Mice with an increased glucocorticoid receptor gene dosage show enhanced resistance to stress and endotoxic shock

Abstract

Targeted mutagenesis of the glucocorticoid receptor has revealed an essential function for survival and the regulation of multiple physiological processes. To investigate the effects of an increased gene dosage of the receptor, we have generated transgenic mice carrying two additional copies of the glucocorticoid receptor gene by using a yeast artificial chromosome. Interestingly, overexpression of the glucocorticoid receptor alters the basal regulation of the hypothalamo-pituitary-adrenal axis, resulting in reduced expression of corticotropin-releasing hormone and adrenocorticotrope hormone and a fourfold reduction in the level of circulating glucocorticoids. In addition, primary thymocytes obtained from transgenic mice show an enhanced sensitivity to glucocorticoid-induced apoptosis. Finally, analysis of these mice under challenge conditions revealed that expression of the glucocorticoid receptor above wild-type levels leads to a weaker response to restraint stress and a strongly increased resistance to lipopolysaccharide-induced endotoxic shock. These results underscore the importance of tight regulation of glucocorticoid receptor expression for the control of physiological and pathological processes. Furthermore, they may explain differences in the susceptibility of humans to inflammatory diseases and stress, depending on individual prenatal and postnatal experiences known to influence the expression of the glucocorticoid receptor.

FIG. 1

Isolation of a YAC carrying the Gr gene. (A) Structure of the unmodified YAC YGR4 and the shortened YAC YGR290. The position of the Gr gene on the YAC and its exon-intron structure (enlarged) is indicated. (B) Restriction analysis of YGR4 by digestion with the rare-cutting enzymes SfiI and NotI, PFGE, and Southern blotting with probes for GR exons 2 and 8. Alignment of fragments is shown in panel A. (C) PFGE and Southern blot analysis of YACs obtained after B1 fragmentation. The position of the original YAC YGR4 and its shortened derivative YGR290 are indicated by an arrow.

FIG. 2

Expression analysis of YGR transgenic mice. (A) Determination of the YAC copy number in YGR mice by Southern blot analysis of tail DNA from WT (wt) mice and one transgenic mouse from each generation (F₁ and F₂). (B) Analysis of transgene expression in the hippocampus of a C57BL/6 mouse, an FVB/N mouse, and a YGR mouse by RT-PCR and RFLP. PCR products were cut with RsaI and analyzed by Southern blotting. (C) Analysis of transgene expression in various tissues by RT-PCR and RFLP. br, whole brain; hyp, hypothalamus; co, cortex; pit, pituitary; ad, adrenal; thy, thymus; sp, spleen; li, liver; lu, lung; w, wild type; Y, YGR transgenic mice. (D) Western blot analysis of GR protein expression in the hippocampus. wt, wild type.

FIG. 3

Expression analysis of genes involved in HPA axis regulation. (A) Immunohistochemistry of CRH in the median eminence of WT mice; (B) immunohistochemistry of CRH in the median eminence of YGR mice; (C) in situ hybridization of POMC in the anterior pituitary of WT mice; (D) in situ hybridization of POMC in the anterior pituitary of YGR mice; (E) immunohistochemistry of ACTH in the anterior pituitary of WT mice; (F) immunohistochemistry of ACTH in the anterior pituitary of YGR mice. 3rd, third ventricle; ME, median eminence; AL, anterior lobe of the pituitary; NIL, intermediate lobe of the pituitary; PL, posterior lobe of the pituitary.

FIG. 4

Consequences of restraint stress on hormone secretion in WT (wt) and YGR mice. (A) Serum corticosterone levels. Basal levels, levels after 40 min of restraint stress, and levels 20 min after the removal of the stressor are shown. (B) Serum ACTH levels at the same time points as described for panel A. Statistical significance was determined by the Student t test (n ≥ 5).

FIG. 5

Glucocorticoid-induced apoptosis of primary thymocytes of WT (wt) and YGR mice. (A) Flow cytometric analysis of thymocytes cultivated for 9 h in the presence of 10⁻⁸ M dexamethasone (dex) and stained with PI. Analysis by gating the cells in region 2 on the basis of their DNA content (FL2-A) and the granulation (SSC) pattern is exemplified. (B) Dose-response curves of dexamethasone-treated thymocytes from four individual mice, two WT and two YGR, are depicted. Cells were cultivated for 9 h in the absence (con) or presence of various concentrations of the GR agonist dexamethasone or after treatment with the GR antagonist ZK112,339 (10⁻⁶ M). The degree of apoptosis was determined as described in Materials and Methods and plotted against the concentration of dexamethasone.

FIG. 6

LPS-induced inflammation and endotoxic shock in WT (wt) and YGR mice. (A) LPS injection (4 mg/kg intraperitoneally) and analysis of IL-6 levels in the serum at the given time points. The difference at 3 h is highly significant. (B) Survival after injection of a high dose of LPS (40 mg/kg intraperitoneally). The percentage of surviving mice was determined at 24-h intervals (for WT mice, n = 6; for YGR mice, n = 8).