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
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ABSTRACT
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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.
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INTRODUCTION
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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.
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RESULTS
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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. 1
). In contrast, SOCS-2
mRNA levels were only modestly increased by LIF and IL-1ß, while CIS
mRNA levels were not significantly altered (Fig. 1
). 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. 1
and 2
). SOCS-3 mRNA levels remained elevated
after stimulation with both cytokines for as long as 8 h (Figs. 1
and 2
). The effective concentration range of LIF was 0.110 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. 3
).

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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. 2A ) or
10 ng/ml IL-1-ß (Fig. 2B ) for 0.58.0 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 0.0110.0 ng/ml LIF for 45 min and with
0.0110.0 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. 1 and 2 ). 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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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. 4
).
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. 4B
). In experiments with LIF
injection, untreated control animals were also killed at 0 min (Fig. 4A
). As described previously (10), systemic LIF injection caused a
significant rise of serum ACTH and corticosterone levels above baseline
(Fig. 4A
). 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. 4A
). 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. 4B
). 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.
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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. 5
). 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 (0.110.0 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.
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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. 6
, 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. 6C
).

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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.
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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. 7
).
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DISCUSSION
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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.
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MATERIALS AND METHODS
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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 (0.110.0 ng/ml) was
then added for a subsequent 2448 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
manufacturers instructions. Autoradiographs were exposed to Kodak
Biomax MS film for 1224 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 (19610 bp; GeneBank
accession number U88328; 20-bp primers), the murine SOCS-2 cDNA
(211940 bp; GeneBank accession number U88327; 20-bp primers), and the
murine CIS cDNA (181740 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
manufacturers 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 812 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 (15762 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
manufacturers 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.
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ACKNOWLEDGMENTS
|
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We gratefully acknowledge technical advice from Drs. T. Prezant
and X. Zhang (Cedars-Sinai-Medical Center, Los Angeles, CA).
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FOOTNOTES
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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 139/11) 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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