Timing of CRH Repression Figure 1. Simplified representation of the hCRH-luc promoter and known response elements. Schematic representation of the composite hCRH proximal promoter, as present in the reporter construct. Although only the known nGRE and CRE have been indicated, many response elements have been identified within the used reporter construct, such as a functional estrogen response element half site [28], and several putative AP1 sites [14,25]. In addition, some of the listed factors act on sequences that are not present in reporter construct [29]. doi:10.1371/journal.pone.0004327.g001 of activation of the cAMP and glucocorticoid signaling pathways may vary considerably, and this is known to affect responses at the level of neuroendocrine secretion [17], we tested the hypothesis that effective repression of the cAMP-induced hCRH proximal promoter depends on the relative timing of GR activation in the At-T20 cell-line. Results Dexamethasone pre- or simultaneous co-treatment with FSK FSK treatment led to a robust and progressive stimulation of the CRH-promoter activity that was evident for luciferase induction from 1 hour to at least 5 hours (fig. 2A). In line with previous reports [14,18], simultaneous DEX co-treatment strongly sup- pressed the FSK-induced stimulation of the hCRH-promoter activity (fig. 2A). DEX co-treatment resulted in up to 75% repression of the FSK-induced promoter activity after 3 hours treatment (fig. 2B). To test our hypothesis that the order of activation of both signaling cascades affects the level of GR- mediated repression, we initiated the DEX treatment at different time points prior to or after initiation of the 3-hours FSK treatment (fig. 2C). We compared the DEX-induced repression in the different groups to the simultaneous co-treatment group, which was set at 100% repression. Two hours of DEX pre- treatment resulted in a significantly increased repression, suggest- ing that a slower mechanism requiring de novo protein synthesis is responsible for the additional repression (data not shown). Activation of the GR up to one hour prior to FSK treatment did not affect the levels of repression (fig 2C, first three time points). Of note, DEX treatment alone (0.1 mM) did not suppress the basal activity of the CRH-promoter, indicating that basal CRH drive is not governed by CREB/CRE dependent mecha- nisms (data not shown). Dexamethasone treatment applied after FSK When DEX was applied after forskolin stimulation of the CRH promoter, the time-window separating both treatments was of great consequence for the level of repression (fig. 2C). A 10 minutes delay in the onset of DEX treatment relative to the FSK treatment resulted in a loss of 20% repression. Strikingly, a 30 minutes delay resulted in a 50% loss of GR-mediated repression, indicating the importance of the relative time of treatment. Clearly, the reduced time of DEX exposure is not proportional to the loss of GR-mediated repression pointing to a ‘GR resistance’. Because FSK treatment induces a progressive increase of the CRH-luc promoter activity over a period of at least 5 hours (fig. 2A) we assume that FSK-induces binding of CREB to the promoter over that period. However, the first hour following FSK treatment is critical for the GR to mediate effective repression. To assess whether FSK treatment alters the translocation properties of the GR to the nucleus, we quantified GR- immunoreactivity in the different conditions. The data show that DEX treatment induces maximal nuclear GR-immunoreactivity (GR-ir) as early as 10 minutes after treatment (figure 3). No difference in nuclear GR-ir was observed between the 10 and 30 minutes DEX treatment groups, suggesting that the ‘GR resistance’ is not due to delayed translocation to the nucleus (fig. 3A). In addition, FSK treatment did not influence transloca- tion dynamics of the GR although it is known that PKA activation can modulate the steroid sensitivity by enhancing DNA binding properties of GR [19]. In sum, the translocation data provide evidence that GR should be capable of modulating gene transcription as early as 10 minutes after treatment and that FSK treatment does not interfere with translocation related mechanisms. Promoter specificity Posttranslational modification such as phosphorylation is known to affect DNA binding properties, transcriptional activation and stability of numerous nuclear receptors including GR [19]. Although translocation to the nucleus was not affected by FSK treatment, we tested whether FSK influenced the transcriptional activity of the GR in these cells. We measured the effect of FSK and DEX co-treatment on a positively regulated promoter (a PLoS ONE | www.plosone.org 2 January 2009 | Volume 4 | Issue 1 | e4327