Human Corticotropin-releasing Hormone Gene Research Articles

AB0027 Impact of additional genetic markers of rheumatoid arthritis on pattern of functional stress responses

BackgroundGrowing evidence supports the hypothesis that alteration of the stress response and interactions between the autonomic nervous system, the hypothalamus-pituitary-adrenal axis and the immune system contributes to the pathogenesis of...

Functional stress response in patients with rheumatoid arthritis: Impact of genetic factors

It is still incompletely understood how the stress response contributes to the pathogenesis of rheumatoid arthritis (RA). To characterize neuroimmune interactions, common variants in the genes of the β2-adrenergic receptor (beta2AR) and corticotropin releasing hormone (CRH) were studied together with functional stress responses in RA patients. An allele-specific PCR was used to determine the polymorphisms of the beta2AR (position 16, 27, 164), as well as the polymorphic sequences in the 5′ flanking region of the human CRH gene in RA patients (n = 310) and controls (n = 305). The autonomic response upon stressful stimuli was investigated studying heart rate variability, and the dynamics of blood glucose levels, CRH, and cortisol production under insulin hypoglycemia (IHT). There was a highly significant distortion in the distribution of the beta2AR polymorphism at codon 16 between RA patients and controls (p = 0.00001). It was also revealed a significant decrease of parasympathetic activity in patients with homozygosity for Gly 16 compared to Arg16Gly RA patients. In addition, the CRH promoter polymorphisms exerted a significant influence on the stress response of RA patients undergoing IHT. The integrated cortisol response to hypoglycemia was significantly lower in RA patients bearing the A1B1 allele compared to the A2B2 (p = 0.016). Polymorphisms of the beta2AR and CRH are associated with disturbed functional stress reactivity on various levels in RA patients. Further studies are warranted to determine the role of genetic factors on stress response in the disease process of RA.

Effects of neurosteroids on the human corticotropin-releasing hormone gene

Increased activity of hypothalamic-pituitary-adrenal (HPA) axis and hypersecretion of corticotropin-releasing hormone (CRH) are known to be important factors in pathogenesis of some stress-related diseases. Some neurosteroids exert anxiolytic and antidepressant effects probably by inhibition of HPA axis activity. The aim of our study was to find out if neurosteroids can directly affect human CRH gene transcription. The effect of allopregnanolone (ALLO), allotetrahydrodeoxycorticosterone (THDOC), pregnenolone (PGL), PGL sulfate (PGL-S), dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEA-S) on CRH expression was determined in differentiated Neuro-2A cells stably transfected with plasmid containing a fragment of human CRH promoter (-663 to+124bp) linked to the chloramphenicol acetyltransferase (CAT) reporter gene. It was found that PGL (0.3–30μΜ), ALLO (1–30μΜ) and THDOC (1–30μΜ) present in the culture medium for 5 days in the concentration-dependent manner inhibited CRH-CAT activity. These neurosteroids also inhibited forskolin-stimulated CRH gene transcription with similar potency. In contrast, PGL-S, DHEA and DHEA-S in a concentration from 0.01 to 10μΜ had no effect on basal and forskolin-stimulated CRH activity. Further experiments revealed that wortmannin (an inhibitor of phosphatidylinositol 3-kinase; PI3-K) at concentrations of 0.01 and 0.02μΜ did not change the inhibitory effect of ALLO (3μΜ) and PGL (1μΜ) on CRH gene transcription. Moreover, ALLO (3μΜ) and PGL (1μΜ) present in the culture medium for 5 days did not change the amount of active, phosphorylated form of protein kinase B (PKB, Akt) and extracellular signal-regulated kinase (ERK). The obtained results indicate that PGL, ALLO and THDOC inhibited basal and forskolin-induced CRH gene promoter activity in the differentiated Neuro-2A cells and that these effects did not depend on the activation of PI3-K/Akt and ERK-MAPK pathways.

Estrogen receptor-α and -β regulate the human corticotropin-releasing hormone gene through similar pathways

Corticotropin-releasing hormone (CRH) plays a central role in controlling stress response. In this study, we aimed to identify the regulatory effect of estrogen receptor (ER) on CRH and the underlying mechanism. We investigated the regulation of CRH mRNA in the BE(2)-C cell line, a human neuroblastoma cell line which express endogenous CRH. Quantitative reverse transcriptase-polymerase chain reactions showed that in the presence of estradiol overexpressing ER α or ER β in BE(2)-C cells increased the transcription of CRH. Chromatin immunoprecipitation assays in this cell line also showed that both ER α and ER β can be recruited to the CRH promoter with the treatment of estradiol. However, electrophoretic mobility shift assays did not show direct binding between estrogen receptors and two estrogen response elements (ERE) half sites in the CRH promoter. To clarify the regulatory mechanism, site-directed mutagenesis and reporter gene assay in the CHO cell line were used. When the ERE half sites and the cAMP regulatory element (CRE) in the CRH promoter were disrupted, ER-mediated up-regulation of CRH promoter activity reduced. Between the two ERE half sites studied, the − 316 ERE half site contributed more to the constitutive CRH expression induced by ER. In summary, our results confirm the stimulation of ER α and ER β on CRH expression and demonstrate the important roles of the ERE half sites and CRE for the action of ER α and ER β.

Sequence Variants of the CRH 5′‐Flanking Region

Recently, studies in adult rheumatoid arthritis patients have shown an association with four single-nucleotide polymorphisms (SNPs) in the 3.7-kb regulatory region of human corticotropin-releasing hormone (hCRH) gene located at positions -3531, -3371, -2353, and -684 bp. Three of these novel polymorphisms are in absolute linkage disequilibrium, resulting in three combined alleles, named A1B1, A2B1, and A2B2. To study whether the described polymorphic nucleotide sequences in the 5' region of the hCRH gene interfere with binding of nuclear proteins, an electric mobility shift assay (EMSA) was performed. At position -2353 bp, a specific DNA protein complex was detected for the wild-type sequence only, possibly interfering with a binding site for the activating transcription factor 6 (ATF6). In contrast, no difference could be detected for the other SNPs. However, at position -684, a quantitative difference in protein binding due to cAMP incubation could be observed. To further investigate whether these SNPs in the CRH promoter are associated with an altered regulation of the CRH gene, we performed a luciferase reporter gene assay with transiently transfected rat pheochromocytoma cells PC12. Incubation with 8-Br-cAMP alone or in combination with cytokines enhanced significantly the promoter activity in PC12 cells. The promoter haplotypes studied exhibited a differential capacity to modulate CRH gene expression. In all our experiments, haplotype A1B1 showed the most pronounced influence on promoter activity. Taken together, our results demonstrate a differential binding capacity of nuclear proteins of the promoter polymorphisms resulting in a different gene regulation. Most probably the SNP at position -2,353 plays a major role in mediating these differences.

Pituitary adenylate cyclase-activating polypeptide (PACAP) mimics neuroendocrine and behavioral manifestations of stress: Evidence for PKA-mediated expression of the corticotropin-releasing hormone (CRH) gene

The physiologic response to stress is highly dependent on the activation of corticotropin-releasing hormone (CRH) neurons by various neurotransmitters. A particularly rich innervation of hypophysiotropic CRH neurons has been detected by nerve fibers containing the neuropeptide PACAP, a potent activator of the cAMP-protein kinase A (PKA) system. Intracerebroventricular (icv) injections of PACAP also elevate steady-state CRH mRNA levels in the paraventricular nucleus (PVN), but it is not known whether PACAP effects can be associated with acute stress responses. Likewise, in cell culture studies, pharmacologic activation of the PKA system has stimulated CRH gene promoter activity through an identified cAMP response element (CRE); however, a direct link between PACAP and CRH promoter activity has not been established. In our present study, icv injection of 150 or 300 pmol PACAP resulted in robust phosphorylation of the transcription factor CREB in the majority of PVN CRH neurons at 15 to 30 min post-injection and induced nuclear Fos labeling at 90 min. Simultaneously, plasma corticosterone concentrations were elevated in PACAP-injected animals, and significant increases were observed in face washing, body grooming, rearing and wet-dog shakes behaviors. We investigated the effect of PACAP on human CRH promoter activity in αT3-1 cells, a PACAP-receptor expressing cell line. Cells were transiently transfected with a chloramphenicol acetyltransferase (CAT) reporter vector containing region − 663/+124 of the human CRH gene promoter then treated for with PACAP (100 nM) or with the adenylate cyclase activating agent, forskolin (2.5 μM). Both PACAP and forskolin significantly increased wild-type hCRH promoter activity relative to vehicle controls. The PACAP response was abolished in the CRE-mutant construct. Pretreatment of transfected cells with the PKA blocker, H-89, completely prevented both PACAP- and forskolin-induced increases in CRH promoter activity. Furthermore, CREB overexpression strongly enhanced PACAP-mediated stimulation of hCRH promoter activity, an effect which was also lost with mutation of the CRE. Thus, we demonstrate that icv PACAP administration to rats under non-stressed handling conditions leads to cellular, hormonal and behavioral responses recapitulating manifestations of the acute stress response. Both in vivo and in vitro data point to the importance of PACAP-mediated activation of the cAMP/PKA signaling pathway for stimulation of CRH gene transcription, likely via the CRE.

Calcium/calmodulin kinase IV pathway is involved in the transcriptional regulation of the corticotropin-releasing hormone gene promoter in neuronal cells

Although corticotropin-releasing hormone (CRH) plays a pivotal role in the regulation of the hypothalamo-pituitary-adrenal axis, the mechanism of CRH gene expression in the neuronal cell is not completely understood. In this study, we examined the transcriptional regulation of human CRH gene 5'-promoter, using a human BE(2)C neuroblastoma cell line expressing intrinsic CRH. In particular, we focused on the involvement of calmodulin kinases (CaMKs), which are known to play an important role in excitation-induced gene expression through the rise in intracellular calcium in the central nervous system. RT-PCR analysis confirmed the expression of CaMK as well as CRH mRNA in BE(2)C cells. When we introduced approximately 1.1 kb of the 5'-promoter region of the human CRH fused with luciferase reporter gene into the cells, a substantial transcriptional activity was observed, and this was further increased by the activation of the cAMP/PKA pathway. We then examined the effect of activation of CaMKs by introducing the expression vectors of each kinase, revealing a potent stimulatory effect of CaMKIV, but no effect of CaMKII. Depolarization of the cells caused an increase in CRH promoter activity, which was completely abolished by the treatment with the CaMK antagonist K252a. Interestingly, KCREB, a dominant negative form of CREB, antagonized the effect of the CaMKIV-mediated effect. Altogether, we conclude that not only the cAMP/PKA but also the calcium/CaMKIV signaling pathway is involved in the regulation of CRH gene expression. Furthermore, CREB is thought to be involved in CaMK- as well as cAMP/PKA-mediated CRH gene expression. Since the CRH gene is expressed in the neuronal cells of the hypothalamus, the calcium/CaMKIV signaling pathway may play an important role in the excitation-mediated regulation of CRH synthesis.

AP-2α modulates human corticotropin-releasing hormone gene expression in the placenta by direct protein–protein interaction

Since AP-2α induces the expression of hPL, hCG and other syncytiotrophoblast-specific marker genes in cytotrophoblast cells during in vitro differentiation, we have examined whether AP-2α also induces hCRH gene expression during differentiation of cytotrophoblast cells. Infection of human cytotrophoblast cells in vitro with an adenovirus expressing AP-2α resulted in a twofold increase in hCRH mRNA levels, while infection with an adenovirus expressing a dominant/negative mutant of AP-2α inhibited basal hCRH mRNA levels by 40% and completely blocked the induction of hCRH mRNA by AP-2α. Transient transfection studies in AtT-20 and HepG2 cells indicated that the induction of hCRH mRNA levels was due, at least in part, to transcriptional activation of the hCRH gene. Gel mobility shift and immunoprecipitation assays strongly suggest that AP-2α induces hCRH gene expression by interacting with CREB and not by binding directly to the hCRH promoter.

The Transcription of Corticotropin-Releasing Hormone in Human Endometrial Cells Is Regulated by Cytokines

Corticotropin-releasing hormone (CRH), a hypothalamic neuropeptide, is also produced in the human endometrium where it participates in local inflammatory phenomena associated with the decidualization of endometrial stroma and the implantation of the fertilized egg. The inflammatory cytokines interleukin 1 (IL-1), IL-6 and leukemia inhibitory factor (LIF) appear to be the dominant local regulators of these intrauterine inflammatory processes. In the present study we have examined the direct interactions between cytokines and CRH in the endometrium. For this purpose we have measured the effects of IL-1, IL-6 and LIF on the activity of CRH promoter inserted in human endometrial cells in culture. Homologous transient transfection experiments were conducted employing a 0.9-kb fragment of the 5′ flanking region of the human CRH gene coupled to the luciferase reporter gene, using Ishikawa human endometrial cells. We have found that IL-1β increased the activity of CRH gene promoter, in a time- and dose-dependent manner. This effect was antagonized by the IL-1 receptor antagonist IL-1ra and blocked completely by the cyclo-oxygenase inhibitor indomethacin. Similarly, IL-6 increased the activity of CRH promoter in a dose-dependent fashion, an effect partially reversed by indomethacin. LIF did not have any apparent effect. In conclusion, our data suggest that IL-1 and IL-6 exert a strong stimulatory effect on the expression of endometrial CRH. This effect is most probably mediated via prostaglandins. Based on these data we hypothesize that in the human endometrium interleukins, prostaglandins and CRH form a local network regulating the inflammatory phenomena taking place within the uterine cavity.

Composite Glucocorticoid Regulation at a Functionally Defined Negative Glucocorticoid Response Element of the Human Corticotropin-Releasing Hormone Gene

Composite Glucocorticoid Regulation at a Functionally Defined Negative Glucocorticoid Response Element of the Human Corticotropin-Releasing Hormone Gene

Composite glucocorticoid regulation at a functionally defined negative glucocorticoid response element of the human corticotropin-releasing hormone gene.

Glucocorticoid-dependent negative feedback of the hypothalamic-pituitary-adrenal axis is mediated in part through direct inhibition of hypothalamic CRH gene transcription. In the present study, we sought to further localize and characterize glucocorticoid receptor (GR) and AP-1 interactions at a functionally defined negative glucocorticoid response element (nGRE) of the CRH promoter. Transient transfection studies in mouse corticotroph AtT-20 cells demonstrated that internal deletion of the nGRE (-278 to -249 nucleotides) within the context of 1 kb of the intact CRH promoter resulted in decreased 8-BrcAMP stimulation and glucocorticoid-dependent repression of CRH promoter activity. The nGRE conferred transcriptional activation by both cAMP and overexpressed c-jun or c-fos AP-1 nucleoproteins as well as specific glucocorticoid-dependent repression to a heterologous promoter. A similar profile of regulation was observed for the composite GRE derived from mouse proliferin promoter. The CRH nGRE was clearly distinct from the consensus cAMP response element (CRE) at -224 nucleotides, which increased basal activity and cAMP responsiveness of a heterologous promoter but did not confer glucocorticoid-dependent repression. High-affinity binding sites for both GR and AP-1 nucleoproteins were identified at adjacent elements within the nGRE. Mutations that disrupted either GR or AP-1 binding activity were associated with loss of glucocorticoid-dependent repression. These results are consistent with a composite mechanism of glucocorticoid-dependent repression involving direct DNA binding of GR and AP-1 nucleoproteins at discrete adjacent sites within the CRH promoter.

C/EBP activates the human corticotropin-releasing hormone gene promoter

The purpose of these studies was to identify whether transcription factors, associated with cytokine signalling, affected promoter activity of the corticotropin releasing hormone (CRH) gene. Fragments of a 3.6 kb sequence of the human CRH gene promoter were amplified by PCR and ligated upstream of a CAT reporter. These constructs were transfected into a variety of cell lines, either alone or together, with transcription factor expression vectors. Basal activity of a 3070 bp CRH promoter fragment was only seen in neuronal and lymphoblastoid cell lines. Promoter activity was increased by the transcription factors C/EBP β (NF-IL6) and more strongly, by C/EBP δ (NF-IL6 β). Increased CRH promoter activity following phorbol ester treatment was inhibited by a dominant negative NF-IL6 mutant, showing that the effects of phorbol ester were principally mediated by C/EBP. Moreover, the inverse changes in the expression of CRH in the hypothalamus and spleens of arthritic rats were paralleled by similar inverse changes in NF-IL6 β expression in these organs. These data show that some transcription factors associated with cytokine signalling can also activate the CRH promoter.

Endometrial corticotropin-releasing hormone. Its potential autocrine and paracrine actions.

Corticotropin-releasing hormone (CRH) is expressed at several peripheral tissues including normal epithelial cells of human and rodent uterus. However, its biological role is unknown in both species. To clarify this role we studied the regulation of CRH promoter in endometrial cells. We performed homologous transfection experiments in Ishikawa cells, a human endometrial cell line, using a 0.9-kb fragment of the 5'-flanking region of human CRH gene coupled to luciferase. We found that the activity of the 5'-flanking region of the CRH gene is stimulated by cAMP and EGF and inhibited in a receptor-mediated, dose-dependent fashion by estradiol and dexamethasone. The antiglucocorticoid RU 486 acted as a glucocorticoid agonist suppressing CRH gene activation, whereas progesterone was devoid of any activity. Prostaglandin E2 and interleukins-1 and -6 stimulated CRH activation, and the prostanoid inhibitor indomethacin suppressed it, most probably by inhibiting endogenous prostaglandins. These findings suggest that endometrial CRH gene expression may be under the negative control of estrogens and glucocorticoids and under the positive control of PGE2, IL-1, and IL-6. Considering the involvement of CRH in proinflammatory phenomena, we postulate that endometrial CRH, in association with uterine prostanoids and cytokines, may participate in intrauterine inflammatory processes of early pregnancy, such as decidualization and blastocyst implantation.

Localization of a negative glucocorticoid response element of the human corticotropin releasing hormone gene

Corticotropin releasing hormone (CRH) plays a primary role in mediating suprapituitary activation of the hypothalamic-pituitary-adrenal axis and is an important physiologic target of negative feedback regulation by glucocorticoids. We sought to define cis-acting regions of the CRH promoter responsible for cAMP-dependent activation and glucocorticoid-dependent repression of CRH promoter activity. In transiently transfected AtT-20 cells, cAMP-dependent transcriptional activation was mediated largely through a classical, consensus, cAMP-response element (CRE) at −224 bp. Dexamethasone (DEX) produced a specific 2–3-fold repression of cAMP-stimulated, but not basal, CRH promoter activity. Using a series of 5′ nested deletions, dexamethasone-dependent repression of cAMP-stimulated CRH promoter activity was localized to promoter sequences between −278 and −249 bp. Specific, high-affinity binding of glucocorticoid receptor (GR) DNA-binding domain to this promoter region was observed using an eletrophoretic mobility shift assay (EMSA). We conclude that (i) cAMP dependent activation of the CRH promoter is mediated primarily by the CRE at −224 bp, (ii) glucocorticoid-dependent repression is specific for the CRH promoter, and not a generalized effect of glucocorticoid signaling or interference with the protein kinase A (PKA) signaling pathway, (iii) a highly conserved region between −278 and −249 bp is critical for glucocorticoid dependent repression, and (iv) GR is capable of interacting directly with this functionally defined negative glucocorticoid response element of the CRH promoter.

Transcription of the human corticotropin-releasing hormone gene in NPLC cells is correlated with Z-DNA formation.

The intron of the corticotropin-releasing hormone (corticoliberin; CRH) gene contains a sequence of over 100 bp of alternating purine/pyrimidine residues. We have used binding of a Z-DNA-specific antibody in metabolically active, permeabilized nuclei to study the formation of Z-DNA in this sequence at various levels of transcription. In the NPLC human primary liver carcinoma cell line, activation of cAMP-dependent pathways increased the level of transcription, while adding glucocorticoids inhibited transcription of the CRH gene. These cells respond in a manner similar to hypothalamic cells. Z-DNA formation in this sequence was detected at the basal level of transcription, as well as after stimulation with forskolin. Inhibition of transcription by dexamethasone abolished Z-DNA formation. Z-DNA formation in the WC gene (c-myc) was affected differently in the same experiment. Thus, changes in Z-DNA formation in the CRH gene are gene specific and are linked to the transcription of the gene.

Regulation of the promoter of the human corticotropin-releasing hormone gene in transfected human endometrial cells.

Corticotropin-releasing hormone (CRH) is expressed in several peripheral tissues, including normal epithelial cells of the human and rodent uterus. However, the biological role of endometrial CRH is known in neither species. As a first step to clarify this role, we studied the regulation of CRH promoter in endometrial cells. We performed homologous transfection experiments in Ishikawa cells, a human endometrial cell line, using a 0.9-kb fragment of the 5'-flanking region of the human CRH gene coupled to luciferase. Transfected cells were exposed for 18 h to 8-bromo cyclic adenosine monophosphate, forskolin, epidermal growth factor, steroids (estradiol, progesterone, and the synthetic glucocorticoid dexamethasone and their antagonists), and prostaglandin E2; then the activity of the luciferase reporter was determined in the cell lysates. We found that the activity of the 5'-flanking region of the CRH gene was stimulated by cyclic adenosine monophosphate and epidermal growth factor and inhibited in a receptor-mediated, dose-dependent fashion by estradiol and dexamethasone. The antiglucocorticoid RU 486 acted as a glucocorticoid agonist, suppressing the CRH gene activation, while progesterone was devoid of any activity. Prostaglandin E2 stimulated the CRH activation, and the prostanoid inhibitor indomethacin suppressed it, most probably by inhibiting endogenous prostaglandins. These findings suggest that endometrial CRH gene expression may be under the negative control of estrogens and glucocorticoids and under the positive control of prostaglandin E2.

Sexual dimorphism of stress response and immune/ inflammatory reaction: the corticotropin releasing hormone perspective

This review higlghts key aspects of corticotropin releasing hormone (CRH) biology of potential relevance to the sexual dimorphism of the stress response and immune/inflammatory reaction, and introduces two important new concepts based on the regulatory potential of the human (h) CRH gene: (1) a proposed mechanism to account for the tissue-specific antithetical responses of hCRH gene expression to glucocorticolds, that may also explain the frequently observed antithetical effects of chronic glucocorticoid administration in clinical practice and (2) a heuristic diagram to illustrate the proposed modulation of the stress response and immune/ inflammatory reaction by steroid hormones, from the perspective of the CRH system.

Hormonal regulation of human corticotropin-releasing hormone gene expression: implications for the stress response and immune/inflammatory reaction

EXPERIMENTAL support for the hypothesis that ACTH secretion is controlled by hypothalamic factors (1) was obtained in 1955 (2, 3). In 1981, a 41-amino acid C-terminal amidated peptide from ovine hypothalami stimulating pituitary ACTH release was identified in vitro and characterized (4). The biologically active form of this peptide, designated corticotropin-releasing hormone (CRH), and also frequently referred to as corticotropin-releasing factor (CRF), was synthesized and found to have potent ACTH-releasing actions in vivo (5). CRH is the only known permissive factor for the anterior pituitary release of ACTH in man (5, 6). It acts in synergy with arginine vasopressin (AVP) and, perhaps, other factors to regulate pituitary ACTH secretion and ultimately the activity of the pituitary-adrenal axis (7–9). Since its discovery, it has become evident that the role of CRH is much wider than initially thought. Coordination of the behavioral and physical components of the stress response and regulation of the immune/inflammatory reaction were shown to be major roles of this neuropeptide (10, 11).

Hormonal Regulation of Human Corticotropin-Releasing Hormone Gene Expression: Implications for the Stress Response and Immune/Inflammatory Reaction

EXPERIMENTAL support for the hypothesis that ACTH secretion is controlled by hypothalamic factors (1) was obtained in 1955 (2, 3). In 1981, a 41-amino acid C-terminal amidated peptide from ovine hypothalami stimulating pituitary ACTH release was identified in vitro and characterized (4). The biologically active form of this peptide, designated corticotropin-releasing hormone (CRH), and also frequently referred to as corticotropin-releasing factor (CRF), was synthesized and found to have potent ACTH-releasing actions in vivo (5). CRH is the only known permissive factor for the anterior pituitary release of ACTH in man (5, 6). It acts in synergy with arginine vasopressin (AVP) and, perhaps, other factors to regulate pituitary ACTH secretion and ultimately the activity of the pituitary-adrenal axis (7–9). Since its discovery, it has become evident that the role of CRH is much wider than initially thought. Coordination of the behavioral and physical components of the stress response and regulation of the immune/inflammatory reaction were shown to be major roles of this neuropeptide (10, 11).

The cAMP-responsive element in the corticotropin-releasing hormone gene mediates transcriptional regulation by depolarization.

Membrane depolarization is a critical element of neuronal signaling. In this study, the biochemical and molecular mechanisms involved in transcriptional regulation of the corticotropin-releasing hormone (CRH) gene by depolarization were investigated. In PC-12 cells, potassium-induced membrane depolarization increased expression of a CRH-reporter construct in a cAMP-dependent manner. This synergistic activation was mediated via calcium influx, predominantly via L-type calcium channels, and calmodulin. RNase protection assays demonstrated increased levels of CRH-reporter transcripts in stably transfected cells after treatment with cAMP and potassium, with the induced transcripts initiating at the major transcription initiation site of the human CRH gene. At the genomic level, the CRH cAMP-responsive element conferred both positive cAMP and synergistic cAMP/depolarization regulation to a heterologous promoter. Additionally, DNase I protection assays demonstrated similar nuclear protein/DNA binding profiles across the cAMP-responsive element after treatment of PC-12 cells with potassium or potassium/cAMP. These results support a model in which the protein(s) binding to the cAMP-responsive element integrates signals initiated by multiple pathways (cAMP and calcium) and transmits that integrated signal to the basal transcription machinery, resulting in increased levels of gene expression.