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J Biol Chem, Vol. 274, Issue 42, 30059-30065, October 15, 1999


The Role of SOCS-3 in Leptin Signaling and Leptin Resistance*

Christian BjørbækDagger §, Karim El-HaschimiDagger §, J. Daniel Frantz, and Jeffrey S. FlierDagger parallel

From the Dagger  Department of Medicine, Division of Endocrinology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215 and  Joslin Diabetes Center and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We earlier demonstrated that leptin induces expression of SOCS-3 mRNA in the hypothalamus. Furthermore, transfection data suggest that SOCS-3 is an inhibitor of leptin signaling. However, little is known about the regulation of SOCS-3 expression by leptin and the mechanism by which SOCS-3 inhibits leptin action. We here show that in CHO cells stably expressing the long form of the leptin receptor (CHO-OBRl), leptin induces transient expression of endogenous SOCS-3 mRNA but not of CIS, SOCS-1, or SOCS-2 mRNA. SOCS-3 protein levels were maximal after 2-3 h of leptin treatment and remained elevated at 20 h. Furthermore, in leptin-pretreated CHO-OBRl cells, proximal leptin signaling was blocked for more than 20 h after pretreatment, thus correlating with increased SOCS-3 expression. Leptin pretreatment did not affect cell surface expression of leptin receptors as measured by 125I-leptin binding assays. In transfected COS cells, forced expression of SOCS-3 results in inhibition of leptin-induced tyrosine phosphorylation of JAK2. Finally, JAK2 co-immunoprecipitates with SOCS-3 in lysates from leptin-treated COS cells. These results suggest that SOCS-3 is a leptin-regulated inhibitor of proximal leptin signaling in vivo. Excessive SOCS-3 activity in leptin-responsive cells is therefore a potential mechanism for leptin resistance, a characteristic feature in human obesity.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Leptin is a 16-kDa hormone derived from adipose tissue that acts on specific regions of the brain to regulate food intake, energy expenditure, and neuroendocrine function (1-5). Leptin is structurally related to cytokines (6) and acts on receptors that belong to the cytokine receptor superfamily (7). Several different leptin receptor isoforms exists including a long form (OBRl), which is highly expressed in regions of the hypothalamus (8-10). In vitro and in vivo studies demonstrate that leptin activates cytokine-like signal transduction via the long form of the leptin receptor (9, 11, 12). Upon leptin stimulation, intracellular Janus tyrosine kinases (JAKs) are activated via transphosphorylation and phosphorylate tyrosine residues on the long form leptin receptor and on signal transducers and activators of transcription (STAT)1 proteins (13, 14). Phosphorylated STAT proteins dimerize and translocate to the nucleus to activate gene transcription (15, 16). Lack of functional leptin in lepob/lepob mice or of the intracellular domain of the long form of the leptin receptor in db/db mice produces severe obesity (1, 8, 17). Although rare cases with mutations in the leptin and the leptin receptor genes causing extreme obesity in humans have been described (18, 19), most humans with obesity have resistance to leptin that has yet to be explained. Potential mechanisms for leptin resistance include defects in transport of leptin across the blood brain barrier, defects in leptin signal transduction in leptin receptor-expressing neurons in the hypothalamus, and antagonism of leptin's physiologic actions at one or more steps beyond the initial leptin-responsive neurons.

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 (20-23). CIS and SOCS are relatively small proteins containing a central SH2 domain and a conserved ~40-amino acid-long C-terminal SOCS-box (24). The SH2 domain of SOCS is thought to bind to phosphorylated tyrosine residues on JAK proteins (22, 25), while the SOCS-box may play a role in preventing degradation of SOCS proteins (25, 26). Members of the cytokine superfamily including leptin, interleukin-6, interferon-gamma , leukemia-inhibitory factor, erythropoietin (EPO), and growth hormone induce transcription of one or more of the cis or socs genes in vivo and in vitro, and when expressed in cell lines, CIS and SOCS proteins inhibit signaling and biological activities of cytokines (20, 21, 27-31). These results suggest that CIS and SOCS proteins can function as inducible intracellular negative regulators of cytokine signal transduction.

We have earlier demonstrated that leptin specifically induces expression of SOCS-3 mRNA in regions of the hypothalamus that express the long form of the leptin receptor (29). In addition, forced expression of SOCS-3 blocks leptin receptor-mediated signal transduction in mammalian cell lines. Furthermore, in the Agouti mouse, a model characterized by hyperleptinemia and resistance to both central and peripheral leptin administration, basal SOCS-3 mRNA levels are increased in those hypothalamic nuclei that express SOCS-3 in normal animals after leptin administration (29). We have thus identified a potential negative feedback circuit connecting peripheral leptin to expression of an inhibitor of leptin signaling in leptin-responsive hypothalamic neurons.

Little is known, however, about how leptin regulates SOCS-3 expression and by what mechanism SOCS-3 inhibits leptin signal transduction. We therefore examined the regulation and function of endogenous SOCS-3 in CHO cells stably expressing the long form of the leptin receptor. We found that leptin induces SOCS-3 mRNA and SOCS-3 protein expression in CHO-OBRl cells. Furthermore, brief leptin pretreatment of CHO-OBRl cells induces subsequent leptin resistance at a proximal leptin-signaling step, which correlates with increased SOCS-3 protein expression. In transfected COS cells, leptin induces association of SOCS-3 with JAK2, and forced expression of SOCS-3 attenuates leptin-induced JAK2 tyrosine phosphorylation. These results are consistent with SOCS-3 playing an important role in negative regulation of proximal leptin signal transduction in vivo.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Recombinant mouse leptin was obtained from Lilly. 125I-Leptin was purchased from NEN Life Science Products. Mammalian expression vectors for double-HA-tagged murine CIS, SOCS-2, and SOCS-3 (29) were kind gifts from Dr. J. D. Frantz and Dr. S. E. Shoelson (Joslin Diabetes Center, Boston). The expression vectors encoding murine long leptin receptor and JAK2 were obtained as described earlier (14). 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). All reagents for cell culture and transfection were from Life Technologies, Inc. The JAK2 and phosphotyrosine (4G10) antibodies were from Upstate Biotechnology, Inc. (Lake Placid, NY). The leptin receptor antibody was generated as described by Bjørbæk et al. (29). The monoclonal HA antibody (12CA5) was from Roche Molecular Biochemicals. TNF-alpha was purchased from Sigma.

Cell Culture and Transient Transfection-- CHO cells stably expressing murine long (OBRl) or short (OBRs) form leptin receptors were generated as described earlier by Bjørbæk et al. (14). Cells were grown in Ham's F-12 medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 10 µg/ml streptomycin at 37 °C in 5% CO2. COS cells were grown in Dulbecco's modified Eagle's medium (low glucose) supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 10 µg/ml streptomycin at 37 °C in 5% CO2. All cells were serum-deprived for 12-15 h prior to stimulation with hormones. For Western blotting experiments, cells were grown in 10-cm dishes. COS cells were transfected with a total of 20 µg of plasmid DNA using 80 µl of LipofectAMINE. Cells were harvested by rinsing in ice-cold phosphate-buffered saline and scraping into 1.0 ml of ice-cold lysis buffer A (1% Nonidet P-40, 0.5% Triton X-100, 10% glycerol, 150 mM NaCl, 2 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 50 mM Tris-HCl, pH 7.4). Lysates were clarified by centrifugation at 23,000 × g for 15 min, and supernatants were immunoprecipitated as described below. For 125I-leptin binding assays, CHO cells were grown in six-well plates. Prior to tracer binding assays, COS cells were grown in six-well plates and transfected with a total of 2.0 µg of plasmid DNA using 10 µl of LipofectAMINE per well.

Immunoprecipitation and Immunoblotting-- Immunoprecipitations were performed as described earlier by Bjørbæk et al. (14). Briefly, clarified lysates were incubated at 4 °C with antibodies, 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 A, the samples were subjected to SDS-polyacrylamide gel electrophoresis. 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 with antibodies, nitrocellulose membranes were washed, and targeted proteins were detected using ECL as described by the manufacturer (Amersham Pharmacia Biotech).

Nuclear Extraction and Electrophoretic Mobility Shift Assay (EMSA)-- Nuclear extractions were done as described earlier (32, 33). Briefly, cells were grown to near confluence and serum-deprived 12-15 h prior to stimulation with hormones. After treatment, cells were rinsed once with 2 ml of ice-cold Tris-buffered saline (TBS) and then scraped into 1.0 ml of ice-cold TBS, transferred to a 1.5-ml Eppendorf tube, and pelleted by centrifugation at 1500 × g at 4 °C for 5 min. The pellets were then resuspended in 400 µl of ice-cold buffer C (40 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) by gentle pipetting in a yellow tip. The cells were allowed to swell on ice for 15 min, after which 25 µl of 10% Nonidet Nonidet P-40 were added, and the tube was vortexed for 10 s. Samples were then centrifuged for 30 s at 14,000 × g, and the nuclear pellets were resuspended in 25 µl of ice-cold buffer D (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) by vigorous rocking at 4 °C for 30 min. The nuclear extracts were finally clarified by centrifugation at 14,000 × g for 20 min and stored at -80 °C until further use. Five µg of nuclear extracts (determined by Bradford protein assay; Bio-Rad) were added to binding buffer (final volume of 20 µl; 13 mM HEPES, pH 7.9, 65 mM NaCl, 1 mM dithiothreitol, 0.15 mM EDTA, 8% glycerol, 50 mg/ml poly(dI-dC), and 0.01% Nonidet P-40), which included 100,000 cpm of the 32P-labeled double-stranded oligonucleotide probe, SIE-mutant 67 (33), and incubated for 15 min at room temperature. The probe was generated by annealing two oligonucleotides (5'-CGCTCCATTTCCCGTAAATCAT-3' and 5'-CGCTCATGATTTACGGGAAATG-3') followed by a fill-in reaction of the 5-base overhangs using T7 polymerase (Life Technologies) and [alpha -32P]dNTPs (each 222 TBq/mmol, 740 MBq/ml) (NEN Life Science Products). Unincorporated nucleotides were removed by using a G25 Quick Spin column (Roche Molecular Biochemicals). Samples were loaded onto a 5% nondenaturing polyacrylamide gel (39:1 acrylamide:bis) containing 2.5% glycerol in 0.5× 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 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 (Roche Molecular Biochemicals) using a vacuum system from Amersham Pharmacia Biotech. Membranes were then subjected to UV cross-linking and prehybridized for 1 h in QuickHyb solution (Stratagene, La Jolla, CA) at 68 °C. The CIS, SOCS-1 and SOCS-2 probes were DNA fragments of the entire coding regions of the genes. The SOCS-3 probe was a 450-base pair DNA fragment generated by reverse transcriptase-polymerase chain reaction using murine hypothalamic RNA as template. The probes were labeled with [alpha -32P]dCTP (222 TBq/mmol, 740 MBq/ml) (NEN Life Science Products) by random priming (Life Technologies), boiled for 5 min, and incubated with the membrane in 12 ml of QuickHyb solution at 68 °C for 15 h. Membranes were washed three times with 2× SSC, 0.1% SDS at room temperature and two times with 0.2× SSC, 0.1% SDS at 60 °C, and finally placed in a PhosphorImager cassette for 12-15 h.

125I-Leptin Binding Assays-- COS cells were transfected as described above, serum-deprived for 12-15 h, and incubated with 100,000 cpm of 125I-leptin in Dulbecco's modified Eagle's medium containing 0.1% of bovine serum albumin at 4 °C for 4 h, in the presence or absence of 200 nM unlabeled leptin. CHO-OBRl cells were grown to confluence in six-well plates, serum-deprived for 12-15 h, and treated or not treated with 50 nM leptin for 1 h. Some cells were then cooled to 4 °C and subjected to 125I-leptin binding as described below. Other cells were washed four times in warm F-12 medium and incubated at 37 °C for 1.5, 3, 6, and 24 h. Available cell surface leptin receptors were determined by incubation with 100,000 cpm of 125I-leptin in F-12 medium containing 0.1% of bovine serum albumin for 4 h at 4 °C to prevent internalization, in the presence or absence of 200 nM unlabeled leptin. COS and CHO cells were then washed four times with ice-cold binding medium and scraped into 1 ml of lysis buffer (1% Nonidet P-40, 0.5% Triton X-100, 1N NaOH). The radioactivity in the lysates was measured in a gamma -counter. Specific binding was determined by subtracting the radioactivity bound in the presence of 200 nM unlabeled leptin (nonspecific binding) from the radioactivity bound in the absence of 200 nM unlabeled leptin.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Induction of SOCS-3 mRNA by Leptin in CHO Cells Stably Expressing the Long Form of the Leptin Receptor-- We have previously demonstrated in rodents that peripheral injection of leptin induces SOCS-3 mRNA, but not CIS, SOCS-1, or SOCS-2 mRNA, in regions of the hypothalamus expressing the long form of the leptin receptor (29). To further study the regulation and function of SOCS-3 in relation to leptin signaling, we used mammalian cell lines expressing leptin receptors. We first tested the ability of leptin to induce endogenous CIS and SOCS mRNA in CHO cells stably expressing the long form of the leptin receptor (CHO-OBRl). As demonstrated by Northern blotting, leptin did stimulate SOCS-3 mRNA expression at 1 h but did not affect CIS, SOCS-1, or SOCS-2 mRNA levels in these cells at 1, 2, or 4 h (Fig. 1). The time course of SOCS-3 mRNA expression after leptin treatment was investigated further in CHO-OBRl cells (Fig. 2A). Leptin induced an ~5-fold increase in SOCS-3 mRNA at 30 and 60 min after treatment. The SOCS-3 mRNA levels returned to base line 2 h after treatment and remained at base line after 6 and 20 h of continuous leptin exposure. In addition, leptin had no effect on SOCS-3 mRNA levels in CHO cells stably expressing the short form of the leptin receptor (Fig. 2B).


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  		Fig. 1.   Leptin induces SOCS-3, but not CIS, SOCS-1, or SOCS-2, mRNA in CHO cells stably expressing the long form of the leptin receptor. Serum-deprived cells were stimulated with 100 nM leptin for 0, 1, 2, and 4 h. Total RNA was then isolated and subjected to Northern blot analysis as described under "Experimental Procedures." Ten µg of total RNA was loaded in each lane. The CIS and SOCS-1 probes did detect positive RNA controls on the same gels (not shown).


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  		Fig. 2.   Leptin induces SOCS-3 mRNA in CHO cells stably expressing the long, but not the short, isoform of the leptin receptor. A, Northern blot of SOCS-3 mRNA after stimulation of CHO-OBRl cells with leptin. Serum-deprived cells were stimulated with 100 nM leptin for different periods of time. Total RNA was isolated and subjected to Northern blot analysis for SOCS-3 mRNA expression as described under "Experimental Procedures." This experiment was performed twice. B, Northern blot of SOCS-3 mRNA from CHO-OBRs cells stimulated or not with 100 nM leptin for 60 min.

Leptin Induces SOCS-3 Protein Expression in CHO-OBRl Cells-- We first generated SOCS-3 antiserum as described under "Experimental Procedures." This antiserum specifically recognized SOCS-3 as determined by Western blotting of lysates from COS cells transiently transfected with SOCS-3 expression vectors (Fig. 3A), and this antibody did not cross-react with CIS or SOCS-2 proteins (data not shown). We next examined endogenous SOCS-3 protein expression after leptin treatment of CHO-OBRl cells. Cells were serum-deprived for 12-15 h and stimulated with leptin for various periods. As demonstrated by Western blotting using SOCS-3 antiserum of SOCS-3 immunoprecipitates, leptin treatment induced SOCS-3 protein expression by ~4-fold at 2-3 h (Fig. 3, B and C). Detectable base-line levels of SOCS-3 protein were seen in all experiments, consistent with the detectable base-line levels of SOCS-3 mRNA as shown above. At 20 h of leptin treatment, SOCS-3 protein levels were still elevated in the cells, although reduced as compared with the maximal levels seen at 2-6 h.


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  		Fig. 3.   Leptin induces SOCS-3 protein expression in CHO-OBRl cells. A, Western blot using SOCS-3 anti-serum of clarified lysates from COS cells transfected with empty vector or HA-tagged SOCS-3 expression vectors. B, time course of SOCS-3 protein expression in CHO-OBRl cells. CHO-OBRl cells were serum-deprived for 15 h and stimulated with 100 nM leptin for various periods of time. Shown is a Western blot using SOCS-3 antiserum of SOCS-3 immunoprecipitates. C, quantification of SOCS-3 protein expression in CHO-OBRl cells. Autoradiograms of Western blots were analyzed by laser scanning densitometry (Molecular Dynamics), and shown are expression levels relative to unstimulated cells (equal to 100%). This experiment was performed two or three times at each time point. Shown are means ± S.E.

Leptin Pretreatment of CHO-OBRl Cells Causes Leptin Resistance in Proximal Leptin Receptor Signaling-- Forced expression of SOCS-3 in mammalian cell lines blocks leptin-induced signal transduction (29). In order to test whether CHO-OBRl cells are leptin-resistant under conditions where endogenous SOCS-3 protein levels are high, we pretreated cells with leptin for 1 h and then carefully washed the cells to remove leptin from the medium. At different times after the leptin pretreatment, we tested the ability of freshly applied leptin to induce intracellular signaling. As demonstrated by Northern blotting, leptin was unable to induce SOCS-3 mRNA for up to 24 h after leptin pretreatment (Fig. 4A). On the other hand, in leptin-pretreated cells, fetal calf serum retained the ability to induce SOCS-3 mRNA (Fig. 4B). These results suggest that leptin pretreatment of CHO-OBRl cells causes leptin-resistant signaling at a step upstream of the socs-3 gene.


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  		Fig. 4.   Leptin pretreatment of CHO-OBRl cells blocks activation of SOCS-3 mRNA by leptin. A, serum-deprived CHO-OBRl cells were pretreated or not with 100 nM leptin for 1 h followed by washing four times with serum-free medium at 37 °C. Three, 6, and 24 h later, cells were stimulated or not with 100 nM leptin for 45 min, and RNA was isolated. Shown is a Northern blot of SOCS-3 mRNA. This experiment was performed two times. B, CHO-OBRl cells were pretreated or not with leptin as above and later treated or not with 20% fetal calf serum for 45 min. Shown is a Northern blot of SOCS-3 mRNA. This experiment was performed twice.

Induction of socs genes by cytokines has been reported to require STAT activation (23). Indeed the promoter of the cis gene contains several STAT binding sites (34). Moreover, STAT3 DNA binding activity is increased in hypothalamus of leptin-treated mice (12). We therefore measured activation of STAT DNA binding activities by leptin in CHO-OBRl cells using an EMSA specific for STAT1 and STAT3 (12, 33). As shown in Fig. 5A, leptin rapidly induces activation of STAT DNA binding activities with maximal levels detected after ~5 min of leptin treatment. We next tested whether leptin had the ability to activate STAT DNA-binding activity after leptin pretreatment. As demonstrated by EMSA, leptin was unable to activate STAT for up to 24 h after leptin pretreatment (Fig. 5B). On the other hand, in the same leptin-pretreated cells, TNF-alpha retained a full ability to activate STAT (Fig. 5C). These results suggest that leptin pretreatment of CHO cells causes blockade of leptin signaling at a step upstream of STAT activation.


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  		Fig. 5.   Leptin pretreatment of CHO-OBRl cells blocks activation of STAT by leptin. A, shown is a time course of STAT DNA binding activity after leptin treatment of CHO-OBRl cells by EMSA. Cells were serum-deprived for ~15 h and stimulated with 100 nM leptin for various periods of time. Nuclear extracts were isolated and subjected to EMSA specific for STAT1 and STAT3 using the m67 probe as described under "Experimental Procedures." B, leptin pretreatment blocks the ability of leptin to induce activation of STAT DNA binding activities in CHO-OBRl cells. Serum-deprived cells were pretreated or not with 100 nM leptin for 1 h followed by washing four times with serum-free medium at 37 °C. After various periods of time, cells were stimulated or not with 100 nM leptin for 10 min, and nuclear extracts were isolated. Shown is an EMSA assay using the m67 probe. The right lane is a positive control demonstrating that nonpretreated cells did retain the ability to activate STAT by leptin after an extended time of serum deprivation. C, activation of STAT DNA binding activities by TNF-alpha after leptin pretreatment of CHO-OBRl cells. Cells were pretreated with leptin as described for B, and 3 h later cells were treated with nothing, 10 ng/ml TNF-alpha , or 100 nM leptin for 10 min. Shown is an EMSA using the m67 probe. All experiments were performed two or three times. The arrows indicate the migration of the STAT·DNA complexes.

Proximal leptin signaling involves tyrosine phosphorylation of the leptin receptor by JAK kinases (14, 35). As shown in Fig. 6A, leptin treatment of serum-deprived CHO-OBRl cells rapidly induces receptor tyrosine phosphorylation as determined by anti-phosphotyrosine blotting of leptin receptor immunoprecipitates. Receptor phosphorylation was maximal after ~5-7 min of treatment and returned to near undetectable levels after 30 min. We next examined whether leptin pretreatment of CHO-OBRl cells affects subsequent stimulation of leptin receptor phosphorylation. As shown in Fig. 6B, pretreatment with 3 or 100 nM leptin for 1 h blocked the ability of fresh leptin to induce receptor phosphorylation 1.5 h after pretreatment. Under these conditions, we showed by Western blotting analysis that SOCS-3 protein levels were increased at the time of the addition of fresh leptin (data not shown). The observed leptin-induced leptin resistance could be due to down-regulation of leptin receptors on the cell surface. We therefore measured 125I-leptin binding to intact cells at 4 °C after leptin pretreatment to determine relative leptin receptor surface expression. As shown in Fig. 6C, binding of tracer leptin was not significantly affected by prior leptin treatment as measured 1.5-24 h after leptin pretreatment. Collectively, these data demonstrate that leptin pretreatment of CHO-OBRl cells result in blockade of proximal leptin signaling without affecting cell surface leptin receptor expression.


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  		Fig. 6.   Leptin pretreatment of CHO-OBRl cells blocks leptin-induced leptin receptor phosphorylation without affecting receptor cell surface expression. A, leptin induces leptin receptor tyrosine phosphorylation. CHO-OBRl cells were serum-deprived and stimulated with 100 nM leptin for various periods of time. Shown is a Western blot using anti-phosphotyrosine antibodies of leptin receptor immunoprecipitates. This experiment was performed five times. B, leptin pretreatment blocks the ability of leptin to induce leptin receptor phosphorylation. CHO-OBRl cells were pretreated with nothing or 3 or 100 nM leptin for 1 h followed by four washes with serum-free medium and incubated at 37 °C for 1.5 h. Cells were then treated or not with 100 nM leptin for 7 min. Shown is a Western blot using anti-phosphotyrosine antibodies of leptin receptor immunoprecipitates. This experiment was performed three times. C, leptin pretreatment does not affect cell surface leptin receptor expression. CHO-OBRl cells were serum-deprived and pretreated or not with leptin for 1 h followed by four washes with serum-free medium at 37 °C and incubated for various periods of time. Cells were then washed with ice-cold serum-free medium with 0.1% bovine serum albumin and subjected to 125I-leptin binding at 4 °C with or without competition with 200 nM cold leptin as described under "Experimental Procedures." Shown is specific binding relative to cells that were not pretreated with leptin (equal to 100%). This experiment was performed three times or more for each time point. Shown are means ± S.E.

Forced Expression of SOCS-3 Does Not Affect Leptin Receptor Surface Expression in Transfected COS Cells-- Recent data suggest that CIS negatively affects EPO receptor signaling by binding to the EPO receptor and thereby targeting the EPOR-CIS complex for proteolytic degradation (36). Under conditions where SOCS-3 blocks leptin-induced leptin receptor tyrosine phosphorylation in transfected COS cells, we earlier showed that forced expression of SOCS-3 in COS cells does not affect total OBRl protein expression by Western blotting (29). However, since the majority of leptin receptors may exist in intracellular compartments, we decided to examine whether forced SOCS-3 expression affects leptin receptor cell surface expression. Under conditions of similar expression of CIS, SOCS-2, and SOCS-3 (Fig. 7A), we found that expression of SOCS-3 did not affect the number of short or long form leptin receptors on the cell surface of transfected COS cells as determined by 125I-leptin binding assays at 4 °C (Fig. 7B). These results are therefore consistent with the results described above using CHO-OBRl cells, suggesting that SOCS-3 blocks leptin signaling at an early signaling step without affecting leptin receptor expression.


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  		Fig. 7.   Forced expression of SOCS-3 in COS cells does not affect cell surface expression of leptin receptors. A, CIS, SOCS-2, and SOCS-3 are expressed at similar levels in transfected COS cells. Shown is a Western blot using anti-HA antibodies of HA immunoprecipitates from COS cells transiently transfected with either empty vector, HA-CIS, HA-SOCS-2, or HA-SOCS-3 expression vectors. CIS migrates as three bands with molecular masses ranging from ~35 to 40 kDa. SOCS-2 and SOCS-3 migrate as single bands of 29 and 32 kDa, respectively. B, cell surface expression of leptin receptors in transfected COS cells. Cells were transiently transfected with OBRs or OBRl expression vectors together with empty vector or CIS, SOCS-2, or SOCS-3 expression vectors. Gray bars show nonspecific 125I-leptin binding (competed with 200 nM cold leptin), and black bars show noncompeted 125I-leptin binding. Shown are means ± S.E.

SOCS-3 Inhibits Leptin-induced Tyrosine Phosphorylation of JAK2, and SOCS-3 Associates with JAK2 in a Leptin-dependent Manner-- SOCS proteins are thought to inhibit cytokine signaling by binding directly to JAK family members and, by an as yet unknown mechanism, inhibit JAK tyrosine kinase activity (22). We were not able to detect leptin-induced tyrosine phosphorylation of JAK isoforms in CHO-OBRl cells by using a variety of JAK antibodies and large amounts of cells (data not shown). This may be due to insufficient sensitivity in our assays or to activation of yet unidentified tyrosine kinases by OBRl in these cells. However, it has been shown earlier that the leptin receptor can activate JAK2 upon ligand binding in other cell lines (14, 35). We therefore decided to test whether SOCS-3 attenuates induction of JAK2 tyrosine phosphorylation by leptin in transfected COS cells. Under transfection conditions where the expression of HA-tagged CIS, SOCS-2, and SOCS-3 proteins are similar (Fig. 7A), activation of JAK2 phosphorylation by leptin was inhibited by SOCS-3, but not by CIS or SOCS-2, as demonstrated by anti-phosphotyrosine blotting of JAK2 immunoprecipitates (Fig. 8, A and B). Expression of SOCS-3 did not significantly affect the expression of JAK2 in these cells (Fig. 8A, lower panel). We next tested specific interaction between SOCS-3 and JAK2. After transient expression of OBRl and JAK2 together with HA-tagged CIS, SOCS-2, or SOCS-3, COS cells were stimulated or not with leptin for 5 min. As demonstrated by Western blotting using anti-JAK2 antibodies of HA-immunoprecipitates, JAK2 co-immunoprecipitates with SOCS-3, but not with CIS or SOCS-2, in samples from leptin-treated cells (Fig. 9).


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  		Fig. 8.   SOCS-3 inhibits leptin-induced JAK2 tyrosine phosphorylation in transfected COS cells. A, Western blot of JAK2 tyrosine phosphorylation in COS cells. Cells were transiently transfected with expression vectors encoding OBRl and JAK2 together with empty vector or HA-CIS, HA-SOCS-2, or HA-SOCS-3 expression vectors. The left lane represents cells not transfected with JAK2 cDNA. After 15 h of serum starvation, cells were stimulated or not with 100 nM leptin for 5 min. Shown are Western blots using anti-phosphotyrosine antibodies of JAK2 immunoprecipitates (top panel) and JAK2 immunoblots of the same membrane (bottom panel). B, quantification of JAK2 tyrosine phosphorylation in COS cells. Autoradiograms of Western blots were analyzed by laser scanning densitometry (Molecular Dynamics), and shown are phosphorylation levels relative to unstimulated vector-transfected cells (equal to 100%). Gray bars depict unstimulated levels, while black bars show the leptin-stimulated levels. Shown are means ± S.E.


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  		Fig. 9.   Leptin induces association of JAK2 with SOCS-3 in COS cells. Cells were transiently transfected with expression vectors encoding OBRl and JAK2 together with empty vector or HA-CIS, HA-SOCS-2, or HA-SOCS-3 expression vectors. After 15 h of serum starvation, cells were stimulated or not with 100 nM leptin for 5 min. Shown are Western blots using anti-JAK2 antibodies of HA immunoprecipitates. This experiment was performed two times and under conditions of similar expression of CIS, SOCS-2, and SOCS-3 as shown in Fig. 7A.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have demonstrated that leptin pretreatment induces leptin resistance in CHO cells stably expressing the long form of the leptin receptor. In these cells, as well as in the hypothalamus of rodents (29), leptin induces expression of SOCS-3, a proposed inhibitor of leptin signaling (29). In CHO-OBRl cells, SOCS-3 protein levels remained elevated for more than 20 h after leptin treatment, thus correlating with the observed leptin resistance resulting from prior leptin exposure to the cells. We also show that the leptin-induced leptin resistance occurs at a signaling step involving inhibition of leptin receptor tyrosine phosphorylation without affecting receptor surface expression. In transfected cells, forced expression of SOCS-3 attenuates leptin-induced tyrosine phosphorylation of JAK2. Furthermore, JAK2 co-immunoprecipitates with SOCS-3 in a leptin-dependent manner. These results strongly suggest that SOCS-3 acts as an inducible negative regulator of proximal leptin receptor signaling.

CIS belongs to the same family of proteins as SOCS and is also a negative regulator of cytokine signaling (20). In contrast to SOCS, however, CIS is reported to associate directly with phosphorylated receptor tyrosine residues, possibly preventing STAT proteins from binding to these sites (20). In addition, the expression of CIS does not affect EPO or interleukin-3 receptor phosphorylation (20) and does not interact with JAK proteins (22). Down-regulation of EPO receptor signaling by CIS may therefore involve competition between CIS and STAT5 for binding to the same tyrosine residue on the receptors, thereby reducing activation of STAT5 proteins (37). Recent data also show that CIS is ubiquitinated and that proteasome inhibitors prolong EPO receptor signaling as well as the interaction of EPO receptors with ubiquitinated CIS proteins (36). These results suggest that CIS may also inhibit EPO signaling by targeting the EPO receptor (and CIS) for proteolytic degradation. In addition, phosphotyrosine phosphatases may inhibit EPO receptor signaling by dephosphorylation of the EPO receptor (38). Thus, several mechanisms are involved in down-regulation of EPO receptor signaling.

In serum-deprived CHO-OBRl cells, leptin-induced tyrosine phosphorylation of the leptin receptor is transient and returns to nearly undetectable levels within 30 min of leptin treatment. This decline appears to occur earlier than the rise in SOCS-3 protein levels after leptin treatment. Although our data are consistent with the possibility that SOCS-3 is involved in the leptin resistance in the hours after leptin pretreatment, it is not clear that SOCS-3 plays a significant role in the rapid down-regulation (minutes) of leptin receptor tyrosine phosphorylation after leptin stimulation of serum-deprived cells. These cells do express detectable base-line amounts of SOCS-3, and this low level may be sufficient to influence leptin signaling. This possibility is consistent with the recent finding that very low levels of SOCS-1 and SOCS-3 are able to attenuate cytokine signaling (31, 39). It is also possible that low SOCS-3 levels are induced as early as ~30 min after leptin treatment of the CHO-OBRl cells and that this is sufficient to play a role in the observed rapid dissemination of leptin receptor phosphorylation after leptin treatment of serum-deprived cells.

Rapid down-regulation of leptin signaling may, however, involve other proteins and pathways in addition to SOCS-3. A recent paper suggests that the phosphotyrosine phosphatase SHP-2 is a negative regulator of leptin receptor-induced STAT3 signaling (40). However, in these studies, SHP-2 did not affect tyrosine phosphorylation of OBRl or of STAT3, suggesting that SHP-2 might regulate other unidentified signaling pathways. Further studies are needed to clarify the identity of other phosphotyrosine phosphatases involved in dephosphorylation of OBRl after leptin binding and whether SOCS-3 plays a role in this process. Down-regulation of leptin receptor signaling may also involve internalization and degradation of leptin receptors (41), and it is unknown whether SOCS-3 plays a role in this process as suggested for CIS and the EPO receptor (36). We did not detect ubiquitination of SOCS-3 in COS or CHO cells. In addition, neither forced expression of SOCS-3 in COS cells nor leptin-induced expression of SOCS-3 in CHO-OBRl cells altered leptin receptor cell surface expression. Finally, down-regulation of leptin signaling may also involve processes at specific downstream signaling steps, including the recently identified family of activated STAT inhibitors, PIAS, which inhibits the DNA binding activity of activated STAT proteins (42).

SOCS-3 mRNA is induced by leptin, growth hormone, leukemia-inhibitory factor, ciliary neurotrophic factor, interleukin-6, and other cytokines in various tissues, and forced expression of SOCS-3 in mammalian cell lines inhibits signal transduction by leptin, growth hormone, interleukin-6, leukemia-inhibitory factor, prolactin, and ciliary neurotrophic factor (27-29, 39, 43, 44). This raises the question of possible cross-talk between different receptor signaling systems. For example, does SOCS-3 induced by one cytokine receptor inhibit signaling by other cytokine receptors in the same cell? Supporting this possibility are results using M1 leukemia cells. Pretreatment of these cells with interferon-gamma , which induces SOCS-1, blocks leukemia-inhibitory factor signaling (43). On the other hand, we have shown here that in CHO-OBRl cells, which are resistant to leptin treatment and have high SOCS-3 protein levels, serum is able to induce SOCS-3 mRNA. This shows that a factor in serum has the ability to induce SOCS-3 mRNA and suggests that this factor is not inhibited by SOCS-3. Alternatively, induced SOCS-3 proteins may not be free in the cytoplasm to act on other receptors that are normally inhibited by SOCS-3. Some data suggest that SOCS-2 may interact directly with the insulin-like growth factor-1 receptor and possibly regulate its function (45). Furthermore, SOCS-1 has been shown to inhibit Tec tyrosine kinases (46). These results suggest that the inhibitory function of SOCS proteins may extend beyond that of JAKs and cytokine receptors. It is, however, unknown whether insulin-like growth factor-1 receptor or other receptor tyrosine kinases like the insulin receptor can induce cis or socs genes. We have attempted to address the question of cross-talk via SOCS by searching for other cytokines capable of inducing SOCS-3 mRNA in CHO cells. We tested several cytokines that have been demonstrated to induce SOCS-3 mRNA in other cells or tissues, including leukemia-inhibitory factor, interleukin-1, interleukin-6, TNF-alpha , growth hormone, and interferon-gamma (21). Unfortunately, none of these factors were able to induce SOCS-3 mRNA levels in CHO cells as determined by Northern blotting (data not shown). This may in part be explained by lack of appropriate receptors in these cells. However, we were able to activate STAT DNA binding activities, but not to induce SOCS-3 mRNA, with TNF-alpha in these cells. Since TNF-alpha is capable of inducing SOCS-3 mRNA in some cells (21), one or more pathways in addition to STAT activation may be required to induce socs-3 gene transcription by TNF-alpha , and such pathways are lacking in CHO cells.

Most obese humans as well as most animal models of obesity are characterized by leptin resistance (47). Since SOCS-3 is an inhibitor of leptin signaling, excessive SOCS-3 activity in leptin-responsive cells is a potential mechanism for leptin resistance. Increased hypothalamic SOCS-3 protein levels could arise from the high circulating leptin levels observed in most obese individuals or from unidentified factors that are up-regulated in obesity and capable of inducing SOCS-3 levels in leptin-responsive neurons. Recently, a number of papers have reported leptin signaling in peripheral tissues (48-52). Increased levels of SOCS-3 in peripheral tissues may therefore result in leptin resistance at these sites. In addition, elevation of SOCS-3 expression by leptin or by other factors in peripheral tissues may cause resistance to other hormones and cytokines that are inhibited by SOCS-3.

In conclusion, we have demonstrated that SOCS-3 protein expression is induced by leptin and that SOCS-3 is a negative regulator of proximal leptin signaling. Furthermore, our data show that SOCS-3 binds to JAK2 in a leptin-dependent manner and suggest that SOCS-3 attenuates leptin receptor signaling by inhibiting JAK-induced tyrosine phosphorylation of the receptor and of JAK itself by a mechanism that remains unknown and requires further studies. Increased SOCS-3 levels in central or peripheral leptin-responsive cells may play a role in leptin resistance, a common feature of human obesity.

    ACKNOWLEDGEMENTS

We especially thank Dr. Steven Shoelson (Joslin Diabetes Center, Boston) for providing the purified SOCS-3 protein used to produce the SOCS-3 antiserum and for expression vectors encoding CIS, SOCS-2, and SOCS-3. We also thank Ryan Buchholz for excellent technical assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant DK R37 28082 and a grant from Lilly (to J. S. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

parallel To whom correspondence should be addressed: Division of Endocrinology, Dept. of Medicine, Beth Israel Deaconess Medical Center, 99 Brookline Ave., Research North, Boston, MA 02215. Tel.: 617-667-2151; Fax: 617-667-2927; E-mail: jflier@caregroup.harvard.edu.

    ABBREVIATIONS

The abbreviations used are: STAT, signal transducers and activators of transcription; EPO, erythropoietin; TNF, tumor necrosis factor; CHO, Chinese hamster ovary; EMSA, electrophoretic mobility shift assay; HA, hemagglutinin.

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RESULTS
DISCUSSION
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