Department Of Molecular Medicine (A.F.-M., E.R.-B., G.N.), Karolinska Institute, 17176 Stockholm, Sweden; Health Science Center, Pharmacology Section (L.F., C.N.), Las Palmas de Gran Canaria University, 35080 Las Palmas de Gran Canaria, Spain; The Walter and Eliza Hall Institute of Medical Research and The Cooperative Research For Cellular Growth Factors, Victoria 3050, Australia (J.-G.Z.); and Department Of Chemistry, National University, Bogota, Colombia (A.U.)
Address all correspondence and requests for reprints to: Amilcar Flores Morales, Ph.D., Centrum for Molecular Medicine, CMM, L8:01, Karolinska Hospital, 171 76 Stockholm, Sweden. E-mail: Amilcar. Flores{at}molmed.ki.se
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ABSTRACT |
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INTRODUCTION |
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The nature of cytokine actions is often dependent on the kinetics of
JAK/STAT activation. For example, growth arrest of malignant lymphoma
cells by IFN has been related to a prolonged rather than transient
activation of STAT1 (7), while continuous STAT5b
activation by the male pattern of GH secretion is essential for the
liver sex differentiation in rodents (8). Cytokines often
exert their actions concomitantly with stressful situations to the
cell. Therefore, it is important to establish whether stress-induced
cellular pathways can modulate cytokine signaling, as this interaction
can change the outcome of cytokine actions. For these reason we have
undertaken to investigate if the cellular response to stress can
modulate the JAK/STAT pathway.
Cellular stress initiates a complex cascade of stress-inducible enzymes
and transcription factors in an attempt to adapt to changes in the
immediate environment (9, 10). The nature of the cellular
response depends on the type and dose of stress. The correct folding of
proteins in the endoplasmic reticulum can be altered by certain types
of stressful stimuli, and this generates a powerful cellular reaction
known as the unfolded protein response (UPR). The UPR can be triggered
by a variety of agents including glycosylation inhibitors, reducing
agents, glucose starvation, hypoxia, viral particles, growth factor
depletion, and perturbation of intracellular calcium homeostasis
(11). These agents provoke the accumulation of unfolded
proteins in the endoplasmic reticulum, which in turn leads to
inhibition of protein translation initiation. This occurs through
activation of protein kinases that specifically phosphorylate the
-subunit of eukaryotic translation initiation factor 2 (eIF-2
). A
small increase in the phosphorylation of eIF-2
rapidly results in
inhibition of the initiation of protein synthesis. In addition to
changes in the translational machinery, the UPR selectively activates
transcription of genes encoding endoplasmic reticulum resident
chaperones (e.g. Bip/GRP78) that perform diverse roles to
maintain endoplasmic reticulum function and facilitate protein folding.
The UPR also triggers the transcriptional activation of the
transcription factor C/EBP homology protein (CHOP), also known as
growth arrest and DNA damage protein 153 (GADD153) (12).
CHOP is not normally expressed in cells but is induced by a variety of
stress insults. This factor can heterodimerize with members of the bZIP
family of transcription factors and, in doing so, alters gene
expression and regulates both cell death and tissue regeneration after
stress (13).
Previous investigations concerning the effects of chemical agents on the duration of JAK 2/STAT5 signals have led us to hypothesize a possible relation between cellular response to stress and the kinetics of JAK2/STAT5 activation by GH. In the present study we have shown that the induction of UPR can prolong the GH-induced JAK/STAT signaling pathway. Our findings suggest that novel mechanisms to control the transient activation of JAK2 and STAT5 proteins exist. Possibly endoplasmic reticulum stress may induce effects on SOCS actions other than the inhibition of its expression. These findings imply that cellular responses to endoplasmic reticulum stress may affect the outcome of cytokine stimulation.
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RESULTS |
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To substantiate that BAPTA-AM activates an UPR response in BRL cells,
experiments were performed to analyze the rate of protein translation
and the expression of the transcription factor CHOP (11).
As shown in Fig. 2, BAPTA-AM caused a
rapid inhibition of protein synthesis within the first hour after
treatment. GH added in combination with BAPTA-AM caused no effect
compared with the drug alone. Changes in protein translation during
stress are, in many cases, related to
phosphorylation of eIF-2
, which inhibits binding
of Met-tRNA to the initiation complex. As shown in Fig. 2B
, treatment
with BAPTA-AM induced the phosphorylation of eIF-2
at serine 51.
Analogous with the protein synthesis results, GH did not have any
marked influence on the BAPTA-AM effect on eIF-2
phosphorylation.
Interestingly, BAPTA-AM as well as GH, caused a rapid increase in CHOP
mRNA (Fig. 2C
), and an additive effect was observed at the 30- and
45-min time point with the combined treatment. The effect of GH is
greater than the effect of BAPTA plus GH at the 60- and 90-min time
point. Taken together, these findings show that BAPTA-AM induces
endoplasmic reticulum stress, and that GH might regulate the cellular
responses downstream of CHOP.
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We next studied the effects of BAPTA-AM on GH-induced SOCS mRNA. SOCS
mRNA levels were increased in the presence of BAPTA-AM (Fig. 5). GH in combination with BAPTA-AM
caused higher SOCS induction than GH alone. The most likely
interpretation is that BAPTA-AM causes a translational arrest that
blocks the production of SOCS proteins normally destined to attenuate
GH signals. A failure in SOCS synthesis would result in the
accumulation of GH-induced transcripts including SOCS mRNAs.
Alternatively, the mechanism that actively degrades SOCS mRNA may be
sensitive to stress interference.
Endoplasmic Reticulum Stress Does Not Inhibit GH-Dependent
Expression of SOCS Proteins but Regulates Its Turnover
We wanted to test the hypothesis that cellular stress-induced
inhibition of protein translation would result in the reduction of SOCS
proteins in response to GH and, consequently, in the prolongation of GH
signaling. Therefore, the levels of SOCS-1, -2, and -3 proteins were
analyzed after BAPTA-induced stress in the presence or absence of GH.
As shown in Fig. 6A, GH treatment of
BRL-4 cells induced the expression of SOCS-1 and -2. SOCS-1 induction
was maximal after 30 min with GH treatment while SOCS-2 showed maximal
induction after 1 h of GH stimulation. When BAPTA-AM was added in
combination with GH, it did not modify the expression of SOCS-1
protein. BAPTA-AM blocked the GH effects on SOCS-2 at 2 h of
treatment, which correlates with the effects of this endoplasmic
reticulum stressor on GH-induced JAK2/STAT5 activity. Interestingly,
despite the finding that BAPTA-AM rapidly inhibits protein synthesis
(see Fig. 2A
), the de novo GH induction of SOCS proteins was
not affected. This suggests that translation inhibition is not enough
to prolong GH signaling and that other signals activated by
BAPTA-AM-induced endoplasmic reticulum stress are necessary. This
observation is supported by experiments in which the negative
regulation of GH-induced STAT5 DNA binding activity is not affected by
short-term inhibition of protein translation by CHX (Fig. 6B
). Despite
the fact that treatment of BRL-4 cells with CHX for 1 h resulted
in a 95% inhibition of protein translation (data not shown), there is
no evidence for the prolongation of GH-induced STAT5 DNA binding
activity by GH (Fig. 6B
). As previously reported (2),
long-term inhibition of protein translation (4 h) was necessary to
prevent down- regulation of GH-induced JAK2/STAT5 activity.
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DISCUSSION |
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The discovery of the SOCS family has provided a new aspect of cytokine receptor regulation that we believe is especially relevant in the present study (3). SOCS proteins function in novel ways to suppress signal transduction pathways. SOCS are SH2 and SOCS box-containing proteins that bind either JAK or cytokine receptors and serve as negative regulators. Transcription of SOCS genes rapidly increases in response to cytokines, both in vitro and in vivo. This suggests that they may act in a classic negative feedback loop to regulate cytokine signal transduction (3). Overexpression experiments have shown that SOCS-1, -2, and -3 and CIS are able to inhibit GH-induced STAT5 activation (5). SOCS-1 has been shown to directly bind and inhibit JAK2, while the SOCS-3 and -2 and CIS mechanism of action remains to be determined (18). The N-terminal and SH2 domains, but not the C-terminal (SOCS homology domain) (6, 18, 19) domain of SOCS proteins, seem to be required for their inhibitory action, which suggests that binding to phosphotyrosine residues in the GHR/JAK2 complex is important. Bound SOCS may act in various ways that are not yet defined, e.g. as a direct kinase inhibitor (20), as an adaptor protein to promote association to phosphotyrosine phosphatases (21, 29), or to target the activated receptor for internalization for protein degradation through the ubiquitin/proteasome system (28). The potential role of SOCS in the regulation of GH responsiveness encouraged us to study SOCS expression in relation to pharmacological agents that induce cellular stress or interfere with a variety of signaling pathways.
Our finding that GH induces SOCS-1, -2, and -3 and CIS gene expression
in BRL-4 cells, as well as in primary rat hepatocytes (4),
is consistent with the notion that SOCS proteins serve as molecules
that turn off GH signals. SOCS-2 mRNA increased steadily in the
presence of GH, while SOCS-1 and -3 and CIS mRNA levels were
transiently induced by GH with a maximal level at 1 h after GH
treatment. The similar kinetics between SOCS-1 and -3 and CIS
regulation may reflect the presence of similar types of GH response
elements in the promoters of these genes. It has previously been shown
that CIS and SOCS-3 gene promoters contain STAT response elements that
are responsible for induction of these genes by erythropoietin
(21) and leukemia inhibitory factor, respectively
(22). STAT-mediated regulation of CIS and SOCS-3 gene
expression would be in agreement with the transient time course of STAT
activation by GH. The transient kinetics of GH-induced SOCS-1 and -3
and CIS expression speaks against their involvement in the long-term
negative regulation of GHR signaling. In this sense it would be more
reasonable if SOCS-2 were involved since its mRNA level is steadily
elevated in the presence of GH (Fig. 4A). In this context, it is also
relevant to note that GH-induced SOCS mRNAs fall rapidly in the absence
of GH (Fig. 4B
), indicating that SOCS mRNAs have a rapid turnover.
We have previously shown that transcriptional inhibition results in the prolongation of GH-induced STAT5 DNA binding activity (2). Since H7 blocks a broad spectrum of rapidly induced mRNAs by diverse stimuli, including those induced by cytokine receptor activation (14), it is likely that the mechanism of action of H7 is related to the inhibition of the GH-induced transcriptional activation of SOCS genes. A mechanistic explanation for this has recently been described; H7 is a strong inhibitor of phosphorylation of the tandem repeats found in the C-terminal domain of RNA polymerase II (23). Furthermore, GH-stimulated STAT5 DNA binding activity is prolonged by DRB, which is a more specific inhibitor of RNA polymerase II phosphorylation. Phosphorylation of RNA polymerase II C-terminal domain by a kinase activity associated with the transcription initiation complex is considered a necessary step in the establishment of a progressive, rather than an abortive, transcription complex (24). The PLC inhibitor D609 is also able to prolong the activation of GH-induced JAK2/STAT5 pathway (2). Nevertheless, it is unlikely that the effects of D609 are related to PLC inhibition as we were not able to detect GH-dependent induction of PLC activity in BRL-4 cells (data not shown). D609 inhibits the transcription of SOCS1, -2, and -3 and CIS genes (data not shown), which further supports the notion that GH-induced SOCS mRNA is necessary for the long-term down-regulation of the JAK2/STAT5 signaling pathway in the continued presence of GH.
The endoplasmic reticulum stressors, BAPTA-AM, DTT, and A23187, were
able to prolong the duration of JAK2/STAT5 activation in the presence
of GH. In the case of BAPTA-AM, it prolongs GH signaling independently
of the levels of SOCS mRNAs. Furthermore, BAPTA-AM increased, in
combination with GH, the levels of SOCS-1, -2, and -3 and CIS mRNA to a
level higher than that by GH alone. On the premise that GH-activated
JAK2/STAT5 activity is down-regulated by SOCS, one may postulate that
endoplasmic reticulum stress blocks SOCS actions. The absence of a
negative feedback regulation to the GHR and the resultant continued
activation of JAK2 will result in a prolonged activation of
transcription, which would increase the levels of GH-regulated
transcripts, including levels of SOCS and CHOP mRNAs. Interestingly,
the stress-induced phosphorylation of eIF-2 and the translation
arrest are independent of GH. We have also noticed that CHOP mRNA, in
addition to being increased by BAPTA-induced stress, is also
GH-regulated. The functional significance of these findings merits
further evaluation as CHOP has been shown to have important regulatory
actions on the transcriptional activity of stress-regulated
transcription factors of the bZIP family, such as JUNs and C/EBPs
(25, 26).
The use of BAPTA-AM in BRL-4 cells can be regarded as a model for the UPR. A variety of signals can trigger this response, including oxidative and osmotic stress, viral infection, hypoxia, and amino acid starvation. In consideration of the putatively short life span of SOCS mRNA, the UPR signal could influence the cellular level of SOCS by preventing SOCS translation. Thus, stress-induced translational arrest, if sufficiently long, will reduce SOCS protein levels, resulting in a prolonged duration of JAK/STAT activation. Accordingly, we have previously shown that long-term incubation with CHX (up to 4 h) prevents desensitization of GH-induced JAK2 and STAT5 signaling (2).
There seems to be an additional and more rapid mechanism whereby stress
influences the deactivation of the JAK2/STAT5 pathway. Blocking protein
synthesis by treating BRL-4 cells with CHX during 1 h before GH
stimulation does not prolong GH-induced STAT5 DNA binding activity
within the time frame studied here. In contrast, BAPTA-AM, which also
induced rapid translational arrest, was able to prolong GH-induced
JAK2/STAT5 signaling pathway. When we performed immunodetection of SOCS
proteins in BAPTA-AM-stressed cells, no major effects were observed
regarding the GH induction of SOCS-1 and SOCS-2 proteins.
Interestingly, a reduction in the level of SOCS-2 was evident at 2
h of combined treatment of GH with BAPTA-AM. To assign the effects
observed in the activity of the JAK2/STAT5 pathway to the changes in
SOCS-2 levels is speculative. It may be possible that other members of
the SOCS family, such as CIS, are specifically targeted by signals from
endoplasmic reticulum stress and are responsible for the prolongation
of JAK/STAT signal. Another alternative is that components downstream
of the SOCS pathway are sensitive to the UPR. A mechanism of
interference between the UPR and the proteasome degradation of
ER-associated proteins has recently been described in yeast
(27). Based on its homology with the Von Hippel-Lindau
domain (17), it has been suggested, but not proven,
that SOCS may act as part of an ubiquitin-ligase complex promoting the
ubiquitination of cytokine receptor/JAK complexes. Ubiquitination of
the phenylalanine 346 in the intracellular domain is necessary for GHR
internalization (28). Thus, it may be that SOCS negatively
regulates GHR signaling by promoting ubiquitination, internalization,
and proteasome degradation of the GHR/JAK2 complex. Accordingly, the
inhibition of proteasome activity by MG132 blocks the long-term
negative regulation of STAT5 DNA binding activity in the continuous
presence of GH. Although the complex nature of the UPR offers a large
number of putative targets that needs to be further investigated
(27), it may be speculated that endoplasmic reticulum
stress acts to interfere with the SOCS actions on the ubiquitination,
internalization, or the proteosomal degradation of the GHR/JAK2
complex. However, there is evidence against the involvement of receptor
internalization for the negative regulation of the JAK/STAT pathway.
Mutation of phenylalanine 346 inhibits the GHR internalization but has
no effect on the GH- induced STAT5-mediated transcriptional
activity (28). It is also worth noting that the rapid and
partial (60% of control) down-regulation of GH-stimulated STAT5
binding activity (Fig. 6C
) was not affected by proteasome inhibition.
Alternatively, the stress signal may target a mechanism unrelated to
the SOCS actions, such as the deactivation of the GHR/JAK2 complex by a
phosphotyrosine phosphatase. It has been recently reported that the
tyrosine phosphatase SHP-2 is a target for the GHR/JAK2 complex, and
mutations in the receptor that inhibit this association prolong the GHR
signal (29). The sensitivity of this mechanism to the UPR
requires further analysis. The results of the treatments with BAPTA-AM
and MG132 show that partial short-term down-regulation can be
dissociated from long-term resistance to JAK/STAT activation induced by
continued treatment with GH. Both short-term down-regulation and
long-term resistance may represent different phenomenon and therefore
distinctive mechanisms (e.g. dephosphorylation or
ubiquitination) may exist to govern both processes.
The duration of JAK/STAT signaling is likely to be an important factor
in regulating the biological outcome of cytokine stimulation. Transient
activation of liver STATs has been linked to physiologically
sex-specific actions of GH (8), while growth arrest of
malignant lymphoma cells by IFN- has been related to a prolonged,
rather than transient, activation of STAT1 (7). STATs are
known to interact with several transcription factors such as nuclear
factor-
B (30), nuclear receptors (31), and
SMA and MAD-related proteins (SMADs) (32), interactions
that may be of relevance for the regulation of the cellular stress
response. A stress-induced reduction in the negative regulation
the JAK/STAT signaling pathway, thought to be dependent on a disrupted
SOCS action, may be of functional significance. The regulation of the
JAK/STAT pathway by cellular stress could play a role in apoptosis
(33), and such a mechanism may be related to the
pathological consequences of stress (34).
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MATERIALS AND METHODS |
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Cell Culture
Buffalo rat liver cells (BRL) stably transfected with the rat
GHR cDNA, designated BRL-4 cells and previously shown to respond to hGH
(2), were cultured in DMEM supplemented with 10% FCS, 50
U/ml penicillin, and 50 µg/ml streptomycin. Cell culture reagents
were obtained from Life Technologies, Inc. (Gaithersburg,
MD). Treatment of the cells with hGH and/or inhibitors is specified in
the figure legends and in Results. All experiments were
performed at least three times.
Analysis of mRNA Expression
Total RNA was isolated using TRIZOL Reagent (Life Technologies, Inc.), according to the protocol supplied by the
manufacturer. mRNA levels corresponding to SOCS1, -2, -3, CIS, and
glyceraldehyde-3-phosphate-dehydrogenase (35) gene
expression were measured in total RNA samples. This was done using a
solution hybridization/RNase protection assay.
Transcript-specific 35S-labeled cRNA probes were
transcribed in vitro from the respective cDNA vector
construct, as described previously (4). Specific mRNA was
quantitated by comparison with a standard curve obtained from
hybridizations to known amounts of in vitro synthesized
mRNA. The concentration of nucleic acid samples was measured
spectrophotometrically. Samples were analyzed in triplicate, and the
results are expressed as picograms of specific mRNA per µg total RNA.
To permit accurate comparison of specific mRNA levels between different
treatments, the samples of interest were always analyzed at the same
time.
Preparation of Nuclear Extracts
BRL-4 cells were grown to confluence in 100-mm culture dishes
and serum starved for 16 h before the addition of inhibitors
and/or hGH. After treatment, the cells were washed twice with cold PBS,
and nuclear extracts were prepared essentially as described
(2). In brief, the cells from 100 mm-diameter dishes were
collected by centrifugation, resuspended in hypotonic buffer [10
mM Tris-HCl, pH 7.4, 10 mM NaCl, 6
mM MgCl2, 1 mM DTT, 0.4
mM phenylmethylsulfonyl fluoride (PMSF), 10 mM
NaF, 1 mM
Na3VO4, and 1 µg/ml
pepstatin, aprotinin, and leupeptin] equal to approximately 3 times
the packed cell volume and incubated on ice for 10 min. The cells were
lysed with 20 strokes in a Dounce homogenizer (pestle B), and the
nuclei were collected by centrifugation and resuspended in 3 volumes of
high salt buffer (20 mM HEPES, pH 7.9, 420 mM
NaCl, 20% glycerol, 1.5 mM MgCl2,
0.2 mM EDTA, 1 mM DTT, 1 mM
Na3VO4, and protease
inhibitors). After 30 min at 4 C, the pellets were removed by
centrifugation, and supernatants containing the nuclear proteins were
stored at -70 C until use. The protein concentration was measured
using Bradford assay (Bio-Rad Laboratories, Inc.,
Richmond, CA) using BSA as standard.
EMSA
EMSA was performed according to standard protocols
(13). The binding reactions were performed by
preincubating 8 µg nuclear extract with 2 µg of poly (dI-dC) in 20
µl buffer containing 20% Ficoll, 60 mM HEPES, pH 7.9, 20
mM Tris, pH 7.9, 0.5 mM EDTA, and 5
mM DTT for 10 min at room temperature.
32P-end-labeled double-stranded SPI.GLE1
(TGTTCTGAGAAATA) (36) was added, and the mixture was
incubated for 10 min at room temperature. The samples were
electrophoresed on 4.5% nondenaturing polyacrylamide gels in 0.25
x TBE (1 x TBE; 0.09 M Tris-borate, 2 mM
EDTA) at 150 V at room temperature. The radioactive pattern was
visualized by autoradiography and quantified by
PhosphorImager scanning (Fuji Photo Film Co., Ltd., Stamford, CT).
Metabolic Labeling
Exponentially growing cells were treated with GH and BAPTA-AM as
described in Fig. 2, rinsed with methionine-free DMEM, and incubated
for 30 min with methionine-free medium supplemented with
trans-35S-methionine (50
µCi/ml) (NEN Life Science Products, Boston MA) for 30
min at 37 C. After washing three times with PBS, cells were harvested
in lysis buffer (150 mM NaCl, 50
mM Tris·HCl, pH 7.5, 0.05% SDS, 1% Nonidet
P-40, 1 mM benzamidine, 1
mM EDTA, 1 mM PMSF). The
protein concentration of the extracts was measured using Bradford assay
(Bio-Rad Laboratories, Inc.) with BSA as standard. An
aliquot of lysate containing equal amounts of protein was precipitated
with trichloroacetic acid and counted in a scintillation counter.
Western Blot and Immunoprecipitation
BRL-4 cells were grown to near confluence in 100-mm cultured
dishes and treated as described in the figure legends. Thereafter,
cells were washed three times with ice-cold PBS and harvested in 1 ml
RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 150
mM NaCl, 5 mM EDTA, 1 mM PMSF, 1
mM Na3VO4, 1
mM DTT, and protease inhibitors). Aliquots from cleared
lysates containing 30 µg protein were subjected to electrophoresis on
SDS-PAGE. Separated proteins were electroblotted to polyvinylidene
difluoride membranes (Millipore Corp. Bedford, MA).
Detection of JAK2 and STAT5 was performed by Western blotting with
anti-JAK2 antibody directed against murine JAK2 (Upstate Biotechnology, Inc. Lake Placid, NY) and monoclonal anti-STAT5
antibody (Transduction Laboratories, Inc., Lexington, KY)
as described (2), respectively. JAK2 phosphorylation was
determined by immunoprecipitation with antiphosphotyrosine monoclonal
antibody (PY20, Transduction Laboratories, Inc.), followed
by Western Blot with anti-JAK2. Phosphorylation of eIF-2 at Serine
51 was detected by Western blotting with a specific antibody against
the phosphorylated form of the protein (Research Genetics, Inc., Huntsville, AL), as previously described
(37). SOCS-1 levels were determined by immunoprecipitation
of 800 µg of cell extracts with monoclonal 4H1 raised against the
N-terminal domain of SOCS-1, followed by Western blot with the same
antibody. SOCS-2 levels were estimated by immunoprecipitation of 1 mg
of whole-cell extracts with 1 µg monoclonal antibody (4H8) raised
against SOCS-2, followed by Western blot with monoclonal
antibody 2D6 anti-SOCS-2. SOCS-3 levels were determined by
inmunoprecipitation of 800 µg of cell extracts with rabbit
anti-SOCS-3 polyclonal antiserum raised against the full-length murine
SOCS-3, followed by Western blot with 1B2 anti-SOCS-3 monoclonal
antibody raised against N-terminal domain of murine SOCS-3. All
antibodies against SOCS proteins were a kind gift from Dr. Douglas J.
Hilton.
Statistics
The data are presented as the mean ± SD
(standard deviation of the mean). Statistical comparisons for each of
the treated samples with the control sample (vehicle) were tested using
the paired t test. The significance of differences among
groups was determined using ANOVA with multiple comparisons among
means, computed by using the Student-Newman-Keuls test. Statistical
significance was reported if P < 0.05 was
achieved.
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FOOTNOTES |
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Abbreviations: BAPTA-AM,
1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid
(acetoxymethyl)ester; CHOP, C/EBP homology protein; CHX,
cycloheximide; CIS, cytokine-induced SH2 protein; DRB,
5,6-dichloro-1-ß-D-ribofuranosylbenzimadole; DMSO,
dimethylsulfoxide; DTT, dithiothreitol; eIF-2, eukaryotic
translation initiation factor-2
; GHR, GH receptor; JAK, Janus
kinase; IFN, interferon; PMSF, phenylmethylsulfonyl fluoride; SOCS,
suppressors of cytokine signaling; STAT, signal transducer and
activator of transcription; UPR, unfolded protein response.
Received for publication April 19, 2001. Accepted for publication June 4, 2001.
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REFERENCES |
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