Feedback Inhibition of Leptin Receptor/Jak2 Signaling via Tyr1138 of the Leptin Receptor and Suppressor of Cytokine Signaling 3

Sarah L. Dunn, Marie Björnholm, Sarah H. Bates, Zhibin Chen, Matthew Seifert and Martin G. Myers, Jr.

Division of Metabolism, Endocrinology and Diabetes (S.L.D., M.B., S.H.B., M.G.M.), Department of Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109-0638; and Research Division (Z.C., M.S.), Joslin Diabetes Center, Boston, Massachusetts 02215

Address all correspondence and requests for reprints to: Martin G. Myers, Jr., M.D., Ph.D., Division of Metabolism, Endocrinology and Diabetes, Department of Medicine, University of Michigan Medical School, 1150 West Medical Center Drive, 4301 MSRB 3, Box 0638, Ann Arbor, Michigan 48109-0638. E-mail: mgmyers{at}umich.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Leptin is an adipocyte-derived hormone that communicates the status of body energy stores to the brain to regulate feeding and energy balance. The inability of elevated leptin levels to adequately suppress feeding in obesity suggests attenuation of leptin action under these conditions; the activation of feedback circuits due to high leptin levels could contribute to this leptin resistance. Using cultured cells exogenously expressing the long form of the leptin receptor (LRb) or an erythropoietin receptor/LRb chimera, we show that chronic stimulation results in the attenuation of LRb signaling and the establishment of a state in which the receptor is refractory to reactivation. Mutation of LRb Tyr1138 (the site that recruits signal transducer and activator of transcription 3) alleviated this feedback inhibition, suggesting that signal transducer and activator of transcription 3 mediates the induction of a feedback inhibitor, such as suppressor of cytokine signaling 3 (SOCS3), during chronic LRb stimulation. Indeed, manipulation of the expression or activity of the LRb-binding tyrosine phosphatase, SH2-domain containing phosphatase-2, by overexpression of wild-type and dominant negative isoforms or RNA interference-mediated knockdown did not alter the attenuation of LRb signals. In contrast, SOCS3 overexpression repressed LRb signaling, whereas RNA interference-mediated knockdown of SOCS3 resulted in increased LRb signaling that was not attenuated during chronic ligand stimulation. These data suggest that Tyr1138 of LRb and SOCS3 represent major effector pathways for the feedback inhibition of LRb signaling. Furthermore, we show that mice expressing an LRb isoform mutant for Tyr1138 display increased activity of the leptin-dependent growth and immune axes, suggesting that Tyr1138-mediated feedback inhibition may regulate leptin sensitivity in vivo.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
LEPTIN, THE POLYPEPTIDE product of the ob gene, is an endocrine signal released from adipocytes to regulate body energy balance (1, 2). Stimulation of the long form of the leptin receptor (LRb), the signaling isoform that is expressed primarily on neurons of the hypothalamus (3, 4), suppresses food intake and increases energy investment in numerous physiological processes during periods when energy stores are abundant (2, 5). Resistance to feeding suppression by leptin occurs in common forms of obesity and may arise from receptor desensitization due to chronic stimulation by the high levels of circulating leptin observed in obese individuals.

Although itself devoid of enzymatic activity, LRb mediates intracellular signals via an associated Jak2 tyrosine kinase. Jak2 associates directly with specific motifs on the intracellular tail of the receptor (6) and phosphorylates tyrosine residues on itself and LRb. Tyrosine autophosphorylation sites on Jak2 and tyrosine phosphorylation sites on the intracellular tail of LRb mediate signaling by recruiting SH2 domain-containing proteins. Whereas the identity and function of most Jak2 autophosphorylation sites are not known, phosphorylated Tyr985 on LRb binds the phosphatase SHP-2 (SH2-domain containing phosphatase-2) (7, 8, 9) and mediates activation of the MAPK pathway as well as binding suppressor of cytokine signaling-3 (SOCS3) to inhibit signal transducer and activator of transcription 3 (STAT3) signaling (9, 10, 11). Phosphorylated Tyr1138 on LRb recruits the latent cytoplasmic transcription factor STAT3, which is then phosphorylated by Jak2 to mediate the translocation of STAT3 into the nucleus to activate gene transcription (9, 12, 13, 14). The Tyr1138-> STAT3 pathway mediates transcription of numerous genes, including the anorectic (appetite-suppressing) neuropeptide proopiomelanocortin and the SOCS3 (9, 15).

Insensitivity to the appetite-suppressing effects of leptin in obesity suggests the importance of understanding the mechanisms that limit LRb signaling. We and others have previously studied the role of Tyr985 in inhibition of LRb signaling using the amplitude of STAT3-driven reporter activity as the surrogate for LRb activity (7, 8, 9). The data from these studies demonstrated that a Tyr985-dependent pathway inhibits STAT3 reporter activity during prolonged receptor activation. Multiple mechanisms have been proposed to mediate this Tyr985-dependent inhibition, including the binding of SHP-2 to Tyr985 and subsequent SHP-2-mediated dephosphorylation of the LRb/Jak2 signaling complex (7, 8) and the Tyr985-mediated binding of the SOCS3 protein that accumulates during LRb->STAT3 signaling (9, 11, 16); most data suggest a central role for SOCS3 (11, 17, 18, 19). A similar mechanism appears to operate for related cytokine receptors (17, 18, 20, 21, 22, 23, 24, 25). All of these previous studies, including our own, however, are limited by their sole reliance upon STAT3 reporter- or DNA-binding assays. Importantly, no investigators have directly assayed the role of Tyr985 (or related motifs on other receptors) on Jak2 and ERK phosphorylation, let alone at the endogenous levels of Jak2 and SOCS proteins that are required to observe feedback inhibition in our hands. Hence, potential differences in mechanisms of feedback inhibition for other LRb signals (e.g. ERK and Jak2 activation) may have been overlooked. Furthermore, a role for Tyr1138->STAT3 signaling in feedback inhibition of LRb has not been examined, because mutation of the STAT3-binding Tyr1138 impairs STAT3 reporter activation (9, 11).

In the present study, we have thus investigated the mechanisms that attenuate numerous aspects of signaling by the intracellular domain of LRb during chronic ligand stimulation. We demonstrate that the Tyr1138-> STAT3 signaling pathway is the dominant inhibitor of Jak2 and ERK signaling during prolonged activation of the intracellular domain of LRb and that this inhibition likely proceeds via SOCS3. Indeed, the increased activity of the neuroendocrine growth and immune axes in mice expressing a LRb isoform mutant for Tyr1138 suggests that this pathway may attenuate leptin signaling in intact animals as well as in cultured cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Attenuation of LRb Signaling during Prolonged Ligand Stimulation
To examine the attenuation of leptin receptor signaling during prolonged ligand stimulation, human embryonic kidney (HEK)293 cells stably expressing an erythropoietin (Epo) receptor extracellular domain/LRb intracellular domain chimera (ELR) (HEK293/ELR cells) were exposed to ligand for 0–24 h and phosphorylation of receptor, Jak2, ERK, and STAT3, was analyzed (Fig. 1AGo, lanes A–F). We employ ELR in place of native LRb because it expresses more robustly than LRb, facilitating the analysis of signaling by the LRb intracellular domain as well as avoiding any potential effects of short LR forms. As previously reported, brief (30 min) treatment with ligand stimulated the phosphorylation of ELR, Jak2, ERK, and STAT3 (9). The phosphorylation of STAT3 peaked after 30–60 min of stimulation, declined to approximately 80% of these levels after 4 h of stimulation, and remained at approximately 60% of peak levels after 24 h of stimulation (see graph, Fig. 1AGo). In contrast, the phosphorylation of Jak2, ELR, and ERK were more transient. Jak2 tyrosine phosphorylation declined rapidly to 40–50% of peak levels at 1–4 h of stimulation and declined to below 30% at 8–24 h of stimulation. ELR and ERK phosphorylation declined to 20–30% of peak levels by 2–4 h of treatment and to 0–5% of peak values after 4–8 h of stimulation. ERK activation is biphasic, with an initial peak of activity between 5–10 min of activation, followed by a chronic activation from 10 min onward (26). Note that we have specifically avoided examining ERK phosphorylation during the acute phase of activation (i.e. 5–10 min) to eliminate the potential for confusing the deactivation of acute activity with the process of chronic signal attenuation. To ensure that this observed signal attenuation was not secondary to the depletion of ligand from the medium, we also examined signal strength after a 15-min reapplication of ligand subsequent to the initial period of exposure [Fig. 1AGo, lanes B'–F'. [Note: lanes B'–F' do not represent a restimulation after the original 24 h, but rather each of these lanes represents the effects of a 15-min restimulation after the corresponding period on the left of the panel, i.e. lane B' represents a 30-min stimulation (as in lane B) followed by an additional 15 min, C' represents 1 h (as in C) plus 15 min, and so on]. This reapplication failed to significantly increase the phosphorylation of Jak2, ELR, ERK, and STAT3 (each increased by ~5% of peak values upon restimulation after a 24-h initial treatment), suggesting that chronic ligand exposure had rendered the intracellular domain of LRb refractory to restimulation.



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Fig. 1. Attenuation of ELR/LRb Signaling during Chronic Stimulation

Quiescent HEK293/ELR (A) or HEK293/LRb (B) cells were stimulated with Epo or leptin for the indicated times (0–24 h). Cells were then either lysed (lanes A–F) or restimulated with ligand for 15 min (which yields a maximal Jak2 response from unstimulated cells) (lanes B'–F'). Cell lysates were immunoprecipitated (IP) with {alpha}Jak2 or {alpha}ELR or loaded directly to SDS-PAGE for immunoblotting (IB) with the indicated antibodies. The migration of detected proteins is indicated to the right of the panels. Each blot is representative of at least three independent experiments. For panel A, phosphorylation of the various proteins was quantified at each time point and graphed as a percent of the phosphorylation detectable after 30 min of stimulation. The phosphorylated species in question are as marked, with open symbols representing initial stimulation and solid symbols being values after the 15 min restimulation.

 
Although the extracellular domain of the Epo receptor in ELR would not be expected to alter the signaling of the LRb intracellular domain of ELR, we nonetheless analyzed the time course of signaling to Jak2, STAT3, and ERK in cells expressing the native LRb (HEK293/LRb, Fig. 1BGo) to explore the possibility that the extracellular domain of LRb plays any role in signal attenuation. Because the lower expression of LRb compared with ELR makes the detection of LRb phosphorylation difficult in the absence of Jak2 overexpression, we were unable to examine the phosphorylation of the receptor itself in these cells. Whereas it is possible to detect the phosphorylation of LRb during cotransfection with Jak2, overexpression of Jak2 and other signaling molecules with ELR or LRb abrogates signal attenuation during chronic ligand treatment (data not shown). As with ELR, LRb-mediated STAT3 phosphorylation remained elevated to 75–80% of peak (30 min) values even after 8–24 h of stimulation, whereas decreased phosphorylation of Jak2 and ERK were detectable after 1.0 h of leptin stimulation and decreased to less than 10% of peak values after 8 h of treatment (Fig. 1BGo). Levels of Jak2, STAT3, or ERK phosphorylation changed by less than 10% of peak values upon reapplication of leptin after an 8–24 h initial stimulation. Thus, these data suggest that the attenuation of signals mediated by the intracellular domain of LRb is accompanied by the induction of a state during which the receptor/cell is refractory to reactivation. The finding that ELR and LRb mediate signal attenuation and the establishment of the refractory state similarly confirms that the motifs controlling these processes reside in the intracellular tail of LRb. The finding that Jak2 and ERK phosphorylation is attenuated more rapidly than that of STAT3 suggests either that different inhibitory processes controls the attenuation of STAT3 or that, once phosphorylated, the dephosphorylation of STAT3 occurs more slowly, although reactivation remains blocked after chronic stimulation.

Establishment and Maintenance of a Refractory Period after Prolonged Stimulation Requires Tyr1138 of LRb
We further probed the nature of the refractory period during which ELR could not be reactivated after chronic ligand stimulation by removing the original ligand-containing media from chronically treated HEK293/ELR cells and replacing it with fresh media followed by readdition of ligand after various times of recovery (Fig. 2AGo). The tyrosine phosphorylation of Jak2 was not different than baseline after prolonged (12 h) ligand treatment. Furthermore, removal of the original ligand-containing media for as long as 8 h failed to permit the reactivation of Jak2 upon ligand reapplication; Jak2 phosphorylation remained at or below baseline after the readdition of ligand at each time point. ERK phosphorylation was reduced to 10–20% of peak levels after 12 h of stimulation and was not changed by restimulation after removal of the initial ligand stimulus for 1 h, although some stimulation to approximately 40% of peak levels was observed after recovery periods of greater than 2 h. As in Fig. 1Go, above, and as reported by others (27), STAT3 phosphorylation remained close to maximal (90–95%) after the initial 12-h stimulation. Importantly, however, STAT3 phosphorylation decreased to 60–65% of peak levels after a 1-h removal of the initial stimulus and did not increase in response to a brief (15 min) reapplication of ligand. Indeed, STAT3 phosphorylation dropped to 35–40% of peak levels by 4–8 h after the media change, in spite of the reapplication of ligand for 15 min. Overall, these data are consistent with the induction of an inhibitor of signaling by the intracellular domain of LRb during prolonged receptor activation to mediate feedback inhibition of LRb signaling. The finding that STAT3 phosphorylation remained high after the 12-h stimulation but decreased after the removal of ligand and was refractory to reactivation by brief exposure to ligand suggests that STAT3 phosphorylation may continue at a low rate in cells where Jak2 activity is present at low levels (as during chronic stimulation with ligand), but that a lower rate of dephosphorylation of STAT3 than that of Jak2 and ERK [due, for example to the Jak2-specific dephosphorylation of molecules such as PTP1B (28, 29, 30)] may result in its continued phosphorylation until the ultimate removal of ligand. Certainly, as for Jak2 and ERK, the reactivation of STAT3 remains inhibited after chronic stimulation, suggesting that although mechanisms of dephosphorylation may differ, STAT3 is subject to the same feedback inhibition as Jak2 and ERK.



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Fig. 2. Refractoriness of ELR to Reactivation after Chronic Stimulation

Quiescent HEK293/ELR cells (A) or HEK293 cells stably expressing the indicated ELR isoforms (B–D) were stimulated with Epo for 12 h. Ligand was then removed and fresh media added to the cells for the indicated period of time before the readdition of Epo for 15 min and subsequent lysis. Cell lysates were immunoprecipitated (IP) with {alpha}Jak2 or loaded directly to SDS-PAGE for immunoblotting (IB) with the indicated antibodies. The migration of detected proteins is indicated to the right of the panels. Each blot is representative of at least three independent experiments.

 
To determine the nature of the signaling pathway by which the intracellular domain of LRb mediates this feedback inhibition, we examined the process of feedback inhibition for ELR variants containing mutations at one or more of the key intracellular tyrosine phosphorylation sites on LRb (Fig. 2Go, B–D). ELRL985 and ELRS1138 contain single-point mutations at each of the two known sites of LRb tyrosine phosphorylation (Tyr985 and Tyr1138, respectively) whereas ELRTriple contains point mutations of all three intracellular tyrosine residues of LRb, including the nonphosphorylated Tyr1077 (9). Each of these mutant ELR isoforms were stably expressed in HEK293 cells. The phosphorylation of Jak2 after prolonged signaling by ELRL985was attenuated to baseline and was refractory to reactivation (<20% of peak levels) in a manner similar to that observed for ELR. In contrast, the phosphorylation of Jak2 remained at approximately 80% of those observed during acute stimulation during prolonged activation of ELRTriple and ELRS1138 and furthermore, were increased to about 200% of peak levels after restimulation of these receptors, suggesting a role for Tyr1138 of LRb in mediating feedback inhibition of LRb signaling during prolonged stimulation. ERK phosphorylation was reduced to approximately 65% of ELRT maximal levels after 12 h of stimulation of this receptor and further reduced to 10% of peak levels after changing media for 1 h and restimulating, potentially consistent with a role for Tyr985 in ERK activation (9). Consistent with the retained phosphorylation of Jak2 during chronic ligand exposure in cells expressing ELRS1138, ERK phosphorylation remained close to 100% after 12 h of stimulation and remained at 80% of peak levels after the 1-h washout and restimulation.

Interestingly, STAT3 phosphorylation in HEK293/ELRL985 cells remained elevated to 95% of peak levels after 12 h and remained high (120% of peak levels) after media washout and restimulation, suggesting potentially decreased attenuation of STAT3 signaling after mutation of Tyr985 and consistent with the idea that Tyr985 may play a role specifically in the inhibition of STAT3 signaling. Because Tyr1138 is required for the activation of STAT3 by the intracellular domain of LRb, it was not feasible to quantitatively examine the kinetics of STAT3 phosphorylation during signaling by ELRTriple and ELRS1138.

Overexpression of SOCS3, Not SHP-2, Inhibits ELR Signaling
The finding that the STAT3 binding site on the intracellular domain of LRb mediates feedback inhibition of Jak2 and ERK signaling suggests the possibility that SOCS3 [which is transcriptionally regulated by STAT3 and which has been shown to interact with and inhibit signaling by the intracellular domain of LRb (9, 11)] may mediate this inhibition. Others have also suggested that SHP-2 may be involved in mediating feedback inhibition of LRb signaling (7, 8). To examine the potential role for SHP-2 and SOCS3, we investigated the function of these molecules in ELR signaling in HEK293 cells. To probe a potential inhibitory role of SHP-2 in ELR signaling, we transiently transfected SHP-2 or a catalytically inactive mutant of SHP-2 [SHP-2C/S, containing a substitution mutation of Cys459 in the active site to serine (31)] in HEK293/ELR cells (Fig. 3Go). The high-level overexpression of SHP-2 requires transient transfection, but signaling by transiently expressed ELR is difficult to detect in the absence of cooverexpressed Jak2 due to the inability to transfect all cells in a given population. Because Jak2 overexpression renders the function of Tyr985 (the SHP-2 binding site) and the effects of SHP-2 difficult to detect (9, 10), and abrogates feedback inhibition (presumably by overwhelming the feedback inhibition system) (data not shown), we overexpressed hemagglutinin (HA)-tagged STAT3 with SHP-2 isoforms in stable HEK293/ELR cells to examine the phosphorylation of {alpha}HA-immunoprecipitated HA-STAT3 in the transfected population of cells. This analysis demonstrated that the approximately 9-fold overexpression of SHP-2 achieved by this method failed to decrease the amplitude of ERK or STAT3 phosphorylation (100–110% of the levels measured in control cells) during a 30-min stimulation; nor did the expression of SHP-2C/S increase the phosphorylation of STAT3 (98% of control cell levels), as would be expected if SHP-2 were to dephosphorylate elements of the ELR/Jak2 signaling complex. Indeed, overexpression of SHP-2C/S inhibited ERK activation (57% of levels in control cells), as we and others have previously shown (10, 31); the incomplete blockade of ERK signaling by SHP-2C/S may be due to the presence of untransfected cells in the total cell population (10).



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Fig. 3. Overexpression of SHP-2 Isoforms in ELR Signaling

HEK293/ELR cells were cotransfected with STAT3-HA plus the indicated SHP-2 isoform, made quiescent, and incubated in the absence (–) or presence (+) of Epo for 30 min before lysis. Cell lysates were immunoprecipitated (IP) with the indicated antibodies or loaded directly to SDS-PAGE for immunoblotting (IB) with the indicated antibodies and exposed to Kodak BioMax MR film. The migration of detected proteins is indicated to the right of the panels. Each blot is representative of at least three independent experiments. WT, Wild type.

 
In contrast to our inability to detect inhibition of STAT3 or ERK phosphorylation by SHP-2 overexpression at low/stable levels of receptor and endogenous levels of Jak2 and ERK expression, we and others have previously detected inhibition of ELR signaling by the expression of exogenous SOCS3 with overexpressed ELR plus Jak2, suggesting very robust inhibition of ELR signaling by SOCS3 (11, 32, 33) (Fig. 4AGo). Overexpression of HA-SOCS3 abrogated the ELR-mediated phosphorylation of Jak2 (13% of control), ELR (38% of control), and STAT3 (<5% of control).



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Fig. 4. SOCS3 in ELR Signaling

A, HEK293 cells were transfected with ELR and Jak2 plus SOCS3 or control plasmid and made quiescent overnight before stimulation with Epo for 15 min. Cell lysates were immunoprecipitated (IP) with the indicated antibodies or loaded directly to SDS-PAGE for immunoblotting (IB)with the indicated antibodies. B, HEK293 cells were transfected with the indicated ELR and Jak2 isoforms and made quiescent overnight before stimulation with Epo for 15 min. Cell lysates were incubated with GST-SOCS3, and the bound proteins were resolved by SDS-PAGE for immunoblotting with {alpha}PY. The migration of detected proteins is indicated to the right of the panels. Each blot is representative of at least three independent experiments.

 
The finding (Fig. 3Go, above) that overexpression of SHP-2 or SHP-2C/S does not significantly alter ELR-mediated STAT3 phosphorylation is consistent with data previously published by ourselves and others suggesting that SHP-2 does not mediate inhibition of STAT3 signaling by LRb or related receptors (9, 10, 19). These data are also consistent with the finding (see Fig. 2Go), that the SHP-2 binding site, Tyr985, does not appear to participate in the attenuation of ELR signaling to Jak2 and ERK during chronic stimulation. The finding that SOCS3 inhibits ELR signaling is also consistent with previous work from ourselves and others (9, 10, 33). We and others, however, have previously demonstrated a role for Tyr985 of LRb (and homologous sites in related receptors) in binding to SOCS3 and mediating the inhibition of STAT3-mediated reporter activity by SOCS3 (10, 16, 17, 18, 34). In this light, it appears confusing initially that Tyr985 does not participate in feedback inhibition of ELR during chronic leptin signaling if, indeed, SOCS3 mediates this feedback inhibition. One reasonable explanation for this conundrum is the presence of an alternative site of interaction between SOCS3 and the LRb/Jak2 signaling complex; indeed, we previously showed that whereas Tyr985 mediates inhibition of STAT3 reporter activity at very low levels of SOCS3, inhibition by higher levels of SOCS3 does not require Tyr985 (10). To address the possibility of a second binding site for SOCS3 within the activated LRb signaling complex, we employed a bacterial glutathione S-transferase (GST)-SOCS3 fusion protein to precipitate phosphorylated proteins from Epo-stimulated HEK293 cells that had been transiently transfected with ELR or ELRL985 plus Jak2 or Jak2Y570F (a constitutively active mutant of Jak2) (35). Precipitated proteins were resolved by SDS-PAGE and detected by immunoblotting with {alpha}-phosphotyrosine ({alpha}PY) (Fig. 4BGo). Whereas GST alone failed to bind phosphorylated ELR or Jak2 (data not shown), GST-SOCS3 bound ELR from cells overexpressing ELR plus either of these Jak2 isoforms. In contrast, tyrosine-phosphorylated ELRL985 was not detectably associated with GST-SOCS3, whereas phosphorylated Jak2 and Jak2Y570F associated with GST-SOCS3 from cells expressing ELR or ELRL985, suggesting that SOCS3 may interact directly with Jak2 as well as with phosphorylated Tyr985 of LRb, as previously suggested (35, 36, 37).

Contribution of Endogenous SOCS3 to ELR Signal Attenuation in HEK293 Cells
It thus seemed reasonable to examine the role for endogenous SOCS3 in feedback inhibition of ELR signaling to Jak2 and ERK phosphorylation. To accomplish this, we obtained small interfering RNAs (siRNAs) to silence SOCS3 and SHP-2 gene expression to further define the mechanisms of LRb signal attenuation (Fig. 5Go). siRNAs directed against the SOCS3 and SHP-2 mRNAs were transfected into HEK293/ELR cells using an optimized transfection method (see Materials and Methods, optimization data not shown). SHP-2 expression, which was detectable at similar levels in cells treated in the absence or presence of ligand, was reduced by 80% upon treatment with SHP-2 RNA interference (RNAi). Although SOCS3 protein was not detected before stimulation, Western blotting detected induction of SOCS3 protein in HEK293 cell lysates after 8 h of ligand treatment; SOCS3 siRNA rendered SOCS3 protein undetectable by immunoblotting (Fig. 5AGo). Using the six-well plate format for transfection of siRNAs recommended by the manufacturer (Mirus Corp., Madison, WI), detection of endogenous STAT3 and Jak2 phosphorylation was difficult due to the small amount of protein available in the sample, forcing us to overexpress these signaling proteins to detect their activation (Fig. 5Go, B and C). Unfortunately, the overexpression of Jak2, even at very low levels, increases signal strength to the point that feedback inhibition of this signal is not readily detectable (our unpublished data); we thus examined only the level of Jak2 phosphorylation in {alpha}Jak2 immunoprecipitates after a 15-min stimulation (Fig. 5BGo). Whereas the expression of siRNA directed at SHP-2 had little effect on the phosphorylation of Jak2 (115% compared with no siRNA controls), the expression of SOCS3-directed siRNA modestly increased the level of Jak2 phosphorylation in this assay to 170% of no siRNA controls, perhaps due to the knock down of basal SOCS3 expression in these cells. Similarly, when the tyrosine phosphorylation of HA-STAT3 was examined, no long-term signal attenuation was detected either in control cells or in cells treated with siRNA (12-h stimulation levels 105–110% of 2 h in each case) (Fig. 5CGo); this is consistent with the modest attenuation of STAT3 phosphorylation during chronic signaling observed in Fig. 1Go, A and B. Although no effect of SHP-2-directed siRNA was evident in the phosphorylation of HA-STAT3 (70–90% of levels in control cells; see Fig. 5DGo), transfection of the SOCS3-directed siRNA increased the amplitude of HA-STAT3 phosphorylation by 2.5-fold at 2 and 12 h of stimulation. This result suggests an important role for SOCS3 in regulating the phosphorylation of STAT3 during signaling by the intracellular domain of LRb.



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Fig. 5. Effects of RNAi-Mediated Gene Silencing of SOCS3 and SHP-2 Expression on LRb Signal Attenuation

HEK293/ELR cells (0.3 x 106 cells per well in six-well plates) were transfected with 100 nM RNAi to the indicated target (SHP-2 or SOCS3) or no RNAi (–) using Mirus transfection reagents as detailed in Materials and Methods. Cells were placed 24 h later into serum-free media overnight. A, Cells were treated with Epo for 8–12 h (+) or left untreated (–) before lysis and analysis by immunoblotting (IB) for SHP-2 or SOCS3. B, Cells were transfected with 100 nM RNAi plus pcDNA3Jak2 (0.5 µg) and made quiescent, as above, before incubation in the absence or presence of Epo for 15 min and lysis. Lysates were immunoprecipitated (IP) with {alpha}Jak2, and precipitated proteins were resolved by SDS-PAGE for immunoblot analysis with {alpha}PY. Note high basal levels of activity due to overexpression of Jak2 protein. C, HEK293/ELR cells were transfected with 100 nM of the indicated RNAi (or control) plus HA-STAT3 (0.5 µg) and made quiescent, as above, before incubation in the absence or presence of Epo for the indicated time and lysis. Cell lysates were immunoprecipitated with {alpha}HA or loaded directly to SDS-PAGE for immunoblotting with the indicated antibodies. The migration of detected proteins is indicated to the right of the panels. Each panel is representative of at least three independent experiments. D, Levels of phosphorylated STAT3 (relative to total HA-STAT3) and phosphorylated ERK were quantified from panel C, duplicate measurements were averaged, and values were plotted relative to the values for control 2-h stimulated values for the various siRNA species tested.

 
We were able to detect the phosphorylation of endogenous ERK from HEK293/ELR cells in these assays, however, eliminating the necessity to overexpress this signaling molecule and risk masking feedback inhibition (Fig. 5CGo). Indeed, attenuation of ERK phosphorylation was detectable in control cells after 12 h of stimulation (31% of levels observed at 2 h); similar attenuation was observed during expression of SHP-2-directed siRNA (reduced to 28% after 12 h), again suggesting that SHP-2 does not mediate feedback inhibition during signaling by the intracellular domain of LRb. Somewhat surprisingly, we did not detect decreased ERK phosphorylation during treatment with SHP-2 RNAi, suggesting that incomplete (80%) knockdown of SHP-2 may permit normal ERK signaling in these cells. In contrast, not only was the increased amplitude of ELR-stimulated ERK phosphorylation evident in cells expressing siRNA that was directed against SOCS3 (3.2-fold compared with no siRNA), but also the increased amplitude was maintained at 12 h of stimulation (108% of 2 h with SOCS3 siRNA and 3.4-fold higher than control cells after a 2-h stimulation), suggesting that this treatment had abrogated feedback inhibition of signaling by the intracellular domain of LRb.

Evidence for Increased Signaling by LRbS1138 in Vivo
Taken together, the preceding data suggest that the STAT3 binding site, Tyr1138, mediates feedback inhibition of LRb signaling in cultured cells. The finding that siRNA-mediated knockdown of the STAT3 target gene, SOCS3, increases the amplitude of signals activated by the intracellular domain of LRb and blocks feedback inhibition on these signals suggests that the Tyr1138->STAT3->SOCS3 pathway may represent an important mechanism of feedback inhibition on LRb signaling in intact animals. We have previously generated s/s mice in which the wild-type leptin receptor gene (lepr) was replaced by an isoform containing a point mutation of Tyr1138; homologous targeting of the mutant LRbS1138 into the endogenous lepr locus ensured expression of LRbS1138 at precisely physiological levels (15). These s/s animals are obese, at least in part, due to the requirement for the Tyr1138->STAT3 signal to mediate transcription of the anorectic (appetite-suppressing) neuropeptide, proopiomelanocortin, in LRb-expressing neurons in the hypothalamus (15). The neuroendocrine signal to the reproductive axis is preserved in s/s mice (as opposed to infertile db/db mice that entirely lack LRb), suggesting the presence of Tyr1138->STAT3-independent functions of LRb. We reasoned that if Tyr1138 indeed mediates feedback inhibition of LRb signaling in intact animals, it should be possible to detect the hyperactivation of some such function. We thus more closely examined the function of the neuroendocrine growth axis (whereby hypothalamic GHRH mediates the pulsatile release of GH from the pituitary, which in turn promotes the synthesis of IGF-I in peripheral tissues) in these animals (Fig. 6Go, A and B). Leptin is thought to promote the activity of this neuroendocrine growth axis (38, 39). Indeed, whereas db/db mice exhibited decreased linear growth compared with control animals on the same genetic background, s/s animals display increased linear growth, suggesting hyperactivity of the growth axis. Because measurement of the hypothalamic release of GHRH and the pituitary release of GH is problematic in mice due to the large serum volume required to measure GH and the pulsatile nature of GH release, we measured the circulating levels of IGF-I to assess the activity of this axis; as for linear growth, total IGF-I levels were decreased by approximately 27% in db/db animals compared with control littermates, whereas s/s mice displayed levels more than 1.5-fold higher than those observed in their littermate controls. Similar increases in circulating IGF-I levels in s/s compared with +/+ mice are observed on other genetic backgrounds (data not shown).



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Fig. 6. Increased Activity of the Neuroendocrine Growth Axis in Mice Expressing LRbS1138

Snout-anus length (A) and serum IGF-I concentrations (B) were determined for 20-wk-old male mice. Mice heterozygous (s/+) or homozygous (s/s) for LRbS1138 were on the segregating 129Sv;C57Bl/6 genetic background and were compared with littermate controls, whereas db/db mice on the congenic C57Bl/6 background were compared with nonlittermate db/+ animals on the same background; C57Bl/6 +/+ littermates to db/+ and db/db animals were not used due to the effect of the homozygous misty gene on animal size (see Materials and Methods). C, Male C57Bl/6J mice (6 wk of age) of the indicated genotype were killed, and thymuses were dissected for the determination of thymocyte numbers as described in Materials and Methods. Data are means ± SEM; n >5 for all determinations; *, P < 0.02 vs. control by Student’s t test.

 
Leptin furthermore controls the function of the immune system, increasing the proliferation of immune cells and promoting an inflammatory immune response (40, 41). To examine the function of the immune system in s/s mice, we examined the cellularity of the thymus in C57Bl/6J +/+, s/s, and db/db mice (Fig. 6CGo). As expected (40, 41), fewer thymocytes were found in the thymus of db/db mice (41% of +/+ levels). In contrast, thymic cellularity was increased in s/s mice (163% of +/+ levels), suggesting increased leptin-mediated immune signaling in s/s mice. Thus, although further studies will be required to conclusively demonstrate the molecular mechanism of this in vivo activation, these data demonstrate that the disruption of the Tyr1138->STAT3 axis that mediates feedback inhibition of LRb signaling in cultured cells results in the stimulation of thymocyte production and hyperactivity of the neuroendocrine growth axis in intact animals, consistent with the role for this pathway in feedback inhibition in vivo.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the present study we have investigated the attenuation of signaling by the intracellular domain of LRb during chronic ligand stimulation. We show that signaling, as measured primarily by Jak2 and ERK phosphorylation, decreases almost to baseline after approximately 4–8 h of stimulation and is poorly reactivated upon the readdition of ligand. Furthermore, this period, during which the receptor is refractory to restimulation, persists for several hours after the removal of the initial stimulus. These findings suggest the induction of a stable inhibitor of signaling by the intracellular domain of LRb via activation of LRb signals. The finding that similar refractory periods are induced during signaling by ELR and the native LRb suggests that the signal that mediates LRb-induced feedback inhibition derives from the intracellular domain of LRb. Indeed, the finding that Jak2 and ERK phosphorylation remain elevated to near peak levels after 12 h of stimulation in receptors that lack Tyr1138 of LRb (the STAT3 binding site) suggests that this feedback inhibitor is transcriptionally induced by STAT3 during LRb signaling.

Our previous work has demonstrated that the transcription of the inhibitory SOCS3 protein is induced via the Tyr1138->STAT3 pathway during signaling by the intracellular domain of LRb (9), suggesting to us that SOCS3 might mediate this feedback inhibition on LRb signaling. On the surface, this hypothesis appeared to contradict our previous finding and the findings of others that Tyr985 of LRb and homologous sites on related receptors represents a high-affinity binding site for SOCS3 during signaling by IL-6 receptor family members and that this site is critical for inhibition of signaling by SOCS3 (10, 16, 17, 18, 34). Our present data indicate that Tyr985 is not required for the attenuation of Jak2 and ERK phosphorylation during prolonged signaling by the intracellular domain of LRb. That previous studies of feedback inhibition by SOCS3 and Tyr985 (or related sites on similar receptor) relied upon STAT3 assays to measure inhibition (11, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) represented a major reason for us to undertake the present study, however, and suggested the possibility that multiple SOCS3 binding sites might exist within the activated LRb signaling complex. Indeed, our present data suggest that bacterially expressed GST-SOCS3 requires Tyr985 of LRb to mediate direct binding to the intracellular domain of LRb but that binding to tyrosine-phosphorylated Jak2 does not require Tyr985. These data are consistent with our previous observation that Tyr985 of LRb is not required for the SOCS3-mediated blockade of Jak2 tyrosine phosphorylation (35), and with data from other laboratories suggesting multiple binding sites for SOCS3 within activated cytokine receptor complexes (36, 37). Consistently, SOCS3 overexpression attenuated all ELR-mediated signals that we examined, even in the presence of overexpressed Jak2. In contrast, the overexpression of SHP-2 or the dominant-negative SHP-2C/S failed to significantly alter ELR signaling except for the previously documented positive role of SHP-2 in ERK activation.

Furthermore, whereas our previous data showed that Tyr985 mediated the inhibition of STAT3 reporter activity at low levels of SOCS3 overexpression, Tyr985 was not required at high levels of SOCS3 overexpression (10). Taken together, these observations suggested that SOCS3 likely interacts with the activated LRb signaling complex via Tyr985 of LRb and via another site(s) on the LRb-associated Jak2 molecule, and that SOCS3 might thus mediate this Tyr1138-dependent, Tyr985-independent feedback inhibition of Jak2 and ERK phosphorylation during prolonged receptor signaling.

Although it is clear that SOCS3 overexpression can inhibit multiple aspects of signaling by the intracellular domain of LRb and other receptors, it had never been directly shown that SOCS3 does, in fact, mediate feedback inhibition of LRb signaling. We thus employed an RNAi-mediated knockdown strategy to test the role of SOCS3 (and SHP-2) in feedback inhibition of signaling by the intracellular domain of LRb. Although we were somewhat limited in terms of signal detection by the small amount of protein that could be derived from the small-scale format required for optimal RNAi-mediated knockdown, compounded by the abrogation of feedback inhibition by the overexpression of signaling molecules, such as Jak2, to enhance signal detection, we were nonetheless able to demonstrate that RNAi-mediated knockdown of SOCS3 (but not SHP-2) expression enhanced the tyrosine phosphorylation of Jak2 and STAT3. Furthermore, because we were able to detect endogenous ERK phosphorylation (and hence attenuation of the ERK signal) in these assays, we were able to demonstrate that knockdown of SOCS3 not only acutely enhanced ERK phosphorylation (>3-fold), but blocked the attenuation of this signal after prolonged stimulation of the receptor. Thus, the Tyr985-independent, Tyr1138-dependent feedback inhibition of ERK (and presumably Jak2) signaling that we have defined in this study is likely to be secondary to the accumulation of SOCS3 during prolonged activation of the intracellular domain of LRb.

Our data also indicate that endogenous SOCS3 expression inhibits the tyrosine phosphorylation of STAT3, as RNAi-mediated knockdown of SOCS3 increases the amplitude of STAT3 phosphorylation. It is also clear that the mechanism(s) by which STAT3 is inhibited during prolonged stimulation of LRb are at least somewhat different than the mechanism(s) by which Jak2 and ERK are attenuated. Not only do a variety of previous data from our laboratory and others demonstrate that Tyr985 of LRb is a central mediator of STAT3 signal repression (consistent with the increased phosphorylation of STAT3 by ELRL985 during ligand washout after prolonged stimulation in this study), but STAT3 phosphorylation is attenuated with prolonged kinetics compared with the relatively brief time course of Jak2 and ERK dephosphorylation. One hypothesis that is consistent with all of these data (Fig. 7Go) is that whereas the binding of SOCS3 to Tyr985 mediates inhibition of STAT3 signaling, SOCS3 interaction at this site is not involved in the attenuation of Jak2 and ERK signaling. Rather, in this model, the feedback inhibition of Jak2 and ERK signaling is inhibited by the interaction of SOCS3 with Jak2; because Jak2 activity is required for the phosphorylation and activation of STAT3 during LRb signaling, the interaction of SOCS3 with Jak2 likely also impacts STAT3 activity. Although the molecular mechanism of SOCS3-mediated inhibition is not well understood, reasonable mechanisms include the direct binding of proteins and subsequent inhibition of signaling activity (e.g. Jak2 kinase activity), sequestration of the signaling complex, or proteasome-mediated degradation of SOCS3-bound signaling complexes. There are unfortunately few data to directly address these possibilities, and the available data are somewhat contradictory: For example, although the SOCS box of SOCS3 appears to associate with the ubiquitination machinery, this SOCS box is not required for inhibition of cytokine receptor signaling by overexpressed SOCS3 (37).



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Fig. 7. A Model of LRb Signal Transduction Activation and Attenuation

Acute stimulation of ELR/LRb signaling (left panel) results in the tyrosine phosphorylation and activation of Jak2 and the subsequent phosphorylation of Tyr985 and Tyr1138 of the intracellular tail of LRb, with rapid recruitment of SHP-2 and STAT3 leading to the activation of ERK and gene transcription, respectively. STAT3 activation drives the transcription of SOCS3. During more lengthy stimulation of the receptor (right panel), SOCS3 accumulates in the cell and binds to Tyr985 and Jak2. We and others have previously shown that the binding of SOCS3 to Tyr985 mediates some inhibition of STAT3 reporter activity. Here, we show that the inhibition of Jak2 and ERK phosphorylation during chronic stimulation is mediated by SOCS3 independently of Tyr985, and, therefore, is presumably mediated by the binding of SOCS3 directly to Jak2. It is not clear whether this binding of SOCS3 to Jak2 mediates direct effects upon STAT3 activity or only indirect effects via the regulation of Jak2 activity.

 
The induction of SOCS3 expression by prolonged LRb signaling constitutes a model of leptin resistance that mimics the effects of chronic stimulation on the leptin receptor during hyperleptinemic obese states, and information gained from the study of this cell culture model may be pertinent to our understanding of the causal factors contributing to leptin resistance in vivo. The potential importance of LRb-promoted SOCS3 accumulation is suggested by the leptin sensitivity of mice haploinsufficient for SOCS3 or null for SOCS3 in the brain (42, 43). Whereas current data available from these models do not address the specific neuronal type(s) affected by decreased SOCS3 expression because neuropeptide mRNA expression does not differ between these mice and controls, our current results suggest that SOCS3 is likely to limit all LRb-mediated signals and thus inhibit leptin action in all LRb-expressing neurons.

We have closely examined the phenotype of animals expressing LRbS1138 and uncovered evidence of increased leptin action in the growth and immune systems of these animals. Although these animals are clearly not globally leptin sensitive (as the lack of STAT3-mediated melanocortin action in these animals renders them hyperphagic and obese) (15), the finding that the lack of STAT3 signaling in these animals actually increases thymic cellularity and the activity of the neuroendocrine growth axis suggests that STAT3 signaling may suppress leptin action in vivo as in the cultured cells examined in this study.

In summary, our present data outline that STAT3 signaling by LRb, as well as mediating important physiological actions of leptin, mediates feedback inhibition of LRb signaling during prolonged stimulation by inducing expression of SOCS3. We propose a model whereby SOCS3 attenuates LRb signaling at multiple sites within the activated LRb signaling complex. This mechanism of feedback inhibition may be important in intact animals as well as in cultured cells and may thus contribute to the leptin resistance associated with obesity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Antibodies and Reagents
Rabbit {alpha}LRb antisera ({alpha}ELR) was raised against a bacterial GST fusion protein containing the 100 COOH-terminal amino acids of murine LRb (amino acids 1075–1174) (9). Polyclonal antibodies were raised in rabbits against the following synthetic peptides: {alpha}Jak2 (amino acids 758–770 of murine Jak2); {alpha}PY-STAT3 (amino acids 699–711 of murine STAT3 containing phosphorylated Tyr705). Sera were affinity purified by passing through an Affigel-10/15 column (Bio-Rad Laboratories. Hercules, CA) conjugated to the corresponding peptide antigen followed by acid elution. Peptides for antibody production were synthesized by Boston Biomolecules (Woburn, MA). Injection and bleeding protocols for antibody production were performed by Covance Research Products (Denver, PA). Monoclonal 4G10 antibody was used for detection of phosphotyrosine residues (Upstate Biotechnology, Inc., Lake Placid, NY).

Affinity-purified horseradish peroxidase (HRP)-conjugated secondary antibodies, monoclonal and polyclonal {alpha}HA, and polyclonal {alpha}SOCS3(M20) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Immunoblotting antibodies directed against SHP-2 and the phosphorylated (activated) form of ERK [{alpha}ERK(P)] were purchased from New England Biolabs (Beverly, MA). Rabbit antimouse antibody was purchased from Calbiochem (San Diego, CA). Chemiluminescence reagents were purchased from Pierce Endogen (Rockford, IL). 125I-labeled protein A was from ICN Biochemicals, Inc. (Los Angeles, CA). Recombinant mouse Epo was purchased from Cardinal Health (Dublin, OH). BSA fraction V, geneticin, and recombinant murine leptin were purchased from Sigma-Aldrich (St Louis, MO). Protein A sepharose and protein G sepharose were purchased from Amersham Biosciences (Piscataway, NJ).

cDNAs
The chimeric ELR cDNA in the pcDNA3 vector has been described previously (9). This chimera consists of the extracellular domain of the Epo receptor joined to the intracellular domain of the long form of the leptin receptor, LRb; the junction occurs at an engineered silent AflII site at the 3'-end of the transmembrane domain-encoding sequence (9). pcDNA3Jak2 and pcDNA3Jak2Y570F, pcDNA3SOCS3, pc DNA3STAT3HA, and pcDNA3LRb have also been described (6, 10, 35). Constructs encoding SHP-2 and the catalytically inactive SHP-2C/S (C459->S) have been described previously (44).

Cell Culture and Transfections
HEK293 cells were grown in DMEM containing 450 mg/ml glucose and 10% fetal bovine serum (FBS) at 5% CO2, 37 C. Cells were transfected with cDNA for various receptor constructs using LipofectAMINE (Invitrogen, Carlsbad, CA). Transfected clones were selected in DMEM supplemented with 10% FBS and 750 mg/ml geneticin. Clones that stably expressed ELR isoforms (293/ELR) or LRb (293/LRb) on the cell surface were identified by 125I-labeled Epo or leptin binding before experimentation, as described previously (6).

Cell Stimulation and Lysis
Subconfluent cells were made quiescent by removal of media containing FBS and addition of DMEM containing 0.5% BSA. The following day, cells were treated with Epo (10 U/ml), or leptin (100 ng/ml) for times indicated in figure legends. Cells were lysed in ice-cold lysis buffer as described (9), and cellular debris was removed by centrifugation at 16,000 x g at 4 C for 15 min. Whole-cell lysates were analyzed for protein concentration using a Bradford Protein assay (Bio-Rad Laboratories), and equal protein was added to 15 µl of respective antibody for immunoprecipitation at 4 C overnight. Protein A-sepharose was added the following day and mixed with the antibody/lysate solution for 1 h. Immune complexes were collected by brief centrifugation and washed three times in lysis buffer before denaturation in 30 µl Laemmli buffer (9). Alternatively, equivalent amounts of protein from cell lysates were diluted directly in Laemmli buffer for the direct analysis of lysates by SDS-PAGE and immunoblotting.

Immunoblotting
Proteins were analyzed by SDS-PAGE. Proteins were transferred to nitrocellulose membranes (Schleichler & Schuell, Keene, NH) in Towbin buffer containing 0.02% sodium dodecyl sulfate and 20% methanol. Membranes were blocked overnight at 4 C or for 1 h at room temperature in block buffer (wash buffer containing 3% BSA). Membranes were incubated in primary antibody in block buffer for 1 h at room temperature, followed by washing three times in wash buffer. Polyclonal antibodies were detected by incubation for 1 h in block buffer containing 125I-labeled protein A. Monoclonal primary antibodies were detected by incubation in rabbit antimouse antibody for 1 h preceding the protein A. For chemiluminescent detection, primary antibody was followed by incubation in appropriate HRP-labeled secondary antibody or HRP-linked protein A. Proteins were visualized by exposure on Kodak X-AR film (Eastman Kodak, Rochester, NY) except as noted in the figure legends. Quantitation of immunoblots was performed using QuantityOne software (Bio-Rad).

RNAi
Short interfering RNAs (siRNAs) complementary to the SOCS3 and SHP-2 genes were designed using custom SMARTPools from Dharmacon (Lafayette, CO). For transfection, HEK293 cells were plated at a density of 0.3 x 106 cells per well in six-well culture plates. On the following day, cells were transfected with 100 nM siRNA using TransIT-TKO transfection reagent (Dharmacon). Where cotransfection with cDNA was required, a total of 1 µg DNA was mixed with LT1 transfection reagent (Mirus, Madison, WI) before addition of siRNA/TKO mixture described above. Media was removed 24 h later and replaced with serum-free media; treatment with Epo and cell lysis was performed the following day.

GST Pulldown
GST-tagged SOCS3 was prepared as described elsewhere (45). Transiently transfected HEK293 cell lysates were prepared as described above and incubated for 30 min with GST-SOCS3 conjugated to sepharose beads or an equal volume of unconjugated beads. Beads were washed three times in lysis buffer before addition of 50 µl Laemmli buffer. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose for immunoblotting.

IGF-I and Thymocyte Determinations in Mice
Animals homozygously expressing LRbS1138 (s/s) and their heterozygous (s/+) and wild-type littermates were generated by intercrossing of s/+ animals within our own colony of mixed background (129Sv;C57Bl/6) animals or our more recent colony of C57Bl/6 (n = 6 generations backcrossed) animals. C57Bl/6 db/+ animals purchased from Jackson Laboratories (Bar Harbor, ME) were intercrossed in our colony to generate db/db animals and control db/+ littermates; ±/+ mice were discarded due to the effect of the homozygous misty mutation (used by in repulsion to db by Jackson to distinguish +/+ from db/+ animals) on animal size. Animals were euthanized by CO2 inhalation at 20 wk of age, and snout-anus length was measured with a micrometer before decapitation and the collection of trunk blood for hormone assays. Serum (20 µl) was subjected to enzyme immunoassay analysis for total IGF-I using kit DSL-10–2900 from Diagnostic System Laboratories (Webster, TX).


    ACKNOWLEDGMENTS
 
We thank Trevor A. Dundon and Walter H. Stearns for invaluable assistance.


    FOOTNOTES
 
This work was supported by National Institutes of Health Grants R01 DK57768 and DK56731 and a mentor-based postdoctoral fellowship from the American Diabetes Association (to M.G.M.) and a grant from The Swedish Foundation for International Cooperation in Research and Higher Education (to M.B.).

Present address for S.L.D.: Division of Endocrinology, Children’s Hospital Boston, New Research Building, 1 Blackfan Circle, Boston, Massachusetts 02215.

First Published Online December 16, 2004

Abbreviations: ELR, Epo-LRb receptor; Epo, erythropoietin; FBS, fetal bovine serum; GST, glutathione S-transferase; HA, hemagglutinin; HEK, human embryonic kidney; HRP, horseradish peroxidase; LRb, long form of the leptin receptor; PY, phosphotyrosine; RNAi, RNA interference; siRNA, small interfering RNA; SOCS, suppressor of cytokine signaling; SHP-2, SH2-domain containing phosphatase-2; STAT, signal transducer and activator of transcription.

Received for publication September 7, 2004. Accepted for publication December 8, 2004.


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