Glucocorticoid Receptor Knockout Mice Research Articles

The glucocorticoid receptor is required for experimental adrenocorticotrophic hormone‐induced hypertension in mice

1. In the present study, we have (i) measured basal blood pressure by telemetry in wild-type (WT) and glucocorticoid receptor knockout (GRKO) mice; (ii) investigated whether or not adrenocorticotrophic hormone (ACTH) can induce hypertension in GRKO mice; and (iii) investigated the effect of mineralocortocoid receptor blockade on the cardiovascular physiology of GRKO mice. 2. Male WT and GRKO mice were treated with ACTH (2mg/kg per day s.c.) or spironolactone (100mg/kg per day s.c.) for 1-2weeks. Blood pressure (BP) was measured using a radiotelemetry system. Urinary Na:K, blood glucose concentrations and haematocrit were also measured during the treatment period. 3. Baseline systolic blood pressure (SBP) was higher in GRKO mice (126±4 mmHg, mean±SEM, n=11) than WT mice (114±2mmHg, n=10; P<0.05). There was no significant difference in baseline haematocrit, blood glucose and urine Na:K ratio in WT and GRKO mice. ACTH raised SBP in WT (135±8mmHg, n=8; P<0.05), but not in GRKO mice (113±9mmHg, n=6). Spironolactone treatment did not alter SBP. 4. Basal SBP was higher in GRKO than WT mice; ACTH raised blood pressure in WT, but not GRKO mice; and spironolactone did not alter BP in GRKO or WT mice. These data, together with a previous study showing both ACTH and corticosterone are increased in GRKO mice, show that the GR is required for the development of ACTH-induced hypertension in mice. Increased endogenous ACTH levels in this model might contribute to the increased basal SBP in GRKO mice, possibly through residual fragments of GR. Mineralocorticoid receptors do not appear to play a critical role in maintaining BP in glucocorticoid receptor deficient mice.

The role of the glucocorticoid receptor in inflammation and immunity

Glucocorticoids are potent immunosuppressive agents with complex actions on immune cells evoking the following effects: inducing apoptosis, changing differentiation fate, inhibition of cytokine release, inhibition of migration and other features. Distinct molecular mechanisms of the glucocorticoid receptor (GR) contribute to different anti-inflammatory effects. Recently inflammatory models have been investigated using conditional knockout and function selective mice shedding light on critical cell types and molecular mechanisms of endogenous and therapeutic GC actions. Here we review the multiple effects of GCs on major immune cells, dendritic cells, myeloid cells and B- and T-lymphocytes and give a summary of studies using conditional GR knockout mice.

The glucocorticoid receptor in the distal nephron is not necessary for the development or maintenance of dexamethasone-induced hypertension

Glucocorticoids are used as a treatment for a variety of conditions and hypertension is a well-recognized side effect of their use. The mechanism of glucocorticoid-induced hypertension is incompletely understood and has traditionally been attributed to promiscuous activation of the mineralocorticoid receptor by cortisol. Multiple lines of evidence, however, point to the glucocorticoid receptor as an important mediator as well. We have developed a mouse model of glucocorticoid-induced hypertension, which is dependent on the glucocorticoid receptor. To determine the site(s) of glucocorticoid receptor action relevant to the development of hypertension, we studied glucocorticoid-induced hypertension in a mouse with a tissue-specific knockout of the glucocorticoid receptor in the distal nephron. Although knockout mice had similar body weight, nephron number and renal histology compared to littermate controls, their baseline blood pressure was mildly elevated. Nevertheless, distal nephron glucocorticoid receptor knockout mice and controls had a similar hypertensive response to dexamethasone. Urinary excretion of electrolytes, both before and after administration of glucocorticoid was also indistinguishable between the two groups. We conclude that the glucocorticoid receptor in the distal nephron is not necessary for the development or maintenance of dexamethasone-induced hypertension in our model.

The role of pituitary glucocorticoid receptors in hypothalamic-pituitary-adrenal axis regulation following chronic social stress

One of the main physiological systems mediating adaptation to stressful stimuli is the hypothalamic-pituitary-adrenal (HPA) axis. Negative feedback control through glucocorticoids (GCs) is an important mechanism to restore homeostasis after HPA axis activation. GCs act via two receptor types, the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). While MRs have been shown to regulate basal and circadian GC levels, GRs play a modulatory role in stressful situations, both acute and chronic. However, the contribution of different physiological subregions in negative feedback control is largely unknown. In this study we investigate the role of negative feedback via GCs on the level of the pituitary by utilizing pituitary-specific conditional GR knockout mice. Following 3 weeks of chronic social defeat, we observed an increase in anxiety-related behavior as well as elevated basal corticosterone levels in control mice. Mice with a GR deficiency in the pituitary show a diminished reaction to chronic stress as indicated by reduced corticosterone levels both basal and after acute stress. These findings are supported by behavioral data that showed decreased anxiety-like behaviour in knockouts compared to controls. In conclusion, we suggest that a lack of pituitary GRs results in a protective effect towards neuroendocrine and behavioral consequences of chronic stress. We hypothesize that this might be due to alterations in postnatal development of GR knockout mice.

The Role of the Forebrain Glucocorticoid Receptor in Acute and Chronic Stress

Previous work has implicated the forebrain glucocorticoid receptor (GR) in feedback regulation of the hypothalamic-pituitary-adrenocortical (HPA) axis. The present series of experiments used male mice with a targeted forebrain-specific GR knockout (in which forebrain includes the prefrontal cortex, hippocampus, and basolateral amygdala) to determine the role of forebrain GR in HPA axis regulation after stress. The data indicate that the forebrain GR is necessary for maintaining basal regulation of corticosterone secretion in the morning, confirming its role in HPA axis regulation. Our data further indicate that the forebrain GR is necessary for negative feedback after both mild and robust acute psychogenic stressors but not hypoxia, a systemic stressor. In contrast, forebrain-specific GR knockout and control mice exhibit equivalent HPA axis hyperactivity and facilitation after chronic variable stress, suggesting that changes in forebrain GR are not essential for chronic stress-induced pathology. These studies provide novel and definitive evidence that the forebrain GR selectively contributes negative feedback regulation of HPA axis responses to psychogenic stressors. Moreover, the data indicate that chronic stress-induced alterations in HPA axis function are mediated by mechanisms independent of the forebrain GR. Overall, the data are consistent with an essential role of the forebrain GR in coordinating endocrine responses to stimuli of a psychological origin.

Peripheral T Cells Are the Therapeutic Targets of Glucocorticoids in Experimental Autoimmune Encephalomyelitis

High-dose glucocorticoid (GC) therapy is widely used to treat multiple sclerosis (MS), but the underlying mechanisms remain debatable. In this study, we investigated the impact of GC administration on experimental autoimmune encephalomyelitis using different GC receptor (GR)-deficient mutants. Heterozygous GR knockout mice were less sensitive to dexamethasone therapy, indicating that the expression level of the receptor determines therapeutic efficacy. Mice reconstituted with homozygous GR knockout fetal liver cells showed an earlier onset of the disease and were largely refractory to GC treatment, indicating that the GR in hematopoietic cells is essential for the beneficial effects of endogenous GCs and dexamethasone. Using cell-type specific GR-deficient mice, we could demonstrate that GCs mainly act on T cells, while modulation of macrophage function was largely dispensable in this context. The therapeutic effects were achieved through induction of apoptosis and down-regulation of cell adhesion molecules in peripheral T(H)17 and bystander T cells, while similar effects were not observed within the spinal cord. In addition, dexamethasone inhibited T cell migration into the CNS, confirming that peripheral but not CNS-residing T lymphocytes are the essential targets of GCs. Collectively, our findings reveal a highly selective mechanism of GC action in experimental autoimmune encephalomyelitis and presumably multiple sclerosis.

T-cell glucocorticoid receptor is required to suppress COX-2-mediated lethal immune activation.

Glucocorticoids, acting through the glucocorticoid receptor, potently modulate immune function and are a mainstay of therapy for treatment of inflammatory conditions, autoimmune diseases, leukemias and lymphomas. Moreover, removal of systemic glucocorticoids, by adrenalectomy in animal models or adrenal insufficiency in humans, has shown that endogenous glucocorticoid production is required for regulation of physiologic immune responses. These effects have been attributed to suppression of cytokines, although the crucial cellular and molecular targets remain unknown. In addition, considerable controversy remains as to whether glucocorticoids are required for thymocyte development. To assess the role of the glucocorticoid receptor in immune system development and function, we generated T-cell-specific glucocorticoid receptor knockout mice. Here we show that the T-cell is a critical cellular target of glucocorticoid receptor signaling, as immune activation in these mice resulted in significant mortality. This lethal activation is rescued by cyclooxygenase-2 (COX-2) inhibition but not steroid administration or cytokine neutralization. These studies indicate that glucocorticoid receptor suppression of COX-2 is crucial for curtailing lethal immune activation, and suggest new therapeutic approaches for regulation of T-cell-mediated inflammatory diseases.

Methylation and cell memory

It has been known for more than 20 years that DNA methylation at CpG dinucleotides is associated with gene silencing in eukaryotes. The preservation of CpG methylation patterns through cell division suggests that they could be important in generating cell ‘memory’ and hence in embryonic cell determination and differentiation. Several lines of evidence support this hypothesis, but a clear example has been lacking. Thomassin et al. 1xGlucocorticoid-induced DNA methylation and gene memory during development. Thomassin, H. et al. EMBO J. 2001; 20: 1974–1983CrossRef | PubMed | Scopus (176)See all References1 now provide an elegant model system in their study of the liver-specific tyrosine aminotransferase (Tat) gene, and Stancheva et al. 2xLoss of the maintenance methyltransferase, xDnmt1, induces apoptosis in Xenopus embryos. Stancheva, I. et al. EMBO J. 2001; 20: 1963–1973CrossRef | PubMed | Scopus (68)See all References2 investigate the role of DNA methylation in amphibian embryonic development.Tat transcription in the liver is triggered at birth by hypoglycemia. This response is mediated by transcription factors that cooperate with the glucocorticoid receptor, interacting with glucocorticoid responsive units (GRUs) located in the Tat enhancer. Thomassin et al. show that glucocorticoid treatment of cultured rat hepatoma cells induces rapid (<1 hour) remodelling of chromatin and recruitment of transcription factors to the Tat enhancer, but also slower (2–3 day) demethylation of CpGs within one of the GRUs. In contrast to transcriptional activation of Tat, which requires continuous glucocorticoid treatment, this CpG demethylation is stable. Furthermore, a second glucocorticoid stimulus, delivered to the cells even one month later, induces Tat transcription more strongly and rapidly. This suggests that demethylation provides the cells with a memory that sensitizes them to the transcription-activating signal.How do these in vitro observations compare with the situation in the embryo? The authors show that the Tat GRU is demethylated, specifically in the liver, following an increase in embryonic corticosteroids a few days before birth. Furthermore, GRU demethylation is prematurely induced by glucocorticoid treatment of cultured fetal hepatocytes. However, unlike hepatoma cells, glucocorticoid stimulation does not immediately trigger Tat transcription in hepatocytes and the embryo. This is induced at birth, when corticosteroids are low, perhaps by factors interacting with the Tat proximal promoter. Thus, this paper suggests that, at least in the Tat gene, DNA demethylation is not sufficient for tissue-specific expression, but contributes to it by preparing the gene for a rapid response to inducing factors. Glucocorticoid receptor knockout mice do not activate the Tat gene at birth, showing the importance of this gene sensitization.Stancheva et al. have previously shown in Xenopus that antisense depletion of the maintenance methyltransferase, xDnmt1, leads to premature expression of certain embryonic transcripts. They now show that phenotypic abnormalities in xDnmt1-depleted embryos are associated with the appearance of apoptotic cells. Experimental inhibition of apoptosis leads to the growth of abnormal undifferentiated cellular masses. These results confirm the importance of DNA methylation for embryogenesis. Cell differentiation activates an apoptotic pathway to eliminate cells in which appropriate methylation is not maintained. An interesting observation is that the genes prematurely activated by xDnmt1-depletion are involved in the early determinative events of embryonic germ-layer patterning. In future, experiments similar to those of Thomassin et al. might shed light on the specific role of DNA methylation in these events.

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

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.

Stress-free T-cell development: glucocorticoids are not obligatory

A role for glucocorticoids in thymopoiesis has been suggested by studies using glucocorticoid receptor (GR) anti-sense transgenic mice, glucocorticoid synthesis inhibitors and GR antagonists. Unfortunately, no consensus has been reached on exactly how glucocorticoids influence T-cell development. The most recent approach, using GR knockout (GR(-/-)) mice, indicates that GR signaling is, in fact, dispensable in this entire process.

Spatial learning deficits in mice with a targeted glucocorticoid receptor gene disruption.

Previous studies in rats using the Morris water maze suggested that the processing of spatial information is modulated by corticosteroid hormones through mineralocorticoid and glucocorticoid receptors in the hippocampus. Mineralocorticoid receptors appear to be involved in the modulation of explorative behaviour, while additional activation of glucocorticoid receptors facilitates the storage of information. In the present study we used the water maze task to examine spatial learning and memory in mice homozygous and heterozygous for a targeted disruption of the glucocorticoid receptor gene. Compared with wild-type controls, homozygous and heterozygous mice were impaired in the processing of spatial but not visual information. Homozygous mutants performed variably during training, without specific platform-directed search strategies. The spatial learning disability was partly compensated for by increased motor activity. The deficits were indicative of a dysfunction of glucocorticoid receptors as well as of mineralocorticoid receptors. Although the heterozygous mice performed similarly to wild-type mice with respect to latency to find the platform, their strategy was more similar to that of the homozygous mice. Glucocorticoid receptor-related long-term spatial memory was impaired. The increased behavioural reactivity of the heterozygous mice in the open field points to a more prominent mineralocorticoid receptor-mediated function. The findings indicate that (i) the glucocorticoid receptor is of critical importance for the control of spatial behavioural functions, and (ii) mineralocorticoid receptor-mediated effects on this behaviour require interaction with functional glucocorticoid receptors. Until the development of site-specific, inducible glucocorticoid receptor mutants, glucocorticoid receptor-knockout mice present the only animal model for the study of corticosteroid-mediated effects in the complete absence of a functional receptor.

Characterization of Adrenal Chromaffin Progenitor Cells in Mice

Publisher Summary In this chapter several studies have been summarized to reveal the pattern of expression of several candidate markers in the embryonic adrenal gland of wild-type mice. It has been suggested that Phox 2 is a marker for both differentiated and presumptive catecholaminergic adrenal chromaffin cells prior to reaching detectable levels of tyrosine hydroxylase (TH) immunoreactivity. In addition to TH, Phox 2, peanut agglutinin (PNA) and neuropeptide Y (NPY) are detectable in all postnatal chromaffin cells. Noradrenergic chromaffin cells possess the full repertoire of chromaffin cell functions except the capacity to synthesize adrenaline. Adult noradrenergic chromaffin cells exhibit at least three markers that they do not share with adrenergic chromaffin cells, GAP43, the adhesion molecule L1, and vesicular monoamine transporter-2 (VMAT2). The data presented in the chapter provide a useful basis for further analyzing the phenotypes of adrenal chromaffin cells in glucocorticoid-receptor knockout mice. Furthermore, the results suggest that one marker found predominantly or exclusively in mature adrenergic chromaffin cells in mice, chromogranin B can be detected in chromaffin progenitor cells prior to the expression of other key markers for the adrenergic phenotype, phenylethano1amine-Nmethyltransferase (PNMT), and secretogranin II. This suggests that differentiative properties of adrenergic chromaffin cells can be regulated sequentially and are probably, in part, independent from glucocorticoid signaling.