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J. Biol. Chem., Vol. 277, Issue 32, 28384-28393, August 9, 2002
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From the
Received for publication, December 9, 2001, and in revised form, May 9, 2002
Secretion of growth hormone (GH) in adult
male rats is characterized by high peak and undetectable trough levels,
both of which are required for male-specific pattern of liver gene
expression and GH-induced phosphorylation of STAT5. The present study
suggests that regulation of GH receptor (GHR) levels in rat hepatoma
cells by repeated GH stimulation determines GH responsiveness via the JAK2/STAT5 pathway. A short exposure to GH rapidly reduced GHR levels
which resulted in an equal desensitization of the JAK2/STAT5 pathway.
Recovery of GH-induced STAT5 phosphorylation correlated with the
time-dependent recovery of GHR levels during incubation in
the absence of GH. Acute GH also induced phosphorylation of ERK1/2 and
Akt, and this induction was also inhibited by prior exposure to GH.
However, unlike the JAK2/STAT5 pathway, the effect of GH to activate
the MEK/ERK and phosphatidylinositol 3-kinase/Akt pathways did not
recover following prolonged incubation in the absence of GH. Thus, GH
administration desensitizes the JAK2/STAT5 pathway, possibly because of
down-regulation of GHR, whereas an additional post-receptor mechanism
is required for the prolonged refractoriness of the MEK/ERK and
phosphatidylinositol 3-kinase/Akt pathways toward a second GH
stimulation. Our study suggests that both receptor and post-receptor
mechanisms are important in GH-induced homologous desensitization.
It is still controversial whether growth hormone
(GH)1-induced desensitization
of GH signaling is the result of down-regulation of GH-receptor (GHR)
levels or post-receptor signaling pathways. Elucidation of the
processes of this homologous desensitization may further our
understanding of the regulation of GH action, as well as the possible
effects of GH on intracellular signaling molecules that are also
utilized by other hormones, cytokines, and growth factors.
GH is secreted in a pulsatile fashion to promote growth and diverse
metabolic actions (1, 2). In young adult male rats, GH is released in
~1-h pulses with peak serum concentrations of 150-400 ng/ml, and
interpulse intervals of 2 h or more, where serum GH concentrations
are negligible (3). In female rats, GH is secreted more frequently,
resulting in the continuous presence of GH in the circulation, with
peaks of 50-150 ng/ml (2-5). The sexually dimorphic pattern of GH
secretion provides an important mechanism for transcriptional
regulation of sexually dimorphic genes in the liver.
Growth hormone signaling cascades include Janus kinase 2 (JAK2) and
signal transducers and activators of transcription (STAT) family
transcription factors (including STAT1, STAT3, STAT5A and STAT5B; Refs.
6-9), phosphatidylinositol 3-kinase (PI 3-kinase) and extracellular
signal-regulated kinases 1 and 2 (ERK1/2) (10-14). GH may induce
phosphorylated (P)-ERK1/2 via the indirect association of JAK2 to
growth factor binding protein 2 (Grb2)-Son of Sevenless (SOS) complexes
through SH2 containing Shc proteins (12, 15, 16) or via
trans-phosphorylation of the epidermal growth factor receptor by JAK2
(17), which can then recruit the Grb2-SOS complex. These pathways can
then lead to activation of the Ras-Raf-MEK-ERK pathway (18). GH may
also activate ERK1/2 via a PI 3-kinase activity (10, 19), which can be
activated by JAK2 via both insulin receptor substrate
1-dependent and -independent mechanisms (19, 20).
Although GH activates ERK in the hepatocyte when either injected into
animals or when added directly to cultured hepatocytes (18, 21), it is
not known whether repeated GH pulses, as is normally present in
vivo, cause multiple spikes of activated (phosphorylated) ERK.
Desensitization and resensitization are critical processes regulating
GH actions on target tissues. In hypophysectomized rats, a second GH
exposure can trigger a full hepatic response of STAT5 tyrosyl
phosphorylation (PY-STAT5) if it follows the first injection by 4 h (7, 22). In a rat hepatocyte-derived cell line, full responsiveness
to succeeding GH stimulation via the JAK2/STAT5 pathway requires a
minimum of 2.5 h without GH (23). These studies indicate that the
liver and liver-derived cells undergo an obligatory recovery period
after stimulation by a GH pulse (23).
Two general mechanisms may underlie the requirement for a recovery
period for resensitization of GH-induced signaling. First, the recovery
period could allow restoration of a short term decrease of cell-surface
GHR following a cycle of GH binding, receptor internalization, and
degradation or recycling. Alternatively, the period may be required to
reset GH-activated intracellular signaling pathways.
Important post-receptor mechanisms of desensitization of GH may involve
changes in the cellular expression of members of the family of
suppressor of cytokine signaling, or cytokine-inducible SH2 proteins
(24-30). Another post-receptor mechanism for the post-GH pulse
recovery period may be the regulation of phosphatase activity (31).
However, for this to be an operative mechanism, a
time-dependent induction followed by reduction of
phosphatase activity coincident with the inhibition and then recovery
of GH responsiveness would be required, and has not yet been demonstrated.
It is clear that GH induces rapid internalization of GHR, but there is
conflicting evidence concerning whether GHR is degraded following
internalization (23, 32-35). The GHR may either be recycled back to
the cell surface or be degraded in the proteasome or lysosome (36-38).
In the present study, using rat hepatoma H4IIE (H4) cells, both
desensitization of the JAK2/STAT5 pathway following initial GH
stimulation and the recovery of GH sensitivity of this pathway during a
GH-free incubation period were strongly correlated with reduction and
recovery, respectively, of GHR levels. Therefore we hypothesize that,
with repeated GH applications, a major mechanism of desensitization and
resensitization of GH signaling and JAK2/STAT5 activation was the
result of reduction and recovery of GHR levels. However, following a
1-h exposure to GH, even 16 h in its absence was insufficient to
obtain a recovery of the GH-induced pathways of MEK/ERK and PI
3-kinase/Akt. This lack of recovery of MEK/ERK and PI 3-kinase/Akt
signaling indicates that recovery of GHR and JAK2/STAT5 signaling was
insufficient for recovery of all GHR signaling capabilities. There must
be at least one additional post-receptor desensitization mechanism that
results in reduced GH signaling even when GHR levels are restored.
Materials--
Bovine GH (bGH; lot APF10325C) was kindly
provided by Dr. A. F. Parlow, Pituitary Hormones and Antisera
Center, Harbor-UCLA Medical Center (Torrance, CA) and the National
Institutes of Health NIDDK National Hormone & Pituitary Program. Fetal
bovine serum, calf serum, and horse serum were purchased from
Invitrogen. Enhanced chemiluminescent detection reagents
were obtained from Amersham Biosciences. Other materials were
purchased from Sigma and Fisher unless otherwise noted.
Antibodies--
Anti-STAT5 monoclonal antibody (raised against
amino acids 451-649 of sheep STAT5A shared by STAT5A and STAT5B) was
purchased from Transduction Laboratories (Lexington, KY). Thus, this
antibody reacts with both STAT5A and STAT5B. A mouse anti-STAT5B
(raised against the unique C terminus of murine STAT5B) monoclonal
antibody and a rabbit anti-phosphotyrosine-STAT5 polyclonal antibody
(raised against the phosphopeptide around C-terminal Tyr-694 of murine STAT5A that is conserved in both STAT5A and STAT5B of human, sheep, and
rat) were obtained from Zymed Laboratories (San Francisco, CA). A
rabbit anti-JAK2 peptide antiserum, directed at residues 758-776 of
murine JAK2, was purchased from Upstate Biotechnology, Inc. (Lake
Placid, NY). Two anti-GHR antisera were raised in rabbits against
peptides of the cytoplasmic domain of the GHR: anti-GHRcyt3728 (GHR3728) against residues 317-620 of the human GHR cytoplasmic domain
and anti-GHRcytAL37 (GHR-AL37) against residues 271-620 fused to
glutathione S-transferase as described (39, 40). The
following antibodies were purchased from New England Biolabs (Beverly, MA): rabbit antibodies for ERK; MEK1/2; Akt; P-ERK1/2, which
can detect dual phosphorylation of Thr-183 and Tyr-185 of rat ERK1/2;
P-MEK1/2, which can detect dual phosphorylation of Ser-217 and Ser-221
or single phosphorylation of Ser-217; and P-Akt, which can detect Akt1
only when phosphorylated at Ser-473 and Akt2 and Akt3 only when
phosphorylated at equivalent sites. Secondary antibodies including the
peroxidase-linked sheep anti-mouse serum and the peroxidase-linked
donkey anti-rabbit serum were obtained from Amersham Biosciences. Other
secondary antibodies such as the donkey anti-goat IgG were obtained
from Santa Cruz Biotechnology.
Cell Culture--
Rat H4-II-E (H4) hepatoma cells were cultured
in Swim's medium supplemented with 10% serum (5% horse serum, 3%
newborn calf serum, and 2% fetal calf serum) and 2 µg/ml gentamycin
sulfate (41). At ~50% confluence, cells were removed from serum and maintained in serum-free (and bovine serum albumin-free) medium for
48 h prior to the start of experimental treatments. For the washing experiments, following the first exposure to GH the medium was
removed by aspiration, the cells were gently rinsed twice with
phosphate-buffered saline, followed by addition of GH-free, serum-free
Swim's medium.
Protein Extraction--
Hormone treatment (detailed in the text)
was terminated by rinsing the cells once with 20 °C TBS (10 mM Tris-HCl, pH 7.4, 150 mM NaCl), cells were
collected in 20 °C SDS lysis buffer (1% SDS, 10 mM
Tris-HCl, pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 50 mM sodium fluoride, 0.5 mM
Na3VO4) and boiled for 5 min, and the cell
lysates were rigorously homogenized. One volume of 4× Laemmli sample
buffer (8% SDS, 250 mM Tris-HCl, pH 6.8, 40% glycerol, 4% Electrophoresis and Immunoblotting--
The protein
lysates in Laemmli sample buffer were then subjected to 9-12%
gradient SDS-PAGE. Western transfer of proteins was performed as
described previously, except for the use of Protran membrane from
Schleicher & Schuell (BA 85; Ref. 40). The membranes were blocked in
0.4% milk, 5% bovine serum albumin in TBS, 0.7% Tween 20, pH 8.0. Immunoblotting was performed with the antibodies at the following
dilutions: anti-STAT5 (1:500), anti-STAT5B (1:5000), anti-PY-STAT5
(1:5000), anti-GHR-AL37 (1:2000), anti-JAK2 (1:2000), anti-IR (1:1000),
anti-ERK1/2 (1:1000), anti-Akt (1:1000), anti-MEK1/2 (1:1000),
anti-P-ERK1/2 (1:1000), anti-P-Akt (1:1000), anti-P-MEK (1:1000) with
horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary
antibodies (1:2000). Incubation with rabbit primary antisera was at
4 °C overnight, whereas incubation with mouse monoclonal antibodies
was at room temperature for 1 h. Washing times after primary and
secondary antibodies were at room temperature for 10 and 20 min,
respectively. All primary antibodies were used in 0.7% Tween 20 in
TBS, pH 8.0, with 0.02% azide. Detection of bound antibodies by
enhanced chemiluminescence and stripping and reprobing of blots were
accomplished according to the manufacturer's suggestions. All blots
measuring phosphorylated proteins were reprobed at least twice, the
second probing using an antibody to detect total amounts
(phosphorylated and non-phosphorylated) of the same protein. These
repeat probings were to ensure even loading from lane to lane and an
unchanging amount of the total protein following experimental
treatments; several of the reprobed Westerns are included in the figures.
Densitometric and Statistical Analysis--
Enhanced
chemiluminescent images of immunoblots were analyzed by scanning
densitometry. Multiple exposures of each blot were used to obtain
gray-scale images of each chemiluminescent band and were quantified
with the Scion Image Analysis program (release beta 2) from Scion Corp.
(Frederick, MD). All data were analyzed by analysis of variance using
the InStat statistical program (version 3) by GraphPad Software, Inc.
(San Diego, CA).
Kinetics of GH-induced Phosphorylation of STAT5--
The
kinetics of GH-induced tyrosyl phosphorylation of STAT5 (PY-STAT5)
in H4 cells was examined. Because plasma GH levels can vary between 0 and 400 (or higher) ng/ml in young adult male rats and between 50 and
150 ng/ml in young adult female rats (4, 5), bGH at concentrations of
50-500 ng/ml were used. The highest concentration of bGH, 500 ng/ml,
resulted in a large induction of PY-STAT5 at all points tested between
5 and 60 min following its addition to the cell culture media (Fig.
1A), compared with vehicle-treated control cells (lane 10). This
large induction diminished after 60 min, returning toward basal values
by 90 min (Fig. 1A). Addition of bGH at various doses
resulted in maximum levels of PY-STAT5 that were clearly
dose-dependent. Designating the 30-min time point of the
500 ng/ml concentration of bGH as 100%, the maximal effects of bGH at
100 and 50 ng/ml at 30 min were ~60 and 50%, respectively (Fig.
1B and additional data not shown). When multiple time-course
experiments were averaged, it is evident that peak PY-STAT5 was
achieved most rapidly at 500 ng/ml and was slowest at 50 ng/ml (Fig.
1C).
GH Rapidly Reduced Immunoreactive GHR--
The relative levels of
immunoreactive GHR before or following GH treatment were determined by
Western blot analysis. Using an antibody against the cytoplasmic domain
of human GHR (GHR3728), which cross-reacts with rat GHR (42), treatment
with bGH (500 ng/ml) for 0-60 min resulted in reduction of GHR levels
by ~75% (Fig. 2A). Using a
second, independently raised antibody against a different yet
overlapping cytoplasmic domain of human GHR (GHR-AL37; Ref. 42), the
levels of GHR following 0-60-min treatment with bGH at different doses
were also determined. These experiments confirmed the earlier studies
that GHR levels were reduced following bGH at 50, 100, and 200 ng/ml,
as well as with 500 ng/ml for 5-60 min, with maximum reductions to
~20-30% of the control levels at 60 min at all concentrations of
bGH tested (Fig. 2B). Similar to induction of PY-STAT5, the
fastest reduction of GHR levels occurred in H4 cells treated with the
highest concentration (500 ng/ml) of bGH.
Reduction in GH Signaling following 1-h GH Pretreatment followed by
a 3-h Wash-out Period to Mimic the in Vivo Interpulse
Interval--
Growth hormone is secreted in ~1-h pulses in young
adult male rats, with interpulse intervals of non-detectable GH greater than 2 h. To mimic this temporal pattern of GH exposure, H4 cells were pretreated with bGH at 50, 100, 200, and 500 ng/ml for 1 h,
after which the GH was washed away and the cell incubated for 3 h
in GH-free, serum-free medium. Compared with untreated cells, those
treated with bGH (500 ng/ml) for 1 h followed by 3 h in GH-free, serum-free medium possessed levels of immunoreactive GHR that
were ~50% of that in the untreated sample (Figs.
3 (row 1,
lane 7 versus lane
6) and 4A). Because
this is higher than the amount following GH for 60 min without the
3 h in GH-free, serum-free medium (Figs. 2B and 3
(row 1, lane 7 versus lane 5)), it suggests that
there was a partial recovery of GHR levels during the 3-h wash-out
period (see the next section). At the beginning of the second bGH
application, and at each treatment time point thereafter, there were
lower GHR levels following the second versus the first
exposure to bGH for 5-60 min. Thus, after a 1-h GH treatment and
3 h in GH-free, serum-free media, there was a lower level of GHR,
and cellular GHR could be further decreased by a second exposure to 500 ng/ml bGH (Figs. 3 and 4A). Similar results were obtained
with bGH at 50 ng/ml (Fig. 4B) and at 100 and 200 ng/ml (data not shown). The GH-induced reduction in GHR levels was faster at
500 versus 50 ng/ml, but achieved a similar percentage
reduction by 60 min (Fig. 4, A versus
B). Additionally, following the first and the second GH
applications at 500 ng/ml, the GH-induced reduction of GHR resulted in
lines that were parallel to each other (Fig. 4, A and
B) suggesting that the rate of loss of GHR was determined by
the concentration of GH in the media, regardless of initial levels of
GHR. Finally, when the same experiments were evaluated for JAK2 protein
levels, they remained approximately constant (Fig. 3, row
2).
Because repeated applications of GH are capable of activating multiple
cycles of STAT5 phosphorylation in vivo, we studied whether
GH-induced changes in the levels of GHR affected the ability of a
second exposure of GH to induce PY-STAT5. Following a 1-h exposure to
500 ng/ml bGH followed by a 3-h incubation in GH-free, serum-free
medium, PY-STAT5 levels returned to basal (Fig. 3, row
3, lane 7 versus
lane 6). The second application of 500 ng/ml bGH
for 5 min induced tyrosyl phosphorylation of STAT5 to ~60% of that
induced by 5 min bGH alone (Figs. 3 (row 3) and
5). The decreased ability of the second
bGH exposure to induce PY-STAT5 compared with the first bGH was evident
at all bGH concentrations tested (500 ng/ml shown in Fig. 5;
data not shown for 50, 100 and 200 ng/ml bGH). Even though there was a
reduced maximal effect of the second exposure to bGH on PY-STAT5, there
were similar kinetics of bGH-induced tyrosyl phosphorylation of STAT5
and PY-STAT5 dephosphorylation for both exposures to GH (Fig. 5). This
suggests no significant changes in the activity of a phosphatase to
dephosphorylate STAT5B between the first and the second exposure to
GH.
The same cell extracts were analyzed for changes in protein levels and
mobility of STAT5B. The STAT5B protein normally appeared as a doublet
in the Western blot because of serine phosphorylation of STAT5B
resulting in retarded mobility (Fig. 3, row 4)
(23). GH-induced STAT5B activation is associated with dual
phosphorylation of STAT5B at both serine and tyrosine residues,
resulting in a third band (highest band) with the slowest mobility and
a loss of the lowest band (23). Following the first bGH treatment, the
intensity of the highest band became greater than that of the second
band; by 3 h following GH removal, STAT5B had been dephosphorylated and had returned to the basal state (Fig. 3, row 4). Consistent with the PY-STAT5 data,
following the second GH exposure for 5-60 min, the intensity of the
highest band was approximately equal to or only slightly greater than
that of the second band (Fig. 3, row 4) instead
of the preponderance of the highest (tyrosyl-phosphorylated STAT5B)
band following the initial GH application. This implies, by measurement
of gel retardation, a lesser ability of GH to induce phosphorylation of
STAT5B following the second versus the first GH exposure,
similar to the lesser induction of PY-STAT5 as measured by
phosphorylation-specific antibodies.
Total STAT5B protein levels were the same or increased, and certainly
not decreased, following the second versus the first GH
exposure (Fig. 3, row 4). Thus, the lower
GH-induced PY-STAT5 resulting from the second versus the
first GH exposure could not be explained by loss of STAT5B protein.
Time-dependent Recovery of GHR and GH-induced
PY-STAT5--
When H4 cells were treated with bGH (500 ng/ml) for
1 h, the bGH was washed away, and the cells were incubated in
GH-free, serum-free media for various time intervals, there was a
recovery of both GHR levels and the ability of GH to induce PY-STAT5.
As described previously, GHR levels were reduced to ~20-30% of the control levels following 1 h of bGH treatment (Fig.
2B). Following a 1-h interval in GH-free, serum-free media,
GHR levels increased negligibly and were ~30% of that in untreated
cells (Fig. 6B). Following 2, 3, 6, and 16 h in GH-free, serum-free media, the cellular levels
of GHR recovered to ~40, 60, 80, and 100%, respectively, of that in
cells not treated with GH (Fig. 6B).
There was little remaining PY-STAT5 after 1 h of GH followed by
1 h in GH-free, serum-free media, and phosphorylation of STAT5 could still be induced by 20 min of GH, but only to ~20% of that induced by 20 min of GH without GH pretreatment and washing (Fig. 6,
A and B). The ability of GH for 20 min to induce
PY-STAT5 increased with increasing times of GH-free, serum-free
incubation. Following 2, 3, 6, and 16 h, the ability of GH to
induce PY-STAT5 recovered to ~40, 60, 80, and 90%, respectively, of
that induced by 20 min of GH without GH pretreatment and washing (Fig.
6, A and B). Thus, the time course of recovery of
GHR was nearly identical to the recovery of the ability of GH to induce
PY-STAT5 (Fig. 6B).
As a washing control, H4 cells were washed without any prior GH
exposure and then incubated for 3 h in GH-free, serum-free media.
Simply washing alone, without GH pretreatment, had no significant effect on GHR levels and in the ability of GH to induce PY-STAT5 (Fig.
6, A and C). Conversely, when GH was maintained
in the medium for 4 h, with no recovery period in GH-free,
serum-free media, there was no recovery in GHR levels, which were
reduced to 10% of that in untreated cells, or in the ability of a
second GH exposure to induce PY-STAT5, which was also reduced to
~10% (Fig. 6, A and C).
Kinetics of GH-induced Phosphorylation of ERK1/2,
MEK1/2, and Akt--
The kinetics of GH-induced
phosphorylation of ERK (P-ERK), MEK (P-MEK), and Akt (P-Akt) were also
examined in H4 cells. Following addition of bGH (500 ng/ml), P-ERK1/2
was detected within 5 min, was maximal at 10-20 min, and decreased by
30 min, returning to basal, unmeasurable levels by 60 min (Fig.
7A, row
1). At lower concentrations of bGH, P-ERK1/2 levels were
increased to a lesser extent and not as rapidly. When comparing
different concentrations of GH, the maximal induction of P-ERK1 was 29, 44, and 82% at 50, 100, and 200 ng/ml, respectively, of the P-ERK1
induced by 500 ng/ml bGH (Fig. 7A and data not shown). When
multiple time-course experiments were averaged, it is evident that peak
P-ERK was achieved most rapidly at 500 ng/ml and was slowest at 50 ng/ml (Fig. 7B). Therefore, bGH at 50-500 ng/ml induced
P-ERK1/2, with greater maximum phosphorylation, and shorter times to
maximum phosphorylation at the higher doses of GH.
Addition of bGH at 100, 200, or 500 ng/ml to H4 cells also induced
phosphorylation of Akt (P-Akt; Fig. 7C; data shown for 500 and 100 ng/ml), whereas induction of P-Akt by 50 ng/ml bGH was barely
detectable (data not shown). Because MEK1/2 are direct upstream kinases
for ERK1/2, the kinetics of P-MEK1/2 induced by bGH were, as expected,
similar to that of P-ERK1/2, although induction of P-MEK1/2 was not as
extensive. GH-induced P-MEK1/2 was maximum between 10 and 20 min, and
decreased after 30 min (Fig. 8,
row 3).
Reduction in GH-induced Phosphorylation of ERK1/2,
MEK1/2, and Akt following 1-h GH Pretreatment followed by a
3-h Wash-out Period to Mimic the in Vivo Interpulse Interval--
When
H4 cells were pretreated with bGH for 1 h followed by 3 h in
GH-free, serum-free media, the GH-induced phosphorylation of both
ERK1/2 and MEK1/2 (Fig. 8, rows 1 and
3, respectively) were greatly diminished compared with that
induced by bGH prior to any pretreatment. When multiple similar
experiments were analyzed, a more rapid increase in phosphorylation of
ERK-1 was evident with increasing bGH concentrations for the first
addition of bGH (Fig. 9,
A-D). In addition, all bGH concentrations (50, 100, 200, and 500 ng/ml) resulted in desensitization of the ERK1/2 pathway to a
second bGH exposure. The second exposure to bGH never achieved greater
than 10-15% of the P-ERK1/2 induced by the first exposure (Fig. 9,
A-D).
To control for equal loading and to determine whether the effects on
P-ERK1/2 and P-MEK1/2 were dependent upon changes in the total amounts
of ERK1/2 and MEK1/2 protein, all blots were re-analyzed for the total
cellular amounts of ERK1/2 and MEK1/2 protein that were not
significantly affected, and certainly were not reduced (Fig. 8,
rows 2 and 4, respectively). Thus, the
decreased ability of the second GH exposure to induce phosphorylation
of ERK1/2 and MEK1/2 was not the result of decreases in the amounts of
these proteins, but of desensitization of GH signaling through the
MEK-ERK pathway.
Growth hormone induction of PI 3-kinase activity may be important for
ERK1/2 induction (19), so the ability of multiple GH exposures to
activate (phosphorylate) the PI 3-kinase substrate Akt was determined.
The first exposure to GH induced P-Akt, which was most noticeable at 5 and 10 min (Fig. 10, row
1). Following exposure to bGH for 1 h, followed by
3 h in GH-free, serum-free medium, there was no significant
induction of P-Akt by a second exposure to bGH. There may have been a
slight increase in total cellular Akt protein following GH pretreatment
and the 3-h wash-out period, but there was certainly no reduction of
total Akt (Fig. 10, row 2). Insulin, which can
induce P-Akt via the PI 3-kinase pathway in H4 cells (Fig. 10,
lane 14), was fully capable of stimulating phosphorylation of Akt at these time points (Fig. 10, lane
13). Therefore, similar to the MEK/ERK pathway, the first
bGH application followed by a 3-h incubation period, led to
refractoriness of P-Akt induction by a second exposure to bGH.
Lack of a Time-dependent Recovery of GH-induced
P-ERK--
There was no significant recovery of GH-induced ERK1/2
phosphorylation, even following many hours in GH-free, serum-free
medium, following the initial GH exposure. As indicated in Fig.
11A, GH administration for
20 min resulted in induction of P-ERK1/2 whether the cells were not
previously washed (Fig. 11A, lane
1) or whether the cells had been washed but without GH
pretreatment, and then placed into serum-free medium for 3 h (Fig.
11A, lane 9). However, following
1 h of GH, washing and 1, 2 (Fig. 11A), or 3 h
(Fig. 8) of GH-free, serum-free medium, GH was unable to induce
phosphorylation of ERK1/2. Surprisingly, even after 6 or 16 h in
GH-free, serum-free medium, GH was still unable to significantly induce
P-ERK1/2 (Fig. 11A). There were no changes in total ERK1/2
protein at any of the times tested. Additionally, even at these longer
time points, insulin, another inducer of P-ERK1/2 in these cells, is
fully active in inducing phosphorylation of ERK1/2 (Fig.
11B). Thus, this lack of recovery was not the result of
changes in ERK1/2 protein levels, the ability of ERK1/2 to be
phosphorylated by other hormones/growth factors, in this case insulin,
or the washing procedure. It was the prior exposure to GH that resulted
in refractory GH signaling. Compared with the recovery of GH-inducible
PY-STAT5 (data from Fig. 6C shown again in Fig.
11C for comparison), this suggests that the recovery of
GH-induced signaling through the ERK1/2 pathway was much delayed.
The data presented in this study suggest that addition of GH to H4
hepatoma cells induces degradation of GHR, which was resynthesized during the GH-free period. This degradation and resynthesis of GHR was
paralleled by desensitization and resensitization, respectively, of
GH-induced STAT5 phosphorylation. In the first experiments, as
expected, an initial exposure to GH resulted in a time- and concentration-dependent increase in PY-STAT5. Clearly shown
here is that GH addition also resulted in a rapid loss of
immunoreactive GHR and the rate of this loss was dependent upon the
concentration of GH added. However, by 60 min following GH addition,
GHR was reduced to ~25% of untreated levels by all concentrations of
GH used (50, 100, 200, or 500 ng/ml).
Several recent studies focused on the mechanisms of desensitization of
GH signaling by either pulsatile or continuous GH stimulation (7, 27,
38, 43, 44). Because of difficulties in directly measuring GHR levels,
it has not been clear whether or not short periods of GH exposure
(0-60 min) rapidly reduce GHR levels. Using Western blot analysis, the
altered intensity of GHR may be caused by reasons other than a change
in total cellular GHR protein levels. For example, spreading of the GHR
protein band on Western blots may be caused by GHR tyrosyl
phosphorylation and gel retardation of a fraction of GHRs, which occurs
rapidly following GH stimulation. Additionally, in IM-9 lymphocytes, GH
mediates internalization and translocation of GHR from a detergent
soluble to an insoluble cellular fraction (45). This results in a loss
of GHR detectable by Western blot analysis of the detergent extract,
making it difficult to determine whether there is reduction in total
GHR or loss of the detergent-extractable GHR (45). The present study
may have avoided these problems by boiling the harvested cells in 2%
SDS buffer, thereby including all subcellular compartments containing GHR (34).
GH-induced covalent modification of GHR could affect binding of GHR
antibodies. However, our two polyclonal antibodies for GHR were raised
against two overlapping, large segments of the cytoplasmic domain of
human GHR. Therefore, it is unlikely that GH-induced receptor
phosphorylation affects binding affinity of these two antisera against
GHR. Even if there were reduced binding affinity of GHR antibodies to
the phosphorylated GHR, it is unlikely that the reduced intensity of
GHR bands in samples treated for 45-90 min with GH would be the result
of this decreased affinity. Most of the tyrosyl-phosphorylated GHR
should have been dephosphorylated by 30-60 min (46), and inducible
serine/threonine phosphorylation of GHR has not been demonstrated.
Thus, the present study clearly indicates that GH exposure rapidly
reduced total GHR levels, as detected by Western blot analysis with our
two separate GHR antibodies, and that the rate of this reduction is
dependent upon the concentration of GH added.
It is not clear whether a GH-inducible or a constitutive
phosphatase is responsible for dephosphorylating tyrosyl-phosphorylated GHR/JAK2 and PY-STAT5. It is proposed that a GH-inducible, labile phosphatase may regulate the process of desensitization and
resensitization (31). Other studies suggest that
desensitization of the GH-induced JAK2/STAT5B pathway
requires de novo synthesis of an inhibitory protein,
possibly a phosphatase (31). In IM-9 lymphocytes, desensitization of
GH-stimulated JAK2 tyrosyl phosphorylation does not affect interferon- In the present study, a 10-fold higher concentration of bGH (500 versus 50 ng/ml) induced a 10-fold higher PY-STAT5 levels at
5 min, and also reduced GHR levels more rapidly, likely by more rapid
binding and increased GHR saturation at the higher GH concentration.
The slower reduction of GHR levels following a lower GH dose was also
supported by the corresponding delay in peak levels of P-ERK1. The data
presented here indicate that GH treatment reduced GHR levels to ~20%
of that in the untreated cells following a 60-min treatment with GH,
and GHR remained low (~10%) with constant GH for 4 h.
Consistent with the loss of GHR, PY-STAT5 levels following 4-h
treatment with GH were ~10% of the peak PY-STAT5 levels induced by
20-min bGH treatment. Thus, there was an approximately equal decrease
in GHR levels and in GH-inducible STAT5 tyrosyl phosphorylation,
implying a cause and effect relationship.
When hepatoma cells were exposed to GH for 60 min, washed, and allowed
to recover in GH-free, serum-free media, immunoreactive GHR was still
decreased to ~50% and a second application of GH further reduced GHR
levels. The rate of loss of GHR was greater in both the first and
second exposures to GH when 500 ng/ml GH was used as compared with 50 ng/ml. The slopes of the lines obtained from the rates of decrease of
GHR were approximately equal, suggesting that the rates of loss of GHR
in response to GH addition were equivalent following either the first
or the second exposure to GH. Thus, although there was less GHR at the
beginning of the second GH exposure, the mechanism of GHR loss seemed
to be similar based on the equal rates of GHR loss.
Significantly, when hepatoma cells were exposed to GH for 60 min,
washed, and allowed to recover in GH-free, serum-free media, the
reduction of GHR resulted in reduced GH-stimulated tyrosyl phosphorylation of STAT5, which can be measured by a phosphorylated tyrosine-specific STAT5 antibody. Less PY-STAT5 was induced by the
second GH exposure than by the first. GH-stimulated tyrosyl phosphorylation of STAT5 can also be measured by a change in mobility of STAT5B on polyacrylamide gels. Retardation of STAT5B is a sensitive indicator of PY-STAT5B. In the first exposure to GH, a larger percentage of STAT5B was retarded, whereas following the second exposure to GH, a lesser percentage of total STAT5B was retarded.
The rates of increase of PY-STAT5 following addition of GH were
approximately equivalent for both the first and the second GH
exposures. The slopes of the lines obtained from the rates of decrease
of PY-STAT5, presumably because of dephosphorylation of PY-STAT5, were
also approximately equal. Thus, although there is less GHR present at
the beginning of the second GH exposure, these data imply that the
rates of STAT5 phosphorylation and dephosphorylation were equivalent
following the first and the second exposures to GH. This suggests that
the phosphatase(s) necessary for PY-STAT5 dephosphorylation was present
in approximately equivalent amounts in untreated cells or in cells
treated with GH for 60 min and then allowed to recover in GH-free,
serum-free media.
The rate-limiting step(s) for resensitization of hepatocytes following
stimulation by a GH pulse is not well defined (23, 31). In the present
study, following a 1-h GH application, subsequent incubations with
GH-free, serum-free media for 1-16 h resulted in a
time-dependent recovery of immunoreactive GHR levels. This is most likely because of an increase in total cellular GHR levels, probably by de novo synthesis. Covalent modifications such
as phosphorylation and ubiquitination may be less likely to be found in
GHR following extended GH-free intervals. Therefore, the increase in
immunoreactive GHR levels in whole cell lysates should accurately reflect total cellular GHR levels. Although interpulse intervals with
non-detectable GH levels in the young adult male rats are ~3 h, GHR
levels in the H4 cells recovered to only approximately half of the
control levels following a 3-h incubation in GH-free, serum-free media.
We speculate that the rate of in vivo GHR
synthesis may be high enough for a full recovery during a 3-h period.
With cultured H4 cell incubations in GH-free, serum-free media, the lack of hormones may result in a slower synthesis rate of GHR, resulting in a longer time course of recovery. Additionally, in vivo exposure to GH following a pulse of secretion is most likely different from the full 1-h constant exposure used in the present studies. In vivo peaks of GH are just that, peaks, unlike
the "plateau" of constant GH used in the present studies.
Significant to this discussion, the gradual increase in GHR levels
correlated well with the recovery of GH signaling as measured by
GH-induced PY-STAT5. This suggests that resensitization of the
GH/JAK2/STAT5 pathway may be dependent on the recovery of GHR levels
during the GH-free interval. The JAK2/STAT5 pathway is one of the major
signaling pathways utilized by GH in the liver, and we have now found
that under several different experimental conditions (the data
presented here and in Ref. 42), there is a direct 1:1 correlation in
the amount of immunoreactive GHR and the ability of GH to induce
tyrosyl phosphorylation of STAT5.
However, when GH-induced phosphorylation of ERK1/2 is examined in H4
cells, the correlation between GHR levels and GH-induced signaling is
limited. Like induction of PY-STAT5, GH induction of P-ERK1/2 is
greater in magnitude and reaches a peak more rapidly when using higher
concentrations of GH. GH addition also results in stimulation of the
kinases upstream of ERK1/2, MEK 1/2. Similarly, GH addition results in
stimulation of a separate signaling pathway, the PI 3-kinase pathway or
a similar pathway, resulting in phosphorylation of Akt. Additionally,
like the decrease in GH-induced PY-STAT5, when H4 cells were pretreated
with GH for 60 min, washed, and allowed to recover for 3 h in
GH-free, serum-free media, the ability of GH to induce P-ERK1/2,
P-MEK1/2, and P-Akt was greatly diminished. This was true at all
concentrations used for the first and the second GH exposures (50, 100, 200, and 500 ng/ml).
Unlike GH-induced PY-STAT5, the present study demonstrated
persistent homologous desensitization of GH-induced phosphorylation of
ERK1/2 and Akt, and possibly PI 3-kinase activity, which is one pathway
resulting in phosphorylation of Akt (48). When H4 cells were pretreated
with GH, washed, and allowed to recover in GH-free, serum-free media,
the ability of GH to induce P-ERK1/2, P-MEK1/2, and P-Akt were
decreased to a much greater extent than the reduction of GHR.
Additionally, the desensitization of these signaling pathways to a
second GH exposure persisted for as long as 16 h in GH-free,
serum-free media, long after GHR had returned to pretreatment levels
and at a time that GH-induced PY-STAT5 had fully recovered. Insulin,
another inducer of both ERK1/2 and Akt activity in H4 cells, was still
able to stimulate phosphorylation of ERK1/2 and Akt following removal
of GH and incubation in GH-free, serum-free media. Thus, GH homologous
desensitization of MEK/ERK and Akt activation was profoundly less able
to recover as compared with GHR and the JAK/STAT5B pathway. For the
loss of GH induction of P-ERK1/2 and P-Akt, there is the requirement of
prior exposure to GH, which causes the desensitization, rather than
just the washing, of cells. The MEK/ERK and PI 3-kinase/Akt pathways
are shared pathways with insulin, whereas the JAK/STAT5B pathway is more specific to GH. It is therefore interesting to speculate whether
the insulin-like effect of GH, which only occurs after long periods of
GH deficiency and is lost after a single exposure to GH (49-53), may
be related to this loss of GH inducible MEK/ERK and PI 3-kinase/Akt signaling.
Our study suggests that repeated GH stimulation may induce
desensitization via the JAK2/STAT5 pathway by down-regulation of GHR,
and that resensitization of the JAK2/STAT5 pathway paralleled the
recovery of GHR levels. However, other post-receptor mechanisms may be
important in the refractoriness of the ERK1/2 and Akt pathways to a
second GH stimulation. This refractoriness is not a deficiency in
activable GHR, JAK2, or STAT5B, because they are fully activable by
6-16 h, whereas P-ERK1/2 and P-Akt are still resistant to a second GH
activation. Thus, both receptor and post-receptor mechanisms are
important in GH-induced homologous desensitization, but these mechanisms differ for different GH-sensitive signaling pathways.
We thank A. Keeton, M. Amsler, W. Bennett,
and J. Jiang for helpful and insightful discussions and suggestions. We
are grateful to A. F. Parlow (Pituitary Hormones and Antisera
Center, Harbor-UCLA Medical Center, Torrance, CA) and to the
National Institutes of Health NIDDK National Hormone & Pituitary
Program for the gift of bGH.
*
This work was supported by National Institutes of Health
Grant DK40456 and a grant from the American Diabetes Association (both
to J. L. M.) and by National Institutes of Health Grants DK46395 and DK58259 and a Department of Veterans Affairs merit review
award (all to S. J. F.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom all correspondence should be addressed: Dept. of Pathology,
Division of Molecular and Cellular Pathology, Volker Hall, G019, 1670 University Blvd., University of Alabama at Birmingham, Birmingham, AL
35294-0019. Tel.: 205-934-4921; Fax: 205-975-1126; E-mail:
messina@path.uab.edu.
Published, JBC Papers in Press, May 22, 2002, DOI 10.1074/jbc.M111723200
The abbreviations used are:
GH, growth hormone;
ERK1/2, extracellular signal-regulated kinases 1 and 2;
Grb2, growth
factor-binding protein 2;
GHR, growth hormone receptor;
JAK2, Janus
kinase 2;
MEK, mitogen-activated protein kinase kinase;
PI 3-kinase, phosphatidylinositol 3-kinase;
STAT, signal transducers and
activators of transcription;
SOS, Son of Sevenless;
bGH, bovine growth
hormone;
TBS, Tris-buffered saline;
IFN-
Growth Hormone-induced Differential Desensitization of STAT5,
ERK, and Akt Phosphorylation*
,
, and**
Department of Pathology, Division of
Molecular and Cellular Pathology, the § Department of
Medicine, Division of Endocrinology and Metabolism, and the
¶ Department of Cell Biology, University of Alabama at Birmingham,
and the Birmingham Veterans Affairs Medical Center,
Birmingham, Alabama 35294
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, 0.02% bromphenol blue) was added to three volumes of whole cell lysates, and the solution was boiled for an
additional 5 min and stored at 
80 °C until subjected to
polyacrylamide gel electrophoresis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Kinetics of GH-induced PY-STAT5. H4
cells were treated as described under "Experimental Procedures" and
at the times and concentrations of bGH indicated in the figures. Whole
cell lysates were prepared by boiling in 1% SDS buffer, and Western
blots were performed with an antibody for PY-STAT5. A and
B, representative Western blots. A, bGH at 500 ng/ml. C, densitometric analysis of the Western blots from
similar experiments (n = 3) were performed to quantify
PY-STAT5 levels at the time (x axis) and bGH concentrations
indicated. The data are expressed as mean ± S.E. The maximal
effect of each concentration of bGH was set to 100%, and the effects
of different times of GH treatment were expressed compared with those
concentrations' maximal effect (100%). At 500 ng/ml the maximal
effect of bGH on PY-STAT5 was at 10 min, whereas at 50, 100, and 200 ng/ml bGH the maximal effect of bGH on PY-STAT5 was at 30 min.
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Fig. 2.
Kinetics of GH-induced GHR reduction.
Western blots were performed as described in Fig. 1 and as indicated,
except for the use of the antibodies for GHR. A,
representative Western blot using the GHR antibody GHR3728.
B, densitometric analysis of the Western blots from similar
experiments (n = 3) were performed to quantify GHR
levels. The GHR-AL37 antibody was used as was bGH at the concentrations
indicated. The data are expressed as mean ± S.E. The GHR levels
in untreated cells were arbitrarily set to 100%, and the effects of
different concentrations and times of treatment with bGH were expressed
compared with GHR measured in untreated cells.
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Fig. 3.
Levels of GHR and GH-induced PY-STAT5
following GH pretreatment. Western blots were performed as
described in the legend for Fig. 1, except for the use of antibodies
for GHR (GHR-AL37), JAK2, PY-STAT5, and total STAT5B. The Western blots
are representative of three independent experiments at the times and
concentrations of bGH indicated. Two different exposures of the
PY-STAT5 probing are shown as rows A and
B. GH1, first GH exposure at 500 ng/ml;
GH2, second exposure to GH (at the same concentration, 500 ng/ml, and the times indicated) following the 1-h first exposure to bGH
and 3 h in GH-free, serum-free media.
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Fig. 4.
Levels of GHR following GH pretreatment at 50 or 500 ng/ml plus a 3-h wash-out period. A and
B, densitometric analysis of the Western blots from three
experiments similar to those in Fig. 3, row 1,
using 500 ng/ml bGH, and from similar experiments using a different
dose (50 ng/ml bGH) were performed. The data are expressed as
means ± S.E. The GHR levels in untreated samples were arbitrarily
set to 100%. GH1 (solid line), first
GH exposure; GH2 (dashed line), second
exposure to GH (at the same concentration and the times indicated)
following the 1-h first exposure and 3 h in GH-free, serum-free
media.
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Fig. 5.
Levels of GH-induced PY-STAT5 following GH
pretreatment plus a 3-h wash-out period. A,
densitometric analysis of the Western blots from three experiments
similar to those in Fig. 3, row 3, at 500 ng/ml
bGH. The data are expressed as means ± S.E. GH1
(solid line), first GH exposure; GH2
(dashed line), second exposure to GH (at the same
concentration and the times indicated) following the 1-h first exposure
and 3 h in GH-free, serum-free media. The PY-STAT5 levels
following 10 min of 500 ng/ml bGH was arbitrarily set to 100%.
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Fig. 6.
Levels of GHR and GH-induced PY-STAT5
following GH pretreatment and various durations of GH-free
incubation. Western blots were performed as described in Figs. 1
and 2 and as indicated with the use of GHR-AL37 and PY-STAT5
antibodies. A, representative Western blot. GH1,
first GH exposure at 500 ng/ml for the time indicated; GH2
(when applicable), second exposure to GH for 20 min following the 1-h
first exposure and varying times in GH-free, serum-free media. The
different times of GH-free, serum-free media (designated
SFM) vary between 1 and 16 h. In lanes
9 and 10, cells were treated with bGH
continuously for 240 min as an additional control and then treated with
(lane 9) or without (lane
10) bGH again for 20 min, respectively. Also in
lane 8, cells were not pretreated with GH, but
were washed and then incubated in fresh GH-free, serum-free media for
3 h before bGH addition for 20 min. B and C,
densitometric analysis of the Western blots from three experiments
similar to those in A were performed to quantify GHR and
PY-STAT5 levels. In B, GHR levels are indicated with the
filled diamonds and solid
line. Cells were pretreated with bGH for 1 h and the
reduction in GHR was measured (at time 0 of serum-free media). GHR then
increased following incubation in fresh GH-free, serum-free media for
the times indicated (at 1, 2, 3, 6, and 16 h of serum-free media).
Additionally, in B, PY-STAT5 levels following 20 min of GH
are indicated by the open circles and
dashed line. The PY-STAT5 levels were measured
following pretreatment with 1-h GH and then incubation in fresh
GH-free, serum-free media for the times indicated (at 1, 2, 3, 6, and
16 h of serum-free media) prior to the 20 min bGH treatment. The
data are expressed as mean ± S.E. The GHR levels in untreated
cells and the PY-STAT5 levels following 20 min of GH alone in untreated
cells were both arbitrarily set to 100%. In C, different
times of GH treatment (500 ng/ml bGH) with or without washing and 0 or
3 h in fresh GH-free, serum-free media.
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Fig. 7.
Levels of GH-induced P-ERK and P-Akt.
Western blots were performed as described in Fig. 1 and as indicated,
except for the use of antibodies for P-ERK1/2 and P-Akt. A
and C, representative Western blots with the bGH
concentrations and treatment times indicated or insulin (1 × 10 
7 M). B, densitometric analysis
of the Western blots from three independent experiments using different
doses of bGH was performed to measure times to peak P-ERK1 levels
(x axis) at the bGH concentrations indicated. The data are
expressed as mean ± S.E. The maximal effect for each
concentration of bGH was set to 100%, and the effects of different
times of GH treatment were expressed compared with that
concentrations' maximal effect. The levels following 20 min of GH at
500, 200, and 100 ng/ml, or 30 min of GH at 50 ng/ml, were the times
for maximal P-ERK1 levels at that concentration and were set to
100%.
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Fig. 8.
Phosphorylation of ERK and MEK following GH
pretreatment plus a 3-h wash-out period. Western blots were
performed as described in Fig. 1 and as indicated, except for the
use of antibodies for P-ERK1/2, total ERK1/2, P-MEK1/2, and total
MEK1/2. A representative Western blot is presented. GH1,
first GH exposure at 200 ng/ml; GH2, second exposure to GH
(at the same concentration, 200 ng/ml, and the times indicated)
following the 1-h first exposure to bGH and 3 h in GH-free,
serum-free media.
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Fig. 9.
Induction of P-ERK1/2 following GH
pretreatment plus a 3-h wash-out period. A-D,
densitometric analysis of autoradiographs from multiple experiments
(n = 3) similar to that represented in Fig. 8 were
performed to quantify P-ERK1 levels following different concentrations
of bGH. GH1 (solid line), first GH
exposure; GH2 (dashed line), second GH
exposure after the first (at the same concentration) for 1 h
followed by 3 h in GH-free, serum-free media. The data are
expressed as mean ± S.E. The P-ERK1 levels following 30 min of GH
at 50 ng/ml, and 20 min of GH for all other doses, were arbitrarily set
to 100%.
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Fig. 10.
Induction of P-Akt following GH pretreatment
plus a 3-h wash-out period. Western blots were performed as
described in the legend for Fig. 1, except for the use of the
antibodies for P-Akt and total Akt. GH1, first GH exposure
at 500 ng/ml; GH2, second exposure to GH (at the same
concentration, 500 ng/ml and the times indicated) following the 1-h
first exposure to bGH and 3 h in GH-free, serum-free media.
Insulin was added for 5 min at 1 × 10 7
M.
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Fig. 11.
Induction of P-ERK1 and PY-STAT5 following
GH pretreatment and various durations of GH-free incubation.
A, representative Western blot performed as described in the
legend for Fig. 1, except for the use of the antibody for P-ERK1/2.
GH1, first GH exposure at 500 ng/ml for 20 or 60 min;
GH2 (when applicable), second exposure to GH for 20 min
following the 1-h first exposure and varying times in GH-free,
serum-free media (SFM). B, representative Western
blot performed as described in A, except comparing the
effects of GH alone, insulin (1 × 10 7
M) alone, or insulin following GH pretreatment for 60 min
and then 16 h in GH-free, serum-free media. C,
densitometric analysis of Western blots from three similar experiments,
as represented in A, with incubation in GH-free, serum-free
media for 1, 2, 3, 6, and 16 h. The P-ERK1 levels induced by 20 min of 500 ng/ml bGH following 1-h pretreatment with 500 ng/ml bGH and
the various periods of incubations in GH-free, serum-free media
(x axis) were expressed as a percentage of the normal P-ERK1
induction by 20 min bGH in non-bGH-pretreated cells. The data are
expressed as mean ± S.E. and indicated by the filled
triangles and dashed line.
Additionally, replicated from Fig. 6C for comparison,
PY-STAT5 levels following 20 min of bGH and the various periods of
incubations in GH-free, serum-free media are indicated by the
open circles and solid
line.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(IFN-)-stimulated phosphorylation of JAK2 (47), suggesting that if there is a de novo synthesized inhibitor
or phosphatase, it must be specific for the GHR/JAK2, but not the IFN-/JAK2 pathway. Alternatively, if GH-induced desensitization of
its own pathway is achieved by down-regulating GHR, it is simple to
envision how the JAK2 response to IFN- can be preserved. The present
data demonstrate a rapid down-regulation of GHR by GH exposure and a
constitutive, rather than GH-inducible, protein-tyrosine phosphatase is
probably sufficient to dephosphorylate and inactivate this pathway.
Thus, our work supports the hypothesis that desensitization following a
GH pulse is the result of the reduction of GHR at the cell surface
following GH binding and receptor internalization/degradation.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
, interferon .
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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