Division of Cell and Molecular Biology Department of Biology Boston University Boston, Massachusetts 02215
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ABSTRACT |
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INTRODUCTION |
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One unique aspect of GH action that has emerged from rodent model
studies is the responsiveness of target tissues to GHs sexually
differentiated plasma profile. In adult male rats, GH is secreted by
the pituitary in an intermittent manner, to give pulses of GH in plasma
that peak at 200 ng/ml every 3.54 h, whereas in females GH is
secreted more frequently, resulting in the continuous presence of GH in
circulation at
2030 ng/ml (15). Pulsatile GH is more effective
than continuous GH in promoting weight gain associated with long bone
growth (16, 17, 18); however, the underlying mechanisms by which the
temporal plasma profile of GH regulates this important physiological
response are not well understood. Liver metabolic function, in
particular cytochrome P450 (CYP)-catalyzed steroid and foreign chemical
metabolism, also exhibits sexual dimorphism in response to the sexually
dimorphic plasma GH profiles (19, 20). P450 enzymes 2C11 and 2C12 are
steroid hydroxylases that are exclusively expressed in male and female
rat liver, respectively, and have served as prototypic examples of
sexually dimorphic, GH plasma pattern-regulated liver gene products
(21). In vivo models have established that
CYP2C11 gene transcription can be induced in
hypophysectomized (GH-depleted) rats given GH in a pulsatile manner
that mimics the intact male GH profile, whereas the same gene is
suppressed and CYP2C12 is activated when GH is given in a
continuous, female-like pattern (22, 23).
Pulsatile GH, but not continuous plasma GH, has recently been shown to activate STAT5 in rat liver, suggesting that this STAT may serve as a direct transcriptional activator of male-specific, GH pulse-activated genes such as CYP2C11 (12). Indeed, STAT5 contributes to GH regulation of CYP3A10, which is a male-specific, GH-dependent steroid 6ß-hydroxylase expressed in hamster liver (24). GH also activates two other STATs in liver tissue, STAT1 and STAT3 (11, 13, 25) but, in contrast to STAT5, the activation of these other signaling molecules by GH is largely independent of its temporal plasma profile (13). GH-induced tyrosine phosphorylation of each STAT is followed by phosphorylation of the STAT on serine or threonine residues (13). Tyrosine phosphorylation is necessary for STAT5 DNA-binding activity, but the role of serine/threonine phosphorylation is less well understood. While the hypophysectomized rat liver model used in these earlier studies has proven very useful for elucidation of these responses of STATs in an intact liver and in a physiological context, mechanistic questions relating to the GH pattern-dependence of STAT5 activation, deactivation of STAT5 after termination of a GH pulse, and the apparently slow desensitization of the GH receptor/JAK2 kinase/STAT5 pathway in liver cells exposed to GH continuously (12) have been more difficult to address.
Recently, an immortalized rat hepatocyte-derived cell line, CWSV-1, was
described as responding to continuous GH treatment by induction of
insulin-like growth factor I, steroid 5-reductase, and GH receptor
mRNA (26). In the present study, CWSV-1 cells are characterized with
respect to their expression of a functional JAK/STAT pathway that is
shown to respond to GH rapidly and in a manner similar to intact liver
in vivo. We describe a GH pulse-responsive STAT5 protein,
STAT5b, that undergoes both tyrosine- and serine/threonine
phosphorylation, and we provide evidence for a role of a
pervanadate-sensitive tyrosine phosphatase in the rapid deactivation of
STAT5b after termination at a GH pulse. STAT5b deactivation is also
shown to be facilitated by a serine/threonine phosphorylation reaction,
thereby resetting the JAK/STAT signaling apparatus in a manner that
enables STAT5b to respond to a subsequent GH pulse-induced activation
event.
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RESULTS |
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Analysis of CWSV-1 cell extracts on 510% gradient SDS gels, followed
by anti-STAT5 Western blotting, revealed that STAT5 band 0 was
comprised of two distinct components, both of which were
substantially depleted in cells treated with high GH concentrations
(500 ng/ml) and were converted to the more slowly migrating band 2. The
intensities and relative levels of these two closely spaced
constitutive STAT5 bands were not affected by treatment with several
phosphoprotein phosphatases, including calf intestinal phosphatase, a
general phosphatase (see Fig. 3A, below, and data not shown). The
relationship between these two constitutive STAT5 bands is not known;
however, both correspond to STAT5b isoforms, insofar as they both react
with the STAT5b-specific antibody used in these experiments (see
Materials and Methods). Neither band reacted with a
STAT5a-specific antibody, which revealed a single band in both
untreated and GH-treated CWSV-1 cells that migrated close to the
position of STAT5b band 2 (data not shown).
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Further support for these STAT5b band identifications is provided
by an analysis of the STAT5b banding patterns on a high resolution gel,
which clearly separates band 1 and band 1a (Fig. 3B). GH treatment
initially yielded the tyrosine-phosphorylated band 1, which was
subsequently converted to the tyrosine +
serine/threonine-phosphorylated band 2 (lanes 13). In cells
pretreated with H7, the basal level of band 1a was lowered (lane 4),
consistent with our identification of this protein as a serine- or
threonine-phosphorylated STAT5b form. GH stimulation of the
H7-pretreated cells resulted in the accumulation of STAT5b band 1 at 10
min (lane 5), followed by its slow conversion to STAT5b band 2
(lane 6). That the conversion of band 1 to band 2 is slowed
down, but is not fully inhibited, by H7 suggests two possibilities: 1)
the serine/threonine kinase active on STAT5b band 1 is only partially
inhibited by H7; 2) two distinct kinases can carry out the
serine/threonine phosphorylation reaction, but only one of these
kinases is inhibited by H7 under the conditions of these experiments.
The serine/threonine-phosphorylated band 1a also appeared with longer
time GH treatment (lane 6). This may arise by phosphotyrosine
dephosphorylation of band 2 (c.f., phosphotyrosine
dephosphorylation preceding phosphoserine/threonine dephosphorylation,
below). Finally, both STAT5b phosphorylation reactions were blocked by
the tyrosine kinase inhibitor genistein (lanes 79), supporting our
conclusion that GH-induced STAT5b tyrosine phosphorylation precedes
serine/threonine phosphorylation.
Indistinguishable STAT5b-DNA EMSA mobilities were exhibited by
untreated and H7-treated CWSV-1 cell extracts (Fig. 2A). This contrasts
with the appearance of a new, lower mobility complex (EMSA complex II)
when activated STAT5 present in liver nuclear extracts was converted to
its monophosphorylated (phosphotyrosine) form by in vitro
treatment with PP2A (13). In agreement with this observation,
STAT5b-DNA complex II was not formed after PP2A treatment of GH
pulse-activated CWSV-1 extracts but was formed when GH pulse-activated
liver nuclear extracts were treated and analyzed in parallel (data not
shown). The absence of complex II in the case of CWSV-1 cells may
reflect a subtle difference between the STAT5b molecules present in the
two systems, or alternatively, may result from the presence in liver
but not in CWSV-1 cells of a novel protein that is a component of STAT5
DNA complex II.
Kinetics of GH Activation
The time course of STAT5b activation in CWSV-1 cells was
investigated as shown in Fig. 4A. At 50 ng/ml GH, STAT5b
activation was faint or absent at 5 min, became detectable at 10 min,
and was maximal by 4560 min. Similarly, Western blot analysis
demonstrated the appearance of the diphosphorylated STAT5b band 2
initially at 20 min and then maximally at 45 min (Fig. 2C
). The time
required for STAT5b activation was progressively shortened as the
concentration of GH was increased from 50 ng/ml to 2000 ng/ml (Fig. 4A
). Since the dissociation constant (Kd) for the GH-GH
receptor complex measured in intact cells is 100 pM (
2
ng/ml) (29), it is expected that even the lowest concentration of GH
used in these experiments, 50 ng/ml, should be saturating with respect
to GH receptor binding. The apparent requirement of a high GH
concentration for rapid STAT5b activation could, in part, be due to a
low-level, constitutive phosphotyrosine phosphatase that
dephosphorylates one of the components required for this response
(e.g. JAK2 kinase, GH receptor, or STAT5b itself), thereby
antagonizing the effects of GH. This possibility was tested by
pretreating CWSV-1 cells with the phosphotyrosine phosphatase inhibitor
sodium pervanadate (60 µM for 1 h), followed by GH
stimulation. At both 20 ng/ml GH and 50 ng/ml GH, the kinetics of
STAT5b activation were not affected by the presence of pervanadate
(Fig. 4B
). Thus, the comparatively slow rate of STAT5b activation seen
at the lower GH concentrations is not due to reversal of the effects of
GH by the action of a pervanadate-sensitive phosphotyrosine
phosphatase.
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STAT5b Is Rapidly Deactivated upon Removal of GH, but Can Be
Reactivated by Repeated GH Pulsation
In rat liver in vivo, STAT5 is exclusively localized in
the cytosol until it becomes activated by a GH pulse, which induces
nuclear translocation of the STAT in close association with GH-induced
tyrosine phosphorylation (12). In order for STAT5 to respond to
sequential GH pulses, as has been shown to occur in rat liver in
vivo, a mechanism must exist for rapid termination of a STAT5
response after each GH pulse. This could involve STAT
dephosphorylation, perhaps associated with recycling of the STAT
protein back to the cytosol, or perhaps deactivation by degradation
followed by new STAT5 protein synthesis. We first investigated whether
CWSV-1 cells respond to repeated GH pulses by repeated cycles of STAT5b
activation. Cells were incubated with GH in a manner that mimicked the
physiological, male plasma GH pattern, where GH pulses of 1 h in
duration are followed by a GH-free period of
2.5 h (overall
3.5-h
pulse frequency, measured peak to peak). The GH concentration was set
at 50 ng/ml for these cellular studies to approximate the average GH
concentration across the 1-h hormone pulse in vivo
(c.f. peak GH concentration in plasma
200 ng/ml) (17).
Upon addition of GH, STAT5b activation was faint at 5 min, maximal at
45 min, and still robust at 60 min (Fig. 5A
, lanes
25). Removal of GH at 60 min (corresponding to termination of a GH
pulse) led to a complete loss of STAT5b DNA-binding activity within
3060 min (Fig. 5A
, lane 6; also see Fig. 6A
, lane 3),
and this was associated with a corresponding disappearance of STAT5b
band 2 detected by Western blotting (Fig. 5B
, lane 6). Addition of a
fresh aliquot of GH 2.5 h after the first GH pulse resulted in the
reactivation of STAT5b2 (Fig. 5
, lanes 811). A
third pulse of GH resulted in a third cycle of STAT5b activation (lanes
1417). Thus, STAT5b undergoes repeated cycles of activation and
deactivation in response to a male pattern of pulsatile GH
stimulation.
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Role of Phosphotyrosine Phosphatase in the Deactivation of STAT5b
After Termination of a GH Pulse
Conceivably, the requirement of 2.53 h for full
responsiveness of STAT5b to a second GH pulse (Fig. 6A
) may reflect
degradation of one or more components of the GH receptor/JAK2/STAT5b
pathway, followed by a need for new protein synthesis. To investigate
this possibility, CWSV-1 cells were incubated with the protein
synthesis inhibitor cycloheximide (10 µg/ml), after which cells were
given two pulses of GH, each 1 h in duration and separated by a
3-h interpulse interval. This experiment was carried out at a
concentration of GH, 500 ng/ml, which resulted in a near complete
conversion of STAT5b to its tyrosine-phosphorylated, activated form
(band 2; cf. Fig. 3B
, lane 3). Cycloheximide given 1 h
before GH did not decrease the intensity of the response of STAT5b to a
GH pulse (data not shown). Cycloheximide pretreatment at either 10
µg/ml (Fig. 6B
) or 50 µg/ml (data not shown) also did not decrease
the response of STAT5b to a second GH pulse. Thus, the comparatively
slow resetting of the GH-activated STAT5b pathway does not result from
a time requirement for new protein synthesis.
To ascertain whether phosphotyrosine phosphatase activity is required
to reset the STAT5b GH signaling pathway, cells were treated with the
membrane-permeable phosphotyrosine phosphatase inhibitor pervanadate
(33). While pervanadate had no discernible effect on the kinetics of
STAT5b activation (Fig. 4B), it markedly slowed the deactivation of
STAT5b after termination of a GH pulse. This was evident from the
prolongation by pervanadate of STAT5b DNA-binding activity during the
interpulse interval after removal of GH (Fig. 7A
) and
from the persistence of STAT5b band 2 on Western blots of these same
cell extracts (Fig. 7B
). Incubation of CWSV-1 cells with pervanadate
alone induced a very slight activation of STAT5b (Fig. 8
, lane 1). This suggests that the prolongation of the
STAT5b signal through the interpulse interval in pervanadate-treated
cells (Fig. 7A
) may be due to inhibition of the deactivation of
existing, activated STAT5b molecules, rather than continued activation
by a constitutively activated JAK2 (cf. Ref.34). The fact
that STAT5b band 2 persists in the presence of
pervanadate, and is not converted by a phosphoserine/threonine
phosphatase to band 1, demonstrates that STAT5b phosphotyrosine
dephosphorylation precedes phosphoserine/threonine dephosphorylation.
This conclusion is supported by our finding that the
phosphoserine/threonine phosphatase inhibitors, okadaic acid and
calyculin, did not block STAT5b deactivation (Fig. 7
, A and C) nor did
they block the dephosphorylation leading to loss of STAT5b band 2 after
GH removal (Fig. 7B
, and data not shown). We conclude, therefore, that
STAT5b tyrosine dephosphorylation obligatorily precedes
phosphoserine/threonine dephosphorylation and is a key step in the
STAT5b deactivation pathway.
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Role of Serine/Threonine Phosphorylation in STAT5b Deactivation
Pathway
GH-induced activation of JAK2 tyrosine kinase is followed by
activation of serine/threonine phosphorylation carried out by an
unidentified GH-responsive kinase. Targets of this latter kinase
activity include tyrosine- phosphorylated STAT5b, as well as STAT1 and
STAT3 (13). To investigate the role of serine/threonine phosphorylation
in STAT5b deactivation, CWSV-1 cells were treated with the
serine/threonine kinase inhibitor H7. H7 significantly inhibited the
decline in STAT5b DNA-binding activity after termination of a GH pulse
(cf. Fig. 7A, lanes 5, 6; H7 vs. corresponding no
drug controls). This protective effect of H7 was less complete than
that provided by pervanadate, suggesting that serine/threonine
phosphorylation of one or more protein factors (including, perhaps,
serine/threonine phosphorylation of STAT5b itself) facilitates, but is
not absolutely required for, STAT5b phosphotyrosine dephosphorylation.
Similar protective effects, albeit to differing extents, were observed
in experiments using structurally diverse serine/threonine kinase
inhibitors, including H8 (Fig. 7C
and Table 1
). The effectiveness of H7
with respect to kinase inhibition was confirmed by its partial
inhibition of the accumulation of STAT5b band 2 after GH treatment
(Fig. 3B
, lanes 46 vs. 13). Conceivably, a more complete
inhibition by H7 of the GH-stimulated accumulation of STAT5b band 2
might have resulted in a more complete inhibitory effect on STAT5b
deactivation. Interestingly, in contrast to the inhibitory effect of
pervanadate on a second cycle of STAT5b activation (Fig. 7A
, lanes 79
vs. lane 6; see above), H7 did not interfere with the repeat
activation of STAT5b by a second GH pulse (Fig. 7A
; note increase in
STAT5b activity in H7 samples from lane 6 to lanes 79). This is
consistent with our earlier observation that although GH induces
phosphorylation of STAT5b on both tyrosine and serine/threonine,
tyrosine phosphorylation alone is sufficient to activate STAT5bs
DNA-binding activity (13).
Female Pattern of Continuous GH Exposure Leads to the Loss of
Activated STAT5b Complex
To investigate whether STAT5b responds in a differential manner to
continuous as compared with intermittent GH stimulation, CWSV-1 cells
were exposed to 50 ng/ml GH continuously for times up to 24 h.
Figure 8 shows that STAT5b was activated maximally during the first 45
min, but within several hours the level of STAT5b DNA-binding activity
declined to 1520% of the peak level. This reduced level of activated
STAT5b was maintained for at least 24 h. The same time-dependent
suppression of activated STAT5b was obtained at 5 ng/ml GH and at 500
ng/ml (data not shown).
Continuous GH treatment of CWSV-1 cells in the presence of pervanadate
delayed the initial phase of STAT5b deactivation by 12 h,
indicating a role for a phosphotyrosine phosphatase in this
deactivation. A role for a serine/threonine kinase was also apparent
from the finding that H7 maintained activated STAT5b at close to its
initial level for at least 4 h in the presence of continuous GH.
The apparent effectiveness of pervanadate for a shorter period, only
2 h after GH addition (i.e. 3 h after pervanadate
addition to the cells), reflects the toxicity associated with
pervanadate treatment at longer exposure times. Longer term studies on
the effects of phosphatase inhibition on the responses to continuous GH
could not be carried out due to the toxicity of these compounds, which
became clearly evident by
34 h in the case of pervanadate and
after 56 h in the case of H7.
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DISCUSSION |
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GH Receptor Activation in CWSV-1 Cells
GH initiates cellular responses by dimerization of the plasma
membrane-bound GH receptor (35, 36), leading to dimerization associated
with autoactivation of JAK2 kinase. The G120R mutant of hGH binds hGH
receptor with a high affinity (Kd = 0.3 nM),
but only interacts with the receptor via site 1 of the GH molecule,
thus blocking the receptor dimerization event required for signaling
(29). G120R-hGH failed to activate STAT5b in CWSV-1 cells, in accord
with its inactivity in other GH-responsive systems (37, 38) and
consistent with a requirement for receptor dimerization. It should be
noted, however, that when administered to hypophysectomized rats, G120R
hGH is poorly bound by rGH receptor and may interact with PRL receptors
(39). While GH, but not PRL, is presently shown to activate STAT5b in
CWSV-1 cells, GH and PRL both activate STAT5b in the human
hematopoietic cell line U17 (40) and in transfected COS-7 cells (41).
Non-liver-derived cell lines, especially ones reconstituted by
transfection of essential signaling components, may exhibit receptor
couplings different from those normally found in hepatocytes. In this
regard, the present findings using CWSV-1 cells closely reflect the
in vivo rat liver model, where STAT5 (primarily STAT5b) is
specifically activated by GH but not PRL (12).
GH binds to its receptor with a Kd of 0.1 nM
(2.2 ng/ml) (29), suggesting that the receptor would be saturated in
CWSV-1 cells even at the lowest GH concentrations used in this study
(2050 ng/ml). While this was apparently the case (cf.
similar maximal level of activated STAT5b at 502000 ng/ml GH; Fig. 4A), the kinetics of STAT5b activation were rather slow at the lower GH
concentrations (2045 min required for maximal STAT5b activation at
50125 ng/ml GH). The GH concentration-dependence of these kinetics
was unaffected by the presence of dexamethasone, which in 3T3-F442A
cells can down-regulate cell surface expression of GH receptor (32).
The kinetics of STAT5b activation were also unaffected by the
phosphotyrosine phosphatase inhibitor pervanadate, and thus the slow
rate of STAT5b activation at lower GH concentrations does not result
from a high basal rate of tyrosine dephosphorylation of one or more
components of the pathway (JAK2, GH receptor, STAT5b). These kinetics
are similar to those seen in GH receptor-transfected cells (42) and
most likely reflect the GH concentration-dependence of receptor binding
leading to STAT5b activation per se, which at physiological
GH concentrations (50200 ng/ml) appears to be slow. Indeed, GH
activation of liver STAT5 in hypophysectomized rats in vivo
is not detected until 10 min after intraperitoneal hormone injection
(13).
STAT5b Phosphorylation and Dephosphorylation
In rat liver in vivo, GH-induced STAT5b tyrosine
phosphorylation is followed by phosphorylation of STAT5b on serine or
threonine (13). GH also induced formation of a tyrosine +
serine/threonine-diphosphorylated STAT5b in CWSV-1 cells (Fig. 3;
STAT5b band 2), although the presence of a high basal level of
serine/threonine-phosphorylated STAT5b in these cells (band 1a) made it
difficult to establish whether this diphosphorylated STAT5b resulted
from GH-induced tyrosine phosphorylation of preexisting band 1a or
whether it reflects tyrosine phosphorylation of STAT5b band 0, followed
by a secondary, GH-stimulated serine/threonine phosphorylation
reaction, as occurs in liver in vivo (13). Support for the
latter hypothesis comes from two observations: 1) STAT5b band 1 appears
as a transient intermediate along the pathway that forms STAT5b band 2
(Fig. 3B
, lanes 13); 2) formation of the diphosphorylated STAT5b band
2 is inhibited by the serine/threonine kinase inhibitor H7, and this
results in accumulation of the tyrosine-phosphorylated band 1 (Fig. 3B
, lane 5). However, given the high basal level of STAT5b band 1a in
CWSV-1 cells, it is still possible that this preexisting
serine/threonine- phosphorylated STAT5b form may also serve as a
substrate for the GH-induced tyrosine phosphorylation reaction. The
source of the endogenous serine/threonine-phosphorylated STAT5b band 1a
is unknown; levels of this STAT5b form were greatly decreased in
H7-pretreated cells (Fig. 3B
, lane 4), but could not be reduced by
overnight culture of the cells in the absence of insulin or other
additives in the RPMI culture medium (data not shown).
Phosphotyrosine dephosphorylation, rather than STAT5b protein degradation, appears to be the primary mechanism for termination of a STAT5b signal. This is indicated by the ability of the phosphotyrosine phosphatase inhibitor pervanadate to block the rapid deactivation of STAT5b after termination of a GH pulse and is supported by our finding that the recycling of STAT5b and its response to a subsequent GH pulse are unaffected by protein synthesis inhibition. Hepatocytes contain numerous phosphotyrosine phosphatases associated with various intracellular compartments, including membranes, cytosol, and the nucleus (43, 44). The SH2-domain-containing phosphotyrosine phosphatases SHP1 and SHP2, in particular, can play both positive and negative regulatory roles in the action of other cytokines and growth factors that signal via JAK kinases (45, 46, 47) and could participate in STAT5b deactivation by binding to tyrosine-phosphorylated signaling molecules either at the level of the GH receptor/JAK2 kinase complex or at the level of nuclear STAT5b. While the present study shows that STAT5b phosphotyrosine dephosphorylation obligatorily precedes phosphoserine/threonine dephosphorylation, STAT5b tyrosine dephosphorylation appears to be facilitated by a prior serine/threonine phosphorylation step. Whether this involves serine/threonine phosphorylation of STAT5b itself or of some other signaling factor cannot be established with certainty (also see below). Further studies are required to elucidate the mechanisms of STAT5b deactivation, including any regulatory effects that GH may have on the phosphotyrosine phosphatase(s) that contribute to this process.
The kinase that catalyzes the secondary, GH-induced phosphorylation of
STAT5b on serine or threonine has not been identified, but could
correspond to mitogen-activated protein kinase, which is also activated
by GH (25) and can catalyze serine phosphorylation of other STAT
proteins (47). Tyrosine phosphorylation, but not serine/threonine
phosphorylation, is required for STAT5bs DNA-binding activity (Fig. 2A). Although not tested directly, GH-induced STAT5b serine/threonine
phosphorylation may contribute to STAT5bs transcriptional activation
potential, as has been shown in other STAT systems (48, 49), including
interleukin 2-activated STAT5 (50). In addition, the present study
suggests that a serine/threonine kinase activity (perhaps the
GH-activated serine/threonine kinase that acts on STAT5b) sensitizes
STAT5b to deactivation by tyrosine dephosphorylation, as indicated by
the inhibitory effects of several serine/threonine kinase inhibitors on
STAT5b deactivation. This intriguing observation raises several
possibilities, including: 1) the phosphotyrosine phosphatase(s) that
deactivate tyrosine-phosphorylated STAT5b may have a preference for
STAT5b molecules that are also serine or threonine phosphorylated; 2)
inhibition of serine/threonine phosphorylation prevents nuclear
translocation of STAT5b, thus shielding the STAT protein from nuclear
phosphotyrosine phosphatases that may catalyze its tyrosine
dephosphorylation and deactivation; and 3) serine/threonine
phosphorylation, perhaps catalyzed by a GH-regulated kinase, may
activate the phosphotyrosine phosphatase that deactivates STAT5b.
However, the fact that H7 treatment not only prolongs STAT5b band 1,
but also prolongs the diphosphorylated band 2 (Fig. 7B
, lanes 12 and 13
vs. 2 and 3) argues against the first two possibilities and
thus lends support to possibility 3.
A low constitutive activation of STAT5b in the absence of GH could be detected when CWSV-1 cells were incubated with pervanadate, which in other cell models can induce tyrosine phosphorylation of multiple proteins, including JAK2 (33, 34). STAT5b was activated in pervanadate-treated CWSV-1 cells to only a small extent, whereas STAT3 and STAT1 were significantly activated under the same treatment conditions, as demonstrated by SIE probe EMSA with supershift analysis using anti-STAT antibodies (C. A. Gebert and D. J. Waxman, unpublished experiments). This difference between the STATs may reflect the preferential association of STAT3 (and perhaps also STAT1) with JAK2, as compared with STAT5b, which apparently requires a more direct interaction with the COOH-terminal cytoplasmic domain of the GH receptor to undergo activation (51). Whether the low basal level of activated STAT5b in pervanadate-treated cells results from its interaction with a JAK2 kinase/GH receptor complex, or perhaps with another tyrosine kinase, is not known. These hormone-independent stimulatory effects of pervanadate on STAT activation suggest that cellular phosphotyrosine phosphatases may in part serve to reverse the effect of adventitious STAT activation resulting from random association with tyrosine kinases, cytokine receptors, or other signaling molecules, thus maintaining a minimal level of tyrosine-phosphorylated STAT in the absence of ligand.
Response of STAT5b to GH Pulses: STAT5b Recycling
The ability of CWSV-1 cells to respond to repeated pulses of GH
with repeated cycles of STAT5b activation and deactivation is
consistent with in vivo studies in GH pulse-treated
hypophysectomized rats and with the finding that in intact adult male
rats nuclear translocation of activated liver STAT5 is intermittent and
coincides with the occurrence of a plasma pulse of GH (12). STAT5 is
thus activated for about 1 h of each 3.5- to 4-h GH pulse/GH
trough cycle. It will be interesting to determine whether the
intermittent activation of liver STAT5 leads to intermittent
transcription of GH pulse-responsive genes. These genes are likely to
include male-specific CYP genes such as CYP2C11, whose
transcription is induced by GH pulses in adult male rat liver and is
suppressed by continuous GH treatment in adult female rats (22, 23).
Indeed, a functional STAT5-binding site has been identified in the
5'-flank of CYP3A10, a GH-regulated, male-specific hamster
gene (24). In hypophysectomized rats given pulsatile GH replacement,
CYP2C11 expression could only be detected in animals given
six or fewer GH pulses per day, corresponding to an interpulse interval
of 2.753 h. By contrast, treatment with seven GH pulses per day,
which results in a shortening of the interpulse interval by only 35
min, fully blocked the activation of CYP2C11 (17). In the
present study, CWSV-1 cells also required an interpulse interval of
3 h for complete responsiveness to a second pulse of GH, but in
contrast to the CYP2C11 gene transcription response in the
in vivo model (17), STAT5b activation could be partially
restored with shorter interpulse intervals, with the strength of the
resultant STAT5 response being roughly linear in relation to the length
of the interpulse interval.
The mechanism underlying this requirement of a relatively long
interpulse interval (up to 3 h) for full restoration of STAT5bs
GH pulse responsiveness is unknown. It does not reflect time required
for resynthesis of one or more of the signaling proteins involved in
the STAT5 pathway, as demonstrated using the protein synthesis
inhibitor cycloheximide. Rather, the extended time interval for
restoration of full GH responsiveness may be dictated by the time
needed for one or more of the following events: 1) recycling of GH
receptor back to the plasma membrane after its internalization to
various intracellular compartments, such as the golgi and the nucleus
(52); 2) phosphotyrosine dephosphorylation of signaling proteins such
as JAK2, GH receptor, or STAT5b itself. Such a dephosphorylation
requirement is supported by our finding that whereas pervanadate
prolongs STAT5b activation after termination of a GH pulse, subsequent
GH pulses do not further increase the level of activated STAT5b (Fig. 7A); and 3) translocation of deactivated STAT5b from the nucleus back
to the cytosol. That STAT5b is likely to recycle to the cytoplasm
after GH pulse activation is indicated by the absence of a protein
synthesis requirement for repeat GH pulse activation of STAT5b, even
when the cells are exposed to a concentration of GH (500 ng/ml) (Fig. 6B
) that results in a near quantitative phosphorylation of the cells
STAT5b pool (Fig. 3B
). On the other hand, STAT5b recycling per
se seems unlikely to be rate limiting with respect to a second GH
pulse response since the 3-h interpulse interval requirement is also
seen in cells treated with GH under conditions (50 ng/ml) where only a
portion of the cellular STAT5b pool becomes phosphorylated at any given
point in time. Interestingly, despite significant differences in the
rate of STAT5b activation at low vs. high GH
concentrations, similar maximal levels of activated STAT5b were
detected by EMSA under both conditions of cell treatment (Fig. 4A
).
This maximal level detected at
45 min after hormone treatment was
not altered by pervanadate treatment, a finding that is consistent with
our observation that STAT5b dephosphorylation is not initiated until
4060 min after addition of GH to the cells (C. A. Gebert and D. J.
Waxman, unpublished experiments).
Finally, continuous exposure of CWSV-1 cells to GH, in a manner that mimics the female plasma GH pattern, led to a down-regulation of the STAT5 response that became apparent within 2 h of continuous GH treatment and was maintained for at least 24 h. This response was substantial, albeit only 8085% complete under the conditions of these cell culture experiments, and generally correlates with the down-regulation of liver STAT5 in vivo in hypophysectomized rats given GH continuously (12). The persistence of activated STAT5b at a low level for 24 h or longer apparently results from continued GH-induced signaling, rather than from an inhibition of phosphotyrosine phosphatase activity, since removal of GH after either 1 h, 2 h, 4 h, or 24 h of continuous hormone exposure leads to a rapid and complete loss of activated STAT5b (C. A. Gebert and D. J. Waxman, unpublished experiments). Whether the prolonged, low level activation of STAT5b in continuous GH-treated cells is mediated by JAK2 kinase or by another tyrosine kinase is unknown.
In conclusion, CWSV-1 cells are shown to provide a useful cell culture model for study of the effects of plasma GH patterns on the activation and deactivation of STAT5b. Further investigation should help elucidate additional mechanistic details that underlie this pathway, including the role of serine/threonine kinase(s) and the contributions of phosphotyrosine phosphatases to STAT5b deactivation and its recycling back to the cytosol. GH-activated STAT5b analyzed on Western blots can be seen to contain as many as five bands in H7-pretreated cells, suggesting additional complexities in this system that remain to be identified.
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MATERIALS AND METHODS |
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For GH and/or inhibitor treatments, 1 ml of fresh medium was used per
60-mm tissue culture dish on the day of the experiments. GH (dissolved
in PBS containing 0.1% BSA and kept on ice until use) was added in
volumes of 10 µl using schedules described in each individual
experiment. Data shown are generally representative of at least three
independent experiments. Rat GH, hGH, and PRL were hormonally pure
preparations obtained from the National Hormone and Pituitary Program,
NIDDK. The hGH mutant Gly 120 ->Arg (hGH-Gl20R) was provided by Drs.
J. Wells and G. Fuh (Genentech, South San Francisco, CA). GH used in
each experiment was rGH unless indicated otherwise. Kinase and
phosphatase inhibitors used in this study were used at concentrations
and under conditions shown in Table 1. Inhibitors were prepared as
100x stock solutions and stored at -20 C, with the exception of
pervanadate, which was prepared fresh daily as a 100x stock containing
6 mM Na3VO4 and 5.7 mM
H2O2 (final nominal concentration, 60
µM pervanadate). Most of the inhibitors shown in Table 1
exhibited little or no apparent toxicity to the cells in the time frame
of the experiments; H7, H8, and HA1077 were significantly toxic to the
cells after
5 h incubation, while pervanadate toxicity become
apparent by
34 h.
Total Cell Extracts
Cells were washed once with ice-cold PBS and then scraped with
100 µl of lysis buffer (20 mM HEPES buffer, pH 7.9, 1%
Triton X-100, 20% glycerol, 20 mM NaF, 1 mM
each EDTA, EGTA, Na3VO4,
Na2P2O7, dithiothreitol, 0.5
mM phenylmethanesulfonyl fluoride, and 1 µg/ml each
pepstatin, antipain, and leupeptin). The crude extract was aspirated
ten times through a 27 gauge needle, adjusted to 150 mM
NaCl, and centrifuged at 15,000 x 30 min at 4 C. Supernatants
were stored in liquid N2 until analysis. Protein
concentrations were determined using the Bio-Rad Dc detergent protein
assay kit.
EMSA
Total cell extracts (10 µg) were made up to a total volume of
5 µl in lysis buffer, then added to 8 µl of gel-shift buffer (5%
glycerol, 1.25 mM MgCl2, 625 µM
EDTA, 625 µM dithiothreitol, 12.5 mM Tris, pH
7.5) plus 1 µl containing 2 µg of
poly(deoxyinosinic-deoxycytidylic)acid (Boehringer Mannheim,
Indianapolis, IN) and incubated 10 min at room temperature.
Double-stranded, 32P-labeled oligonucleotide probe (1 µl,
10 fmol) was then added to give a total volume of 15 µl. Incubation
was continued for 20 min at room temperature and then 10 min on ice,
followed by addition of 2 µl loading dye (30% glycerol, 0.25%
bromophenol blue, 0.25% xylene cyanol). The incubation mixture was
loading onto an acrylamide gel (5.5% acrylamide, 0.07% bis-acrylamide
(National Diagnostics, Atlanta, GA) in 0.5x TBE (44.5 mM
Tris, 44.5 mM boric acid, 5 mM EDTA), which had
been prerun at 4 C for 30 min at 100 V. The gel was run at 100 V in
0.5 x TBE, first for 20 min at 4 C and then for an additional 160
min at room temperature. Cooling the samples for a minimum of 10 min
followed by electrophoresis into the gel at 4 C maximized the amount of
specific STAT5-DNA gel-shift complex and minimized the formation of
more rapidly migrating nonspecific complexes. After electrophoresis of
the samples into the gel for 20 min at 4 C, the gel apparatus was moved
to room temperature to increase the speed of protein migration. Cold
probe competitions were carried out using up to a 200-fold molar excess
of unlabeled DNA probe added in a volume of 1 µl just before addition
of the 32P-labeled DNA probe. For supershift analysis,
antibody was added 10 min after the labeled DNA probe. Antibodies used
were anti-STAT5b (Santa Cruz Biotechnology, Santa Cruz, CA;
sc-835), anti-STAT3 (Santa Cruz, sc-482), anti-STAT1 (Transduction
Labs, Lexington, KY; G16920), as described previously (13). Gels were
exposed to phosphorimager plates for 16 h followed by quantitation
using a Molecular Dynamics PhosphorImager and ImageQuant software
(Sunnyvale, CA).
The STAT5/mammary gland factor response element of the rat ß-casein promoter (nucleotides -101 to -80), 5'-GGA-CTT-CTT-GGA-ATT-AAG-GGA-3' (sense strand) was used for EMSAs. The probe was end labeled with 32P on one strand, annealed to the antisense strand, and then gel purified. STAT1 and STAT3 DNA-binding activity was assessed using a c-fos gene SIE probe as described elsewhere (13).
Immunoprecipitation, Western Blot, and Other Analyses
Immunoprecipitation with anti-phosphotyrosine antibody
PY-20 and treatment with the phosphatases PTP1B and PP2A were as
described (13). For Western blotting, total cell extracts were
electrophoresed through Laemmli SDS-polyacrylamide gels (7.5% gels or
510% gradient gels) run at constant current and a starting voltage
of 75 V, with cross-over to constant voltage at 170 V. In some cases
(Fig. 3B), 6.5% gels were run overnight at a constant current of 6 mA
to maximize resolution of STAT5b bands 1 and 1a. Gels were
electrotransferred to nitrocellulose and probed with anti-STAT5
antibodies. Blocking and probing conditions were as described (12).
Detection on x-ray film was by enhanced chemiluminescence using
Amersham ECL reagents (Amersham, Arlington Heights, IL). Anti-STAT5a
antibody was from Santa Cruz (catalog no. sc-1081). Anti-STAT5b
antibody (Santa Cruz, catalog no. sc-835) was used in all experiments
shown unless noted otherwise. This antibody was shown to be
STAT5b-specific under our Western blotting conditions by analysis of
extracts of Cos 1 cells transiently transfected with either mouse
STAT5a cDNA or mouse STAT5b cDNA, kindly provided by Dr. Alice Mui
(DNAX Corp., Palo Alto, CA) (data not shown) (53). The
unphosphorylated, cDNA-expressed STAT5a migrated distinctly slower than
STAT5b, i.e. at a position close to the GH-activated,
diphosphorylated STAT5b band 2 (see Results), whereas
cDNA-expressed STAT5b comigrated with the major STAT5 form present in
untreated CWSV-1 cells, supporting our identification of the
GH-activated STAT5 protein presented in CWSV-1 cells as STAT5b.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Supported in part by NIH Grant DK-33765 (to D.J.W.).
1 As shown in Fig. 3B, below, STAT5b band 1a
(serine/threonine-phosphorylated form) migrates somewhat slower than
STAT5b band 1 (tyrosine-phosphorylated form) on Western blots of SDS
gels. In most cases, however, these two bands could not be
distinguished electrophoretically and are therefore marked as
band 1/1a on each of the figures. In untreated cell extracts
(e.g. Fig. 1B
, lane 1) this band actually corresponds to the
serine-threonine-phosphorylated band 1a, whereas in GH-stimulated
extracts (e.g. Fig. 1B
, lane 2), it corresponds to a mixture
of band 1 and band 1a.
2 The somewhat lower STAT5b DNA-binding activity
and lower level of STAT5b band 2 observed after the second GH pulse as
compared with the first GH pulse in this experiment and in the one
shown in Fig. 7 may reflect the 2.5-h interpulse interval used in this
experiment, which is shorter than the optimal 3-h interval identified
in Fig. 6A
.
Received for publication October 4, 1996. Revision received December 24, 1996. Accepted for publication December 30, 1996.
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REFERENCES |
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