1 The Department of Pediatrics and Yale Child Health Research Center 06520-8081 and the 2 Yale Liver Center, Yale University School of Medicine, New Haven, Connecticut; 3 The Department of Pediatrics, Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213; 4 The Departments of Medicine, Cell Biology, and Physiology, University of Alabama at Birmingham, Birmingham, Alabama 35294; and 5 National Hormone and Peptide Program, Harbor-University of California at Los Angeles Medical Center, Torrance, California 90509
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ABSTRACT |
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Cytokines may cause an acquired
growth hormone (GH) resistance in patients with inflammatory diseases.
Anabolic effects of GH are mediated through activation of STAT5
transcription factors. We have reported that TNF- suppresses hepatic
GH receptor (GHR) gene expression, whereas the cytokine-inducible
SH2-containing protein 1 (Cis)/suppressors of cytokine signaling
(Socs) genes are upregulated by TNF-
and IL-6 and inhibit
GH activation of STAT5. However, the relative importance of these
mechanisms in inflammatory GH resistance was not known. We
hypothesized that IL-6 would prevent GH activation of STAT5 and that
this would involve Cis/Socs protein upregulation. GH ± LPS was
administered to TNF receptor 1 (TNFR1) or IL-6 null mice and wild-type
(WT) controls. STAT5, STAT3, GHR, Socs 1-3, and Cis
phosphorylation and abundance were assessed by using immunoblots, EMSA,
and/or real time RT-PCR. TNF-
and IL-6 abundance were assessed by
using ELISA. GH activated STAT5 in WT and TNFR1 or IL-6 null mice. LPS pretreatment prevented STAT5 activation in WT and TNFR1 null mice; however, STAT5 activation was preserved in IL-6 null mice. GHR abundance did not change with LPS administration. Inhibition of STAT5
activation by LPS was temporally associated with phosphorylation of
STAT3 and upregulation of Cis and Socs-3 protein in WT and TNFR1 null
mice; STAT3, Cis, and Socs-3 were not induced in IL-6 null mice. IL-6
inhibits hepatic GH signaling by upregulating Cis and Socs-3, which may
involve activation of STAT3. Therapies that block IL-6 may enhance GH
signaling in inflammatory diseases.
STAT5; STAT3; suppressors of cytokine signaling; endotoxin; cytokines
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INTRODUCTION |
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GROWTH HORMONE (GH) resistance, characterized by normal GH secretion with impaired production of GH target genes including IGF-I, is an important complication of acute and chronic inflammatory diseases (3). Consequences include linear growth failure, skeletal muscle wasting, poor wound healing, and abnormalities in carbohydrate and lipid metabolism (2). Binding of GH to the GH receptor (GHR) activates JAK2 kinase, leading to phosphorylation and nuclear translocation of STAT5 transcription factors and upregulation of anabolic target genes including IGF-I (33). Potential mechanisms of cytokine induced GH resistance include downregulation of the GHR itself, and upregulation of postreceptor inhibitory proteins including members of the recently described suppressors of cytokine signaling (SOCS) family (13, 27).
We have recently reported that TNF- signaling mediates
downregulation of hepatic Ghr RNA expression by LPS, via
inhibition of Sp1/Sp3 Ghr gene transactivators
(13). A significant reduction in GHR abundance could then
impair GH signaling. Several groups have demonstrated that
cytokine-inducible SH2-containing protein 1 (Cis) and Socs 1-3 can
inhibit GH signaling by preventing GHR/JAK2-dependent phosphorylation of STAT5b (7, 27, 28). Under
physiological conditions, Cis/Socs proteins may function in a
GH-dependent negative feedback manner; their importance in regulating
postnatal growth has been confirmed in transgenic and null mice.
Transgenic overexpression of Cis resulted in impaired STAT5
phosphorylation and stunted growth, whereas the Socs-2 null
mouse exhibited significantly increased size compared with wild-type
(WT) littermates (24, 25). Because of embryonic or
perinatal lethality, Socs-1 and Socs-3 null mice
have not been informative with respect to regulation of postnatal
growth (21, 31). Several inflammatory cytokines including
TNF-
and IL-6 can upregulate Cis and Socs
1-3 hepatic RNA expression (8). This may occur
via STAT5 (Cis) or STAT3 (Socs-3) cis elements in
their respective promoters (1, 35). Recent reports
(18, 28) have also identified posttranscriptional mechanisms of regulation involving control of protein translation, as
well as mechanisms involving protein stability and proteasomal degradation. However, the relative importance of
cytokine-dependent alterations in Ghr or Cis/Socs 1-3 abundance in
modulating hepatic GH signaling was not known.
Intraperitoneal LPS administration induces hepatic production of
several inflammatory cytokines, including TNF-, IL-1
, and IL-6,
and is established as an experimental model of the acute phase response
(14-17). It has recently been reported that LPS administration reduced GH-dependent hepatic STAT5b activation in a
time-dependent manner (4, 22). Moreover, LPS, IL-6, and,
to a lesser extent, TNF-
, have each been shown to induce murine
hepatic Socs-1, -2, and -3 RNA expression
(6, 8, 22). Importantly, in these studies, intact TNF
signaling was not required for IL-6 to elicit maximal initial induction
of Socs-1, -2, and -3 RNA and SOCS-3 protein
(6). Moreover, TNF-
, IL-1
, or IL-6 treatment of
primary rat hepatocytes has reproduced the in vivo induction of
Socs 1-3 RNA, and TNF-
and IL-1
have been shown
to inhibit GH upregulation of IGF-I in vitro (8, 12, 34,
37). We hypothesized that IL-6 would be required for LPS to
inhibit hepatic STAT5b activation by GH and that this would involve
upregulation of Cis/Socs proteins. In this study, we have confirmed
that inhibition of STAT5b activation by LPS is temporally associated
with upregulation of Cis and Socs-3 and that this is dependent on
intact IL-6 signaling.
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MATERIALS AND METHODS |
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Materials.
Eight-week-old male C57BL/6J WT, TNFR1 null (Tnfrsfla, cat. no. 002818)
and IL-6 null (B6.129s6-IL6tm1Kopf, cat. no. 002650) mice were obtained
from Jackson Laboratory (Bar Harbor, ME). LPS, Escherichia
coli 026:B6, and human GH were obtained from Sigma (St. Louis,
MO). STAT5b (cat. no. sc-835), STAT3-P04 (cat. no. sc-8059), CIS (cat.
no. sc-1529), SOCS 1-3 (cat. no. sc-9023, 7007, and 7009), ACTIN
(cat. no. sc-1615), and SH-PTP1 (cat. no. sc-287) polyclonal antibodies
were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A
STAT5-P04 (phosphotyrosine 699 of STAT5b, cat. no. s4308) antibody was
obtained from Sigma. The GHR AL-47 polyclonal antibody that recognizes
the cytoplasmic domain of the GHR has previously been described
(38). The Renaissance chemiluminescence kit was obtained
from New England Nuclear (Wilmington, DE). The digoxigenin
(DIG) EMSA kit was obtained from Boehringer-Mannheim (Indianapolis, IN). The NE-PER kit for preparation of liver protein was
obtained from Pierce. TRIzol for preparation of RNA was
obtained from GIBCO-BRL. An ELISA kit for determination of tissue
TNF- and IL-6 abundance was obtained from R&D Systems (Minneapolis, MN). Human GH levels in sera were determined in samples obtained 30 min
after intraperitoneal administration of human GH utilizing a previously
described assay (19).
Oligonucleotides. EMSA oligonucleotides were synthesized by GIBCO-BRL (Rockville, MD). The following synthetic oligonucleotide was used for the consensus STAT5 EMSA: STAT5s: 5'-GGACCTCTTGGAATTAAGGGA-'3. A double-stranded oligonucleotide for EMSA was generated by annealing this synthetic oligonucleotide with the complimentary sequence.
GH and LPS treatments. Endotoxemia was induced by a single intraperitoneal LPS injection (1 µg/g body wt). GH treatment consisted of a single intraperitoneal injection (3 mcg/gm or 0.3 µg/g body wt) administered 30 min before harvesting of livers. Control mice were injected with an equal volume of sterile saline. Three hours after LPS treatment, livers were harvested for preparation of nuclear and cytoplasmic proteins and total RNA. The study protocol was approved by the Yale Animal Care and Use Committee.
RT-PCR analysis of Ghr, Cis, and Socs-3 gene expression.
Total RNA was extracted by using TRIzol (GIBCO Life Technologies,
Gaithersburg, MD). Concentration and purity were confirmed by
spectrophotometry (model DU-640B; Beckmann Instruments, Palo Alto, CA).
RNA was stored at 80°C. The following primers and probes were used
for the TaqMan RT-PCR assay (synthesized by PE Biosystems):
mGhr: forward, 5'-GGATCTTTGTCAGGTCTTCTTAACCT-'3; reverse,
5'-CAAGAGTAGCTGGTGTAGCCTCACT-'3; probe,
5'-TGGCAGTCACCAGCAGCAGCATTTT-'3; mCis: forward,
5'-GCCTTCCCAGATGTGGTCA-'3; reverse, 5'-CCGGGTGTCAGCTGCAC-'3; probe,
5'-CCTTGTGCAGCACTATGTGGCCTCC-'3; mSocs-3: forward,
5'-GCTCCAAAAGCGAGTACCAGC-'3; reverse, 5'-AGTAGAATCCGCTCTCCTGCAG-'3;
probe, 5'-TGGTGAACGCCGTGCGCAA-'3; rGAPDH: proprietary (PE Biosystems,
Foster City, CA). Total Ghr transcripts were amplified as previously
reported (12). TaqMan real-time quantitative PCR assay was
performed on an ABI Prism 7700 Sequence Detection System, according to
the manufacturer's protocol (Applied Biosystems). Amplification of
GAPDH was performed to standardize the quantification of target cDNA,
allowing relative quantitation by using the ABI Prism 7700 (SDS
software). Briefly, 2.0, 1.0, 0.5, and 0.25 µl of synthesized mouse
liver cDNA were amplified in triplicate for both GAPDH and each of the
target genes to create a standard curve. Likewise, 2.0 µl of cDNA was amplified in triplicate in all isolated mouse liver samples for each
primer/probe combination and GAPDH. Each sample was supplemented with
both respective forward and reverse primers, fluorescent probe, and
made up to 50 µl by using TaqMan Master-Mix (Applied Biosystems).
Each target probe was amplified in a separate 96-well plate. All
samples were incubated at 50°C for 2 min and at 95°C for 10 min and
then cycled at 95°C for 15 s and 60°C for 1 min for 40 cycles.
After the standard curves were calculated, the input amounts of cDNA of
the unknown samples were calculated for all target probes and GAPDH.
After the input amount of the probe sample was normalized to the GAPDH
expression, the relative amount of the expressed RNA species in each
unknown sample was calculated.
Preparation of nuclear and cytoplasmic proteins, EMSA, and immunoblot analysis. Nuclear and cytoplasmic proteins were prepared from mouse liver utilizing the NE-PER kit as per manufacturer's recommendations (Pierce). Oligonucleotide probes were end-labeled, and EMSA was performed utilizing the DIG-EMSA kit as per manufacturer's recommendations (Boeringher-Mannheim). In supershift assays, 2 µg of polyclonal antibody were added to the gelshift reaction for 1 h after preincubation of the probe and nuclear proteins for 20 min on ice. Nuclear (20 µg) or cytosolic (40-50 µg) proteins were also loaded onto 7.5 or 12% SDS-PAGE (Bio-Rad, Hercules, CA) and subjected to electrophoresis and electrotransfer onto nitrocellulose membranes. For the Ghr immunoblot, cytoplasmic proteins (100 µg) were immunoprecipitated by using Sepharose A beads before immunoblotting. Briefly, protein samples were suspended in 100 µl immunoprecipitation buffer [20 mM HEPES, pH 7.5, 1% IDEAL CA-630 (Sigma), 10% glycerol, 2.5 mM EGTA, 2.5 mM EDTA, 1 µg/ml PMSF, leupeptin, aprotinin, and pepstatin, and 1 mM NaF]. Samples were then incubated with 3 µl Ghr antibody or preimmune rabbit serum with mixing at 4°C for 6 h, followed by incubation with 20-µl protein A beads (Pierce) with end-over-end mixing at 4°C overnight. Protein bead complexes were collected by centrifugation at 1,000 g at 4°C for 5 min and then washed three times with 500 µl immunoprecipitation buffer. Proteins were eluted by boiling samples for 5 min in 40 µl 1× sample buffer (75 mM Tris, pH 6.8, 2.5% SDS, 12.5% glycerol, and 3% 2-mercaptoethanol, containing bromophenol blue dye). Uniformity of protein loading and transfer was assessed by using Ponceau staining and by reprobing immunblots for SH-PTP1 (nuclear proteins) or ACTIN (cytosolic proteins). Nitrocellulose membranes were blocked overnight at 4°C in 20 mM Tris · HCl, 150 mM NaCl, 5% nonfat dry milk, 0.1% Tween-20 [Tris saline (TS) complete]. Blots were incubated at room temperature for 1 h with STAT5b, STAT5-P04, STAT3-P04, GHR AL-47, ACTIN, or SH-PTP1 (1:1,000) or CIS or SOCS-1, 2, or 3 (1:200) antibodies in TS complete. Blots were washed and then incubated with anti-rabbit horseradish peroxidase conjugate antibody (1:2,000 to 1:5,000; Santa Cruz Biotechnologies) for 1 h at room temperature. Immune complexes were detected by using the New England Nuclear Chemiluminescence Plus Western blotting kit and Kodak Biomax light film. Band signal intensity was quantified by densitometry (Molecular Dynamics, Sunnyvale, CA).
ELISA.
Liver tissue TNF- or IL-6 abundance was determined by using
cytoplasmic protein extracts and an ELISA kit as per manufacturer's recommendations (R&D Systems).
Statistical analysis. Data were expressed as the means ± SD of experiments with at least three independent treatments per group. Differences among experimental groups were analyzed by ANOVA. Where there were differences among the groups (P < 0.05), these were subjected to post hoc comparisons by using the unpaired Students t-test. P < 0.05 was considered to be significant.
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RESULTS |
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LPS pretreatment inhibits STAT5b activation and DNA binding.
Recent studies have demonstrated that LPS administration would inhibit
GH activation of STAT5b in rat liver (4, 22, 36). This was
time dependent with maximal inhibition of STAT5b activation observed
from 3-4 h after the administration of a single intraperitoneal dose of LPS. Intrahepatic production of several cytokines, which may
modulate GH signaling, including TNF-, IL-1
, and IL-6, are increased within 1-3 h after LPS administration to WT mice. As a
basis for studies in TNFR1 and IL-6 null mice, we initially determined
whether LPS pretreatment would inhibit STAT5b activation in C57BL/6
mice. GH ± LPS was administered to WT mice, and liver nuclear
proteins were isolated. Total and tyrosine phosphorylated STAT5b
nuclear levels were determined by immunoblotting. The primary GH dose
used in this study was in the range of what has typically been
reported in the literature (e.g., from 1 to 3 µg/g) in other studies
examining the effect of LPS on hepatic GH signaling. For example, Mao
et al. (22) used 1.5 µg/g GH and 1 µg/g LPS and demonstrated upregulation of CIS and SOCS-3 RNA, which temporally preceded inhibition of STAT5 activation. The authors stated that the GH
dose used represented a 10-fold higher amount than would be expected to
yield a maximal hepatic response in intact rodents. They reasoned that
a supramaximal GH dose was more appropriate for this type of study, to
ensure that observed changes in signaling were not due to altered
hormone delivery to tissue receptors. Two other recent papers examining
LPS regulation of liver GH action used similar doses i.e., Defalque et
al. (12) used 3 µg/g GH, whereas Wang et al.
(36) used 1.5 µg/g GH.
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Ghr protein abundance is modestly reduced by short-term LPS
treatment.
One mechanism that could account for the LPS-dependent inhibition of GH
signaling would involve downregulation of the Ghr itself. Two recent
reports (12, 22) utilizing short-term (3-6 h) low (1 µg/g)- or high (7.5 µg/g)-dose LPS administration have demonstrated
either no change in hepatic GHR protein levels or a significant
reduction in GH binding, respectively. We have recently reported
(13) that longer-term (12 h) treatment with low-dose LPS
(1 µg/g) reduced hepatic Ghr gene expression via
TNF--dependent inhibition of binding of Sp1/Sp3 transcription
factors to adjacent cis elements in the Ghr gene L2
promoter. We therefore examined Ghr gene expression in this
model. As shown in Fig. 2A,
short-term LPS treatment led to a modest reduction in Ghr
RNA levels in WT mice. Immunoblots were then performed to
determine whether the observed reduction in RNA expression was
associated with an alteration in Ghr abundance. As shown in Fig.
2B, total Ghr protein abundance was also modestly reduced,
by ~30%, although this difference was not statistically significant
(n = 3). Precipitation with preimmune rabbit serum
confirmed that the identified Ghr complex was specific (see lane
3 of Fig. 2B). Therefore, the observed reduction in STAT5 activation was likely due primarily to induction of postreceptor signaling inhibitors.
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Induction of Cis and Socs-3 protein is temporally associated with
inhibition of STAT5b activation.
Several reports have now detailed postreceptor mechanisms by which Cis
and Socs 1-3 can inhibit GH activation of STAT5b in liver
(27). We therefore determined Cis and Socs 1-3
protein expression in response to LPS pretreatment in WT mice to
determine whether this was associated with inhibition of STAT5b
activation. As shown in Fig. 3, Cis and
Socs-3 proteins were significantly upregulated by LPS pretreatment.
Interestingly, despite prior reports demonstrating LPS-dependent
upregulation of Socs-1 and Socs-2 RNA expression,
an increase in Socs-1 or Socs-2 protein was not observed (data not
shown) (6, 8). These data indicated that upregulation of
Cis and Socs-3 was likely involved in the observed inhibition of STAT5b
activation by LPS.
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LPS upregulation of hepatic TNF- and IL-6 abundance.
Several prior studies have reported upregulation of TNF-
and IL-6 in
serum after intraperitoneal LPS administration to WT and TNFR1 or IL-6
null mice (17, 29). Because both TNF-
and IL-6 have
previously been shown to upregulate Cis and Socs-3 in liver, we
determined abundance of these cytokines in mice after LPS
administration. Serum levels of these cytokines are typically assumed
to reflect local hepatic expression in this acute-phase model. For
TNFR1 null mice, LPS administration was shown to yield a two- to
threefold reduction in upregulation of serum IL-6 compared with WT mice
(29). Conversely, for IL-6 null mice, LPS administration was shown to yield a threefold higher serum level of TNF-
compared with WT mice (17). These alterations in LPS-dependent
cytokine expression compared with WT could contribute to differences in acute-phase gene regulation. However, it is possible that serum levels
of TNF-
and IL-6 could also reflect synthesis in other tissues
stimulated by intraperitioneal LPS administration, such as peritoneal
macrophages. Therefore, we utilized ELISA to determine hepatic tissue
abundance of TNF-
and IL-6 in WT and TNFR1 and IL-6 null mice after
LPS administration. As shown in Fig. 4,
LPS administration upregulated liver TNF-
(60 ± 9.2 pg/mg) and
IL-6 (71.5 ± 16 pg/mg) abundance in WT mice. Both of these
cytokines were also significantly upregulated by LPS in TNFR1 null mice (TNF-
, 40 ± 2.8 pg/mg; IL-6, 44.6 ± 9.3 pg/mg). Although
these were lower than in WT mice, this difference was not statistically different. LPS administration increased TNF-
abundance to 33 ± 9.9 pg/mg in IL-6 null mice. This difference was significantly less
than in WT mice. These data confirmed that LPS administration upregulated hepatic TNF-
and IL-6 abundance in WT mice. Moreover, IL-6 upregulation in TNFR1 null mice and TNF-
upregulation in IL-6
null mice were reduced by ~50% compared with WT mice. Studies were
then performed to determine the relative importance of TNF-
vs. IL-6
in upregulating Cis/Socs-3 and inhibiting STAT5 activation.
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IL-6 mediates LPS-dependent inhibition of STAT5b activation.
A recent report has described a temporal association between
LPS-dependent upregulation of IL-6 and Socs-3 and inhibition of GH activation of IGF-I (36). However, whether
inflammatory cytokines would have redundant effects on GH signaling in
vivo, or whether a single cytokine, such as TNF- or IL-6 would be
required for LPS inhibition of STAT5b activation was not known. To test this, the effect of LPS pretreatment on hepatic STAT5b activation in
TNFR1 and IL-6 null mice was determined. As shown in Fig.
5, GH treatment (for 30 min)
significantly increased both total and tyrosine phosphorylated STAT5b
nuclear levels in TNFR1 or IL-6 null mice. LPS pretreatment (for 3 h) inhibited STAT5b activation in TNFR1 null mice to a degree
comparable with that seen in WT mice (Fig. 1A). In contrast
to this, STAT5 activation was preserved despite LPS pretreatment in
IL-6 null mice. This was not dependent on the dose of GH used, because
STAT5 activation was preserved with either high (3 µg/g)- or low (0.3 µg/g)-dose GH. These data demonstrated that LPS inhibition of STAT5b
activation required intact IL-6 signaling. Because Cis and Socs-3 have
been shown to inhibit GH-dependent activation of STAT5, we then
determined whether upregulation of these proteins differed between
LPS-treated TNFR1 and IL-6 null mice.
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IL-6 signaling is required for upregulation of Cis and Socs-3.
Several inflammatory cytokines including TNF- and IL-6 have been
shown to upregulate Cis and Socs 1-3 RNA
expression (8). However, a recent in vivo study
(6) of hepatic regeneration demonstrated that IL-6 alone
could induce Cis or Socs 1-3 RNA expression
in the absence of intact TNF signaling, whereas the converse was not
true. However, the duration of Socs-3 induction was
significantly less after the administration of IL-6 alone vs. partial
hepatectomy, in which multiple cytokines including TNF-
were also
induced (6). Whether IL-6-dependent upregulation of Cis
and Socs-3 was necessary and sufficient to acutely inhibit hepatic GH
signaling was not known. As shown in Figs. 3 and
6, Cis and Socs-3 proteins were
upregulated by LPS in WT and TNFR1 null mice (note that for Socs-3
upregulation by LPS/GH in TNFR1 null mice, n = 2). By
comparison, Cis or Socs-3 upregulation in LPS-treated IL-6 null mice
was either not detected or significantly reduced compared with TNFR1
null mice. Taken together, these data indicated that IL-6 inhibited
hepatic GH signaling via upregulation of Cis and Socs-3 protein
abundance. We then determined whether the observed alterations in Cis
and Socs-3 protein abundance were associated with differences in RNA
expression.
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IL-6 mediates LPS activation of STAT3 and upregulation of Socs-3
RNA expression.
In most experimental systems reported to date, cytokine upregulation of
Cis or Socs-3 in hepatocytes has been reported at the RNA level, and is assumed to correlate with corresponding changes
in protein abundance (8). Recently, the administration of
IL-6 alone has been shown to upregulate both Socs-3 RNA and protein in
liver (6). Interestingly, whereas TNF- administration was also shown to upregulate Socs-3 RNA expression (although
to a fourfold lesser degree) in that study, no Socs-3 protein was detected. Therefore, we determined Cis and Socs-3
RNA expression in TNFR1 and IL-6 null mice after LPS
administration. As shown in Fig.
7A, Socs-3 RNA
expression was upregulated to a significantly greater degree in
LPS-treated TNFR1 null (16 ± 2 ng) vs. IL-6 null (4.1 ± 2.5 ng) mice, although it was upregulated in both vs. controls. By
comparison, absolute Cis RNA expression did not differ
between LPS-treated TNFR1 null (8.4 ± 4.6 ng) and IL-6 null
(9.8 ± 4.2 ng) mice, although the upregulation compared with control was greater for the TNFR1 null mice (ninefold vs. twofold). These data indicated that the observed difference in Socs-3
protein abundance between LPS-treated TNFR1 null and IL-6 null
mice was due to a difference in RNA expression, whereas the observed
difference in Cis protein abundance might also be due to effects of
IL-6 on Cis protein translation.
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DISCUSSION |
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Signal-transduction pathways that mediate GH-dependent activation of STAT5 transcription factors in liver have been extensively studied and are now well defined (9). Until recently, the factors that negatively regulated GH signaling were less well understood (21, 27). In particular, whereas evidence of GH resistance had been observed in inflammatory diseases and in experimental models of inflammation, the specific factors that mediate cellular resistance to GH had not been identified. Identification of these factors will be a critical step in designing therapies to selectively restore GH signaling in inflammatory conditions. In this study, we have demonstrated that IL-6 upregulation of Cis and Socs-3 in liver can significantly inhibit GH activation of STAT5b.
The relative importance of TNF- vs. IL-6 in suppressing GH signaling
in inflammatory conditions has been difficult to determine. Transgenic
mice that overexpress either TNF-
or IL-6 exhibit stunted growth
with evidence of GH resistance and reduced IGF-I synthesis (11,
20). The overproduction of TNF-
and IL-6 that occurs in
experimental colitis has also been associated with GH resistance; this
was partially reversed by treatment with either anti-TNF-
or
anti-IL-6 antibodies, although anti-IL-6 was more effective (2,
3). The role that alterations in Ghr or Cis/Socs protein
abundance may play in causing inflammatory GH resistance has not been
determined, although potential mediators, such as Socs-3, have been
shown to be upregulated in patients with inflammatory bowel disease and
in experimental colitis (32).
GH signaling could be inhibited by cytokines at the receptor or postreceptor level. We have recently reported (13) that LPS administration led to a 70% reduction in hepatic Ghr gene expression 12 h later and that this was due to TNF-dependent reduction in DNA binding of Ghr gene promoter Sp1/Sp3 transactivators. In a model utilizing an eightfold higher dose of LPS than in the present study, reduced IGF-I activation associated with a reduction in total liver GH binding was demonstrated 6 h after treatment (12). However, another group, (22) which used the same LPS dose as in the present study for 4 h, did not observe a reduction in Ghr abundance. Importantly, in a recent report (30) describing GH resistance in experimental chronic uremia, although hepatic Ghr RNA levels were reduced, this was not associated with a reduction in Ghr protein abundance or GH binding. In the present study, in which samples were isolated 3 h after low-dose LPS administration, we observed a modest (~30%) reduction in Ghr RNA expression and protein expression. Therefore, it was likely that primarily postreceptor mechanisms accounted for the observed differences in STAT5b activation in this model. However, downregulation of the Ghr may play a significant role with longer-term exposure to LPS in chronic inflammatory conditions. This is currently being investigated.
A significant advance in understanding postreceptor modulation of GH signaling has come with cloning of members of the SOCS family including Cis and Socs 1-3 (21). In vitro studies (27, 28) have demonstrated that each of these proteins can inhibit GH signaling by preventing STAT5b phosphorylation in association with the activated GHR:JAK2 complex. Stunted growth in Cis transgenic mice and the high-growth phenotype of the Socs-2 null mouse have confirmed their importance in regulating postnatal growth (24, 25). Recent studies (6) have demonstrated that Cis/Socs 1-3 are positive acute-phase hepatic genes that are potently upregulated by LPS and inflammatory cytokines, in particular IL-6. Moreover, a temporal association was observed between maximal inhibition of GH activation of STAT5b or IGF-I by LPS and upregulation of IL-6 protein and Socs-3 RNA expression (22, 36). Therefore, the Cis/Socs genes represented good candidates for inflammatory inhibitors of GH action. However, direct evidence for this in vivo was lacking, and regulation of hepatic Cis and Socs 1-3 protein abundance by LPS had not been characterized.
In the present study, GH-dependent STAT5b activation was abolished in
WT or TNFR1 null mice in which LPS activated STAT3 and upregulated CIS
and SOCS-3 RNA and protein. STAT5b activation was preserved in IL-6
null mice in which STAT3 activation was prevented and CIS/SOCS-3
protein were not upregulated. Taken together, these data support a
molecular mechanism of GH resistance in which IL-6 activation of STAT3
leads to upregulation of Cis and Socs-3, and
inhibition of STAT5b phosphorylation by the GHR:JAK2 complex. This is
consistent with a recent report (36) using a similar model
of the LPS-induced hepatic acute-phase response, in which inhibition of
IGF-I upregulation by GH was temporally associated with elevated IL-6
levels and Socs-3 RNA expression. Regulation of the murine
Cis and Socs-3 genes by cytokines and STAT
transcription factors has been characterized in several prior reports
(1, 23, 35). STAT5a has been identified as a critical
regulator of Cis; however, in the present study,
upregulation of CIS occurred under conditions in which STAT3, but not
STAT5, was activated (35). This is consistent with a
recent report (10) in which GH-dependent upregulation of
Cis expression was preserved in STAT5b null mice, indicating
that alternate transcription factors, potentially including STAT3, can
also upregulate Cis. The STAT1/STAT3 cis element that has
been characterized in the mouse Socs-3 proximal promoter
could mediate the SOCS-3 upregulation we observed (1). However, although IL-6 has been shown to upregulate the rat
Socs-3 promoter in hepatocytes, this effect was not mapped
to the known STAT1/STAT3 elements (26). Therefore,
regulation of Cis or Socs-3 expression by GH or
IL-6 and associated STAT transcription factors is complex and appears
to be dependent on the specific tissue and experimental conditions
used. It should be noted that studies utilizing hepatocytes in culture
have also identified a potential role for IL-1 in upregulating
Socs-3 RNA and inhibiting GH-induced expression of the
ALS gene (5). Taken together, these data add to
the growing body of evidence supporting a critical role for CIS/SOCS
proteins in modulating GH signaling in inflammatory conditions
(22). This may provide for several therapeutic options, including targeted blockade of specific cytokines or individual CIS/SOCS proteins, in addressing complications of inflammatory GH resistance.
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ACKNOWLEDGEMENTS |
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The excellent technical assistance of Carol Williams, Himmat Bajwa, Jennifer Hicks, and Lisa Difedele is gratefully acknowledged.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-02700, the Charles H. Hood Foundation, and the Yale Child Health Research Center (to L. A. Denson).
This work was presented, in part, in abstract form at the 2001 annual meeting of the North American Society of Pediatric Gastroenterology, Hepatology, and Nutrition.
Address for reprint requests and other correspondence: L. A. Denson, Yale Child Health Research Center, 464 Congress Ave., P.O. Box 208081, New Haven, CT 06520-8081 (E-mail: lee.denson{at}yale.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.
First published January 2, 2003;10.1152/ajpgi.00178.2002
Received 13 May 2002; accepted in final form 23 December 2002.
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