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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}, 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
(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. 1Go, 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. 1aGo). 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.

 
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. 1bGo). 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. 2Go, a–d). 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. 2aGo). 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. 2bGo). LIF stimulated SOCS-3 mRNA 9.3-fold ± 1, representing 100% SOCS-3 gene induction in the corresponding densitometric quantification (Fig. 2cGo). (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.

 
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. 2dGo). 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. 3Go). 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. 1Go), 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. 3Go). 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.

 
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. 4aGo). (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.

 
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. 4aGo). 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. 4bGo). 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. 4cGo). 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. 5aGo). 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. 5bGo). 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 1.5–2 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. 5cGo). 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-{gamma}-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).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). 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. 2Go, 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 2–4 h (Fig. 4bGo), consistent with the time course of cAMP-induced SOCS-3 promoter activity and SOCS-3 gene expression (Figs. 1aGo and 2aGo). In contrast, CREB, which is rapidly activated through a PKA-dependent serine phosphorylation (26), does not bind to this AP-1 element (Fig. 4bGo), 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. 1bGo). 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. 2Go, 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. 1Go and 2Go). 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. 3Go), 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. 3Go). 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. 4Go). 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. 4aGo), and its mutation significantly reduced (Bu)2 cAMP- and LIF+(Bu)2cAMP-induced SOCS-3 promoter activity (Fig. 4bGo) 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 1.5–2.5 h abrogated LIF-induced JAK2 kinase activity, STAT3 binding to the POMC promoter, and rat POMC promoter activity (Fig. 5Go). 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-{gamma} (IFN{gamma})-induced STAT1 and STAT3 DNA-binding activity in mononuclear cell cultures and T cells (40). Finally, in macrophages, VIP and PACAP inhibit IFN{gamma}-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{gamma}-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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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-{gamma}-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 8–12 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{gamma}, interferon-{gamma}; 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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 ABSTRACT
 INTRODUCTION
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 DISCUSSION
 MATERIALS AND METHODS
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