Steroid receptor coactivator-1 is necessary for regulation of corticotropin-releasing hormone by chronic stress and glucocorticoids

Abstract

Adaptation to stress in vertebrates occurs via activation of hormonal and neuronal signaling cascades in which corticotropin-releasing hormone (CRH) plays a central role. Expression of brain CRH is subject to strong, brain-region specific regulation by glucocorticoid hormones and neurogenic intracellular signals. We hypothesized that Steroid Receptor Coactivator 1 (SRC-1), a transcriptional coregulator of the glucocorticoid receptor, is involved in the sensitivity of CRH regulation by stress-related factors. In the brains of SRC-1 knockout mice we found basal CRH mRNA levels to be lower in the central nucleus of the amygdala. Hypothalamic CRH up-regulation after chronic (but not acute) stress, as well as region-dependent up- and down-regulation induced by synthetic glucocorticoids, were significantly attenuated compared with wild type. The impaired induction of the crh gene by neurogenic signals was corroborated in AtT-20 cells, where siRNA and overexpression experiments showed that SRC-1 is necessary for full induction of a CRH promoter reporter gene by forskolin, suggestive of involvement of transcription factor CREB. In conclusion, SRC-1 is involved in positive and negative regulation of the crh gene, and an important factor for the adaptive capacity of stress.

Fig. 1.

SRC-1 affects basal CRH mRNA expression in a region specific manner. (A) Autoradiograms showing CRH mRNA signal in the brain at the level of PVN and CeA. (B) CRH mRNA is lower in the CeA in SRC-1 knockout mice under basal conditions (n = 6–8). Two-way ANOVA indicates a significant effect for brain region (F_1,26 = 130.1; P < 0.0001) and an interaction for genotype x brain region (F_1,26 = 9.60; P < 0.01). **, P < 0.01, comparison with vehicle treated group.

Fig. 2.

CRH expression is resistant to dexamethasone treatment in SRC-1 knockout mice. (A) Down-regulation of CRH expression is observed in the PVN of wild type mice but not in SRC-1 knockout animals (n = 3–5). Two-way ANOVA indicates a significant interaction between genotype and treatment (F_1,13 = 6.63; P < 0.05). (B) Up-regulation in the CeA occurs only in wild type mice. Two-way ANOVA indicates a significant effect of genotype and an interaction effect (F_1,13 = 7.74; P < 0.05). Data are expressed as percentage of vehicle treated wild type mice. *, P < 0.05 and **, P < 0.01, comparisons with vehicle treated group.

Fig. 3.

Adaptation to chronic rat stress depends on SRC-1. (A) Acute rat exposure leads to a robust corticosterone response, which is not different between the genotypes (n = 7–10). Two-way ANOVA, time effect: F_3,54 = 13.87; P < 0.0001. (B) After 10 days the mice had habituated to the stressor but still showed a significant response to the stressor of rat exposure. Two-way ANOVA indicates a significant effect for time (F_3,54 = 9.67; P < 0.0001) and for genotype (F_1,54 = 5.42; P < 0.05). (C) CRH mRNA expression in the PVN is increased after chronic stress in wild type but not knockout mice (n = 7–19). Data are expressed as percentage of nonstressed wild type mice. Two-way ANOVA shows significance for treatment (F_1,41 = 5.42; P < 0.05) and an interaction between treatment and genotype (F_1,41 = 8.85; P < 0.01). (D) In PVN, the CRH hnRNA response to acute restraint is not affected by prior rat stress or genotype. (E) Rat stress does not lead to significant changes in expression of CRH in CeA in either genotype. Two-way ANOVA only shows the genotype effect also observed in untreated animals because of >2-fold lower expression of CRH mRNA in SRC-1 knockout mice (F_1,41 = 93.93; P < 0.001; see also Fig. 1).

Fig. 4.

Forskolin-stimulated induction of the CRH promoter depends on SRC-1. (A) Knockdown of SRC-1 with siRNA dose-dependently attenuates induction of the hCRH promoter by forskolin, as tested by 1-way ANOVA (F_2,8 = 11.53; P < 0.005). *, P < 0.05, **, P < 0.01, compared with control condition, Tukey's posthoc test. (B) The 2 main SRC-1 splice variants both increase forskolin-stimulated induction of the hCRH promoter as indicated by 1-way ANOVA (F_2,13 = 156.1, P < 0.001). **, significantly different from control condition, P < 0.01, Tukey's posthoc test.