 |
INTRODUCTION |
SOCS-11 is an
intracellular SH2 domain-containing protein that was initially
isolated by screening a retroviral cDNA library for inhibitors of
IL-6 signaling (1). It is also known as STAT-inducible STAT inhibitor-1
(SSI-1) (2) or JAK-binding protein (JAB) (3). SOCS-1 defines a family
of eight proteins, SOCS-1 to -7 and CIS (cytokine-inducible
SH2-containing protein), each of which contains a central SH2 domain
and a C-terminal SOCS box (4).
In vitro studies have shown that the expression of SOCS-1,
-2, -3, and CIS is inducible by a number of cytokines and that, when
constitutively overexpressed, each SOCS protein can act to switch off
cytokine-induced responses by inhibiting the Janus kinase/signal
transducers and activators of transcription (JAK/STAT) pathway (1-3).
The latter studies are difficult to interpret because not only are the
SOCS proteins grossly overexpressed, but they are also expressed in a
temporally inappropriate manner, being present prior to the onset of
signaling rather than induced as a consequence of signal transduction,
which occurs physiologically.
Although both SOCS-1 and SOCS-3 act to suppress signal transduction
from a similarly broad spectrum of cytokines in vitro, including IL-6, IL-4, growth hormone, IFN
/
, and IFN
(1-3, 5-8), analyses of mice deficient in either SOCS-1 or SOCS-3 have indicated that each may play a relatively specific role in
vivo. Mice lacking SOCS-3 die during embryogenesis apparently from
erythrocytosis, suggesting that SOCS-3 is critical in regulating fetal
liver erythropoiesis (9). SOCS-1 deletion results in mice that die,
before weaning, of a complex multiorgan disease (10-12). Although the
primary defect in mice lacking SOCS-3 has not been defined, the fatal
neonatal disease characteristic of SOCS-1-deficient mice requires
IFN
, because this pathology does not develop in SOCS-1-deficient
mice that also lack the IFN
gene (13).
Interferons are fundamental components of the immune system. The
predominant function of interferons is to confer cellular resistance to
viral infections, although other immunomodulatory functions have also
been described (14, 15). IFN
is produced by activated T lymphocytes
and natural killer cells during viral infections and initiates an
antiviral program in target cells by up-regulating the expression of
genes that mediate the anti-viral response, including those encoding
MHC class I antigens (14, 15). Similar to other cytokines, interferons
activate the JAK/STAT pathway (16), and studies in vivo have
shown that STAT1 is critical for mediating biological responses to
IFN
(17, 18). Elevated levels of IFN
can be toxic, and it is
clear that both IFN
production and responses to this cytokine must
be tightly regulated in order to achieve a balance between beneficial
and harmful effects (19-21). Other cytokines including IL-4, IL-10,
and IL-13 contribute to some extent to this regulation by antagonizing
IFN
functions (22-24). In addition, negative regulators such as
SOCS proteins act to limit signal transduction in response to
IFN
.
At least in vitro, SOCS-1 expression is induced by IFN
and overexpression of SOCS-1 inhibits IFN
signaling (6, 7). Clearly,
IFN
is an essential mediator of the fatal disease seen in mice
lacking SOCS-1 (13), but the precise mechanism is unclear. This report
compares IFN
signaling in wild-type and SOCS-1-deficient mice
in vivo and finds that both biochemical and biological
responses to IFN
are attenuated by SOCS-1.
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EXPERIMENTAL PROCEDURES |
Maintenance of Mice and Injection of
IFN
--
SOCS-1
/
IFN
/
mice were
generated as described previously and were maintained on a mixed 129/Sv
and C57Bl/6 genetic background (10, 13). For biological studies,
neonatal mice were given daily intraperitoneal injections of 3, 0.3, or
0 µg recombinant murine IFN
(rmIFN
, PeproTech, Rocky Hill, NJ)
in 0.05 ml of saline from birth and were sacrificed when moribund or at
21 days of age.
Immunoprecipitation and Western Blotting of Liver
Proteins--
For biochemical studies, adult mice weighing an average
of 29 g were given a single intraperitoneal injection of either
0.2 ml of saline, 2 µg of rmIFN
(PeproTech, Rocky Hill, NJ) or 10 µg of recombinant murine IL-6 (a gift from Dr. Richard Simpson, Ludwig Institute for Cancer Research, Melbourne, Australia). Mice were
sacrificed by cervical dislocation at the indicated times after
injection. Livers were dissected, immediately frozen in liquid
nitrogen, and stored at
70 °C. Frozen livers were pulverized with
a hammer and homogenized in RIPA buffer (1% v/v Triton X-100, 1% w/v
sodium deoxycholate, 0.1% w/v SDS, 150 mM NaCl, 10 mM Tris, pH 7.5) containing 10 µg/ml leupeptin (Auspep,
Parkville, Australia), 1 mM iodoacetic acid, 50 µg/ml
soybean trypsin inhibitor, 20 µg/ml aprotinin, 1 mM
phenylmethylsulfonyl fluoride, 0.5 mM EGTA (Sigma Chemical
Co.) and 1 mM pervanadate. Pervanadate was prepared by adding hydrogen peroxide (20 mM final) to 1 mM
sodium orthovanadate (Sigma) and incubating at room temperature for
30 min. Lysates were cleared by centrifugation and protein
content was quantitated using a Coomassie Protein Assay Reagent
(Pierce, Rockford IL).
For immunoprecipitations, liver lysates (12-20 mg of protein) were
first precleared with protein G-Sepharose (0.060 ml, suspended 1:1 in
RIPA buffer; Amersham Pharmacia Biotech, Uppsala, Sweden) for 1.5 h, then incubated with 4 µg of anti-STAT1 antibody (Transduction Laboratories, Lexington, KY) and protein G-Sepharose (0.06 ml, suspended 1:1 in RIPA buffer) for 2 h or overnight. Unbound
protein was separated from the immunoprecipitate by washing 2-4 times in Nonidet P-40 lysis buffer (150 mM NaCl, 1% (v/v)
Nonidet P-40, 50 mM Tris, pH 8.0) containing 10 µg/ml
leupeptin, 1 mM iodoacetic acid, 50 µg/ml soybean trypsin
inhibitor, 20 µg/ml aprotinin, 1 mM phenylmethylsulfonyl
fluoride, 0.5 mM EGTA, and 1 mM pervanadate. Bound protein was eluted from the Sepharose in 0.03 ml of 2× SDS loading buffer (115 mM Tris-HCl, pH 6.8, 1.8% (w/v) SDS,
18.2% (v/v) glycerol, 2.5% (v/v) 2-mercaptoethanol, bromphenol blue) at 95 °C for 5 min, centrifuged at 13,000 rpm for 5 min, and the supernatant was loaded on SDS-PAGE gels. Eluates were split prior to
SDS-PAGE to allow quantitation of protein loading. Proteins were
fractionated by SDS-PAGE under reducing conditions and then electrophoretically transferred to PVDF-Plus membranes (Micron Separations Inc., Westborough, MA). Membranes were blocked with 10%
skim milk for a minimum of 1 h and then incubated either for 2 h with anti-STAT1 antibody (1:250 dilution, Transduction
Laboratories), or overnight with anti-phosphorylated STAT1 antibody
(1:500, New England Biolabs, Beverly, MA). Antibody binding was
visualized with either horseradish peroxidase-conjugated anti-rabbit or
anti-mouse-Ig Fc fragment-specific (Jackson ImmunoResearch Laboratories
Inc., West Grove, PA) and the SuperSignal West Pico chemiluminescent substrate (Pierce Chemical Co.). Results shown are representative of
two experiments.
Hepatocyte Isolation--
Hepatocytes were harvested from
10-week-old mice as described previously (25). Briefly, livers were
perfused retrogradely via the inferior vena cava using HBSS without
calcium and magnesium and then with the same medium containing 0.5 mM EDTA. EDTA was then removed by flushing the liver with
HBSS without calcium and magnesium. Hepatocytes were released by
perfusing with HBSS without magnesium but containing 5 mM
CaCl2 and 0.05% collagenase IV (Sigma). Viable hepatocytes
were purified using a Percoll density gradient (Amersham Pharmacia
Biotech, Uppsala, Sweden), and the resulting cell pellet was washed
three times by 50 × g centrifugation in RPMI 1640 medium containing 10% v/v fetal calf serum and 5 × 10
5 M 2-mercaptoethanol (RF10 medium). Cells
were plated in 10-cm cell culture dishes (Corning, NY) in the same
medium at a density of 7.5 × 105 cells per plate and
were allowed to adhere overnight at 37 °C.
Electrophoretic Mobility Shift Assays--
Adherent hepatocytes
were stimulated for 10 min at 37 °C with 5 ng/ml murine IFN
(PeproTech) in RF10 medium, after which the cytokine was washed from
the plates, and the cells were cultured in fresh RF10 at 37 °C for
the remainder of the indicated time. Hepatocytes were harvested from
the plates using a rubber policeman and were washed once with cold
phosphate-buffered saline containing 1 mM pervanadate. Cell
pellets were snap frozen on dry ice, and high salt nuclear extracts
were prepared as described (26). Electrophoretic mobility gel shift
assays were performed on 10 µg of nuclear protein using the m67
oligonucleotide as described (27). To identify DNA-binding complexes,
protein samples were preincubated with 2 µg of antibody specific for
either STAT1 (Transduction Laboratories) or STAT3 (Santa Cruz
Biotechnology Inc., Santa Cruz, CA) before binding to the oligonucleotide.
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RESULTS |
SOCS-1 Is Required for Regulating Responses to IFN
in
Vivo--
The neonatal mortality in SOCS-1 knockout mice has been
shown to be dependent on IFN
, because mice lacking functional genes for both SOCS-1 and IFN
are viable and healthy (13). We wished to
use SOCS-1
/
mice to assess the extent to which SOCS-1
regulates IFN
signal transduction; however, such studies are
complicated by the endogenous production of IFN
in vitro
and in vivo and compounded by the neonatal morbidity and
mortality of SOCS-1
/
mice. To overcome these problems
we took advantage of the observation that SOCS-1
/
IFN
/
mice, which fail to produce IFN
, are healthy
and survive to adulthood.
Previous studies have documented the toxicity of IFN
when
administered to newborn mice (19). Further, the pathology induced by
IFN
treatment of wild-type mice is similar to that seen in SOCS-1
/
mice (13). The phenotype of
SOCS-1
/
mice may be due to increased circulating levels
of IFN
, increased sensitivity of cells to the effects of IFN
because of unregulated signaling, or a combination of both. To
determine whether mice are more sensitive to IFN
in the absence of
SOCS-1, newborn SOCS-1
/
IFN
/
and
SOCS-1+/+ IFN
/
mice received daily
intraperitoneal injections of IFN
. These mice were monitored and
were sacrificed when moribund. Mice lacking SOCS-1 were substantially
more sensitive to the toxicity of IFN
. Injections of 3 µg of
IFN
induced morbidity within 2 days in SOCS-1
/
IFN
/
mice, whereas this dose was tolerated for ~3
weeks in SOCS-1+/+ IFN
/
mice (Fig.
1). Differences in sensitivity were also
seen in response to the administration of 0.3 µg of IFN
. In
contrast to SOCS-1
/
IFN
/
mice, which
were all moribund by 3 weeks of age and developed the same pathological
features as SOCS-1
/
mice (Fig. 1 and data not shown),
SOCS-1+/+ IFN
/
mice all remained healthy
after receiving this dose of IFN
(Fig. 1).

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Fig. 1.
Mice lacking SOCS-1 are more sensitive to the
effects of exogenous IFN . Neonatal mice
were given daily intraperitoneal injections of IFN from birth. Mice
were administered either 3 µg of IFN (top panel), 0.3 µg of IFN (middle panel) or saline (bottom
panel), and their health was monitored daily. n = number of mice/group.
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|
IFN
-induced STAT Activation Is Prolonged in Mice Lacking
SOCS-1--
To assess whether SOCS-1 regulates the intensity or
duration of responses to IFN
, the activation of downstream signaling molecules was assessed in mice lacking SOCS-1. The organ we chose to
examine in these studies was the liver because it is severely affected
both in SOCS-1
/
mice and in mice injected with IFN
.
SOCS-1
/
IFN
/
, SOCS-1+/+
IFN
/
, and SOCS-1+/+
IFN
+/+ mice were given intraperitoneal injections of
IFN
and the subsequent activation of STAT1 in the liver was
monitored at various times thereafter. STAT1 phosphorylation was
evident in all mice within 15 min of IFN
injection, with the
response continuing for at least 2 h but declining to basal levels
by 4 h in SOCS-1+/+ IFN
/
and
SOCS-1+/+ IFN
+/+ mice (Fig.
2A). However, phosphorylation
of STAT1 was prolonged in mice lacking SOCS-1 and was maintained until
8 h after injection. This prolonged response was specific to
IFN
, because there was no difference in the kinetics of STAT1 or
STAT3 phosphorylation induced by IL-6 in mice lacking SOCS-1 (Fig.
2B and data not shown). SOCS-1 appeared to be important for
regulating the duration of the response to IFN
, but not the
intensity, because peak levels of STAT1 phosphorylation did not differ
substantially between mice of different genotypes.

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Fig. 2.
IFN -induced STAT1
phosphorylation is prolonged in SOCS-1 /
IFN / mice.
SOCS-1+/+ IFN +/+, SOCS-1+/+
IFN / and SOCS-1 /
IFN / mice were injected with 2 µg of IFN
(A) or 10 µg of IL-6 (B) and sacrificed at the
times indicated. Liver lysates were prepared and STAT1 protein
immunoprecipitated with an anti-STAT1 antibody. Duplicate blots were
incubated with either an antibody specific for phosphorylated STAT1
( -STAT1-P, left panels) or STAT1 (right
panels).
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SOCS-1 Is Required for Regulating Responses to IFN
in
Hepatocytes in Vitro--
The in vivo experiments described
above indicate a role for SOCS-1 in regulating IFN
-induced responses
in the liver but may be complicated by factors such as clearance of
IFN
from the circulation. We therefore wished to examine IFN
responses in vitro using primary hepatocyte cultures. To
assess the sensitivity of hepatocytes to IFN
, primary hepatocyte
cultures were established from SOCS-1
/
IFN
/
and SOCS-1+/+
IFN
/
mice. Hepatocytes were stimulated for 10 min
with a pulse of IFN
, after which time cytokine was washed from the
cells, and the response was allowed to decay over varying periods. Gel
shift analysis was used to monitor the DNA binding activity of STAT dimers throughout the time course. In both cell types, the response to
IFN
peaked at 30 min after stimulation. The response decayed rapidly
in SOCS-1+/+ IFN
/
cells and was
virtually undetectable at 2 h poststimulation (Fig. 3A). In contrast, the activity
of STAT dimers was prolonged in SOCS-1
/
IFN
/
cells, persisting beyond 3 h after IFN
treatment (Fig. 3B).

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Fig. 3.
IFN -induced STAT
activation is prolonged in SOCS-1 /
IFN / hepatocytes.
Electrophoretic mobility shift assays of extracts from primary
hepatocytes derived from SOCS-1+/+ IFN /
(A) or SOCS-1 / IFN / mice,
stimulated for 10 min with 5 ng/ml IFN and harvested at the times
indicated. In panel C, extracts from 10 min time points were
pretreated with antibodies specific for either STAT1 or STAT3 before
binding to the m67 oligonucleotide. Addition of an anti-STAT1 antibody
abolished the formation of the two DNA/protein complexes and resulted
in a supershifted complex, indicated by *. Pretreatment with an
anti-STAT3 antibody did not affect the intensity of the lower
DNA-protein complex, but the intensity of the upper band was
reduced (bands indicated by arrows).
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|
Supershift analyses (Fig. 3C) identified STAT1 homodimers as
the predominant STAT dimers formed in response to IFN
(Fig. 3,
A and B, lower band), with a smaller amount of
STAT1/STAT3 homodimers also being activated (Fig. 3, A and
B, upper band). Similar to the in vivo studies,
only the duration of the response to IFN
was altered in
SOCS-1
/
cells and not the intensity. Other experiments
were performed in which hepatocytes were exposed to the stimulus for
the entire time course rather than just a pulse. Active STAT dimers
persisted for longer in these experiments but did not decay in cells
lacking SOCS-1 throughout an 8-h culture period, whereas STAT dimer
formation in SOCS-1+/+ IFN
/
cells had
decayed to a baseline level by 5 h of culture (data not shown).
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DISCUSSION |
IFN
is critical for mediating the multiorgan pathology of
SOCS-1
/
mice, because mice lacking both SOCS-1 and
IFN
are viable and healthy (13). However, the specific relationship
between SOCS-1 and IFN
has yet to be defined. Is the phenotype of
SOCS-1
/
mice due to increased sensitivity of cells to
IFN
in the absence of SOCS-1 or to increased production of IFN
?
Both appear to contribute to the phenotype. A previous study has shown
that circulating levels of IFN
are increased in mice lacking SOCS-1
(12). The data presented here clearly establish that in the absence of
SOCS-1, mice and cells isolated from them are more sensitive to the
effects of IFN
. Neonatal mice lacking SOCS-1 were substantially more sensitive to the toxic effects of administered IFN
. Further, we have
directly established a biochemical basis for this hypersensitivity and
have shown that SOCS-1 attenuates signal transduction pathways in
vivo, consistent with in vitro data. STAT1 activation
was prolonged in the liver of SOCS-1
/
IFN
/
mice after injection of IFN
. This increased
sensitivity in vivo was paralleled in vitro by
prolonged STAT1 activation in primary hepatocytes in response to
IFN
.
Although forced expression of SOCS-1 has been shown to limit the
intensity of STAT activation in response to cytokine (1-3), SOCS-1
expression is temporally inappropriate in these studies. In the present
study, which relies on more physiological expression, SOCS-1 appears to
be important for limiting the duration of responses to cytokine and not
the magnitude of the response. This result is consistent with the
current model of SOCS action in which SOCS expression is induced by
cytokine and then functions to switch off the cytokine-induced signal
(1). The relative contributions to the SOCS-1
/
phenotype of increased IFN
production and prolonged IFN
responses are difficult to delineate. In situations in which IFN
is limiting, for instance in SOCS-1
/
IFN
+/
mice,
pathological changes including uncontrolled macrophage activation are
still evident and may result predominantly from inappropriate and
prolonged signaling in response to more normal levels of circulating
IFN
(28). Further, serum IFN
is undetectable in some
SOCS-1
/
mice (data not shown). Our data suggest that
hypersensitivity to IFN
may be driving the typical
SOCS-1
/
phenotype seen in these mice.
Although the JAK/STAT pathway has been well characterized using both
primary and continuous cell lines, the kinetics of JAK/STAT activation
in vivo following cytokine stimulation has not been comprehensively studied. In the course of this study we have developed assays to monitor the phosphorylation of STATs from mouse liver, resulting in a detailed study of the kinetics of IFN
signaling in vivo.
The phenotype of SOCS-1
/
mice demonstrates a specific
non-redundant role for SOCS-1 in determining the rate at which
interferon signaling is attenuated. The lack of effect on IL-6
signaling in vivo may indicate that other SOCS proteins can
compensate for the absence of SOCS-1 in other cytokine pathways.
Whereas IFN
signaling is clearly prolonged by several hours in
SOCS1
/
mice, STAT1 activation is still switched off,
albeit later than normal. This residual negative regulation of signal
transduction may be mediated by other members of the SOCS family or by
other inhibitory mechanisms such as phosphatases (29) and the protein inhibitor of activated STAT (PIAS) (30). The results presented in this
study using wild-type and SOCS-1-deficient cells provide a benchmark
against which cells from other compound-knockout mice can be compared.