cAMP Neuropeptide Agonists Induce Pituitary Suppressor of Cytokine Signaling-3: Novel Negative Feedback Mechanism for Corticotroph Cytokine Action
Corinne Bousquet,
Vera Chesnokova,
Anastasia Kariagina,
Audrey Ferrand and
Shlomo Melmed
Department of Medicine (C.B., V.C., A.K., S.M.), Cedars-Sinai
Research Institute-University of California Los Angeles School of
Medicine, Los Angeles, California 90048; and Institut National de
la Santé et de la Recherche Médicale (INSERM) U531 (C.B.,
A.F.), Centre Hospitalo-Universitaire Rangueil, 31403 Toulouse,
France
Address all correspondence and requests for reprints to: Dr. Shlomo Melmed, Academic Affairs, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Room 2015, Los Angeles, California 90048. E-mail:
melmed{at}csmc.edu
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ABSTRACT
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The hypothalamo-pituitary-adrenal (HPA) axis maintains a
homeostatic response to stress, infection, or neoplasia. Inflammatory
cytokines, including leukemia inhibitory factor (LIF), stimulate the
HPA axis either directly at the pituitary corticotroph, or indirectly
through induction of CRH or sympathetic noradrenergic neurons, and
mediate the immuno-neuroendocrine interface. Unrestrained HPA axis
activation leads, however, to immunosuppression. Because suppressor of
cytokine signaling-3 (SOCS-3) is a potent inhibitor of LIF-activated
HPA axis, and dynamic interactions between hypothalamus-derived
cAMP-inducing neuropeptides and proinflammatory cytokines occur at the
corticotroph level, we investigated SOCS-3 expression in response to
peptides that stimulate cAMP including CRH, pituitary adenylate
cyclase-activating polypeptide, and epinephrine.
(Bu)2cAMP mediates induction of SOCS-3 promoter activity
(6.7-fold ± 0.5, P < 0.001) and SOCS-3 gene
expression (4-fold ± 0.8, P < 0.005) in a
PKA-dependent manner. LIF and cAMP-inducing agents are additive on
SOCS-3 promoter activity (22-fold ± 2.6, LIF +
(Bu)2cAMP vs. 7.3-fold ± 0.6, LIF
alone, P < 0.05) and on SOCS-3 transcription
(11.3-fold ± 2.1, LIF + (Bu)2cAMP vs.
9.3-fold ± 1, LIF alone, P < 0.05),
suggesting alternate pathways for LIF and cAMP-mediated corticotroph
signaling. Similarly, LIF and CRH or pituitary adenylate
cyclase-activating polypeptide are additive for SOCS-3 promoter
activity and transcription (P < 0.05). Whereas
signal transducer and activator of transcription 3 binding to
the SOCS-3 promoter mediates LIF action, several SOCS-3 promoter
regions containing cAMP-responsive elements are required for cAMP-PKA
effect. Thus, both classes of POMC-inducing agents, cytokines as well
as cAMP-inducing central peptides, regulate SOCS-3, providing a further
level of negative HPA axis control during inflammation. These results
indicate a sensitive intracellular autoregulation of corticotroph
function.
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INTRODUCTION
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THE HYPOTHALAMO-PITUITARY-ADRENAL
(HPA) axis is a central integrative system that governs stress
homeostasis by controlled peptide and steroid hormone secretion
(1, 2). Central and peripherally derived stressors
activate the neuroendocrine cascade in hypothalamic CRH-expressing
parvocellular neurons in the paraventricular nucleus (PVN)
(3). Under the control of brain stem and higher limbic
brain structures, the PVNs respond with CRH release into the venous
system, to act on pituitary corticotroph cells expressing POMC and
circulating ACTH. ACTH induces the antiinflammatory adrenal cortisol,
which antagonizes the action of HPA stressors, including immune
responses, which activate the HPA axis during infection or
inflammation. Immunoregulating cytokines play a pivotal role in the
bidirectional communication between the immune and neuroendocrine
systems (1, 4). During inflammation and infection,
proinflammatory cytokines, such as IL-6, leukemia inhibitory factor
(LIF), IL-1, or TNF
, produced by peripheral inflammatory monocytic
cells, or centrally in the hypothalamus and pituitary, directly or
indirectly mediate central hypothalamic and hypophyseal
neuroendocrine responses (1, 5).
We previously demonstrated that LIF potently stimulates in
vitro and in vivo pituitary POMC expression and ACTH
secretion, and synergizes with CRH action in corticotrophs
(6, 7, 8). Both independent and dependent LIF and CRH
signaling pathways converge at the level of the POMC promoter to
synergistically stimulate gene transcription (7, 9, 10).
By binding to its receptor (LIFR), LIF induces heterodimerization
between LIFR and gp130 subunits, with subsequent JAK-STAT (janus
kinase-signal transducer and activator of transcription)
pathway activation (5). STAT3 is the major STAT protein
activated by LIF in murine corticotroph cells and is critical for LIF
activation of the HPA axis (11). LIF-induced STAT3 binds
directly to the POMC promoter, or also mediates c-fos and
JunB transcription and binding to the POMC promoter (9).
In contrast, CRH binds to its seven transmembrane receptor, induces
cAMP and intracellular calcium, and subsequently activates several
transcription factors including Nurr, cAMP-responsive element binding
protein (CREB), c-fos, and JunB, which activate the POMC
promoter (12, 13, 14, 15).
Prolonged HPA axis activation leads to excess glucocorticoid
production and to a state of immune suppression that predisposes to
infection or tumor progression. A fine regulation of the HPA axis
activation is therefore essential to maintain appropriate
immuno-neuroendocrine homeostasis. CRH action is suppressed by
glucocorticoids, which negatively regulate hypothalamic CRH production
and also decrease CRH-induced pituitary POMC expression (4, 16). SOCS-3 (suppressor of cytokine signaling) acts as a potent
negative regulator of cytokine signaling (17) and of
LIF-induced POMC and ACTH activation (5). By inducing the
JAK-STAT3 pathway, LIF stimulates both POMC and SOCS-3 gene
transcription, SOCS-3, acting via an intracellular autocrine loop,
negatively regulates LIF signaling (18, 19) by directly
binding to the JAK/LIFR/gp130 (20) complex and inhibiting
JAK kinase activity (21).
In a physiological setting, corticotroph cells are exposed
simultaneously to hypothalamus-derived neuropeptides and to peripheral
proinflammatory cytokines, which both interact to produce ACTH
responses. Induction of POMC expression and ACTH secretion therefore
results from synergistic signals between hypothalamic cAMP agonists and
the gp-130 cytokine family (7, 10, 22). The potent
amplification of hypothalamic neuropeptide-induced HPA activation by
proinflammatory cytokines is controlled in a negative feedback loop by
the cytokine-dependent production of hypothalamic and pituitary SOCS-3
(23). We therefore investigated whether hypothalamic
neuropeptides also negatively impact on cytokine-induced HPA axis
activation by inducing SOCS-3. This mechanism would provide a further
level of control and negative regulation of the HPA axis during
inflammation. Our results demonstrate that, in addition to cytokines,
cAMP agonists, alone or in combination with LIF, stimulate SOCS-3
promoter activity and SOCS-3 gene expression, thus providing a further
level of negative autocrine POMC regulation.
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RESULTS
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(Bu)2cAMP Analog Stimulates SOCS-3 Promoter
Activity
Because SOCS-3 is a potent inhibitor of cytokine-mediated
HPA axis activation (5), and because dynamic interactions
occur between hypothalamus-derived cAMP-inducing neuropeptides and
proinflammatory cytokines at the level of the pituitary corticotroph
cells (1), we investigated whether SOCS-3 expression was
up-regulated by peptides that stimulate the cAMP pathway (Fig. 1
, a and b). The -2,757/+929 fragment of
the mouse SOCS-3 promoter was previously cloned and shown to drive
expression of the luciferase reporter in AtT20 cells (19).
(Bu)2cAMP (5 mM) modestly stimulated
(3.1-fold ± 0.6) SOCS-3 promoter activity after 3 h
incubation with SOCS-3 promoter-transfected cells, whereas LIF was
already maximally effective at this time point (6.3-fold ± 1.6),
as compared with 6 h and 24 h LIF treatment (7.3-fold ±
0.6 and 5.7-fold ± 0.6, respectively) (Fig. 1a
). However, longer
incubation with (Bu)2cAMP (up to 24 h)
showed increased SOCS-3 promoter activity (6.7-fold ± 0.5 at
6 h and 9.2-fold ± 0.3 at 24 h). Interestingly,
cotreatment with 1 nM LIF and 5 mM
(Bu)2cAMP additively (9.7-fold ± 2.8) at
3 h, or synergistically (22-fold ± 2.6 and 25.8-fold ±
4.2) at 6 h and 24 h, respectively, induced SOCS-3
promoter activation.

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Figure 1. cAMP Analogs and cAMP Agonists Stimulate SOCS-3
Promoter Activity
The -2,757/+929 fragment of the SOCS-3 promoter was fused to the
luciferase reporter gene in the pGL3 vector (Promega Corp.) and transiently transfected in AtT20 cells
(19 ). Cells were stimulated with 1 nM LIF
± 5 mM (Bu)2cAMP for 3, 6, or 24 h (a)
or, with 1 nM LIF ± 5 mM 8-br-cAMP, 10
nM CRH, 10 µM epinephrine (EpiN), or 100
nM PACAP (b) for 6 h. Luciferase activity was measured
in cell lysates in the presence of the luciferin substrate. Results are
expressed as fold-induction over control (control = nonstimulated
cells = 1). *, P < 0.05 and **,
P < 0.01 for LIF + cAMP agonist treatment
vs. LIF treatment. None of the agonists used stimulated
luciferase activity in pGL3-transfected AtT20 cells.
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To confirm these results with other cAMP analogs or natural
cAMP-inducing neuropeptides, SOCS-3 promoter-transfected AtT20 cells
were stimulated for 6 h with 1 nM LIF and/or 5
mM 8-bromo-cAMP (8-br-cAMP), 10 nM CRH, 10
µM epinephrine, or 100 nM pituitary adenylate
cyclase-activating polypeptide (PACAP) (Fig. 1b
). Whereas these
cAMP-inducing agents alone weakly induced SOCS-3 promoter activity
(1.7-fold ± 0.2 for 8-br-cAMP, 1.4-fold ± 0.1 for CRH,
1.4-fold ± 0.2 for epinephrine, and 1.3-fold ± 0.1 for
PACAP), an at least additive effect was observed when they were
coincubated with LIF (12.2-fold ± 2.3 for 8-br-cAMP,
10.8-fold ± 1.8 for CRH, 10.3-fold ± 1.8 for epinephrine,
and 10.2-fold ± 1 for PACAP). These results therefore demonstrate
that cAMP mediates stimulation of the SOCS-3 promoter activity, and
that cAMP-inducing agents enhance LIF action on this promoter.
cAMP Analogs Stimulate SOCS-3 Gene Expression
To confirm that cAMP-inducing agents stimulate SOCS-3 gene
expression, we performed Northern blots using a probe recognizing
SOCS-3 mRNA (Fig. 2
, ad). Cells were
first stimulated with 5 mM (Bu)2cAMP
for up to 5 h, and total RNA was extracted. SOCS-3 gene expression
was up-regulated as early as 0.5 h of treatment and persisted up
to 4 h with maximal stimulation at 2 h (Fig. 2a
). Cells were
therefore costimulated for 2 h with 1 nM LIF and/or 5
mM (Bu)2cAMP, 5 mM
8-br-cAMP, 100 nM PACAP, 10 nM CRH, or 10
µM epinephrine (Fig. 2b
). LIF stimulated SOCS-3 mRNA
9.3-fold ± 1, representing 100% SOCS-3 gene induction in the
corresponding densitometric quantification (Fig. 2c
).
(Bu)2cAMP, PACAP, epinephrine, CRH, and 8-br-
cAMP induced (P
0.02) SOCS-3 gene expression after
2 h (46.5% ± 9.3, 21.6% ± 6.1, 24.8% ± 4.3, 24.4% ± 6.6,
30% ± 9 of LIF effect). An additive effect was observed on SOCS-3
mRNA expression when cells were coincubated with both LIF and each cAMP
analog (130% ± 10.6, 125% ± 9.2, 123% ± 9.3, 114% ± 2.6, 127%
± 5, respectively; P < 0.05 vs. LIF
alone), confirming the results observed in the luciferase assay using
the SOCS-3 promoter.

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Figure 2. LIF and cAMP Agonists Additively Induce SOCS-3 mRNA
Expression
a and b, Total RNA (5 µg), extracted from nonstimulated (C), or from
1 nM LIF ± 5 mM
(Bu)2cAMP-treated AtT20 cells for 0.5, 1, 2, 3, 4, or
5 h (a), or from 1 nM LIF ± 5 mM
(Bu)2cAMP, 100 nM PACAP, 10 µM
EpiN, 10 nM CRH, or 5 mM 8-br-cAMP-treated
AtT20 cells for 2 h (b), were separated on a 1%
agarose/formaldehyde gel and transferred to nitrocellulose membrane.
Hybridization was performed with a 32P-labeled
double-stranded DNA probe corresponding to the SOCS-3 or ß-actin
coding sequence, as previously described (18 ). c, Northern
blot signals for SOCS-3 mRNA were analyzed by quantitative densitometry
and normalized for ß-actin. The relative increase (fold-induction) of
LIF ± cAMP agonists-induced SOCS-3 mRNA vs.
control (no treatment) was calculated from three independent
experiments (n = 3), and plotted as % of LIF effect (LIF
induction = 100%). *, P < 0.05 for LIF +
cAMP agonist treatment vs. LIF treatment. d, Northern
blot was performed as in panel b with 20 µg total RNA extracted from
murine pituitaries (8 animals per group) injected ip with 5 µg CRH or
vehicle.
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cAMP-mediated induction of SOCS-3 transcription was further confirmed
in vivo by ip injecting 5 µg CRH in C57BL6J mice. Eight
hours after CRH injection, total pituitary RNA was extracted and pooled
from eight mice, and SOCS-3 expression was assessed by Northern blot
(Fig. 2d
). A striking induction of SOCS-3 mRNA by CRH was observed in
the pituitary of CRH-injected mice, as compared with vehicle-injected
animals, which strengthens the in vitro observations showing
cAMP-inducing neuropeptides stimulating SOCS-3 expression.
cAMP-Mediated Stimulation of SOCS-3 Promoter Activity Is PKA
Dependent
cAMP analogs, as well as the cAMP-inducing neuropeptides CRH,
PACAP, and epinephrine, stimulate gene transcription through PKA
(24, 25), a serine/threonine kinase that activates CREB-,
fos-, and Jun-related factors (26, 27). Using wild-type
and dominant negative forms of PKA, we therefore investigated the PKA
dependency of cAMP-mediated induction of SOCS-3. Both wild-type and
mutated forms of PKA cDNA were transiently cotransfected in AtT20 cells
with murine SOCS-3 promoter-luciferase, and activity was measured after
LIF and/or (Bu)2cAMP, 8-br-cAMP, CRH, PACAP, and
epinephrine treatments (Fig. 3
). Further
induction of SOCS-3 promoter activity was observed when wild-type
PKA-transfected cells were stimulated with 8-br-cAMP, CRH, PACAP, and
epinephrine, alone or in combination with LIF, as compared with
nontransfected cells (Fig. 1
), whereas LIF alone did not further
enhance SOCS-3 promoter activity in these cells. Furthermore,
transfection of a dominant negative PKA form reduced cAMP-mediated
induction of SOCS-3 promoter activity by approximately 50%, and
restored SOCS-3 promoter activity to LIF-induced levels when cells were
stimulated with both LIF and each respective cAMP-inducing agent
(11.4-fold ± 0.9 for LIF + (Bu)2cAMP,
9.7-fold ± 1.4 for LIF + 8-br-cAMP, 9.4-fold ± 1.5 for LIF
+ CRH, 8.1-fold ± 1.1 for LIF + PACAP, and 7.4-fold ± 0.5
for LIF + epinephrine, vs. 7.4-fold ± 0.3 for LIF
alone) (Fig. 3
). These results therefore demonstrate that cAMP
induction of SOCS-3 promoter activity is a PKA-mediated event.

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Figure 3. PKA Transduces cAMP-Mediated Activation of the
SOCS-3 Promoter Activity
The -2,757/+929 fragment of the SOCS-3 promoter was fused to the
luciferase reporter gene and transiently transfected in AtT20 cells
(19 ). cDNA (0.5 µg) encoding either wild-type or a
mutated form of PKA was cotransfected with the SOCS-3 promoter plasmid.
Cells were then stimulated with 1 nM LIF ± 5
mM (Bu)2cAMP, 5 mM 8-br-cAMP, 10
nM CRH, 100 nM PACAP, or 10 µM
EpiN for 6 h. Luciferase activity was measured in cell lysates in
the presence of the luciferin substrate. Results are expressed as
fold-induction over control (control = nonstimulated cells =
1). *, P < 0.05 for treatment with wild-type PKA
cDNA-transfected cells vs. mutated PKA cDNA-transfected
cells.
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Delineation of cAMP-Responsive Elements on the Murine
SOCS-3 Promoter
To explore whether a specific DNA motif on the
murine SOCS-3 promoter transduces cAMP-induced SOCS-3 promoter
activity, luciferase constructs comprising progressive 5'-deletions of
the SOCS-3 promoter (19) were transiently transfected in
AtT20 cells, and the corresponding luciferase activity was measured
after treatment with (Bu)2cAMP for 6 h (Fig. 4a
). (Bu)2cAMP
stimulated SOCS-3 promoter activity 6.2-fold ± 0.8 when the full
SOCS-3 promoter (-2,757/+929) was used (clone 6), whereas a weak
effect was observed with the empty vector lacking the SOCS-3 promoter
(pGL3) (2-fold ± 0.2). Progressively deleting the SOCS-3 promoter
reduced (Bu)2cAMP-induced SOCS-3 promoter
activity, between constructs 6T1 (-1,862/+929) and 6T2 (-855/+929)
(6.2-fold ± 0.6 and 5-fold ± 0.3, respectively,
P = 0.05), between constructs 6T2 and 6T3 (-159/+929)
(5-fold ± 0.3 and 4.2-fold ± 0.2, respectively;
P = 0.09), or between constructs 6T3 and 6T4
(-61/+929) (4.2-fold ± 0.2 and 3.4-fold ± 0.3,
respectively; P = 0.03). We therefore conclude that
different SOCS-3 promoter regions mediate cAMP induction of this
promoter and that several DNA-binding elements might be involved.

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Figure 4. Identification of cAMP-Responsive Regions on the
SOCS-3 Promoter
a, Progressive 5'- deletions of the SOCS-3 promoter were fused to the
luciferase reporter gene and transiently transfected in AtT20 cells
(19 ). Cells were stimulated with 5 mM
(Bu)2cAMP for 6 h. Luciferase activity was measured in
cell lysates in the presence of the luciferin substrate. Results are
expressed as fold-increase over control (no treatment, control =
1) (n 3). b, Clone 8 and clone 8-AP-1 mut (-106
gTgAcTAA- 99 mutated to AAgcTTAA)
were transiently transfected in AtT20 cells. Cells were stimulated with
1 nM LIF, 5 mM (Bu)2cAMP
or LIF+(Bu)2cAMP for 6 h and corresponding
luciferase activity was measured. Results are expressed as % of LIF-,
(Bu)2cAMP-, or LIF+(Bu)2cAMP-induced
SOCS-3 promoter activity (100%). *, P < 0.01 for
clone 8-AP-1 mut vs. clone 8 (n 3). c, A
32P-labeled double-stranded oligonucleotide
corresponding to region -116/-86 of the SOCS-3 promoter (5'-gTg cAg
AgT AgT gAc TAA AcA TTA cAA gAA g-3') was used as probe. EMSA was
performed using 10 µg nuclear extracts from treated AtT20 cells.
Cells were either untreated (C) or treated with 5
mM (Bu)2cAMP for 1, 2, 4,
or 6 h. Using 10 µg nuclear extracts from 2 h
(Bu)2cAMP-treated AtT20 cells, competition was
performed with either 100-fold molar excess of cold -116/-86
double-stranded oligonucleotide (self), of cold mutated -116/-86
double-stranded oligonucleotide (mut: 5'-gTg cAg AgT
cgg
gcA TTA AcA TTA cAA gAA
g-3'), or with 1 µg anti-c-fos (c-fos Ab), anti-JunB (JunB
Ab), anti-phospho-CREB (P-CREB), or anti-c-fos + anti-JunB
(c-fos + JunB Ab) antibodies.
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PKA-mediated cAMP action is transduced through activation of
transcription factors such as CREB, Jun, or fos, which bind to
consensus DNA-binding sequences (26, 27). Using the
MatInspector program www.gsf.de/cgi-bin/matsearch2.pl, we noted
several of these elements, designated as CRE or AP-1, present on
different regions of the SOCS-3 promoter. An AP-1 motif is contained
within nucleotides (-105/-99) of the SOCS-3 promoter. This element is
present in the clone 8 construct (-273/+160), which shows high
(Bu)2cAMP-induced SOCS-3 promoter activity
(8.2-fold ± 0.5) (Fig. 4a
). This AP-1 motif was therefore mutated
in clone 8 (clone 8-AP-1 mut), transiently transfected in AtT20 cells,
and the corresponding LIF-, (Bu)2cAMP-, and
LIF+(Bu)2cAMP-induced SOCS-3 promoter activity
was measured (Fig. 4b
). Mutation of the (-105/-99) AP-1 site reduced
(Bu)2cAMP- and
LIF+(Bu)2cAMP-induced SOCS-3 promoter activity by
38% and 31% (P < 0.01), respectively, whereas the
LIF effect was not altered. These results confirm a role for the
(-105/-99) AP-1 element in
(Bu)2cAMP-induced SOCS-3 promoter
activity.
To investigate whether (Bu)2cAMP induces specific
CREB, Jun, or fos-related transcription factor binding on the SOCS-3
promoter, we performed an EMSA using the -116/-86 region, which
comprises the (-105/-99) AP-1 site (Fig. 4c
). An inducible complex
was observed as early as 1 h after (Bu)2cAMP
treatment and was maximal at 2 and 4 h. Specificity of this
complex was demonstrated by showing abrogation with 100-fold molar
excess of cold probe (self), but not with the same probe mutated at the
AP-1 site (mut). Furthermore, c-fos and JunB, but not
phospho-CREB, were shown to be components of this complex, as addition
of c-fos or JunB antibody alone reduced the complex
formation, while both antibodies together abrogated the complex. A
phospho-CREB antibody did not alter binding. We therefore conclude that
Jun and fos-related transcription factors bind to the -105/-99
(TgAcTAA) element, which might transduce a component of the observed
cAMP-mediated SOCS-3 activation.
cAMP-Induced SOCS-3 Inhibits LIF Corticotroph Action
We previously demonstrated that LIF activation of POMC gene
transcription and ACTH secretion is transduced through LIF-induced JAK2
kinase activity and STAT3 binding to the POMC promoter
(9). To explore whether cAMP-induced SOCS-3 is
functionally active for inhibition of cytokine signaling, we used AtT20
cells pretreated for up to 3 h with 5 mM
(Bu)2cAMP and then further treated with 1
nM LIF. To explore LIF-induced JAK2 kinase activity, a
kinase assay was performed with anti-JAK2 immunoprecipitates and showed
a potent induction of JAK2 kinase activity when AtT20 cells were
stimulated for 5 min with LIF, but not with
(Bu)2cAMP (Fig. 5a
). However, cell pretreatment for up to
3 h with 5 mM (Bu)2cAMP markedly
reduced LIF-induced JAK2 kinase activity with maximal suppression after
2 h of cell pretreatment with (Bu)2cAMP. LIF-induced
STAT3 binding to the POMC promoter was explored in an EMSA using
nuclear extracts from (Bu)2cAMP-pretreated AtT20
cells (Fig. 5b
). Whereas specific complexes were not observed in the
nonstimulated or 0.5 h (Bu)2cAMP-treated
cells, 0.5 h LIF cell treatment, without
(Bu)2cAMP pretreatment (0), induces a specific
complex that was abrogated after addition of 100-fold molar excess cold
wild-type probe, but not with the cold mutated probe. Furthermore,
abrogated LIF-induced STAT3 complex formation was observed when
cells were preincubated for 1.52 h with
(Bu)2cAMP. We therefore conclude that AtT20 cell
pretreatment with (Bu)2cAMP prevents LIF induction of
the JAK-STAT3 pathway and therefore contributes to antagonize
LIF corticotroph action. This was confirmed in a luciferase assay using
the rat POMC promoter fused to the luciferase reporter (7)
and transiently transfected in AtT20 cells (Fig. 5c
). As previously
described (9), 6 h LIF treatment stimulated rat POMC
promoter activity 4.6-fold ± 0.2 (100%), whereas cell
pretreatment with 5 mM (Bu)2cAMP markedly
attenuates LIF-induced POMC promoter activity maximally at 2.5 h
(59% ± 8), 2 h (57% ± 3), and 1.5 h (60% ± 2) of cell
pretreatment. Because (Bu)2cAMP induces SOCS-3 in
these cells, we hypothesized that cAMP-mediated inhibition of LIF
action is SOCS-3 dependent.

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Figure 5. Cell Pretreatment with (Bu)2cAMP
Inhibits LIF-Induced JAK2 Kinase Activity, STAT3 Binding to the POMC
Promoter, and POMC Promoter Activity
a, JAK2 kinase activity was measured after AtT20 cell treatment
for 5 min with 1 nM LIF, with or without pretreatment with
5 mM (Bu)2cAMP. Upper
panel, Protein lysates were immunoprecipitated with an
anti-JAK2 antibody and used in a kinase assay with
32P- -ATP. Proteins were then separated in 10% SDS-PAGE,
and corresponding JAK2-incorporated radioactivity was analyzed by
autoradiography of the dried gel. Lower panel, To
confirm equal anti-JAK2 immunoprecipitation in each reaction tube,
anti-JAK2 immunoprecipitates were directly assessed in a Western blot
experiment using the anti-JAK2 antibody. b, A 32P-labeled
double-stranded oligonucleotide corresponding to region -407/-374 of
the POMC promoter
(5'--407TAGTGATATTTACCTCCAAATGCCAGGAAGGCAG-374-3')
was used as probe, as previously described (9 ). EMSA was
performed using 15 µg nuclear extracts from treated AtT20 cells.
Cells were either untreated (C) or treated with 5 mM
(Bu)2cAMP for 0.5 h [(Bu)2cAMP
0.5], or pretreated with 5 mM
(Bu)2cAMP for 0, 0.5, 1, 1.5, 2, 2.5, or 3
h, and then treated with 1 nM LIF for 0.5 h. Using 15
µg nuclear extracts from 0.5 h LIF-treated AtT20 cells,
competition was performed with 100-fold molar excess of cold
(-407/-374) wild-type probe (self). c, Rat POMC promoter was fused to
the luciferase reporter gene (9 ) and transiently
transfected in AtT20 cells (19 ). Cells were pretreated or
not for the indicated times with 5 mM
(Bu)2cAMP and then stimulated for 6 h with 1
nM LIF. Luciferase activity was measured in cell lysates
and results were expressed as percent of LIF-induced POMC promoter
activity without (Bu)2cAMP pretreatment (100%).
*, P < 0.05 and **, P < 0.01 for
(Bu)2cAMP-pretreated and LIF-stimulated AtT20
cells vs. LIF-only stimulated cells (n 3).
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DISCUSSION
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We previously demonstrated potent LIF induction of
STAT1/3-dependent SOCS-3 promoter activity and SOCS-3 gene expression
(18, 19). We here describe a novel regulation of SOCS-3
transcription that involves activation of the cAMP/PKA pathway. Because
SOCS-3 negatively impacts on cytokine signaling, hypothalamic
neuropeptides that stimulate cAMP transduction might therefore act as
potential negative regulators of the proinflammatory cytokine-mediated
activation of the HPA axis.
Interestingly, LIF-induced SOCS-3 promoter activity is maximal as early
as 3 h after LIF exposure whereas (Bu)2cAMP
is more potent at 6 h and up to 24 h of stimulation (Fig. 1
).
Similarly, SOCS-3 gene expression is already stimulated at 30 min of
LIF treatment (18), whereas
(Bu)2cAMP maximal activity occurs in
vitro at 2 h and in vivo at 8 h (Fig. 2
, a
and d). Different signalings are indeed required for both agents
activity. LIF induces STAT3 tyrosine phosphorylation, which occurs
rapidly within 15 min of LIF stimulation (11). Conversely,
cAMP-induced fos- and Jun-related transcription factors, the production
of which depend on a transcription/translation mechanism, bind
maximally to the SOCS-3 promoter -105/-99 AP-1 site within 24 h
(Fig. 4b
), consistent with the time course of cAMP-induced SOCS-3
promoter activity and SOCS-3 gene expression (Figs. 1a
and 2a
). In
contrast, CREB, which is rapidly activated through a PKA-dependent
serine phosphorylation (26), does not bind to this AP-1
element (Fig. 4b
), but might transduce cAMP-mediated induction of
c-fos and JunB. Binding of cAMP-induced CREB to another CRE
or AP-1 element on the SOCS-3 promoter cannot, however, be
excluded.
(Bu)2cAMP induction of SOCS-3 promoter activity
was further confirmed using 8-br-cAMP and endogenous cAMP-inducing
neuropeptides, CRH, epinephrine, and PACAP (Fig. 1b
). CRH, epinephrine,
and PACAP are hypothalamic-derived factors that stimulate POMC
transcription in AtT20 cells (24, 25). After inflammation
or infection, CRH and PACAP are induced in the PVN of the hypothalamus
(1, 28), and epinephrine is induced in noradrenergic
neurons of the brain sympathetic system (29). CRH and
noradrenergic neurons of the central stress system innervate and
stimulate each other by positive feedback, such that activation of one
system leads to activation of the other (30). These
neuropeptides directly or indirectly stimulate pituitary POMC
transcription and ACTH secretion. Strikingly, CRH injection induces
mouse pituitary SOCS-3 gene expression in vivo, which
gives a physiological relevance for SOCS-3 induction by CRH.
Furthermore, CRH, PACAP, and epinephrine induced similar effects on
SOCS-3 promoter activity and gene transcription as observed with
(Bu)2cAMP (Fig. 2
, b and c). SOCS-3 promoter
activity and gene expression were not as potently induced by the cAMP
analogs alone as by LIF, but, surprisingly, a consistent additive
effect was observed between the cAMP-inducing ligands and LIF
(Figs. 1
and 2
). Furthermore, enhanced SOCS-3 induction by CRH,
epinephrine, PACAP, and 8-br-cAMP was observed when the wild-type PKA
cDNA was cotransfected in AtT20 cells (Fig. 3
), which confirmed a PKA
mediation of their effects and indicated alternate transduction
pathways for LIF- and cAMP-mediated induction of SOCS-3. Cell
transfection with a dominant negative form of PKA consistently
abrogated cAMP analog-induced, but not LIF-induced, SOCS-3 promoter
activation, which supports the STAT dependence of LIF action
(19) (Fig. 3
). PKA mediates CRH, epinephrine, or PACAP
action on pituitary POMC and other genes (24, 25, 31, 32).
To inhibit endogenous PKA, we used a dominant negative PKA form, rather
than a chemical inhibitor of PKA such as H89, because of the potential
nonspecificity and toxicity of H89.
Using progressive deletions of the SOCS-3 promoter, we showed that
different regions are cAMP responsive, in contrast to the
STAT-dependent LIF action, which depends on a single STAT binding site
on the SOCS-3 promoter (19) (Fig. 4
). This is consistent
with the presence of multiple cAMP-responsive DNA binding elements or
CRE/AP-1 sites on the SOCS-3 promoter. The SOCS-3 promoter -105/-99
AP-1 site was chosen for EMSA, as this region showed potent
responsiveness to (Bu)2cAMP in terms of SOCS-3
promoter activity (clone 8, Fig. 4a
), and its mutation significantly
reduced (Bu)2 cAMP- and LIF+(Bu)2cAMP-induced SOCS-3
promoter activity (Fig. 4b
) and because it is localized only 35 bp
upstream of the LIF-induced STAT binding element. Demonstration of
c-fos and JunB binding to this AP-1 site identifies these
transcription factors as potential mediators of cAMP-induced SOCS-3
promoter activity, but does not exclude involvement of other
transcription factors at this AP-1 site or at other AP-1 sites of the
SOCS-3 promoter. Fos and Jun-related transcription factors are
activated not only by the cAMP/PKA pathway (33, 34, 35), but
also by MAPK-activating signals (36). In our system, MAPK
involvement in cAMP induction of SOCS-3 was excluded by showing the
ineffectiveness of the MEKK1 inhibitor, PD98059, to inhibit
cAMP-mediated action (data not shown). Interestingly, recent reports
describe a MAPK-mediated activation of SOCS-3 transcription by PMA or
bFGF (37). However, in this latter study, the responsive
elements on the SOCS-3 promoter were not yet identified.
To investigate whether cAMP-induced SOCS-3 is functionally relevant for
inhibition of cytokine induction of the JAK-STAT pathway, AtT20 cells
were pretreated with (Bu)2cAMP to induce SOCS-3
and then stimulated with LIF. LIF induction of the JAK-STAT3 pathway
was assessed in a JAK2 kinase assay, in an EMSA using the recently
described STAT3 binding site on the POMC promoter (9), and
in a luciferase assay using the rat POMC promoter driving expression of
the luciferase reporter. Pretreatment with
(Bu)2cAMP for 1.52.5 h abrogated LIF-induced
JAK2 kinase activity, STAT3 binding to the POMC promoter, and rat POMC
promoter activity (Fig. 5
). These kinetics are concordant with
(Bu)2cAMP induction of SOCS-3 mRNA expression and
with the stability of the SOCS-3 protein, which is rapidly degraded by
the proteasome pathway in AtT20 cells (21).
Interestingly, cAMP-mediated down-regulation of cytokine-induced
JAK-STAT has been extensively described.
(Bu)2cAMP attenuated STAT1 activation in rat
stellate cells stimulated with PDGF/BB without reducing STAT1 protein
levels, and subsequently blocked cell proliferation (38).
8-Br-cAMP inhibited IL-6-induced JAK1 kinase activity, STAT3
tyrosine phosphorylation, and STAT3 DNA-binding activity in mononuclear
cells, by requiring new RNA and protein synthesis (39).
8-Br-cAMP also inhibited both interferon-
(IFN
)-induced STAT1 and
STAT3 DNA-binding activity in mononuclear cell cultures and T cells
(40). Finally, in macrophages, VIP and PACAP inhibit
IFN
-induction of JAK1/JAK2/STAT1 phosphorylation, thereby acting as
peripheral antiinflammatory agents (41).
The cAMP signaling pathway is typically triggered by noncytokine
ligands, and inhibition of the cytokine-mediated JAK-STAT activation by
this pathway adds a level of complexity to the multiple cross-talks
already described between major endocrine signaling routes. Strikingly,
SOCS-3 might be involved in cAMP-mediated down-regulation of JAK-STAT
activity, although further experiments, using cells that overexpress a
gp130 subunit mutated at the SOCS-3 binding site (Y757) and in which,
subsequently, SOCS-3 cannot act as a negative regulator of JAK kinase
activity (20), are required to confirm this.
SOCS-3-mediated inhibition of the cytokine-induced JAK-STAT pathway has
thus far only been described for SOCS-3 induced by other cytokines
(42, 43, 44), or recently by the MAPK pathway
(37). Therefore, we here describe a novel regulation of
SOCS-3 expression by the cAMP pathway. Recently, PACAP was shown not to
stimulate macrophage SOCS-3 gene mRNA, although PACAP inhibits the
IFN
-induced JAK/STAT pathway in these cells (41). In
this study, however, we observed a significant effect of PACAP
on SOCS-3 gene expression, which was more striking when the SOCS-3
promoter-luciferase construct was used in the luciferase assay, when
cells were coincubated with LIF, or when the wild-type form of PKA was
cotransfected. The observed differences might result from the use of
different cytokines, cell lines, or peptide concentrations.
In the periphery, cAMP-inducing agents act as antiinflammatory agents
by inhibiting cytokine-induced JAK/STAT pathway and therefore
cytokine-mediated inflammation (41). In the anterior
pituitary, by inducing SOCS-3, these neuropeptides might counteract the
cytokine-induced HPA axis. Cytokines that stimulate corticotroph POMC
expression and ACTH secretion are produced peripherally or locally, in
the hypothalamus or pituitary, after infection or inflammation
(1). By stimulating the HPA axis, they antagonize their
own peripheral proinflammatory action. Excessive HPA axis stimulation
leads to immunosuppression and, therefore, increased susceptibility to
infection. Hypothalamic cAMP-inducing neuropeptides, such as CRH,
PACAP, or epinephrine, the production of which is also enhanced after
stress stimuli, might therefore, by stimulating SOCS-3 expression,
counterbalance the excessive stimulatory effect of cytokine on the HPA
axis and maintain immuno-neuroendocrine homeostasis.
 |
MATERIALS AND METHODS
|
---|
Culture of AtT20 Cells
AtT20 cells were grown in DMEM supplemented with 10% FBS, 2
mM L-glutamine, 100 U/ml streptomycin, 100 U/ml
penicillin, and 0.25 µg/ml Amphotericin (Life Technologies, Inc., Gaithersburg, MD).
Transient Transfection and Luciferase Assay
AtT20 cells were plated in 2-ml dishes (100,000 cells per well)
and allowed to adhere for 16 h. Cells were transiently transfected
as described previously (10) with 0.5 µg per well of the
murine SOCS-3 promoter (-2,757/+929), or of 5'-deleted constructs of
the SOCS-3 promoter (19), or of the rat POMC promoter
(7), fused to the luciferase reporter gene, and with 0.5
µg of pSV-lacZ (Promega Corp., Madison, WI), expressing
ß-galactosidase as an internal transfection control. Twenty-four
hours after transfection, cells were treated with 1 nM LIF
and/or 5 mM (Bu)2cAMP, 5
mM 8-br-cAMP, 100 nM PACAP, 10 nM
CRH, or 10 µM epinephrine in triplicate for up to 24
h. Cells were then lysed in lysis buffer and subjected to assay for
luciferase (10) or ß-galactosidase activity
(Promega Corp.). Transfection efficiency was also tested
by a dot-blot with genomic DNA extracted from each well. Hybridization
of the corresponding membrane with a ß-galactosidase gene probe
confirmed equal expression of this gene in each well.
JAK2 Kinase Assay
AtT20 cells were plated in 10-ml dishes (500,000 cells per dish)
and allowed to adhere for 48 h. Cells were pretreated or not with
5 mM (Bu)2cAMP for the indicated times and then
treated with 1 nM LIF for 5 min. Cell lysis and
immunoprecipitation were performed as previously described
(21) for 2 h at 4 C using 5 µl/immunoprecipitation
of the polyclonal anti-JAK2 antibody (Upstate Biotechnology, Inc., Lake Placid, NY). Immunoprecipitates were washed twice in
50 mM HEPES, pH 7.5, 150 mM NaCl, 10
mM EDTA, 10 mM
Na2P2O7,
100 mM NaF, 2 mM
Na3VO4, 1% NP40, and three
times in TBS (25 mM Tris, pH 7.5, 150 mM NaCl).
Immunoprecipitates were then dried and 20 µl of kinase buffer were
added (10 mM MnCl2, 5 mM
MgCl2, 0.1 mM
Vn2+, 10 mM Tris, pH 7.4). Twenty
microliters of 32P-
-ATP (3.75
µM, 10 µCi/point) were then added and samples were
incubated for 10 min at 37 C. Reactions were stopped by the addition of
19.3 µl of 100 mM Tris, pH 6.8, 12% SDS, and 7.7 µl of
bromophenol blue/ß-mercaptoethanol (vol/vol). Proteins were then
separated on a 10% SDS-PAGE. Proteins were fixed in the gel, which is
then dried and exposed for autoradiography.
EMSA
For nuclear extract preparations, AtT20-treated cells were
harvested in cold PBS buffer. Nuclear extracts were prepared as
described (45). Final concentrations were typically 3
µg/µl, as determined by protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). For EMSA, a double-stranded DNA
oligonucleotide was used as a probe. Fifteen micrograms of nuclear
extracts and 10 pg 32P-end-labeled DNA probe
(50,000 cpm) were used per reaction. Nuclear extracts were preincubated
for 15 min at room temperature in 20 µl of binding buffer (10
mM HEPES, pH 7.9, 80 mM NaCl, 1 mM
EDTA, 1 mM dithiothreitol, 10% glycerol) with 2 µg of
poly(dI-dC). 32P-Labeled probe was added, and the
binding reaction was allowed to proceed at room temperature for 20 min.
In competition experiments, 100-fold molar excess of unlabeled cold
competitor oligonucleotide was added to the preincubation reaction.
Antibody competitions were performed using 1 µg of each antibody,
added to the preincubation reaction for 1 h at 4 C. The
protein-DNA complexes were resolved on a 6% polyacrylamide gel in
0.5x Tris borate/EDTA. Gels were dried and autoradiographed.
Northern Blot
Total RNA were prepared from AtT20 cells or from mouse
pituitaries using TRIzol reagent (Life Technologies, Inc.). Five micrograms of RNA/lane were size fractionated under
denaturing conditions using formaldehyde-agarose (1%) gel, transferred
to nitrocellulose, and hybridized using
32P-labeled SOCS-3 (18) or
ß-actin cDNA.
Animal Injection
Female C57BL6J mice from 812 wk of age were injected ip with 5
µg rat CRH or vehicle per animal (Bachem/Peninsula Laboratories, Inc., San Carlos, CA). Immediately after
death, pituitaries were extracted.
Animal studies were conducted in accord with the principles and
procedures outlined in "Guidelines for Care and Use of Experimental
Animals."
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Dr. G. S. McKnight
(mcknight@u.washington.edu) for providing the cDNA encoding for
the wild-type and dominant negative form of PKA, and to Dr. C. J.
Auernhammer for providing the murine SOCS-3 promoter luciferase
constructs.
 |
FOOTNOTES
|
---|
This work was supported by NIH Grant R01-DK-50238 and the Doris Factor
Molecular Endocrinology Laboratory.
Abbreviations: 8-br-cAMP, 8-Bromo-cAMP; CREB, cAMP response
element binding protein; HPA, hypothalamo-pituitary-adrenal; IFN
,
interferon-
; JAK, janus kinase; LIF, leukemia-inhibitory factor;
LIFR, LIF receptor; PACAP, pituitary adenylate cyclase-activating
polypeptide; PVN, paraventricular nucleus; SOCS-3, suppressor of
cytokine signaling-3; STAT, signal transducer and activator of
transcription.
Received for publication December 19, 2000.
Accepted for publication August 3, 2001.
 |
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