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Department of Medicine (C.B., J.K.E., K.E.-H., J.K., R.S.A., S.H., J.S.F.), Division of Endocrinology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215; and Department of Neurology (J.K.E.), Beth Israel Deaconess Medical Center, and Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02215
Address all correspondence and requests for reprints to: Jeffrey S. Flier, Division of Endocrinology, Department of Medicine, Beth Israel Deaconess Medical Center, 99 Brookline Avenue, Research North, Boston, Massachusetts 02215. E-mail: jflier{at}caregroup.harvard.edu
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) (9), the leukemia inhibitory factor receptor subunit, and
gp130 (10). The CNTF receptor subunit is expressed throughout the
brain (9, 11, 12), and specific localization of CNTFR has been
demonstrated in the arcuate nucleus (Arc) and other parts of the
hypothalamus (13), regions known to be involved in regulation of body
weight (14). In rodents, it has been noted that peripheral
administration of CNTF results in fever and transient reduction in food
intake (13, 15, 16, 17, 18). Recent data also show that peripherally
administered CNTF reduces the body weight of ob/ob and
db/db mice (13), suggesting that CNTF and leptin may act
through similar central pathways, possibly via specific nuclei in the
hypothalamus (13, 19). Leptin, an adipocyte-derived hormone, is known to act on specific brain regions to regulate food intake, energy expenditure, and neuroendocrine function (20, 21, 22, 23, 24). Leptin acts on receptors that belong to the cytokine-receptor superfamily, which includes the IL-6 receptor (IL-6R), gp130, and the LIF receptor (LIFR) (25, 26). In vitro and in vivo studies demonstrate that leptin activates cytokine-like signal transduction by stimulating the JAK-STAT pathway via a long leptin receptor isoform (27, 28, 29, 30), which is highly expressed in regions of the hypothalamus (26, 27, 31, 32). Lack of functional leptin or of long-form leptin receptors produces severe obesity in rodents and humans (20, 26, 33, 34, 35, 36, 37).
Cytokine stimulation induces members of the STAT transcription factor family to dock onto receptor phosphotyrosines, enabling their own tyrosine phosphorylation by JAK tyrosine kinase family members (see Ref. 38 for review). Subsequently, STAT proteins translocate to the nucleus and bind to conserved genomic regulatory sequences to provide a rapid means of activating gene transcription (38). Recently, a new family of cytokine-inducible inhibitors of signaling has been identified, including CIS (cytokine-inducible sequence), SOCS-1 (suppressor of cytokine signaling), SOCS-2, and SOCS-3 (39, 40, 41, 42). Most, if not all, members of the cytokine superfamily (including leptin, IL-6, LIF, erythropoietin, and GH) induce transcriptional activation of one or more of the cis or socs genes in vivo and in vitro (39, 40, 41, 42, 43, 44, 45). This activation is thought to occur via activation of the JAK-STAT signaling pathway (39, 40, 41, 42). These results suggest that CIS and SOCS isoforms may act as part of an intracellular negative feedback loop, which results in switching off or dampening cytokine signaling.
Because CNTF activates the JAK-STAT signal-transduction pathway and can affect body weight after peripheral administration, we tested the possibility that CNTF could activate the expression of cis or socs genes in the hypothalamus, and we compared it to that of leptin. We found that CNTF strongly induced SOCS-3 mRNA in the Arc of the hypothalamus, a region known to express SOCS-3 mRNA after leptin treatment and to be a key target of leptin action. These data are consistent with the role of Arc as a key site for the regulation of body weight by CNTF. In addition, we also show that SOCS-3 is an inhibitor of CNTF signal transduction.
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-interferon
activated-sequence luciferase reporter construct (GAS-luc)
was a kind gift from Dr. L. Stancato and Dr. R. Pine (Sphinx
Pharmaceuticals, Durham, NC). The SOCS-3 antiserum was generated by
injection of purified SOCS-3 protein into rabbits (Quality Controlled Biochemicals, Inc., Hopkinton, MA). The purified and
refolded bacterially expressed full-length mouse SOCS-3 protein used
for antiserum production was kindly provided by Dr. R. Shigeta and Dr.
S. E. Shoelson (Joslin Diabetes Center). The affinity
purified goat anti-SOCS-3 antibody (C-terminus) was purchased from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All
reagents for cell culture were from Gibco BRL
(Gaithersburg, MD).
Animals and histology
Male ob/ob mice (age, 10 weeks) and male Sprague
Dawley rats (250–350 g) were purchased from The Jackson Laboratory (Bar Harbor, ME) and Taconic Farms, Inc. (Germantown, NY), respectively. The animals and
procedures used were in accordance with the guidelines and approval of
the Harvard Medical School and Beth Israel Deaconess Institutional
Animal Care and Use Committees. For RT-PCR experiments, mice were
deeply anesthetized by inhalation of Metofane (Mallinckrodt, Inc. Veterinary, Inc., Mundelein, IL) and then
decapitated. The skull was reflected from the brain, and hypothalamus
and cerebellum were isolated by snap freezing in liquid nitrogen.
Samples from liver and kidney were also taken. For in situ
experiments, animals were deeply anesthetized with ip injection of
chloral hydrate (7%; 350 mg/kg) and perfused transcardially with
diethylpyrocarbonate-treated saline, followed by
neutral-buffered formalin (10%). Coronal sections were cut at 30 µm
(rats, 1:5 series; mice, 1:4 series).
Cell culture and transient transfection
Astrocytes were grown in DMEM (high glucose) supplemented with
10% FCS, 100 U/ml penicillin, and 10 µg/ml streptomycin at 37 C in
5% CO2. Cells were serum-deprived for 12–15 h before
stimulation with hormones. For luciferase and ß-galactosidase assays,
cells were grown in 6-well plates and transfected with a total of 2.0
µg plasmid DNA using 15 µl Lipofectamine per well. Forty-eight
hours post transfection, cells were lysed in 500 µl of 25
mM glycylglycine, 15 mM MgSO4, 4
mM EGTA with 1% Triton X-100, and 2 mM
dithiothreitol (DTT) (lysis buffer A) and were assayed as described
below. For Western blotting experiments, cells were grown in 10-cm
dishes. Cells were harvested by rinsing in ice-cold PBS and scraping
into 1.0 ml ice-cold lysis buffer B (1% Nonidet P-40, 0.5% Triton
X-100, 10% glycerol, 150 mM NaCl, 2 mM
Na3VO4, 1 mM phenylmethylsulfonyl
fluoride (PMSF), 5 µg/ml leupeptin, 5 µg/ml aprotinin, 50
mM Tris-HCl, pH 7.4). Lysates were clarified by
centrifugation at 23,000 x g for 15 min, and
supernatants were immunoprecipitated as described below.
Luciferase and ß-galactosidase assay
After lysis, 50-µl aliquots were used for the luciferase
assay. Briefly, 150 µl 0.75 mM luciferin (Molecular Probes, Inc., Eugene, OR) and 150 µl assay buffer (lysis
buffer A + 15 mM K2HPO4, 6
mM ATP, 3 mM DTT, pH 7.6) were injected
simultaneously and measured for 20 sec by a Luminometer (LB 9501, EG&G
Berthold, Bad Wildbad, Germany). ß-galactosidase activities were
determined in 20-µl samples using Galacton (Tropix, Inc., Bedford,
MA), as described by the manufacturer, and were measured for 5 sec by
the Luminometer.
Immunoprecipitation and immunoblotting
Immunoprecipitations were performed as described earlier by
Bjørbæk et al. (46). Briefly, clarified lysates were
incubated at 4 C with SOCS-3 antiserum, together with protein A-agarose
beads (1:15 dilution of a 50% slurry in 1% Nonidet P-40, 0.5% Triton
X-100, 10% glycerol, 150 mM NaCl, 50 mM
Tris-HCl, pH 7.4) for 15 h. After three washes in ice-cold buffer
B, the samples were subjected to 12% SDS-PAGE. Proteins were then
transferred to nitrocellulose membranes and blocked in 10% dry-milk in
20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05%
Tween 20. After incubation of membranes with anti-SOCS-3 antibodies
(Santa Cruz Biotechnology, Inc.) and washing, targeted
proteins were detected using enhanced chemiluminescence, as described
by the manufacturer (Amersham International,
Buckinghamshire, UK).
Nuclear extraction and electrophoretic mobility shift assay
(EMSA)
Nuclear extractions were done as described earlier (47).
Briefly, astrocytes were grown to confluence in 6-well dishes and
serum-deprived 12–15 h before stimulation with hormones. After
treatment, cells were rinsed once with 2 ml ice-cold Tris-buffered
saline and then scraped into 1.0 ml ice-cold Tris-buffered saline,
transferred to a 1.5-ml tube (Eppendorf, Brinkmann Instruments, Inc., Westbury, NY) and pelleted by centrifugation at
1500 x g at 4 C for 5 min. The pellets were then
resuspended in 400 µl ice-cold buffer C [40 mM HEPES (pH
7.9), 10 mM KCl, 0.1 mM EDTA, 0.1
mM EGTA, 1 mM DTT, 0.5 mM PMSF] by
gentle pipetting in a yellow tip. The cells were allowed to swell on
ice for 15 min., after which 25 µl 10% Nonidet NP-40 were added and
the tube vortexed for 10 sec. Samples were then centrifuged for 30 sec
at 14,000 x g, and the nuclear pellets were
resuspended in 25 µl ice-cold buffer D [20 mM HEPES (pH
7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM
EGTA, 1 mM DTT, 1 mM PMSF] by vigorous rocking
at 4 C for 30 min. The nuclear extracts were finally clarified by
centrifugation at 14,000 x g for 20 min and stored at
-80 C until further use. Five micrograms of nuclear extracts
(determined by Bradford protein assay, Bio-Rad Laboratories, Inc., Hercules, CA) were added to binding buffer [final
vol was 20 µl: 13 mM HEPES (pH 7.9), 65 mM
NaCl, 1 mM DTT, 0.15 mM EDTA, 8% glycerol, 50
mg/ml poly (dI-dC), and 0.01% NP-40], which included 100,000 cpm of
the 32P-labeled double-stranded oligonucleotide probe
[SIE-mutant 67 (48)], and were incubated for 15 min at room
temperature. The probe was generated by annealing two oligonucleotides:
5'-CGCTCCATTTCCCGTAAATCAT-3' and 5'-CGCTCATGATTTAC-GGGAAATG-3',
followed by a fill-in reaction of the five base overhangs using T7
polymerase (Gibco BRL) and
32P--deoxynucleotide triphosphates (each 222 TBq/mmol,
740 MBq/ml; NEN, Boston, MA). Unincorporated nucleotides were
removed by using a G25 Quick Spin column (Boehringer Mannheim, Indianapolis, IN). Samples were loaded onto a 5%
nondenaturing polyacrylamide gel (39:1, acrylamide:bis) containing
2.5% glycerol in 0.5 x Tris-Borate-EDTA buffer and run for
1.5 h at 220 V at 4 C. After drying, gels were placed in a
PhosphorImager cassette (Molecular Dynamics, Inc.,
Sunnyvale, CA) for 12–15 h.
Northern blot analysis
RNA was extracted from hypothalami or cells
(Tel-Test Inc., Friendswood, TX), and 15 µg of total RNA
(determined by UV absorbance corroborated by ethidium bromide-stained
integrity gels) were resolved on 1% agarose gels containing 37%
formaldehyde. Electrophoresis was performed at 75 V for 2 h. Gels
were then treated with 50 mM NaOH, 10 mM NaCl
for 15 min, and 0.1 M Tris (pH 7.5) for 15 min before
transfer to nylon membranes (Boehringer Mannheim) using a
vacuum system from Amersham Pharmacia Biotech (Piscataway,
NJ). Membranes were then subjected to UV cross-linking and
prehybridized for 1 h in QuickHyb solution
(Stratagene, La Jolla, CA) at 68 C. The SOCS-3 probe was
generated by RT-PCR using murine hypothalamic RNA. The PCR
product was then ethanol precipitated to remove buffer and free
nucleotides, labeled with 32P--deoxycycidine
triphosphate (222 TBq/mmol, 740 MBq/ml; NEN) by random-priming
(Gibco BRL), boiled for 5 min, and incubated with the
membrane in 12 ml QuickHyb solution at 68 C for 15 h. Membranes
were washed three times with 2 x standard sodium citrate (SSC),
0.1% SDS at room temperature, and two times with 0.2 x SSC,
0.1% SDS at 60 C, and finally, placed in a PhosphorImager cassette for
12–15 h.
Quantification of CIS and SOCS mRNAs by RT-PCR
Total RNA purification and subsequent cDNA synthesis
(Stratagene) was done in parallel from all tissue samples.
Preliminary PCR experiments showed that the rate of amplification was
linear for cis, socs-1, socs-2, and
socs-3 when applying less than 28 PCR-cycles. We chose 25
cycles for the PCR quantification, as described earlier by Bjørbæk
et al. (49). The following primers were used for specific
PCR amplification of cis, socs-1,
socs-2, and socs-3 cDNAs: CIS-A:
5'-ctggagctgcccgggccagcc-3' and CIS-B: 5'-caaggctgaccacatctggg-3'
(400-bp product); SOCS-1A: 5'-ccactccgattaccggcgcatc-3' and SOCS-1B:
5'-gctcctgcagcggccgcacg-3' (350 bp product); SOCS-2A:
5'-aagacgtcagctggaccgac-3' and SOCS-2B: 5'-tcttgttggtaaaggcagtccc-3'
(300-bp product); SOCS-3A: 5'-accagcgccacttcttcacg-3' and SOCS-3B:
5'-gtggagcatcatactgatcc-3' (450-bp product). Each 50-µl PCR reaction
was carried out with 5.0 µl cDNA as template. The assay conditions
were: 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5
mM MgCl2, 0.01% gelatin, 0.2 mM
deoxynucleotide triphosphates, 20 pmol of each primer, 2.5 U
Taq polymerase (Stratagene), and 1.0 µl
-32P-deoxycycidine triphosphate (29.6 TBq/mmol, 370
MBq/ml; NEN). The mixture was overlaid with 25 µl mineral oil; and
after initial denaturation at 96 C for 3 min, the samples were
subjected to 25 cycles of amplification: denaturation at 95 C for 1
min, annealing at 60 C for 1 min, and extension at 72 C for 45 sec. Ten
microliters of the reaction were then combined with 5 µl sequencing
stop solution and heated to 85 C for 5 min before loading 4 µl onto a
4% urea-acrylamide gel. Electrophoresis was carried out at 60 W of
constant power for 2 h, before the gels were transferred to filter
paper, dried, and finally subjected to 32P quantification
by PhosphorImager analysis.
In situ hybridization histochemistry
Mouse SOCS-3 sense and antisense 35S-RNA probes were
generated as described earlier by Bjørbæk et al. (45). The
protocol for in situ hybridization histochemistry was a
modification of that previously reported (50, 51, 52) and performed as
described earlier (45, 49). Briefly, tissue sections were mounted onto
slides and stored in desiccated boxes at -20 C. Hybridization
solution, including the 35S-labeled SOCS-3 RNA probe, was
applied to each slide, and sections were incubated for 12–16 h at 56
C. Slides were then rinsed in 2 x SSC and treated with
ribonuclease A (Boehringer Mannheim) for 30 min. Sections
were then rinsed in decreasing concentrations of SSC, followed by
dehydration in graded ethanol. Slides were dipped in NTB2 photographic
emulsion (Eastman Kodak Co., Rochester, NY) and stored at
4 C for 2 weeks. The emulsion-dipped slides were finally developed with
Dektol developer (Eastman Kodak Co.) and counterstained
with thionin. Sections were analyzed using a Zeiss Axioplan light
microscope (Carl Zeiss, Inc., Thornwood, NY).
Photomicrographs were produced using a Kodak DCS camera and printed on
a dye sublimation printer.
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. This was confirmed by
Northern blot analysis (Fig. 1B). No effects on CIS, SOCS-1, or SOCS-2
mRNA were detected in this tissue (Fig. 1A). As we have demonstrated
earlier (45), leptin treatment caused a modest increase of SOCS-3 mRNA
but not of CIS, SOCS-1, or SOCS-2 mRNA (Fig. 1A). PhosphorImager
analysis demonstrated a 6- to 15-fold and a 2-fold increase in SOCS-3
mRNA after CNTF and leptin treatment, respectively. Similar effects on
SOCS-3 mRNA were seen 1 or 3 h after treatments (data not
shown).
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, A and
B). A significant proportion of the SOCS-3-expressing cells in this
region were of neuronal nature, as determined by the characteristic
clustering of silver grains. The ependymal lining of all ventricles,
the median eminence, and the subfornical organ also exhibited
high-intensity specific signals. Similar results were obtained in
ob/ob mice (data not shown).
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). After iv
injection of murine leptin (1 µg/g BW), a marked increase in specific
hybridization was detected in the Arc and the dorsomedial hypothalamic
nucleus (DMH), as we have demonstrated earlier (45). No specific
signals were detected in the subfornical organ or in the ependymal
lining of the ventricles. The Arc and the DMH are those regions of the
hypothalamus in which the long form of the leptin receptor is most
highly expressed (32, 49). The hybridization pattern, obtained in the
Arc with CNTF, overlaps with those regions expressing neuropeptide Y
(NPY), POMC, cocaine- and amphetamine-regulated transcript (CART), and
agouti-related protein (AGRP), all of which are peptides involved in
regulation of feeding that are regulated by leptin in vivo
(53, 54, 55, 56, 57, 58).
CNTF activates SOCS-3 mRNA and SOCS-3 protein in astrocytes and
forced expression of SOCS-3 inhibits CNTF-induced signal
transduction
CNTF has been shown to activate STAT proteins in neuronal,
cortical precursors, and glial cells (13, 59, 60, 61, 62). Furthermore,
activation of STAT proteins is thought to be necessary for induction of
CIS and SOCS mRNA levels (39, 40, 41, 42). To demonstrate that CNTF has the
capability to directly induce socs-3 gene expression, we
used a CNTF-responsive astrocyte cell line. We first measured
activation of the JAK-STAT pathway in the astrocytes using an EMSA
specific for activated STAT1 and STAT3 (48). As shown in Fig. 3A, CNTF induced robust DNA binding
activities of STAT1 and STAT3 hetero- and homodimers. As determined by
Northern blot analysis of RNA isolated from astrocytes, CNTF treatment
resulted in strong induction of SOCS-3 mRNA (Fig. 3B). Furthermore,
CNTF treatment of these cells generated a robust increase in cellular
SOCS-3 protein levels, as determined by Western blotting of SOCS-3
immunoprecipitates (Fig. 3C). Leptin did not activate STAT DNA binding
activity or induce SOCS-3 mRNA in these cells (data not shown), most
likely due to lack of leptin receptor expression, since we did not
detect specific 125I-leptin binding to the cells (data not
shown).
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, SOCS-3 (but not CIS or SOCS-2)
inhibited CNTF-induced activation of a STAT-responsive luciferase
reporter construct in transfected astrocytes. We also observed that
baseline promoter activities were reduced by forced expression of
SOCS-3 in these cells. The reason for this result is unclear, but
others studying SOCS-1 signaling have published similar results on
baseline promoter activity (41).
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), but no effects on CIS,
SOCS-1, or SOCS-2 mRNAs were detected. In kidney, CNTF had a strong
effect on both SOCS-2 and SOCS-3 mRNA levels (Fig. 5). In the liver,
however, all the tested genes, including cis,
socs-1, socs-2, and socs-3, were
induced after CNTF treatment (Fig. 5). Leptin did not affect CIS or
SOCS mRNA levels in cerebellum, in kidney, or in liver, suggesting that
these tissues do not express significant levels of the long form of the
leptin receptor.
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Our data also suggest that SOCS-3 is a CNTF-inducible inhibitor of CNTF signaling. Leptin and GH also preferentially induced SOCS-3 mRNA after hormone administration to rodents (44, 45), and forced expression of SOCS-3 in mammalian transfection models completely blocked leptin- (45), GH- (44), and LIF-induced (43) signal transduction. These and other data suggest that CIS and SOCS family members are acting as negative feedback regulators of cytokine signaling (41, 42). However the exact mechanism by which this occurs is presently unclear. SOCS proteins are thought to bind to JAK isoforms and, by an unknown mechanism, prevent JAK from phosphorylating the receptor and STAT proteins that interact with phosphorylated tyrosine residues on the receptor (41, 42). In contrast, CIS seems to associate directly with phosphorylated receptor sites, possibly preventing STAT proteins from binding to these sites (39, 67). In addition, CIS does not affect erythropoietin- or IL-3 receptor phosphorylation (39). A recent paper suggests that SOCS-2 may interact directly with the insulin-like growth factor-1 receptor and possibly regulate its function (68). These results suggest that the function of SOCS proteins may not be restricted to inhibition of cytokine receptors. Because the leptin receptor, GH receptor, and LIFR all activate JAK2 activity and SOCS-3 mRNA and belong to the same subfamily of cytokine-receptors (43, 44, 45, 69, 70, 71, 72, 73), it is possible that SOCS-3 may have some specificity for inhibition of JAK2 and that SOCS-3 acts in a similar manner to inhibit CNTF, LIF, GH, and leptin signaling.
In the brain, dense SOCS-3 hybridization signals were detected in the
median eminence and the subfornical organ after CNTF administration. In
addition, robust signals were seen in the ependymal lining of all
ventricles. These results raise the possibility that CNTF may reach the
cerebrospinal fluid, possibly via diffusion through the fenestrated
vessels in the circumventricular organs. Cells within the
circumventricular organs are clearly also highly responsive to
peripheral administration of CNTF. Once in the cerebrospinal fluid,
CNTF may induce SOCS-3 mRNA expression by activation of CNTF receptors
expressed on the ependymal cells, which comprise the lining of the
ventricles. Alternatively, once in the cerebrospinal fluid, CNTF may
bind to soluble CNTFR proteins, which have been reported to be
present at this site (74). The soluble CNTF-CNTFR complex may then
act on cells that normally do not respond to CNTF (2). Lack of specific
hybridization signals in the ependymal lining after leptin treatment
may be attributable to low expression of the long form of the leptin
receptors at this site or may be a consequence of limited transport of
leptin into the cerebrospinal fluid. Our SOCS-3 in situ
experiments suggest that a significant number of the cells activated by
CNTF in the Arc are neurons, a result which is consistent with data
demonstrating expression of CNTFR in neurons (11, 12). However, our
results from the astrocyte cell line suggest that some SOCS-3-positive
cells in this region may be of glial origin. In addition, these results
are also consistent with the possibility that nonneuronal cells
comprising the ependymal lining of the ventricles and glial populations
within the circumventricular organs are direct targets of peripherally
administered CNTF.
A recent study used in situ hybridization to examine changes
in the tis-11 early-response gene in the hypothalamus of
mice after peripheral administration of CNTF and leptin (13). The
hypothalamic distribution of tis-11 mRNA was similar after treatment
with CNTF and leptin. In contrast to the induction of SOCS-3 mRNA by
CNTF in the ependymal lining, the circumventricular organs, and the
arcuate hypothalamic nucleus observed in the present study, tis-11 mRNA
was only detected in the Arc after CNTF administration. The SOCS-3
hybridization pattern seen after leptin treatment overlapped with the
expression pattern of the long form of the leptin receptor, including
strong SOCS-3 hybridization signals in the Arc and the dorsomedial
hypothalamus, suggesting a direct action of leptin on these regions
(45). In addition to detecting expression of CNTF receptor mRNA in
the arcuate, Gloaguen et al. (13) also reported expression
of CNTF receptor mRNA in the paraventricular hypothalamus. We did
not detect stimulation of socs-3 gene expression in the
paraventricular hypothalamus by CNTF, suggesting that CNTF receptors
were not activated in this region. Using immunohistochemistry for Fos,
another immediate early gene product, we have reported
leptin-induced Fos activation in hypothalamic regions, including
the dorsomedial hypothalamus (75, 76). CNTF treatment did not produce
activation of cells in the dorsomedial hypothalamus but did activate
regions of the Arc in a similar manner to leptin. These data suggest
that the effects of these two peptides on body weight involve both
overlapping and distinct hypothalamic neuronal circuits. This, in part,
may be explained by differences between the ability of CNTF and leptin
to gain access to different regions of the hypothalamus.
Systemic administration of CNTF to ob/ob and db/db mice reduces their food intake and body weight (13). These data suggest that some pathways that are activated by CNTF in the central nervous system are parallel to pathways activated by leptin. Based on our data, it is possible that this effect of CNTF occurs via CNTF receptors expressed in neurons that also express the long form of the leptin receptor, thereby regulating one or more neuropeptides that are regulated by leptin. CNTF has also been shown to affect body weight in mice rendered obese by being fed a high-fat diet (13). These mice were leptin resistant, further demonstrating that CNTF can bypass leptin resistance and supporting the possibility that CNTF acts on pathways similar to those activated by leptin. We have suggested earlier that elevated activity of SOCS-3 in the hypothalamus may play a role in central leptin resistance (45). This hypothesis may contradict the above results and those presented in this paper, because increased protein levels or activity of SOCS-3, under conditions of leptin resistance, might inhibit both leptin and CNTF signaling in neurons expressing both leptin and CNTF receptors. However, several possibilities might explain these findings. First, it is yet unknown whether CNTF receptors are expressed on leptin receptor-positive neurons. It is possible that CNTF receptors are expressed on subpopulations of NPY, POMC, AGRP, or CART neurons that do not express leptin receptors. Additional anatomic double-labeling studies are clearly needed to address this question. Second, it is not known whether leptin receptor-induced SOCS-3 proteins, under conditions of leptin-resistant obesity, can inhibit CNTF receptors in the same cell. Possible cross-talk between leptin and CNTF receptor systems at the level of SOCS-3 could be addressed in vivo by prior pretreatment of rodents with CNTF or leptin, to see if subsequent leptin or CNTF signaling were attenuated. Third, SOCS-3 activity may not be elevated in hypothalamic nuclei in leptin-resistant states, including diet-induced obesity in rodents.
In peripheral tissues, including kidney and liver, CNTF also induced
robust activation of SOCS-3 mRNA. However, in these tissues, we also
detected strong increases in the mRNA levels of several other
socs genes. In particular, CNTF activated CIS, SOCS-1,
SOCS-2, and SOCS-3 mRNA in liver tissue. It is unclear how CNTF
activates several different cis and socs genes in
a tissue-dependent manner. CNTF receptors have been found in
skeletal muscle (2), and some of the effects observed might therefore
be caused by direct actions of CNTF on peripheral tissues.
Alternatively, it is also possible that administered CNTF might bind to
soluble CNTFR components released from skeletal muscle and then act
on diverse cell types that normally do not respond to CNTF because of
lack of CNTFR (2, 74). Finally, CNTF may induce expression of
other cytokine-like factors, which act through different receptors to
induce other SOCS isoforms. Our result that CIS and SOCS-2 mRNA were
induced in liver tissue but neither CIS nor SOCS-2 had any effect on
CNTF signaling, may support this possibility. Recently, several new
members of the socs gene family have been cloned (43, 77).
This raises the possibility that one or more of these genes may be
activated by CNTF or leptin in the central nervous system and/or in the
periphery, and that these gene products could also play a role in
attenuating CNTF and leptin signal transduction.
Several lines of evidence suggest that CNTF may play an important role in the response to injury in the central nervous system (2). Because the expression of CNTF has been shown to increase in the region of a mechanically lesioned site in the nervous system (8), it is possible that SOCS-3 mRNA and SOCS-3 protein may also be induced locally in cells that are CNTF responsive. SOCS-3 protein may thus act as a negative modulator of this response. Further studies are clearly needed to address these issues.
In conclusion, we have demonstrated that induction of SOCS-3 mRNA occurs rapidly, after peripheral CNTF administration, in regions of the hypothalamus that are known to be involved in regulation of body weight. We also show that SOCS-3 is a negative regulator of CNTF signal transduction. Inhibition of SOCS-3 protein expression or function is a potential target for development of drugs aimed at enhancing CNTF activity, either after neuronal injury or in combination with CNTF administration aimed at reducing body weight.
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Acknowledgments |
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Footnotes |
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Received September 29, 1998.
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component as a soluble mediator of CNTF responses. Science 259:1736–1739
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 P. Trayhurn and C. Bing Appetite and energy balance signals from adipocytes Phil Trans R Soc B, July 29, 2006; 361(1471): 1237 - 1249. [Abstract] [Full Text] [PDF]
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 E. Papathanassoglou, K. El-Haschimi, X. C. Li, G. Matarese, T. Strom, and C. Mantzoros Leptin receptor expression and signaling in lymphocytes: kinetics during lymphocyte activation, role in lymphocyte survival, and response to high fat diet in mice. J. Immunol., June 15, 2006; 176(12): 7745 - 7752. [Abstract] [Full Text] [PDF]
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 B. Emery, H. S. Cate, M. Marriott, T. Merson, M. D. Binder, C. Snell, P. Y. Soo, S. Murray, B. Croker, J.-G. Zhang, et al. Suppressor of cytokine signaling 3 limits protection of leukemia inhibitory factor receptor signaling against central demyelination PNAS, May 16, 2006; 103(20): 7859 - 7864. [Abstract] [Full Text] [PDF]
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 M. V. Kokoeva, H. Yin, and J. S. Flier Neurogenesis in the Hypothalamus of Adult Mice: Potential Role in Energy Balance Science, October 28, 2005; 310(5748): 679 - 683. [Abstract] [Full Text] [PDF]
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 J. W. Bullen Jr, M. Ziotopoulou, L. Ungsunan, J. Misra, I. Alevizos, E. Kokkotou, E. Maratos-Flier, G. Stephanopoulos, and C. S. Mantzoros Short-term resistance to diet-induced obesity in A/J mice is not associated with regulation of hypothalamic neuropeptides Am J Physiol Endocrinol Metab, October 1, 2004; 287(4): E662 - E670. [Abstract] [Full Text] [PDF]
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 J. F. Kelly, C. F. Elias, C. E. Lee, R. S. Ahima, R. J. Seeley, C. Bjorbaek, T. Oka, C. B. Saper, J. S. Flier, and J. K. Elmquist Ciliary Neurotrophic Factor and Leptin Induce Distinct Patterns of Immediate Early Gene Expression in the Brain Diabetes, April 1, 2004; 53(4): 911 - 920. [Abstract] [Full Text] [PDF]
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R. Pal and A. Sahu Leptin Signaling in the Hypothalamus during Chronic Central Leptin Infusion Endocrinology, September 1, 2003; 144(9): 3789 - 3798. [Abstract] [Full Text] [PDF]
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C. J. Auernhammer, N. B. Isele, F. B. Kopp, G. Spoettl, N. Cengic, M. M. Weber, G. Senaldi, and D. Engelhardt Novel Neurotrophin-1/B Cell-Stimulating Factor-3 (Cardiotrophin-Like Cytokine) Stimulates Corticotroph Function via a Signal Transducer and Activator of Transcription-Dependent Mechanism Negatively Regulated by Suppressor of Cytokine Signaling-3 Endocrinology, April 1, 2003; 144(4): 1202 - 1210. [Abstract] [Full Text] [PDF]
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 C. J. Greenhalgh and D. J. Hilton Negative regulation of cytokine signaling J. Leukoc. Biol., September 1, 2001; 70(3): 348 - 356. [Abstract] [Full Text] [PDF]
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A. W. Roberts, L. Robb, S. Rakar, L. Hartley, L. Cluse, N. A. Nicola, D. Metcalf, D. J. Hilton, and W. S. Alexander Placental defects and embryonic lethality in mice lacking suppressor of cytokine signaling 3 PNAS, July 31, 2001; 98(16): 9324 - 9329. [Abstract] [Full Text] [PDF]
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S. P. Kalra Circumventing leptin resistance for weight control PNAS, April 10, 2001; 98(8): 4279 - 4281. [Full Text] [PDF]
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A. Colson, A. Le Cam, D. Maiter, M. Edery, and J.-P. Thissen Potentiation of Growth Hormone-Induced Liver Suppressors of Cytokine Signaling Messenger Ribonucleic Acid by Cytokines Endocrinology, October 1, 2000; 141(10): 3687 - 3695. [Abstract] [Full Text] [PDF]
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 C. J. Auernhammer and S. Melmed Leukemia-Inhibitory Factor--Neuroimmune Modulator of Endocrine Function Endocr. Rev., June 1, 2000; 21(3): 313 - 345. [Abstract] [Full Text]
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B. Emanuelli, P. Peraldi, C. Filloux, D. Sawka-Verhelle, D. Hilton, and E. Van Obberghen SOCS-3 Is an Insulin-induced Negative Regulator of Insulin Signaling J. Biol. Chem., May 19, 2000; 275(21): 15985 - 15991. [Abstract] [Full Text] [PDF]
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 D. Krebs and D. Hilton SOCS: physiological suppressors of cytokine signaling J. Cell Sci., January 8, 2000; 113(16): 2813 - 2819. [Abstract] [PDF]
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C. Bjorbak, K. El-Haschimi, J. D. Frantz, and J. S. Flier The Role of SOCS-3 in Leptin Signaling and Leptin Resistance J. Biol. Chem., October 15, 1999; 274(42): 30059 - 30065. [Abstract] [Full Text] [PDF]
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C. Bjorbak, H. J. Lavery, S. H. Bates, R. K. Olson, S. M. Davis, J. S. Flier, and M. G. Myers Jr. SOCS3 Mediates Feedback Inhibition of the Leptin Receptor via Tyr985 J. Biol. Chem., December 15, 2000; 275(51): 40649 - 40657. [Abstract] [Full Text] [PDF]
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S. E. Nicholson, D. De Souza, L. J. Fabri, J. Corbin, T. A. Willson, J.-G. Zhang, A. Silva, M. Asimakis, A. Farley, A. D. Nash, et al. Suppressor of cytokine signaling-3 preferentially binds to the SHP-2-binding site on the shared cytokine receptor subunit gp130 PNAS, June 6, 2000; 97(12): 6493 - 6498. [Abstract] [Full Text] [PDF]
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