Down-Regulation of Liver JAK2-STAT5b Signaling by the Female Plasma Pattern of Continuous Growth Hormone Stimulation
Carol A. Gebert,
Soo-Hee Park and
David J. Waxman
Division of Cell and Molecular Biology Department of
Biology Boston University Boston, Massachusetts 02215
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ABSTRACT
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The suppression of male-specific, GH
pulse-induced, liver transcription in adult female rats has been
linked to the down-regulation of STAT5b activation by the female plasma
pattern of near-continuous GH exposure. The mechanism underlying this
down-regulation was studied in the rat liver cell line CWSV-1, where
continuous GH suppressed the level of activated (tyrosine-
phosphorylated) STAT5b to approximately 1020% of the maximal GH
pulse-induced STAT5b signal within 3 h. In contrast to the robust
JAK2 kinase-dependent STAT5b activation loop that is established by a
GH pulse, JAK2 kinase signaling to individual STAT5b molecules was
found to be short lived in cells treated with GH continuously.
Moreover, maintenance of the low-level STAT5b signal required ongoing
protein synthesis and persisted for at least 7 days provided that GH
was present in the culture continuously. Increased STAT5b DNA-binding
activity was observed in cells treated with the proteasome inhibitor
MG132, suggesting that at least one component of the GH receptor
(GHR)-JAK2-STAT5b signaling pathway becomes labile in response to
continuous GH treatment. The phosphotyrosine phosphatase inhibitor
pervanadate fully reversed the down-regulation of STAT5b DNA-binding
activity in continuous GH-treated cells by a mechanism that involves
both increased STAT5b activation and decreased STAT5b
dephosphorylation. Moreover, the requirement for ongoing GH stimulation
and active protein synthesis to maintain STAT5b activity in continuous
GH-treated cells were both eliminated by pervanadate treatment,
suggesting that phosphotyrosine dephosphorylation may be an obligatory
first step in the internalization/degradation pathway for the GHR-JAK2
complex. Finally, the sustaining effect of the serine kinase inhibitor
H7 on GH pulse-induced JAK2 signaling to STAT5b was not observed in
continuous GH-treated cells. These findings suggest a model where
continuous GH exposure of liver cells down-regulates the STAT5b pathway
by a mechanism that involves enhanced dephosphorylation of both STAT5b
and GHR-JAK2, with the latter step leading to increased
internalization/degradation of the receptor-kinase complex.
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INTRODUCTION
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Cytokine and growth factor-induced cell surface receptor
aggregation is coupled to the activation of transcriptional events in
the nucleus by several interdependent signaling pathways, including the
mitogen-activated protein (MAP) kinase (1, 2) and JAK-STAT (Janus
kinase-signal transducer and activator of transcription)
pathways (3, 4). Recent studies on cytokine-induced signaling have
focused on the biochemical events that occur in ligand-naïve
cells shortly after ligand stimulation. These events are particularly
relevant to situations characterized by a pulsatile plasma profile for
the cytokine or growth factor, as occurs during interleukin release in
response to acute stress (5, 6) or PRL release in response to suckling
(7, 8). In other cases, including many therapeutic applications,
stimulation of a cytokine receptor may be continuous (9, 10). The
polypeptide hormone GH is a unique example of a cytokine/growth factor
that circulates in the blood and stimulates target tissues in either of
two, temporally distinct plasma profiles regulated by hypothalamic
stimulatory and inhibitory release factors. In the rat, GH is released
from the pituitary gland in a pulsatile manner in males and in a
near-continuous fashion in females (11, 12). Plasma GH pulses in male
rats are approximately 1 h in duration with peak blood plasma
concentration of 200 ng/ml, followed by an approximately 2.5-h period
where GH is undetectable in plasma (<2 ng/ml). By contrast,
circulating GH levels in female rats are more continuous, and typically
range from about 2040 ng/ml. These sex-dependent plasma GH patterns
lead to distinct, sex-dependent patterns of gene expression in the
liver, a major target organ of GH action, as exemplified by the
patterns of transcription of the sex-dependent steroid hydroxylase
P450 genes CYP2C11 (male-specific expression) and
CYP2C12 (female-specific expression) (13, 14, 15). This implies
that GH can activate two distinct intracellular signaling pathways via
the plasma membrane-bound GH receptor (GHR) and in a manner that is
dependent on the temporal pattern of plasma GH stimulation.
In the adult male rat, GH pulses stimulate an intracellular signaling
pathway that is dependent on the tyrosine kinase JAK2 and the signaling
molecule and transcriptional activator STAT5b. This pathway involves
the repeated activation of STAT5b by sequential plasma GH pulses, a
process that occurs in male but not female rat liver (16) and is
required for the sexual dimorphism of liver gene expression and of
whole body growth rates (17). A continuous GH-activated nuclear factor,
termed GHNF, which is distinct from the GH-activated STATs and
whose DNA-binding activity is high in female, as compared with male,
liver nuclear extracts, has also been described (18). However, the
intracellular signaling pathway(s) and events that are stimulated in
hepatocytes in response to continuous GH exposure (19) and that may
activate this or other factors or contribute to the suppression of
STAT5b signaling (16) are poorly understood.
In vivo studies in hypophysectomized rats have
established that the time interval between plasma GH pulses is a
critical factor in the ability of liver cells to reset their signaling
systems to respond to subsequent GH pulses. Physiological GH dose
replacement at six pulses per 24-h period leads to restoration of a
normal male pattern of liver gene expression, whereas no such
restoration occurs when the GH pulse interval is shortened slightly, by
increasing the GH frequency to seven pulses/24 h (20). Continuous GH
exposure both suppresses the male transcriptional response and at the
same time induces (or activates) the GH-activated nuclear factor GHNF
in association with the transcriptional activation of female-expressed
liver genes (14, 18). Suppression of the male transcriptional response
in continuous GH-treated male rats (14, 15) appears to be due to the
loss of activated, nuclear STAT5b, which first becomes evident within
several hours of continuous GH treatment in vivo, but which
requires several days to be complete (16). The cellular mechanism
underlying this down-regulation of the STAT5b pathway in liver cells
exposed to GH continuously is presently unknown and is the subject of
the present study, carried out in the GH-responsive rat liver cell line
CWSV-1 (21, 22, 23). These cells respond to a pulse of GH with a rapid
increase in the cellular level of tyrosine-phosphorylated, activated
STAT5b molecules (
10 min), which subsequently undergo phosphorylation
on serine or threonine, dimerization, nuclear translocation (24), and
DNA binding (22). Evidence is provided in support of a model where
continuous GH exposure down-regulates GH signaling to STAT5b through
the action of phosphotyrosine phosphatase(s) that deactivate both
STAT5b and the GHR-JAK2 signaling complex. The latter step is proposed
to lead to enhanced degradation of the receptor-kinase complex,
necessitating ongoing protein synthesis and the continual generation of
fresh GHR-JAK2 complexes to maintain a low level of activated STAT5b in
continuous GH-treated liver cells.
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RESULTS
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Continuous GH Exposure Stimulates Low Levels of Activated
STAT5b
GH given as a continuous infusion to hypophysectomized rats
suppresses STAT5b activation over a period of hours to days (16). To
determine whether the cultured liver cell model CWSV-1 also responds to
continuous GH with a decrease in STAT5b activation, cells were treated
with GH for periods up to 7 days. Electrophoretic mobility shift assay
(EMSA) revealed that activated STAT5b levels fall from their peak level
at approximately 45 min to about 1020% of the peak level within
about 2 h of continuous GH treatment (Fig. 1A
). This decrease of STAT5b EMSA
activity to a lower steady-state level in continuous GH-treated cells
is termed phase 2 STAT5b signaling to distinguish it from the initial
GH pulse activation event, which is termed phase 1 STAT5b signaling
(23). The low STAT5b EMSA activity stimulated by continuous GH could be
maintained for at least 7 days at both 500 ng/ml GH (Fig. 1A
) and at 50
ng/ml (cf., female plasma GH level of
2040 ng/ml GH). This
low-level STAT5b EMSA activity was reduced by about half at a
continuous GH concentration of 5 ng/ml (data not shown), in accord with
the reported dissociation constant (Kd) of GHR for GH of
about 2 ng/ml (25).

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Figure 1. Down-Regulation of STAT5b Signaling in Phase 2,
Continuous GH-Treated CWSV-1 Cells
Panel A, CWSV-1 cells were treated continuously with 500 ng/mL GH for
the indicated times. Culture media was changed and fresh GH was added
every other day (i.e. on days 2, 4, and 6, in the case
of the 7-day continuous GH samples, and on day 2 in the case of the
4-day GH sample). In the case of cells treated with GH for 7 days,
cells were trypsinized and then passaged on day 4 at a 1:4 split into
media containing fresh GH. Cell extracts were prepared and then
analyzed by EMSA using the STAT5-binding ß-casein probe. Shown is a
graph displaying EMSA data combined from several experiments. In each
case, the STAT5b EMSA signal intensity is graphed as a percentage of
the signal obtained 45 min after initiation of a GH pulse, which
corresponds to the maximum intensity signal during GH pulse-induced
phase 1 signaling. The basal level of phase 2 STAT5b EMSA activity seen
in these samples corresponds to approximately 20% of the maximal
STAT5b signal at 45 min. The basal level was similar in experiments
carried out at 50 ng/ml GH, while at 5 ng/ml GH the STAT5b signal was
reduced to about 10%. In experiments carried out with later passages
of CWSV-1 cells, continuous GH at 50 or 500 ng/ml induced a STAT5b
response that corresponded to about 10% of the 45-min peak (cf. Fig. 5D and STAT5b Western blot shown in Fig. 1B ). Inset,
EMSA of extracts from cells treated with GH for the times indicated. In
data not shown, supershift analysis using STAT5 form-specific anti-COOH terminal
region antibodies (Santa Cruz Biotechnology; STAT5a antibody sc-1081
and STAT5b antibody sc-835) confirmed that the GH-induced gel shift
complex contains STAT5b but not other GH-activatable liver STATs (22 31 ). Panel B, Anti-STAT5b Western blot of CWSV-1 extracts, comparing
relative amounts and STAT5b SDS-PAGE banding pattern in response to an
initial GH pulse (lane 9) and longer-term, continuous GH phase 2
signaling (lanes 1012). Also shown is the same blot reprobed with
anti-JAK2 antibody (lanes 1317). The multiple STAT5b bands correspond
to a pair of (unresolved) unphosphorylated bands (bands 0),
tyrosine-phosphorylated STAT5b (band 1), which comigrates in the
Western blot shown with the constitutively serine (or
threonine)-phosphorylated STAT5b (band 1a), and tyrosine +
serine/threonine-diphosphorylated STAT5b (band 2), as characterized and
described in detail elsewhere (22 ). STAT5b bands 1 and 2 are both
active when assayed by EMSA using the ß-casein DNA probe, as shown by
phosphatase treatment (31 ), and by using H7 to inhibit the
serine/threonine phosphorylation associated with conversion of band 1
to band 2 (22 ). Band 2 is absent from lane 8 and is just barely visible
in lanes 1012. Panel C, Anti-STAT5b Western blot of extracts from
CWSV-1 treated with 500 ng/ml GH for times ranging up to 8 h.
Lanes 2125 show immunoprecipitation of cell extract (150 µg) using
agarose-conjugated antiphosphotyrosine monoclonal antibody PY99. Lanes
1820, direct STAT5b Western blot analysis of samples shown in lanes
2123 (20 µg cell extract/lane).
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Continuous GH-treated CWSV-1 cell extracts were analyzed by Western
blotting to determine whether continuous GH exposure decreases the
cellular level of STAT5b or JAK2 protein. No significant change in the
abundance of either protein was seen after GH treatment (Fig. 1B
, lanes
1012 vs. 8; lanes 1517 vs. 13). Moreover, the
low-level STAT5b EMSA activity during phase 2 is associated with a
correspondingly low level of tyrosine + serine (threonine)
diphosphorylated STAT5b, detected on Western blots of continuous
GH-treated cell extracts (Fig. 1B
, STAT5b band 2, lanes 1012
vs. 9). Antiphosphotyrosine immunoprecipitation using
monoclonal antibody PY99 followed by STAT5b Western blotting confirmed
the presence of tyrosine-phosphorylated STAT5b (band 1) and tyrosine +
serine/threonine diphosphorylated STAT5b (band 2) in the continuous
GH-treated cells at relative ratios similar to those induced by a pulse
of GH (Fig. 1C
, lanes 2325 vs. 22).
The Low Level of STAT5b EMSA Activity during Phase 2 Is Dependent
on Continuous Signaling by JAK2 Kinase
The low level of STAT5b EMSA activity seen in
CWSV-1 cells exposed to GH continuously could reflect 1) ongoing
signaling by JAK2 kinase, but at a lower level than after an initial GH
pulse, or 2) residual activated STAT5b molecules that remain from the
phase 1 pulse but are not deactivated by phosphotyrosine phosphatases
or degraded. To distinguish between these two possibilities, we
examined the effects of GH removal and of tyrosine kinase inhibition on
STAT5b EMSA activity during phase 2. In the first experiment, CWSV-1
cells were incubated with GH for 24 h (Fig. 2A
, lanes 15), after which GH was
removed from the culture medium. GH removal led to a total loss of
STAT5b EMSA activity within 30 min (lane 6 vs. 5). Thus, GH
must be continually present to maintain a phase 2 STAT5b EMSA signal.
Readdition of GH after a GH-free interval of 30 min, 1 h, or
3 h led to reactivation of STAT5b, albeit only to the level seen
during phase 2 signaling (lanes 7, 9, and 11 vs. lane 5).
Thus, during phase 2, CWSV-1 cells respond to GH with weak activation
of STAT5b compared with that seen when cells are treated with a GH
pulse. This suggests that the cells responsiveness to GH addition has
been intrinsically altered by continuous GH exposure.

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Figure 2. Continued GHR-JAK2 Signaling Is Required to
Maintain Low-Level STAT5b EMSA Activity during Phase 2
Panel A, CWSV-1 cells were treated with 500 ng/ml GH for times ranging
from 45 min up to 24 h (lanes 25). Twenty four hours after GH
exposure, the cells were washed with PBS, fresh medium without GH was
added, and the cells were incubated for an additional 30 min, 1 h,
or 3 h (lanes 6, 8, and 10), as shown in the step diagram
below panel A. GH was added after each of these time intervals
(X hr) and EMSA activity was assayed 45 min later (lanes 7, 9, and 11).
Cell extracts were prepared and analyzed by EMSA. Note total loss of
STAT5b EMSA signal upon GH removal (lanes 6, 8, and 10). Panel B, Cells
were treated with 500 ng/ml GH for 3 h (lane 13), at which point
500 µM genistein or 2 µM staurosporin
was added and the cells incubated for an additional 15 min with GH +
the indicated kinase inhibitor (lanes 14 and 15). Cell extracts were
analyzed by EMSA. The STAT5b EMSA signal shown in lane 13 is already
low (since it corresponds to a phase 2 signal), which in the
reproduction shown may make it difficult to compare with the signal in
lanes 14 and 15, which is undetectable. Panel C, JAK2
immunoprecipitation followed by Western blot detection with
antiphosphotyrosine antibody 4G10 of samples from the same experiment
shown in Fig. 1C . JAK2 is seen to be tyrosine phosphorylated in
continuous GH-treated cells, albeit at a much lower level than after
the initial GH pulse (lanes 1820 vs. 17) (upper
gel). Also shown is a reprobing of the same gel with anti-JAK2
(lower gel). Small differences in JAK2
immunoprecipitation efficiency and in JAK2 protein mobility between
lanes are apparent in this experiment.
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In a separate experiment, CWSV-1 cells were incubated with GH for
3 h, long enough to establish phase 2 signaling, and then were
treated with either the tyrosine kinase inhibitor genistein or the
general kinase inhibitor staurosporin. EMSA of STAT5b activity
demonstrated that the phase 2 STAT5b signal was lost within 15 min
(Fig. 2B
, lanes 14 and 15 vs. 13). We conclude that the low
STAT5b EMSA activity in continuous GH-treated cells is dependent on
continued signaling via a GH-activated tyrosine kinase. Since genistein
and staurosporin both cause a complete loss of STAT5b EMSA activity
within 15 min, the lifespan of an activated STAT5b molecule during
phase 2 may be shorter than during phase 1, which we have estimated to
be 2530 min long (i.e. three decay half-lives at
t1/2 = 8.8 min) (23).
Immunoprecipitation of JAK2 tyrosine kinase, followed by
antiphosphotyrosine 4G10 Western blotting verified that continuous GH
activates (phosphorylates) JAK2, albeit at a much reduced level
compared with that achieved after a GH pulse (Fig. 2C
, lanes 1820
vs. 17). Thus, JAK2 is likely to be the tyrosine kinase
involved in the ongoing phase 2 GH signaling to STAT5b. The maintenance
of a low level of phosphorylated JAK2 for at least 8 h in
continuous GH-treated cells (Fig. 2C
, lane 20) contrasts with the
transient phosphorylation of JAK2 after a GH pulse (23, 24) and is
consistent with our conclusion that STAT5b EMSA activity is maintained
at a low level during GH phase 2 as a consequence of an ongoing, low
level signaling by JAK2 kinase to STAT5b.
Phase 2 Is Characterized by Increased Phosphotyrosine Phosphatase
Action
We next investigated whether the low level of STAT5b EMSA activity
in continuous GH-treated cells is the result of elevated
phosphotyrosine phosphatase activity associated with dephosphorylation,
and thus deactivation, of either GHR, JAK2, or STAT5b. To test this
hypothesis, continuous GH-treated cells were incubated with the
phosphotyrosine phosphatase inhibitor pervanadate. Figure 3A
shows that pervanadate dramatically
increased STAT5b EMSA activity within 30 min, fully restoring it to the
maximum level seen after a GH pulse (i.e. during phase 1
signaling). By contrast, pervanadate had no effect on the rate of
STAT5b activation at low GH concentrations or on the maximal STAT5b
EMSA signal induced by a pulse of GH (22). In addition, pervanadate
alone added to GH-naive CWSV-1 induced only a marginal activation of
STAT5b (cf. Fig. 8, lane 1, of Ref. 22). The reversal by pervanadate of
the down-regulated STAT5b signal seen in continuous GH-treated cells
therefore suggests that phase 2 signaling is associated with increased
phosphotyrosine phosphatase action on the GHR/JAK2/STAT5b pathway,
leading to a significant decrease (8090%) in the steady-state level
of activated STAT5b.

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Figure 3. Influence of Pervanadate and Genistein on Phase 2
GH Signaling
CWSV-1 cells, incubated with 500 ng/ml GH for 3 h, were treated
with 60 µM pervanadate either in the absence or presence
of 500 µM genistein. Extracts prepared at the indicated
time points after pervanadate addition were analyzed by EMSA. Panel A,
PhosphorImager (Molecular Dynamics, Sunnyvale, CA) quantitation
of STAT5b activities obtained by EMSA. Genistein was added to the
GH-containing culture dishes 5 min before pervanadate, 5 or 15 min
after pervanadate, or not at all, as indicated. Individual plates were
analyzed for STAT5b EMSA activity at times ranging from 5 to 30 min
after pervanadate addition, as shown. Pervanadate only (no gen) and
GH only (no pervanadate or genistein) sets of samples (control)
were also analyzed. GH was present throughout the course of the
experiment. Panel B, Shown are EMSA gels from a set of experiments
similar to the one shown in panel A, except that GH was incubated with
the cells continuously for 3 h, and then was removed from the
culture medium 5 min after pervanadate addition (pervan at 2:55;
panel B2). In panel B3, pervanadate was added as in panel B2 and
genistein was added 5 min later, at the time when GH was withdrawn. In
the absence of GH, the STAT5b EMSA signal is lost within 15 min (panel
B1, lanes 3 vs. 2) (cf. Fig. 2 ), whereas the STAT5b
signal increases approximately 5-fold in intensity in the presence of
pervanadate (panel B2) or about 2- to 2.5-fold in the presence of
genistein + pervanadate (panel B3).
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The stimulatory effect of pervanadate on STAT5b activity seen in these
experiments could be due to the inhibition by pervanadate of the
phosphatase(s) that dephosphorylate JAK2 and/or GHR, leading to
sustained activation of the GHR-JAK2 signaling complex. Alternatively,
it could be due to a more direct inhibition of STAT5b
dephosphorylation. To determine the relative contributions of these two
mechanisms, continuous GH-treated cells were incubated with pervanadate
in combination with the JAK2 tyrosine kinase inhibitor genistein. Since
the rates of cellular uptake of these two inhibitors might differ,
genistein was added to the cells either 5 min before or 5 min after the
addition of pervanadate, as shown in the schematic below Fig. 3A
. In
both cases, genistein reduced the pervanadate-stimulated increase in
activated STAT5b from 5-fold to only 22.5 fold, as seen in the EMSAs
quantitated in Fig. 3A
. Addition of genistein 15 min after pervanadate
(at which time the STAT5b EMSA signal was already increased
3.8-fold
above its low basal level) blocked further increases in the
accumulation of activated STAT5b (Fig. 3A
). Since genistein fully
blocks new tyrosine kinase signaling to STAT5b (22), our finding that
genistein reduced the pervanadate-stimulated increase by about half
(Fig. 3A
) suggests that approximately 50% of the
pervanadate-stimulatory effect results from pervanadate inhibition of
JAK2 or GHR dephosphorylation, and that the balance of the pervanadate
effect is due to its inhibition of STAT5b dephosphorylation. Thus, the
major suppression of STAT5b signaling seen in continuous GH-treated
liver cells reflects an increase in pervanadate-sensitive
phosphotyrosine phosphatase activity toward both GHR-JAK2 and
STAT5b.
Interestingly, if GH was removed from the culture medium 5 min after
pervanadate addition, then the 5-fold increase in STAT5b EMSA signal
was still observed (Fig. 3
, panel B2 vs. B1). Again,
genistein was able to reduce the pervanadate-stimulated increase in
STAT5b activity to only about 2- to 2.5-fold (panel B3). This suggests
that pervanadate is able to effect its dramatic stimulation of phase 2
STAT5b activity, restoring a normal phase 1 GH pulse response, even
without the recruitment by GH of new GHR-JAK2 complexes.
STAT5b Signaling during Phase 2 Differs from Phase 1 Signaling in
its Response to the Serine/Threonine Kinase Inhibitor H7
H7 and other serine kinase inhibitors can prolong STAT5b activity
in CWSV-1 cells after a GH pulse (22, 23). In contrast, H7 did not
prolong phase 2 signaling after removal of the continuous GH stimulus.
Figure 4A
presents an experiment where
CWSV-1 cells were incubated with GH for 3 h, to establish STAT5b
phase 2 signaling, at which time H7 was added. GH was removed from the
culture medium 30 min later. EMSA showed the STAT5b signal was lost
within 30 min of GH removal (Fig. 4A
, lanes 68 vs. lanes
4, 5), just as in the absence of H7 (cf. Fig. 2A
, lane 6). By contrast,
H7 could prolong a phase 1 STAT5b signal at approximately 6065% of
its initial level for a full 1.5 h after termination of a GH pulse
(Fig. 4B
, lanes 1214). Thus, the requirement of an H7-sensitive
serine/threonine kinase activity for termination of GH-induced STAT5b
EMSA activity seen in GH pulse-treated cells (22, 23) is abolished once
phase 2 becomes established.

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Figure 4. Serine/Threonine Kinase Inhibitor H7 Does Not
Prolong Phase 2 Signaling
Panel A, CWSV-1 cells were incubated with GH for 2.5 h, at which
time 200 µM H7 was added to the culture medium. GH was
removed 30 min later, but H7 was retained in the medium. Samples were
taken for EMSA at 3 h, i.e. just before GH
withdrawal (lane 5), and at several times points thereafter (lanes
68), as illustrated in the step diagram. Extracts were
analyzed for STAT5b DNA-binding by EMSA. Panel B, CWSV-1 cells were
incubated with 200 µM H7 30 min before GH addition and
maintained in culture medium after the withdrawal of GH at 1 h.
Extracts taken at the times indicated were analyzed by EMSA. Panel C,
CWSV-1 cells were incubated with GH for 3 h, at which time 200
µM H7 was added to the medium. Samples were taken at
times ranging up to 4 h later and analyzed by EMSA. Note slow
increase in STAT5b activity with prolonged H7 treatment.
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Interestingly, when continuous GH-treated cells were incubated in the
presence of H7 for several hours, the activated STAT5b signal increased
several-fold (Fig. 4C
, lanes 1921 vs. lanes 17 and 18).
This suggests that a serine/threonine kinase-activated mechanism may
contribute to the lower level of STAT5b EMSA activity during phase 2.
Prolonged H7 treatment did not, however, increase STAT5b EMSA activity
to the same extent as did pervanadate (cf. Fig. 3
). This suggests that
this serine/threoine kinase mechanism contributes to down-regulation of
STAT5b activity in a minor way, compared with the effects of
phosphotyrosine phosphatases.
Addition of 20 µM PD98059, a MEK kinase inhibitor
that blocks GH-induced signaling to MAP kinase (26), also did not block
the loss of STAT5b EMSA activity upon GH removal during phase 2, nor
did it inhibit STAT5b activation during phase 2 when added to cells
incubated in the presence of continuous GH (data not shown). Thus, the
MAP kinase pathway, which can be activated by GH (27, 28), does not
contribute to the activation or deactivation of STAT5b during phase 2
GH signaling.
Proteasome Inhibitor MG132 Elevates Activated STAT5b Levels during
Phase 2 Signaling
When CWSV-1 cells were incubated with GH for 3 h to establish
phase 2 signaling, and then were treated with the proteasome inhibitor
MG132, STAT5b EMSA activity increased approximately 2-fold within
1 h, and then slowly declined (Fig. 5A
, lanes 47 vs. 3). This
effect was less dramatic than, but additive with, the
pervanadate-stimulatory effect (panel B, lanes 1122 and panel D).
This suggests that proteasome degradation of one or more factors
required for STAT5b activation occurs during phase 2 GH signaling,
thereby giving a higher steady-state level of STAT5b EMSA activity when
MG132 is added to the cells. Figure 5C
shows, however, that STAT5b
protein (bands 0, 1, 1a, and 2, collectively) and JAK2 protein are not
significantly increased by MG132 treatment (lanes 2730 vs.
lanes 25, 26). Finally, when GH was removed 5 min after MG132 addition,
the activated STAT5b EMSA signal disappeared (data not shown). Since GH
must be continuously present to observe MG132s stimulatory effect,
MG132 most likely exerts its effect on GHR-JAK signaling to STAT5b, and
not at the level of STAT5b dephosphorylation.

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Figure 5. Effect of Proteasome Inhibitor MG132 on STAT5b
Activation
Panel A, CWSV-1 cells were incubated with GH for times up to
3 h (lanes 13), at which time 50 µM MG132 was
added to the culture medium. Extracts were analyzed by EMSA at times
ranging up to 2.5 h after MG132 addition (lanes 47). Shown is an
EMSA of STAT5b activity. Panel B, Shown is the EMSA of an experiment
similar to that in panel A, except that the 3-h continuous GH-treated
cells were treated with MG132 alone (lanes 1114), with 60
µM pervanadate alone (lanes 1518), or with MG132 +
pervanadate (lanes 1922). Note that panel B presents a lighter
exposure than the one shown in panel A. Panel C, Western blot of
samples from the same treatments shown in panel B probed with a mixture
of anti-JAK2 and anti-STAT5b antibodies. The multiple, differentially
phosphorylated STAT5b bands, labeled 02, are identified in the legend
to Fig. 1B . The two unphosphorylated STAT5b forms, bands 0, partially
resolve in this gel. Of these, the lower band is preferentially lost in
the pervanadate-treated samples in association with the conversion of
STAT5b to the active, diphosphorylated band 2 (lanes 3134
vs. duplicate controls shown in lanes 25 and 26). MG132
slightly increases the intensity of band 2, which migrates just below
the lower of the two nonspecific (n.s.) bands, which originate from the
anti-JAK2 antibody. MG132 does not have a significant effect on the
total JAK2 or STAT5b immunoreactivity. Panel D, STAT5b EMSA bands from
panel B were integrated using PhosphoImager and graphed as a percentage
of the STAT5b EMSA activity at 45 min. The error bar on the 45-min
sample point represents the SD of three data points. Note
that the phase 2 level of STAT5b activity in this experiment, and in
the one shown in Fig. 6 , is approximately 10% of the initial GH pulse
signal (vs. 20% in Fig. 1A ). UT, No inhibitors added
at 3 h; VO, addition of pervanadate at 3 h; MG, addition of
MG132 at 3 h (vertical arrow).
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Cycloheximide Eliminates the STAT5b Signal during Phase 2
Cycloheximide inhibition experiments have shown that new protein
synthesis is not required for CWSV-1 cells to respond repeatedly to
sequential, physiological pulses of GH under conditions where STAT5b is
quantitatively converted to its nuclear, tyrosine-phosphorylated form
(22). This implies that STAT5b is recycled from the nucleus back to the
cytosol after termination of a GH pulse, and that the other components
required for phase 1 signaling (GHR and JAK2) are either present in the
cell in excess or are effectively reutilized. In contrast,
cycloheximide treatment of CWSV-1 cells already in phase 2 completely
eliminated STAT5b EMSA activity within 1.5 h (Fig. 6A
, lanes 58 vs. lane 4).
This indicates that at least one essential component of the
GHR/JAK2/STAT5b pathway is labile during phase 2 GH signaling. Western
blot analysis showed, however, that the cycloheximide-sensitive protein
component is neither JAK2 nor STAT5b (Fig. 6C
, lanes 2729
vs. lanes 25 and 26).

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Figure 6. Loss of STAT5b Phase 2 EMSA Activity upon
Cycloheximide Treatment and Reversal by Pervanadate
Panel A, CWSV-1 cells were treated with GH for up to 3 h (lanes
14), at which time 10 µg/ml cycloheximide was added to the culture
medium for an additional 12.5 h (lanes 58). Shown is an EMSA
indicating the loss of STAT5b activity by 1.52 h after cycloheximide
addition (lanes 58 vs. control in lane 4). The strong
initial GH pulse/phase 1 signal is seen in lanes 2 and 3. Panel B, An
experiment similar to that in panel A was performed except that
continuous GH-treated samples were additionally treated with
cycloheximide (lanes 1214), pervanadate (lanes 1518), or
pervanadate + cycloheximide (lanes 1922). Samples were analyzed for
STAT5b activity by EMSA. Panel C, Western blot of samples shown in
panel B probed with a mixture of anti-JAK2 and anti-STAT5b antibodies,
as detailed in the legend to Fig. 5C . The loss of STAT5b activity
with 1.5 h cycloheximide treatment seen in panels A, B, and
D is not accompanied by any decrease in total JAK2 or STAT5b
immunoreactivity. Panel D, EMSA bands shown in panel C were integrated
using a PhosphoImager and graphed as a percentage of the STAT5b
activity at 45 min. The pervanadate-only (VO) and the untreated data
points are from the same samples shown in Fig. 5 .
|
|
Our working hypothesis to explain both the effects of
cycloheximide during phase 2 and the pervanadate-stimulatory response
(Figs. 3
and 5
) is that dephosphorylation of the GHR-JAK2 signaling
complex occurs rapidly during phase 2 signaling and is followed by GHR
internalization and degradation (see Fig. 7
, below). A clear prediction of this
model is that blockage of the initial dephosphorylation step by
pervanadate treatment will eliminate the protein synthesis requirement
for continuous GH signaling to STAT5b. Indeed, while cycloheximide
eliminated the phase 2 STAT5b EMSA signal within 2 h (Fig. 6B
, lanes 1214 vs. 11), it had no significant effect in
pervanadate-treated cells (Fig. 6B
, lanes 1922 vs.
1518). Pervanadate treatment fully restored the maximal STAT5b
activity seen after a GH pulse (Fig. 6D
). This effect of pervanadate is
associated with the formation of activated STAT5b, band 2 (tyrosine +
serine/threonine diphosphorylated STAT5b) (Fig. 6C
, lanes 3032).

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Figure 7. Model for Down-Regulation of GHR-JAK2 Cycle and
STAT5b Activation Loop in Continuous GH-Treated Liver Cells
GH-induced assembly of GHR-JAK2 signaling complexes is proposed to
establish a STAT5b activation loop, whereby multiple STAT5b molecules
become activated by phosphorylation of Tyr699, followed by
serine or threonine phosphorylation (not shown) and nuclear
translocation. Continuous GH exposure is proposed to down-regulate the
overall pathway of STAT5b activation through enhanced phosphotyrosine
dephosphorylation of activated STAT5b (step A) and of activated
GHR-JAK2 signaling complexes (step B) (thick arrows). The
latter step is proposed to lead to GHR internalization and degradation
(step C). This gives rise to the observed requirement for new protein
synthesis, which can be blocked by cycloheximide (step D), and to a
continual need for GH to be present to stimulate assembly of new
GHR-JAK2 signaling complexes (step E).
|
|
 |
DISCUSSION
|
---|
The activation of liver STAT5b in response to plasma GH
pulses in adult male rats is an essential component in the GH
pulse-induced expression of a male pattern of liver gene expression
(16, 17). By contrast, in female rat liver, which is exposed to plasma
GH nearly continuously, STAT5b is activated only at a very low level
(16), an observation that helps to explain the near absence, in female
liver, of GH pulse-activated, STAT5b-inducible gene products. Earlier
studies found that GHR (29, 30), JAK2, and cytoplasmic STAT5b (24, 31)
are expressed in female rat liver at levels similar to (JAK2, STAT5b)
or higher than (GHR) those of males. This suggests that a deficiency in
one of these components does not account for the lack of a robust liver
STAT5b response to GH in the female. The present study investigates the
mechanism that underlies this apparent down-regulation of the STAT5b
signaling pathway in continuous GH-treated liver cells. These studies
were carried out in the GH-responsive rat liver cell line CWSV-1 (21),
which responds to physiological GH pulses with a repeated pattern of
JAK2-catalyzed STAT5b tyrosine phosphorylation, followed by
serine/threonine phosphorylation, nuclear translocation, and then
phosphotyrosine dephosphorylation (22, 23, 24). Continuous GH treatment of
CWSV-1 cells is presently shown to result in a low level of activated
STAT5b (
1020% of the maximal EMSA signal observed after a GH
pulse) that can persist for at least 7 days. This low-level STAT5b EMSA
activity results from ongoing, low-level signaling from JAK2 tyrosine
kinase to STAT5b and is termed phase 2 GH signaling. This signaling
pathway, like that which characterizes phase 1 signaling in response to
a GH pulse, is likely to involve dimerization of GHR by GH and
recruitment of JAK2 to the receptor complex, followed by entry into an
activation loop, in which the GHR-JAK2 complex recruits, activates, and
then releases tyrosine-phosphorylated STAT5b (Fig. 7
). In contrast to
the STAT5b activation loop that characterizes phase 1 GH pulse
signaling (23), however, the GHR-JAK2 complexes formed during phase 2
are proposed to retain their activity and remain in the activation loop
for a much shorter period of time, thereby explaining the apparent
cessation of GHR-JAK2 signaling that results in STAT5b deactivation
within 1530 min of GH removal or tyrosine kinase inhibition (Fig. 2
).
Lower Steady-State Level of Activated STAT5b in Continuous
GH-Treated Cells
The much lower level of STAT5b EMSA activity that is maintained by
continuous GH (phase 2), as compared with that which is induced by
pulsatile GH (phase 1), may be explained by one or more of the
following: 1) one of the factors required for formation of a functional
GHR-JAK2-STAT5b activation loop becomes limiting at the cytoplasmic
face of the plasma membrane, where STAT activation is believed to
occur; 2) increased phosphotyrosine phosphatase activity shortens the
half-life of tyrosine-phosphorylated STAT5b molecules, decreasing the
steady-state pool size of activated STAT5b (Fig. 7
, step A); and 3)
increased phosphotyrosine phosphatase activity shifts the equilibrium
from phosphorylated (active) to dephosphorylated (inactive) GHR-JAK2
complexes (step B). In this latter situation, fewer GHR-JAK2 complexes
would signal to STAT5b at any given point in time, and fewer STAT5b
activation cycles would occur per GHR dimerization event. If
dephosphorylation of the GHR-JAK2 signaling complex leads to its
dissociation and/or internalization and degradation (step C), this
could explain several of the requirements and characteristics of phase
2 signaling identified in this study. These include a need for: 1)
ongoing protein synthesis, necessary to replenish degraded components
(step D), and 2) a continuous GH presence, necessary to dimerize and
activate fresh GHR-JAK2 complexes (step E). More complicated models can
be envisaged. These include 1) continuous GH induction of a
signal-inhibitory protein belonging to the recently described SOCS/CIS
family (32, 33, 34) (see below) and 2) continuous GH activation of a SIRP
family protein (35, 36) which recruits a phosphotyrosine phosphatase
such as SHP2 to the GHR-JAK2 complex.
Cell surface GHR levels are more abundant in liver cells exposed to GH
continuously (female rat liver) as compared with intermittently (male
rat liver) (29, 30). This sex difference in GHR levels reflects the
stimulation of GHR expression by continuous GH in females, a response
also seen in cultured CWSV-1 cells (21), coupled with the GH
pulse-induced internalization of cell surface GHR that occurs in males
(37, 38). Thus, there appears to be an abundance, rather than a
shortage, of cell surface GHR on CWSV-1 cells treated with GH
continuously compared with cells exposed to GH pulses. The other two
protein components, JAK2 and STAT5b, are also present in CWSV-1 cells
during phase 2 GH signaling at a level similar to untreated cells or
cells in phase 1 signaling (Fig. 1B
), so these proteins are also not
likely to be limiting in continuous GH-treated cells. However, GHR
(39), JAK2 (24, 40), and STAT5b can also be found in intracellular
compartments, such as the nucleus, so that Western blot measurements of
total cellular levels of these components might not necessarily be
indicative of their functional availability for phase 2/continuous GH
signaling to STAT5b. For example, GHR-JAK2 signaling complex components
may become sequestered or otherwise inhibited from forming a functional
complex after continuous GH exposure. Further studies of the effects of
continuous GH on the intracellular localization of each of these
components will be required to address this point.
One potential inhibitory mechanism for STAT5b activity in continuous
GH-treated cells, noted above, could involve the cytokine-inducible
suppressor CIS. This STAT5-inducible negative feedback regulator binds
to the tyrosine- phosphorylated erythropoietin and interleukin 3
receptors to inhibit STAT5 phosphorylation and downstream
STAT5-dependent transcriptional responses (32, 33), perhaps by a
mechanism that involves enhanced receptor degradation (41). Recent
studies identify a family of CIS-related proteins, also termed SOCS,
JAB, or SSI proteins, which are rapidly transcribed in a variety of
cell types in response to cytokines and growth factors, and which
function as negative regulators of cytokine signaling, probably by a
range of mechanisms, including binding to, and inhibition of JAK
kinases as well as cytokine/growth factor receptors (42, 43, 44, 45).
Conceivably, one or more of these cytokine signaling-inhibitory
proteins could be induced by continuous GH and contribute to the
observed down-regulation of the STAT5b activation pathway in female rat
liver [cf. recent report that GH can induce SOCS3 mRNA in mouse liver
(34)]. Since SOCS/CIS protein expression is transcriptionally induced
by STATs, and given that the transcriptional activity of STATs is
modulated by serine phosphorylation (46, 47), the increase in STAT5b
EMSA activity that is observed upon longer-term H7 treatment (Fig. 4C
)
could conceivably reflect an inhibition of GH-induced CIS/SOCS
transcription.
Experiments using the phosphotyrosine phosphatase inhibitor pervanadate
and the JAK2 kinase inhibitor genistein indicated that enhanced
phosphotyrosine phosphatase activity toward both JAK2 and STAT5b makes
an important contribution to the approximately 8090% decrease in
STAT5b activation seen when CWSV-1 cells are treated with GH
continuously. By contrast, phosphotyrosine phosphatase activity appears
to be low during the first 20 min of a GH pulse, as judged from the
absence of an effect of the inhibitor pervanadate on the net rate of
accumulation of tyrosine-phosphorylated STAT5b in cells treated with
low concentrations of GH (22). Distinct phosphotyrosine phosphatases
are probably responsible for dephosphorylation of STAT5b and
dephosphorylation of the GHR-JAK2 complex, since these deactivations
occur during distinct periods of time. STAT5b dephosphorylation occurs
at a steady rate from 2090 min after GH pulse stimulation, while
dephosphorylation of JAK2 only begins after about 45 min (23).
Moreover, STAT5b, but probably not GHR-JAK2, may be dephosphorylated
while still in the nucleus (P. A. Ram and D. J. Waxman,
unpublished experiments). The SH2 domain-containing phosphotyrosine
phosphatase SHP-1 is rapidly activated approximately 3- to 4-fold by a
pulse of GH and can bind to GH-activated STAT5b (24) and to JAK2 kinase
(48), suggesting that it could be involved in one of these
dephosphorylation events. SHP-1 translocates from the cytosol to the
nucleus in response to a pulse of GH, suggesting that STAT5b may be
directly dephosphorylated in the nucleus by SHP-1 to terminate a phase
1 GH signal (24); however, further studies are required to establish
whether SHP-1 plays a direct catalytic role in STAT5b
dephosphorylation, either after a GH pulse or in continuous GH-treated
cells.
GH must be present continuously to sustain the low-level signaling to
STAT5b during phase 2 (Fig. 2
), suggesting that there is an ongoing
requirement to recruit and activate fresh GHR-JAK2 complexes.
Nevertheless, activation of STAT5b could proceed even in the absence of
GH once pervanadate was added to the cells (Fig. 3B
). This suggests, as
a working hypothesis, that exit of the GHR-JAK2 complex from the
STAT5b activation loop requires phosphotyrosine phosphatase action
(Fig. 7
, step B). According to this proposal, when phosphatase activity
is inhibited by pervanadate, this loop continues to activate STAT5b
molecules unabated, even if GH-stimulated entry of new GHR-JAK2
complexes ceases. Our proposal that GHR-JAK2 dephosphorylation is an
obligatory first step along the pathway leading to receptor degradation
(step C) is further supported by the observation that the need for
ongoing protein synthesis to sustain a phase 2 STAT5b EMSA signal (Fig. 6A
) (cf. Fig. 7
, step D) is eliminated in pervanadate-treated cells
(Figs. 6B
, 6C
). This proposal is also consistent with the finding that
GHR molecules that undergo GH-induced ubiquitination, which has been
linked to receptor internalization (49), are not tyrosine
phosphorylated (50).
Destruction of the GHR-JAK2 Signaling Complex
Treatment of CWSV-1 cells with a pulse of GH is proposed to induce
a STAT5b activation loop, where each activated GHR-JAK2 complex can
sequentially recruit, phosphorylate, and release multiple STAT5b
molecules (23). If JAK2 becomes dephosphorylated and deactivated, then
its intrinsic autophosphorylation activity might, perhaps, return it
directly to the activation loop. Similarly, if GHR were to be
deactivated by dephosphorylation of its cytoplasmic
phosphotyrosine-binding sites for STAT5b (51, 52, 53), then so long as the
receptor remains in a dimeric complex associated with JAK2, JAK2 may
rephosphorylate and thereby reactivate the receptors STAT5b-binding
sites (Fig. 7
, upper arrow of GHR-JAK2 cycle). A high
phosphotyrosine phosphatase level might therefore be expected to
inhibit, but not totally eliminate, reentry of GHR-JAK2 complexes into
the STAT5b activation loop. Our finding that the low steady-state level
of STAT5b EMSA activity during phase 2 GH signaling can in large part
be ascribed to elevated phosphotyrosine phosphatase activity is
consistent with this model. However, the situation is likely to be more
complicated, as indicated by our finding that phase 2 GH signaling to
STAT5b requires the constant presence of GH, that it depends on new
protein synthesis, and that it can proceed at an approximately 2-fold
higher level when proteasome degradation is blocked. Together, these
observations suggest that, in addition to the down-regulatory effects
of phosphotyrosine phosphatase(s), the GHR-JAK2 complex is actively
disassembled or degraded in continuous GH-treated cells. This
degradation likely proceeds via ubiquitination leading to receptor
internalization and degradation (49). Dissociation leading to
degradation of the inactivated GHR-JAK2 complex may rapidly follow its
dephosphorylation, as proposed above, since when pervanadate was added
to CWSV-1 cells just 30 sec after GH removal, the dramatic increase in
activated STAT5b levels in response to pervanadate (Fig. 3A
) was not
observed (data not shown).
The protein synthesis inhibitor cycloheximide eliminated the
continuous GH-activated STAT5b EMSA signal within 90 min (Fig. 6
).
Since neither STAT5b protein nor JAK2 is depleted from the cells under
these circumstances (Fig. 6C
), GHR may be the component that is
down-regulated and degraded during phase 2, thus explaining the
requirement for continued protein synthesis to maintain signaling to
STAT5b (Fig. 7
, step D). The inhibitory effect of cycloheximide on GH
signaling to STAT5b during phase 2 contrasts with its protective
effect on signaling to STAT5b at the conclusion of phase 1 (23), which
apparently blocks transition of the cells from GH phase 1 to phase 2
signaling. Furthermore, the absence of a protein synthesis requirement
for reactivation of STAT5b by repeated GH pulses given to CWSV-1 cells
(22) indicates that all of the components of the receptor-kinase
activation pathway are stable molecules during phase 1, or that they
are present in sufficient excess, such that they are not depleted by
just two GH pulses. By contrast, the GHR-JAK2 complex appears to become
more labile in cells treated with GH continuously than in GH
pulse-treated cells, thus contributing to the down-regulation of
JAK2-STAT5b signaling, which characterizes female rat liver.
The serine/threonine kinase inhibitor H7 prolongs the activated
STAT5b signal induced by a GH pulse (22, 23) but did not prolong the
activated STAT5b signal in continuous GH-treated CWSV-1 cells once GH
was removed (Fig. 4
). In the case of a GH pulse, H7 apparently can both
prevent the inhibition of new GHR-JAK2 complex assembly and sustain
JAK2 signaling to STAT5b. Neither of these effects is seen when H7 is
added to CWSV-1 cells at the conclusion of a GH pulse (23). Our finding
that H7 has no immediate effect when added to CWSV-1 cells during phase
2 continuous GH signaling (Fig. 4A
) is consistent with the suggestion
that both H7-sensitive inhibitory mechanisms are initiated early after
exposure of the cells to GH. For H7 to have an effect when added to
cells after the initial GH treatment, it needs to be incubated with the
cells for times longer than the half-lives of any inhibitory
molecule(s) that may have already been induced by the initial GH
treatment. Indeed, with prolonged H7 incubation, an elevation of
activated STAT5b levels was observed (Fig. 4C
). This effect of
prolonged H7 treatment could occur at the level of the phosphatase
responsible for the low level STAT5b signal, e.g. by
inhibiting its ongoing transcription, or perhaps it may occur via H7s
effects on the assembly of new GHR-JAK2 complexes. Further study will
be required to address this and other questions regarding the
mechanisms whereby continuous GH down-regulates GHR-JAK2 signaling via
the STAT5b pathway.
 |
MATERIALS AND METHODS
|
---|
CWSV-1 Cell Culture and GH Treatment
Culture of CWSV-1 cells, treatment with GH, alone or in
combination with inhibitors, and preparation of cell extracts were
performed using the general methods described elsewhere (22). For GH
and/or inhibitor treatments, cells were incubated in 1.5 ml of fresh
medium per 60-mm dish on the day of the experiment. Rat GH (dissolved
in PBS containing 0.1% BSA and kept on ice until use) and inhibitors
were added in volumes of 15 µl. Rat GH was a hormonally pure
preparation obtained from Dr. A. F. Parlow, National Hormone and
Pituitary Program, NIDDK (rGH-B-14-SIAFP, BIO), and was added to the
cells to give a final concentration of 500 ng/ml unless specified
otherwise. GH was removed from the cells by aspiration of the medium
(1.5 ml), after which the cells were washed with PBS (3 ml) and then
incubated with fresh medium (1.5 ml) in the absence of GH.
General Experimental Design
GH was added to CWSV-1 cell culture medium and the dishes were
typically incubated for 3 h, sufficient time to achieve the low
level phase 2' STAT5b signaling (see Results). At that
time, inhibitors were added to the culture medium as detailed in the
figure legends. Except where indicated, GH was maintained in the
culture medium during the entire course of the experiment. Data points
shown in each experimental set represent separate culture dishes that
had been treated identically up to the point where the cells were
harvested and cell extracts were prepared. The experimental design for
each study is illustrated by a step diagram, where large steps
represent addition or removal of GH, and small steps represent addition
or removal of inhibitors. Times at which individual cell culture dishes
were harvested for analysis of STAT5b activity by EMSA are represented
in the step diagram by vertical arrows. Time intervals shown
in the EMSA and Western blot data are related to the time of GH
addition to the culture medium, or to the time of inhibitor addition,
as noted in each figure. For example, in the EMSA gel shown in Fig. 2B
, lanes marked 3h and 15' (lanes 2 and 3) correspond to samples taken
3 h after the initiation of GH treatment (lane 2) and 3 h
after GH treatment followed by a 15 min further incubation with GH +
genistein (lane 3). Data shown are representative of results obtained
in at least three independent experiments.
Inhibitors
Inhibitors were obtained from Sigma Chemical Co.( St. Louis,
MO), except for MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal), which
was from Peptides International (Louisville, KY). Inhibitors were
prepared as 100x stock solutions in dimethylsulfoxide (genistein,
staurosporin H7, MG132) or in water (cycloheximide, pervanadate) and
kept frozen in aliquots at -20 C. Final concentrations were: genistein
(500 µM), pervanadate (60 µM), H7 (200
µM), cycloheximide (10 µg/ml), and MG132 (50
µM). Where used, final dimethylsulfoxide concentrations
were generally 1% (vol/vol), which in control experiments not
presented did not alter the intensity of STAT5b EMSA activity in
response to a GH pulse or after continuous GH exposure (phase 2 STAT5b
signal). Pervanadate stock solution was prepared by incubating equal
volumes of 12 mM sodium vanadate (freshly dissolved in
water) and 12 mM hydrogen peroxide at room temperature for
20 min before use.
Other Methods
Biochemical analyses, including EMSA, Western blotting, and
immunoprecipitation, were carried out as previously described (22).
Antiphosphotyrosine monoclonal antibody PY99 conjugated to agarose
beads, used for immunoprecipitation, and an anti-STAT5b-specific
antibody (sc-835) used for Western blotting were obtained from Santa
Cruz Biotechnology (Santa Cruz, CA). Antiphosphotyrosine antibody 4G10
(Upstate Biotechnology, Inc., Lake Placid, NY), and anti-JAK2 antibody
used for Western blotting (QCB, Inc., Hopkinton, MA) were from the
indicated sources. Immunoprecipitation of JAK2 was carried out using
antibody generously provided by Dr. C. Carter-Su (University of
Michigan).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. David J. Waxman, Department of Biology, Boston University, 5 Cummington Street, Boston, Massachusetts 02215. email: djw@bio.bu.edu
Supported in part by NIH Grant DK-33765 (to D.J.W.).
Received for publication September 11, 1998.
Revision received November 3, 1998.
Accepted for publication November 4, 1998.
 |
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