Glucocorticoid Receptor Deficient Mice GRhypo Confirm a Major Role for the GR in Physiology

In order to study in vivo the role of the glucocorticoid receptor in physiology and development the GR-gene was disrupted by homologous

FIGURE 1 The regulatory system of the hypothalamus-pituitary-adrenal (HPA) axis. Glucocorticoids released after stimuli such as stress lead to physiological responses in a variety of organs. The HPA axis is a complex regulatory circuit that controls this release of glucocorticoids from the adrenal gland. Homeostasis is achieved by feedback inhibition of several of the involved factors in the hypothalamus and the anterior pituitary. These include corticotropin-releasing factor (CRF), arginine vasopressin (AVP), proopiomelanocortin (POMC), adrenocor-ticotrope hormone (ACTH), and prolactin (PRL). Signals from the hippocampus and the amygdala as well as from the immune system exert further influences on the control of the HPA axis.

FIGURE 1 The regulatory system of the hypothalamus-pituitary-adrenal (HPA) axis. Glucocorticoids released after stimuli such as stress lead to physiological responses in a variety of organs. The HPA axis is a complex regulatory circuit that controls this release of glucocorticoids from the adrenal gland. Homeostasis is achieved by feedback inhibition of several of the involved factors in the hypothalamus and the anterior pituitary. These include corticotropin-releasing factor (CRF), arginine vasopressin (AVP), proopiomelanocortin (POMC), adrenocor-ticotrope hormone (ACTH), and prolactin (PRL). Signals from the hippocampus and the amygdala as well as from the immune system exert further influences on the control of the HPA axis.

recombination in ES-cells using an insertion strategy (Cole et al., 1995). We obtained mutant mice lacking functional GR-protein and found that the majority of homozygous mutants dies around birth due to atelectasis of the lungs. This demonstrates that transcriptional control of lung development and function crucially depends on glucocorticoid signalling via the GR. However, the exact cause of atelectasis and the primary targets of the GR in the lung still remain to be defined. As these mutants carry a hypomorphic allele of the glucocorticoid receptor gene the mouse strain is now called GRhypo to distinguish it from the newly generated GRnull mice (see next section).

Because the majority of homozygous mutants do not reach adulthood, the main focus of the initial experiments was the analysis of development and gene expression during embryogenesis. In liver of newborn GRhypo/hypo mice, for example, impaired expression of gluconeogenic enzymes was observed. This was most prominent for mRNA-expression of tyrosine aminotransferase (TAT) and serine dehydrogenase (SDH), although expression was not completely abolished for any of the genes analyzed. The residual expression may result from activation of other signaling pathways, e.g., by glucagon, since expression of these genes is also dependent on CREB and related transcription factors (Ruppert et al., 1990).

Regulation of glucocorticoid homeostasis is achieved by the HPA axis, which ensures fast adaption of corticosterone levels via a negative feedback circuit. Regulation is thought to be mainly mediated by GR localized in the anterior lobe of the pituitary and the paraventricular nucleus (PVN) of the hypothalamus. Thus, it was important to see how this system would respond to the lack of feedback control. As the HPA axis is fully established by day E16.5, the major components of the system could already be followed in embryos (Reichardt and Schutz, 1996). When corticosterone levels were measured in newborn GRhypo/hypo mice it became immediately evident that the system was out of equilibrium, as this hormone was nearly threefold elevated. This is probably due to an increase in the respective trophic hormone, namely ACTH, which was more than 10-fold elevated in GRhypo/hypo mice with respect to wild-type littermates. Next we analyzed mRNA and peptide expression of relevant components of the HPA axis to see if the changes in hormone concentrations were the result of altered gene transcription. In the anterior pituitary of GRhypo/hypo embryos we found a strong upregulation of proopiomelanocortin (POMC) mRNA starting around E16.5 of development, indicating that at least in part the high levels of ACTH are due to an increased transcriptional rate. However, CRH, the major releasing hormone for ACTH, also showed increased mRNA expression in the paraventricular nucleus of the hypothalamus, as well as elevated peptide levels in the median eminence. Interestingly, AVP, another releasing hormone for ACTH, was only moderately elevated compared to expectations from previous studies using adrenalectomy (Kretz etal., 1999). This suggests that the major targets for transcriptional regulation by the GR are the genes encoding CRH and POMC.

From human pathology it is known that increased ACTH levels lead to alterations in the adrenal gland. For example, in Cushing's syndrome, pituitary tumors result in a massive overproduction of ACTH, and this is associated with adrenal hyperthrophy and hyperplasia (Miller and Blake Tyrrel, 1995). Interestingly, a comparable phenotype was found in the adrenals of GRhypo/hypo mice. The whole adrenal gland of the mutants is enlarged and the cortex shows massive signs of hyperplasia and hypertrophy. Because of the strong proliferation of the cortex, no defined central medulla but rather scattered patches of chromaffin cells are found in GRhypo/hypo mice. Additionally, mRNA expression of several steroidogenic enzymes is increased, which may also contribute to the elevated corticosterone levels measured in the serum of the mutants.

Approximately 20% of the GRhypo/hypo mice survive to adulthood (Cole et al., 1995). This allowed study of at least some physiological functions of the GR in adult mice, although the number of animals available was limited. However, caution must be taken in the interpretation of these data, because the survivors represent only a distinct and small subgroup of the homozygous mutant with probably special characteristics (see next section). Initial studies on the role of the GR in the brain were undertaken to analyze the influence of the GR on the processing of spatial information. These experiments suggested that loss of the GR led to impairment of spatial learning along with increased motor activity (Oitzl et al., 1997). In addition, the electrophysiological properties of hippocampal CA1 neurons, such as voltage-gated Ca currents and responses to serotonin and carbachol, were found to be altered (Hesen et al., 1996). These studies illustrate the value of GR-deficient mice for this type of analysis, although it became clear from the sometimes conflicting results that brain-specific GR-mutants as well as mutants carrying point mutations in the GR are necessary to confirm and extend the results obtained in this initial approach.

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