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Endocrinology Vol. 140, No. 5 2035-2043
Copyright © 1999 by The Endocrine Society

ARTICLES

Activation of SOCS-3 Messenger Ribonucleic Acid in the Hypothalamus by Ciliary Neurotrophic Factor1

Christian Bjørbæk, Joel K. Elmquist, Karim El-Haschimi, Joseph Kelly, Rexford S. Ahima, Stanley Hileman and Jeffrey S. Flier

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ciliary neurotrophic factor (CNTF) is a neurocytokine expressed in glial cells that acts on brain cells to promote gene expression, survival, and differentiation. When administered systemically, CNTF reduces food intake and body weight in rodents. Genes encoding suppressors of cytokine signaling (SOCS) are induced by cytokines that activate membrane receptors in the same class as those that are activated by CNTF. We therefore examined the ability of CNTF to induce expression of socs genes in brain and peripheral tissues of rats and mice. Peripheral CNTF administration to ob/ob mice rapidly induced SOCS-3 messenger RNA (mRNA) in hypothalamus, as determined by Northern blotting and quantitative RT-PCR, but had no effect on cytokine-inducible sequence (CIS), SOCS-1, or SOCS-2 mRNA. In situ hybridization histochemistry of hypothalamus from ob/ob mice and normal rats demonstrated that CNTF induced SOCS-3 mRNA in the arcuate nucleus (Arc). Strong hybridization signals were also detected in the ependymal lining of the ventricles and the subfornical organ. This hybridization pattern was distinct from that resulting from peripheral leptin treatment with overlapping hybridization patterns only in the Arc. CNTF also induced expression of CIS, SOCS-1, SOCS-2, and SOCS-3 mRNA in the liver, and SOCS-2 and SOCS-3 mRNA in the kidney. CNTF induced SOCS-3 mRNA and SOCS-3 protein levels in an astrocyte cell line. Transient expression of SOCS-3, but not CIS or SOCS-2, inhibited CNTF-induced signal transduction in astrocytes. In conclusion, SOCS-3 mRNA is specifically induced by CNTF in regions of the hypothalamus that are both overlapping and distinct from that induced by leptin. Similar to leptin, the Arc is likely to be a direct target of CNTF, and this region may play a role in the body weight-reducing effects of CNTF. SOCS-3 is a negative regulator of CNTF signal transduction, and inhibitors of SOCS-3 function may enhance endogenous CNTF signaling after neuronal injury or enhance the body weight-reducing effect of CNTF after peripheral administration.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CILIARY NEUROTROPHIC factor (CNTF) is a neurocytokine normally expressed in glial cells that acts on both neural and nonneural cells to promote gene expression, survival, and differentiation (1, 2). CNTF was initially identified as a trophic factor that induced survival of embryonic ciliary ganglion neurons (3), but it was later shown to belong to the superfamily of cytokines that includes interleukin (IL)-6, leukemia inhibitory factor (LIF), and leptin (4, 5, 6, 7). The expression of CNTF messenger RNA (mRNA) has been shown to increase in the region of a mechanically lesioned site in the nervous system (8). This evidence suggests that CNTF may play an important role in the response to neuronal injury (2). CNTF activates intracellular signal transduction by binding to a membrane-bound heterotrimeric receptor complex consisting of a ligand-specific subunit (CNTFR{alpha}) (9), the leukemia inhibitory factor receptor subunit, and gp130 (10). The CNTF{alpha} receptor subunit is expressed throughout the brain (9, 11, 12), and specific localization of CNTFR{alpha} 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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant mouse leptin and human CNTF were obtained from Eli Lilly & Co. (Indianapolis, IN). The immortalized human astrocytes were a kind gift from Dr. A. Muruganandam and Dr. D. Stanimirovic, Institute for Biological Sciences, National Research Council, Canada. Mammalian expression vectors for murine CIS, SOCS-2, and SOCS-3 (45) were kind gifts from Dr. J. D. Frantz and Dr. S. E. Shoelson (Joslin Diabetes Center, Boston, MA). The CMV-lacZ reporter construct was from CLONTECH Laboratories, Inc. (Palo Alto, CA). The {gamma}-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-{alpha}-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-{alpha}-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 {alpha}-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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Activation of SOCS-3 mRNA by CNTF and leptin in hypothalamus from ob/ob mice
Ad libitum-fed male ob/ob mice, 7–8 weeks old, were injected ip with 10 µg recombinant human CNTF, or 100 µg recombinant mouse leptin, or saline. Two hours later, total RNA was purified from hypothalami; and quantitative 32P-RT-PCR for CIS, SOCS-1, SOCS-2, and SOCS-3 mRNAs was performed. CNTF treatment resulted in a robust increase of SOCS-3 mRNA in the hypothalamus, as shown in Fig. 1AGo. This was confirmed by Northern blot analysis (Fig. 1BGo). No effects on CIS, SOCS-1, or SOCS-2 mRNA were detected in this tissue (Fig. 1AGo). 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. 1AGo). 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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  		Figure 1. Activation of SOCS-3 mRNA in hypothalamus of ob/ob mice after CNTF or leptin administration. Ob/ob mice were given a single ip injection of 10 µg recombinant human CNTF or 100 µg recombinant murine leptin or saline. Hypothalami were isolated 2 h after injection. A, Autoradiograms of 32P-labeled RT-PCR products of CIS, SOCS-1, SOCS-2, and SOCS-3 mRNA, amplified under conditions of limiting numbers of cycles. Each lane represents one mouse. B, Northern blot analysis of SOCS-3 mRNA in hypothalami from ob/ob mice treated with saline or CNTF, as described above. Equal amounts (10 µg) of total RNA were loaded onto the denaturing agarose gel. Each lane represents one mouse.

 
SOCS-3 mRNA is differentially activated by CNTF and leptin in the hypothalamus
To identify and compare specific regions of the hypothalamus in which CNTF and leptin induces SOCS-3 mRNA, we performed in situ hybridization histochemistry. In normal rats given a single iv injection of recombinant human CNTF (0.25 µg/g BW), a marked increase in specific hybridization was detected in the Arc, compared with saline-injected rats (Fig. 2Go, 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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  		Figure 2. Distribution of SOCS-3 mRNA in rat hypothalamus by in situ hybridization histochemistry after iv CNTF or leptin administration. Shown are dark-field photomicrographs of emulsion-dipped slides of rat hypothalamic sections hybridized with SOCS-3 antisense 35S-labeled RNA probes, 1 h after pyrogen-free saline (PFS) (A) or CNTF (0.25 µg/g; B) or leptin (1.0 µg/g; C) administration. cDMH, Caudal DMH; 3v, third ventricle. Bar, 0.50 mm. Similar hybridization patterns were also obtained in ob/ob mice after peripheral CNTF or leptin administration (data not shown). We did not detect any specific hybridization signals using SOCS-3 sense RNA probes (data not shown) (45 ).

 
The SOCS-3 hybridization pattern obtained after CNTF treatment was different from that obtained after leptin treatment (Fig. 2CGo). 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. 3AGo, 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. 3BGo). 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. 3CGo). 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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  		Figure 3. CNTF activates STAT DNA binding activities and induces SOCS-3 mRNA and SOCS-3 protein levels in astrocytes. Human immortalized astrocytes were grown to confluence and serum-deprived 12 h before stimulation with hormones. A, Activation of STAT DNA binding activity by CNTF, as demonstrated by EMSA. Cells were stimulated with nothing or with CNTF (0.1 µg/ml) for 15 min. Nuclear extracts were isolated, and EMSAs were performed as described in Materials and Methods. A-, B-, and C-arrows indicate STAT3/STAT3, STAT3/STAT1, and STAT1/STAT1 dimers, respectively (28 48 ). B, CNTF induces SOCS-3 mRNA, as demonstrated by Northern blotting. Cells were stimulated with nothing or with CNTF (0.1 µg/ml) for 45 min. Total RNA was isolated, and 15 µg from each sample were subjected to Northern blot analysis with a 32P-labeled mouse SOCS-3 DNA probe. C, CNTF induces SOCS-3 protein, as demonstrated by Western blotting. Astrocytes were stimulated or not with CNTF (0.1 µg/ml) for 60 min. Clarified lysates were subjected to immunoprecipitation with SOCS-3 antiserum and were detected by Western blotting using affinity-purified anti-SOCS-3 antibodies. All experiments were performed twice.

 
CIS and SOCS proteins are thought to act as negative regulators of cytokine signaling (39, 40, 41, 42, 43, 44, 45). To determine the effects of SOCS-3 on CNTF signaling, we examined the effect of SOCS-3 on CNTF-induced transcription in astrocytes. As shown in Fig. 4Go, 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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  		Figure 4. SOCS-3 is an inhibitor of CNTF-induced signal transduction. Astrocytes were grown in 6-well dishes and were transfected with GAS-luc and CMV-lacZ reporter plasmids, together with empty vector (Vec), CIS, SOCS-2, or SOCS-3 expression vectors, as described in Materials and Methods. Cells were treated or not with 0.1 µg/ml CNTF for 5 h. Luciferase activities were measured as described in Materials and Methods. Differences in transfection efficiency were eliminated by normalizing luciferase activities to ß-galactosidase activities from the same samples. The ß-galactosidase activities did not vary with CNTF treatment. This experiment was performed twice, each in triplicate. Shown is the mean ± SE of one experiment.

 
CNTF activates multiple SOCS and CIS isoforms in cerebellum, kidney, and liver
We next tested whether peripherally administered CNTF acted on other regions of the brain or on peripheral tissues. In the cerebellum, CNTF specifically induced SOCS-3 mRNA, as determined by quantitative RT-PCR (Fig. 5Go), 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. 5Go). In the liver, however, all the tested genes, including cis, socs-1, socs-2, and socs-3, were induced after CNTF treatment (Fig. 5Go). 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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  		Figure 5. Activation of CIS and SOCS mRNAs in cerebellum, kidney, and liver of ob/ob mice after CNTF or leptin administration. RT-PCR of CIS, SOCS-1, SOCS-2, and SOCS-3 mRNA, isolated from various tissues from ob/ob mice given a single ip injection of 10 µg recombinant human CNTF or 100 µg recombinant murine leptin or saline. Tissues were isolated 2 h after injection. Shown are autoradiograms of 32P-labeled RT-PCR products amplified from RNA isolated from cerebellum, kidney, and liver, under conditions of limiting numbers of cycles. Each lane represents one mouse.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated that peripheral administration of recombinant CNTF to normal rats and ob/ob mice induces SOCS-3 mRNA in specific areas of the hypothalamus. In addition to the circumventricular organs and the ependymal lining of the ventricles, in situ hybridization of brain sections from CNTF-treated rats detected significant SOCS-3 mRNA expression in the Arc of the hypothalamus. The region of the Arc expressing SOCS-3 mRNA overlaps with that expressing several neuropeptides involved in regulation of body weight, including NPY, POMC, AGRP, and CART (57, 58, 63, 64). In addition, SOCS-3 mRNA is also induced in this nucleus by leptin (45), which is known to regulate expression of each of these neuropeptides in vivo (53, 54, 55, 56, 57, 58). These results are consistent with the possibility that the anorexia noted after peripheral injection of CNTF into animals and humans (13, 17, 65) may involve regulation of one of more of these neuropeptides. Indeed, a recent paper suggests that the anorexia produced by CNTF in rats involves suppression of NPY mRNA in the hypothalamus (66).

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{alpha} proteins, which have been reported to be present at this site (74). The soluble CNTF-CNTFR{alpha} 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{alpha} 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{alpha} receptor mRNA in the arcuate, Gloaguen et al. (13) also reported expression of CNTF{alpha} 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{alpha} 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{alpha} components released from skeletal muscle and then act on diverse cell types that normally do not respond to CNTF because of lack of CNTFR{alpha} (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.


   Acknowledgments
 
We thank Dr. A. Muruganandam and Dr. D. Stanimirovic, Institute for Biological Sciences, National Research Council, Canada, for providing the human astrocyte cell line. Special thanks to Dr. Dan Frantz, Dr. Ron Shigeta, and Dr. Steven Shoelson (Joslin Diabetes Center) for providing the purified SOCS-3 protein and for the expression vectors encoding CIS, SOCS-2, and SOCS-3.


   Footnotes
 
1 This work was supported by NIH Grant DK-R37–28082 and a grant from Eli Lilly & Co. (to J.S.F.) and NIH Grant MH-56537 (to J.K.E.). Back

Received September 29, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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