Role of glucocorticoids and cAMP-mediated repression in limiting corticotropin-releasing hormone transcription during stress

Abstract

The role of glucocorticoids and the repressor isoform of cAMP response element (CRE) modulator (CREM), inducible cAMP early repressor (ICER), in limiting corticotropin-releasing hormone (CRH) transcription during restraint stress were examined in both intact and adrenalectomized rats receiving glucocorticoid replacement. CRH primary transcript, measured by intronic in situ hybridization, increased after 30 min of restraint and returned to basal levels by 90 min, despite the persistent stressor. The decline was independent of circulating glucocorticoids, because adrenalectomized rats displayed an identical pattern. ICER mRNA in the hypothalamic paraventricular nucleus (PVN) increased after 30 min and remained elevated for up to 4 h in a glucocorticoid-independent manner. Western blot and electrophoretic mobility shift assay analyses showed increases in endogenous ICER in the PVN of rats subjected to restraint stress for 3 h. Chromatin immunoprecipitation assays showed the recruitment of CREM by the CRH CRE in conjunction with decreases in RNA polymerase II (Pol II) binding in the PVN region of rats restrained for 3 h. These data show that stress-induced glucocorticoids do not mediate the limitation of CRH transcription. Furthermore, the ability of CREM to bind the CRH CRE and the time relationship between elevated CREM and reduced Pol II recruitment by the CRH promoter suggest that inhibitory isoforms of CREM induced during stress contribute to the decline in CRH gene transcription during persistent stimulation.

Figure 1.

Changes in CRH hnRNA in the PVN during 4 h of restraint stress, measured by intronic in situ hybridization. Rats were placed in plastic restrainers and killed by decapitation at the times indicated. Images are representative of five rats per experimental group. Data points are the mean ± SE of integrated density (Int Density) values in five rats per group. ^*p < 0.01 compared with basal; ^**p < 0.001 compared with basal. Error bars represent SEM.

Figure 2.

Glucocorticoids do not mediate the rapid decline in CRH transcription during stress. A, B, Plasma corticosterone (Cort; A) and CRH hnRNA levels in the PVN (B) during the course of 3 h of restraint stress in sham-operated (Sham) and adrenalectomized (ADX) rats receiving constant levels of corticosterone via subcutaneous implants. Rats were killed by decapitation at the indicated time points. Data points are the mean ± SE of the values obtained in five rats per experimental group. ^*p < 0.001 compared with basal; ^#p < 0.05 compared with basal sham-operated rats. Int Density, Integrated density. Error bars represent SEM.

Figure 3.

Changes in CREM/ICER mRNA measured by in situ hybridization using either a riboprobe recognizing all forms of CREM (CREM mRNA) or a 48-mer oligoprobe specific for ICER (ICER mRNA). A, Representative images from film autoradiography of one of three rats per experimental group for CREM mRNA and five rats for ICER mRNA. B, Data points are the mean ± SE of the pooled data, expressed as percentage of basal values. Basal integrated density values were 7983 and 1019 arbitrary units for CREM mRNA and ICER mRNA, respectively. ^*p < 0.001 compared with basal. C, Cellular localization of CREM mRNA in PVN sections of rats subjected to restraint stress for 3 h. Double-labeling in situ hybridization using ³⁵S-labeled CREM cRNA probes (bright green grains) and digoxigenin-labeled CRH cRNA probes shows ³⁵S-CREM staining (bright grains) overlaying digoxigenin-labeled parvicellular CRH neurons. 3rd V, Third ventricle. C, Bottom, Higher magnification of the area enclosed in the square. The white arrowheads indicate some of the CRH neurons labeled with CREM. CREM staining in control nonstressed rats was no different from background staining [12.3 ± 1.8 and 9.6 ± 1.4 grains/cell (data not shown)]. Error bars represent SEM.

Figure 4.

Increases in CREM expression during stress are independent of glucocorticoids. Changes in CREM mRNA during restraint stress in sham-operated (Sham) or adrenalectomized (ADX) rats receiving constant low levels of corticosterone (Cort) replacement via subcutaneous implants are shown. A, Representative autoradiographic images of three rats per experimental group. B, Data points are the mean ± SE of the integrated density (Int Density) values in three rats per experimental group. ^*p < 0.001 versus basal in sham-operated and adrenalectomized rats; treatment had no effect (adrenalectomy). Error bars represent SEM.

Figure 5.

Restraint stress leads to sequential increases in phospho-CREB and CREM protein in the hypothalamic PVN region. A, Western blot analysis of phospho-CREB and CREM in nuclear extracts of microdissected PVN of rats under basal conditions and after 15 min or 3 h of restraint stress. Data were obtained from pooled hypothalamic tissue from 10 rats per experimental group. Protein loading in the gel was verified by tubulin levels, as shown in the bottom row. B, Semiquantitative analysis of the ICER bands, expressed as percentage change from basal values. Error bars represent SEM.

Figure 6.

A, Gel-shift assay and supershift of CRH CRE-radiolabeled oligonucleotides and nuclear proteins from microdissected PVNs from 10 rats per group of control rats or rats subjected to restraint stress for 15 min or 3 h. The black arrow indicates the major shifted band that is supershifted with phospho-CREB antibody. The white arrow shows a faster-migrating band that was supershifted by the CREM antibody. The image is representative of three similar experiments. The semiquantitative analysis of this band in the presence or absence of phospho-CREB and CREM antibodies is shown in B. Ab, Antibody. Data are expressed as fold-change from basal without antibody.

Figure 7.

Restraint stress induces recruitment of phospho-CREB and CREM by the CRH promoter in the hypothalamic PVN region. A, ChIP assays using phospho-CREB, CREM, and Pol II antibodies and cross-linked DNA from the microdissected hypothalamic PVN region of control rats and rats subjected to restraint stress for 30 min or 3 h. Similar increases in CREM recruitment by 3 h are shown in B. B, PCR for the CRH promoter in the pooled remaining cross-linked DNA for this experiment (Pool), immunoprecipitated with an unrelated antibody against a polypeptide corresponding to the C terminus of the vasopressin V_1b receptor, yielded no PCR band. C, No PCR product was found using primers against the V_1b receptor on DNA immunoprecipitated with CREM antibody. D, PCR for the CRH coding region (CR) performed on the immunoprecipitants in A showed nonspecific bands, which did not change with stress. Gel images are representative of the results in three experiments. Ab, Antibody; p-CREB, phospho-CREB; V_1bR, vasopressin V_1b receptor.