guo, and S. Mohan Evidence that Sensitivity to Growth Hormone (GH) Is Growth Period and Tissue Type Dependent: Studies in GH-Deficient lit/lit Mice Endocrinology, September 1, 2003; 144(9): 3950 - 3957. [Abstract] [Full Text] [PDF]
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N. Balthasar, P.-F. Mery, C. B. Magoulas, K. E. Mathers, A. Martin, P. Mollard, and I. C. A. F. Robinson Growth Hormone-Releasing Hormone (GHRH) Neurons in GHRH-Enhanced Green Fluorescent Protein Transgenic Mice: A Ventral Hypothalamic Network Endocrinology, June 1, 2003; 144(6): 2728 - 2740. [Abstract] [Full Text] [PDF]
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 F. R. Simon, J. Fortune, M. Iwahashi, and E. Sutherland Sexual dimorphic expression of ADH in rat liver: importance of the hypothalamic-pituitary-liver axis Am J Physiol Gastrointest Liver Physiol, September 1, 2002; 283(3): G646 - G655. [Abstract] [Full Text] [PDF]
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L. Gonzalez, J. G. Miquet, A. I. Sotelo, A. Bartke, and D. Turyn Cytokine-Inducible SH2 Protein Up-Regulation Is Associated with Desensitization of GH Signaling in GHRH-Transgenic Mice Endocrinology, February 1, 2002; 143(2): 386 - 394. [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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G. S. Tannenbaum, H. K. Choi, W. Gurd, and D. J. Waxman Temporal Relationship Between the Sexually Dimorphic Spontaneous GH Secretory Profiles and Hepatic STAT5 Activity Endocrinology, November 1, 2001; 142(11): 4599 - 4606. [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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P. A. Ram and D. J. Waxman Role of the Cytokine-inducible SH2 Protein CIS in Desensitization of STAT5b Signaling by Continuous Growth Hormone J. Biol. Chem., December 8, 2000; 275(50): 39487 - 39496. [Abstract] [Full Text] [PDF]
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