Transcriptional Regulation of Hypothalamic Corticotropin-Releasing Factor Gene

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Transcriptional Regulation of Hypothalamic CorticotropinReleasing Factor Gene Kazunori Kageyama and Toshihiro Suda Contents I. Introduction II. Regulatory Elements on Hypothalamic CRF Gene A. Cyclic AMP (cAMP) response element-binding protein (CREB) B. Inducible cAMP-early repressor (ICER) C. Estrogen D. Glucocorticoid receptor E. Activator protein 1 (fos and Jun) F. Suppressor of cytokine signaling (SOCS)-3 III. Conclusion Acknowledgments References

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Abstract Corticotropin-releasing factor (CRF) plays a central role in regulating stress responses. Forskolin or pituitary adenylate cyclase-activating polypeptide stimulates adenylate cyclase and then increases intracellular cAMP levels in hypothalamic cells. Activation of the protein kinase A pathway leads to binding of cAMP response element (CRE)-binding protein (CREB) on the CRF promoter. Forskolin-stimulated CRF gene transcription is mediated by CRE on the CRF 50 promoter region. Inducible cAMP-early repressor suppresses a stress response via inhibition of the cAMP-dependent CRF gene. Glucocorticoid-dependent repression of cAMP-stimulated CRF promoter activity is mediated by both nGRE and SRE in hypothalamic cells. Interleukin (IL)-6 produced in the hypothalamus stimulates the CRF gene. Suppressor of cytokine signaling-3, which is induced by a cAMP stimulant and IL-6, is involved in the negative regulation of CRF gene expression in hypothalamic cells. Such complex mechanisms would contribute to stress responses and homeostasis in the hypothalamus. ß 2010 Elsevier Inc. Department of Endocrinology and Metabolism, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori, Japan Vitamins and Hormones, Volume 82 ISSN 0083-6729, DOI: 10.1016/S0083-6729(10)82016-3

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I. Introduction Corticotropin-releasing factor (CRF) plays a central role in regulating stress responses (Suda et al., 1985; Vale et al., 1981). Moreover, CRF coordinates neuroendocrine, behavioral, autonomic, and immune responses, and controls the hypothalamic–pituitary–adrenal (HPA) axis during stressful periods. In the hypothalamic paraventricular nucleus (PVN) of the brain, CRF is produced in response to stress. CRF and arginine vasopressin (AVP) neurons in the parvocellular region of the PVN project to the external zone of the median eminence (Gonzalez-Hernandez et al., 2006; Seasholtz et al., 1988). Also, CRF and AVP in parvocellular PVN neurons exert synergistic effects on adrenocorticotropic hormone (ACTH) secretion from the anterior pituitary (AP; Gillies et al., 1982; Mouri et al., 1993). On the other hand, ACTH stimulates the release of glucocorticoids from the adrenal glands (Whitnall, 1993). Circulating glucocorticoids are critical to recovery from stress conditions because they inhibit the hypothalamic PVN production of CRF and the pituitary production of ACTH, thereby ensuring that serum levels of glucocorticoids are appropriate to the stress experienced (Whitnall, 1993). Pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to modulate hypothalamic CRF gene expression in vivo (Grinevich et al., 1997). PACAP induces intracellular cAMP production in hypothalamic 4B cells and stimulates CRF gene transcription via the cAMP-dependent pathway. Limbic structures, such as the extended amygdala and the bed nuclei of the stria terminalis, are identified as innervation sites of PACAP neurons, suggesting an important role in stress responses (Kozicz and Arimura, 2002; Piggins et al., 1996). Indeed, nerve fibers containing PACAP connect to CRF neurons (Hannibal et al., 1995; Legradi et al., 1998). Other studies also suggest that PACAP stimulates the CRF gene via the cAMP/protein kinase A (PKA) signaling pathway (Agarwal et al., 2005). Activation of the PKA pathway, causing phosphorylation of cAMP response element (CRE)-binding protein (CREB), acts on the CRF promoter (Itoi et al., 1996; Seasholtz et al., 1988; Spengler et al., 1992). A functional CRE on the 50 -promoter region takes part in regulating CRF gene expression (Seasholtz et al., 1988; Spengler et al., 1992).

II. Regulatory Elements on Hypothalamic CRF Gene Computer analysis of the proximal CRF promoter reveals several possible binding sites for transcriptional factors, such as CRE, activator protein 1 (AP-1) protein (Fos/Jun) binding sites, half glucocorticoid regulatory element (GRE), and half estrogen-responsive element (ERE; Yao and Denver, 2007).

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A. Cyclic AMP (cAMP) response element-binding protein (CREB) Forskolin or PACAP stimulates adenylate cyclase and then increases intracellular cAMP levels in hypothalamic 4B cells (Fig. 16.1). Forskolin increases CRF transcriptional activity in hypothalamic cells (Fig. 16.2) in agreement with previous studies using other cells (Seasholtz et al., 1988; Spengler et al., 1992; Yamamori et al., 2004). Activation of the PKA pathway leads to binding of CREB to the CRE on the CRF promoter in hypothalamic cells as well as in human placental cells (Cheng et al., 2000). The forskolin-stimulated activity of CRF gene transcription is reduced in 4B cells transfected with a mutant construct, CRF-233Mtluc, in which the CRE element (TGACGTCA) is mutated (TGGATCCA), or with a deletion mutant construct of the CRF gene promoter, CRF-220luc (Fig. 16.3). Therefore, the forskolin-induced CRF gene transcription is mainly mediated by CRE, which includes 220 to 233 base pairs (bp), on the CRF 50 -promoter region in hypothalamic cells. A functional CRE on the 50 -promoter region is important for increasing CRF gene expression 1000

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Figure 16.1 Effects of forskolin and PACAP on cAMP production in 4B cells. * P < 0.05, ** P < 0.005 (compared with control [C]). Cells were preincubated for 20 min with medium containing 0.1 mM 3-isobutyl-1-methylxanthine, followed by the addition of forskolin (Fsk) or PACAP. The level of intracellular cAMP was measured by cAMP EIA. (Reproduction from Kageyama et al. (2007) with permission of the publisher.) Copyright 2007, Society for Endocrinology.

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Figure 16.2 Effects of forskolin on CRF 50 -promoter activity in 4B cells. Control cells treated with medium alone are indicated as (C). *P < 0.05 (compared with control [C]). Time-dependent changes in forskolin-induced CRF 50 -promoter activity (left panel). Cells were incubated with medium containing 10 mM forskolin. Dose-dependent changes in forskolin-induced CRF 50 -promoter activity (right panel). Cells were incubated for 2 h with medium containing 0.01–10 mM forskolin (Fsk). (Reproduction from Kageyama et al. (2008) with permission of the publisher.) Copyright 2008, Editrice Kurtis srl.

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Figure 16.3 Effects of CRE deletion on forskolin-induced CRF 50 -promoter activity in 4B cells. (A) Schematic representation of CRE in the CRF promoter. (B) Cells were transfected with full-length (CRF-907luc), deleted (CRF-220luc or CRF-233luc), or mutant (CRF-233Mtluc) promoter constructs, and then incubated for 2 h with 10 mM forskolin alone (Fsk) or vehicle (C). * P < 0.05 (compared with forskolin alone [Fsk] in CRF-907luc transfected cells). þ P < 0.05 (compared with each control). (Reproduction from Kageyama et al. (2008) with permission of the publisher.) Copyright 2008, Editrice Kurtis srl.

(Seasholtz et al., 1988; Spengler et al., 1992). In addition, King et al. demonstrated a second response element by cAMP between 125 and 118 bp, a caudal-type homeobox response element on the CRF promoter (King et al., 2002). Therefore, both the CRE and the caudal-type homeobox response element on the CRF promoter contribute to a cAMP-associated increase in the expression of the CRF gene (Cheng et al., 2000; King et al., 2002).

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B. Inducible cAMP-early repressor (ICER) Inducible cAMP-early repressor (ICER) is a cAMP-inducible member of the CRE modulator (CREM) family and a repressor isoform of CREM (Foulkes et al., 1991; Molina et al., 1993). CREM, CREB, and activating transcription factor 1 (ATF-1) bind to CRE promoter elements (Lalli and Sassone-Corsi, 1994), and ICER acts as a competitive inhibitor of CRE-dependent transcription (Foulkes et al., 1991). ICER may then suppress a stress response via inhibition of the cAMP-dependent CRF gene. Forskolin induces an increase in ICER protein levels in hypothalamic cells, and transfection of the ICER decreases forskolin-induced CRF 50 -promoter activity (Liu et al., 2006). Therefore, considering that CRF plays a central role in controlling the HPA axis in stress, the induction of ICER may influence the suppression of a stress response via regulation of the CRF gene.

C. Estrogen Estrogens acting centrally, including the pituitary corticotrophs and hypothalamus, can modulate stress responses (Nakano et al., 1991), and direct estrogenic regulation of CRF gene expression has been demonstrated in various tissues (Dibbs et al., 1997; Vamvakopoulos and Chrousos, 1993). Estrogen regulates the HPA axis by stimulation of CRF gene expression in the hypothalamus in vivo, since high levels of estrogen replacement increases basal levels of CRF mRNA in the PVN of ovariectomized rats (Ochedalski et al., 2007). A physiologically relevant dose, 10 nM of estradiol (E2), stimulates both CRF gene transcription (Fig. 16.4A) and mRNA (Ogura et al., 2008) expression in hypothalamic 4B cells. E2 and diarylpropionitrile (DPN), an estrogen receptor (ER) b agonist, increase CRF gene transcriptional activity (Fig. 16.4B). Therefore, ERb activation by estrogens induces the transcription of the CRF gene in hypothalamic cells. Treatment with both E2 and forskolin shows an additive effect on the CRF promoter activity (Fig. 16.4C). Therefore, in addition to cAMP, the presence of other signal pathways may be indicated in the activation of CRF by estrogens. ERb often antagonizes the effect of ERa on gene regulation (Liu et al., 2002) and stimulates CRF transcriptional activity in HeLa cells (Miller et al., 2004). ERE half-sites are contained on the CRF promoter (Chen et al., 2008) and are suggested to be involved in the stimulation of the CRF gene via ERb (Vamvakopoulos and Chrousos, 1993). Therefore, direct estrogenic transcriptional regulation of the CRF gene in hypothalamic 4B cells suggests that EREs may be of functional significance via ERb in the PVN.

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Figure 16.4 Effects of E2 on CRF 50 -promoter activity in 4B cells. Control cells treated with medium alone are indicated as (C). * P < 0.05 (compared with [C]). þ P < 0.05, (compared with E2 or Fsk alone). (A) Time-dependent changes in E2-induced CRF 50 -promoter activity. Cells were incubated with medium containing 10 or 100 nM E2. (B) Dose-dependent changes in E2-induced CRF 50 -promoter activity. Cells were incubated for 2 h with medium containing 1–100 nM E2, 10 nM PPT, or 10 nM DPN. (C) Effects of E2 on forskolin-induced CRF 50 promoter activity. Cells were incubated for 2 h with medium alone (C) or medium containing 500 nM forskolin (Fsk) and/or 100 nM E2. (Reproduction from Ogura et al. (2008) with permission of the publisher.) Copyright 2008, Elsevier.

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D. Glucocorticoid receptor The HPA axis is regulated by a negative feedback mechanism. Hypothalamic parvocellular neurons are known to express glucocorticoid receptors, and glucocorticoids regulate CRF gene expression directly in the hypothalamus. Repression by glucocorticoids occurs through inhibition of CRF gene transcription in a subclone of AtT-20 cells (Malkoski and Dorin, 1999). The CRF promoter does not contain a classical consensus GRE; however, there are a number of regions in the sequence where glucocorticoid receptors are able to bind (Guardiola-Diaz et al., 1996). Malkoski and Dorin (1999) demonstrated glucocorticoid regulatory regions on the CRF promoter. By using a series of 50 -nested deletions, they demonstrated that dexamethasone-dependent repression of cAMP-stimulated CRF promoter activity is localized to promoter sequences between 278 and 249 bp (Fig. 16.5). High-affinity binding of the glucocorticoid receptor DNA-binding domain to this promoter region was observed with an electrophoretic mobility shift assay. Therefore, this region would contribute to the inhibition of CRF promoter activity by glucocorticoids as a negative GRE (nGRE). We demonstrated that other promoter regions are involved in the inhibitory regulation of CRF gene expression in hypothalamic 4B cells (Fig. 16.5). The glucocorticoid suppression of cAMP-stimulated CRF promoter activity is also involved in the CRF promoter sequences between 248 and 233 bp in hypothalamic cells (Fig. 16.5). Serum response element (SRE) is included in this region and would thus contribute to the negative response to glucocorticoids, because the glucocorticoid receptor can bind to SRE and inhibit promoter activation by antagonizing the function of positive transcription (Karagianni and Tsawdaroglou, 1994). Therefore, in addition to nGRE, the glucocorticoid suppression of cAMPstimulated CRF promoter activity may also be caused by the SRE in hypothalamic 4B cells.

E. Activator protein 1 (fos and Jun) In addition to the PKA pathway, protein kinase C (PKC) is involved in the regulation of forskolin-induced CRF gene expression because a PKC inhibitor inhibits forskolin-induced CRF promoter activity. Activation of the PKC pathway leads to binding of AP-1 (Fos/Jun proteins) to AP-1-binding sites on the CRF promoter. Binding sites for both glucocorticoid receptor and AP-1 nucleoproteins have been shown at adjacent elements within the nGRE (Malkoski and Dorin, 1999) because mutations that disrupted either glucocorticoid receptor or AP-1-binding activity cause a similar loss of glucocorticoid-dependent repression. These results suggest that the nGRE functions as a composite

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Figure 16.5 Effects of nGRE or SRE deletion on the dexamethasone suppression of CRF 50 -promoter activity in 4B cells. (A) Schematic representation of nGRE or SRE in the CRF promoter. (B) Cells transfected with a full-length (CRF-907luc), deleted (CRF-233luc, CRF-248luc, or CRF-278luc), or mutant (CRF-278Mtluc or CRF-248Mtluc) promoter construct, were preincubated for 30 min with medium containing dexamethasone (Dex, 100 nM) or vehicle (C), followed by the addition of 10 mM forskolin for 2 h. Data are presented as relative activity, and luciferase activity in response to forskolin alone (C) was set at 100% in all transfected cells. Experiments were conducted in triplicate, and the means of three independent experiments are shown. * P < 0.05 (compared with forskolin alone [C] in all transfected cells). þ P < 0.05 (compared with Dex in CRF-907luc). þþ P < 0.05 (compared with Dex in CRF-907luc and CRF-278luc). þþþ P < 0.05 (compared with Dex in CRF-907luc, CRF-278luc, and CRF-248luc). (Reproduction from Kageyama et al. (2008) with permission of the publisher.) Copyright 2008, Editrice Kurtis srl.

regulatory element, involving direct DNA binding of the glucocorticoid receptor and AP-1 nucleoproteins. King et al. proposed that transcription factor differences among cells cause negative or positive regulation because CREB and Fos were detected in AtT-20 while CREB and Jun were found

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in placental cells (King et al., 2002). Furthermore, glucocorticoids can inhibit CREB and Fos in the PVN ( Jacobson et al., 1990; Legradi et al., 1997). The modified expression of transcription factors in cells changes the binding to nGRE and/or CRE, and then the promoter activity, resulting in the suppression of CRF gene transcription.

F. Suppressor of cytokine signaling (SOCS)-3 Following inflammatory stresses, interleukin (IL)-1 and IL-6 stimulate the HPA axis. IL-6 is an important mediator of the interaction between the neuroendocrine and immune systems. IL-6 is coexpressed with CRF and AVP in the supraoptic nucleus and PVN neurons (Ghorbel et al., 2003), and plays an important role in regulating both CRF and AVP in the hypothalamus. For example, IL-6 increases CRF gene expression and secretion in the PVN (Navarra et al., 1991; Vallieres and Rivest, 1999). Forskolin or PACAP increases IL-6 mRNA expression and protein levels in the hypothalamic cells. In addition, the stimulatory effects of PACAP on CRF promoter activity are significantly inhibited by treatment with anti-IL-6 monoclonal antibody in hypothalamic cells (Fig. 16.6). Therefore, endogenous IL-6 production would be involved in the PACAPinduced CRF gene transcription in an autocrine manner in hypothalamic 4B cells. Considering the delayed response to IL-6 and the partial inhibition of PACAP effects induced by anti-IL-6 antibody, IL-6 may be important for sustaining the activity of CRF and AVP genes. In fact, the CRF promoter contains multiple nuclear factor (NF)-kB and Nurr1-binding sites in response to cytokines. Thus, it is possible that IL-6, produced in the hypothalamus, stimulates CRF and AVP gene expression. Suppressor of cytokine signaling (SOCS)-3 acts as a potent negative regulator of cytokine signaling and suppresses cytokine-induced proopiomelanocortin gene transcription and ACTH secretion in corticotrophs (Auernhammer et al., 1999; Krebs and Hilton, 2000). IL-6 stimulates the Janus kinase/signal transducers and activators of transcription ( JAK/STAT) signaling pathway, while IL-6-induced SOCS-3 acts as a negative regulator and inhibits STAT phosphorylation by JAK at the receptor complex (Ram and Waxman, 1999; Schmitz et al., 2000). SOCS-3 would also be involved in the negative regulation of CRF gene expression in the hypothalamus. In fact, SOCS-3 was found to be regulated by IL-6 and via the cyclic AMP/protein kinase A pathway in hypothalamic cells (Fig. 16.7). SOCS-3 knockdown increased IL-6- or forskolin-induced CRF gene transcription and mRNA levels (Fig. 16.8). Therefore, SOCS-3, which is induced by a cAMP stimulant and IL-6, is involved in the negative regulation of CRF gene expression in hypothalamic cells.

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Figure 16.6 Effects of anti-IL-6 Ab on PACAP-induced CRF 50 -promoter activity in 4B cells. *P < 0.05, **P < 0.005 (compared with control [C]). þþ P < 0.005 (compared with 100 nM PACAP). # P < 0.05 (compared with 10 nM PACAP). Cells were preincubated with medium containing anti-IL-6 Ab or control IgG for 30 min, followed by the addition of 10 or 100 nM PACAP or vehicle for 2 h. Cells treated with control IgG are indicated as C. (Reproduction from Kageyama et al. (2007) with permission of the publisher.) Copyright 2007, Society for Endocrinology.

III. Conclusion Forskolin or PACAP stimulates adenylate cyclase and then increases intracellular cAMP levels in hypothalamic 4B cells. Activation of the PKA pathway leads to binding of CREB on the CRF promoter in hypothalamic cells (Fig. 16.9). Forskolin-stimulated CRF gene transcription is mediated by CRE, which included from  220 to 233, on the CRF 50 -promoter region. ICER acts as a competitive inhibitor of CRE-dependent transcription and then suppresses a stress response via inhibition of the cAMPdependent CRF gene. E2 may enhance the activation of the CRF gene in stress. Glucocorticoid-dependent repression of cAMP-stimulated CRF promoter activity is mediated by both nGRE and SRE in hypothalamic cells. IL-6 produced in the hypothalamus stimulates CRF gene expression (Fig. 16.9). SOCS-3, induced by a cyclic AMP stimulant and IL-6, would

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Figure 16.7 Effects of JAK and PKA inhibitors on SOCS-3 mRNA levels. Control cells treated with medium alone are indicated as (C). Experiments were conducted in triplicate, and the means of three independent experiments are shown. Statistical analyses were performed using one-way ANOVA, followed by post hoc tests. *P < 0.05 (compared with control [C]). þ P < 0.05 (compared with forskolin [Fsk]). (A) Effects of JAK inhibitor I on IL-6-stimulated SOCS-3 mRNA levels. Cells were preincubated for 30 min with medium containing 1 mM JAK inhibitor I ( JAK I) or vehicle, followed by the addition of 100 ng/ml IL-6 or vehicle for 6 h. (B) Effects of H89 on forskolin-stimulated SOCS-3 mRNA levels. Cells were preincubated for 30 min with medium containing 1 mM H89 or vehicle, followed by the addition of 10 mM forskolin (Fsk) or vehicle for 2 h. (Reproduction from Kageyama et al. (2009) with permission of the publisher.) Copyright 2009, Society for Endocrinology.

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Figure 16.8 Effects of SOCS-3 on the regulation of CRF gene in 4B cells. Experiments were conducted in triplicate, and the means of three independent experiments are shown. Statistical analyses were performed using unpaired t-test. * P < 0.05 (compared with control [siControl]). The 4B cells, seeded into 12-well plates at a density of 2  104 cells/well, were incubated for 24 h in 1 ml of culture medium containing siRNA for either control (siControl) or SOCS (siSOCS). (A–C) Effects of SOCS-3 on the regulation of CRF 50 -promoter activity. After transfection, the cells were retransfected with a CRF promoter construct, and then incubated with vehicle (A), 10 mM forskolin (Fsk) for 2 h (B) or 100 ng/ml IL-6 for 24 h (C). (Reproduction from Kageyama et al. (2009) with permission of the publisher.) Copyright 2009, Society for Endocrinology.

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Figure 16.9 A schematic model of transcriptional regulation of hypothalamic CRF gene. Forskolin or PACAP stimulates adenylate cyclase, and then intracellular cAMP levels in hypothalamic 4B cells. Activation of the PKA pathway leads to binding of CREB to CRE on the CRF promoter. ICER acts as a competitive inhibitor of CREdependent transcription, and then suppresses the stimulation of cAMP-dependent CRF gene. Glucocorticoids-dependent repression of cAMP-stimulated CRF promoter activity is mediated by both nGRE and SRE. IL-6, produced in the hypothalamus, stimulates CRF gene expression. SOCS-3, induced by a cyclic AMP stimulant and IL-6, would be involved in the negative regulation of CRF gene expression in the hypothalamus.

be involved in the negative regulation of CRF gene expression in hypothalamic cells. Such complex mechanisms would contribute to stress responses and homeostasis.

ACKNOWLEDGMENTS This work was supported in part by Health and Labour Science Research Grants (Research on Measures for Intractable Diseases) from the Ministry of Health, Labor, and Welfare of Japan.

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