The Protective Side of the Mineralocorticoid Receptor

The mineralocorticoid receptor (MR), like other steroid receptors, is a ligand-dependent transcription factor. In addition, MR associated with the plasma membrane have rapid nongenomic effects and may interact with other membrane receptors (1, 2). For many years the primary physiological function of the MR was thought to be the promotion of the vectorial transport of electrolytes within the kidney tubule when activated by aldosterone (3). However, it was also recognized very early that the MR is selectively expressed throughout the body, including the brain, with the highest concentration in the hippocampus. Normal activation of MR in the rat brain has a positive role in maintaining neuronal function and survival (4). However, inappropriate MR activation by a combination of aldosterone excess and a high salt intake is associated with cardiac, renal, and vascular, including cerebrovascular damage (5) as well as increased activation of the sympathetic nervous system and salt appetite, central nervous system functions (6). The diversity and complexity of MR function has been studied using a wide variety of tools including genetically modified animal models, primary cells, and immortalized cells. MR are expressed in undifferentiated human and mouse embryonic stem (ES) cells and persist during differentiation (7). The paper in this issue of Endocrinology by Munier et al. (8) describes the expression of MR and their role in the survival of ES cells differentiated in vitro to form neurons. MR expression was increased 5-fold during differentiation and high levels were maintained in the ES-derived neurons (9). Two proximal promoters, P1 and P2, drive MR expression with the P1 being stronger, including in undifferentiated mouse embryonic stem cells. Overexpression in mice of the human MR driven by its P1 proximal promoter leads to cardiomyopathy and arrhythmias (10). When mouse ES cells were stably transfected with either the human MR P1 or P2 promoter driving the expression of green fluorescent protein (GFP), basal GFP expression was weaker in the P2-human (h) MR- than P1-hMR-ES cells and correlated with MR expression from the P2 promoter (9). P2 promoter activity was enhanced by physiological levels of aldosterone, 1 nm; the P1 promoter was significantly less sensitive, requiring 100 nm aldosterone for similar activation. GFP reporter gene transcripts were 7- and 5-fold greater in the P1-GFP-ES- and P2-GFP-ES-derived differentiated neurons, respectively, compared with that of the parent undifferentiated ES cells. These ES and differentiated neurons also expressed the glucocorticoid receptor (GR). This is important for the model because GR play an indispensable role in MR-dependent transcription as they form GR-MR heterocomplexes necessary for efficient nuclear trafficking of the MR (11). Studies of animal models of MR overexpression or inactivation confirm the significance of MR function in the brain. Global deletion of the MR, but not GR, in mice decreases the number of hippocampal granule cells (12). Targeted deletion of the MR in the limbic system also demonstrated that the MR is involved in the maintenance of cellular and structural integrity of neurons required for normal cognition, behavior, and endocrine control, rather than central nervous system development (13). Although global and targeted MR−/− mice have impaired learning abilities (13), overexpression of the MR in the forebrain improves memory (14). The hippocampus is the major site of neurogenesis in adult dogs and hippocampal neurons, as well as MR, decrease with age (15). In addition to their importance in maintaining normal stress response, memory, behavior, MR are important for neuronal survival in the hippocampus of rats and increased survival of rat primary cortical neurons in response to mild injury (4) and hippocampal neurons after cerebral transient global ischemia (14). One mechanism by which the MR influences limbic function is by inhibiting apoptosis (16). The ES cell-derived models of neuronal differentiation described by Munier et al. in this issue of Endocrinology (8) provide important insights into the effect of the MR in the process of differentiation and neuronal survival. ES cells from wild-type and transgenic mice overexpressing the MR under the control of the P1-hMR promoter were induced to differentiate into mature neurons. Levels of endogenous mouse MR mRNA were similar in the undifferentiated wild-type and P1-hMR transgenic ES cells and were increased similarly by differentiation into neurons. Total MR protein expression in ES-derived neurons was significantly higher than in the undifferentiated ES cells, with protein expression significantly greater in P1-hMR-derived neurons than wild type. The expression of the differentiation markers MAP2 and β-tubulin III in P1-hMR neurons was also significantly higher. Proliferation rates of both cells were similar, but the ratio of cleaved caspase-3 to caspase-3, a measure of apoptosis, was lower in the P1-hMR neurons. Expression of the antiapoptotic proteins B-cell leukemia (Bcl2) and BclxL were similar in both undifferentiated ES cells and was unchanged by differentiation in the P1-hMR-derived neurons. However, Bcl-2-associated X protein (Bax) and Bcl-2-antagonist/killer protein expression was very significantly reduced by differentiation in the wild-type neurons and the ratio of Bcl2/Bax in the P1-hMR-derived neurons was double that of the wild-type. The affinity of the MR for aldosterone and the physiological glucocorticoids, cortisol or corticosterone, is similar (17, 18). MR in circumventricular areas of the brain and the brain stem are thought to be activated by aldosterone and mediate effects on fluid, electrolyte, and cardiovascular homeostasis, whereas hippocampal MR are activated by the physiological glucocorticoids and have a variety of important actions unrelated to electrolyte transfer (4, 6, 19, 20). MR-mediated effects are largely independent of the agonist ligand in transport epithelia such as that of the renal tubule. Thus, deficiency or inhibition of the 11β-hydroxysteroid dehydrogenase 2 enzyme responsible for the inactivation of endogenous cortisol or corticosterone and normally expressed in aldosterone target cells of the kidney produces apparent mineralocorticoid excess due to MR activation by the more abundant endogenous glucocorticoid. However, MR-mediated effects on trophic functions in hippocampal neurons in rats are ligand dependent (4, 21, 22). Derangement of the raphe-hippocampal serotonin system by adrenalectomy is significantly corrected by MR occupation by corticosterone, but not aldosterone, and aldosterone interferes with the ameliorative effects of corticosterone replacement in this model (21). The chronic intracerebroventricular infusion of very low concentrations of aldosterone, but not similar amounts of corticosterone, produces hypertension (23). The concomitant infusion of corticosterone with aldosterone in this model abrogates the hypertension of centrally infused aldosterone (6, 23). Little is certain about the ligand specificity or function of the significant number of MR in the cerebellum and cerebral cortex. The differentiation of both types of ES cells into neurons significantly increased MR expression and lowered the apoptotic index with a greater effect in the P1-hMR neurons that express more total MR. Knockdown of the MR expression with small interfering RNA significantly decreased markers of differentiation as well as antiapoptotic molecules. The ES cells were differentiated into neurons in media containing fetal calf serum, which contains cortisol and aldosterone; charcoal-dextran-extracted fetal calf serum, which is devoid of steroids did not support neuronal differentiation. Although it was most likely cortisol, it is unclear which steroid was responsible for the MR-mediated neuronal differentiation and positive pro-/antiapoptotic protein expression. It is possible that the balanced combination of both aldosterone and cortisol at physiological concentrations produced the optimal growth and differentiation (12). Unfortunately, supraphysiological concentrations of steroids were used for the studies to determine ligand specificity. Addition of MR-saturating concentrations of corticosterone (100 nm) to the culture media significantly increased the anti-/proapoptotic ratio of Bcl2/Bax only in P1-hMR neurons, whereas MR-saturating concentrations of aldosterone had no effect. In neurons derived from the wild-type ES, corticosterone decreased the anti- to proapoptotic ratio and aldosterone had no effect. Addition of physiological concentrations of corticosterone, aldosterone, or both to the charcoal-extracted serum would have clarified which MR ligand was important for neuronal differentiation from ES cells. These novel studies and the model described by Munier et al. (8) in this issue permit the dissection of mechanisms of neuronal integrity mediated by the MR that are not possible in intact animals and enhance our knowledge of the role of the MR in neuronal differentiation from stem cells. This paradigm should be very useful in the study of differential gene expression mediated by MR, depending on its ligand, and may allow for the identification of specific coactivator or corepressor proteins that interact differently with the MR bound to aldosterone, cortisol, or corticosterone. Most exciting, the model may facilitate the identification MR-mediated neurotrophic mechanisms that might be enhanced to modify the response to neuronal injury, oxidative damage, depression, and aging in the brain.