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Am J Physiol Gastrointest Liver Physiol 291: G35-G44, 2006. First published March 30, 2006; doi:10.1152/ajpgi.00550.2005
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HORMONES AND SIGNALING

Tumor necrosis factor inhibits growth hormone-mediated gene expression in hepatocytes

Tamer Ahmed,1,* Gladys Yumet,1,* Margaret Shumate,1 Charles H. Lang,1,2 Peter Rotwein,3 and Robert N. Cooney1,2

Departments of 1Surgery and 2Cellular and Molecular Physiology, The Pennsylvania State University-College of Medicine, Hershey, Pennsylvania; and 3Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon

Submitted 5 December 2005 ; accepted in final form 21 March 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Growth hormone (GH) stimulates STAT5 phosphorylation by JAK2, which activates IGF-I and serine protease inhibitor 2.1 (Spi 2.1) transcription, whereas STAT5 dephosphorylation by protein tyrosine phosphatases (PTPs) terminates this signal. We hypothesized that the inhibitory effects of TNF on GH signaling and gene transcription were responsible for hepatic GH resistance. CWSV-1 hepatocytes were treated with TNF, pervanadate (a PTP inhibitor), or both, before GH stimulation. Total and tyrosine-phosphorylated JAK2, STAT5, ERK1/2, SHP-1 and SHP-2, IGF-I, and Spi 2.1 mRNA levels were measured. GH stimulated STAT5 and ERK1/2 phosphorylation, IGF-I, and Spi 2.1 mRNA expression. TNF attenuated JAK2/STAT5 and ERK1/2 phosphorylation and IGF-I and Spi 2.1 mRNA expression following GH stimulation. SHP-1 and SHP-2 protein levels were unaltered by TNF or GH, and the GH-induced increase in SHP-1 PTP activity was not further increased by TNF. In TNF-treated cells, pervanadate restored STAT5 and ERK1/2 phosphorylation to control levels following GH stimulation but did not restore IGF-I or Spi 2.1 mRNA induction. Cells transfected with a Spi 2.1 promoter-luciferase vector demonstrate a 50-fold induction in luciferase activity following GH stimulation or cotransfection with a constitutively active STAT5 vector. TNF prevented the induction of Spi 2.1 promoter activity by GH and the STAT5 construct. We conclude that TNF does not inhibit GH activity by inducing SHP-1 or -2 expression and that correction of GH signaling defects in TNF-treated cells by pervanadate does not restore GH-induced gene expression. The inhibitory effects of TNF on GH-mediated gene transcription appear independent of STAT5 activity and previously identified abnormalities in JAK2/STAT5 signaling.

growth hormone resistance; STAT5; insulin-like growth factor I; serine protease inhibitor 2.1

GROWTH HORMONE (GH) is an important anabolic peptide that stimulates the expression of multiple genes regulating cell growth, intermediary metabolism, and protein synthesis (15, 24, 51). Binding of GH to the transmembrane GH receptor (GHR) results in the formation of a multiprotein GHR complex involving the tyrosine kinase JAK2 (9, 20, 37, 41). The activated GHR-JAK2 complex stimulates signaling via the JAK/STAT, MAPK, and other pathways (21–22, 36–39; for a review, see Ref. 20; see also Fig. 1). Tyrosine phosphorylation of STAT5 by this complex results in the dimerization and nuclear translocation of STAT5. Binding of STAT5 dimers to specific DNA sites called {gamma}-activated sequences (GAS) activates the transcription of the GH-inducible genes, such as IGF-I and serine protease inhibitor 2.1 (Spi 2.1) in liver (3, 5, 13, 52, 53). Activation of the MAPK pathway by GH stimulates ERK-1 and -2 phosphorylation and serine phosphorylation of STAT5, which influences STAT5-mediated gene transcription (36–39). Dephosphorylation of STAT5 and other signaling proteins by protein tyrosine phosphatases (PTPs) terminates GH signaling. Figure 1 provides a schematic diagram of the GH signaling pathways discussed in the manuscript.


Figure 1
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  		Fig. 1. Schematic diagram of growth hormone (GH) signaling. 1) Binding of GH to the GH receptor (GHR) forms an activated GH-(GHR)2-JAK2 complex. 2) JAK2 phosphorylates STAT5 to stimulate STAT5 dimerization and nuclear translocation. JAK2 also activates the MAPK pathway resulting in phosphorylation and nuclear translocation of ERK 1 and 2. 3) Phosphorylated STAT5 binds to specific DNA sequences to activate GH-inducible gene expression. 4) Dephosphorylation of STAT5 by SHP-1 protein tyrosine phosphatase (PTPase) terminates the GH signal. MEK, MAPK kinase; Spi2–1, serine protease inhibitor 2.1.

 
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 (1, 15, 24, 51). Consequently, GH administration has been suggested as adjunct therapy to nutritional support to prevent the erosion of lean body mass observed in patients with catabolic diseases such as sepsis (7, 50). Unfortunately, the administration of recombinant human GH (rhGH) does not fully restore the reductions in circulating IGF-I observed during sepsis (11, 12). The inflammatory cytokine TNF-{alpha} has been identified as an important mediator of GH resistance during sepsis (10, 14, 55). Intravenous injection of TNF-{alpha} in healthy animals results in a 30–40% reduction in circulating and tissue IGF-I levels (14). Furthermore, treatment of septic rats with a specific TNF antagonist, TNF-binding protein, prevents the catabolism of muscle protein, ameliorates the inhibition of gastrocnemius protein synthesis, and attenuates the reductions in plasma IGF-I observed during abdominal sepsis (10, 55). Experiments performed in CWSV-1 hepatocytes demonstrate that pretreatment with TNF inhibits both the duration of STAT5 phosphorylation and the induction of IGF-I mRNA following GH stimulation (55). TNF-mediated inhibition of JAK2 activation and/or termination of GH signaling by induction of STAT5 PTP activity represent potential mechanisms for this observation. The cytosolic PTPs SHP-1 and SHP-2 have been identified as prospective regulators of GH signaling (8, 25a, 41, 45). Pulsatile GH activity is terminated by nuclear SHP-1 in CWSV-1 hepatocytes (41). However, SHP-2 has also been implicated in the regulation of STAT5-mediated signaling (8, 45, 46). Although TNF-mediated alterations in STAT5 phosphorylation and activity could explain the inhibitory effects of TNF on GH-mediated gene expression, they could also be caused by direct effects on GH-inducible promoter activation or by other mechanisms.

The current study examines the effect of TNF on SHP-1 and SHP-2 expression and JAK2 phosphorylation and whether inhibition of PTP activity by pervanadate (PV) ameliorates the inhibitory effects of TNF on GH signaling and gene expression. We found no evidence for increased SHP-1 or SHP-2 expression in TNF-treated hepatocytes. The transient increase in SHP-1 PTP activity following GH administration was not influenced by pretreatment with TNF. Although pretreatment with PV prevented the inhibitory effects of TNF on STAT5 and ERK phosphorylation following GH, it did not restore the induction of IGF-I or Spi 2.1 mRNA by GH. In addition, TNF inhibited the induction of Spi 2.1 promoter activity by both GH and a constitutively active STAT5 vector. Collectively, these results suggest that the inhibitory effects of TNF on GH-induced gene expression are independent of STAT5 activity and that previously identified abnormalities in JAK2/STAT5 signaling are not the primary cause of hepatic GH resistance.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials and plasmids. rhGH (Pharmacia and Upjohn, Stockholm, Sweden) was used in all experiments. TNF-{alpha} was obtained from R & D Systems (Minneapolis, MN). Polyclonal GHR antibody was obtained from W. R. Baumbaugh (American Cyanamid, Princeton, NJ) and used at a dilution of 1:250 (42, 55). Rabbit polyclonal STAT5b antibody (sc-835 and sc-835x; Santa Cruz Biotechnology, CA) and PY20 phosphotyrosine antibody conjugated with horseradish peroxidase (BD Transduction Laboratories, San Diego, CA) were used for immunoblot analyses. Monoclonal antibodies to SHP-1 and SHP-2 were purchased from BD Transduction Laboratories (Lexington, KY). Rabbit polyclonal JAK2 and phospho-JAK2 antibodies from Upstate Cell Signaling Solutions (Lake Placid, NY) were used for immunoblot analyses. Polyclonal p44/42 MAPK antibody and phospho-p44/42 MAPK antibody (specific for Thr183 and Tyr185) that recognize ERK1 and ERK2 were obtained from Cell Signaling (New England Biolabs, Beverly, MA). The rat IGF-I cDNA, the IGF-I promoter luciferase constructs #5, 7, 8, and 9, and the STAT5CA expression vector were previously described (43, 52, 55). The oligonucleotide (5'- ACG ATG CTG AGC ACC C-3') was used to measure Spi 2.1 expression (3). pcDNA 3.1 was commercially acquired from Invitrogen (Carlsbad, CA).

The Spi 2.1 promoter luciferase construct was created by PCR amplification of rat genomic DNA (BD Biosciences, San Diego, CA) using custom primers 5'-TGT GGC AGA ATC AAA AAG CCT GCA-3' and 5'-GGT GTG TTG TTA TCC CCA GT GCACA-3' (Integrated DNA Technologies, Coralville, IA) to amplify sequences from –1059 to +8 in the proximal 5'-flanking region of the Spi 2.1 gene. PCR products were purified by gel electrophoresis, cloned into pCR II-Blunt-TOPO plasmid vector, then transformed into Escherichia coli using the Zero Blunt TOPO PCR Cloning kit (Invitrogen). DNA sequencing was used to confirm the Spi promoter sequence. The Spi 2.1 promoter region (–1059 to +8) was then excised and cloned into the pGL3 luciferase vector (Promega, Madison, WI) and reverified by restriction enzyme digestion and DNA sequencing.

Hepatocytes. CWSV-1 hepatocytes were cultured as previously described (25, 30, 43, 54, 55). For this study, CWSV-1 cells were grown in chemically defined RPMI (RPCD) media to 70–80% confluency. TNF-treated cells were incubated with 10 ng/ml TNF-{alpha} for 4 h and then 500 ng/ml rhGH were added for the indicated time periods. Cells were treated with PV at 60 µM for 60 min before GH stimulation based on the time course of GH signaling (55) and sustained STAT5b EMSA activity for 1 h after GH treatment with this concentration of PV (16).

SHP1 PTP assay. SHP-1 PTP activity was measured using a commercially available chromogenic assay kit (kit 17–125; Upstate Cell Signaling Solutions, Charlottesville, VA). CWSV-1 cell extracts (100 µg protein) were immunoprecipitated using monoclonal antibody to SHP-1 (BD Transduction Laboratories), then incubated with 100 µM of tyrosine phosphopeptide (NH2-RRLIEDAEpYAARG-COOH) for 30 min at 30°C. The reaction was terminated by the addition of 100 µl malachite green solution and allowed an additional 15 min for color development. Phosphatase activity (pmol of Pi released in a 30-min assay) was measured at 660 nm in a microtiter plate reader using a free phosphate standard curve (200–2,000 pmol of Pi) as described in the manufacturer's methods.

Northern blot analysis. The relative abundances of IGF-I and Spi2.1 mRNA were determined by Northern blot analysis as previously described (58, 64). 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 (43, 55). In liver, the exon 1-derived 7.5-kb transcript of IGF-I mRNA represents the predominant (80%) IGF-I mRNA species. For the detection of Spi 2.1, an oligonucleotide complementary to its reactive center was used as a probe (3, 43). The Spi 2.1 oligonucleotide was 5'-labeled with T4 polynucleotide kinase and [{gamma}-32P]ATP, then purified with mini Quick Spin oligo columns according to the manufacturer's instructions (Roche Diagnostics, Indianapolis, IN). Prehybridization and hybridization for oligonucleotide probes were performed as previously reported (10, 43, 55). After exposure of the completed blots to film, the autoradiographs were scanned using a Hewlett-Packard ScanJet 5300C model scanner. Northern blots were stripped and reprobed with the 18S ribosomal subunit message to confirm uniform loading of RNA as previously described (43, 55). 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. Whole cell lysates were prepared from cells grown in culture dishes after washing 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 dishes and incubated on ice for 30 min (51, 64). Lysed cells were collected without scraping and cleared by centrifugation at 13,000 rpm for 5 min. Supernatants were snap frozen in liquid nitrogen and stored at –70°C. Nuclear extracts were prepared as previously described (43, 55). Protein determination was by the Bradford method using a commercial protein assay reagent (Bio-Rad, Hercules, CA).

Western blot analysis and immunoprecipitation. For the detection of total protein, equal amounts of protein were electrophoresed on a 7.5% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Immobilon P; Millipore, Bedford, MA), using standard electroblotting procedures. For the detection of phosphorylated proteins, cell lysates (100–500 µg for cytosolic protein and 10–50 µg for nuclear protein) were immunoprecipitated, and immunocomplexes were resolved using SDS-PAGE. Total SHP-1 and SHP-2 and total and phosphorylated STAT5b were measured by Western blot analysis (43, 55). For detection of total and phosphorylated JAK2, the protocol from Upstate Cell Signaling Solutions was followed. Total and phosphorylated ERK1/ERK2 were measured according to the manufacturer's guidelines (Cell Signaling). Antibody reactions were visualized using ECL-Plus (Amersham Pharmacia Biotech, Piscataway, NJ). The intensity of antibody reactions was analyzed using Scion Image for Windows.

Transient transfection. CWSV-1 cells were plated in RPCD medium at a density of 500,000 cells/60-mm dishes 24 h before transfection. Plasmids used for transfection were purified using a Qiagen System column (Qiagen, Valencia, CA). Briefly, cells were transfected with 0.5 µg of luciferase construct, 0.5 µg of pcDNA3 or pcDNA-STAT5CA, and 0.1 µg of pRSV-beta-galactosidase using the Effectene reagent (Qiagen) according to the manufacturer's directions. Medium was removed 18–20 h after transfection, and the cells were plated with fresh medium ± TNF-{alpha} at 10 ng/ml. Cells were then stimulated with rhGH at 500 ng/ml after 4 h of TNF pretreatment. Twenty hours later, the cells were washed with PBS and lysed in 400 µl of 1x lysis buffer (Promega). Cell extracts (20 µl) were assayed for luciferase activity (Promega's luciferase assay reagent) using a luminometer (Berthold). Another 20 µl was assayed for beta-galactosidase activity (19), and 10 µl of extract was used to determine total protein concentration (Bradford Assay, Bio-Rad). Luciferase activity was reported as relative light units and was normalized to both beta-galactosidase activity and total protein concentration initially. Each data point represents the mean of triplicate measurements for an experiment repeated at least three times. Initial experiments are reported as fold induction (normalized luciferase activity), and subsequent experiments are reported as luciferase activity per milligram of protein after demonstrating the validity of this technique.

Statistical methods. 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 performed by ANOVA followed by the Tukey-Kramer or Student-Newman-Keuls posttest using Instat GraphPad 5.02 (San Diego, CA). Differences among means were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
SHP1 and SHP2 expression. To determine whether pretreatment with TNF induced the expression of protein tyrosine phosphatases SHP-1 or SHP-2, the relative abundance of these proteins was measured in nuclear extracts from CWSV-1 cells (±10 ng/ml TNF) following GH stimulation. As shown in Fig. 2A, neither TNF nor GH significantly influenced the total amount of either SHP-1 or SHP-2 protein. The effect of the PTP inhibitor PV on SHP-1 and SHP-2 protein was also examined. PV did not significantly alter either SHP-1 or SHP-2 protein levels in nuclear extracts from CWSV-1 cells (Fig. 2B). GH alone transiently increased SHP-1 activity at 30 min (40% increase; P < 0.01 vs. all other time points). Preincubation of hepatocytes with TNF before GH did not alter the GH-induced increase (Fig. 2C).


Figure 2
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  		Fig. 2. Effect of TNF-{alpha} on SHP-1 and SHP-2 expression in hepatocytes. A: CWSV-1 hepatocytes were treated with 10 ng/ml TNF for 4 h and then incubated with rhGH (500 ng/ml) and harvested at either 5 or 60 min following GH stimulation. Nuclear protein was isolated and immunoblotted with antibody to SHP-1 or SHP-2 as described in MATERIALS AND METHODS. B: hepatocytes were treated with TNF (10 ng/ml), pervanadate (PV; 60 µM, 1 h), or both before GH stimulation as described in MATERIALS AND METHODS. Nuclear protein was immunoblotted with anti-SHP-1 and anti-SHP-2. Blots are representative of experiments done at least 3 times. C: cells ± TNF (10 ng/ml, 4 h) were treated with rhGH (500 ng/ml) and harvested at 5, 30, and 60 min after GH stimulation. SHP-1 PTP activity was measured in cell extracts as described in MATERIALS AND METHODS. Results are presented as pmol Pi released in a 30-min assay minus background activity recorded for the buffer alone. Values are means ± SE; n = 8–10 for the 8 experimental groups. aP values are statistically similar to each other; bP < 0.01 vs. all other groups.

 
GH signaling via the JAK/STAT and MAPK pathways. To determine whether TNF influenced STAT5 phosphorylation by JAK2, we examined the effects of TNF pretreatment on the time course of JAK2 phosphorylation and total JAK2 following GH stimulation. As shown in Fig. 3, pretreatment with TNF had no effect on the relative abundance of total JAK2. However, the ratio of phosphorylated to total JAK2 was decreased at several time points in TNF-pretreated cells (P < 0.01 vs. GH, 15 min; P < 0.05 vs. GH, 30 min) following GH stimulation.


Figure 3
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  		Fig. 3. Total and phosphorylated JAK2 in TNF-treated cells. Cells ± TNF (10 ng/ml, 4 h) were treated with rhGH (500 ng/ml) and harvested at 2, 5, 10, 15, and 30 min after GH stimulation. A: cell lysate immunoblotted with anti-phosphotyrosine JAK2 antibody. B: cell lysate immunoblotted with anti-JAK2 antibody. C: densitometry data for phosphorylated JAK2 were normalized to total protein, presented as relative densitometry units (RDU), and expressed as means ± SE. aP < 0.01 vs. GH alone at 15 min; bP < 0.05 vs. GH alone at 30 min. Blots are representative of experiments done at least 3 times.

 
The effects of TNF and PV on STAT5 phosphorylation were examined 60 min after GH stimulation. This time point was chosen based on previous studies that showed that preincubation with TNF inhibited the duration of the tyrosine phosphorylation (activated state) for STAT5 (55). To investigate whether TNF-induced changes in GH signaling were caused by increased PTP activity, we used PV to inhibit phosphatase activity. As shown in Fig. 4, STAT5 phosphorylation was markedly increased in extracts from GH-stimulated cells (P < 0.05 vs. untreated cells). When compared with TNF-pretreated cells stimulated with GH, a 40% reduction in STAT5 phosphorylation was observed (P < 0.05 vs. GH alone). Inhibition of PTP activity with PV restored STAT5 phosphorylation by GH in TNF-pretreated cells to control levels (P < 0.05 vs. TNF/GH and untreated). There were no changes in the relative abundance of total STAT5 in cells treated with TNF, PV, or GH (Fig. 4) to explain this observation.


Figure 4
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  		Fig. 4. Effect of TNF and PV on STAT5 phosphorylation. CWSV-1 hepatocytes were pretreated ±TNF (10 ng/ml, 4 h), then PV (60 µM) was added for 1 h, and cells were stimulated with rhGH (500 ng/ml). Cells were harvested 60 min after GH. Phosphorylated and total STAT5 were measured as described in MATERIALS AND METHODS. Densitometry data for phosphorylated STAT5b were normalized to total protein, presented as RDU and expressed as means ± SE. aP < 0.05 vs. untreated; bP < 0.05 vs. GH alone; cP < 0.05 vs. TNF/GH and untreated. Blots are representative of experiments done at least 3 times.

 
Next, we examined the effects of TNF and PV on tyrosine and threonine phosphorylation of ERK1 and 2 proteins 5 min after GH stimulation. TNF alone did not significantly alter basal ERK phosphorylation, whereas PV alone and in combination with TNF increased basal ERK1/2 phosphorylation by fourfold (Fig. 5). GH stimulation resulted in a 2.5-fold increase in the phosphorylation status of ERK1/2 (P < 0.05 vs. untreated) and pretreatment with TNF inhibited GH-induced ERK1/2 phosphorylation by 70% (P < 0.05 vs. GH and GH/PV). PV restored the GH-induced tyrosine and threonine phosphorylation of ERK1/2 in TNF-treated cells to control plus GH levels (Fig. 5). Neither TNF, PV, nor GH altered the relative abundance of total ERK1/2 proteins (Fig. 5).


Figure 5
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  		Fig. 5. Effect of TNF-{alpha} and PV on ERK1/2 levels in CWSV-1 cells. Cells were treated with 10 ng/ml of TNF-{alpha} for 4 h, incubated with PV (60 µM) for 1 h, then stimulated with 500 ng/ml of rhGH for 5 min. Total and phosphorylated ERK1/2 were measured as described in MATERIALS AND METHODS. Densitometry data for phosphorylated ERK1/2 were normalized to total protein, presented as RDU, and expressed as means ± SE. aP < 0.05 vs. untreated; bP < 0.05 vs. GH alone and GH/PV. Blots are representative of experiments done at least 3 times.

 
GH-mediated gene expression in TNF- and PV-treated cells. To determine whether the restoration of GH signaling via the STAT and MAPK pathways by PV in TNF-treated cells restored GH-mediated gene expression, we measured IGF-I and Spi 2.1 mRNA content in GH-treated cells with or without TNF and PV. As shown in Fig. 6, neither TNF nor PV alone significantly altered basal IGF-I mRNA levels. GH stimulated the induction of IGF-I mRNA by approximately fourfold (P < 0.001 vs. untreated). Pretreatment with TNF significantly inhibited the induction of IGF-I mRNA by GH (P < 0.001 vs. GH). However, PV was not able to prevent the inhibitory effects of TNF on the induction of IGF-I mRNA by GH.


Figure 6
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  		Fig. 6. Induction of IGF-I mRNA by growth hormone. Cells were treated with 10 ng/ml of TNF-{alpha} for 4 h, incubated with 60 µM PV for 1 h, then stimulated with 500 ng/ml of rhGH 20 h. Northern blot analysis was performed as described in MATERIALS AND METHODS. Densitometry data for IGF-I mRNA were normalized to 18S rRNA message and expressed as means ± SE. aP < 0.001 vs. untreated; bP < 0.001 vs. GH alone. Blots are representative of experiments done at least 3 times.

 
The experiment was repeated using another GH-inducible gene Spi 2.1 to determine whether these results were specific to IGF-I or a more generalized effect on GH-inducible gene expression. In this study, basal levels of Spi 2.1 mRNA were not affected by TNF or PV alone (Fig. 7). However, the 10-fold increase in Spi 2.1 mRNA observed in GH-stimulated cells (P < 0.01 vs. untreated) was inhibited by pretreatment with TNF (P < 0.01 vs. GH alone). Although PV appeared to increase the induction of Spi 2.1 by GH, it was unable to restore Spi 2.1 mRNA levels in cells treated with the combination of TNF and GH (Fig. 7).


Figure 7
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  		Fig. 7. Induction of Spi 2.1 mRNA by GH. CWSV-1 cells were treated with 10 ng/ml of TNF-{alpha} for 4 h, incubated with PV (60 µM) for 1 h, then stimulated with 500 ng/ml of rhGH for 20 h. Northern blot analysis was performed as described in MATERIALS AND METHODS. Densitometry data for Spi 2.1 mRNA were normalized to 18S rRNA message and expressed as means ± SE. aP < 0.01 vs. untreated; bP < 0.01 vs. GH alone. Blots are representative of experiments performed at least 3 times.

 
Effect of TNF on the IGF-I promoter activity. Transient transfections were performed in CWSV-1 cells to examine the effects TNF, GH, and constitutively active STAT5 on IGF-I promoter activity. First, cells were transfected with IGF-I promoter constructs containing increasing amounts of the 5'-IGF-I promoter sequence (Fig. 8A). Construct 8 (–395 to +328) demonstrated the highest basal promoter activity. However, none of these promoter constructs demonstrated a reproducible increase in luciferase activity following GH stimulation. The remainder of the experiments was performed using IGF-I promoter construct 7 (–823 to +328). After transfection with the IGF-I vector and beta-galactosidase with or without STAT5/CA, cells were pretreated with TNF for 4 h and stimulated with GH for 20 h. As shown in Fig. 8B, basal promoter activity of construct 7 was not significantly altered by treatment with TNF or GH alone. However, cotransfection with a constitutively active STAT5 vector (STAT5/CA) resulted in a four- to fivefold induction of IGF-I promoter activity (P < 0.001 vs. empty vector). GH stimulation did not significantly increase the STAT5/CA-dependent activation of the IGF-I promoter. However, pretreatment with TNF reduced the STAT5/CA-mediated activation of IGF-I promoter activity by 40% (P < 0.01 vs. STAT5/CA vector).


Figure 8
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  		Fig. 8. IGF-I promoter activity in CWSV-1 hepatocytes. A: CWSV-1 cells were transfected with IGF-I promoter luciferase constructs 5, 7, 8, and 9 containing increasing amounts of the 5'-IGF-I promoter sequence. After 18–20 h, cells were stimulated with GH (500 ng/ml) for 20 h, then harvested and assayed for luciferase activity as described in MATERIALS AND METHODS. B: CWSV-1 cells were transfected with empty vector (pcDNA3) or pcDNA-STAT5/CA and pRSV-beta-galactosidase as described in MATERIALS AND METHODS. At 18–20 h after transfection, cells were treated ±TNF (10 ng/ml) for 4 h, then stimulated with GH (500 ng/ml) for 20 h. Cell extracts were assayed for luciferase and beta-galactosidase activity, and total protein concentrations were determined. Luciferase activity in these experiments is normalized to both beta-galactosidase activity and total protein and reported as relative luciferase activity or fold induction (normalized luciferase activity). Values are means ± SE; n = 3–4 for the experimental groups. aP < 0.001 vs. empty vector; bP < 0.01 vs. STAT5/CA vector.

 
Regulation of Spi 2.1 promoter activity by GH, TNF, and STAT5/CA. The Spi 2.1 promoter luciferase vector (Spi-pGL3) was transfected into CWSV1 cells, and the effects of pretreatment with TNF, GH stimulation, and cotransfection with STAT5/CA were examined. As shown in Fig. 9A, TNF alone did not significantly alter basal Spi 2.1 promoter activity. In contrast, GH stimulation significantly stimulated Spi 2.1 promoter activity treatment (P < 0.001 vs. control, TNF, and TNF + GH). However, pretreatment of cells with TNF significantly inhibited the induction of Spi 2.1 promoter activity by GH (Fig. 9A). Likewise, the increase in Spi 2.1 promoter activity observed with STAT5/CA cotransfection ± GH was significantly attenuated by pretreatment with TNF (Fig. 9B). Collectively, these results suggest that the inhibitory effects of TNF on GH-inducible gene transcription are independent of STAT5 phosphorylation/activation.


Figure 9
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  		Fig. 9. Spi 2.1 promoter activity in CWSV-1 hepatocytes. A: CWSV-1 cells were transfected with the Spi 2.1 promoter luciferase construct or the empty vector as described in MATERIALS AND METHODS. At 18–20 h after transfection, cells were treated ±TNF (10 ng/ml) for 4 h, then stimulated with GH (500 ng/ml) for 20 h. aP < 0.001 vs. control, TNF, and TNF/GH. B: hepatocytes were cotransfected with the Spi 2.1 and pcDNA-STAT5 CA promoter luciferase constructs. At 18–20 h after transfection, cells were treated ±TNF (10 ng/ml) for 4 h, then stimulated with GH (500 ng/ml) for 20 h. aP < 0.001 vs. control; bP < 0.001 vs. control, GH alone. Cell extracts were assayed for luciferase activity, and total protein concentrations were determined. Luciferase activity is reported as relative luciferase units (RLU) normalized to total protein (luciferase units/ug protein). Values are means ± SE; n ≥ 3 for each experimental group.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Several lines of evidence suggest that sepsis-induced hepatic GH resistance is caused by postreceptor defects in GH signaling. In liver from LPS-treated rats stimulated with GH, reductions in phosphorylated/total JAK2 and STAT5 were observed (34). A simultaneous increase in suppressor of cytokine signaling (SOCS-3) expression was also noted, and SOCS-mediated impairment in GH signaling was hypothesized as the cause of hepatic GH resistance (34). In support of this hypothesis, decreased STAT5 phosphorylation and reduced STAT5 DNA binding to the GH response element were observed in liver tissue from LPS-treated rats following GH administration (4). Likewise, the duration of STAT5 phosphorylation/activation was decreased, and IGF-I mRNA levels were reduced in TNF-pretreated, GH-stimulated CWSV-1 hepatocytes (55). Collectively, these results suggest that sepsis-induced alterations in GH signaling are responsible for the associated reductions in GH-induced gene expression (4, 34, 55).

The duration of STAT5 tyrosine phosphorylation following GH stimulation is regulated by JAK2-mediated phosphorylation and PTPase-mediated dephosphorylation (41). The observation that GH-mediated STAT5 phosphorylation was normal at 5 min but diminished at 60 min in TNF-treated hepatocytes originally suggested the induction of PTP activity by TNF as a potential mechanism for hepatic GH resistance. The cytosolic PTPases SHP-1 and SHP-2 have been implicated in the regulation of GH signaling (16, 41, 45, 46). SHP-2 induces the formation of a multiprotein GHR, JAK2, SHP-2, SIRP complex in fibroblasts (25a, 45). However, SHP-2 activation appears to have a positive effect on GH-induced c-fos gene expression (45). In CWSV-1 hepatocytes, GH stimulates the nuclear translocation and binding of SHP-1 to phosphorylated STAT5, suggesting a role in STAT5 dephosphorylation/deactivation (41). In the current study, the relative abundance of SHP-1 and -2 proteins was not altered by TNF, GH, or PV (Fig. 2). Therefore, increased amounts of SHP-1 or SHP-2 protein per se do not explain the reduction in STAT5 phosphorylation following GH stimulation in TNF-treated cells. The transient induction of SHP-1 PTP activity observed following GH administration was similar to that observed by Ram and Waxman (41) in CWSV-1 hepatocytes, but a superinduction of SHP-1 PTP activity was not seen in hepatocytes treated with both GH and TNF. Consequently, a change in SHP-1 PTP activity by TNF cannot explain the inhibitory effects of TNF on GH signaling via the JAK/STAT pathway.

GH-induced tyrosine phosphorylation of STAT5 by JAK2 also represents a critical step in the activation of GH-mediated gene transcription (20, 21, 37, 40, 41, 51). Although JAK2 activity was not directly measured in this study, TNF pretreatment caused a slight reduction in the magnitude of JAK2 phosphorylation following GH stimulation. This finding suggests that TNF-mediated reductions in JAK2 phosphorylation/activation may contribute to the defects in STAT5 phosphorylation observed in TNF-treated, GH-stimulated cells.

PV (a PTP inhibitor) increases the duration of GH-mediated JAK2/STAT5 signaling by inhibiting STAT5 dephosphorylation (16). The current study is unique in the use of PV to investigate the relationship between the duration of GH signaling and GH-mediated gene expression in TNF-treated hepatocytes. The effects of PV on STAT5 phosphorylation were examined initially based on the critical role of STAT5 in the regulation of IGF-I and Spi 2.1 gene transcription (3, 4, 13, 28, 52, 53). As shown in Fig. 4, the reduction in STAT5 phosphorylation observed in TNF-treated cells was restored to control plus GH levels when PV was added before GH stimulation. There were no changes in the relative abundance of total STAT5. Although TNF did not increase SHP-1 PTP activity per se, the inhibition of PTP activity by PV significantly ameliorated the inhibitory effects of TNF on the duration of JAK2/STAT5 signaling.

Next, we examined the effects of TNF and PV on GH signaling via the MAPK pathway. The MAPK pathway is activated by GH in CWSV-1 hepatocytes, and administration of the MAPK inhibitor PD-98059 caused a 20% reduction in GH-inducible IGF-I mRNA expression (43). Activation of the MAPK pathway by GH may occur via JAK2-dependent or RAS-like GTPase-mediated pathways (31, 57). In either case, activation of MAPK kinase by GH results in the phosphorylation of ERK on Thr183 and Tyr185 (36–39). The phosphorylation of ERK stimulates nuclear translocation and activation of several transcription factors including STAT5a (Ser725, Ser779), STAT5b (Ser730), and Elk-1 (22, 36–39). Although tyrosine phosphylation of STAT5b is required for GH-mediated gene transcription, mutation of STAT5-Ser730 to alanine reduces GH-stimulated gene expression by 50% (36). This suggests that serine phosphorylation of STAT5b by ERK1/2 is necessary to achieve full transcriptional activation by GH.

To determine whether the inhibitory effects of TNF were due to impaired GH signaling via the MAPK pathway, we examined the effects of TNF and PV on the activation of ERK1 and 2 by GH. The antibody that was used to detect phosphorylated ERK only recognizes the activated form of ERK1/2 where both Thr183 and Tyr185 are phosphorylated. As shown in Fig. 5, the 2.5-fold induction of ERK1/2 phosphorylation/activation by GH was significantly inhibited by TNF. This is the first study to show an inhibitory effect of TNF on the GH signaling via the MAPK pathway. PV increased basal ERK1/2 phosphorylation and restored the ERK phosphorylation in TNF + GH-treated cells to control + GH levels. There was no change in the relative abundance of total ERK proteins under these experimental conditions. These findings suggest that PTPases are also involved in ERK1/2 dephosphorylation. Although TNF does not appear to induce PTPase activity, the inhibitory effects of TNF on JAK2 phosphorylation/activation represent a potential mechanism by which TNF interferes with serine phosphorylation of STAT5b and, consequently, prevents full transcriptional activation by GH.

To investigate whether TNF-mediated alterations in GH signaling were responsible for the inhibitory effects of TNF on GH-meditated gene expression, we measured IGF-I and Spi 2.1 mRNA in PV-treated cells. Although PV corrected the TNF-induced defects in STAT5 and ERK1/2 phosphorylation, it had no effect on either IGF-I or Spi 2.1 gene expression. These results are the first to suggest that the inhibitory effects of TNF on GH-inducible gene expression are independent of sepsis-induced alterations in JAK/STAT and MAPK signaling.

The role of STAT5 in the regulation of GH-mediated hepatic gene expression has been characterized for IGF-I, Spi 2.1, IGF binding protein-3 (IGFBP-3), and acid labile subunit (ALS) (3, 5, 6, 28, 44, 49, 51). Studies in STAT5b knockout mice suggest that STAT5b is required for basal and GH-induced expression of hepatic IGF-I (13). STAT5b dominant negative adenoviral transfection completely inhibits the induction of IGF-I, IGFBP-3, and ALS by GH in liver tissue (52). Although the promoter elements responsible for basal IGF-I transcription are contained in the 5'-untranslated region (UTR), the exact mechanisms of IGF-I activation by GH remain poorly characterized (2, 35, 48, 49). The identification of a GH-regulated DNAse-I hypersensitive site (HS7) in intron 2 of the IGF-I gene suggests that alterations in chromatin structure are required for GH activation (5, 47). More recently, tandem STAT5 binding sites were identified in HS7 by chromatin immunoprecipitation assays, which showed that GH-induced STAT5 binding to these GHRE sites was required for activation of IGF-I transcription (53). In contrast to IGF-I, the STAT5 binding sites for GH activation are located in the 5'-UTR of the Spi 2.1 and ALS genes (3, 6, 28).

Given the location of the GH response elements in intron 2 of the IGF-I gene, it is not surprising that the 5'-IGF-I promoter constructs (which do not include HS7/intron 2) were not significantly activated by GH alone. However, cotransfection of the constitutively active STAT5 vector resulted in a fivefold increase in IGF-I promoter activity that was inhibited by pretreatment with TNF. Analysis of the 5'-UTR of the IGF-I promoter has identified several potential STAT5 consensus sites (–1698, –565, and –400) that might explain this observation. However, the inhibitory effects of TNF on IGF-I expression appear to be independent of STAT5 phosphorylation/activation status. This finding is supported by the observation of Hong-Brown et al. (23) that impaired IGF-I expression in skeletal muscle from GH-treated rats after cecal ligation and puncture was not associated with alterations in STAT5 phosphorylation.

The rat Spi 2.1 promoter luciferase vector was used to further characterize the inhibitory effects of TNF on GH-mediated gene expression. GH activation of the Spi 2.1 promoter is mediated by two GAS sites (–149 to –115) in the 5'-UTR of the Spi 2.1 promoter (3). However, DNase I footprinting analysis of the Spi 2.1 promoter identified a GAGA-box region that correlates with DNase I sensitivity and is required for gene expression, suggesting an important role for GH-dependent chromatin remodeling in Spi 2.1 gene transcription (44). Although LPS-mediated inflammation and TNF are known to inhibit the induction of Spi 2.1 mRNA by GH, the relationship between impaired STAT5 signaling and gene expression has not previously been evaluated (4). Other studies have examined the effects of IL-1 on GH signaling and GH-inducible gene expression (6, 43). In CWSV-1 hepatocytes, IL-1beta had no effect on STAT5 or MAPK signaling but inhibited the induction of both IGF-I and Spi 2.1 mRNA by GH (43). However, in H4IIE hepatocytes, the inhibitory effects of IL-1 on ALS expression were attributed to increased SOCS3 expression and decreased STAT5 binding to GAS sequences in the ALS promoter (6).

The current study does not identify exact mechanisms responsible for the inhibitory effects of TNF on GH-inducible gene expression. However, our results suggest that TNF-induced abnormalities in PTP activity and JAK2/STAT5 signaling do not represent the primary cause of hepatic GH resistance. Furthermore, the inhibitory effects of TNF on GH-mediated gene transcription appear to be independent of STAT5 phosphorylation/activation. The possibility of inhibitory cross-talk between the STAT5b and NF-{kappa}B signaling pathways has been suggested by Luo and Yu-Lee (33). In this study, the inhibitory effects of STAT5b transfection on the induction of interferon regulatory factor-1 promoter by TNF were reversed by cotransfection with the transcriptional coactivator p300/CREB-binding protein (CBP) (33). Transcriptional coactivators and corepressors may act as bridging molecules between transcription factors or influence histone acetylation to regulate gene transcription (18, 32). Several transcriptional coactivators have been implicated in the regulation of GH and STAT5-mediated gene expression including ying yang 1, the glucocorticoid receptor, and p300/CBP (4, 18, 32). Additional studies will be required to determine the exact mechanisms by which TNF inhibits GH-mediated gene expression.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by National Institutes of Health Grants GM-55639A (to R. N. Cooney), GM-38032 (to C. H. Lang), and T32-GM-64332 (to T. Ahmed and G. Yumet).


   ACKNOWLEDGMENTS
 
The authors thank H. Isom (Dept. Microbiology, Penn State College of Medicine) for providing the CWSV1 cells and Dr. D. Noonan (Univ. of Kentucky) for providing the pRSV-beta-galactosidase plasmid.


   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.

* T. Ahmed and G. Yumet contributed equally to this work. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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