Pituitary Corticotroph SOCS-3: Novel Intracellular Regulation of Leukemia-Inhibitory Factor-Mediated Proopiomelanocortin Gene Expression and Adrenocorticotropin Secretion

Christoph J. Auernhammer, Vera Chesnokova, Corinne Bousquet and Shlomo Melmed

Division of Endocrinology and Metabolism Cedars-Sinai Research Institute-UCLA School of Medicine Los Angeles, California 90048


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
As pituitary leukemia-inhibitory factor (LIF) mediates neuroimmune signals to the hypothalamo-pituitary-adrenal axis, we tested the role of intracellular SOCS-3 in corticotroph function. SOCS-3, a cytokine-inducible protein of the suppressor of cytokine signaling (SOCS) family, is expressed in the murine pituitary in vivo. After ip injection of LIF (5.0 µg/mouse) or interleukin-1ß (0.1 µg/mouse) pituitary SOCS-3 mRNA was stimulated 9-fold and 6-fold, respectively. Also, in corticotroph AtT-20 cells LIF and interleukin-1ß both potently stimulated SOCS-3 mRNA expression. In AtT-20 cells, stable overexpression of SOCS-3 inhibits basal and LIF-stimulated ACTH secretion in comparison to mock-transfected AtT-20 cells (basal: 4426 ± 118 vs. 4973 ± 138 pg/ml, P < 0.05; LIF-induced: 5511 ± 172 vs. 9308 ± 465 pg/ml, P < 0.001). Stable overexpression of SOCS-3 cDNA in AtT-20 cells also resulted in a significant 50% decrease of LIF-induced POMC mRNA levels (P < 0.05) and POMC promoter activity (P < 0.001), respectively. Western blot analysis revealed an inhibition of LIF-stimulated gp130 and STAT-3 phosphorylation in SOCS-3 overexpressing AtT-20 cells. Thus, SOCS-3 inhibits the Janus kinase (JAK) and signal transducers and activators of transcription (STAT) pathway, which is known to mediate LIF-stimulated ACTH secretion and POMC gene expression. In conclusion, SOCS-3 functions as an intracellular regulator of POMC gene expression and ACTH secretion, acting as a negative feedback mediator of the cytokine-mediated neuro-immuno-endocrine interface.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although hypothalamic hormones regulate anterior pituitary function (1, 2), pituitary-derived cytokines play an important role in modulating pituitary hormone secretion and pituitary tumorigenesis (2, 3). We have shown leukemia-inhibitory factor (LIF) (4, 5, 6) to act as an autocrine/paracrine determinant of pituitary corticotroph function (7, 8, 9, 10, 11, 12, 13). In vivo, hypothalamic and pituitary LIF gene expression is increased by lipopolysaccharide and interleukin-1ß (IL-1ß), respectively (7, 8). Corticotroph ACTH secretion and POMC mRNA expression are potently stimulated by LIF in vivo and in vitro (9, 10). The signaling pathway of LIF in the corticotroph cell involves gp130 receptor subunit signaling (11), phosphorylation of signal transducer and activator of transcription (STAT)-1 and STAT-3 (12), and distal synergy with CRH (13). Recently, a family of cytokine-inducible inhibitors of signaling has been described: suppressors of cytokine signaling (SOCS), JAK binding protein (JAP), STAT-induced STAT inhibitors (SSI), and cytokine-inducible SH2 protein (CIS) (14, 15, 16, 17, 18, 19, 20). SOCS-1/JAB/SSI-1 suppresses the IL-6-induced tyrosyl phosphorylation of gp130, Jak2, and STAT-3 in M1 cells and inhibits IL-6-induced cell differentiation (14, 15, 16). Human CIS-3/SSI-3, which has a 90% nucleotide and 97% amino acid homology to murine SOCS-3 (14, 18, 19), inhibits STAT-3 phosphorylation and cell differentiation in LIF-treated M1 cells (18, 19). LIF-inducible SOCS-3 expression in the murine pituitary and in corticotroph AtT-20 cells is reported herein. Overexpression of SOCS-3 in AtT-20 cells inhibited LIF-induced gp130 and STAT-3 phosphorylation, ACTH secretion, POMC mRNA expression, and POMC promoter activity. This study characterizes SOCS-3 as an intracellular regulator of POMC gene expression and ACTH secretion, indicating its role in the negative feedback control of the cytokine-mediated neuro-immuno-endocrine interface.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cytokine-Induced SOCS-3 Gene Expression in AtT-20 Cells
We investigated the effect of LIF and IL-ß on the mRNA expression of SOCS-3, SOCS-2, and CIS, in the AtT-20 murine corticotroph cell line. LIF and IL-1ß potently induced SOCS-3 mRNA expression (Fig. 1Go). In contrast, SOCS-2 mRNA levels were only modestly increased by LIF and IL-1ß, while CIS mRNA levels were not significantly altered (Fig. 1Go). The time course of SOCS-3 gene induction by LIF and IL-1ß differed. After LIF stimulation SOCS-3 mRNA peaked at 30 min, while IL-1ß did not increase SOCS-3 mRNA until 120 min (Figs. 1Go and 2Go). SOCS-3 mRNA levels remained elevated after stimulation with both cytokines for as long as 8 h (Figs. 1Go and 2Go). The effective concentration range of LIF was 0.1–10 ng/ml, while IL-1ß caused a significant increase of SOCS-3 mRNA only at higher concentrations of 1.0 and 10.0 ng/ml (Fig. 3Go).



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Figure 1. Effect of LIF and IL-1ß on SOCS-3, SOCS-2, and CIS mRNA Expression in AtT-20 Cells

Cells were untreated, or incubated with 10 ng/ml LIF or 10 ng/ml IL-1ß, respectively. Total RNA was extracted from the cells after 30, 60, and 120 min of incubation. Northern blot analysis was performed with 20 µg total RNA per lane. Membranes were stripped and reblotted for ß-actin.

 


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Figure 2. Time Course of LIF- and IL-1ß-Induced SOCS-3 mRNA Expression in AtT-20 Cells

Cells were untreated (0 h) or incubated with 10 ng/ml LIF (Fig. 2AGo) or 10 ng/ml IL-1-ß (Fig. 2BGo) for 0.5–8.0 h. Northern blot analysis was performed with 20 µg total RNA per lane as indicated. Top, SOCS-3 mRNA; bottom, ß-actin mRNA.

 


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Figure 3. Dose-Dependency of LIF and IL-1ß on SOCS-3 mRNA Expression in AtT-20 Cells

Cells were incubated with 0.01–10.0 ng/ml LIF for 45 min and with 0.01–10.0 ng/ml IL-1ß for 120 min, respectively. The time point of expected peak SOCS-3 mRNA expression was chosen for each cytokine, in accordance with the different time-course of LIF- and IL-1ß-induced SOCS-3 gene expression (Figs. 1Go and 2Go). Northern blot analysis was performed with 20 µg total RNA per lane as indicated. Top, SOCS-3 mRNA; bottom, ß-actin mRNA.

 
Cytokine-Induced SOCS-3 Gene Expression in Hypothalamus and Pituitary in Vivo
To evaluate the regulation of pituitary SOCS-3 expression in vivo, Northern blot analysis was performed on total RNA derived from hypothalamic and pituitary tissue of C57BL/6 mice. Low levels of SOCS-3 mRNA were detectable in hypothalamic and pituitary tissue of untreated control mice (Fig. 4Go). Injection of PBS alone did not alter ACTH and corticosterone serum levels (8), nor were changes in pituitary SOCS-3 mRNA expression observed in these control animals (Fig. 4BGo). In experiments with LIF injection, untreated control animals were also killed at 0 min (Fig. 4AGo). As described previously (10), systemic LIF injection caused a significant rise of serum ACTH and corticosterone levels above baseline (Fig. 4AGo). After LIF injection SOCS-3 mRNA expression in the pituitary increased dramatically (9-fold) as early as 30 min, while only a modest increase was observed in the hypothalamus (3-fold) (Fig. 4AGo). Systemic administration of IL-1ß also resulted in a several-fold increase of SOCS-3 mRNA in the pituitary (6-fold) 60 min after IL-1ß injection (Fig. 4BGo). IL-1ß also modestly increased hypothalamic SOCS-3 mRNA after 60 min (2-fold).



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Figure 4. SOCS-3 mRNA Expression in Hypothalamus and Pituitary in Vivo

A, C57BL/6 mice were injected ip with 5 µg LIF (n = 10 per group). Northern blot analysis was performed with total RNA (20 µg per lane) derived from hypothalamic and pituitary tissue. The lanes show total RNA from untreated controls, and treated mice, killed 30, 60, and 120 min after LIF administration, respectively. Top, SOCS-3 mRNA; bottom, ß-actin mRNA. Plasma ACTH and corticosterone levels were measured, respectively. *, P < 0.05; **, P < 0.01. B, C57BL/6 mice were injected ip with 0.1 µg IL-1ß or PBS, respectively (n = 7 per group). Northern blot analysis was performed with total RNA (25 µg per lane) derived from hypothalamic and pituitary tissue. The lanes show total RNA from untreated controls, and mice, killed 30, 60, and 120 min after ip injection of PBS or IL-1ß, respectively. Top, SOCS-3 mRNA; bottom, ß-actin mRNA.

 
Effect of SOCS-3 Overexpression on LIF-Induced ACTH Secretion and POMC Gene Expression in AtT-20 Cells
To evaluate, whether LIF-induced expression of SOCS-3 in the corticotroph cell might act as a negative feedback regulator on cytokine-induced ACTH secretion and POMC gene expression, AtT-20 cells were stably transfected with a SOCS-3 sense-pCR3.1 vector construct (AtT-20-S), or mock-transfected with the pCR3.1 vector alone (AtT-20-M). LIF-induced ACTH secretion at 24 and 48 h was significantly suppressed in AtT-20-S, which overexpressed SOCS-3, as compared with ACTH secretion in AtT-20-M cells (Fig. 5Go). Baseline ACTH secretion was also modestly reduced in AtT-20-S compared with AtT-20-M cells, which may reflect inhibition of autocrine LIF action on basal ACTH secretion in AtT-20-S cells overexpressing SOCS-3. After normalization of LIF-stimulated ACTH increase in AtT-20-M and AtT-20-S cells to their respective baseline ACTH secretion, AtT-20-S cells still exhibited reduced (P < 0.01) relative increase of ACTH secretion in comparison to AtT-20-M cells.



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Figure 5. Effect of SOCS-3 Overexpression on LIF-Stimulated ACTH Secretion

Mock-transfected AtT-20-M cells and SOCS-3 over-expressing AtT-20-S cells were incubated with LIF (0.1–10.0 ng/ml) for 24 and 48 h, respectively. ACTH values are shown as mean ± SE. The depicted experiment is representative of four independently performed experiments, each performed with n = 6 wells per treatment group. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The inset shows a Northern blot analysis of total RNA derived from AtT-20-M and AtT-20-S cells hybridized with a specific probe for SOCS-3. The upper band (3.2 kb) represents endogenous SOCS-3 mRNA, while the lower band (1.1 kb) shows the exogenous, transfected SOCS-3 transcript.

 
We have previously shown that LIF also stimulates POMC mRNA expression in vivo and in vitro (9, 10) and enhances POMC promoter activity (11, 12, 13). In AtT-20-S cells overexpressing SOCS-3, the relative increase of LIF-induced POMC mRNA expression was reduced by 56% (P < 0.05), in comparison to AtT-20-M cells (Fig. 6Go, A and B). To further investigate whether the observed suppression in POMC gene expression in AtT-20-S cells is due to decreased POMC transcriptional activity, we transiently cotransfected AtT-20-M and AtT-20-S cells with a -706/+64 rat POMC promoter-luciferase construct. LIF stimulation of cotransfected AtT-20-M cells resulted in 4.24 ± 0.26-fold increase of POMC promoter activity, while only a 2.14 ± 0.23-fold increase of POMC promoter activity was observed in AtT-20-S cells (P < 0.001) (Fig. 6CGo).



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Figure 6. Effect of SOCS-3 Overexpression on LIF-Stimulated POMC mRNA Expression and POMC Promoter Activity

A and B, Mock-transfected AtT-20-M cells and SOCS-3 overexpressing AtT-20-S cells were incubated with LIF (10.0 ng/ml) for 24 h. A, Northern blot signals for POMC were analyzed by quantitative densitometry and normalized for ß-actin. The relative increase of POMC mRNA after LIF stimulation was calculated from four independently performed experiments. *, P < 0.05. B, Northern blot analysis was performed with 20 µg total RNA per lane derived from untreated vs. LIF-treated AtT-20-M and AtT-20-S cells. The depicted experiment is representative of four independently performed experiments. Top, POMC mRNA; bottom, ß-actin mRNA. C, Luciferase activity of a (-706/+64) rat POMC promoter-luciferase construct was measured in basal and LIF-stimulated AtT-20-M and AtT-20-S cells. LIF-stimulated luciferase activity was normalized to the unstimulated control in AtT-20-M and AtT-20-S cells, respectively. Relative induction of luciferase activity after LIF stimulation was calculated from five independently performed experiments. Each experiment was performed with n = 6 wells per group. ***, P < 0.001.

 
Effect of SOCS-3 Overexpression on LIF-Induced gp130 and STAT-3 Phosphorylation in AtT-20 Cells
As SOCS-3 may inhibit LIF-induced ACTH secretion and POMC gene expression by gp130 activation and subsequent Jak-STAT cascade, we tested these intracellular signaling pathways. Western blot analysis revealed suppression of LIF-induced gp130 and STAT-3 phosphorylation in the AtT-20-S cells, compared with AtT-20-M cells (Fig. 7Go).



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Figure 7. Effect of SOCS-3 mRNA Overexpression on LIF-Stimulated gp130 and STAT-3 Phosphorylation in AtT-20 Cells

Mock-transfected AtT-20-M cells and SOCS-3 overexpressing AtT-20-S cells were untreated (0 min) or incubated with 10 ng/ml LIF for 5 and 10 min, respectively. Immunoprecipitation of the cell protein lysate was performed with anti-gp130 (Fig. 7AGo) or anti-STAT-3 (Fig. 7BGo) antibody, respectively, followed by Western blot analysis with anti-pTyr antibody (top lane). Equal loading was confirmed by reblotting the stripped membrane with anti-gp130 (Fig. 7AGo) or anti-STAT-3 (Fig. 7BGo) antibody, respectively (bottom lane). A representative blot from three independently performed experiments is shown.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We show herein that SOCS-3 is the predominant cytokine-regulated gene of the SOCS/JAB/SSI/CIS-family in the pituitary in vivo, and in the corticotroph AtT-20 cells in vitro. In corticotroph AtT-20 cells, SOCS-3 is induced by LIF and IL-1ß, in contrast to bone marrow cells, where LIF and IL-1ß also induce SOCS-2 and CIS (14). These findings support cell type-specific expression of the SOCS family genes, i.e. the same cytokine may cause different expression patterns in different cell types (14). The observed in vivo effects of LIF and IL-1ß on pituitary SOCS-3 gene expression correlate well with the in vitro pattern in corticotroph AtT-20 cells, i.e. both in vivo and in vitro, IL-1 exhibited a slower temporal induction of SOCS-3 mRNA in comparison to LIF. Activation of STAT-3 and STAT-5 has been shown to play an essential role in the expression of SSI-1 and CIS, respectively (16, 21). IL-1 stimulates different genes of the SOCS/JAB/SSI/CIS family (14), although the Jak-STAT pathway has not been shown to be involved in its signaling mechanisms. On the other hand, we found that IL-1 stimulates autocrine LIF gene expression in AtT-20 cells (8). Therefore, the different time course of LIF- and IL-1ß-stimulated SOCS-3 gene expression in AtT-20 cells might be due to IL-1 acting through autocrine pituitary LIF on SOCS-3 gene expression. The modest increase of hypothalamic SOCS-3 mRNA expression after systemic injection of either cytokine may also be a reflection of the blood-brain barrier allowing only a small fraction of the injected cytokines to act at the hypothalamic level (22), while the anterior pituitary is fully exposed to systemic cytokines.

Our results demonstrate that LIF is a strong inducer of pituitary SOCS-3 expression. Human CIS-3/SSI-3 has a 90% nucleotide and 97% amino acid homology to murine SOCS-3 (14, 18, 19). Expression of CIS-3/SSI-3 in M1 cells inhibits LIF-stimulated STAT-3 phosphorylation and prevents growth arrest, normally observed after LIF stimulation in M1 cells (18, 19). Also, in the corticotroph cell, STAT-3 is phosphorylated by LIF, mediating its signaling pathway (12). Therefore, we reasoned that LIF-induced expression of SOCS-3 in the corticotroph cell might act as a negative feedback regulator on cytokine-induced ACTH secretion and POMC gene expression. Stable transfection of AtT-20 cells with SOCS-3 caused a decrease in LIF-induced ACTH secretion, POMC mRNA expression, and POMC promoter activity in these cells, in comparison to mock-transfected controls. Overexpression of SOCS-3 in AtT-20 cells also inhibits LIF-induced gp130 and STAT-3 phosphorylation in the corticotroph cell, and might therefore mediate its suppressive effects by inhibiting the JAK-STAT pathway. Other important signals for the corticotroph cell (i.e. glucocorticoids and CRH) do not appear to induce SOCS-3 (data not shown).

In summary, SOCS-3 mRNA is expressed in hypothalamus and pituitary, and LIF and IL-1ß are powerful stimuli of corticotroph SOCS-3 mRNA expression in vivo and in vitro. LIF-stimulated increase in ACTH secretion, POMC promoter activity, and POMC gene expression can be suppressed by overexpression of SOCS-3, possibly mediated by an inhibitory effect of SOCS-3 on gp130 and STAT-3 phosphorylation. Thus, pituitary corticotroph SOCS-3 is an intracellular regulator of cytokine-mediated POMC gene expression and ACTH secretion, acting as a cytokine-induced negative feedback mediator on the hypothalamo-pituitary-adrenal (HPA) axis. Cytokine-induced activation of the HPA axis in response to stress or inflammation is highly plastic (1, 3). Several mechanisms support a fast "on" and "off" response of the cytokine-induced activation of the HPA-axis, e.g. concomitant expression of the IL-1 receptor antagonist in hypothalamus and pituitary (23, 24). Cytokine-induced expression of SOCS-3 provides a new intracellular mechanism of short-loop negative feedback for cytokine-induced activation of the HPA axis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Recombinant murine IL-1ß, DMEM, glutamine, deoxyribonucelase I (DNase I), Superscript II, Taq Polymerase, Trizol, RadPrime random priming kit, Lipofectin, Lipofectamine, and G418, were purchased from GIBCO (Gaithersburg, MD). Recombinant murine LIF was from R&D Systems (Minneapolis, MN). Sodium orthovanadate, phenylmethylsulfonylfluoride, aprotinin, leupeptin, and protein A-Sepharose were from Sigma Chemical Co. (St. Louis, MO). Polyclonal anti-STAT3 antibody and antiphosphotyrosine antibody PY20 were from Santa Cruz Biotechnology (Santa Cruz, CA). The gp-130 antibody was from Upstate Biotechnology (Lake Placid, NY). Hybond-N+ membrane, ECL immunodetection system and ThermoSequenase were from Amersham (Cleveland, OH). GeneAmp PCR System 9600 and Ampliwax PCR Gem 100 were from Perkin-Elmer (Foster City, CA). The QiaexII gel extraction kit was from QIAGEN (Valencia, CA). The pCR3.1 vector was from Invitrogen (Carlsbad, CA). ProteinScript T7 kit was from Ambion (Austin, TX). QuickHyb Rapid was from Stratagene (La Jolla, CA). Kodak Biomax MS film was from Kodak (Rochester, NY).

Cell Culture of AtT-20 Cells
AtT-20/D16v-F2 cells were obtained from the American Tissue Culture Collection (Rockville, MD), and cultured as described previously (8). For mRNA expression studies, AtT-20 cells were preincubated for 16 h in serum-free DMEM (supplemented with 0.1% BSA) and then incubated in fresh serum-free DMEM with or without recombinant murine LIF and recombinant murine IL-1ß reconstituted in PBS containing 0.1% BSA. For ACTH secretion studies, AtT-20-M and AtT-20-S cells (1 x 104/well) were incubated for 48 h in DMEM supplemented with 10% FBS and for a further 48 h in serum-depleted DMEM (supplemented with 1% FBS). Fresh serum-depleted DMEM with or without added LIF (0.1–10.0 ng/ml) was then added for a subsequent 24–48 h. ACTH in the supernatant was measured with a commercial RIA (Diagnostic Products Corp., Los Angeles, CA), as described previously (8).

Northern Blot Analysis
Total RNA extraction and Northern blot analysis were performed as described previously (8). Briefly, total RNA was extracted with Trizol, electrophoresed in a 1% agarose, 6.4% formaldehyde gel, and transferred to Hybond-N+ nylon membrane. Prehybridization and hybridization were performed with QuickHyb Rapid following the manufacturer’s instructions. Autoradiographs were exposed to Kodak Biomax MS film for 12–24 h. Probe labeling was performed by using [32P]dCTP and the random priming kit Rad Prime.

Templates for Probes
Fragments of the murine SOCS-3 cDNA (19–610 bp; GeneBank accession number U88328; 20-bp primers), the murine SOCS-2 cDNA (211–940 bp; GeneBank accession number U88327; 20-bp primers), and the murine CIS cDNA (181–740 bp; GeneBank accession number D31943; 21-bp primers) were obtained by RT-PCR of murine pituitary total mRNA. DNAse I digestion and RT with Superscript II, were performed according to the manufacturer’s instructions. "Hot-start" PCR using Ampliwax PCR Gem 100 and Taq DNA Polymerase was performed on a GeneAmp PCR System 9600 with an initial denaturation step (94 C, 4 min), followed by 40 PCR cycles (denaturation 94 C, 30 sec; annealing 61 C, 30 sec; extension 72 C, 45 sec) and a single elongation step at 72 C for 10 min. PCR products were electrophoresed on a 1.5% agarose gel, and specific bands were gel-purified by Quiaex II. Before using as a template for random priming, the specificity of each RT-PCR product was verified by multiple restriction enzyme analysis. A 0.6-kb fragment of the murine POMC cDNA, encoding the 3'-half of exon 3 of the murine POMC gene was kindly provided by Dr. Malcolm J. Low (Portland, OR). The 1.076-kb mouse ß-actin DECAprobe template was from Ambion.

Animal Experiments
Male C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME) at the age of 8–12 weeks. Recombinant murine LIF (kindly provided by Dr. R. Klupacs, AMRAD, Victoria, Australia) and recombinant murine IL-1ß were dissolved in 0.2 ml sterile PBS and injected ip. Mice were killed 30, 60, and 120 min after LIF administration (5.0 µg/mouse; n = 10, each group) or IL-1ß administration (0.1 µg/mouse; n = 7, each group), respectively. Pituitary and hypothalamic tissues were immediately removed and frozen on dry ice. Trunk blood was collected on ice after sacrifice for measurement of plasma ACTH and corticosterone levels. Plasma ACTH (Nichols Institute Diagnostics, San Juan Capistrano, CA) and plasma corticosterone (ICN Biomedicals Inc., Costa Mesa, CA) were measured by RIA, as described previously (8). All experimental procedures were approved by the Institutional Animal Care and Use Committee.

SOCS-3 Overexpression in AtT-20 Cells
A cDNA product of murine SOCS-3, spanning the entire coding sequence (15–762 bp; GeneBank Accession U88328), was obtained by RT-PCR of murine pituitary mRNA, using the following primers: sense 5'-GCCATGGTCACCCACAGCAAG-3' and antisense 5'-CTTGTGCCATGTGCCTCGGAG-3'. Using TA-cloning, the product was inserted into the pCR3.1 vector, containing a cytomegalovirus (CMV) promoter. Specificity and orientation of the insert was proven by restriction enzyme analysis and subsequently verified by full-length sequencing, using the ThermoSequenase kit. In vitro transcription and translation of the SOCS-3 sense construct with the Proteinscript T7 Kit revealed a protein of the expected size of about 25 kDa (data not shown). Stable transfections of AtT-20 cells with pCR3.1 alone (mock-transfection; AtT-20-M), and pCR3.1/SOCS-3 sense insert (AtT-20-S) were performed with Lipofectin, following standard protocols. Before being used for experiments, polyclonal cells were grown in selection medium with G418 (1 mg/ml) for 4 weeks. G418 (1 mg/ml) was also added to the medium at any time after the selection period.

POMC Promoter Luciferase Assay
Transient transfection of AtT-20-M and AtT-20-S cells with a -706/+64 rat POMC promoter-luciferase construct (0.5 µg) and measurement of luciferase activity were performed as reported previously (12, 13). Briefly, cells were plated at a density of 1 x 105cells per well and incubated for 24 h, after which transfection was performed with Lipofectamine according to the manufacturer’s instructions. Transfected cells were incubated for 24 h before testing. POMC promoter activity was then measured in untreated cells and after stimulation with LIF (10 ng/ml) for 6 h. The relative increase of untreated vs. LIF-stimulated POMC promoter activity was calculated for AtT-20-S and AtT-20-M, respectively.

Immunoprecipitation and Western Blotting
Imunoprecipitation and Western blotting were performed as described (25). Briefly, after incubation, cells were washed with PBS (pH 7.0) and then with lysis buffer (50 mM HEPES, 150 mM NaCl, 10 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 2 mM sodium orthovanadate, pH 7.4). Cells were lysed in 500 µl of lysis buffer containing 1% Triton X-100, 1 mM phenylmethylsulfonylfluoride, 2 µg/ml aprotinin, 20 µM leupeptin. After a 15-min incubation at 4 C, the lysate was collected and centrifuged at 13,000 x g for 10 min at 4 C to remove insoluble material. Proteins of interest were immunoprecipitated with polyclonal anti-STAT3 or gp-130 antibodies coupled to protein A-Sepharose during 2 h at 4 C by gentle rocking. Immune complexes were collected by centrifugation, washed twice with a 30 mM HEPES buffer containing 30 mM NaCl, 0.1% Triton X-100, pH 7.4, and boiled for 5 min in 50 µl of sample buffer (4.5 mM Na2HPO4, 2.7% SDS, 9% glycerol, 10% bromophenol blue/ß-mercaptoethanol). Proteins were then separated by SDS-PAGE and electroblotted onto PVDF membranes. Membranes were incubated overnight at 4 C in saline buffer (20 mM Tris-HCl, 500 mM NaCl, pH 7.4, 0.1% Tween-20) containing 3% BSA and blotted with anti-phosphotyrosine antibody PY20 for 3 h at room temperature. Immunoreactive bands were detected by ECL immunodetection system. To reblot the membranes with either anti-STAT-3 or anti-gp130 antibody, membranes were stripped in 62.5 mM Tris-HCl (pH 6.7), 2% SDS, 100 mM ß-mercaptoethanol for 30 min at 50 C, washed several times in saline buffer, and blotted with the appropriate antibodies.

Statistical Analysis
Statistical analysis was performed by unpaired t test, and P < 0.05 was considered significant. All values are mean ± SE.


    ACKNOWLEDGMENTS
 
We gratefully acknowledge technical advice from Drs. T. Prezant and X. Zhang (Cedars-Sinai-Medical Center, Los Angeles, CA).


    FOOTNOTES
 
Address requests for reprints to: Dr. Shlomo Melmed, Division of Endocrinology & Metabolism, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, B-131, Los Angeles, California, 90048. E-mail: MELMED{at}CSHS.Org

This study was supported by a scholarship of the Deutsche Forschungsgemeinschaft (Au 139/1–1) and by NIH Grant DK-50238.

Received for publication February 9, 1998. Revision received March 20, 1998. Accepted for publication March 31, 1998.


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 INTRODUCTION
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 DISCUSSION
 MATERIALS AND METHODS
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