Departments of 1 Surgery, and 2 Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
During sepsis,
growth hormone (GH) resistance contributes to the catabolism of muscle
protein. To determine the role of tumor necrosis factor (TNF) as a
mediator of GH resistance, we examined the effects of a TNF antagonist
[TNF-binding protein (TNFbp)] on the GH/insulin-like growth factor
(IGF) I system during abdominal sepsis. To investigate potential
mechanisms, the effects of TNF on the IGF-I response to GH and GH
signaling were examined in cultured rat hepatocytes (CWSV-1). Three
groups of rats were studied: Control, Sepsis, and Sepsis + TNFbp.
Liver, gastrocnemius, and plasma were collected on day 5. In
gastrocnemius, neither sepsis nor TNFbp altered the abundance of IGF-I
mRNA. However, septic rats demonstrated an increase in circulating GH
and a reduction in plasma IGF-I concentrations that was ameliorated by
pretreatment with TNFbp. Liver from septic rats demonstrated a 50%
reduction in GH receptor (GHR) and IGF-I mRNA on day 5 that
was attenuated by TNFbp. However, the abundance of GHR protein was not
different in liver from Control, Sepsis, or Sepsis + TNFbp rats.
Consequently, a decreased amount of hepatic GHR does not explain the
GH-resistant septic state. In CWSV-1 hepatocytes, TNF- had no effect
on GHR protein level but inhibited the induction of IGF-I mRNA by GH. Nuclear protein from TNF-treated hepatocytes demonstrated similar levels of phosphorylated signal transducer and activator of
transcription-5 (STAT5) and DNA binding relative to controls 5 min
after GH treatment. However, both of these parameters were decreased
(vs. control) in TNF-treated cells 60 min after GH treatment.
Collectively, these results suggest that TNF mediates hepatic GH
resistance during sepsis by inhibiting the duration of signaling via
the janus kinase-2/STAT5 pathway.
growth hormone/insulin-like growth factor I axis; hepatocytes; janus kinase/signal transducer and activator of transcription
signaling; tumor necrosis factor-
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE METABOLIC RESPONSE TO
INFECTION is associated with negative nitrogen balance and the
catabolism of muscle protein (10). Nutritional support
alone is unable to prevent the loss of muscle protein during infection,
suggesting that other factors induce or regulate this phenomenon
(6, 10). The inflammatory cytokines tumor necrosis factor
(TNF)- and interleukin (IL)-1
have been implicated as mediators
of muscle protein catabolism (11, 34, 55, 56).
Administration of TNF-
or IL-1
antagonists ameliorates the loss
of muscle protein by preventing both the sepsis-induced decrease in
muscle protein synthesis and the increase in myofibrillar protein
degradation (11, 34, 55, 56). However, incubation of
muscle in vitro with buffer containing TNF-
or IL-1
does not
appear to inhibit protein synthesis, suggesting that the effects of
TNF-
on muscle protein synthesis are delayed or indirect in nature
(24, 40).
Several lines of evidence suggest that inflammatory cytokines inhibit
muscle protein synthesis, at least in part, via their effects on the
growth hormone (GH)/insulin-like growth factor (IGF) I axis during
sepsis. First, there is a linear correlation between the rate of
protein synthesis and IGF-I content in gastrocnemius during chronic
abdominal sepsis (34). Second, acute infusion of TNF-
or IL-1
in healthy animals reduces IGF-I levels in both plasma and
skeletal muscle (18, 20). Third, treatment of septic rats
with IL-1 receptor antagonist (IL-1ra) attenuates the inhibitory effects of sepsis on the GH/IGF-I axis and restores muscle protein synthesis to control levels (34). Fourth, both the
infusion of IGF-I in perfused hindlimb preparations from septic rats
(31) and the treatment of septic rats with an
IGF-I/IGF-binding protein-3 complex (49) restores muscle
protein synthesis to control levels.
Under normal conditions, circulating GH binds to transmembrane GH receptors (GHR) in liver, muscle, and other tissues. The activated GHR complex initiates several intracellular signaling cascades including the janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway (57). Although GH has been shown to activate STAT1 and STAT3 in different tissues, STAT5 appears to be preferentially activated in liver tissue (7). Phosphorylated STAT5 dimers translocate to the cell nucleus, where they bind to specific DNA sequences in the promoter region to activate the transcription of GH target genes like serine protease inhibitor 2.1 (23). Although GH stimulates the synthesis and secretion of IGF-I by liver, the exact mechanisms by which GH stimulates IGF-I transcription are still unresolved. However, studies in STAT5b knockout mice suggest that STAT5 is required for both basal and GH-induced expression of hepatic IGF-I (14). Sepsis also appears to impair hepatic GH action, as evidenced by a smaller GH-induced increase in plasma IGF-I in septic patients compared with control subjects (13).
We hypothesized that TNF- mediates the development of "GH
resistance" during abdominal sepsis by inhibiting GH signaling. To
test this hypothesis, we examined the effects of TNF-binding protein
(TNFbp), a potent TNF antagonist (17), on the expression of GHR and IGF-I in muscle and liver from septic rats. We also examined
the effects of TNF-
on JAK/STAT5 signaling and IGF-I gene expression
initiated by GH stimulation of CWSV-1 hepatocytes. The results suggest
that TNF is an important mediator of GH resistance in liver and acts by
inhibiting the duration of GH signaling via the JAK/STAT pathway.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials.
Recombinant human GH (rhGH, Pharmacia and Upjohn, Stockholm, Sweden)
was used in all experiments. The rat TNF- was obtained from R & D
Systems (Minneapolis, MN). TNFbp, a p55-soluble TNF receptor, was a
kind gift of Carl Edwards (Amgen, Thousand Oaks, CA). Plasmid
containing the rat IGF-I cDNA was a kind gift from Peter Rotwein
(Oregon Health Science) (47), and the rat GHR cDNA was
from L. S. Mathews (University of Michigan) (39).
Antibodies included polyclonal GHR antibody, a kind gift from W. R. Baumbagh (American Cyanamid, Princeton, NJ), used at a dilution of
1:250 (46), a rabbit polyclonal STAT5b antibody raised
against a peptide mapping the carboxy terminus (sc-835, Santa Cruz
Biotechnology, CA), and PY20 phosphotyrosine antibody conjugated with
horseradish peroxidase (HRP) from BD Transduction Laboratories (San
Diego, CA).
Animals.
Three groups of male Sprague-Dawley rats (200-250 g; Charles River
Breeding Laboratories, Wilmington, MA) were studied: Control, Sepsis,
and Sepsis + TNFbp. A Control + TNFbp group was not included in all studies, because preliminary results suggested that TNFbp had no
effect on the GH/IGF-I system in control animals (Table 1). Saline (1.0 ml) or TNFbp (1 mg/kg,
1.0 ml) was injected subcutaneously daily with the initial dose
administered 4 h before induction of sepsis. The dose and timing
of TNFbp administration were based on previous work demonstrating
inhibitory plasma levels and biological activity in the model of
abdominal sepsis used in the current study (11).
|
Cell culture experiments.
CWSV-1 cells were obtained from Dr. Harriet Isom (Dept. of
Microbiology, College of Medicine, Pennsylvania State University). CWSV-1 is an SV40 transformed rat hepatocyte cell line that has been
extensively characterized and demonstrates many of the important regulatory mechanisms observed in normal liver tissue (32, 37, 55). CWSV-1 cells were grown in RPCD medium (32, 36,
53) for 48 h. TNF-treated cells were incubated with 10 ng/ml TNF- for 4 h, and then 500 ng/ml rhGH were added for the
indicated time periods.
Northern blot analysis.
The relative abundance of IGF-I and GHR mRNA was determined by Northern
blot analysis (51). Total RNA was isolated from liver
tissue using the ToTally RNA Isolation Kit (Ambion, Austin, TX). CWSV-1
hepatocyte RNA was isolated using the RNeasy Mini Kit (Qiagen,
Valencia, CA). Twenty micrograms of total RNA were separated on a
denaturing agarose-formaldehyde gel and transferred onto a GeneScreen
hybridization transfer membrane (NEN Life Science Products, Boston, MA)
following standard Northern blot techniques. cDNA probes were labeled
using [32P]dCTP and the random primer technique
(Multiprime DNA Labeling System, Amersham Pharmacia Biotech,
Piscataway,NJ) and purified by gel filtration chromatography on G-25
Sephadex Quick Spin columns (TE; Roche Diagnostics, Indianapolis, IN).
For IGF-I, an 800-bp XhoI-EcoRI fragment
corresponding to the rat IGF-I cDNA containing exons 1, 3, 4, 5, and 6 was utilized as a probe (47). The rat IGF-I gene exhibits
alternate splicing in the first exon, resulting in the production of
multiple exon 1-derived transcripts. The liver expresses all of the
exon 1-derived IGF-I mRNA species ranging in size from 7.5 to 0.8 kb,
with the high-molecular-weight species of 7.5 kb representing the
majority (80%) of the IGF-I mRNA present. In the muscle, the 7.5-kb
IGF-I transcript represents almost 100% of IGF-I mRNA
(1). For the GHR, a 600-bp
HincII-HindIII fragment corresponding to the rat
GHR was used as a probe (39). Membranes were prehybridized
in 0.5 M sodium phosphate, pH 7.2, with 1% powdered milk, 1 mM EDTA,
pH 8.0, 2% SDS, 2× Denhardt's solution, and 200 µg/ml fish sperm
DNA. Prehybridization was conducted for 1 h at 65°C.
Hybridization was conducted overnight at 65°C with radiolabeled cDNA
probes. After a washing, the blots were exposed at 70°C for
2-5 days to Kodak X-Omat AR film in a cassette equipped with a Du
Pont Lightning Plus intensifying screen. The exposed autoradiographs
were scanned using a densitometer (model 100A; Molecular Dynamics,
Sunnyvale, CA). Band intensities were determined using the Quantity One
version 2 software (Protein Databases, Bio-Rad Laboratories, Hercules,
CA). Northern blots were stripped and reprobed with the 18S ribosomal
subunit message to confirm the absence of variations in RNA loading. An
oligonucleotide specific for 18S was 5'-end labeled using T4
polynucleotide kinase (Promega, Madison, WI)
5'-GTTATTGCTCAATCTCGGGTG-3'and used as a probe. Data are reported
as relative densitometry units after normalization to 18S
rRNA message.
Preparation of liver membrane extracts. Hepatic membranes were isolated using a modification of Posner et al. (27). Briefly, powdered frozen liver was homogenized in 10 vol of ice-cold 0.3 M sucrose, pH 7.4, by use of a Brinkman polytron type PT-10 at a setting of 5 for 25 s (43). The homogenate was centrifuged for 15 min at 600 g at 4°C. Individual pellets were resuspended in cold 0.3 M sucrose buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and centrifuged at 40,000 g twice for 45 min. The pellets (membrane fraction) were gently homogenized in 0.3 M sucrose, 25 mM Tris · HCl, pH 7.4, 10 mM MgCl2, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml pepstatin A, and leupeptin using a 25-gauge needle and 1-ml syringe assembly.
Isolation of whole cell lysates and nuclear protein.
Cells grown in culture dishes were placed on ice and rinsed three times
with cold PBS. Lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM
MgCl2, 1.0 mM EGTA, 10% glycerol, 1% Triton X-100, 100 mM
NaF, 0.2 mM Na3VO4, 1 mM PMSF, and 10 µg/ml
aprotinin) was added directly to the plates and incubated for 30 min.
Lysates were collected and cleared by centrifugation at 7,000 g for 5 min. Nuclear extracts from CWSV-1 cells were
prepared as previously described (48). Briefly, cells were
washed three times with cold PBS and then scraped in PBS containing 0.5 mM PMSF. Each cell pellet was washed and resuspended in lysis buffer:
10 mM HEPES, pH 7.9, 1 mM EDTA, 60 mM KCl, 0.5% IGEPAL CA-630, 1 mM dithiothreitol (DTT), and 1 mM PMSF. Lysed cells were spun at 1,200 g for 5 min, and nuclei were washed in lysis buffer without detergent. Nuclei were resuspended in buffer containing 250 Tris, pH
7.8, 60 mM KCl, 1 mM DTT, and 1 mM PMSF and then subjected to three
cycles of freezing and thawing. Lysed nuclei were spun for 15 min at
7,000 g, and the supernatant was collected and quickly frozen in liquid nitrogen and stored at 70°C.
Western blot analysis and immunoprecipitation. Equal amounts of protein were electrophoresed on a 7.5 or 4-15% gradient-resolving polacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon P; Millipore, Bedford, MA), by means of standard electroblotting procedures. Membranes were blocked for 1 h at room temperature with 5% nonfat milk in TBS (10 mM Tris, 150 mM NaCl, pH 7.5) for GHR detection. For phosphotyrosine detection of STAT5 and GHR, membranes were blocked overnight at 4°C in 5% BSA in TBS-T (TBS with 0.1% Tween-20) and, for GHR and STAT5 detection, 5% milk in TBS-T.
The membranes were incubated overnight at room temperature with a polyclonal antibody specific for rat GHR (46) or incubated for 1 h at room temperature with a polyclonal antibody against STAT5b. The secondary antibody utilized was linked to horseradish peroxidase (Accurate Chemical and Scientific, Westbury, NY). The wash step was repeated, and the antibodies were visualized using ECL-Plus (Amersham Pharmacia Biotech) according to the manufacturer's instructions. Densitometry scans of the exposed films were performed using a densitometer, and intensity was analyzed using Quantity One version 2 software (PDI, Bio-Rad). Cell lysates (100-500 µg) were immunoprecipitated in HNTG buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 0.2 mM Na3VO4, 0.2 mM PMSF, 2 µg/ml aprotinin), 2 µg of STAT5b antibody, or GHR antibody and 20 µl of agarose-conjugated beads (protein A-agarose; Santa Cruz Biotechnology) overnight at 4°C. Beads were collected and washed three times, and 2× sample Laemmli buffer (Bio-Rad) was added to samples and boiled for 5 min. Immunocomplexes were resolved using SDS-PAGE, and Western blot analysis was performed as described.Electrophoretic mobility shift assay.
Oligonucleotides for gel shift assays were as follows: complementary
strands to the rat -casein promoter 5'-GGA CTT CTT GGA ATT AAG
GGA-3' were labeled independently using T4 kinase (Promega) and
[
-32P]ATP, annealed, and purified on an 18% PAGE
(5). STAT5 consensus and mutant oligonucleotides were used
as cold competitors (Santa Cruz Biotechnology). Nuclear protein from
CWSV-1 cells (3-5 µg) was used in a binding reaction containing
2 µg of poly(dI-dC), 0.5-1.0 ng of probe (50,000 cpm), and 1×
binding buffer (10 mM Tris, pH 7.5, 4% glycerol, 1 mM
MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl)
(44). Reactions were incubated for 30 min at 25°C and
then electrophoresed on a prerun 4% PAGE/0.5× TBE for 2.5 h at
25 mA at 4°C. To supershift complexes, STAT5b antibody (sc-835-X, Santa Cruz Biotechnology) was added to the reactions and incubated for
30 min at 25°C before the addition of the probe. Gels were dried onto
a Whatman paper with a gel dryer (Bio-Rad) and exposed to film.
GH and IGF-I determination. IGF-I concentrations in plasma were determined by radioimmunoassay (RIA), as previously described (34) with the use of an antibody supplied by the National Hormone and Pituitary Program (Rockville, MD). Before the assay was performed, plasma was extracted using an acid-ethanol solution and subsequent cryoprecipitation to remove binding proteins. The eluate was evaporated, and the dried sample was reconstituted with phosphate buffer for IGF-I determination. GH concentrations in plasma were determined using commercially available RIA with a rat GH 125I assay system (Amersham Pharmacia Biotech).
Statistical analysis. Data are expressed as means ± SE for 7-12 animals in each group. Cell culture data represent the results of at least three independent experiments. The Northern blot and immunoblot data are expressed as relative densitometry units. Statistical evaluation of the data was analyzed by analysis of variance (ANOVA) followed by the Tukey-Kramer Multiple Comparison Test or by Student's t-test (two tailed) using Instat GraphPad 5.02 (San Diego, CA). Differences among means were considered significant at P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Role of TNF in mediating sepsis-induced changes in GH/IGF-I axis in
vivo.
The effects of TNFbp and the septic insult on the GH/IGF-I axis were
assessed by evaluating plasma levels of GH and IGF-I (Table 1). Septic
rats demonstrated a twofold increase in circulating GH on day
5 of chronic abdominal sepsis (P < 0.05 vs.
Control and Sepsis + TNFbp). Despite the increase in circulating
GH observed in septic rats, plasma IGF-I levels were decreased by
~30% (P < 0.05 vs. Control). Although treatment
with TNFbp did not influence either GH or IGF-I in control animals, it
significantly attenuated the sepsis-induced changes in plasma GH and
IGF-I levels. To determine whether the effect of sepsis on the GH/IGF-I
axis was seen in both liver and muscle, we examined the expression of
IGF-I in both tissues. In gastrocnemius, neither sepsis nor the
administration of TNFbp (Fig.
1A) altered the relative
abundance of IGF-I mRNA. In contrast, IGF-I mRNA content in liver from
septic rats was decreased on day 5 compared with Control
values, and pretreatment with TNFbp prevented the sepsis-induced
decrease in hepatic IGF-I mRNA (Fig. 1, B and C).
|
|
Effects of TNF- on GH-induced changes in IGF-I under in vitro
conditions.
To further investigate the complex events that occur in vivo during the
septic insult and to dissect the role of individual cytokines, we
examined the effects of TNF-
on IGF-I synthesis by CWSV-1
hepatocytes. Of importance, CWSV-1 hepatocytes synthesize IGF-I mRNA in
response to GH, and GH signaling in this cell line has been extensively
characterized (23, 32, 45). The time course of IGF-I mRNA
expression after GH administration was determined using 500 ng/ml GH
(Fig. 3A). IGF-I mRNA levels
were not significantly altered at 1 h after GH stimulation.
However, a twofold increase in IGF-I mRNA was observed at 24 h,
and a fourfold increase in IGF-I mRNA was noted in CWSV-1 cells after
48 h of incubation with 500 ng/ml rhGH (Fig. 3A). As
shown in Fig. 3B, incubation of hepatocytes with GH for
18 h resulted in a twofold increase in the abundance of IGF-I mRNA
(P < 0.05 vs. Control). Although preincubation of
hepatocytes with TNF-
for 4 h had no effect on basal IGF-I
expression, it prevented the normal induction of IGF-I mRNA observed
after GH administration (P < 0.05 vs. Control + GH; Fig. 3C).
|
|
Activation of the JAK/STAT-signaling pathway represents an
important mechanism by which GH induces target gene transcription.
Binding of GH to its membrane-bound receptor stimulates tyrosine
phosphorylation of receptor-associated JAK2, the cytoplasmic portion of
GHR, and STAT5b in liver (42). Phosphorylated STAT5b translocates to the cell nucleus, where it binds a specific DNA sequence to stimulate target gene transcription. To determine whether
TNF- had an inhibitory effect on the activation of STAT5b by GH, we
measured total and phosphorylated STAT5 in cytoplasmic and nuclear
extracts from CWSV-1 hepatocytes. Total protein was isolated from cell
lysates, and nuclear extracts were harvested 5 and 60 min after
stimulation with GH. Total STAT5b protein levels were not altered in
cell lysates from cells treated with TNF-
or exposed to GH (Fig.
5A). As shown in Fig.
5B, phosphorylated STAT5b was barely detectable in both
control and TNF-treated cells before GH stimulation. However, the
abundance of tyrosine-phosphorylated STAT5b protein in the cytoplasmic
fraction of control cells was increased more than fourfold at both 5 and 60 min after GH stimulation. The abundance of phosphorylated STAT5b
in the cytosolic fraction was also increased more than fourfold in
TNF-treated cells exposed to GH for 5 min. However, the phosphorylation
of STAT5 was decreased by 40% (P < 0.05 vs. GH at 60 min) in TNF-treated cells after the 60-min incubation with GH (Fig.
5B).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
GH is an important mediator of growth, differentiation, and metabolism in many tissues (30). The somatomedin hypothesis proposes that circulating IGF-I is synthesized by the liver in response to GH and is responsible for the effects of GH on postnatal growth and development. Consequently, GH administration has been suggested as an adjunct therapy to nutritional support to prevent the erosion of lean body mass observed in patients with catabolic diseases such as sepsis and burn injury (52). However, in 1988, Dahn et al. (13) demonstrated that neither circulating IGF-I nor urinary nitrogen excretion was improved in septic patients treated with rhGH. The results of that and other studies (12, 21, 29, 34) suggested that systemic infection and other catabolic illnesses are associated with the development of a GH-resistant state.
The inflammatory cytokines (TNF-, IL-1
, and IL-6) have been
proposed as potential mediators of GH resistance. Intravenous injection
of TNF-
and IL-1
in healthy animals results in a 30-40% reduction in circulating and tissue IGF-I levels (18, 20). Furthermore, treatment of septic rats with IL-1 receptor antagonist (IL-1ra) attenuates the effects of sepsis on the GH/IGF-I system (34). Treatment of septic rats with TNFbp attenuates the
catabolism of muscle protein and ameliorates the sepsis-induced
inhibition of gastrocnemius protein synthesis (11). In the
present study, injection of TNFbp ameliorated the increase in
circulating GH and reduction in plasma IGF-I observed in septic rats.
Although septic animals demonstrate reduced food intake on days
1-3, chow consumption returned to normal on days
4-5 and was not influenced by TNFbp administration (9,
11). Furthermore, plasma levels of corticosterone are similar in
septic and control rats on day 5 after chronic abdominal
sepsis (19, 36). Consequently, neither differences in
circulating glucocorticoid levels nor those in food intake appear to
explain the effects of TNFbp on the GH/IGF-I axis in septic animals.
In the present study, TNFbp was administered before the induction of sepsis to ensure that inhibitory levels of the TNF antagonist would coincide with peak plasma TNF levels (9, 11). Although circulating levels of TNF receptor are present after endotoxemia in humans (53), they are preceded by a peak in plasma TNF. Because only a brief exposure to TNF appears to be required to induce GH resistance in hepatocytes, the timing of TNFbp administration may be an important determinant in its ability to ameliorate the hepatic GH resistance.
The decrease in circulating IGF-I observed in septic rats was accompanied by reductions in hepatic IGF-I and GHR mRNA. The ability of TNFbp to ameliorate the inhibitory effects of sepsis on IGF-I and GHR mRNA levels in liver suggests that TNF is an important in vivo mediator of this phenomenon. In contrast to liver, the abundance of IGF-I mRNA in gastrocnemius was not significantly altered by sepsis or TNFbp. This may reflect a relative resistance of muscle to the inhibitory effects of sepsis on IGF-I synthesis compared with liver. Previous studies from our laboratory demonstrate a 30% reduction in IGF-I protein levels in gastrocnemius from septic rats relative to controls. A concomitant 40% reduction in protein synthesis was also observed in gastrocnemius from septic rats. This observation may help to explain the discrepancy between IGF-I mRNA and protein levels observed in gastrocnemius during chronic abdominal sepsis.
The results of several studies suggest that the effects of sepsis and
inflammatory cytokines on the GH/IGF-I axis are mediated by decreased
expression or affinity of the GHR for its ligand (15, 16,
50). In primary rat hepatocytes, both TNF- and IL-1
inhibited the synthesis of IGF-I mRNA in response to GH (50). Defalque et al. (15) found that, in
endotoxin [lipopolysaccharide (LPS)]-treated rats, hepatic GHR mRNA
and GHR-binding activity were reduced 5 h after the insult,
suggesting that the LPS-induced GH resistance is caused by a decreased
expression of hepatic GHR. A reduction in hepatic GHR mRNA was also
observed in LPS-treated wild-type but not LPS-treated TNF receptor
knockout mice (16). In this study, TNF was implicated in
the regulation of GHR expression at the transcriptional level by
inhibiting Sp1 and Sp3 transactivation of the GHR promoter
(16).
To determine whether decreased expression of GHR may be responsible for the reduction in circulating IGF-I observed in septic rats, the relative abundance of GHR protein in hepatic membrane preparations from Control, Sepsis, and Sepsis + TNFbp animals was measured. Neither sepsis nor TNFbp significantly altered the relative amount of GHR protein. Consequently, the GH-resistant state observed with abdominal sepsis does not appear to be caused by a reduction in the amount of hepatic GHR. These results are similar to those observed by Mao et al. (38) in LPS-treated rats in which similar levels of GHR protein were noted in liver from control and LPS-treated rats 4 h after LPS administration. Although differences in the abundance of hepatic IGF-I mRNA were reflected in the circulating IGF-I protein levels, the same relationship between mRNA and protein was not observed for the GHR mRNA and protein. Although the reduction in hepatic GHR mRNA could result in decreased synthesis of GHR protein, the relative abundance of GHR would remain unchanged if GHR stability or degradation were influenced as well. Recent observations suggest that GHR expression may be controlled, at least in part, by translational mechanisms (28). This might explain the discrepancy observed between the mRNA and protein levels in liver under in vivo conditions.
To investigate whether the effects of TNFbp on hepatic GH resistance
were directly attributable to TNF-, we performed additional experiments in a cultured rat hepatocyte model. The CWSV-1 hepatocytes were chosen because the cells express GHR, synthesize IGF-I in response
to GH (32, 37, 54), and transduce GH signals via the
JAK/STAT pathway (22, 32, 45). Incubation of hepatocytes with TNF-
for 4 h completely inhibited the synthesis of IGF-I mRNA induced by GH but had no effects on IGF-I mRNA content under basal, nonstimulated conditions. Treatment of CWSV-1 cells with TNF-
had no effect on the relative abundance of GHR protein. This finding
confirms our in vivo results in septic rats and provides additional
evidence that decreased expression of GHR is not necessary for the
induction of GH resistance by TNF-
. However, there is a significant
reduction in the levels of tyrosine-phosphorylated GHR observed 5 min
after GH administration in TNF-treated cells. These data suggest that
TNF-
attenuates the duration of the JAK/STAT signaling by GH.
The activated GHR complex stimulates multiple signaling pathways
including Ras-mitogen-activated protein kinase, insulin receptor substrate-phosphatidylinositol 3-kinase, and JAK/STAT
(26). Although the mechanism(s) by which GH stimulates
IGF-I synthesis is unknown, mice with targeted deletion of STAT5
demonstrate reduced basal and GH-induced levels of plasma IGF-I
(14). Previous studies suggest that LPS administration
inhibits hepatic GH signaling via the JAK2/STAT5 pathway (3,
38). Therefore, we examined the effects of TNF- on STAT5
tyrosine phosphorylation, nuclear translocation, and the
STAT5-dependent
-casein promoter DNA binding.
The comparable degree of STAT5 activation (STAT5 tyrosine
phosphorylation, nuclear translocation, and DNA binding) observed 5 min
after GH administration in TNF- and control cells shows that TNF-
does not completely prevent GH signaling via the JAK/STAT pathway in
CWSV-1 hepatocytes. However, the reduction in STAT5 activation observed
60 min after GH administration in TNF-treated cells suggests that
TNF-
significantly attenuates the duration of JAK/STAT5 signaling by
GH in these cells. The relative abundance of STAT5 was similar in
cytoplasmic cell lysates from TNF-
and control cells at both 5 and
60 min after GH. Consequently, reductions in total STAT5 do not explain
the decrease in STAT5 activity observed at 60 min after GH
administration in the TNF-treated cells. Instead, the time course of
STAT5 activation-deactivation that was noted suggests that TNF-
pretreatment enhances the termination of GH-induced STAT5 signaling.
Several biochemical events appear to be important in regulating the
duration of GH-induced STAT5 signaling. The activated GHR complex is
internalized by endocytosis and subsequently degraded by the
ubiquitin-proteasome system (2). Administration of the proteasome inhibitor MG132 has been shown to prolong the GH-induced activity of GHR, JAK2, and STAT5 by stabilizing the tyrosine
phosphorylation status of these proteins (2, 23). TNF-
has been shown to activate ubiquitin-dependent proteasome-mediated
proteolytic pathways (35, 41). Thus the inhibitory effects
of TNF-
on GH signaling could be caused by proteasome-mediated
deactivation of the GH signal.
The duration of GH-activated STAT5 signaling also depends on the
activity of JAK2 (which phosphorylates STAT5) as well as the
dephosphorylation of STAT5 by phosphotyrosine phosphatases (reviewed in
Refs. 26 and 27). The phosphorylation status and kinase
activity of JAK2 were not measured in the present study; however, both
the GHR and STAT5 are tyrosine phosphorylated by JAK2
(27). Therefore, the reductions in GHR phosphorylation (5 min) and STAT phosphorylation (60 min) observed in TNF-treated cells
could be explained by reductions in activity of JAK2, because the
relative abundance of JAK2 was unaltered (data not shown). An
alternative explanation is that an increase in phosphotyrosine phosphatase activity is responsible for the inhibitory effects of
TNF- on GH signaling. Phosphotyrosine phosphatase inhibitors block
the deactivation of STAT5b after termination of a GH pulse (23). Dephosphorylation of GHR, JAK2, and STAT proteins is
thought to involve the recruitment of one or more SH2 tyrosine
phosphatases to GHR/JAK2 or JAK/STAT complexes. GH stimulation induces
SHP-1 phosphatase activity, activates nuclear translocation, and
promotes its association with phosphorylated nuclear STAT5b in CWSV-1
cells (23). Consequently, the activity of SHP-1 and other
tyrosine phosphatases represents another important regulatory mechanism for STAT5b signaling which could be influenced by TNF-
.
The recently described suppressors of cytokine signaling (SOCS) have
also been shown to inhibit GH-mediated signaling through interactions
with the cytoplasmic portion of the GHR and/or JAK2 (8, 25,
33). The messenger RNA for cytokine-inducible Sh2 protein (CIS)
or SOCS1-3 is typically found at low levels in different cells or
tissues (4, 8, 33). However, an increase in SOCS mRNA is
commonly observed when cells are exposed to various cytokines or growth
factors, including TNF- and GH (8). Overexpression of
the SOCS proteins in transfected cells inhibits the activation of Spi
2.1 and the acid-labile subunit by GH (3, 4, 25). Furthermore, LPS-treated rats demonstrate a fourfold induction of CIS
mRNA and a tenfold induction of SOCS-3 mRNA in liver when compared with
saline-treated controls (38). A recent study showed that
TNF-
could potentiate the induction of SOCS-3 and CIS by GH in
primary rat hepatocytes (4). Thus TNF-mediated induction of SOCS represents another potentially important mechanism by which
sepsis or inflammatory cytokines could inhibit GH signaling.
In summary, TNF- appears to be an important mediator of the
GH-resistant septic state. Reductions in hepatic GHR are not necessary
to explain the inhibitory effects of sepsis or TNF-
on the induction
of IGF-I synthesis by GH. Although TNF-
does not appear to impair
early GH signaling, this cytokine clearly inhibits the duration of
GH-mediated JAK/STAT signaling. Several regulatory mechanisms could
explain the inhibition of hepatic GH signaling observed in TNF-treated
cells: proteasome-mediated deactivation of the GHR/JAK2 complex,
stimulation of tyrosine phosphatase activity, or the inhibition of
JAK/STAT signaling by SOCS. Additional studies will be necessary to
determine the relative importance of these different biochemical
pathways in regulating hepatic JAK/STAT signaling by TNF-
and GH.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Peter Rotwein (U. of Oregon) and Lawrence S. Mathews (U. of Michigan) for generously providing the IGF-I and GHR plasmids used in this study. We also acknowledge W. R. Baumbach (Monsanto) for providing antibodies to rat GHR, H. Isom (Dept. of Microbiology, Pennsylvania State University College of Medicine) for providing the CWSV-1 cells, and Carl Edwards (Amgen, Thousand Oaks, CA) for providing the TNFbp.
![]() |
FOOTNOTES |
---|
This work was supported in part by National Institute of General Medical Sciences Grants GM-55639A (R. N. Cooney), GM-38032 (CH. Lang), and GM-38032-S1 (G. Yumet).
Address for reprint requests and other correspondence: R. N. Cooney, Dept. of Surgery, Pennsylvania State Univ. College of Medicine, Hershey, PA 17033 (E-mail: rcooney{at}psu.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.
10.1152/ajpendo.00107.2002
Received 8 March 2002; accepted in final form 4 May 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Adamo, ML,
Neuenschwander S,
and
LeRoith D.
Structure, expression, and regulation of the IGF-I gene.
In: Current Directions in Insulin-Like Growth Factor Research, , edited by LeRoith D,
and Raisada MK. New York: Plenum, 1994, p. 1-11.
2.
Alves dos Santos, CM,
van Kerkhof P,
and
Strous GJ.
The signal transduction of the growth hormone receptor is regulated by the ubiquitin/proteasome system and continues after endocytosis.
J Biol Chem
276:
10839-10846,
2001
3.
Bergad, PL,
Schwarzenberg SJ,
Humbert JT,
Morrison M,
Amarasinghe S,
Towle HC,
and
Berry SA.
Inhibition of growth hormone action in models of inflammation.
Am J Physiol Cell Physiol
279:
C1906-C1917,
2000
4.
Boisclair, YR,
Wang J,
Shi J,
Hurst KR,
and
Oii GT.
Role of the suppressor of cytokine signaling-3 in mediating the inhibitory effects of interleukin-1B on the growth hormone-dependent transcription of the acid-labile subunit gene in liver cells.
J Biol Chem
275:
3841-3847,
2000
5.
Carter, R,
Yumet G,
Pena A,
Soprano DR,
and
Soprano KJ.
Transcriptional regulation of c-Jun expression during late G1/S in normal human cells is lost in human tumor cells.
Oncogene
9:
2675-2682,
1994[ISI][Medline].
6.
Cerra, FB,
Siegel JH,
Coleman B,
Border JR,
and
McMenamy RR.
Septic autocannibalism: a failure of exogenous nutritional support.
Ann Surg
192:
570-580,
1980[ISI][Medline].
7.
Chow, JC,
Ling PR,
Qu Z,
Laviola L,
Ciccarone A,
Bistrian BR,
and
Smith RJ.
Growth hormone stimulates tyrosine phosphorylation of JAK2 and STAT5 but not insulin response substrate-1 or SHC proteins in liver and skeletal muscle of normal rats in vivo.
Endocrinology
137:
2880-2886,
1996[Abstract].
8.
Colson, A,
Le Cam A,
Maiter D,
Ederly M,
and
Thiessen J.
Potentiation of growth hormone-induced liver suppressors of cytokine signaling messenger ribonucleic acid by cytokines.
Endocrinology
141:
3687-3695,
2000
9.
Cooney, RN,
Iocono J,
Maish GO,
Smith JS,
and
Ehrlich P.
Tumor necrosis factor mediates impaired wound healing in chronic abdominal sepsis.
J Trauma
42:
415-420,
1997[ISI][Medline].
10.
Cooney, RN,
Kimball SR,
and
Vary TC.
Regulation of skeletal muscle protein turnover in sepsis: mechanisms and mediators.
Shock
7:
1-16,
1997[ISI][Medline].
11.
Cooney, RN,
Vary TC,
Maish GO,
Shumate ML,
and
Eckman R.
TNF binding protein ameliorates alterations in skeletal muscle protein metabolism during sepsis.
Am J Physiol Endocrinol Metab
276:
E611-E619,
1999
12.
Dahn, MS,
and
Lange MP.
Systemic and splanchnic metabolic response to exogenous growth hormone.
Surgery
123:
528-38,
1998[ISI][Medline].
13.
Dahn, MS,
Lange MP,
and
Jacobs LA.
Insulin-like growth factor I production is inhibited in human sepsis.
Arch Surg
123:
1409-1414,
1988[Abstract].
14.
Davey, HW,
Xie T,
McLachlan MJ,
Wilkins RJ,
Waxman DJ,
and
Grattan DR.
STAT5b is required for GH-induced liver IGF-I gene expression.
Endocrinology
142:
3836-3841,
2001
15.
Defalque, D,
Brandt N,
Ketelslegers JM,
and
Thissen JP.
GH insensitivity induced by endotoxin injection is associated with decreased liver GH receptors.
Am J Physiol Endocrinol Metab
276:
E565-E572,
1999
16.
Denson, LA,
Menon RK,
Shaufl A,
Bawa HS,
Williams CR,
and
Karpen SJ.
TNF- downregulates murine hepatic growth hormone receptor expression by inhibiting Sp1 and Sp3 binding.
J Clin Invest
107:
1451-1458,
2001
18.
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 Regul Integr Comp Physiol
269:
R1204-R1212,
1995
19.
Fan, J,
Molina PE,
Gelato MC,
and
Lang CH.
Differential tissue regulation of insulin-like growth factor-I content and binding proteins after endotoxin.
Endocrinology
134:
1685-1692,
1994[Abstract].
20.
Fan, J,
Wojnar M,
Theodorakis M,
and
Lang CH.
Regulation of insulin-like growth factor (IGF-I) mRNA and peptide, and IGF-binding proteins by interleukin-1.
Am J Physiol Regul Integr Comp Physiol
270:
R621-R629,
1996
21.
Frost, RA,
and
Lang CH.
Growth factors in critical illness: regulation and therapeutic aspects.
Curr Opin Clin Nutr Metab Care
1:
195-205,
1998[Medline].
22.
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
23.
Gebert, CA,
Park SH,
and
Waxman DJ.
Termination of growth hormone pulse-induced STAT5b signaling.
Mol Endocrinol
13:
38-56,
1999
24.
Goodman, MN.
Interleukin-6 induces skeletal muscle protein breakdown in rats.
Proc Soc Exp Biol Med
205:
182-185,
1994[Abstract].
25.
Hansen, JA,
Lindberg K,
Hilton DJ,
Nielsen JH,
and
Billestrup N.
Mechanism of inhibition of growth hormone receptor signaling by suppressor of cytokine signaling proteins.
Mol Endocrinol
13:
1832-1843,
1999
26.
Herrington, J,
and
Carter-Su C.
Signaling pathways activated by the growth hormone receptor.
Trends Endocrinol Metab
12:
252-257,
2001[ISI][Medline].
27.
Herrington, J,
Smit LS,
Schwartz J,
and
Carter-Su C.
The role of STAT protein in growth hormone signaling.
Oncogene
19:
2585-2597,
2000[ISI][Medline].
28.
Jiang, H,
and
Lucy MC.
Variants of the 5'-untranslated region of the bovine growth hormone receptor mRNA: isolation, expression and effects on translational efficiency.
Gene
265:
45-53,
2001[ISI][Medline].
29.
Jenkins, RC,
and
Ross RJ.
Acquired growth hormone resistance in adults.
Baillieres Clin Endocrinol Metab
12:
315-329,
1998[ISI][Medline].
30.
Jones, JI,
and
Clemmons DR.
Insulin-like growth factors and their binding proteins: biological actions.
Endocr Rev
16:
3-32,
1995[ISI][Medline].
31.
Jurasinski, CV,
and
Vary TC.
Insulin-like growth factor-I accelerates protein synthesis in skeletal muscle during sepsis.
Am J Physiol Endocrinol Metab
269:
E977-E981,
1995
32.
Kempe, KC,
Isom HC,
and
Greene FE.
Responsiveness of an SV40-immortalized hepatocyte cell line to growth hormone.
Biochem Pharmacol
49:
1091-1098,
1995[ISI][Medline].
33.
Krebs, DL,
and
Hilton DJ.
SOCS: physiological suppressors of cytokine signaling.
J Cell Sci
113:
2813-2819,
2000
34.
Lang, CH,
Fan J,
Cooney RN,
and
Vary TC.
IL-1ra attenuates sepsis-induced alterations in the IGF system and protein synthesis.
Am J Physiol Endocrinol Metab
270:
E430-E437,
1996
35.
Li, YP,
and
Reid MB.
NF-B mediates the protein loss induced by TNF-
in differentiated skeletal muscle myotubes.
Am J Physiol Regul Integr Comp Physiol
279:
R1165-R1170,
2000
36.
Li, YH,
Fan J,
and
Lang CH.
Differential role of glucocorticoids in mediating endotoxin-induced changes in IGF-I and IGFBP-1.
Am J Physiol Regul Integr Comp Physiol
272:
R1990-R1997,
1997
37.
Liao, WS,
Ma KT,
Wodworth CD,
Mengel L,
and
Isom HC.
Stimulation of acute-phase response in simian virus 40-hepatocyte cell lines.
Mol Cell Biol
9:
2779-2786,
1989[ISI][Medline].
38.
Mao, Y,
Ling P,
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.
Matthews, LS,
Enberg B,
and
Norstedt G.
Regulation of rat growth hormone receptor gene expression.
J Biol Chem
264:
9905-9919,
1989
40.
Moldawer, LL,
Svaninger G,
Gerlin J,
and
Lundolm KG.
Interleukin-1 and tumor necrosis factor do not regulate protein balance in skeletal muscle.
Am J Physiol Cell Physiol
253:
C766-C773,
1987
41.
Pallares-Trujillo, J,
Carbo N,
Lopez-Soriano FJ,
and
Argiles JM.
Does the mechanism responsible for TNF-mediated insulin resistance involve the proteasome?
Med Hypotheses
54:
565-569,
2000[ISI][Medline].
42.
Pellegrini, S,
and
Dusanter-Fourt I.
The structure, regulation and function of the Janus kinases (JAKs) and the signal transducers and activators of transcription (STATs).
Eur J Biochem
248:
615-633,
1997[Abstract].
43.
Posner, B,
Kelly PA,
Shiu PC,
and
Friesen HG.
Studies of insulin, growth hormone, and prolactin binding: tissue distribution, species variation, and characterization.
Endocrinology
95:
521-531,
1974[ISI][Medline].
44.
Ram, PA,
Park S,
Choi HK,
and
Waxman DJ.
Growth hormone activation of Stat 1, Stat 3, and Stat 5 in rat liver.
J Biol Chem
271:
5929-5960,
1996
45.
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
46.
Sadeghi, H,
Wang BS,
Lumanglas AL,
Logan JS,
and
Baumbach WR.
Identification of the origin of rat growth hormone binding protein in rat serum.
Mol Endocrinol
4:
1799-1805,
1990[Abstract].
47.
Shimatsu, A,
and
Rotwein P.
Sequence of two rat insulin-like growth factor 1 mRNAs differing within the 5' untranslated region.
Nucleic Acids Res
15:
7196,
1987[ISI][Medline].
48.
Stein, B,
Rahmsdorf HJ,
Steffen A,
Litfin M,
and
Herrlich P.
UV-induced DNA damage is an intermediate step in UV-induced expression of human immunodeficiency virus type 1, collagenase, c-fos, and metallothionein.
Mol Cell Biol
9:
5169-5181,
1989[ISI][Medline].
49.
Svanberg, E,
Frost RA,
Lang CH,
Isgaard J,
Jefferson LS,
Kimball SR,
and
Vary TC.
IGF-I/IGFBP-3 binary complex modulates sepsis-induced inhibition of protein synthesis in skeletal muscle.
Am J Physiol Endocrinol Metab
279:
E1145-E1158,
2000
50.
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
51.
Vary, TC,
Voisin L,
and
Cooney RN.
Regulation of peptide-chain initiation in muscle during sepsis by interleukin-1 receptor antagonist.
Am J Physiol Endocrinol Metab
271:
E513-E520,
1996
52.
Wilmore, D.
Catabolic illnesses: strategies for enhancing recovery.
N Engl J Med
325:
695-702,
1991[Abstract].
53.
Wilson, M,
Blum R,
Dandona P,
and
Mousa S.
Effects in humans of intravenously administered endotoxin on soluble cell-adhesion molecule and inflammatory markers: a model of human diseases.
Clin Exp Pharmacol Physiol
28:
376-380,
2001[ISI][Medline].
54.
Woodworth, CD,
and
Isom HC.
Regulation of albumin gene expression in a series of rat hepatocyte cell lines immortalized by simian virus 40 and maintained in chemically defined medium.
Mol Cell Biol
7:
3740-3748,
1987[ISI][Medline].
55.
Zamir, O,
Hasselgren PO,
Kunkel SL,
Frederick JA,
Higashiguchi T,
and
Fischer JE.
Evidence that tumor necrosis factor participates in the regulation of muscle proteolysis during sepsis.
Arch Surg
127:
170-179,
1992[Abstract].
56.
Zamir, O,
Hasselgren PO,
O'Brien WO,
Thompson RC,
and
Fischer JE.
Muscle protein breakdown during endotoxemia in rats and after treatment with IL-1ra.
Ann Surg
216:
381-387,
1992[ISI][Medline].
57.
Zhu, T,
Goh ELK,
Graichen R,
Ling L,
and
Lobie PE.
Signal transduction via the growth hormone receptor.
Cell Signal
13:
599-616,
2001[ISI][Medline].