GRKO mice express an aberrant dexamethasone-binding glucocorticoid receptor, but are profoundly glucocorticoid resistant

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Abstract

The introduction of a targeted insertion mutation into exon 2 of the gene coding for the glucocorticoid receptor (GR) enabled production of glucocorticoid receptor knock-out (GRKO) mice. GRKO mice on a C57BL/6/129sv mixed genetic background show a variable phenotype, with 90% of −/− mice dying at birth with respiratory insufficiency but 10% of mutant mice surviving to maturity. To investigate the possibility of residual GR expression in surviving GRKO mice we have measured binding of the synthetic glucocorticoid dexamethasone in tissue extracts from adrenalectomized mice. High affinity binding of dexamethasone in protein extracts of liver, kidney, lung and brain from adult GRKO mice is found at levels 30–60% those in wild-type mice, with heterozygotes (+/–) having intermediate levels. PCR and ribonuclease protection analysis showed comparable levels of GR mRNA on the 3′ side of the gene-targeted insertional mutation in exon 2 of the GR gene, with almost no GR mRNA detected from exons 1 and 2 on the 5′ side of the gene-targeted insertional mutation. Western blot analysis using a C-terminal specific GR antibody detects a 39 kDa GR fragment in extracts from adult GRKO mice. Despite the evidence for expression of a ligand-binding domain fragment of the glucocorticoid receptor these mice are profoundly glucocorticoid resistant, with elevated levels of plasma ACTH and corticosterone. Thymocytes from adult and fetal GRKO mice are resistant to dexamethasone-induced apoptosis and cultured fetal hepatocytes from GRKO mice are completely refractory to glucocorticoid induction of the gluconeogenic enzyme glucose-6-phosphatase. Thus although the surviving adult homozygous GRKO mice express a dexamethasone-binding GR fragment, their classic target tissues remain profoundly glucocorticoid insensitive.

Introduction

Glucocorticoids are important in the regulation of many developmental and homeostatic processes such as gluconeogenesis, lung maturation, and responses to stress through interactions in the hippocampus and other parts of the brain (Ballard, 1979, Orth et al., 1992). The majority of these actions are mediated through two specific intracellular receptors, the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR), the latter functioning in epithelia as the physiological receptor for the mineralocorticoid aldosterone (Funder, 1997). In aldosterone target tissues the activity of the glucocorticoid-metabolizing enzyme 11β-hydroxysteroid dehydrogenase type-2 prevents the higher circulating levels of glucocorticoids from activating MR, and thus mimicking aldosterone action (Funder et al., 1988, Edwards et al., 1988). Both GR and MR are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors (Parker, 1993, Mangelsdorf et al., 1995, Beato et al., 1995). Upon activation by ligand, GR and MR homodimerize and translocate to the nucleus where they bind to specific DNA sequences (hormone response elements) to modulate target gene transcription.

To provide further insight into the role of GR and MR in glucocorticoid and mineralocorticoid physiology, the genes for both receptors have been separately disrupted or knocked out in mice using gene targeting (Cole et al., 1995, Berger et al., 1998). In addition, a neural-specific knockout of the GR gene in mice has recently been described (Tronche et al., 1999). All MR-deficient (MRKO) mice die 8–10 days after birth due to an inability to retain renal sodium and water (Berger et al., 1998). The majority of GR-deficient (GRKO) mice (∼90%) die at birth, reflecting retarded lung maturation and atelectasis (Cole et al., 1995, Cole, 1996). GRKO mice show a reduced capacity to activate key gluconeogenic enzyme genes in the liver and impaired negative feedback in the hypothalamus–pituitary–adrenal (HPA) axis, resulting in markedly elevated plasma ACTH and corticosterone levels (Cole et al., 1995, Christoffels et al., 1998). On a C57Bl6/129sv genetic background a small proportion (10%) of GRKO mice survive to maturity but display deficits in hepatic regulation of gene expression and brain function (Cole et al., 1999, Hesen et al., 1996, Oitzl et al., 1997). In contrast, all GRKO mice on the 129sv isogenic genetic background die at birth. Although rare forms of partial glucocorticoid resistance have been reported (Karl and Chrousos, 1993), there are no known human cases of complete glucocorticoid resistance, a condition perhaps incompatible with survival.

In an attempt to explain the variable phenotype of GRKO mice on the C57Bl6/129sv genetic background we have further investigated the disrupted GR gene locus and its expression in surviving adult GRKO mice. These mice were generated by disrupting exon 2 of the GR gene via insertion of a 1.8 kb neomycin resistance gene cassette (Neo). Initial studies by Northern and Western blot analysis, using probes and antibodies directed towards exon 2 encoded sequences, showed no expression of normal GR protein (Cole et al., 1995). Given the variable phenotype in C57Bl6/129sv GRKO mice, the large size of the GR gene (over 100 kb), and examples of residual expression in other gene-targeted mice mutated by insertions (Couse et al., 1995), we measured GR-like glucocorticoid binding activity in GRKO mice. In competitive binding studies with dexamethasone we show that adrenalectomized adult GRKO mice express reduced but still high levels of an aberrant GR-like binding protein. We similarly detected comparable levels of GR mRNAs on the 3′ side of the Neo gene cassette insertion in exon 2, but no mRNA on the 5′ side of the insertion, indicating that transcripts are most probably expressed from the Neo gene promoter. Western blot analysis using a C-terminal GR antibody detects a small truncated LBD-containing GR fragment. Adult and fetal thymic T-cells and fetal hepatocytes from GRKO mice are however profoundly glucocorticoid resistant, reflecting the inability of this mutant GR to trigger transcription despite intact ligand binding.

Section snippets

Mice and adrenalectomy

GRKO mice were generated by gene-targeting as described previously (Cole et al., 1995), by mating GRKO heterozygous (+/–) mice of a combined C57Bl/6/129sv genetic background. Offspring were genotyped for the GR gene locus at three weeks of age by either PCR (see below) or Southern blot analysis of tail DNA as described previously (Cole et al., 1995). Adult GR wildtype, GRKO heterozygous (+/–), and GRKO homozygous (−/−) mice were adrenalectomized at least 1 day before use and maintained on

Dexamethasone binding in cytosolic extracts from adrenalectomized GRKO mice

Extracts from liver, kidney, lung and brain from wild-type, heterozygous (+/–) and GRKO mice specifically bound [3H]dexamethasone as shown in Fig. 1. All incubations contained RU28318 to block binding of dexamethasone to MR. Reduced but significant levels of dexamethasone-binding were seen in extracts from GRKO mice with intermediate levels in tissue extracts from heterozygote (+/–) mice. The level of dexamethasone binding in GRKO mice is 60% of wild-type in lung, 50% in kidney, 40% in brain

Discussion

Gene targeting in mice has been increasingly used to ablate expression of a specific gene in the whole animal to further understand the function of encoded proteins. In most cases this has been performed either by insertional mutagenesis with a marker gene by homologous recombination into an appropriate exon, or by deletion of an exon or gene region. Formal demonstration of ablated expression is normally provided by either PCR, Northern or Western blot analysis, with subsequent analysis then

Acknowledgements

Timothy J. Cole is supported by a Fellowship from the Australian Research Council. Dale I. Godfrey is supported by the National Health and Medical Research Council of Australia and Diabetes Australia. Jarod Purton is supported by a Monash University Postgraduate Award. We would like to thank Prof. George Yeoh for help in establishing fetal hepatocyte cultures, Ms Debbie Ramsey and members of the Baker Institute Biological Research Unit for maintaining mice, and for surgical assistance, Varuni

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