Characterization of a novel gain of function glucocorticoid receptor knock-in mouse

Abstract

Glucocorticoids (GCs) exert profound influences on many physiologic functions by virtue of their diverse roles in growth, development, and maintenance of homeostasis. We previously created a novel gain of function in the human glucocorticoid receptor (hGR), hGRM604L, which is active at GC concentrations 5-10-fold lower than wild-type GR. To gain a greater insight into GC physiology in vivo, we inserted this mutant GR (GRM610L in mice) into mice via homologous recombination. Mice expressing the allele are phenotypically normal with respect to GC function. However, corticosterone levels, ACTH levels, and adrenocortical size are markedly reduced, suggesting they are phenotypically normal because the mutant GR alters the basal regulation of the hypothalamic-pituitary-adrenal axis. We demonstrate via physiologic and immunologic studies that GRM610L mice have increased sensitivity to GCs in vivo. Sensitivity to the actions of endogenous GCs may be an important factor underlying the development of many human diseases including hypertension, obesity, and diabetes. Our model may provide a new and powerful tool for the study of GC physiological and pathological processes in vivo.

FIGURE 1.

GR_M610L knock-in strategy. A, the structure of the 14-kb mouse GR exon 6 region is shown. Exon 6, bearing the M610L mutation, is inserted between flanking loxP sites, which in turn is flanked by a 2.5-kb short arm containing exon 5 and a 6.5-kb long arm containing exons 7 and 8. The neomycin phosphor-reductase cassette is flanked by frt sites as indicated. Gene targeting was assayed via long-range PCR. B, long-range PCR of short arm DNA using the MGRSA-F and MGRSA-R primers. Clones 11, 32, 41, 75, 143, and 149 showed evidence of homologous recombination. This was subsequently confirmed via long-range PCR on the long arm and Southern blotting of EcoRV-digested DNA (data not shown). C, robust expression of GR_M610L RNA in vivo. Total RNA was prepared from kidney from a mouse heterozygous for the GR_M610L allele, GR exons 4–9 were amplified via reverse transcriptase-PCR and then sequenced using the exon 4 forward primer. These primers were chosen as they lie outside sequences utilized in our targeting vector. The wild-type and L610 alleles are identical except at the (indicated) site of the M610L substitution. At this nucleotide, there is evidence for approximately equivalent expression of the two alleles.

FIGURE 2.

Circulating corticosterone and ACTH levels are reduced in GR_M610L mice. A and B, daytime (A) and nighttime (B) plasma corticosterone levels in GR_M610L mice and WT littermates. C and D, daytime (C) and nighttime (D) plasma ACTH levels in GR_M610L mice and WT littermates. GR_M610L heterozygous and homozygous mice have a highly significant decrease in plasma corticosterone and ACTH levels compared with wild-type animals. E and F, corticosterone (E) and ACTH (F) levels in control and GR_M610L littermates after 30-min restraint stress. G, corticosterone levels in control and GR_M610L littermates after 36 h fasting. H, corticosterone levels in control and GR_M610L mice littermates 20 min after ACTH injection (10 μg/kg, intraperitoneal). I, aldosterone levels in control and GR_M610L mice littermates. ^*, p < 0.05; ^**, p < 0.01.

FIGURE 3.

GR_M610L mice have markedly reduced adrenal weight compared with control animals. A, gross appearance of adrenal glands taken from +/+, +/L, and L/L littermates. B, mean adrenal weight of 3–4-month-old female and male GR_M610L mice and wild-type littermates. Adrenal weight is expressed as milligrams/g body weight. GR +/L and L/L mouse adrenals are significantly smaller than those from wild-type littermates in both male and female mice (^*, p < 0.001 versus +/+). C, low (×10) and high (×40) power views of hematoxylin and eosin-stained adrenal tissue sections derived from a +/+ mouse (left panel) and an L/L mouse (right panel). There is marked thinning of the adrenal cortex (bracketed) in GR_M610L mice. D, mean adrenal cortical thickness in +/+ (n = 13), +/L (n = 14), and L/L (n = 13) mice. There was a marked and highly significant reduction in adrenal cortical thickness in GR_M610L mice.

FIGURE 4.

The GR_M610L allele increases sensitivity to exogenous glucocorticoids in vivo. A, baseline blood pressure was recorded for 4 days in an L/L mouse (left panel) or in a +/+ (right panel) littermate. Subsequently, dexamethasone was provided in the drinking water (1 mg/liter) and blood pressure was followed radiotelemetrically. The blood pressure tracing of a representative mouse is shown. The initiation and termination of dexamethasone are indicated. Each data point represents the average of all measurements obtained within a 4-h window. DEX (1 mg/liter) induces a rapid rise in BP with loss of normal diurnal variation in L/L mice, but not in +/+ littermates; and that this rise in BP rapidly reverses following discontinuation of dexamethasone. Subsequently, 10 mg/liter DEX was provided to the +/+ mouse, and a rapid rise in BP was observed. B, 12-h means of MAP in male GR +/+ (n = 6), L/L (n = 6), or +/L (n = 6) littermates before (baseline) and after treatment with DEX (1–10 mg/liter) in the drinking water. MAP values were measured by telemetry. Values were analyzed as 12 h means reflecting the day and night periods and expressed as mean ± S.E.; C, mean ± S.E. values for sleep-to awake BP ratio of GR L/L mice before and after 1 mg/liter DEX treatment.

FIGURE 5.

A, serum testosterone (T) levels in wild-type (+/+, n = 11), heterozygous (+/L, n = 8), and homozygous (L/L, n = 8) mice. B–D, the effect of low-dose dexamethasone (DEX, 0.1 mg/kg intraperitoneally) treatment on serum testosterone levels in wild-type (B, n = 12), heterozygous (C, n = 9), and homozygous (D, n = 6) mice. Data are expressed as mean ± S.E. ^*, p < 0.05 compared with saline treatment.

FIGURE 6.

A, 11β-HSD1 activities in livers of wild-type (+/+, n = 5), heterozygous (+/L, n = 5), and homozygous (L/L, n = 4) mice. B–D, the effects of low-dose dexamethasone (DEX, 0.1 mg/kg intraperitoneally) treatment on 11β-HSD1 activity in wild-type (panel B, n = 7), heterozygous (panel C, n = 7), and homozygous (panel D, n = 8) mice. Data are expressed as mean ± S.E. ^*, p < 0.05 versus saline treatment.

FIGURE 7.

Decreased immune cell counts in wild-type recipients of GR_M610L bone marrow. Irradiated C57/BL6 mice were transplanted with T-cell depleted bone marrow from mice carrying 0, 1, or 2 copies of the GR_M610L allele. Ten mice were transplanted in each group. Ten weeks later (after engraftment), blood was removed via standard retroorbital bleeding techniques, and prepared for fluorescence-activated cell sorter analysis. Cell counts for the indicated cell types were assessed. One-way analysis of variance and Tukey Cramer analysis was used to compare the experimental groups with the wild-type control.