Timing of CRH Repression Figure 2. Relative timing of DEX and FSK treatment determines efficacy of GR-dependent repression of CRH-promoter activity. (2A) FSK-stimulation progressively induces the CRH-promoter activity in the Att20 cells over time. Co-treatment with DEX resulted in a repressed CRH- activity. 2B) CRH-promoter activity expressed as percentage of maximal induction after 3 hours forskolin (FSK) treatment (filled bar). Simultaneous co- treatment with DEX (open bar) resulted in a strong repression of the CRH-promoter activity. (2C) The repression induced by DEX in the co-treatment group was set at 100%. All groups were treated for three hours with FSK. Different time of onset of the DEX treatment relative to the FSK treatment results in a significant loss of repression when DEX treatment is started 10 minutes after FSK treatment (*). FSK treatment leads to a progressive increase in CRH-luc promoter activity over a period of at least 5 hours (inset). Values represent group averages 6 SD. doi:10.1371/journal.pone.0004327.g002 synthetic GRE-containing promoter; TAT3-luc; [20]). FSK co- treatment synergistically induced transcription on an exclusively GRE-containing promoter compared to DEX treatment alone (fig. 4). FSK treatment prior DEX treatment resulted in an increased transcriptional activity of the GR. Likewise, the longer the time of FSK co-treatment the higher the transcriptional activity of the GR. Discussion The current data demonstrate that time-dependent interactions between GR and cAMP/CREB can occur at the level of the CRH gene, where these factors seem to functionally compete for the same promoter. While similar interactions have been described for the secretion of ACTH from the pituitary [17], we now show that cAMP induced ‘GR-resistance’ occurs at the level of a single promoter, and that it is not a global cellular phenomenon, but gene-specific. Using the nGRE that was reported to be functional in these cells as a working model [14], the observed ‘primacy’ effect for transcription factor action at the CRH promoter may be due to the close proximity of the two response elements involved. The spacing of the elements is such that it is unlikely that both GR and CREB may bind simultaneously in an independent manner [16]. Sterical hindrance at the promoter due to the formation of larger protein complexes may be responsible for the importance of timing of stimuli. Alternatively, in view of the dynamic nature of transcription factor-DNA interactions, CREB-mediated chroma- tin remodeling events that disfavor GR-binding may account for the decreased GR efficacy observed after prior cAMP elevations. Interestingly, the analogous dependence of timing of both cAMP and GR that exists for ACTH secretion [17], which obviously is not linked to the activity of the exogenous reporter plasmid, suggests that the phenomenon of time-dependence occurs at multiple genes. Any gene regulated in a parallel manner will allow better hypotheses as to the mechanism that is responsible for the time dependent effects. POMC and CRH seem to depend on the same coregulator molecule, namely SRC-1 [18,21]. In this respect it would be of great interest to also study negative regulation of the endogenous POMC gene in these cells under similar conditions as were used for the CRH reporter construct. Although numerous studies were devoted to understanding the regulation of CRH gene expression in the paraventricular nucleus of the hypothalamus, it is still a topic of debate whether the activated-GR directly acts on the promoter region of the gene or that different mechanisms are responsible for the repression of CRH gene after stress. Bali et al. convincingly demonstrated in organotypic slice cultures that the GR directly acts on the paraventricular neurons to repress FSK-induced activity. Howev- er, the molecular mechanisms underlying this GR-mediated repression are still unknown. Guardiola-Diaz et al. suggested in 1996 that glucocorticoid repression occurs via interactions between the GR and the cAMP-responsive element-binding proteins [15], rather than via direct DNA binding of GR. In contrast, Dorin et al. provided evidence, also in the same cell line as used in present study, that the nGRE in the promoter is essential for repression by glucocorticoids [13]. It would certainly be of interest to study whether CREB phosphorylation status changes as a consequence of GR activation at different time points, and test the hypothesis that it is inversely related with the extent of GR repression. However, while CREB-driven transcription is re- pressed by glucocorticoids on the composite hCRH promoter, it is unaffected on a 56CRE-containing promoter [18]. With the possible caveat that the 56CRE may be not allow detection of subtle changes in CREB function, these data point to gene/ promoter specificity of any direct CREB-GR interactions. On the other hand, FSK-induced PKA can modulate glucocor- ticoid signaling both on the composite hCRH and the exclusively 36GRE-containing promoters. Therefore, PKA activation can determine the transcriptional outcome at glucocorticoid target genes, PLoS ONE | www.plosone.org 3 January 2009 | Volume 4 | Issue 1 | e4327