Departments of 1 Pediatrics, 2 Biochemistry, Molecular Biology and Biophysics, and the 3 Institute of Human Genetics, University of Minnesota, Minneapolis, Minnesota 55455
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Growth hormone (GH) action is attenuated during the hepatic acute-phase
response (APR). To understand this attenuation, we asked whether GH and
cytokine-signaling pathways intersect during an APR. In
hypophysectomized rats treated with lipopolysaccharide (LPS),
accumulation of activated signal transducer and transcription activator
5 (Stat5) in hepatic nuclei in response to GH and its binding to a GH
response element (GHRE) from the serine protease inhibitor (Spi) 2.1 promoter are diminished in a time-dependent manner. Similarly,
accumulation of activated Stat3 in hepatic nuclei in response to LPS
and its binding to a high-affinity sis-inducible element
(SIE) are also diminished by the simultaneous administration of GH. In
functional assays with primary hepatocytes, LPS-stimulated monocyte-conditioned medium (MoCM) inhibits the GH response of Stat5-dependent Spi 2.1 reporter activity but induces Stat3-dependent Spi 2.2 reporter activity, as in an APR. Similar results are obtained when hepatocytes are treated with either tumor necrosis factor- (TNF-
) or interleukin (IL)-1
. TNF-
, IL-1
, and IL-6 also
inhibit GH-induced Spi 2.1 mRNA expression in hepatocytes. Thus
inhibition of the GH signaling pathway during an APR results in reduced
expression of GH-responsive genes.
signal transducers and activators of transcription proteins; rat liver; serine protease inhibitors 2.1 and 2.2; lipopolysaccharide
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
INFLAMMATORY
STIMULI, such as thermal burns or administration of turpentine or
lipopolysaccharide (LPS), result in a series of coordinated changes in
expression of a group of hepatic genes. These alterations, known
collectively as the hepatic acute-phase response (APR), occur in part
at the level of transcription and are important in the restoration and
maintenance of homeostasis after injury (3, 15, 24, 33,
34). These events are mediated predominantly by cytokines
released by monocytes and hepatic macrophages in response to these
stimuli (52). These cytokines include interleukin-6
(IL-6), tumor necrosis factor- (TNF-
), and interleukin-1
(IL-1
) (9, 10). Although cytokines stimulate increased
expression of one subgroup of hepatic genes, expression is decreased in
another subgroup, referred to as negative acute-phase reactants
(30, 31).
Several growth hormone (GH)-responsive genes are among the negative acute-phase reactants, suggesting that factors responding to GH may be specifically targeted during an APR. There is evidence that the suppression of GH action has clinical consequences when inflammation is severe or chronic. Suppression of linear growth is a hallmark of chronic inflammation in childhood, contributing significantly to the morbidity of inflammatory disease for children. For example, in juvenile rheumatoid arthritis, plasma insulin-like growth factor I (IGF-I) and IGF binding protein-3 levels, both of which are under direct GH regulation, are reduced (11). Patients with trauma or sepsis also have decreased IGF-I levels (18, 23, 49). Treating such patients with GH before the development of inflammation may, however, significantly reduce their ability to restore homeostasis. This is one of the proposed mechanisms for the excess mortality in critically ill patients treated with GH (58). Thus the balance between the need to promote homeostasis through the APR and the need for growth of essential tissues is a key survival issue in prolonged or chronic inflammatory illness.
To understand how the APR interferes with the transcription of GH-responsive genes, we evaluated the APR and GH responses of the rat serine protease inhibitor 2 (Spi 2) locus. Spi 2.1 is GH responsive and a negative acute-phase reactant. Its homologue, Spi 2.2, however, is not GH responsive but is a positive acute-phase reactant. During an inflammatory response after turpentine administration, the Spi 2.1 mRNA level decreases to 20% of its normal level in 24 h. Within this period the normally low Spi 2.2 mRNA level increases sevenfold (42, 50, 51). We therefore examined the divergent regulation of these two highly homologous genes in an effort to understand the interaction between inflammatory and GH effects.
After the binding of GH to its receptor, Jak2 from the JAK family of
tyrosine kinases is activated and becomes associated with the receptor
(22, 29, 55, 62, 68). Signal transducers and activators of
transcription (STAT) proteins 1, 3, and 5 are recruited to the receptor
complex and become tyrosine phosphorylated (6, 7, 16, 20, 45, 53,
56, 60, 67). Further phosphorylation of STAT proteins at serine
residues is followed by their dimerization and translocation to the
nucleus. The binding of STAT dimers to interferon -activated sites
(GAS) on target genes results in the induction of transcription of
these genes (17, 19). We delineated a GH response element
(GHRE) in the Spi 2.1 promoter that contains two GAS sites recognized
by Stat5, and we purified a GH-inducible nuclear factor that contains
Stat5 (4, 69). In functional assays, the GHRE sequence is
necessary for the GH-dependent induction of transcription of Spi 2.1 reporter fusion genes in primary rat hepatocytes (4). In
COS7 cells, cotransfection of a Stat5 cDNA expression plasmid, but not
a Stat1 or Stat3 cDNA expression plasmid, resulted in the GH-dependent activation of a GHRE reporter fusion gene, indicating that stimulation of gene expression via the GHRE is Stat5 specific (66).
The APR also involves similar JAK/STAT signaling pathways. Binding of IL-6, a predominant ligand in the induction of positive acute-phase reactants (14, 24) to its receptors, leads to the activation and translocation of Stat3 to the nucleus (2, 5, 45, 47, 48, 63, 64, 71). In primary hepatocyte cultures, treatment with IL-6 results in the activation of Stat3 and expression of Stat3-dependent Spi 2.2-CAT reporter activity and endogenous Spi 2.2 mRNA (5). Thus Stat3 is likely the major mediator of transcription of Spi 2.2 and many other positive APR genes.
Addition of other cytokines released during an APR, such as TNF- and
IL-1
, to hepatocyte cultures results in the inhibition of the
GH-induced mRNA expression of IGF-I (12, 13, 35, 59, 65).
We were therefore interested to find out whether they play a similar
role in the downregulation of other GH-responsive genes, such as Spi
2.1, during an APR.
We hypothesized that the divergence in expression of the Spi 2 genes during an APR is due, in part, to changes in the patterns of activation of STAT proteins in response to competing stimuli. To examine this, we assessed the responses of STAT proteins to GH in whole animals during an APR to determine whether these stimuli are capable of modulating each other's actions through STAT pathways. We also examined their interactions in primary rat hepatocytes under different treatment conditions by utilizing the responses of Spi 2.1 and Spi 2.2 reporter fusion genes and mRNA expression as respective models of Stat5 and Stat3 action.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cytokines.
Human GH was obtained from Eli Lilly (Indianapolis, IN). Human TNF-
(210-TA), rat IL-1
(501-RL), and rat IL-6 (206-IL) were obtained
from R & D Systems (Minneapolis, MN).
Treatment of animals. All animals were handled in accordance with experimental protocols approved by the University of Minnesota Institutional Committee on the Care and Use of Animals. Normal or hypophysectomized male Sprague-Dawley rats (100-125 g body wt) were obtained from the supplier (Harlan Sprague Dawley, Indianapolis, IN). Hypophysectomized rats were observed for 3 wk after arrival to confirm growth failure. Individual animals were injected with LPS (Sigma Chemical, St. Louis, MO) at a dose of 1 mg/150 g body wt and were killed at 1, 2, 3, or 4 h after injection. Nuclei were isolated from freshly excised livers and extracted for proteins according to previously published protocols (6).
A parallel series of hypophysectomized rats was similarly treated with LPS for 1, 2, 3, or 4 h, and, to determine the interactions of GH effects with the APR, each rat also received a dose of GH (30 µg/100 g body wt) 1 h before it was killed. Three different series of rats were subjected to these treatments, and hepatic nuclear extracts were prepared.Oligonucleotides.
The sequences of the Spi 2.1 oligonucleotide, GHRE, and SIE duplexes
are shown in Table 1. The GHRE and SIE
duplexes were extended with the Klenow fragment of Escherichia
coli DNA polymerase I and radiolabeled
deoxy-[-32P]CTP. The extended products were
subsequently purified through Bio-Spin 6 columns (Bio-Rad Laboratories,
Hercules, CA).
|
Electrophoretic mobility shift assays. Electrophoretic mobility shift assays (EMSA), as previously described (4), were carried out with hepatic nuclear proteins extracted from treated animals and the radiolabeled GHRE or the high-affinity sis-inducible element (SIE). Briefly, 5 µg of hepatic nuclear extracts were incubated with 20 fmol of the radiolabeled probe of interest in a buffer containing 20 mM HEPES (pH 7.6), 10% glycerol, 2 mM MgCl2, 5 mM CaCl2, 0.1 mg/ml BSA, 4% Ficoll 400, 1 mM spermidine, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 µg poly dI.dC. Final KCl concentration was adjusted to 50 mM. After 30 min of incubation at 30°C, the binding mixture was loaded onto a nondenaturing, 3.2% glycerol, 5% polyacrylamide gel in 0.5× TBE (45 mM Tris, 45 mM boric acid, and 1 mM EDTA, pH 8.3) and electrophoresed at 150 V for 2.5 h. The gel was dried and exposed to film. Quantitation of the shifted complexes on the film was performed using the Kodak Digital Science ID Image Analysis Software.
Antibodies. All antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Stat5b antibody (sc-835) is a polyclonal antibody generated against amino acids 711-727 at the carboxy terminus of mouse Stat5b. This antibody reacts with both Stat5a and Stat5b. Antibody to Stat3 (sc-7179) is a polyclonal antibody generated against amino acids 50-240 at the amino terminus of human Stat3. Antibody to phosphotyrosine (sc-7020) is a mouse monoclonal antibody specific for detection of phosphotyrosine-containing proteins.
Immunoprecipitations and immunoblots. Immunoprecipitations were performed, as previously reported (25), with hepatic nuclear extracts from treated animals and an antibody to phosphotyrosine. Briefly, aliquots containing 100 µg of nuclear proteins were incubated with 2 µg of phosphotyrosine antibody in RIPA buffer (50 mM Tris · HCl, pH 7.5, 1% Nonidet P-40, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, and 0.1% sodium deoxycholate) containing, in addition, 0.2 mM PMSF, 1 mg/ml aprotinin, and 10 mM NaF. After 1 h of mixing, protein A agarose (120 µg) was added to each mixture, and end-to-end mixing was continued for 16 h at 4°C. The mixtures were centrifuged at 700 g for 5 min at 4°C. The resulting pellets were washed and solubilized in SDS electrophoresis sample buffer, and aliquots were loaded onto a 7.5% SDS-PAGE gel. Immunoblotting with antibodies to either Stat3 or Stat5 was performed as reported previously (4, 6). Positive signals were detected with the enhanced chemiluminescence detection system (Amersham Life Science, Arlington Heights, IL) and quantitated using Kodak Digital Science ID Image Analysis Software.
Plasmid construction.
The constructions of Spi 2.1 (275/+85) and Spi 2.2 (
319/+85) into
the Hind III/Pst I sites of the parent
chloramphenicol acetyltransferase (CAT) reporter plasmid pCAT(An), and
Spi 2.1 (
147/
102)4-TKCAT, containing four copies of the
GHRE, were described previously (5, 69).
Monocyte-conditioned medium.
Monocyte-conditioned medium (MoCM) was prepared according to the method
of Darlington et al. (10). In brief, 20 ml of human blood
were centrifuged at 400 g for 10 min at room temperature. The cell pellet, enriched for leukocytes, was resuspended in RPMI medium (GIBCO BRL, Rockville, MD) and layered over 5 ml of
Ficoll-Hypaque. Polymorphonuclear leukocytes and granulocytes were
sedimented by centrifugation at 400 g for 30 min. Cells
located at the interface of the supernatant and the Ficoll-Hypaque were
isolated by suction, washed twice with PBS, and plated in RPMI medium
plus 10% fetal bovine serum. Monocyte cultures were stimulated to
increase production of APR-mediating factors by the addition of 10 µg/ml of LPS for 48 h. The MoCM was then collected and frozen in
aliquots at 20°C.
Functional assays.
Primary hepatocytes were isolated using the collagenase method,
according to previously published protocols (4). After a
4-h attachment period, cells were transfected with either Spi 2.1-CAT
or Spi 2.2-CAT using Lipofectin reagent (Life Technologies, Grand
Island, NY) in modified Williams E medium with 27.5 mM glucose for
12-14 h. After removal of the Lipofectin and subsequent washing, Matrigel (Life Technologies, Grand Island, NY) at 667 µg/35-mm culture dish was added to the medium, and the hepatocytes were cultured
in the presence or absence of 50 ng/ml GH. For experiments involving
specific cytokines, 1 ng/ml of TNF-, 1 ng/ml of IL-6, or 0.5 ng/ml
of IL-1
was added in addition to GH. At the end of 24 h, all
media were replaced, and fresh GH and/or cytokines were added. At the
end of another 24 h, the cells were harvested and lysed in
Reporter Lysis Buffer (Promega, Madison, WI) for CAT assays. Results of
the assays are expressed as percent conversion of chloramphenicol to
its acetylated forms as determined by phosphor screen autoradiography
(Molecular Dynamics, Sunnyvale, CA). Each experiment was repeated three
times with freshly isolated hepatocytes. At least two different plasmid
preparations of each reporter fusion gene were tested in these experiments.
Northern blots.
For hepatocyte mRNA studies, Matrigel was added to the modified
Williams E medium with 27.5 mM glucose after the attachment period, and
the cells were allowed to rest for 16 h. GH and/or specific
cytokines were then added. After 24 h of culture, the hepatocytes
were harvested and RNA was extracted with TRIzol reagent (GIBCO BRL).
Northern blots prepared with these RNAs were first probed with a Spi
2.1 oligonucleotide complementary to its reactive center
(51) that had been 5'-labeled with T4 polynucleotide kinase and [-32P]ATP. The blots were then probed
simultaneously with cDNAs of Spi 2.2 from its unique 3'-untranslated
region and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). These
cDNAs were radiolabeled using the Oligolabeling Kit (Amersham Pharmacia
Biotech, Piscataway, NJ) and deoxy-[
-32P]CTP. All
quantitations of Spi 2.1 or Spi 2.2 mRNAs were normalized to those of
GAPDH mRNA, as determined by phosphor screen autoradiography.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
LPS treatment of hypophysectomized rats reduces Stat5 binding to
the GHRE during a GH response.
To study the potential interactions between LPS and GH treatments
in whole animals, we examined the effects of treatment with LPS (1 mg/150 g body wt) in two groups of hypophysectomized rats. One group
was given LPS alone and killed at 1, 2, 3, or 4 h after treatment; the second group received, in addition, GH (30 µg/100 g body wt) 1 h before death. The results of EMSA that use
hepatic nuclear proteins extracted from these animals and radiolabeled GHRE are shown in Fig. 1. We have shown
previously (4, 60) that GHRE binds specifically to
activated Stat5 and that administration of GH to an untreated
hypophysectomized rat 1 h before it is killed results in
significant induction of Stat5 binding (lane 2 vs. lane 1). LPS treatment preceding GH administration
diminished this binding in a time-dependent manner, with only 10% of
the expected GH-induced Stat5 binding present 4 h after LPS
treatment (lanes 3-6). A parallel series of rats
treated with LPS alone did not show any Stat5 binding to the GHRE
(lanes 7-10). Therefore, events occurring during the
progression of an APR result in a reduction of GH-induced Stat5
binding.
|
GH treatment reduces Stat3 binding to the SIE during the early
stage of LPS treatment of hypophysectomized rats.
To examine the effects of GH administration on changes in
Stat3 binding during the progression of an APR, we carried out EMSA of
the same hepatic nuclear extracts using radiolabeled SIE. Figure 2 shows that, as previously reported
(4), GH treatment activated Stat1 and Stat3, resulting in
the formation of three complexes containing Stat3 homodimers,
Stat1 homodimers, and Stats 1 and 3 heterodimers (lane
2 vs. lane 1). LPS treatment alone led to maximal
formation of a Stat3 complex 1 h after treatment (lane 7). This binding decreased over the next 3 h to 56% at
4 h after LPS treatment (lanes 8-10). GH
treatment, given at the same time as LPS treatment, diminished the
binding of Stat3 stimulated by LPS within the 1st h by 36% (lane
3 vs. lane 7). However, GH treatment given at later
time points in LPS treatment did not result in any notable differences
in Stat3 binding (lanes 4-6) compared with those
treated with LPS alone (lanes 6-10).
|
Simultaneous treatment with LPS and GH reduces the accumulation of
tyrosine-phosphorylated Stats 3 and 5 in the nucleus.
The decreases in Stat5 binding to the GHRE and Stat3 to the SIE in
extracts from hypophysectomized rats treated with both LPS and GH could
be due to an inhibition of DNA binding or decreases in the amounts of
these STAT proteins in the nuclei. To delineate between these two
possibilities, we performed immunoprecipitations of these extracts with
an antibody to phosphotyrosine, and we probed blots of the resultant
immunoprecipitates with an antibody either to Stat5 (see Fig. 3) or to
Stat3 (see Fig. 4).
|
|
MoCM inhibits the GH response of Spi 2.1-CAT. Our studies in whole animals to this point indicated that the GH-activated and LPS-stimulated STAT pathways interfere with each other and may account, in part, for the reduction in expression of some negative acute-phase reactants during an APR. To better understand this aspect of the complex events that are taking place during an APR and to further dissect the roles of individual cytokines in influencing STAT activation, we concentrated our further investigations on primary hepatocyte culture. The GHRE in the Spi 2.1 promoter has served as an in vitro model system for Stat5 activation (66). Similarly, a Spi 2.2 promoter reporter fusion gene has served as an in vitro model system for Stat3 activation (5, 32). We therefore conducted functional assays utilizing Spi 2.1 and Spi 2.2 promoter reporter fusion genes and studied their responses to different stimuli to dissect aspects of the hormonal milieu generated during an APR.
MoCM has been used as a means for initiating an APR in human hepatoma cells (10). We therefore tested the effects of MoCM on the GH response of Spi 2.1-CAT in hepatocytes (Fig. 5A). In the absence of MoCM, Spi 2.1-CAT exhibited a robust GH response (GH vs. UT), but in its presence, the activity of Spi 2.1-CAT in response to GH was almost completely inhibited. This inhibitory effect was also observed with Spi 2.1 (
|
Both TNF- and IL-1
inhibit the GH response of Spi 2.1-CAT.
By means of radioimmunoassays, we found that MoCM contains TNF-
(487 pg/ml), IL-1
(140 pg/ml), and IL-6 (490 pg/ml), all cytokines known
to be active during an APR (3, 10). We have previously
found that IL-6, at 5 ng/ml, will induce Spi 2.1-CAT activity in
hepatocyte culture, and that addition of IL-6 to GH does not alter the
GH response of Spi 2.1-CAT (5). However, at the lower
concentration of 1 ng/ml, similar to that found in MoCM, IL-6 addition
resulted in only a modest activation of Spi 2.1-CAT. Simultaneous
addition of TNF-
, IL-1
, and IL-6 led to a reduction of this
response (Fig. 6).
|
|
TNF-, IL-1
, and IL-6 inhibit Spi 2.1 mRNA induction by GH in
isolated primary hepatocytes.
To determine whether the in vitro effects of TNF-
, IL-1
, and IL-6
on the response of the Spi 2.1 promoter to GH correlate with their
effects on endogenous Spi 2.1 mRNA expression, we cultured primary
hepatocytes in the presence of GH with or without these cytokines for
24 h. Figure 8, top,
shows a representative Northern blot of RNA extracted from these cells
and probed with a radiolabeled Spi 2.1 oligonucleotide. Figure 8,
bottom, shows the mean values obtained from four similar
blots. GH treatment typically resulted in an 8- to 10-fold induction
(on average) of Spi 2.1 mRNA, expressed as 100% in Fig. 8,
bottom. Addition of either TNF-
(0.5 ng/ml) or IL-1
(0.25 ng/ml) reduced this induction by almost 60%. Treatment with
IL-6, which is capable of inducing Spi 2.1 promoter activity (5), did not result in induction of endogenous Spi 2.1 mRNA. At 0.5 ng/ml, IL-6 treatment actually reduced GH-induced Spi 2.1 mRNA expression by 65%. Simultaneous addition of TNF-
, IL-1
, and
IL-6 completely blocked the GH-stimulated induction of Spi 2.1 mRNA.
Thus TNF-
, IL-1
, and IL-6, given singly or in combination, inhibit Spi 2.1 mRNA induction by GH.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We examined the interactions between GH and LPS stimulation in hypophysectomized rats during the first several hours of an APR. LPS is frequently employed in inducing an APR in an animal. Although its treatment might elicit changes in cytokine levels that are different from more authentic immune responses resulting from chronic inflammations, the information we learn from the use of LPS as a model is still valuable and can guide us in further efforts to understand the interactions between GH signaling and inflammation. Because the effects on GH signaling 1 h after treatment are well documented (60), we administered GH to these LPS-treated animals 1 h before they were killed. We found time-dependent changes in the amounts of GH-responsive phosphorylated Stat5 in the liver nuclei that were caused by events occurring during an APR. These changes were not correlated to changes in the total amount of Stat5 under these conditions, because immunoprecipations of whole cell extracts from livers of these animals with an antibody that recognises both Stat5a and Stat5b (sc-835) indicated that there was little change in the expression level of Stat5 in the liver during the first 4 h after LPS administration (data not shown). However, immunoprecipitations of nuclear extracts with an antibody to phosphotyrosine showed that the amount of GH-responsive phosphorylated Stat5 in the nuclei decreased progressively during the same time period. This decrease, down to ~40% at the end of 4 h, was somewhat less than the decrease in Stat5 binding to the GHRE observed in EMSA, indicating that there may be specific inhibition of Stat5 binding in the nucleus. Recently, two protein inhibitors of activated STAT (PIAS) that specifically block DNA binding activities of either Stat1 or Stat3 (PIAS1 and PIAS3) have been reported (8, 36). Thus it is possible that, during an APR, a similar Stat5-specific inhibitor could be activated to block the DNA binding of Stat5. Taken together, our data suggest that, during an APR, there is interference with the GH/Stat5 signaling pathway and that this interference provides a mechanistic explanation for the negative response of Spi 2.1 expression to an APR.
This mechanistic explanation likely involves inhibition of the phosphorylation, translocation, and/or DNA binding of Stat5, or competition for factors common to both GH and APR signaling pathways. Candidates for interfering with Stat5 activation include the family of suppressers of cytokine signaling (SOCS-1 to SOCS-7) and cytokine-inducible SH2-containing protein (CIS) (39-41), or tyrosine phosphatases (SHP-1 and SHP-2) (28). Several SOCS proteins are involved in the negative feedback loops for both GH (1) and IL-6 (57) signaling. Their transient expression is activated with varying kinetics by different cytokines, and their mechanisms of action range from inhibition of several different JAK tyrosine kinase activities to competition for binding to phosphorylated tyrosine residues on specific receptors (39, 70). For example, SOCS-3 mRNA expression in the livers of hypophysectomized rats is rapidly induced by GH and peaks 30 min after GH treatment (61). Its constitutive expression in 3T3-F442A fibroblasts has been shown to block the GH-induced transactivation of the Spi 2.1 promoter (1).
Recently, LPS treatment of normal rats has been shown to increase the
expression of liver SOCS-2, SOCS-3, and CIS mRNAs and to decrease the
tyrosine phosphorylation of liver Stat5. These correlative data have
led to the hypothesis of a specificity-spillover machanism involving
the induction of SOCS genes by cytokines released in response to LPS,
and subsequent SOCS inhibition of GH signaling (38). In
this report we have shown similar data on the LPS-dependent decrease of
GH-responsive Stat5 phosphorylation and DNA binding in the liver of
hypophysectomized rats. Our data suggest that the hypothesis that
SOCS-induced inhibition alone may account for the downregulation of GH
signaling may be an oversimplification. Hepatocyte studies in our
laboratory indicate that treatment with either TNF- or IL-1
alone, under conditions that led to a reduced GH response of Spi
2.1-CAT, did not result in an increase in expression of either SOCS-2
or SOCS-3 mRNAs in the first 8 h of culture, indicating that other
mechanisms of inhibition are likely at work (data not shown).
A comparison of the time courses of induction of CIS, SOCS-2, and SOCS-3 by seemingly antagonistic stimuli, such as GH (1, 38, 57), indicates that they are remarkably similar. This raises the interesting question of how a gene that is involved in the GH negative feedback loop in the normal state can become a more enduring inhibitor when it is activated during an APR with no change in the kinetics of its induction or the persistence of its mRNA. Whether there is a change in the level or the half-life of its protein product under these different conditions remains to be established. If we assume that there are changes at the protein levels, how then is the GH-signaling pathway selectively inhibited whereas that of IL-6, necessary in an APR, is not? Part of the answer may lie in the discovery of an inhibitor specific for a particular target or step in the GH-signaling pathway. To date, no CIS or SOCS protein has been shown to provide this degree of specificity. GH activates Stat1 and Stat3, in addition to Stat5, in the liver of hypophysectomized rats, whereas LPS treatment results only in the activation of Stat1 and Stat3. This suggests that there is an inhibitory step during the induction of the APR that specifically targets the activation of Stat5 by GH. The results we report here and those recently published (38) support this view.
Another possible mechanism for the interference of the GH-signaling pathway could involve the tyrosine phosphatases SHP-1 and SHP-2. During a GH response, SHP-1 translocates to the nucleus and associates with phosphorylated Stat5b, suggesting that it can participate in the dephosphorylation of nuclear Stat5b (46). At the same time, SHP-1 is also associated with Jak2 and appears to be involved in the attenuation of GH-activated JAK activity (21). On the other hand, SHP-2 is a negative regulator in the IL-6 induction of positive acute-phase reactants and acts by downregulating JAK activity (28). The interactions of these phosphatases during an APR thus may also be involved in the mutual modulation of GH- and LPS-stimulated cytokine signaling.
In hypophysectomized rats treated with LPS alone, we observed maximal binding of Stat3 at 1 h after LPS administration. Interestingly, this initial activation can be modulated by simultaneous administration of GH and may in part account for the GH attenuation of an APR due to thermal injury (26, 27, 44). This modulation may be due, in part, to the simultaneous activation of Stat1 by GH that may sequester some Stat3 into the formation of Stat1/Stat3 heterodimers. It is not clear from current literature what role, if any, this heterodimer plays in vivo. GH administration at 2 h or later after LPS treatment did not alter the amount of activated Stat3 in liver nuclei, indicating that signaling events involving the activation of Stat3 take place within the first 2 h after LPS administration.
GH activation of Stat3 in the hypophysectomized rat does not result in
the transcription of Spi 2.2, a positive acute-phase reactant. The
explanation for this apparent discrepancy is not understood. The amount
of Stat3 activated by GH was less than when the animal was treated with
LPS alone and might contribute to this observation. The simultaneous
activation of Stats 1 and 5 by GH may affect both the amount of Stat3
activated and its eventual binding to the promoters of target genes.
Interactions among different STAT proteins or competition for binding
sites may determine the transcriptional outcome for a responsive gene. For example, Stat5b modulates the Stat1-mediated transcription of
interferon regulatory factor-1 in response to prolactin by protein-protein interactions that do not involve DNA binding
(37). On the other hand, FcR1, an interferon
-responsive gene, contains a GAS region that binds both Stat1
(43) and Stat5 (4), resulting in the
formation of complexes containing both dimers and tetramers of these
respective factors. However, binding of its GAS region to Stat1 alone
is sufficient to initiate transcription (43).
Utilizing Spi 2.1 and Spi 2.2 promoter reporter fusion genes, we
investigated their responses to different stimuli as indicators of
functional Stat5 and Stat3, respectively. MoCM, produced by isolated
human monocytes stimulated with LPS, was able to attenuate the GH
response of Spi 2.1-CAT and at the same time induce Spi 2.2-CAT
activity. The divergent responses of these Spi promoter reporter fusion
genes to MoCM are similar to those observed for their respective
mRNAs during an APR in an intact rat (51). Thus
factors present in MoCM are capable of reproducing an in vivo
observation during an APR. An analysis of MoCM showed that, similar to
previous reports (10), it contains substantial
amounts of IL-6, TNF-, and IL-1
. The reduction of Spi 2.1-CAT
activity in hepatocytes cultured in the presence of GH and either
exogenous TNF-
or IL-1
demonstrated that both TNF-
and IL-1
are capable of inhibiting the GH response of Spi 2.1-CAT. Both TNF-
and IL-1
, at 10 ng/ml, have also been shown to inhibit GH-responsive
expression of IGF-1 and Spi 2.1 mRNAs (54, 59) in
hepatocyte cultures. We now show that, in the presence of even low
concentrations of these two cytokines, at 0.25 or 0.5 ng/ml, both
GH-responsive Spi 2.1 promoter activity and its endogenous mRNA
expression are markedly inhibited.
Spi 2.2 belongs to type II positive acute-phase reactants that require
IL-6, but not IL-1, in their induction; in contrast, type I reactants
require both IL-6 and IL-1 (32). Treatment of hepatocytes
with either TNF- or IL-1
does not result in induction of Spi
2.2-CAT activity. Moreover, their combined addition results in an
inhibition of IL-6-induced Spi 2.2-CAT activity. The inhibitory effects
of these two cytokines on the GH-induced Spi 2.1-CAT activity are more
prominent and are also noted in expression of their endogenous mRNA.
Thus their role in the regulation of the Spi genes during an APR
appears to be primarily inhibitory.
LPS administration to hypophysectomized rats leads to the accumulation of a small amount of phosphorylated Stat5 in the hepatic nucleus. Treatment of hepatocytes with IL-6, an important ligand released during an APR, results in some, albeit low, Spi 2.1-CAT activity. Preliminary data from our laboratory on GHRE mutation studies in hepatocytes indicate that IL-6 induction of Spi 2.1-CAT activity occurs via the GHRE, a sequence that is Stat5 specific (66). Moreover, treatment of rats with complete Freund's adjuvant, another APR stimulant, has resulted in the stimulation of Stat5, in addition to Stat3 (48). Thus, in addition to activating Stat3 (2, 5), IL-6 appears to be capable of activating Stat5 to some extent, resulting in Stat5-specific (GHRE) promoter activity. The extent of this activation, however, does not give rise to the production of endogenous Spi 2.1 mRNA. IL-6 treatment of hepatocytes actually attenuates the accumulation of Spi 2.1 mRNA in response to GH. These conflicting effects of IL-6 on Spi 2.1 expression may indicate posttranscriptional regulation. The attenuation of Spi 2.1 mRNA induction by IL-6 in hepatocytes correlates well with the observation that, in the intact animal, turpentine treatment results in a decreased induction of Spi 2.1 mRNA (51).
In the dynamic situation within a cell, interactions among different
factors and their modulations of each other's functions may determine
the eventual transcriptional outcomes of target genes. Signaling by
competing ligands results in complex events that can be mutually
regulated at multiple steps. Using the hepatocyte model system, we have
shown that IL-6, IL-1, and TNF-
act in concert to reduce the
GH-induced expression of Spi 2.1, a Stat5-mediated gene. We propose
that, during an APR, inhibition specific to the GH/Stat5 signaling
pathway leads to the downregulation of Stat5-mediated GH-responsive
genes such as Spi 2.1.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Lisa Siebenson and Brigit Riley for excellent technical assistance.
![]() |
FOOTNOTES |
---|
This work was performed with the support of National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-32817, the Minnesota Medical Foundation, and the Vikings Children's Fund.
Address for reprint requests and other correspondence: S. A. Berry, Dept. of Pediatrics, Univ. of Minnesota, 420 Delaware St. SE, Box 75, Minneapolis, MN 55455 (E-mail: berry002{at}tc.umn.edu).
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.
Received 28 March 2000; accepted in final form 31 July 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Adams, TE,
Hansen JA,
Starr R,
Nicola NA,
Hilton DJ,
and
Billestrup N.
Growth hormone preferentially induces the rapid, transient expression of SOCS-3, a novel inhibitor of cytokine receptor signaling.
J Biol Chem
273:
1285-1287,
1998
2.
Akira, S,
Nishio Y,
Inoue M,
Wang XJ,
Wei S,
Matsusaka T,
Yoshida K,
Sudo T,
Naruto M,
and
Kishimoto T.
Molecular cloning of APRF, a novel IFN-stimulated gene factor 3 p91-related transcription factor involved in the gp130-mediated signaling pathway.
Cell
77:
63-71,
1994[ISI][Medline].
3.
Baumann, H,
Prowse KR,
Marinkovic S,
Won KA,
and
Jahreis GP.
Stimulation of hepatic acute phase response by cytokines and glucocorticoids.
Ann NY Acad Sci
557:
280-296,
1989[ISI][Medline].
4.
Bergad, PL,
Shih HM,
Towle HC,
Schwarzenberg SJ,
and
Berry SA.
Growth hormone induction of hepatic serine protease inhibitor 2.1 transcription is mediated by a Stat5-related factor binding synergistically to two -activated sites.
J Biol Chem
270:
24903-24910,
1995
5.
Berry, SA,
Bergad PL,
Stolz AM,
Towle HC,
and
Schwarzenberg SJ.
Regulation of Spi 2.1 and 2.2 gene expression after terpentine inflammation: discordant responses to IL-6.
Am J Physiol Cell Physiol
276:
C1374-C1382,
1999
6.
Berry, SA,
Bergad PL,
Whaley CD,
and
Towle HC.
Binding of a growth hormone inducible nuclear factor is mediated by tyrosine phosphorylation.
Mol Endocrinol
8:
1714-1719,
1994[Abstract].
7.
Campbell, GS,
Meyer DJ,
Raz R,
Levy DE,
Schwartz J,
and
Carter-Su C.
Activation of acute phase response factor (APRF)/Stat3 transcription factor by growth hormone.
J Biol Chem
270:
3974-3979,
1995
8.
Chung, CD,
Liao J,
Liu B,
Rao X,
Jay P,
Berta P,
and
Shuai K.
Specific inhibition of Stat3 signal transduction by PIAS3.
Science
278:
1803-1805,
1997
9.
Cooper, AL,
Brouwer S,
Turnbull AV,
Luheshi GN,
Hopkins SJ,
Kunkel SL,
and
Rothwell NJ.
Tumor necrosis factor- and fever after peripheral inflammation in the rat.
Am J Physiol Regulatory Integrative Comp Physiol
267:
R1431-R1436,
1994
10.
Darlington, GJ,
Wilson DR,
and
Lachman LB.
Monocyte-conditioned medium, interleukin-1, and tumor necrosis factor stimulate the acute phase response in human hepatoma cells in vitro.
J Cell Biol
103:
787-793,
1986[Abstract].
11.
Davies, UM,
Jones J,
Reeve J,
Camacho-Hubner C,
Charlett A,
Ansell BM,
Preece MA,
and
Woo PMM
Juvenile rheumatoid arthritis: effects of desease activity and recombinant human growth hormone on insulin-like growth factor 1, insulin-like growth factor binding proteins 1 and 3, and osteocalcin.
Arthritis Rheum
40:
332-340,
1997[ISI][Medline].
12.
Delhanty, PJ.
Interleukin-1 beta suppresses growth hormone-induced mRNA levels and secretion in primary hepatocytes.
Biochem Biophys Res Commun
243:
269-272,
1998[ISI][Medline].
13.
Fan, J,
Char D,
Bagby GJ,
Gelato MC,
and
Lang CH.
Regulation of insulin-like growth factor-I (IGF-I) and IGF-binding proteins by tumor necrosis factor.
Am J Physiol Regulatory Integrative Comp Physiol
269:
R1204-R1212,
1995
14.
Fattori, E,
Cappelletti M,
Costa P,
Sellitto C,
Cantoni L,
Carelli M,
Faggioni R,
Fantuzzi G,
Ghezzi P,
and
Poli V.
Defective inflammatory response in interleukin 6-deficient mice.
J Exp Med
180:
1243-1250,
1994[Abstract].
15.
Fey, GH,
and
Gauldie J.
The acute phase response of the liver in inflammation.
Prog Liver Dis
9:
89-117,
1990[Medline].
16.
Finbloom, DS,
Petricoin EF,
Hackett RH,
David M,
Feldman GM,
Igarashi K,
Fibach E,
Weber MJ,
Thorner MO,
Silva CM,
and
Larner AC.
Growth hormone and erythropoietin differentially activate DNA-Binding proteins by tyrosine phosphorylation.
Mol Cell Biol
14:
2113-2118,
1994[Abstract].
17.
Gebert, CA,
Park SH,
and
Waxman DJ.
Regulation of signal transducer and activator of transcription (STAT) 5b activation by the temporal pattern of growth hormone stimulation.
Mol Endocrinol
11:
400-414,
1997
18.
Ghahary, A,
Fu S,
Shen J,
Shankowsky HA,
and
Tredget EE.
Differential effects of thermal injury on circulating insulin-like growth factor binding proteins in burn patients.
Mol Cell Biochem
135:
171-180,
1994[ISI][Medline].
19.
Gronowski, AM,
Lestunff C,
and
Rotwein P.
Acute nuclear actions of growth hormone (GH): cycloheximide inhibits inducible activator protein-1 activity, but does not block GH-regulated signal transducer and activator of transcription activation or gene expression.
Endocrinology
137:
55-64,
1996[Abstract].
20.
Gronowski, AM,
Zhong Z,
Wen ZL,
Thomas MJ,
Darnell JE,
and
Rotwein P.
In vivo growth hormone treatment rapidly stimulates the tyrosine phosphorylation and activation of stat3.
Mol Endocrinol
9:
171-177,
1995[Abstract].
21.
Hackett, R,
Wang Y,
Sweitzer S,
Feldman G,
Wood W,
and
Larner A.
Mapping of a cytoplasmic domain of the human growth hormone receptor that regulates rates of inactivation of Jak2 and Stat proteins.
J Biol Chem
272:
11128-11132,
1997
22.
Hansen, JA,
Hansen LH,
Wang X,
Kopchick JJ,
Gouilleux F,
Groner B,
Nielsen JH,
Moldrup A,
Galsgaard ED,
and
Billestrup N.
The role of GH receptor tyrosine phosphorylation in Stat5 activation.
J Mol Endocrinol
18:
213-221,
1997[Abstract].
23.
Hawker, FH,
Stewart PM,
Baxter RC,
Borkmann M,
Tan K,
Caterson ID,
and
McWilliam DB.
Relationship of somatomedin-C/insulin-like growth factor I levels to conventional nutritional indices in critically ill patients.
Crit Care Med
15:
732-736,
1987[ISI][Medline].
24.
Heinrich, PC,
Castell JV,
and
Andus T.
Interleukin-6 and the acute phase response.
Biochem J
265:
621-636,
1990[ISI][Medline].
25.
Humbert, JT,
Bergad PL,
Masha O,
Stolz AM,
Kaul S,
and
Berry SA.
Growth hormone action in hypothyroid infant rats.
Pediatr Res
47:
250-255,
2000
26.
Jarrar, D,
Wolf SE,
Jeschke MG,
Ramirez RJ,
DebRoy M,
Ogle CK,
Papaconstaninou J,
and
Herndon DN.
Growth hormone attenuates the acute-phase response to thermal injury.
Arch Surg
132:
1171-1176,
1997[Abstract].
27.
Jeschke, MG,
Chrysopoulo MT,
Herndon DN,
and
Wolf SE.
Increased exoression of insulin-like growth factor-1 in serum and liver after recombinant human growth hormone administration in thermally injured rats.
J Surg Res
85:
171-177,
1999[ISI][Medline].
28.
Kim, H,
and
Baumann H.
Dual signaling of the protein tyrosine phosphatase SHP-2 in regulating expression of acute-phase plasma proteins by interleukin-6 cytokine receptors in hepatic cells.
Mol Cell Biol
19:
5326-5338,
1999
29.
King, APJ,
Tseng MJ,
Logsdon CD,
Billestrup N,
and
Carter-Su C.
Distinct cytoplasmic domains of the growth hormone receptor are required for glucocorticoid- and phorbol ester-induced decreases in growth hormone (GH) binding. These domains are different from that reported for GH-induced receptor internalization.
J Biol Chem
271:
18088-18094,
1996
30.
Koj, A.
Acute-phase reactants: their synthesis, turnover and biological significance.
In: Structure and Function of Plasma Proteins, edited by Allison AC. New York: Plenum, 1974, p. 73-130.
31.
Koj, A,
Gauldie J,
and
Baumann H.
Biological perspectives of cytokine and hormone networks.
In: Acute Phase Proteins: Molecular Biology, Biochemistry, and Clinical Applications, edited by Mackiewicz A,
Kushner I,
and Baumann H. Boca Raton, FL: CRC, 1993, p. 275-288.
32.
Kordula, T,
Rippberger J,
Morella K,
Travis J,
and
Baumann H.
Two separate signal transducer and activator of transcription proteins regulate transcription of the serine protease inhibitor-3 gene in hepatic cells.
J Biol Chem
271:
6752-6757,
1996
33.
Kushner, I.
The phenomenon of the acute phase response.
Ann NY Acad Sci
389:
39-48,
1982[ISI][Medline].
34.
Kushner, I.
The acute phase response: an overview.
Methods Enzymol
163:
373-383,
1988[ISI][Medline].
35.
Lazarus, DD,
Moldawer LL,
and
Lowry SF.
Insulin-like growth factor-1 activity is inhibited by interleukin-1, tumor necrosis factor-
, and interleukin-6.
Lymphokine Cytokine Res
12:
219-223,
1993[ISI][Medline].
36.
Liu, B,
Liao J,
Rao X,
Kushner SA,
Chung CD,
Chang DD,
and
Shuai K.
Inhibition of Stat1-mediated gene activation by PIAS1.
Proc Natl Acad Sci USA
95:
10626-10631,
1998
37.
Luo, G,
and
Yu-Lee L.
Transcriptional inhibition by Stat5.
J Biol Chem
272:
26841-26849,
1997
38.
Mao, Y,
Ling PR,
Fitzgibbons TP,
McCowen KC,
Frick GP,
Bistrian BR,
and
Smith RJ.
Endotoxin-induced inhibition of growth hormone receptor signaling in rat liver in vivo.
Endocrinology
140:
5505-5515,
1999
39.
Matsumoto, A,
Masuhara M,
Mitsui K,
Yokouchi M,
Ohtsubo M,
Misawa H,
Miyajima A,
and
Yoshimura A.
CIS, a cytokine inducible SH2 protein, is a target of the JAK-STAT5 pathway and modulates STAT5 activation.
Blood
89:
3148-3154,
1997
40.
Nicholson, SE,
and
Hilton DJ.
The SOCS proteins: a new family of negative regulators of signal transduction.
J Leukocyte Biol
63:
665-668,
1998[Abstract].
41.
Ohya, K,
Kajigaya S,
Yamashita Y,
Miyazato A,
Hatake K,
Miura Y,
Ikeda U,
Shimada K,
Ozawa K,
and
Mano H.
SOCS-1/JAB/SSI-1 can bind to and suppress Tec protein-tyrosine kinase.
J Biol Chem
272:
27178-27182,
1997
42.
Pages, G,
Rouayrenc JF,
Le Cam G,
Mariller M,
and
Le Cam A.
Molecular characterization of three rat liver serine-protease inhibitors affected by inflammation and hypophysectomy: protein and mRNA analysis and cDNA cloning.
Eur J Biochem
190:
385-391,
1990[Abstract].
43.
Pearse, RN,
Feinman R,
Shuai K,
Darnell JE,
and
Ravetch JV.
Interferon -induced transcription of the high affinity Fc receptor for IgG requires assembly of a complex that includes the 91-kDa subunit of transcription factor ISGF3.
Proc Natl Acad Sci USA
90:
4314-4318,
1993[Abstract].
44.
Petersen, SR,
Jeevanandam M,
Shahbazian LM,
and
Holaday NJ.
Reprioritization of liver protein synthesis resulting from recombinant human growth hormone supplementation in parenterally fed trauma patients: the effect of growth hormone on the acute-phase response.
J Trauma
42:
987-996,
1997[ISI][Medline].
45.
Ram, PA,
Park SH,
Choi HK,
and
Waxman DJ.
Growth hormone activation of Stat 1, Stat 3, and Stat 5 in rat liverdifferential kinetics of hormone desensitization and growth hormone stimulation of both tyrosine phosphorylation and serine/threonine phosphorylation.
J Biol Chem
271:
5929-5940,
1996
46.
Ram, PA,
and
Waxman DJ.
Interaction of growth hormone-activated STATs with SH2-containing phosphotyrosine phosphatase SHP-1 and nuclear JAK2 tyrosine kinase.
J Biol Chem
272:
17694-17702,
1997
47.
Raz, R,
Durbin JE,
and
Levy DE.
Acute phase response factor and additional members of the interferon-stimulated gene factor 3 family integrate diverse signals from cytokines, interferons, and growth factors.
J Biol Chem
269:
24391-24395,
1994
48.
Ripperger, JA,
Fritz S,
Richter K,
Hocke GM,
Lottspeich F,
and
Fey GH.
Transcription factors Stat3 and Stat5b are present in rat liver nuclei late in an acute phase response and bind interleukin-6 response elements.
J Biol Chem
270:
29998-30006,
1995
49.
Ross, R,
Miell J,
Freeman E,
Jones J,
Matthews D,
and
Buchanan C.
Critically ill patients have high basal growth hormone levels with attenuated oscillatory activity associated with low levels of insulin-like growth factor-I.
Clin Endocrinol
35:
47-54,
1991[ISI][Medline].
50.
Schwarzenberg, SJ,
Yoon JB,
Seelig S,
Potter CJ,
and
Berry SA.
Discoordinate hormonal and ontogenetic regulation of four rat serpin genes.
Am J Physiol Cell Physiol
262:
C1144-C1148,
1992
51.
Schwarzenberg, SJ,
Yoon JB,
Sharp HL,
and
Seelig S.
Homologous rat hepatic protease inhibitor genes show divergent functional responses to inflammation.
Am J Physiol Cell Physiol
256:
C413-C419,
1989
52.
Scotte, M,
Hiron M,
Masson S,
Lyoumi S,
Banine F,
Tenier P,
Lebreton JP,
and
Daveau M.
Differential expression of cytokine genes in monocytes, peritoneal macrophages and liver following endotoxin- or turpentine-induced inflammation in rat.
Cytokine
8:
115-120,
1996[ISI][Medline].
53.
Silva, CM,
Lu HW,
and
Day RN.
Characterization and cloning of STAT5 from IM-9 cells and its activation by growth hormone.
Mol Endocrinol
10:
508-518,
1996[Abstract].
54.
Simar-Blanchet, AE,
Legraverend C,
Thissen JP,
and
Le Cam A.
Transcription of the rat serine protease inhibitor 2.1 gene in vivo: correlation with GAGA box promoter occupancy and mechanism of cytokine-mediated down regulation.
Mol Endocrinol
12:
391-404,
1998
55.
Smit, LS,
Meyer DJ,
Billestrup N,
Norstedt G,
Schwartz J,
and
Carter-Su C.
The role of the growth hormone (GH) receptor and JAK1 and JAK2 kinases in the activation of Stats 1,3, and 5 by GH.
Mol Endocrinol
10:
519-533,
1996[Abstract].
56.
Smit, LS,
Vanderkuur JA,
Stimage A,
Han YL,
Luo GY,
Yulee LY,
Schwartz J,
and
Carter-Su C.
Growth hormone-induced tyrosyl phosphorylation and deoxyribonucleic acid binding activity of Stat5A and Stat5B.
Endocrinology
138:
3426-3434,
1997
57.
Starr, R,
Willson TA,
Viney EM,
Murray LJ,
Rayner JR,
Jenkins BJ,
Gonda TJ,
Alexander WS,
Metcalf D,
Nicola NA,
and
Hilton DJ.
A family of cytokine-inducible inhibitors of signalling.
Nature
387:
917-921,
1997[ISI][Medline].
58.
Takala, J,
Ruokonen E,
Webster NR,
Nielsen MS,
Zandstra DF,
Vundelinckx G,
and
Hinds CJ.
Increased mortality associated with growth hormone treatment in critically ill adults.
N Engl J Med
341:
785-792,
1999
59.
Thissen, JP,
and
Verniers J.
Inhibition by interleukin-1 beta and tumor necrosis factor-alpha of the insulin-like growth factor I messenger ribonucleic acid response to growth hormone in rat hepatocyte primary culture.
Endocrinology
138:
1078-1084,
1997
60.
Thomas, MJ,
Gronowski AM,
Berry SA,
Bergad PL,
and
Rotwein P.
Growth hormone rapidly activates rat serine protease inhibitor 2.1 gene transcription and induces a DNA-binding activity distinct from those of Stat1, -3, and -4.
Mol Cell Biol
15:
12-18,
1995[Abstract].
61.
Tollet-Egnell, P,
Flores-Morales A,
Stavreus-Evers A,
Sahlin L,
and
Norstedt G.
Growth hormone regulation of SOCS-2, SOCS-3, and CIS messenger ribonucleic acid expression in the rat.
Endocrinology
140:
3693-3704,
1999
62.
Vanderkuur, JA,
Wang XY,
Zhang LY,
Allevato G,
Billestrup N,
and
Carter-Su C.
Growth hormone-dependent phosphorylation of tyrosine 333 and/or 338 of the growth hormone receptor.
J Biol Chem
270:
21738-21744,
1995
63.
Wegenka, UA,
Buschmann J,
Lutticken C,
Heinrich P,
and
Horn F.
Acute-phase response factor, a nuclear factor binding to acute-phase response elements, is rapidly activated by interleukin-6 at the posttranslational level.
Mol Cell Biol
13:
276-288,
1993[Abstract].
64.
Wegenka, UM,
Lutticken C,
Buschmann J,
Yuan J,
Lottspeich T,
Muller-Esterl W,
Schindler C,
Roeb E,
Heinrich PC,
and
Horn F.
The interleukin-6-activated acute-phase response factor is antigenically and functionally related to members of the signal transducer and activator of transcription (STAT) family.
Mol Cell Biol
14:
3186-3196,
1994[Abstract].
65.
Wolf, M,
Bohm S,
Brand M,
and
Kreymann G.
Proinflammatory cytokines interleukin 1 and tumor necrosis factor
inhibit growth hormone stimulation of insulin-like growth factor I synthesis and growth hormone receptor mRNA levels in cultured rat liver cells.
Eur J Endocrinol
135:
729-737,
1996[ISI][Medline].
66.
Wood, TJJ,
Sliva D,
Lobie PE,
Goullieux F,
Mui AL,
Groner B,
Norstedt G,
and
Haldosen LA.
Specificity of transcription enhancement via the STAT responsive element in the serine protease inhibitor 2.1 promoter.
Mol Cell Endocrinol
130:
69-81,
1997[ISI][Medline].
67.
Wood, TJJ,
Sliva D,
Lobie PE,
Pircher TJ,
Gouilleux F,
Wakao H,
Gustafsson JA,
Groner B,
Norstedt G,
and
Haldosen LA.
Mediation of growth hormone-dependent transcriptional activation by mammary gland factor Stat 5.
J Biol Chem
270:
9448-9453,
1995
68.
Yi, WS,
Kim SO,
Jiang J,
Park SH,
Kraft AS,
Waxman DJ,
and
Frank SJ.
Growth hormone receptor cytoplasmic domain differentially promotes tyrosine phosphorylation of signal transducers and activators of transcription 5b and 3 by activated JAK2 kinase.
Mol Endocrinol
10:
1425-1443,
1996[Abstract].
69.
Yoon, JB,
Berry SA,
Seelig S,
and
Towle HC.
An inducible nuclear factor binds to a growth hormone-regulated gene.
J Biol Chem
265:
19947-19954,
1990
70.
Yoshimura, A,
Ohkubo T,
Kiguchi T,
Jenkins N,
Gilbert D,
Copeland N,
Hara T,
and
Miyajima A.
A novel cytokine-inducible gene CIS encodes an SH2-containing protein that binds to tyrosine-phosphorylated interleukin 3 and erythropoietin receptors.
EMBO J
14:
2816-2826,
1995[Abstract].
71.
Zhong, Z,
Wen Z,
and
Darnell JE.
Stat 3: a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6.
Science
264:
95-98,
1994[ISI][Medline].