Timing of CRH Repression Figure 3. Translocation of the GR occurs within 10 minutes after treatment. (3A) Time course of GR-ir in different treatment groups. DEX alone and FSK + DEX co-treatment, but not FSK alone show nuclear GR staining after 10 minutes treatment. (3B) Control IgG staining show specificity of the GR-specific antibody. (3C) Nuclear quantification of GR-ir after 10 minutes treatment. Values represent average 6SEM. doi:10.1371/journal.pone.0004327.g003 independent of the presence of CREs in the promoter. We postulate that there is no cross-talk between the GR and CREB off the DNA but that PKA activation modulates GR-mediated transcription by changing e.g the phosphorylation status of coregulator proteins. Chromatin immunoprecipitation assays should be used to test the interactions between GR and (phospho-)CREB at the CRH promoter, and demonstrate lack of direct GR-CREB interactions at non-composite GRE-containing promoters. An unexplained phenomenon is that, in contrast to the situation in PVN, glucocorticoids induce, rather than repress, CRH gene expression in the placenta and amygadala [2,22–24]. The opposite effect of GR in these cells may rather relate to differential presence of transcription factors or coactivators such as SRC1a [18,25]. One principle difference in cellular context between CRH containing cells in PVN and other tissues is that activation of the CRH gene in the paraventricular cells often will be accompanied by increased activation of the HPA axis, causing a quick rise in glucorticoid levels and GR activation. However, current data should be interpreted in the context of regulation of the CRH-promoter in the PVN, and do not give insights in the mechanisms governing the cell-specific effects of glucocorticoids on CRH expression. It is well known that acute exogenous steroid treatment effectively suppresses stress-induced expression of CRH mRNA in rats [26]. However, the current study using a model system shows that repression is markedly attenuated if GR activation is initiated with as little as a 10 minutes delay. The critical time- window for effective repression by glucocorticoids may have interesting implications in the control of CRH expression in vivo. The order of activation of both signaling pathways is variable, and depends on the history of stress and glucocorticoid exposure, as well as the circadian and ultradian pulsatility of glucocorticoid levels [27]. Therefore, it is likely that effective GR-mediated repression of the stress-induced CRH mRNA expression will only occur in specific situations. We conclude that the differences in timing of stimulatory and repression signals are of consequence for adaptation of the organism to stress. Materials and Methods Reporter assays 0.16106 cells were transiently transfected in 24-wells plate using Lipofectamine 2000 (Invitrogen, Breda, The Netherlands) accord- ing to the manufacturer’s instructions. Per well, 200 ng of the hCRH-luc reporter plasmid [18] or the 36GRE containing TAT3-luc reporter were transfected. The day after transfection, the cells were treated with 10 mM forskolin (Calbiochem, Darmstadt, Germany) and/or 0.1 mM of the synthetic glucocor- ticoid dexamethasone (DEX) and assayed for luciferase activity. PLoS ONE | www.plosone.org 4 January 2009 | Volume 4 | Issue 1 | e4327