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Joslin Diabetes Center (Y.M., T.P.F., K.C.M., R.J.S.) and Beth Israel-Deaconess Medical Center (P.-R.L., K.C.M., B.R.B., R.J.S.), Harvard Medical School, Boston, Massachusetts 02215; and the Department of Physiology, University of Massachusetts Medical School (G.P.F.), Worcester, Massachusetts 01655
Address all correspondence and requests for reprints to: Robert J. Smith, M.D., Joslin Diabetes Center, Harvard Medical School, One Joslin Place, Boston, Massachusetts 02215. E-mail: robert.smith{at}joslin.harvard.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The immediate signaling events following GH binding to its receptor have only recently been defined (10). In target tissues, such as liver and skeletal muscle, GH binding results in dimerization of the GH receptor and consequent activation of the intracellular receptor-associated tyrosine kinase Janus kinase-2 (JAK2) (11, 12). This is followed by JAK2 autophosphorylation and tyrosine phosphorylation of the GH receptor and members of the signal transducer and activator of transcription (STAT) protein family (13). Phosphorylation of the STAT proteins leads to their dimerization and translocation to the nucleus, where they modulate gene transcription (10, 14). Although studies in cell culture and experimental animals have provided evidence for GH-stimulated tyrosine phosphorylation of a number of cellular signaling proteins, including various STATs (10, 15), insulin receptor substrate-1 (IRS-1), IRS-2, IRS-3 (13, 16, 17), and the SHC proteins (18), STAT5 appears to be the dominant intracellular signaling protein phosphorylated by GH-activated JAK2 in adult rat liver and muscle in vivo (12, 14). GH resistance in catabolic states has been related at least in part to decreased tissue abundance of GH receptors (19, 20, 21). In other hormonal pathways, such as the insulin signaling pathway, disease-related hormone resistance has been shown to result from changes in both receptor abundance and postreceptor signaling responses (22, 23, 24). It is not known whether GH resistance also may result from alterations in postreceptor steps in the GH signaling pathway.
In many experimental studies, infusion of the Gram-negative bacterial cell wall lipopolysaccharide, endotoxin, has been used to generate a model of catabolic disease states. Endotoxin is an important contributor to the fever and tissue catabolic response associated with Gram-negative sepsis (2, 3). In normal humans injected with Escherichia coli endotoxin, plasma IGF-I concentrations were shown to decrease despite a transient, marked increase in GH levels (25), suggesting the development of GH resistance. Studies in experimental animals have demonstrated that infusion of endotoxin results in marked decreases not only in plasma IGF-I concentrations, but also in IGF-I messenger RNA (mRNA) abundance in plasma, liver, and skeletal muscle (26). A decrease in circulating IGF-I levels after endotoxin infusion also was evident in rats treated with exogenous GH (27), further supporting the hypothesis that endotoxin induces a state of GH resistance. Endotoxin has been shown to induce postreceptor insulin resistance characterized by a marked decrease in insulin-stimulated tyrosine phosphorylation of the insulin receptor and the intracellular receptor substrate IRS-1 (24). The possibility that endotoxin leads to abnormalities in the content or tyrosine phosphorylation of JAK2 and intermediates in the GH signaling pathway has not been investigated.
As potential mediators of endotoxin-induced GH resistance, a number of related genes, designated suppressors of cytokine signaling (SOCS), that are believed to have a role in JAK/STAT signal attenuation recently have been identified (28, 29, 30). Interleukin-6, leukemia inhibitory factor, erythropoietin, and leptin, in addition to GH, have been shown to rapidly increase expression of SOCS genes in vivo and in vitro through STAT-mediated transcriptional activation (29, 31, 32, 33, 34, 35, 36), and the protein products of the SOCS genes are thought to serve as negative feedback inhibitors by decreasing JAK/STAT signaling. As the expression of SOCS genes can be stimulated by multiple cytokine pathways, we considered the possibility that increased SOCS gene expression induced by one or more cytokines secreted as part of the response to endotoxin infusion could result in resistance to GH.
In this study we have determined the effects of endotoxin infusion on the tyrosine phosphorylation and tissue content of proteins mediating early steps in the GH signaling pathway in rat liver in vivo, including JAK2, STAT5, and the GH receptor. To investigate SOCS gene expression as a potential molecular mechanism of endotoxin-induced GH resistance, rat complementary DNAs (cDNAs) corresponding to the full coding sequences of three members of this gene family [cytokine-inducible sequence (CIS), SOCS-2, and SOCS-3] were cloned and used as probes to define the levels of their corresponding mRNAs in liver tissue.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endotoxin and GH infusion protocols
Male Sprague Dawley rats (Taconic Farms, Inc.,
Germantown, NY), weighing 180–200 g, were acclimated for 5 days in a
light-controlled room at 24 C, with water and chow available ad
libitum. Nonfasted animals were anesthetized with ketamine (40
mg/kg BW, ip), and under aseptic conditions a SILASTIC brand catheter
(Dow Corning Corp., Midland, MI; id, 0.025 in.; od, 0.047
in.) was implanted in the right jugular vein through a small incision,
tunneled sc to the dorsal interscapular region, and exteriorized by
suturing to a rotating swivel device (Instech Laboratories, Plymouth
Meeting, PA). After recovery from anesthesia, the animals were placed
in individual metabolic cages with the catheter assembly anchored in a
manner enabling free movement around the cage. Water and chow were
provided ad libitum, and the catheters were flushed every 2
days with a small volume of heparinized saline to maintain patency.
Four days after catheter implantation, nonfasted rats were given a bolus iv injection of either endotoxin (1 mg/kg BW) or saline through the jugular venous catheter. This represents a dose of endotoxin that is known to induce insulin resistance but to be sublethal during the planned period of study (24). At various time points after endotoxin or saline administration (0–6 h), the animals were given a bolus injection of either hGH (1.5 mg/kg BW) or saline through the jugular catheter. Previous studies have shown marked stimulation of GH signaling responses in rats after the infusion of recombinant human GH (12, 39). The dose of 1.5 mg/kg used in this study was selected because it is known to be approximately 10-fold greater than the amount required to produce a maximum response, and it is known to result in the same effects on the activation of GH signaling intermediates as lower doses. As described in previous reports on GH signaling (12, 39) and insulin signaling in vivo (24, 37, 40), supramaximal doses of hormones are used in this type of experimental system to assure that observed changes in signaling do not result from altered hormone delivery to tissue receptors. Five minutes after GH or saline injection, the animals were decapitated, and the left lobe of the liver was rapidly removed, frozen in liquid nitrogen, and stored at -80 C for subsequent analysis. It is known from previous in vivo studies that 5 min of GH stimulation results in maximum tyrosine phosphorylation of major GH signaling proteins (12). The animal protocols were in compliance with the Guide for the Care and Use of Laboratory Animals published by the NIH and were approved by the institutional animal care committee.
Immunoprecipitation and immunoblotting
Frozen liver tissue samples were weighed, pulverized under
liquid nitrogen with a stainless steel mortar and pestle, and
homogenized with a Polytron (Brinkmann Instruments, Inc.,
Westbury, NY) in 6 vol buffer containing 20 mM Tris-HCl (pH
7.6), 120 mM NaCl, 1% (vol/vol) Nonidet P-40, 10%
(vol/vol) glycerol, 2 mM sodium orthovanadate, 10
mM sodium pyrophosphate, 1 mM
phenylmethanesulfonylfluoride, 40 µg/ml leupeptin, and 100
mM sodium fluoride. The resulting homogenates were
centrifuged at 200,000 x g for 1 h at 4 C, and
the supernatants were collected and stored in aliquots at -80 C. The
protein concentrations of the tissue extracts were determined with a
Bradford dye binding assay kit (Bio-Rad Laboratories, Inc.
Hercules, CA), using BSA as a standard.
Equal amounts of tissue protein (5 mg) were incubated in the above-described buffer with specific antibodies at the indicated dilutions overnight at 4 C. The antibodies were then adsorbed onto protein A-Sepharose beads (Pierce Chemical Co., Rockford, IL) for 2 h at 4 C, and the resulting immunocomplexes were washed three times by centrifugation and resuspension in a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% (vol/vol) Nonidet P-40, 1 mM EGTA, 0.25% (wt/vol) sodium deoxycholate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 mM sodium fluoride. The washed immunoprecipitates plus additional tissue extracts (200 µg) not subjected to immunoprecipitation were heated in Laemmli buffer with 100 mM dithiothreitol for 5 min at 100 C, resolved by SDS-PAGE, and electroblotted onto nitro-cellulose membranes (Schleicher & Schuell, Inc., Keene, NH). The membranes were blocked with 20 mM Tris buffer (pH 7.4) containing 0.9 g/dl sodium chloride, 15 mM sodium azide, 5% (wt/vol) BSA, and 0.05% (vol/vol) Nonidet P-40 and probed with the indicated antibodies. After washing, specifically bound antibodies were detected with [125I]protein A as previously described (37) and quantitated with a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).
Cloning of rat SOCS
cDNAs2
As rat cDNAs for members of the SOCS gene family were not
available, and the rat sequences for these genes had not been
published, we cloned cDNAs containing the full coding sequences of
three SOCS genes, CIS, SOCS-2, and SOCS-3, by RT of total RNA from rat
liver, followed by PCR amplification with primers corresponding to the
equivalent mouse cDNA sequences. For this purpose, 5-µg aliquots of
total RNA were incubated in 20-µl vol at 42 C with random primers
(Life Technologies, Inc., Gaithersburg, MD) and 100 U
Moloney murine leukemia virus H reverse transcriptase (Superscript,
Life Technologies, Inc.). Using 100-ng cDNA equivalents of
the RT products as template, 30 cycles of PCR were conducted with 18-
to 20-mer primers (Joslin DNA Core Facility, Boston, MA) at 0.15
mM with 2.5 U Pfu DNA polymerase
(Stratagene, La Jolla, CA) and the following parameters:
94 C for 1 min, 55 C for 1 min, and 72 C for 2 min. The resulting cDNAs
were restriction digested and cloned into the pGEX vector
(Amersham Pharmacia Biotech, Piscataway, NJ). Multiple
clones corresponding to each SOCS family mRNA were sequenced several
times in both directions to assure correct definition of the rat
sequence.
Northern blotting
Total liver RNA was isolated by the acid guanidinium
thiocyanate-phenol-chloroform extraction method of Chomcyznski and
Sacchi (41). Final RNA concentrations were determined
spectrophotometrically at 260 nm, and the samples were stored in
aliquots at -80 C. For Northern blotting, 10- to 30-µg aliquots of
denatured RNA from each sample were resolved by electrophoresis in
1.2% agarose-formaldehyde gels. RNA integrity and equality of loading
were confirmed by analysis of ethidium bromide-stained 28S and 18S
ribosomal RNA bands. The RNA then was transferred to GeneScreen
membranes (Life Science Products, Boston, MA) by overnight blotting in
sodium chloride-sodium citrate (SSC) and fixed with a UV cross-linker
(Stratagene, La Jolla, CA). The membranes were
prehybridized for 2–4 h at 42 C in 50% (vol/vol) formamide, 10%
(wt/vol) dextran sulfate, and 1% (wt/vol) SDS.
Hybridization was performed for 14–16 h at 42 C in prehybridization
solution supplemented with 0.1 mg/ml denatured salmon sperm DNA using
specific rat cDNA probes labeled with [32P]deoxy-CTP
(
2 x 106 dpm/ml) by random priming (Multiprime DNA
Labeling Kit, Amersham Pharmacia Biotech, Arlington
Heights, IL). These included an approximately 2000-bp JAK2 cDNA
(provided by Dr. Roy Duhe, NCI-Frederick Cancer Research and
Development Center, Frederick, MD) and an 807-bp CIS cDNA, a 920-bp
SOCS-2 cDNA, and an 831-bp SOCS-3 cDNA cloned in our laboratory. After
hybridization, the membranes were sequentially washed for 5 min with
2 x SSC-0.1% SDS and 1 x SSC-0.1% SDS at 22 C and then
with 0.1 x SSC-0.1% SDS at 42 C. Specific mRNA bands were
identified and quantified using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). Each blot was subsequently
stripped and reblotted with a mouse 18S ribosomal cDNA probe to verify
equal total RNA loading. To determine the time course of the effects of
endotoxin on the different mRNAs, only samples from rats treated for 5
min with GH were analyzed. It can be reliably assumed that mRNA levels
would not be affected by the short period of GH treatment, and this
limited the total number of assays required.
Statistical analysis
Data are presented as the mean ± SEM. All
statistical calculations were performed using QuickStat for MS-DOS
(Lund University, Lund, Sweden). Significant differences among groups
with different times of endotoxin treatment were identified by ANOVA.
When the ANOVA indicated a significant effect, individual groups were
compared with the time zero control by Student’s t test.
The 
levels were adjusted to declare a significant P
value (P < 0.05) based on the number of comparisons
using the Bonferroni correction. Data on GH receptor content, which was
limited to two experimental groups, were analyzed by t test.
Results from immunoblotting and Northern blotting experiments are
expressed as arbitrary densitometry U normalized against an internal
standard included on each blot.
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Results |
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, left panel), which
previously has been shown to represent STAT5 (12). Quantitative
analysis of immunoblots from multiple control rats demonstrated a
7-fold increase in STAT5 tyrosine phosphorylation after GH stimulation
(Fig. 1A, right panel). Treatment with endotoxin had no
effect on the basal level of phosphorylation of STAT5 in the absence of
GH, but resulted in a significantly decreased stimulatory effect of GH
on STAT5 tyrosine phosphorylation (60% of GH-stimulated control value;
P < 0.05).
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(left
panel), STAT5 antibody bound to two protein bands of approximately
95 and 92 kDa. Previous studies from our group and others have
demonstrated that the upper band represents tyrosine-phosphorylated
STAT5, and the lower band represents nonphosphorylated STAT5 (12, 17).
In accordance with this conclusion, GH stimulation resulted in an
increase in the higher molecular mass band and a corresponding decrease
in the lower band in liver from both control and endotoxin-treated
animals. When the total content of STAT5 was determined by quantifying
the densities of the two bands on immunoblots from multiple rats, no
differences were observed in control vs. endotoxin-treated
rats in the basal state or in control vs. endotoxin-treated
rats after GH stimulation (Fig. 1B, right panel). There was
a small, but significant, decrease in the intensity of the combined
STAT5 bands in GH-stimulated vs. basal rats in both the
presence and absence of endotoxin treatment, which was attributed to a
presumed decrease in the affinity of the STAT antibody for
phosphorylated compared with nonphosphorylated STAT5. When considered
together with the results presented in Fig. 1A, these data indicate
that endotoxin administration leads to a decrease in GH-stimulated
liver STAT5 tyrosine phosphorylation that is not explained by altered
tissue content of the STAT5 protein.
To evaluate the time course of the inhibitory effects of endotoxin on
STAT5 tyrosine phosphorylation, rats were injected iv with hGH (1.5
mg/kg) at various time intervals (0.5–6 h) after a bolus iv injection
of endotoxin (1 mg/kg), and liver tissue was removed for analysis 5 min
after GH administration. To provide measures of the effects of GH in
the absence of endotoxin, STAT5 phosphotyrosine levels were determined
in basal and GH-stimulated rats not treated with endotoxin and in
additional rats given a bolus injection of saline and studied in the
basal and GH-stimulated states 6 h later. As shown in a
representative immunoblot (Fig. 2, left panel) and by quantitative results from multiple
animals (Fig. 2, right panel), there was a progressive
time-dependent decrease in GH-stimulated STAT5 tyrosine phosphorylation
throughout the 6-h study period after endotoxin administration.
Although saline controls were not included at each time point, it is
reasonable to conclude that the decrease in STAT5 phosphorylation
resulted from the effects of endotoxin, because the injections were
given to unanesthetized rats via implanted catheters with little
disturbance of the animals. In support of this conclusion, there was no
change in basal or GH-stimulated STAT5 phosphorylation 6 h after
administration of a saline control solution, and a similar lack of
change in control animals was demonstrated 4 h after endotoxin in
the study described in Fig. 1.
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). During the first 1–2 h after the
infusion of endotoxin, there was a trend toward decreased GH-stimulated
tyrosine phosphorylation of JAK2, but this did not reach statistical
significance at any time point compared with the GH-stimulated 0 and
6 h saline control values. There was no change in basal or
GH-stimulated JAK2 phosphorylation in the 6 vs. 0 h
saline controls (Fig. 3A) and no change in 4-h saline-infused animals
in a separate study (data not shown).
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). There was no change in JAK2 during the first
2 h after endotoxin; at the 4 h point, there was a 2-fold
increase in JAK2 protein content, which was sustained 6 h after
endotoxin (P < 0.01). In control rats infused with
saline instead of endotoxin, JAK2 content was unchanged after 6 h
(Fig. 3B), and a similar lack of change in JAK2 content was
demonstrated 4 h after endotoxin in a separate group of animals
(data not shown). When the ratio of tyrosine-phosphorylated JAK2 to
total JAK2 was calculated in individual GH-stimulated rats, a
progressive, time-dependent decrease in endotoxin-treated animals was
evident, such that the relative stoichiometry of JAK2 phosphorylation
was 31% of the level in controls 6 h after endotoxin
administration (P < 0.05; Fig. 4).
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, left
panel). Quantitative analysis of the combined JAK2 transcripts on
Northern blots from multiple animals demonstrated that the increase in
JAK2 mRNA was detectable at 1 h, reached a maximum (40-fold above
control) at 2 h, and was sustained for at least 6 h after a
single dose of endotoxin. There was no change in JAK2 mRNA in
saline-injected control animals studied at the 0 vs. 6
h points. From these data, it can be concluded that the increase in
JAK2 protein in the liver after endotoxin is preceded by an even more
marked increase in JAK2 mRNA.
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(left panel), the GH
receptor was identified as a broad band of approximately 110 kDa.
Quantitation of immunoblots from multiple animals demonstrated no
differences in liver GH receptor content between control and
endotoxin-treated rats (Fig. 6, right panel). Thus, the
decreased effects of GH on STAT5 tyrosine phosphorylation and on the
stoichiometry of JAK2 phosphorylation after endotoxin treatment cannot
be attributed to a decreased abundance of GH receptors. It was not
possible to determine the effects of GH stimulation or endotoxin
pretreatment on GH receptor tyrosine phosphorylation, because of an
apparent decreased effectiveness of the GH receptor antibody in
precipitating receptors from rats after GH administration (data not
shown).
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As shown in Fig. 7A, a single CIS mRNA
transcript of 2.5 kb was identified in rat liver. This mRNA was almost
undetectable in liver from control animals and increased markedly after
endotoxin administration. A significant increase in CIS mRNA was
evident 1 h after endotoxin infusion; this increase reached a
maximum of 4-fold above the control value 2 h after endotoxin. The
SOCS-2 cDNA probe hybridized with a 3.4-kb mRNA transcript, which also
was present at low levels in liver from control rats and increased
after endotoxin (Fig. 7B). The more slowly migrating band evident in
Fig. 7B was not observed in additional experiments and thus is not
thought to represent a SOCS-2 mRNA transcript. Quantitation of the
3.4-kb transcript in multiple animals demonstrated a small, transient
increase in SOCS-2 mRNA, which was significant only at the 1 h
point after endotoxin administration (Fig. 7B, right panel).
SOCS-3 mRNA was most markedly affected by endotoxin treatment. A single
3.2-kb SOCS-3 transcript identified with the rat cDNA probe was present
at a low level in control rat liver, increased approximately 8-fold at
1 h and 10-fold at 2 h after endotoxin treatment, and
remained significantly elevated for at least 6 h after a single
dose of endotoxin (6.4-fold at 6 h). There were no differences in
mRNA levels for these three SOCS members in saline-injected control
animals studied at 0 and 6 h. Thus, coincident with the induction
of GH resistance by endotoxin, there was a marked increase in hepatic
mRNA levels for CIS, SOCS-2, and SOCS-3. These and other members of the
SOCS gene family represent potential mediators of the endotoxin-induced
alterations in GH-stimulated STAT5 and JAK2 tyrosine phosphorylation
described in this study.
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Discussion |
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The binding of GH to its cell surface receptors activates the receptor-associated JAK2 tyrosine kinase (10), which then undergoes autophosphorylation and catalyzes the phosphorylation of tyrosine residues in a number of cellular proteins, including the GH receptor and STAT5 (12, 13). At various time points after endotoxin administration, there was a 15–50% decrease in mean JAK2 tyrosine phosphorylation after GH stimulation, but this did not reach statistical significance because of relatively large interanimal variation in both control and endotoxin-treated rats. By contrast, endotoxin resulted in a highly significant, greater than 2-fold increase in the liver content of JAK2, which became apparent 4 h after endotoxin and persisted through the 6 h point. When the ratio of JAK2 tyrosine phosphorylation to JAK2 protein content was determined in individual animals, a progressive, time-dependent decrease in the mean stoichiometry of JAK2 phosphorylation induced by endotoxin was evident (69% decrease 6 h after endotoxin). Autophosphorylation occurs on multiple tyrosine residues in JAK2 (44), and thus, the decrease in JAK2 phosphorylation could result from decreased phosphorylation of individual sites within JAK2 or an increase in the pool of nonphosphorylated JAK2. Further investigation of these two alternative mechanisms and their implications for JAK2 signaling to STAT5 will probably require the development of an in vitro experimental system in which radioisotopic tracer methodology can be used to map the effects of endotoxin on specific JAK2 tyrosine phosphorylation sites.
The increase in JAK2 protein content induced by endotoxin was preceded by a marked rise in the level of JAK2 mRNA transcripts in the liver. JAK2 mRNA was elevated within 1 h of endotoxin administration, increased 40-fold 2 h after endotoxin, and remained elevated for at least the next 4 h. Although the half-life of JAK2 mRNA in the liver or other mammalian tissues has not been established, it is unlikely that the rapid change in JAK2 mRNA can be explained by mRNA stabilization, and it is probable that this represents an endotoxin-induced increase in JAK2 gene transcription. JAK2 mRNA has been reported to be rapidly up-regulated after mitogenic stimulation of B lymphocytes and, to a lesser extent, T cells (45). However, to our knowledge, the response to endotoxin described in the present paper represents the first example of rapid JAK2 regulation observed in vivo and in a nonlymphocyte cell system. It is possible that the increase in JAK2 gene transcription occurred as a response to a transient decrease in total amount of JAK2 tyrosine phosphorylation induced by endotoxin. Alternatively, the rapid increase in JAK2 mRNA may be indicative of regulatory sequences in the JAK2 promoter that are activated in response to endotoxin or endotoxin-induced proinflammatory cytokines. In future studies, it will be important to investigate the mechanism and physiological importance of JAK2 mRNA up-regulation in catabolic disease states.
GH receptor abundance in the liver is known to be sensitive to nutrient
regulation (19), and recent studies have shown that GH receptor mRNA is
reduced after elective surgery (20). As a potential mechanism to
explain the effects of surgical stress on GH receptor expression,
studies in cultured rat hepatocytes have demonstrated decreased GH
receptor mRNA levels after incubation with tumor necrosis factor- or
interleukin-1 (21, 46). Because endotoxin stimulates the synthesis of
tumor necrosis factor-, interleukin-1, and other cytokines in
vivo, it was important to investigate the possibility that the
altered stoichiometry of JAK2 phosphorylation and decreased STAT5
tyrosine phosphorylation induced by endotoxin might reflect a change in
the amount of GH receptor in the liver. Using a specific GH receptor
antibody preparation, GH receptor levels were shown by
immunoprecipitation and immunoblotting to be unchanged 4 h after
the administration of endotoxin. The endotoxin-induced alterations in
JAK2 and STAT5 were maximal at this time point, and it therefore can be
concluded that they occur independent of changes in the abundance of GH
receptors in the liver. In contrast to our finding of unaltered GH
receptor abundance 4 h after endotoxin, in a recent publication by
Defalque et al. (47), a decreased number of liver GH
receptors was observed 5 h after endotoxin treatment. This
difference may have occurred because of the use of younger animals
(3-week-old females vs. approximately 8-week-old males) as
well as higher doses of endotoxin (2.5- and 7.5-fold higher) in the
Defalque study.
As GH was infused at a high dose that insured saturation of available GH receptors, our data provide strong evidence for postreceptor resistance in the GH signaling pathway. Although it has been suggested that GH resistance in catabolic states may result from a combination of decreased receptor abundance and inhibited postreceptor signaling, these data represent the first demonstration of GH resistance occurring in the absence of a change in receptor abundance. With longer term endotoxin exposure or other sustained stress states that influence nutritional status as well as stimulating inflammatory cytokines, GH receptor levels would be expected to decrease, and compromised GH actions in the liver and other tissues then would result from a combination of decreased receptor abundance and postreceptor GH resistance. Because significant GH effects on hepatic IGF-I mRNA levels do not occur until approximately 24 h after GH administration in normal, nonhypophysectomized rats (data not shown), determinations of such downstream responses were not included in this study of acute GH signaling responses.
As candidate mediators of postreceptor GH resistance, we determined the effects of endotoxin on hepatic mRNAs for members of the recently described SOCS gene family (29, 31, 32, 34). The levels of mRNA for several SOCS genes have been shown to rapidly increase in the liver and other cell types after cytokine receptor activation (29, 35, 36), and the SOCS proteins are hypothesized to function as negative feedback regulators of cytokine signaling (35). We focused on three members of the SOCS gene family (CIS, SOCS-2, and SOCS-3) that are relatively abundant in the liver and responsive to cytokine regulation. As rat cDNAs corresponding to these proteins were not available, we used RNA PCR methods to obtain clones from rat liver and established their full coding sequences. This both provided cDNA probes with assured homology for use in Northern blotting of rat tissues and defined for the first time the sequences of these proteins in the rat. Northern blotting analysis of liver total RNA preparations demonstrated a rapid 4-fold increase in CIS and a 10-fold increase in SOCS-3 mRNA, but only a small, transient increase in SOCS-2 mRNA after the administration of a single dose of endotoxin. SOCS-3 mRNA was not only most markedly affected by endotoxin, but it also remained significantly elevated for at least 6 h after endotoxin administration.
The increases in mRNAs for the SOCS genes correlate temporally with endotoxin-induced resistance to GH-stimulated STAT5 and JAK2 tyrosine phosphorylation, consistent with a role for the SOCS proteins in mediating the effects of endotoxin on GH signaling. In CHO cells cotransfected with an Spi 2.1-CAT reporter construct and the rat GH receptor, overexpression of SOCS-3 and the closely related SOCS-1 protein inhibited GH trans-activation of the Spi 2.1 promoter (35), indicating a capacity of these proteins to negatively regulate GH signaling. By contrast, CIS did not alter GH induction of the Spi 2.1 CAT reporter, and SOCS-2 augmented the effect of GH. Although additional studies are needed in more physiological systems, these findings suggest that specific SOCS gene products may have distinct effects on the actions of GH. SOCS-1 has been shown to directly interact with JAK2 as well as other JAK tyrosine kinases (32, 34), resulting in inhibition of JAK tyrosine kinase activity and JAK-catalyzed tyrosine phosphorylation of STAT proteins (34). It has been suggested that a conserved sequence in the C-terminal region of SOCS-1, which is similar to the autophosphorylation site that regulates JAK tyrosine kinase activity, may interact with the catalytic region of JAK kinases and thus inhibit their activity. Although not yet investigated, similar binding of SOCS-3 to JAK2 could explain the decreased stoichiometry of JAK2 phosphorylation and inhibited STAT5 phosphorylation induced by endotoxin in this study.
GH itself has been shown to induce mRNAs for SOCS-3 and, to a lesser extent, SOCS-1, in mouse liver (35). It is likely that GH-induced SOCS protein expression functions in a negative feedback loop to attenuate GH signaling, as has been suggested for interleukins and other cytokines. Interleukin-6 infusion in mice also leads to a rapid increase in hepatic SOCS mRNAs, including SOCS-1, SOCS-2, SOCS-3, and CIS (29). In bone marrow cells, a broad spectrum of cytokines induce SOCS mRNAs (29), and it is probable that multiple cytokines also regulate hepatic SOCS gene expression. After endotoxin administration, multiple cytokines are elaborated (2), each of which may act in the liver to induce increased transcription of SOCS family mRNAs. We propose that the marked increase in SOCS-3 and other SOCS proteins induced by endotoxin could result not only in feedback inhibition of cytokine signaling, but also in resistance to GH. A phenomenon of specificity-spillover has been described (48), in which high levels of a hormone can result in its binding to receptors for another hormone in addition to its own receptors (e.g. GH binding to PRL as well as GH receptors in patients with acromegaly) (48, 49). Our data suggest that a postreceptor specificity-spillover mechanism involving the inhibition of GH signaling by cytokine-induced SOCS proteins may contribute to the development of GH resistance in catabolic states. In a similar manner, endotoxin-induced SOCS genes may have clinically important inhibitory effects on other cytokine signaling pathways.
Further investigation of the proposed role of SOCS proteins in GH resistance will require determination of the levels of these proteins and their interactions with GH signaling intermediates in rat liver. Although antibodies to several SOCS proteins can be obtained from commercial suppliers, our studies of multiple currently available antibody preparations have demonstrated their interaction with recombinant SOCS proteins at high concentrations, but not with endogenous SOCS proteins in rat tissues. In addition to studies on the CIS, SOCS-2, and SOCS-3 proteins, it will be important to investigate the role of SOCS-1 in endotoxin-induced GH resistance.
In summary, endotoxin infusion results in a time-dependent decrease in GH-stimulated tyrosine phosphorylation of STAT5 in the liver that is not explained by decreased tissue content of the STAT5 protein. This is associated with an increase in JAK2 protein content and a 50% decrease in the amount of GH-stimulated phosphotyrosine per JAK2 in endotoxin-treated animals, which is preceded by a marked increase in JAK2 mRNA. The GH signaling changes after endotoxin infusion are not explained by diminished GH receptor abundance in the liver. SOCS mRNAs, including CIS, SOCS-2, and SOCS-3, are rapidly up-regulated after endotoxin administration, suggesting the involvement of SOCS proteins in endotoxin-induced alterations in the GH signaling pathway.
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Acknowledgments |
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Footnotes |
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2 Nucleotide sequences corresponding to the coding
regions of rat CIS, SOCS-2, and SOCS-3 are available in GenBank under
accession no. AF065161, AF075382, and AF075383, respectively.
Received May 27, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 of the insulin-like
growth factor I messenger ribonucleic acid response to growth hormone
in rat hepatocyte primary culture. Endocrinology 138:1078–1084This article has been cited by other articles:
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  C. J. Amuzie, J. Shinozuka, and J. J. Pestka Induction of Suppressors of Cytokine Signaling by the Trichothecene Deoxynivalenol in the Mouse Toxicol. Sci., October 1, 2009; 111(2): 277 - 287. [Abstract] [Full Text] [PDF]
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 Y. Chen, S. Sood, V. M. R. Krishnamurthy, P. Rotwein, and R. Rabkin Endotoxin-Induced Growth Hormone Resistance in Skeletal Muscle Endocrinology, August 1, 2009; 150(8): 3620 - 3626. [Abstract] [Full Text] [PDF]
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 X. Wang, J. Jiang, J. Warram, G. Baumann, Y. Gan, R. K. Menon, L. A. Denson, K. R. Zinn, and S. J. Frank Endotoxin-Induced Proteolytic Reduction in Hepatic Growth Hormone (GH) Receptor: A Novel Mechanism for GH Insensitivity Mol. Endocrinol., June 1, 2008; 22(6): 1427 - 1437. [Abstract] [Full Text] [PDF]
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T. H. Elsasser, S. Kahl, C.-J. Li, J. L. Sartin, W. M. Garrett, and J. Rodrigo Caveolae Nitration of Janus Kinase-2 at the 1007Y-1008Y Site: Coordinating Inflammatory Response and Metabolic Hormone Readjustment within the Somatotropic Axis Endocrinology, August 1, 2007; 148(8): 3803 - 3813. [Abstract] [Full Text] [PDF]
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 T. A. Ahmed, M. D. Buzzelli, C. H. Lang, J. B. Capen, M. L. Shumate, M. Navaratnarajah, M. Nagarajan, and R. N. Cooney Interleukin-6 inhibits growth hormone-mediated gene expression in hepatocytes Am J Physiol Gastrointest Liver Physiol, June 1, 2007; 292(6): G1793 - G1803. [Abstract] [Full Text] [PDF]
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 Y. Chen, D. Sun, V. M. R. Krishnamurthy, and R. Rabkin Endotoxin attenuates growth hormone-induced hepatic insulin-like growth factor I expression by inhibiting JAK2/STAT5 signal transduction and STAT5b DNA binding Am J Physiol Endocrinol Metab, June 1, 2007; 292(6): E1856 - E1862. [Abstract] [Full Text] [PDF]
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 T. Priego, M. Granado, E. Castillero, A. I. Martin, M A. Villanua, and A. Lopez-Calderon Nitric oxide production by hepatocytes contributes to the inhibitory effect of endotoxin on insulin-like growth factor I gene expression. J. Endocrinol., September 1, 2006; 190(3): 847 - 856. [Abstract] [Full Text] [PDF]
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T. Ahmed, G. Yumet, M. Shumate, C. H. Lang, P. Rotwein, and R. N. Cooney Tumor necrosis factor inhibits growth hormone-mediated gene expression in hepatocytes Am J Physiol Gastrointest Liver Physiol, July 1, 2006; 291(1): G35 - G44. [Abstract] [Full Text] [PDF]
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M. L. Shumate, G. Yumet, T. A. Ahmed, and R. N. Cooney Interleukin-1 inhibits the induction of insulin-like growth factor-I by growth hormone in CWSV-1 hepatocytes Am J Physiol Gastrointest Liver Physiol, August 1, 2005; 289(2): G227 - G239. [Abstract] [Full Text] [PDF]
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 K.-C. Leung, G. Johannsson, G. M. Leong, and K. K. Y. Ho Estrogen Regulation of Growth Hormone Action Endocr. Rev., October 1, 2004; 25(5): 693 - 721. [Abstract] [Full Text] [PDF]
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 E. S. Nylen and B. Muller Endocrine Changes in Critical Illness J Intensive Care Med, March 1, 2004; 19(2): 67 - 82. [Abstract] [PDF]
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 E. M. Dahly, M. E. Miller, P. K. Lund, and D. M. Ney Postreceptor Resistance to Exogenous Growth Hormone Exists in the Jejunal Mucosa of Parenterally Fed Rats J. Nutr., March 1, 2004; 134(3): 530 - 537. [Abstract] [Full Text] [PDF]
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 M. R. Waldron, T. Nishida, B. J. Nonnecke, and T. R. Overton Effect of Lipopolysaccharide on Indices of Peripheral and Hepatic Metabolism in Lactating Cows J Dairy Sci, November 1, 2003; 86(11): 3447 - 3459. [Abstract] [Full Text] [PDF]
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L. Q. Hong-Brown, C. R. Brown, R. N. Cooney, R. A. Frost, and C. H. Lang Sepsis-induced muscle growth hormone resistance occurs independently of STAT5 phosphorylation Am J Physiol Endocrinol Metab, July 1, 2003; 285(1): E63 - E72. [Abstract] [Full Text] [PDF]
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 F. Gu, N. Dube, J. W. Kim, A. Cheng, M. d. J. Ibarra-Sanchez, M. L. Tremblay, and Y. R. Boisclair Protein Tyrosine Phosphatase 1B Attenuates Growth Hormone-Mediated JAK2-STAT Signaling Mol. Cell. Biol., June 1, 2003; 23(11): 3753 - 3762. [Abstract] [Full Text] [PDF]
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L. A. Denson, M. A. Held, R. K. Menon, S. J. Frank, A. F. Parlow, and D. L. Arnold Interleukin-6 inhibits hepatic growth hormone signaling via upregulation of Cis and Socs-3 Am J Physiol Gastrointest Liver Physiol, April 1, 2003; 284(4): G646 - G654. [Abstract] [Full Text] [PDF]
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S.-E. Kong, S. M. Firth, R. C. Baxter, and P. J. D. Delhanty Regulation of the acid-labile subunit in sustained endotoxemia Am J Physiol Endocrinol Metab, October 1, 2002; 283(4): E692 - E701. [Abstract] [Full Text] [PDF]
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Y. E. Timsit and D. S. Riddick Stimulation of Hepatic Signal Transducer and Activator of Transcription 5b by GH Is Not Altered by 3-Methylcholanthrene Endocrinology, September 1, 2002; 143(9): 3284 - 3294. [Abstract] [Full Text] [PDF]
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S. G. Ronn, J. A. Hansen, K. Lindberg, A. E. Karlsen, and N. Billestrup The Effect of Suppressor of Cytokine Signaling 3 on GH Signaling in {beta}-Cells Mol. Endocrinol., September 1, 2002; 16(9): 2124 - 2134. [Abstract] [Full Text] [PDF]
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G. Yumet, M. L. Shumate, P. Bryant, C.-M. Lin, C. H. Lang, and R. N. Cooney Tumor necrosis factor mediates hepatic growth hormone resistance during sepsis Am J Physiol Endocrinol Metab, September 1, 2002; 283(3): E472 - E481. [Abstract] [Full Text] [PDF]
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V. Beauloye, B. Willems, V. de Coninck, S. J. Frank, M. Edery, and J.-P. Thissen Impairment of Liver GH Receptor Signaling by Fasting Endocrinology, March 1, 2002; 143(3): 792 - 800. [Abstract] [Full Text] [PDF]
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 P. A. Gruppuso The Clinical Laboratory Evaluation of GH Responsiveness J. Clin. Endocrinol. Metab., February 1, 2002; 87(2): 466 - 468. [Full Text] [PDF]
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S. P. Tam, P. Lau, J. Djiane, D. J. Hilton, and M. J. Waters Tissue-Specific Induction of SOCS Gene Expression by PRL Endocrinology, November 1, 2001; 142(11): 5015 - 5026. [Abstract] [Full Text] [PDF]
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 C. J. Greenhalgh and D. J. Hilton Negative regulation of cytokine signaling J. Leukoc. Biol., September 1, 2001; 70(3): 348 - 356. [Abstract] [Full Text] [PDF]
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 P. L. Bergad, S. J. Schwarzenberg, J. T. Humbert, M. Morrison, S. Amarasinghe, H. C. Towle, and S. A. Berry Inhibition of growth hormone action in models of inflammation Am J Physiol Cell Physiol, December 1, 2000; 279(6): C1906 - C1917. [Abstract] [Full Text] [PDF]
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A. Colson, A. Le Cam, D. Maiter, M. Edery, and J.-P. Thissen Potentiation of Growth Hormone-Induced Liver Suppressors of Cytokine Signaling Messenger Ribonucleic Acid by Cytokines Endocrinology, October 1, 2000; 141(10): 3687 - 3695. [Abstract] [Full Text] [PDF]
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C. H. Lang, X. Liu, G. J. Nystrom, and R. A. Frost Acute response of IGF-I and IGF binding proteins induced by thermal injury Am J Physiol Endocrinol Metab, June 1, 2000; 278(6): E1087 - E1096. [Abstract] [Full Text] [PDF]
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 L. Terstegen, P. Gatsios, J. G. Bode, F. Schaper, P. C. Heinrich, and L. Graeve The Inhibition of Interleukin-6-dependent STAT Activation by Mitogen-activated Protein Kinases Depends on Tyrosine 759 in the Cytoplasmic Tail of Glycoprotein 130 J. Biol. Chem., June 16, 2000; 275(25): 18810 - 18817. [Abstract] [Full Text] [PDF]
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