NF-kappaB participates in the corticotropin-releasing, hormone-induced regulation of the pituitary proopiomelanocortin gene.

Corticotropin-releasing hormone is a main regulator of mammalian stress response by stimulating pituitary proopiomelanocortin (POMC) gene expression, and thus adrenocorticotropic hormone (ACTH) secretion, which then causes glucocorticoid release from the adrenal. In a recent study in the pituitary corticotroph cell line AtT20, oxidative stress stimulated the activity of nuclear transcription factor B (NF-kappaB), whereas corticotropin-releasing hormone (CRH) inhibited both the constitutive and the oxidative stress-induced NF-kappaB DNA-binding activity. To further investigate the role of NF-kappaB on the CRH-induced pituitary POMC gene activation, AtT20 cells were transiently transfected with a POMC-luciferase construct mutated at an NF-kappaB binding site. After treatment with CRH, intracellular POMC-luciferase activity was significantly higher from the stimulation observed with transfection of the parental POMC-luciferase construct. In agreement with a previous report, CRH inhibited the constitutive NF-kappaB DNA-binding activity in AtT20 cells, as shown by electrophoretic mobility-shift assay, as soon as within 15 min of treatment. These effects of CRH were blocked by the CRH-R1 antagonist CP154,256. Our findings provide evidence that the regulation of corticotroph NF-kappaB activity by CRH is related to the activation of the pituitary POMC gene and, thus, may play an important role in stress response.

Hypothalamic corticotropin-releasing hormone (CRH) 1 acts as a major mediator of the mammalian stress response by stimulating pituitary proopiomelanocortin (POMC) gene expression and adrenocorticotropic hormone (ACTH) secretion that, in turn, stimulates release of glucocorticoid from the adrenal gland (1). CRH stimulates POMC gene transcription in pituitary corticotrophs through cAMP and calcium-mediated events (2). Positive and negative regulation of the POMC gene has been described and shown to be mediated by several transcription factors such as AP-1 (3), Nurr77 (4), Ptx1 (5), and glucocorticoid receptor (6).
The Crh-deficient (Crh Ϫ/Ϫ ) mouse has normal basal circulat- ing ACTH levels, but shows no increase in ACTH secretion following restrain stress (7). This fact suggests that, although CRH is not required for a normal basal pituitary POMC gene expression, it is necessary for the stress-induced regulation of the POMC gene. Activation of the pituitary POMC gene by CRH is mediated by CRHR1, the receptor subtype expressed in corticotroph cells, as exemplified by the inability of Crfr1 Ϫ/Ϫ mice to respond to stress by increased levels of ACTH (8,9). Although it has been well shown that binding of CRH to CRHR1 increases cAMP levels, the cascade of events between cAMP production and POMC gene activation has not yet been elucidated. It has been shown previously that the transcription factor NF-B is expressed in the brain, and its DNA binding activity is inhibited by CRH in hippocampal neurons as well as in a pituitary corticotroph cell line, the AtT20 cells (10). This finding has been associated with the neuroprotective effects of CRH during hypoxia. We have investigated the possibility of a biologically significant role for NF-B expressed in pituitary corticotrophs by studying the role of NF-B in the regulation of the pituitary POMC gene by CRH. We show that inhibition of the pituitary NF-B DNA-binding activity is required for the transcriptional activation of the POMC gene by CRH in corticotroph cells. Therefore, we propose that the CRH-mediated inhibition of NF-B DNA-binding activity is required for the pituitary corticotroph hormonal response to stress.

EXPERIMENTAL PROCEDURES
Cell Culture-The AtT20 mouse pituitary corticotroph cell line was cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml streptomycin, and 100 units/ml penicillin (Invitrogen). On the day of the experiment, the media were replaced with serum-free media, and the cells were treated with CRH, LPS, H 2 O 2 , dexamethasone, antalarmin (a CRH receptor antagonist), the PKA inhibitor H89, or the PKC inhibitor GF109203X, as indicated in the figure legends. CRH and antalarmin were generous gifts of Dr. Chrousos, NICHD, National Institutes of Health, GF109203X was purchased from Calbiochem, and all other chemicals were purchased from Sigma.
Mutagenesis and Plasmid Construction-Site-directed mutagenesis was performed by using the Transformer site-directed mutagenesis kit (Clontech) according to the manufacturer's instructions. The sequence of the NF-B binding site on the POMC promoter was, for the NF-B binding site, GGGAAGCCCC, and for the mutated binding site, GG-GAAGAACC. The mutated plasmid was sequenced for verification.
Transfection of AtT20 Cells and Luciferase-Renilla Assays-AtT20 cells were placed in 24-well plates and allowed to adhere for 24 h. All the transfection studies were performed in phenol-free media. Cells were transiently transfected with a plasmid containing the full-length POMC promoter driving the luciferase reporter gene (kindly provided by Dr. Melmed, UCLA/Cedars Sinai Medical Center) (11) or with a plasmid containing a mutated POMC promoter at the NF-B binding site, as described above. Cells were also co-transfected with a plasmid containing the CMV promoter driving the Renilla reporter gene. After cell lysis, luciferase and Renilla activities were measured by using the Dual luciferase reporter assay system (Promega, Madison, WI), according to the manufacturer's instructions, using a Lumat LB 9501 luminometer (PerkinElmer Life Sciences). * This work was supported by National Institutes of Health Grant RO1 DK04777 (to K. P. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

RNA Extraction and Northern Blot
Hybridization-AtT20 cells were plated in six-well tissue culture dishes and allowed to adhere for 24 h. Cells were transiently transfected with a plasmid containing the fulllength cDNA of human IB␣, subcloned in Invitrogen PCDNA3 vector at the EcoRI site, kindly provided by Dr. Simeonides, Beth Israel Deaconess Medical Center, Boston, MA. Twenty hours later, both transfected and non-transfected cells were treated with either vehicle (saline) or 10 Ϫ7 M CRH. Cells were isolated 18 h later and RNA was extracted, as we have previously reported, by using Trizol reagent (Sigma). RNA (10 g) was separated on a 1.4% formaldehyde agarose gel and transferred to GeneScreen (PerkinElmer Life Sciences) following standard protocols (12). A complementary RNA POMC riboprobe was labeled with [␣-32 P]UTP (PerkinElmer Life Sciences) and T7 polymerase, as previously described (13). Hybridization was carried out at 65°C for at least 16 h with 10 6 cpm riboprobe/lane. The filter was washed (3ϫ for 20 min in 0.1ϫ SSC-10% SDS) and exposed to Kodak XAR 5 film at room temperature for 30 min.
Isolation of Nuclear Extracts-AtT20 cells were harvested, and the cell pellets were lysed in ice-cold hypotonic lysis buffer containing 10 mM HEPES-KOH (pH 7.9), 10 mM KCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.5 mM dithiothreitol, 10 g/ml each of aprotinin, leupeptin, and pepstatin, 1 mM NaF, 1 mM NaVO 4 , and 1% Nonidet P-40 for 10 min. After a brief centrifugation at 3000 ϫ g for 1 min, the cytosolic extracts were collected while the nuclear pellets were lysed in high-salt extraction buffer containing 20 mM HEPES-KOH (pH 7.9), 0.42 M NaCl, 1.5 mM MgCl 2 , 0.3 mM EDTA, 0.5 mM dithiothreitol, 20% glycerol, 0.1% Triton X-100, and 10 g/ml each of aprotinin, leupeptin, and pepstatin. After incubation on ice for 45 min with intermittent vortexing, the nuclear extracts were collected, followed by centrifugation at 14,000 ϫ g for 30 min at 4°C, and their protein concentration was determined with the BCA protein assay kit (Pierce) using bovine serum albumin as a standard. The nuclear extracts were stored at Ϫ80°C until further use.
Electrophoretic Mobility Shift Assay (EMSA)-Nuclear extracts from AtT20 cells were subjected to EMSA analysis. Double-stranded oligonucleotides of the core sequence of the NF-B binding element on mouse immunoglobulin light chain (sense, 5Ј-TCG, ACA GAG GGG ACT TTC CGA GAC GC-3Ј; antisense, 5Ј-TCG AGC CTC TCG GAA, AGT CCC CTC TG-3Ј) were labeled with [ 32 P]dCTP (50 Ci at 3000 Ci/mmol, PerkinElmer Life Sciences) using a Klenow fragment of Escherichia coli, DNA polymerase I (Roche Applied Science). Equal amounts (6 -10 g) of nuclear extracts were incubated in 20 l of binding buffer containing 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.5 mM dithiothreitol, 0.5 mM EDTA, 1 mM MgCl 2 , 4% glycerol, 2.5 g of poly(dI/dC) (Amersham Biosciences) and 2 l of the 32 P-labeled probe for 30 min at room temperature. For the supershift studies, 2 l of antibody (anti-p50, anti-p65, Santa Cruz Biotechnology, Santa Cruz, CA) was added to the reaction mixture and incubated for another 20 min at room temperature before the addition of the 32 P-labeled probe. The DNA-proteinbinding complexes were analyzed by EMSA on a nondenaturing 6% polyacrylamide gel using a Tris/glycine/EDTA buffer. After being dried, the gel was exposed to film at Ϫ70°C.
Statistical Analysis-All experiments were performed at least three times. Data were analyzed by Student's t test and one-way analysis of variance followed by post-hoc multiple comparison tests. Significance was accepted at p Ͻ 0.05.

CRH Inhibits Pituitary NF-B-
We evaluated the effect of CRH on pituitary NF-B DNA-binding activity using AtT20 cells, a pituitary corticotroph cell line. Our findings showed inhibition of the NF-B DNA-binding activity by CRH (Fig.  1A), which is in agreement with a previous report (10), whereas lipopolysaccharide (LPS), an inducer of the NF-B DNA-binding activity in immune cells, had a similar effect in AtT20 cells as well (Fig. 1A). The effect of CRH was apparent within 15 min of treatment and lasted at least 6 h after its addition to the culture (Fig. 1B). This effect of CRH was blocked by CP154,526, a nonpeptide-specific CRHR1 antagonist (Fig. 2). Challenge of the cells with dexamethasone inhibited the NF-B DNAbinding activity, analogous to the effect of dexamethasone on the expression of NF-B in immune cells (Fig. 2). Finally, co-addition of CRH and dexamethasone resulted in a greater inhibition of the NF-B DNA-binding activity than each agent alone (Fig. 2), suggesting that these hormones might activate this transcription factor by means of independent mechanisms.

NF-B-dependent Effect of CRH on Pituitary POMC Gene
Expression-The importance of pituitary NF-B on the CRHinduced regulation of the POMC gene expression was studied by transfecting AtT20 cells with a plasmid containing the POMC promoter coupled to the luciferase reporter gene either intact or mutated at an NF-B binding site. Cells were treated with 10 Ϫ7 M of CRH for 6 h or vehicle (control). As has been shown previously (11), CRH treatment of AtT20 cells transfected with the intact construct resulted in 2.5-3ϫ induction of the transcriptional activity of the POMC gene (Fig. 3A). CRH treatment of the AtT20 cells transfected with the mutated construct resulted in a further increase (6ϫ over the control) of the transcriptional activation of the POMC gene (Fig. 3A). To elucidate the pathway mediating the above effects of CRH, we pretreated AtT20 cells transfected with either the WT or the mutant construct for 1 h with 10 M of H89, PKA, or GF109203X, the PKC inhibitor, before the 6-h treatment with 10 Ϫ7 M CRH. H89 abolished the CRH-induced transcriptional activation of both the WT and the mutated POMC promoter construct (Fig. 3B, left panel).
GF109203X had no effect on the CRH-mediated transcriptional activation of either POMC promoter construct (Fig. 3B,  right panel).
We further investigated the effect of NF-B on the transcriptional activation of the pituitary POMC using AtT20 cells transiently transfected with a plasmid containing the full-length cDNA of IB␣, the cytoplasmic protein that binds NF-B and does not allow its activation. The abundance of POMC mRNA was assessed by Northern blot analysis in cells transfected with IB or in control cells. As shown in Fig. 4, the abundance of POMC mRNA was significantly increased (2.5ϫ) after CRH treatment in the cells overexpressing IB␣. These data suggest that induction of NF-B DNA-binding activity is an important pathway for the CRH-induced stimulation of the pituitary POMC gene expression. DISCUSSION In mammals, activation of the hypothalamic-pituitary-adrenal (HPA) axis constitutes the main endocrine response to stress and is mediated primarily by increased expression of the hypothalamic CRH (14). By binding to CRHR1 on pituitary corticotrophs, CRH stimulates pituitary POMC transcription and thus secretion of ACTH, which leads to glucocorticoid release (15). CRH-induced POMC expression is mediated through the cAMP/protein kinase A pathway and through calcium-dependent events (2). Other regulators of POMC such as leukemia inhibitory factor have been found to act independently of cAMP and may work by stimulation of the transcription factor STAT3 (16 -18).
NF-B is a transcription factor that regulates the expression of a variety of proinflammatory genes. NF-B DNA-binding activity is induced by proinflammatory factors as well as by cellular stresses such as heat and hypoxia (19). NF-B is expressed in the brain; a recent study (10) showed that the hypoxia-induced NF-B DNA-binding activity in hippocampal neurons is inhibited by CRH, suggesting protective effects of CRH on brain cells suffering ischemia-induced damage. This finding provided a mechanism for the suggested neuroprotective effects of CRH. In this report, we demonstrate that induction of pituitary NF-B DNA-binding activity participates in the transcriptional regulation of the POMC gene by CRH, a critical step of the stress response. In a study evaluating the effect of heating stress on NF-B activation in a lung cell line, there was inhibition of the NF-B DNA-binding activity in lung cells that paralleled the degree of stimulation of heat-shock proteins (20). These findings suggested that the inhibition of the NF-B DNA-binding activity may be part of the tissuespecific stress responses.
In previous studies (21), we showed that CRH stimulates the NF-B DNA-binding activity in immune cells. This effect of CRH is attributed to the proinflammatory action of peripheral CRH, and it is unlikely to be mediated by the CRH receptor 1 because a CRHR1-specific antagonist did not block this effect of CRH. On the other hand, the effect of CRH on pituitary NF-B should be mediated by CRHR1, the only shown CRH receptor subtype expressed in corticotroph cells. The above suggests receptor-specific effects of CRH on the regulation of transcription factors such as NF-B, and, furthermore, they raise the hypothesis of differential regulation of NF-B by immune versus physical/psychological stressors.
Our findings suggest that the inhibitory effect of CRH on the NF-B DNA-binding activity in pituitary cells is related to the transcriptional activation of the POMC gene by CRH (Fig. 4). Activation of the POMC gene leads to ACTH secretion, a step necessary for the release of glucocorticoid, the end product of the activated HPA axis. Three putative regulatory elements on POMC promoter have been found to bind CRH-induced transcription factors. The AP-1 site in exon 1 (ϩ41/ϩ47) binds CRHinduced cAMP-response element-binding protein, cfos, and junB. Nurr1 and Nurr77 bind to two specific sequences: Ϫ70/Ϫ63, which corresponds to a pivotal responsive sequence for positive or negative POMC regulation by Nurr and the glucocorticoid receptor, respectively, or Ϫ404/Ϫ383, that recognizes Nurr77 homodimers or heterodimers of the POMC promoter (4,22). We found stimulation of the CRH-mediated transcriptional activation of POMC after mutation of a traditional NF-B binding sequence. There is no overlap of this site with any of the above well characterized sequences on the POMC promoter. In addition, there is no overlap between this sequence and the suggested glucocorticoid responsive element on the POMC promoter. Negative regulation of the POMC gene by glucocorticoid by means of antagonism between the glucocorticoid receptor and factors such as the orphan nuclear receptor Nur77 or the AP-1 has been shown (4,23,24), but to our knowledge, inhibition of the transcriptional stimulation of POMC gene by any other transcription factor has not been reported.
In summary, inhibition of NF-B DNA-binding activity is associated with the transcriptional activation of the POMC gene. It has been well shown that transcriptional activation of the POMC gene is associated with activation of the AP-1 factor (25). Parallel activation of NF-B and AP-1, which leads to increased transcription, has been described for several genes related mainly to activation of the immune system (26). Our findings suggest opposing roles for NF-B and AP-1 in the activation of the POMC gene in corticotroph cells. The contribution of NF-B in the regulation of the induced expression of other pituitary hormones and its physiological significance remains to be determined.