Interleukin-1 inhibits the induction of insulin-like growth factor-I by growth hormone in CWSV-1 hepatocytes

Margaret L. Shumate, Gladys Yumet, Tamer A. Ahmed, and Robert N. Cooney

Department of Surgery, The Pennsylvania State University-College of Medicine, Hershey, Pennsylvania

Submitted 21 September 2004 ; accepted in final form 4 April 2005


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Sepsis results in hepatic "growth hormone (GH) resistance" with reductions in plasma IGF-I despite a two- to fourfold increase in circulating GH. In this study, we examine the effects of IL-1 on GH receptor (GHR) expression, GH signaling (via the JAK/STAT and MAPK pathways), and the induction of gene expression [IGF-I mRNA and serine protease inhibitor (Spi) 2.1] by GH in CWSV-1 hepatocytes. Incubation of cells with IL-1{beta} (10 ng/ml, 24 h) had no effect on the relative abundance of GHR or signaling proteins JAK2, STAT5b, and ERK1/2 in cell lysates. Baseline phosphorylation of GHR, JAK2, STAT5b, and ERK1/2 was minimal. After GH stimulation, tyrosine phosphorylation of GHR, JAK2, STAT5b, and ERK1/2 increased 2- to 10-fold. However, neither the time course nor the magnitude of GHR, JAK2, and ERK1/2 phosphorylation by GH were significantly altered by IL-1. The GH-induced translocation of STAT5b to the nucleus was not prevented by IL-1. Although phosphorylated STAT5 in nuclear extracts from GH + IL-1 cells was decreased by 24% (vs. controls) 15 min after GH stimulation, this did not result in reduced STAT5-DNA binding activity. Pretreatment with IL-1 did not significantly decrease IGF-I mRNA stability. We conclude that IL-1 only minimally affects the time course of JAK2/STAT5 and MAPK signaling by GH. Therefore, an inhibitory effect of IL-1 on IGF-I and Spi 2.1 mRNA synthesis by GH represents the most likely mechanism for IL-1-mediated GH resistance.

growth hormone resistance; sepsis; hepatocytes; janus kinase/signal transducer and activator of transcription signaling; mitogen-activated protein kinase signaling


THE DEVELOPMENT OF GROWTH hormone resistance is one of the major metabolic derangements in patients with sepsis (15, 16, 66). During severe infection, the catabolism of body protein results in multiple complications that prolong recovery and cause death (8, 12, 71, 72). Nutrient intake alone is unable to prevent the loss of lean body mass, suggesting that other factors are important (8). Several lines of evidence implicate the inflammatory cytokines TNF and IL-1 as indirect modulators of muscle protein metabolism during sepsis via the GH/IGF-I system (13, 35, 71, 72). First, sepsis is associated with an increase in inflammatory cytokine production, increased plasma GH levels, and reductions in circulating IGF-I that are reproduced by TNF-{alpha} or IL-1{beta} administration (19, 20, 23). Second, incubation of muscle tissue with IL-1{beta} or TNF-{alpha} in vitro shows no direct effect of either cytokine on muscle protein synthesis (39). Third, in vivo infusion of IL-1 or TNF antagonists attenuates the sepsis-induced alterations in the IGF-I system and prevents the catabolism of protein in gastrocnemius (13, 35). Finally, the role of IGF-I as a mediator of muscle protein synthesis in septic rats has been confirmed in isolated hindlimb perfusions (31). Addition of IGF-I to the perfusate produced a dose-dependent increase in gastrocnemius protein synthesis, which was restored to control levels. Collectively, these results provide evidence that the inhibitory effects of IL-1 and TNF on skeletal muscle protein synthesis during sepsis are mediated indirectly by changes in the GH/IGF-I system.

GH stimulates the synthesis of circulating IGF-I by liver, which upregulates protein synthesis in many tissues and promotes wound healing (73). Although the exact mechanisms by which GH stimulates hepatic IGF-I expression are not completely defined, the JAK2/STAT5 pathway appears to play an important role in regulating this process. Initiation of GH signaling involves the sequential binding of GH with two transmembrane receptors and association of GH-(GHR)2 with JAK2, an intracellular protein kinase that forms an activated multiprotein signaling complex. The activated GHR/JAK2 complex forms high-affinity binding sites for several signaling and regulatory proteins that propagate signaling via the JAK/STAT, MAPK, and phosphatidylinositol 3 (PI3)-kinase pathways. These pathways, in turn, regulate gene transcription and cellular metabolism. The propagation of GH signaling involves a coordinated series of phosphorylation reactions that activate gene transcription. GH stimulates the tyrosine phosphorylation of STAT5b by JAK2 and serine/threonine phosphorylation of STAT5b by the MAPK pathway (9). Phosphorylated STAT5b forms homodimers and heterodimers that translocate to the nucleus and bind DNA sequences in the promoter region of target genes to regulate transcriptional activity (22).

Although the mechanisms responsible for the activation of IGF-I transcription by GH remain incompletely characterized, studies in STAT5b knockout mice suggest that STAT5b is required for both basal and GH-induced expression of hepatic IGF-I (17). STAT5b dominant negative adenoviral transfection was recently shown to inhibit GH-mediated hepatic IGF-I gene transcription (64). The identification of tandem STAT5 binding sites in intron 2 of the IGF gene that mediate activation of IGF-I transcription by GH provides additional evidence for the JAK2/STAT5 pathway in regulating the induction of IGF-I by GH (65). Likewise, activation of acid-labile subunit (ALS) and serine protease inhibitor 2.1 (Spi 2.1) transcription by GH is mediated by STAT5 binding to {gamma}-interferon-activated sequences (GAS) in the promoter region of these genes (4, 40). As with IGF-I, the mediators of change in hepatic gene response during sepsis also modulate ALS and Spi 2.1 expression (3, 5, 55); however, the specific events involved remain to be clarified.

Because IGF-I is responsible for many of GH’s anabolic properties (59), we hypothesized that IL-1 contributes to the development of hepatic GH resistance by inhibiting GH signaling and IGF-I gene transcription. To test this hypothesis, we studied the effects of IL-1 on the activation, propagation, and termination of GH signaling, as well as the induction of IGF-I mRNA and IGF-I mRNA stability in CWSV-1 hepatocytes (32, 36, 38, 68). We also examined the effects of IL-1 on GH-mediated Spi 2.1 expression in our hepatocyte model. Our results suggest that the inhibitory effects of IL-1 on the induction of IGF-I and Spi 2.1 mRNA by GH do not appear to be mediated by reductions in GH receptor, postreceptor defects in JAK/STAT or MAPK signaling. We also showed that inhibition of IGF-I gene expression is not attributed to changes in IGF-I mRNA stability.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Recombinant human GH (rhGH; Pharmacia and Upjohn, Stockholm, Sweden) was used in all experiments. Rat IL-1{beta} was obtained from R&D Systems (Minneapolis, MN). The plasmid containing the rat IGF-I cDNA was a kind gift from Peter Rotwein (Oregon Health Science) (51). The oligonucleotide sequence used as a probe to detect Spi 2.1 is as described by Bergad et al. (3). The cDNA for murine suppressors of cytokine signaling-3 (SOCS-3) was a kind gift from Robyn Starr (The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia) (56). Polyclonal GHR antibody was obtained from W. R. Baumbaugh (American Cyanamid, Princeton, NJ) and used at a dilution of 1:250 (49). Rabbit polyclonal STAT5b antibody (sc-835, Santa Cruz Biotechnology, Santa Cruz, CA) and PY20 phosphotyrosine antibody conjugated with horseradish peroxidase (BD Transduction Laboratories, San Diego, CA) were used for immunoblot analyses. Polyclonal JAK2 antibody and rabbit anti-phospho-JAK2 were obtained from Upstate Biotechnology (Lake Placid, NY). Polyclonal p44/42 MAPK antibody and phospho-p44/42 MAPK antibody that recognize ERK1 and ERK2 were obtained from Cell Signaling, New England Biolabs (Beverly, MA). PD-98059, an inhibitor of MEK-1 (MAPK kinase), was also obtained from Cell Signaling, New England Biolabs.

Cell culture experiments. CWSV-1 hepatocytes, obtained from Dr. Harriet Isom (Dept. of Microbiology and Immunology, College of Medicine, Pennsylvania State University), were cultured as previously described (32, 36). For this study, CWSV-1 cells were grown in RPCD medium for 48 h. IL-1-treated cells were incubated with 10 ng/ml IL-1{beta} for 24 h, and then 500 ng/ml rhGH was added for the indicated time periods. In other experiments, cells were treated for 60 min with 50 µM PD-98059 prior to stimulation with GH.

Northern blot analysis. The relative abundance of IGF-I, Spi 2.1, and SOCS-3 mRNA was determined by Northern blot analysis as previously described (63, 70). 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 used as a probe (51). In liver, the exon 1-derived 7.5-kb transcript of IGF-I mRNA represents the predominant (80%) IGF-I mRNA species. To detect the 1.8-kb Spi 2.1 transcript, a 25-base oligonucleotide sequence was used as the probe (3). For SOCS-3, a 681-bp MluI fragment corresponding to the mouse SOCS-3 cDNA was used as a probe (56). After exposure of the completed blots to film, the autoradiographs were scanned using an HP ScanJet 5300C model scanner. Northern blot analyses were stripped and reprobed with the 18S ribosomal subunit message to confirm uniform loading of RNA as previously described (70). Scans were analyzed using Scion Image for Windows (National Institutes of Health). Data are reported as relative densitometry units after normalization to 18S rRNA message.

Preparation of cell lysates and isolation of nuclear protein. Cell lysates were prepared from cells grown in culture dishes, then 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 to the dishes that were then incubated at 4°C for 30 min. Lysates were cleared from nuclei by centrifugation at 10,000 rpm for 5 min. Supernatants were snap frozen in liquid nitrogen and stored at –70°C (48). Nuclear extracts were prepared as previously described (58, 70).

Western blot analysis and immunoprecipitation. For the detection of total protein, equal amounts of protein were electrophoresed on an 8% polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon P; Millipore, Bedford, MA), using standard electroblotting procedures. For the detection of phosphorylated proteins, cell lysates (100–500 ug) were immunoprecipitated and immunocomplexes were resolved using SDS-PAGE (70). Total and phosphorylated GHR and total and phosphorylated STAT5b were measured by Western blot analysis as described in Yumet et al. (70). Antibody reactions were visualized using ECL-Plus (Amersham Pharmacia Biotech). The intensity of antibody reactions was analyzed using Scion Image for Windows.

To visualize total JAK2, membranes were blocked in PBS containing 3% nonfat milk overnight at 4°C. Incubation continued in a rabbit polyclonal anti-JAK2 diluted in PBS-milk for 3 h at room temperature. The membranes were washed with water, then incubated with secondary antibody-horseradish peroxidase (HRP) conjugate for 1 h at room temperature. For detection of phosphorylated JAK2, membranes were blocked in Tris-buffered saline (TBS) containing 5% milk and 0.05% Tween (TBST) for 30 min at room temperature. Membranes were incubated with the primary anti-phospho-JAK2 in TBST-milk overnight at 4°C. After the wash step, membranes were exposed to secondary antibody-HRP for 1.5 h at room temperature. Membranes were again washed, and the ECL method was used for detection of the antibody reactions.

To detect total or phosphorylated ERK1 and ERK2, membranes were blocked in TBST containing 5% nonfat milk for 1 h at room temperature. After washes with TBST, membranes for total protein were incubated in rabbit polyclonal p44/42 MAPK antibody diluted in TBST with 5% BSA overnight at 4°C. Membranes for detection of phosphorylated ERK1 and ERK2 were incubated in rabbit phospho-p44/42 MAPK antibody diluted in TBST with 5% nonfat milk overnight at 4°C. Membranes were washed as before and incubated with secondary antibody-HRP for 1 h at room temperature. The membranes were washed, and the reactions were detected using the ECL method.

EMSA. Oligonucleotides for gel shift assays were prepared as previously described (6, 70). Complementary strands to the rat {beta}-casein promoter 5'-GGA CTT CTT GGA ATT AAG GGA-3' were labeled independently using T4 kinase (Promega). Nuclear protein from CWSV-1 cells (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 1x binding buffer (10 mM Tris, pH 7.5, 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl) (45). Reactions were incubated for 30 min at 25°C and then electrophoresed on a prerun 4% PAGE/0.5x TBE gel 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 and exposed to film.

IGF-I mRNA stability. The transcriptional inhibitor 5,6-dichloro-{beta}-D-ribofuranosyl-benzimdazole (DRB; 72 µM, Calbiochem, La Jolla, CA) was used to examine the effects of IL-1 on the stability and natural decay kinetics of GH-induced IGF-I mRNA. CWSV-1 cells treated with or without IL-1{beta} (10 ng/ml) for 24 h were stimulated with rhGH (500 ng/ml) for 12 h. DRB (72 µM) or vehicle was added to the cells, and total RNA was isolated at 0, 30, 60, 90, and 120 min following the addition of DRB. The relative abundance of IGF-I mRNA was determined by Northern blot analysis and normalized to 18S rRNA.

Statistical analysis. Data are presented as means ± SE and represent the results of at least three independent experiments. The Northern blot analysis and immunoblot data are expressed as relative densitometry units. Statistical evaluation of the data was analyzed by ANOVA followed by the Tukey-Kramer multiple comparison test using Instat GraphPad 5.02. Differences among means were considered significant at P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
IL-1 inhibits the induction of IGF-I mRNA by GH. To investigate the role of IL-1 in hepatic GH resistance, we initially examined the effects of IL-1{beta} on the induction of IGF-I mRNA by GH. As shown in Fig. 1, A and B, incubation of hepatocytes with rhGH resulted in a twofold increase in the abundance of IGF-I mRNA (*P < 0.05 vs. control). Pretreatment of CWSV-1 cells with IL-1{beta} for 24 h had no effect on basal IGF-I expression. However, preincubation with IL-1{beta} for 24 h significantly inhibited the induction of IGF-I mRNA following GH administration (40% reduction; **P < 0.05 vs. GH). Interestingly, the effects of IL-1 on the regulation of IGF-I by GH appear to be time dependent, because incubation of hepatocytes with IL-1{beta} for 6 h (Fig. 1C) did not significantly influence the induction of IGF-I by GH (14% reduction).



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Fig. 1. IL-1 inhibits the induction of IGF-I mRNA by growth hormone (GH). A: cells were treated with 10 ng/ml IL-1{beta} for 24 h, then stimulated with 500 ng/ml recombinant human (rh)GH for 18 h. Northern blot analysis was performed as described in METHODS. IGF-I mRNA corresponds to the 7.5-kb exon 1-derived transcript. B: densitometry data for IGF-I mRNA were normalized to 18S rRNA message and expressed as means ± SE. *P < 0.05 vs. control; **P < 0.05 vs. GH. C: cells were treated with IL-1{beta} for 6 h, followed by GH for 18 h. Northern blot analysis was then performed.

 
Effect of IL-1 on GH stimulation of Spi 2.1 mRNA expression. Using the same experimental conditions, we examined the effects of IL-1 on expression of Spi 2.1, another GH-responsive, STAT5-mediated gene (3). GH stimulation of hepatocytes elicited a greater than 10-fold increase in expression of Spi 2.1 mRNA compared with baseline (Fig. 2, A and B). Treatment of hepatocytes with IL-1{beta} for 24 h significantly inhibited the induction of Spi 2.1 mRNA (90% reduction) by GH compared with GH stimulation alone (Fig. 2, A and B; *P < 0.001 vs. GH).



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Fig. 2. IL-1 inhibits the induction of serine protease inhibitor 2.1 (Spi 2.1) mRNA by GH. A: cells were treated with 10 ng/ml IL-1{beta} for 24 h, then stimulated with 500 ng/ml rhGH for 18 h. Northern blot analysis was performed as described in METHODS. B: densitometry data for Spi 2.1 mRNA were normalized to 18S rRNA message and expressed as means ± SE. *P < 0.001 vs. GH.

 
Induction of SOCS-3 expression by IL-1 and GH. Stimulation of the SOCS expression by inflammatory cytokines, particularly SOCS-3, has been implicated as a mechanism for GH resistance (5, 37). Therefore, we examined the effects of IL-1{beta} incubation and GH stimulation on SOCS-3 expression in CWSV-1 cells. As shown in Fig. 3A, basal expression of SOCS-3 was minimal. A fivefold increase in SOCS-3 mRNA was noted from 30 to 60 min after GH stimulation, which returned to basal levels by 120 min. IL-1 treatment alone resulted in a transient increase in SOCS-3 mRNA (2-fold) from 120 to 240 min, which decreased to basal levels by 360 min. In Fig. 3B, we examined GH-induced SOCS-3 expression in cells incubated ±IL-1{beta} for 24 h. There was no detectable effect of IL-1 preincubation on the magnitude, timing, or duration of SOCS-3 mRNA expression in response to GH stimulation compared with GH treatment alone. Because the induction of SOCS-3 by IL-1 has the potential to inhibit GH signaling via the JAK/STAT pathway, we subsequently examined the effects of IL-1 on the initiation, propagation, and termination of GH signaling.



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Fig. 3. Induction of suppressors of cytokine signaling-3 (SOCS-3) mRNA expression by IL-1 and GH. A: cells were treated with 500 ng/ml rhGH or 10 ng/ml IL-1{beta} for 30, 60, 120, 240, 360, and 480 min. B: 1 group of cells was pretreated with 10 ng/ml IL-1{beta} for 24 h. Then, both sets of cells were stimulated with 500 ng/ml rhGH for 5–360 min. Northern blot analysis was performed as described in METHODS. Uniformity of loading was confirmed by comparison with 18S rRNA message.

 
Initiation of GH signaling in IL-1-treated hepatocytes. To determine whether IL-1 influenced the initiation of GH signaling, we examined the effects of IL-1{beta} incubation on total GHR levels and the time course of GHR phosphorylation in GH-stimulated cells. Total GHR protein levels were not significantly altered (relative to baseline) by either preincubation with IL-1{beta} or stimulation with GH (Fig. 4A). As shown in Fig. 4B, a relatively low level of tyrosine-phosphorylated GHR was observed under basal conditions. After the addition of rhGH, levels of tyrosine-phosphorylated GHR increased sixfold for more than 15 min and subsequently decreased to basal levels by 60 min. The time course and magnitude of GHR phosphorylation were not significantly altered by IL-1{beta} pretreatment (Fig. 4C).



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Fig. 4. Total and phosphorylated GHR in IL-1 treated cells. Cells were treated with 10 ng/ml IL-1{beta} for 24 h and then stimulated with 500 ng/ml rhGH for 2.5, 5, 10, 15, 30 and 60 min. A: cell lysate immunoblotted with anti-GHR polyclonal antibody. B: lysate immunoprecipitated with anti-GHR and immunoblotted with PY20 (anti-phosphotyrosine) antibody. C: densitometry data for phosphorylated GH receptor (GHR) were normalized to total protein, presented as relative densitometry units (RDU) and expressed as means ± SE. Blots are representative of experiments done >3 times.

 
The tyrosine phosphorylation of JAK2 by the activated GH-GHR2 complex represents a critical step in the initiation of GH signaling (43). Therefore, we measured total JAK2 protein and the time course of JAK2 phosphorylation following GH stimulation in hepatocytes (±IL-1{beta}). As indicated in Fig. 5A, neither IL-1 nor GH influenced the relative abundance of total JAK2 protein in CWSV-1 cells. Stimulation of hepatocytes with GH resulted in a transient (2.5–90 min) induction of tyrosine-phosphorylated JAK2 (Fig. 5B). However, there were no significant differences in either the time course or levels of tyrosine-phosphorylated JAK2 in IL-1{beta}-pretreated cells following GH stimulation (Fig. 5C). Thus IL-1 does not appear to influence the initiation of GH signaling by altering total or tyrosine-phosphorylated JAK2.



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Fig. 5. GH-induced JAK2 phosphorylation in IL-1-treated cells. Cells were treated with 10 ng/ml IL-1{beta} for 24 h and then stimulated with 500 ng/ml rhGH for 2.5, 5, 10, 15, 30, 60, and 90 min. A: cell lysate immunoblotted with anti-JAK2 polyclonal antibody. B: lysate immunoblotted with anti-phospho-JAK2 polyclonal antibody. C: densitometry data for phosphorylated JAK2 were normalized to total protein, presented as RDU, and expressed as means ± SE. Blots are representative of experiments done >3 times.

 
Propagation of the GH signaling via the JAK/STAT pathway. The activated GHR/JAK2 complex propagates GH signaling via the signal transducer and activator of transcription (STAT5) pathway (26, 27, 73). To determine whether IL-1 inhibits the propagation of GH signaling via the STAT5 pathway, we examined the time course of STAT5b phosphorylation, nuclear translocation, and DNA binding following GH stimulation in CWSV-1 hepatocytes (±IL-1{beta}). First, the relative abundance of STAT5b protein was measured in cell lysates and nuclear extracts harvested over time following stimulation with GH. Total STAT5b protein levels were not altered in cell lysates harvested 0 to 90 min following GH stimulation (±IL-1{beta}; Fig. 6A). Before GH stimulation, tyrosine-phosphorylated STAT5b was barely detectable in either control or IL-1{beta}-treated cells (Fig. 6B). After the addition of GH, the relative abundance of tyrosine-phosphorylated STAT5b protein in the lysates increased ~10-fold from 0 to 30 min (P < 0.001 vs. baseline), then gradually decreased. Although slight reductions in the relative abundance of phosphorylated STAT5b were observed in IL-1-treated cells at 30, 60, and 90 min after GH stimulation (25% reduction at 30 min), these differences were not statistically significant (Fig. 6C).



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Fig. 6. Effect of IL-1{beta} on STAT5b levels in lysates of CWSV-1 cells. Cells were treated with 10 ng/ml IL-1{beta} for 24 h and then stimulated with 500 ng/ml rhGH for 2.5, 5, 10, 15, 30, 60, and 90 min. A: cell lysate immunoblotted with anti-STAT5b polyclonal antibody. B: lysate immunoprecipitated with anti-STAT5b and immunoblotted with PY20 antibody. C: densitometry data for phosphorylated STAT5b were normalized to total protein, presented as RDU and expressed as means ± SE. Blots are representative of experiments performed a minimum of 6 times.

 
Before GH stimulation, only low levels of total STAT5b were observed in the nuclear protein fraction (Fig. 7A). Fifteen minutes after GH stimulation, total nuclear STAT5b protein levels were increased twofold in both the GH and IL-1{beta} + GH groups (P < 0.001 vs. baseline). As shown in Fig. 7A, the amount of total STAT5b remaining in the nucleus of IL-1{beta} + GH-treated cells was significantly less (25%) at 60 min relative to the GH group (P < 0.05 vs. GH). Tyrosine-phosphorylated STAT5b was barely detected in the nucleus before GH stimulation in either group (Fig. 7B). However, the relative abundance of phosphorylated STAT5b in the nuclear fraction of GH-stimulated cells increased 14-fold 15 min after exposure to GH. The IL-1{beta}-treated cells demonstrated a 24% reduction in phosphorylated nuclear STAT5b at 15 min after GH (Fig. 7C; *P < 0.05 vs. GH). Levels of phosphorylated STAT5b in the nuclear fraction in both groups decreased to near baseline by 60 min after GH stimulation.



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Fig. 7. Effect of IL-1{beta} on STAT5b levels in the nuclear fraction of CWSV-1 cells. Cells were treated with 10 ng/ml IL-1{beta} for 24 h and then stimulated with 500 ng/ml rhGH for 15 and 60 min. A: nuclear fraction immunoblotted with anti-STAT5b polyclonal antibody. B: nuclear fraction immunoprecipitated with anti-STAT5b and immunoblotted with PY20 antibody. C: densitometry data for phosphorylated STAT5b were normalized to total protein, presented as RDU and expressed as means ± SE. *P < 0.05 vs. GH at 15 min.

 
To determine the functional significance of the reduction in phosphorylated STAT5 observed at 15 min in the nuclear fraction of the IL-1{beta} + GH group, DNA binding activity of nuclear protein extracts was measured using an EMSA to a STAT5 {beta}-casein promoter sequence (6, 70). The STAT5 DNA-binding activity of nuclear protein was increased at both 15 and 60 min after GH treatment of the cells (Fig. 8A). DNA binding by STAT5 was increased twofold (vs. baseline) at 15 min after GH treatment. The DNA binding activity of nuclear protein was diminished but still observed at 60 min. Pretreatment of the cells with IL-1{beta} did not diminish STAT5 DNA-binding activity at 15 or 60 min following GH stimulation (Fig. 8B). The specificity of the STAT5 binding reaction was confirmed by the ability of antibody specific for STAT5b to shift the mobility of the STAT5 DNA complex (Fig. 8A, lane 7). Additionally, the specificity of STAT5 binding conditions in this EMSA was previously demonstrated using cold consensus and mutated competitor sequences as described by Yumet et al. (70). Although the IL-1{beta} + GH group yielded a slightly lower level of phosphorylated STAT5b in the nucleus at 15 min post-GH treatment, this did not significantly affect the DNA-binding activity of STAT5 at that time. Consequently, IL-1 does not appear to inhibit the ability of activated STAT5 to bind to DNA.



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Fig. 8. Effects of IL-1{beta} on GH-induced STAT5-DNA binding activity. CWSV-1 cells were treated with 10 ng/ml IL-1{beta} for 24 h and the stimulated with 500 ng/ml rhGH for 15 and 60 min. A: nuclear extracts (5 ug) were used in an EMSA with a STAT5-labeled probe as described in METHODS. P, probe alone; C, untreated cells. Lane 7: protein from GH after 15 min was used in the presence of anti-STAT5b antibody. The migration of the shifted DNA-protein complex and supershift complex are indicated. The blot is representative of experiments done >3 times. B: densitometry data for the STAT5-DNA complex are expressed as means ± SE.

 
Propagation GH signaling via the MAPK pathway. GH also activates the MAPK pathway to regulate inflammatory and metabolic responses. Serine phosphorylation of STAT5 via the MAPK pathway has been shown to regulate STAT5-mediated gene transcription. To determine whether the MAPK pathway influences GH-induced IGF-I gene expression, we treated cells with PD-98059, an inhibitor of MEK-1 (53). Treatment with PD-98058 resulted in a 20% reduction in the abundance of IGF-I mRNA following GH stimulation (Fig. 9, A and B; *P < 0.05 vs. GH). This indicates that the MAPK-signaling pathway contributes to induction of IGF-I by GH.



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Fig. 9. Contribution of the MAPK pathway to GH-induced IGF-I gene expression. A: cells were treated for 60 min with 50 uM of the MAPK inhibitor PD-98059 (PD), then stimulated with 500 ng/ml GH for 18 h. Northern blot analysis was performed as previously described. B: densitometry data for IGF-I mRNA were normalized to 18S rRNA message and expressed as means ± SE. *P < 0.001 vs. GH.

 
The potential for IL-1 to alter GH-mediated activation of the MAPK cascade was evaluated by measuring total and phosphorylated ERK1 and ERK2 in cells harvested 0–60 min after GH stimulation (62). Neither GH stimulation nor IL-1{beta} pretreatment alters total ERK1 or ERK2 levels relative to controls (Fig. 10A). Low levels of phosphorylated ERK1 and ERK2 were detected in control cells. Phosphorylated ERK1/2 significantly increased (2-fold, from 0 to 15 min) following GH stimulation (Fig. 10B). However, IL-1 did not alter the abundance of GH-mediated phosphorylated ERK1 and ERK2 at any time point (Fig. 10C). Consequently, IL-1 does not appear to influence the amplitude or duration of activated ERK1 and ERK2 in the cytoplasm.



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Fig. 10. Effects of IL-1{beta} on ERK1 and ERK2 levels. Cells were treated with 10 ng/ml IL-1{beta} for 24 h and then stimulated with 500 ng/ml rhGH for 2.5, 5, 10, 15, 30, 60, and 90 min. A: cell lysates immunoblotted with polyclonal p44/42 MAPK antibody for total protein. B: lysates immunoblotted with phospho-p44/42 MAPK antibody for phosphorylated ERK1/2. C: densitometry data for phosphorylated ERK1/2 were normalized to total ERK1/2, presented as RDU, and expressed as means ± SE. Data are representative of experiments done >3 times.

 
With the use of similar experimental conditions, the effects of IL-1 on the time course of GH signaling via the p38 and JNK-MAPK pathways were examined (28, 73). Neither the abundance of total p38 protein nor the induction of phosphorylated p38 by GH was significantly altered by IL-1 (data not shown). In this cell line, GH failed to stimulate phosphorylation of the transcription factor c-Jun, a substrate of JNK. Therefore, no impact of IL-1 on c-Jun activation could be determined. Consequently, these pathways do not appear to play a role in the development of GH resistance from exposure to IL-1.

IGF-I mRNA stability. The effects of IL-1 on IGF-I mRNA stability were examined to determine whether pretreatment with IL-1{beta} increased IGF-I mRNA degradation. As shown in Fig. 11D, cells treated with GH for 12 h (GH) demonstrate stable IGF-I mRNA levels over the 120-min study period. As one would expect, addition of the transcriptional inhibitor DRB (GH + DRB) resulted in a gradual reduction in IGF-I mRNA over time to ~50% of control levels 90 min after the addition of DRB (Fig. 11, A and D). IL-1{beta}-pretreated cells (IL-1{beta}+GH) demonstrate a 35% reduction in the relative abundance of IGF-I mRNA (P < 0.001 vs. GH) at baseline, which decreases slightly over the 120-min study period (Fig. 11, B and D). When DRB is added to IL-1{beta}-pretreated cells (IL-1{beta} + GH + DRB), the relative abundance of IGF-I initially decreases, then stabilizes, suggesting either somewhat increased stability or decreased turnover of IGF-I mRNA in IL-1{beta}-pretreated cells (Fig. 11, C and D).



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Fig. 11. IGF-I mRNA stability in IL-1-treated cells. A: to establish the decay kinetics of GH-induced IGF-I mRNA, CWSV-1 hepatocytes were stimulated with rhGH (500 ng/ml) for 12 h and total RNA was isolated at 0, 30, 60, 90, and 120 min following the addition of 5,6-dichloro-{beta}-D-ribofuranosyl-benzimdazole (DRB; 72 µM) or vehicle. B: cells were pretreated with IL-1{beta} (10 ng/ml) for 24 h. After stimulation with rhGH for an additional 12 h, total RNA was isolated at the time points described. C: cells were pretreated with IL-1{beta} for 24 h, then stimulated with rhGH for 12 h. After the addition of DRB, total RNA was isolated at the time points indicated above. Northern blot analysis was performed as previously described. D: densitometry data for IGF-I mRNA were normalized to 18S rRNA message and were expressed as means ± SE. Data are representative of experiments performed >3 times.

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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The phenomenon of GH resistance was initially described in 1988 by Dahn et al. (16) when septic patients administered rhGH demonstrated neither an improvement in nitrogen balance nor an increase in circulating IGF-I. Since then, numerous studies have implicated the inflammatory cytokines (TNF, IL-1, and IL-6) as potential mediators of GH resistance (13, 30, 35, 71, 72). Intravenous injection of TNF-{alpha} or IL-1{beta} in healthy animals causes a 30–40% reduction in circulating and tissue IGF-I levels, which is similar to that observed during systemic infection (19, 20). Furthermore, the treatment of septic rats with specific IL-1 or TNF antagonists attenuates both the catabolism of muscle and sepsis-induced perturbations in the GH/IGF-I system (13, 35, 71, 72). Although the role of inflammatory cytokines in the genesis of GH resistance seems relatively well established, the molecular mechanisms of GH resistance at the cellular level remain poorly defined.

The lack of GH receptor and IGF-I expression in many immortalized hepatocyte cell lines and the technical limitations of primary hepatocyte studies have been major obstacles to mechanistic studies of cytokine-mediated GH resistance. In the current study, we describe the effects of IL-1 on GH signaling and the induction of IGF-I mRNA by GH using CWSV-1 hepatocytes. CWSV-1 is an SV40-transformed rat cell line that expresses GHR and synthesizes IGF-I in response to GH (32, 36, 68). CWSV-1 cells incubated with IL-1{beta} for 6 h demonstrate a normal twofold induction of IGF-I mRNA following GH stimulation. However, preincubation with IL-1{beta} for 24 h significantly inhibits the induction of IGF-I mRNA by GH. In addition, we demonstrated that CWSV-1 cells express Spi 2.1, a second GH-responsive, STAT5-mediated gene, which is associated with the hepatic acute phase response to inflammatory stimuli (2, 3, 21, 34, 50). IL-1{beta} inhibits the expression of Spi 2.1 under similar experimental conditions. This is the first study that demonstrates an inhibitory effect of IL-1 on two GH-responsive genes, IGF-I and Spi 2.1, in an immortalized hepatocyte cell line.

Previous studies examining the relative abundance of GH receptor in liver tissue from septic rats have yielded conflicting results. Defalque et al. (18) identified a 50% reduction in hepatic GH-binding sites 5–10 h after LPS administration using a bovine GH binding assay. Although reductions in hepatic GHR mRNA have been described following LPS administration or abdominal sepsis, the relative abundance of GHR protein in liver was not influenced by sepsis when measured by immunoblot analysis (37, 70). Because cytokine-mediated reductions in the relative abundance of GH receptor have also been hypothesized as a mechanism for hepatic GH resistance, we began by examining the effects of IL-1 on GHR expression (60, 67). The relative abundance of GHR was similar in IL-1{beta}-treated and control cells in the current study. Therefore, IL-1-mediated reductions in GHR do not explain the inhibitory effects of IL-1 on the induction of IGF-I by GH.

Several studies (3, 37) have identified postreceptor defects in hepatic GH signaling via the JAK/STAT pathway during sepsis or inflammation. The SOCS proteins are cytokine/growth factor-inducible inhibitors of cell signaling via the JAK/STAT pathway (11, 14, 46). Increased SOCS-3 expression by IL-1 has been hypothesized as a potential mechanism for repression of ALS expression in hepatic GH resistance (5). Others have discounted the influence of the SOCS inhibition of Spi 2.1 expression in hepatocyte cultures because IL-1 treatment alone did not result in an increase in SOCS-3 expression that could contribute to early termination of the GH signal (3). Both IL-1 and GH stimulated the transient expression of SOCS-3 mRNA in CWSV-1 cells. However, preincubation with IL-1{beta} for 24 h did not significantly influence either the magnitude or time course of SOCS-3 expression by GH. It has been shown that interactions between the SOCS proteins and JAK2 or GHR inhibit GH signaling via the JAK/STAT pathway (5, 10, 25, 33, 46). Consequently, if the induction of SOCS-3 expression by IL-1 were responsible for hepatic GH resistance, an inhibitory effect on GH signaling via the JAK2/STAT5 pathway should be seen. Therefore, we examined the effects of IL-1 on the relative abundance of signaling proteins and the time course of GH signaling via the JAK2/STAT5 pathway.

The current study is unique in that it meticulously examines the effects of IL-1 on the time course of GH signaling via the JAK2/STAT5 pathway. The relative abundance of JAK2 and STAT5 and the time course of GHR and JAK2 phosphorylation by GH were unaltered by IL-1. Although transient reductions in STAT5b phosphorylation were identified in IL-1{beta}-pretreated cells, the magnitude of these changes does not appear to correlate well with the observed reductions in IGF-I or Spi 2.1 expression. It remains possible that the transient reductions in STAT5 phosphorylation observed in IL-1{beta}-treated cells could influence the induction of these genes by GH. However, one would expect the effects on IGF-I or Spi 2.1 expression to be minimal as well. An alternative explanation is that neither increased SOCS-3 expression nor impaired JAK/STAT signaling is an important regulatory mechanism for IL-1-mediated reductions in GH-inducible target gene expression in CWSV-1 hepatocytes. Our study is the first to suggest that the cytokine-mediated inhibition of the GH/IGF-I axis in hepatocytes is not caused by impaired JAK/STAT signaling.

Unfortunately, few studies to date have carefully characterized the effects of sepsis or inflammation on the time course of GH signaling or examined the relationship between STAT5 phosphorylation and IGF-I expression. Postreceptor defects in hepatic GH signaling via the JAK/STAT pathway have been identified during sepsis and inflammation (3, 37). However, none of these studies conclusively shows that signaling defects are responsible for decreased expression of GH-inducible genes. Although the relative abundance of tyrosine-phosphorylated JAK2 and STAT5 were diminished following GH administration in liver from LPS-treated rats (37), the effects of LPS on GH-mediated gene expression were not evaluated in that study. Preincubation of CWSV-1 cells with TNF attenuates the duration of STAT5 phosphorylation by GH and is associated with decreased IGF-I expression, but cause and effect remain unproven (70). Therefore, although postreceptor defects in GH signaling via the JAK/STAT pathway represent a potential mechanism for decreased IGF-I expression during sepsis, the exact mechanisms remain unproven.

More recently, serine phosphorylation of STAT5 by the "mitogen-activated" or MAPK pathway has been implicated in the regulation of STAT5 transcriptional activation (29). Because the MAPK pathway is one of the major signaling pathways activated by GH (7, 26), we performed several experiments to determine whether IL-1 inhibits GH-induced IGF-I expression by influencing the MAPK pathway. Treatment of CWSV-1 cells with PD-98059 resulted in a 20% reduction (P < 0.05 vs. GH) in IGF-I expression. These results are consistent with the 24% reduction in IGF-I mRNA observed with MAPK kinase inhibition in GH-stimulated primary hepatocytes (53).

Although we did not directly assess serine phosphorylation of STAT5 in the current study, we did examine the effects of IL-1 on the time course of MAPK signaling by measuring the relative abundance and phosphorylation time course of the ERKs 1 and 2 (28, 61, 62). After GH stimulation, phosphorylated ERKs translocate to the cell nucleus where they activate several transcription factors including Elk-1, which stimulates the transcription of c-Fos and c-Jun by GH as well as influences inflammatory responses, cell division, and apoptosis in various cell types. A number of signaling proteins in mitogen- or stress-activated pathways undergoes both serine/threonine and tyrosine phosphorylation resulting in "cross-talk" between the pathways. Because IL-1 did not alter the magnitude or time course of ERK1 or ERK2 phosphoryation, we conclude that IL-1 does not cause GH resistance by altering MAPK signaling. The lack of an IL-1 effect on GH signaling via the p38 MAPK and JNK pathways suggests neither of these MAPK pathways is responsible for IL-1-mediated GH resistance in CWSV-1 hepatocytes. Although the PI3-kinase signaling pathway has also been implicated in the regulation of IGF-I synthesis by GH, we could find no evidence of Akt phosphorylation following GH stimulation in CWSV-1 hepatocytes (data not shown). Consequently, IL-1 does not appear to inhibit GH-inducible gene expression by altering the abundance or activation of either JAK/STAT- or MAPK-signaling proteins.

In summary, incubation of CWSV-1 hepatocytes with IL-1{beta} for 24 h inhibits the induction of IGF-I and Spi 2.1 mRNA by GH. The inhibitory effects of IL-1 do not appear to be caused by reductions in the relative abundance of GHR. The reduction in IGF-I mRNA in cells treated with PD-98059 provides evidence for involvement of the MAPK pathway in the regulation of IGF-I expression by GH. However, because IL-1 did not influence the time course of ERK phosphorylation by GH, this cytokine does not appear to act by inhibiting MAPK signaling. IL-1 was associated with increased SOCS-3 expression and modest reductions in STAT5b phosphorylation. However, STAT5 activation (as measured by EMSA) by GH was not significantly impaired by IL-1. Therefore, the inhibitory effects of IL-1 do not appear to be completely explained by SOCS-3 expression or impaired JAK2/STAT5 signaling. Potential mechanisms for the reduction in IGF-I mRNA observed in IL-1{beta}-treated cells include reductions in IGF-I mRNA synthesis or increased IGF-I mRNA degradation.

The current study provides the first evidence that IGF-I mRNA stability is not significantly decreased by IL-1 pretreatment, and in fact, it may be somewhat stabilized (Fig. 11). On the basis of these results, we conclude that the inhibitory effects of IL-1 on GH-mediated IGF-I expression are caused by its effects on IGF-I promoter activity. The finding that GH stimulation of both IGF-I and Spi 2.1 mRNA was inhibited by IL-1 suggests a more general regulatory mechanism by which inflammatory cytokines suppress JAK2/STAT5-mediated gene expression by GH. The inhibitory effects of IL-1 on GH-inducible Spi 2.1 and ALS promoter activity in transfected primary hepatocytes and H4-II-E rat hepatoma cells support this concept (3, 5). The transcriptional activation of both Spi 2.1 and ALS by GH is regulated by STAT5 binding to GAS sequences in their promoter regions (4, 40). More recently, the transcriptional activation of IGF-I by GH was demonstrated to involve STAT5 binding to GAS-like sequences in intron 2 using chromatin immunoprecipitation (65).

Unfortunately, the current study falls short of identifying the specific mechanisms by which IL-1 inhibits the expression of IGF-I by GH. However, the regulation of IGF-I gene expression, similar to that of other mammalian genes, is controlled by a number of factors including the production and activity of transcription factors, recruitment of transcriptional coactivators, localized remodeling of chromatin structures in DNA-binding and trans-activation sites, and the timely and appropriate degradation of messenger RNA. Recent studies have identified a number of regulatory mechanisms for STAT5-mediated gene expression. The potential for negative regulation is observed in splice variants of STAT5 that exhibit normal phosphorylation and DNA binding but lack trans-activation ability and act as a strong suppressor of wild-type action (24). Hepatocyte-enriched nuclear factor HNF3{beta} and peroxisome proliferator-activated receptor-{alpha} activation were recently shown to inhibit STAT5b-mediated gene expression by GH in HepG2 cells (42, 52). Additionally, the glucocorticoid response element and p300/CREB-binding proteins have been shown to bind STAT5 and influence STAT5-mediated transcription activity (24, 44). The protein inhibitors of activated STATs (PIAS) have also been shown to inhibit STAT-mediated gene activation (54, 57, 69). At least one such protein, PIASy, influences gene expression through its effects on chromatin structure and does not prevent STAT association with DNA (69). Such a PIAS, if recruited by IL-1, could mediate negative regulation by direct protein-protein interaction with STAT5. STAT5 has also been shown to influence remodeling of chromatin structure, leading to transcriptional activation of target genes. Consequently, the histone acetyl transferases, which are involved in chromatin remodeling and act as coactivators to regulate transcriptional activity of STATs, may serve as targets for IL-1 inhibition (47). Finally, negative regulation of GH-mediated gene expression may also be influenced by the ubiquitin-proteasome pathway, which uses GHR internalization and degradation as a mechanism of termination (1, 22, 41). Additional studies will be required to determine the exact mechanisms by which IL-1 inhibits the induction of IGF-I by GH; however, collectively, the evidence suggests a defect in IGF-I gene expression.


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This work was supported in part by National Institute of General Medical Sciences Grants GM-55639 (to R. N. Cooney) and T32-GM-64332 (to G. Yumet and T. Ahmed).


    ACKNOWLEDGMENTS
 
We thank P. Rotwein (U. of Oregon) and R. Starr (The Walter and Elizabeth Hall Institute of Medical Research) for generously providing the IGF-I and SOCS-3 plasmids used in this study. We also acknowledge W. R. Baumbach (Monsanto) for providing antibodies to rat GHR and H. Isom (Dept. of Microbiology, Pennsylvania State University College of Medicine) for providing the CWSV-1 cells.


    FOOTNOTES
 

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.


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 RESULTS
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  1. 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.[Abstract/Free Full Text]
  2. 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]
  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.[Abstract/Free Full Text]
  4. Bergad PL, Shis 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 {gamma}-activated sites. J Biol Chem 270: 24903–24910, 1995.[Abstract/Free Full Text]
  5. Boisclair YR, Wang J, Shi J, Hurst KR, and Ooi GT. Role of the suppressor of cytokine signaling-3 in mediating the inhibitory effects of interleukin-1{beta} on the growth hormone-dependent transcription of the acid-labile subunit gene in liver cells. J Biol Chem 275: 3841–3847, 2000.[Abstract/Free Full Text]
  6. 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: 2675–2782, 1994.[ISI][Medline]
  7. Carter-Su C and Smit LS. Signaling via JAK tyrosine kinases: growth hormone receptor as a model system. Recent Prog Horm Res 53: 61–83, 1998.[Medline]
  8. 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]
  9. 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]
  10. Colson A, Le Cam A, Maiter D, Ederly M, and Thissen J. Potentiation of growth hormone-induced liver suppressors of cytokine signaling messenger ribonucleic acid by cytokines. Endocrinology 141: 3687–3695, 2000.[Abstract/Free Full Text]
  11. Cooney RN. Suppressors of cytokine signaling (SOCS): inhibitors of the JAK/STAT pathway. Shock 17: 83–90, 2002.[CrossRef][ISI][Medline]
  12. Cooney RN, Kimball SR, and Vary TC. Regulation of skeletal muscle protein turnover in sepsis: Mechanisms and mediators. Shock 7: 1–16, 1997.[CrossRef][ISI][Medline]
  13. 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.[Abstract/Free Full Text]
  14. Cooney RN and Yumet G. Cytokine signaling in sepsis: redundancy, crosstalk, and regulatory mechanisms. Crit Care Med 30: 262–263, 2002.[CrossRef][ISI][Medline]
  15. Dahn MS and Lange MP. Systemic and splanchnic metabolic response to exogenous growth hormone. Surgery 123: 528–538, 1998.[CrossRef][ISI][Medline]
  16. Dahn MS, Lange P, and Jacobs LA. Insulin-like growth factor 1 production is inhibited in human sepsis. Arch Surg 123: 1409–1414, 1988.[Abstract]
  17. 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.[Abstract/Free Full Text]
  18. 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.[Abstract/Free Full Text]
  19. 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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  21. Fey GH and Gauldie J. The acute phase response of the liver in inflammation. Prog Liver Dis 9: 89–117, 1990.[Medline]
  22. Gebert CA, Park SH, and Waxman DJ. Termination of growth hormone pulse-induced STAT5b signaling. Mol Endocrinol 13: 38–56, 1999.[Abstract/Free Full Text]
  23. Goodman MN. Interleukin-6 induces skeletal muscle protein breakdown in rats. Proc Soc Exp Biol Med 205: 182–185, 1994.[Abstract]
  24. Groner B, Fritsche M, Stocklin E, Berchotold S, Merkle C, Moriggl R, and Pfitzner E.Regulation of the trans-activation potential of STAT5 through its DNA-binding activity and interactions with heterologous transcription factors. Growth Hormone IGF Res, Suppl B: S15–S20, 2000.
  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.[Abstract/Free Full Text]
  26. Herrington J and Carter-Su C. Signaling pathways activated by the growth hormone receptor. Trends Endocrinol Metab 12: 252–257, 2001.[CrossRef][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.[CrossRef][ISI][Medline]
  28. Hodge C, Liao Stofega MJ, Guan K, Carter-Su C, and Schwartz J. Growth hormone stimulates phosphorylation and activation of Elk-1 and expression of c-fos, egr-1, and junB through activation of extracellular signal-regulated kinases 1 and 2. J Biol Chem 273: 31327–31336, 1998.[Abstract/Free Full Text]
  29. Horvath CM and Darnell JE. The state of the STATs: recent developments in the study of signal transduction to the nucleus. Curr Opin Cell Biol 9: 233–239, 1997.[CrossRef][ISI][Medline]
  30. Jenkins RC and Ross RJ. Acquired growth hormone resistance in adults. Baillieres Clin Endocrinol Metab 12: 315–329, 1998.[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.[Abstract/Free Full Text]
  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.[CrossRef][ISI][Medline]
  33. Krebs DL and Hilton DJ. SOCS: physiological suppressors of cytokine signaling. J Cell Sci 113: 2813–2819, 2000.[Abstract/Free Full Text]
  34. Kushner I. The acute phase response: an overview. Methods Enzymol 163: 373–383, 1988.[ISI][Medline]
  35. 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.[Abstract/Free Full Text]
  36. Liao WS, Ma KT, Woodworth 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]
  37. 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.[Abstract/Free Full Text]
  38. Matthews LS, Enberg B, and Norstedt G. Regulation of rat growth hormone receptor gene expression. J Biol Chem 264: 9905–9919, 1989.[Abstract/Free Full Text]
  39. Moldawer LL, Svaninger G, Gerlin J, and Lundhlm KG. Interleukin-1 and tumor necrosis factor do not regulate protein balance in skeletal muscle. Am J Physiol Cell Physiol 253: C766–C773, 1987.[Abstract/Free Full Text]
  40. Ooi GT, Hurst KR, Poy MN, Rechler MM, and Boisclair YR. Binding of STAT5a and STAT5b to a single element resembling a gamma-interferon-activated sequence mediates the growth hormone induction of the mouse acid-labile subunit promoter in liver cells. Mol Endocrinol 12: 675–687, 1998.[Abstract/Free Full Text]
  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.[CrossRef][ISI][Medline]
  42. Park SH and Waxman DJ. Inhibitory cross-talk between STAT5b and liver nuclear factor HNF3{beta}. J Biol Chem 276: 43031–43039, 2001.[Abstract/Free Full Text]
  43. 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]
  44. Pfitzner E, Jahne R, Wissler M, Stoecklin E, and Groner B. p300/CREB-binding protein enhances the prolactin-mediated transcriptional induction through direct interaction with the transactivation domain of Stat5, but does not participate in the Stat5-mediated suppression of the glucocorticoid response. Mol Endocrinol 12: 1582–1593, 1998.[Abstract/Free Full Text]
  45. 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.[Abstract/Free Full Text]
  46. Ram PA and Waxman DJ. SOCS/CIS protein inhibition of growth hormone-stimulated STAT5 signaling by multiple mechanisms. J Biol Chem 274: 35553–35561, 1999.[Abstract/Free Full Text]
  47. Rascle A and Lees E. Chromatin acetylation and remodeling at the cis promoter during STAT5-induced transcription. Nucleic Acids Res 31: 6882–6890, 2003.[Abstract/Free Full Text]
  48. Rubini M, D’Ambrosio C, Carturan S, Yumet G, Catalano E, Shan S, Huang Z, Criscuolo M, Pifferi M, and Baserga R. Characterization of an antibody that can detect an activated IGF-I receptor in human cancers. Exp Cell Res 251: 22–32, 1999.[CrossRef][ISI][Medline]
  49. 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]
  50. 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.[Abstract/Free Full Text]
  51. 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]
  52. Shipley JM and Waxman DJ. Down-regulation of STAT5b transcriptional activity by ligand-activated peroxisome proliferator-activated receptor (PPAR){alpha} and PPAR{gamma}. Mol Pharmacol 64: 355–364, 2003.[Abstract/Free Full Text]
  53. Shoba LN, Newman M, Liu W, and Lowe WL Jr. LY294002, an inhibitor of phosphatidylinositol 3-kinase, inhibits GH-mediated expression of the IGF-I gene in rat hepatocytes. Endocrinology 142: 3980–3986, 2001.[Abstract/Free Full Text]
  54. Shuai K. Modulation of STAT signaling by STAT-interacting proteins. Oncogene 19: 2638–2644, 2000.[CrossRef][ISI][Medline]
  55. Simar-Blanchet AE, Legraverend C, Thissen JP,and Le Cam A. Transcription of the rat serine protease inhibitor 21 gene in vivo: correlation with GAGA box promoter occupancy and mechanism of cytokine-mediated down-regulation. Mol Endocrinol 12: 391–404, 1998.[Abstract/Free Full Text]
  56. Starr R, Willson TA, Viney EM, Murray LJL, Rayner JR, Jenkins BJ, Gonda TJ, Alexander WS, Metcalf D, Nicola NA, and Hilton DJ. A family of cytokine-inducible inhibitors of signaling. Nature 387: 917–921, 1997.[CrossRef][ISI][Medline]
  57. Starr R and Hilton DJ. Negative regulation of the JAK/STAT pathway. Bioessays 21: 47–52, 1999.[CrossRef][ISI][Medline]
  58. 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]
  59. Stewart CE and Rotwein P. Growth, differentiation, and survival: multiple physiological functions for insulin-like growth factors. Physiol Rev 76: 1005–1026, 1996.[Abstract/Free Full Text]
  60. 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.[Abstract/Free Full Text]
  61. VanderKuur J, Allevato G, Billestrup N, Norstedt G, and Carter-Su C. Growth hormone-promoted tyrosyl phosphorylation of Shc proteins and Shc association with Grb2. J Biol Chem 270: 7587–7593, 1995.[Abstract/Free Full Text]
  62. VanderKuur JA, Butch ER, Waters SB, Pessin JE, Guan KL, and Carter-Su C. Signaling molecules involved in coupling growth hormone receptor to mitogen activated protein kinase activation. Endocrinology 138: 4301–4307, 1987.[CrossRef]
  63. 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.[Abstract/Free Full Text]
  64. Woelfle J, Billiard J, and Rotwein P. Acute control of IGF-I gene transcription by growth hormone through Stat5b. J Biol Chem 278: 22696–22702, 2003.[Abstract/Free Full Text]
  65. Woelfle J, Chia DJ, and Rotwein P. Mechanisms of growth hormone action: identification of conserved STAT5 binding sites that mediate GH-induced insulin-like growth factor-I gene activation. J Biol Chem 278: 51261–51266, 2003.[Abstract/Free Full Text]
  66. Wojnar MM, Fan J, Frost RA, Gelato MC, and Lang CH. Alterations in the insulin-like growth factor system in trauma patients. Am J Physiol Regul Integr Comp Physiol 268: R960–R977, 1995.
  67. Wolf M, Bohm S, Brand M, and Kreymann G. Proinflammatory cytokines interleukin 1 beta and tumor necrosis factor alpha 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.[Abstract]
  68. 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]
  69. Wormald S and Hilton DJ. Inhibitors of cytokine signal transduction. J Biol Chem 279: 821–824, 2004.[Abstract/Free Full Text]
  70. Yumet G, Shumate ML, Bryant P, Lin CM, Lang CH, and Cooney RN. Tumor necrosis factor mediates hepatic growth hormone resistance during sepsis. Am J Physiol Endocrinol Metab 283: E472–E481, 2002.[Abstract/Free Full Text]
  71. 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]
  72. Zamir O, Hasselgren PO, O’Brien WO, Thompson RC, and Fischer EJ. Muscle protein breakdown during endotoxemia in rats and after treatment with IL-1ra. Ann Surg 216: 381–387, 1992.[ISI][Medline]
  73. Zhu T, Goh ELK, Graichen R, Ling L, and Lobie PE. Signal transduction via the growth hormone receptor. Cell Signal 13: 599–616, 2001.[CrossRef][ISI][Medline]




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