Timing Is Critical for Effective Glucocorticoid Receptor Mediated Repression of the cAMP-Induced CRH Gene Siem van der Laan, E. Ronald de Kloet, Onno C. Meijer* Division of Medical Pharmacology, Leiden/Amsterdam Center for Drug Research and Leiden University Medical Center, Leiden, The Netherlands Abstract Glucocorticoid negative feedback of the hypothalamus-pituitary-adrenal axis is mediated in part by direct repression of gene transcription in glucocorticoid receptor (GR) expressing cells. We have investigated the cross talk between the two main signaling pathways involved in activation and repression of corticotrophin releasing hormone (CRH) mRNA expression: cyclic AMP (cAMP) and GR. We report that in the At-T20 cell-line the glucocorticoid-mediated repression of the cAMP- induced human CRH proximal promoter activity depends on the relative timing of activation of both signaling pathways. Activation of the GR prior to or in conjunction with cAMP signaling results in an effective repression of the cAMP-induced transcription of the CRH gene. In contrast, activation of the GR 10 minutes after onset of cAMP treatment, results in a significant loss of GR-mediated repression. In addition, translocation of ligand-activated GR to the nucleus was found as early as 10 minutes after glucocorticoid treatment. Interestingly, while both signaling cascades counteract each other on the CRH proximal promoter, they synergize on a synthetic promoter containing ‘positive’ response elements. Since the order of activation of both signaling pathways may vary considerably in vivo, we conclude that a critical time-window exists for effective repression of the CRH gene by glucocorticoids. Citation: van der Laan S, de Kloet ER, Meijer OC (2009) Timing Is Critical for Effective Glucocorticoid Receptor Mediated Repression of the cAMP-Induced CRH Gene. PLoS ONE 4(1): e4327. doi:10.1371/journal.pone.0004327 Editor: Bernhard Baune, James Cook University, Australia Received October 1, 2008; Accepted November 19, 2008; Published January 29, 2009 Copyright: 2009 van der Laan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The research described was funded by a ZON-MW Vidi grant (016.036.381) to OCM, and the Royal Dutch Academy of Arts and Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: o.meijer@lacdr.leidenuniv.nl Introduction Corticotropin releasing hormone (CRH) is a pivotal signaling molecule in the regulation of the stress response. This neuropep- tide is expressed at high levels in the hypothalamus, from where it coordinates the hormonal and autonomic response to stress, and the central nucleus of the amygdala, where it plays a crucial role in regulating anxiety. Regulation of the expression of CRH is therefore thought to be physiologically important in relation to coping with stress. CRH gene regulation is a complex process that involves multiple activating and repressing transcription factors [1]. Among the often studied factors that can regulate CRH expression are glucocorticoid hormones [2], which in a cell- dependent manner either repress of stimulate the CRH gene. As such, the CRH promoter can be considered a model gene for cell- specific negative regulation of gene expression via glucocorticoids. Cross-talk of intracellular signaling pathways is central to many neuroendocrine control systems [3,4]. The expression and/or secretion of the two main neuroendocrine secretagogues of the hypothalamus-pituitary-adrenal axis (HPA axis) are both stimu- lated by cAMP and suppressed by glucocorticoids, the end- product of the HPA axis: hypothalamic CRH, as well as adrenocorticotrophic hormone (ACTH) from anterior pituitary corticotrophs [5–8]. At the molecular level, these signals are represented by protein kinase A (PKA), the transcription factor cAMP element-binding protein (CREB), and the GR, respectively. The proximal promoter of the human corticotrophin releasing hormone (hCRH) gene contains a canonical, functional cAMP response element (CRE) and a negative glucocorticoid receptor response element (nGRE) (fig. 1). Induction of the hCRH gene by the PKA pathway is mediated by phosphorylation of CREB at serine residue 133 [9,10]. In vivo, Wo¨ lfl et al. showed that binding of CREB to the canonical CRE located at the nucleotide position 2224 (upstream of exon 1) was specifically induced after activation of the PKA pathway with forskolin [11]. Additionally, Kovacs et al. demonstrated that in the hypothalamic parvocellular neurons of rodents subjected to ether stress, CREB phosphorylation was induced in a time course that parallels the increase of CRH heteronuclear RNA levels [12]. In vitro, the At-T20 cell-line is a well-established model system for studying glucocorticoid-induced repression of the hCRH proximal promoter. Nested deletions and site-specific point mutations of the CRE located at nucleotide 2224 resulted in a significant loss of induction by cAMP, demonstrating that CREB binding is necessary for the stimulation of the gene [13]. In parallel, electrophoretic mobility shift assays (EMSA) identified a GR-binding site at position nt 2249 that was indispensable for GR-mediated repression of the cAMP-induced promoter. Internal deletion of the entire nGRE and specific point mutations resulted in a loss of repression by the ligand- activated GR, indicating that DNA binding is essential for the glucocorticoid-induced repression [14]. Of note: while we have taken this nGRE-mode as working model, a separate series of experiments did not find evidence for direct GR binding to the CRH promoter, but rather suggested direct CREB-GR interactions as the cause of GR-mediated reression [15]. The nGRE in the hCRH promoter is separated by as few as 25 bp with the canonical CRE, a distance that clearly permits functional interactions at the promoter [16]. Since, in vivo the order PLoS ONE | www.plosone.org 1 January 2009 | Volume 4 | Issue 1 | e4327