Interdomain interactions in the mineralocorticoid receptor

The potential for interaction between the N-terminal domain and the C-terminal region (hinge and ligand-binding domain) of the mineralocorticoid receptor (MR) was examined using the mammalian-2-hybrid assay. The MR C-terminal region was fused to the GAL4 DNA-binding domain (GAL4-MRC). To examine if the AF-2 is involved in the interaction, as has been reported for other steroid hormone receptors, it was inactivated by point mutation (E962A). The N-terminal domain was fused to the VP16 transactivation domain (VP16-MRNT). In the mammalian-2-hybrid assay both GAL4-MRC and GAL4-MRC(E962A) interact with VP16-MRNT in an aldosterone-dependent manner. The GAL4-MRC(E962A) construct was used in subsequent experiments to examine the AF-2-independent N/C-interaction. The MR antagonist spironolactone inhibits the aldosterone-mediated association of the two domains. GAL4-MRC(E962A) interacts weakly with the GR or AR N-terminal domains in the presence of aldosterone. No dimerization between GAL4-MRC(E962A) and VP16-MRC is observed. Interestingly, cortisol produces a much weaker N/C-interaction than aldosterone, and it is possible that the N/C-interaction may contribute to observed functional differences in the MR bound to the two ligands.     

Vascular responsiveness to serotonin metabolites in mineralocorticoid hypertension

This study characterizes vascular responsiveness to serotonin and its metabolites and to several monoamines that are structurally related to serotonin in deoxycorticosterone acetate (DOCA)-salt hypertension. Mesenteric arteries from normotensive and hypertensive rats were excised and cut into helical strips for isometric force recording. Dose-response curves to serotonin in arteries from hypertensive rats were shifted significantly to the left compared with those in arteries from normotensive rats (ED25: DOCA-treated = 2.4 X 10(-8) M; control = 17.1 X 10(-8) M). Contractile responses to 5-hydroxyindole acetic acid and 5-hydroxytryptophol were greater in mesenteric arteries from hypertensive rats, whereas reactivity to 5-methoxytryptamine and melatonin in arteries from hypertensive rats did not differ from that in arteries from normotensive rats. Mesenteric arteries from both rat groups were unresponsive to the serotonin metabolite N-acetylserotonin. Contractile responses to 5,6-dihydroxytryptamine and 6-hydroxytryptamine were greater in mesenteric arteries from hypertensive rats, whereas responsiveness to 3-hydroxytryptamine in hypertensive arteries did not differ from normotensive values. Contractile responses to serotonin and its metabolites and to the structurally related monoamines were inhibited by the serotonergic antagonist ketanserin. These results demonstrate that vascular sensitivity to serotonin is increased in DOCA-hypertensive rats. Based on the experiments with serotonin metabolites and with other monoamines, the increased responsiveness to these compounds appears to be related to the structural location of hydroxyl and amine moieties.     

The N-terminal domain of the mineralocorticoid receptor modulates both mineralocorticoid receptor- and glucocorticoid receptor-mediated transactivation from Na/K ATPase beta1 target gene promoter

Mineralocorticoid and glucocorticoid hormones activate the expression of the Na/K ATPase β1 through direct binding of the mineralocorticoid receptor (MR) and glucocorticoid receptors (GR) to a mineralocorticoid-and glucocorticoid-responsive element in the β1 promoter region, but activation of the β1 promoter is inhibited by coexpression of both receptors. Here, using a series of mutated and chimeric receptors, we show that the N-terminal region of MR mediates an inhibitory effect on MR and GR activation from the β1 promoter, in CV-1 cells. Deletion of the N-terminal region of MR (1-603) enhanced MR activation four-fold. Activation by chimeric MR, in which the N-terminus of GR replaces the N-terminal region of MR, was threefold that of wild-type MR. In addition, whereas coexpression of wild-type MR and GR was inhibitory, coexpression of chimeric MR and wild-type GR was nearly equal to that of MR. By contrast, mutated GR lacking its N-terminal region (1–420) was less efficient than the wild type in activating this promoter. These results demonstrate that the N-terminal domains of MR and GR have opposite transactivation properties and that MR region 1–603 is indeed inhibitory for both MR- and GR-mediated regulation of the Na/K ATPase β1 gene promoter.     

Calcium sensing receptor gene A986S polymorphism and responsiveness to calcium supplementation in postmenopausal women

Recently several studies in adolescent girls or premenopausal women have implicated the calcium sensing receptor (CASR) gene A986S polymorphism in calcium and bone metabolism. However, the role of this genetic variant in postmenopausal women, specifically the development of osteoporosis, is unknown. This study reports the findings of a randomized, double-blind, placebo-controlled study of healthy postmenopausal women followed for 2 yr while taking placebo or supplementary calcium. Specifically, we examined the relationship between the CASR A986S polymorphism, bone biochemical profile, and bone mineral density at baseline and after 2 yr of treatment. We found no effect of this genetic variant in postmenopausal women at baseline or in response to calcium supplementation. These results are in contrast to those in young or premenopausal women, and they provide no support for an important role for the CASR A986S polymorphism in osteoporosis.     

A polymorphism in the alpha4 nicotinic receptor gene (Chrna4) modulates enhancement of nicotinic receptor function by ethanol

BACKGROUND: Several studies indicate that ethanol enhances the activity of alpha4beta2 nicotinic acetylcholine receptors (nAChR). Our laboratory has identified a polymorphism in the alpha4 gene that results in the substitution of an alanine (A) for threonine (T) at amino acid position 529 in the second intracellular loop of the alpha4 protein. Mouse strains expressing the A variant have, in general, greater nAChR-mediated 86Rb+ efflux in response to nicotine than strains with the T variant. However, the possibility of the polymorphism modulating the effects of ethanol on the 86Rb+ efflux response has not been investigated. METHODS: We have used the 86Rb+ efflux method to study the acute effects of ethanol on the function of the alpha4beta2 nAChR in the thalamus in six different mouse strains. Experiments were also performed on tissue samples taken from F2 intercross animals. The F2 animals were derived from A/J mice crossed with a substrain of C57BL/6J mice that carried a null mutation for the gene encoding the beta2 nAChR subunit. RESULTS: In strains carrying the A polymorphism (A/J, AKR/J, C3H/Ibg), coapplication of ethanol (10-100 mM) with nicotine (0.03-300 microM) increased maximal ion flux when compared with nicotine alone with no effect on agonist potency. In contrast, ethanol had little effect on the nicotine concentration-response curve in tissue prepared from strains carrying the T polymorphism (Balb/Ibg, C57BL/6J, C58/J). Experiments with the F2 hybrids demonstrated that one copy of the A polymorphism was sufficient to produce a significant enhancement of nAChR function by ethanol (50 mM) in animals that were also beta2 +/+. Ethanol had no effect on nicotine concentration-response curves in T/T beta2 +/+ animals. CONCLUSIONS: The results suggest that the A/T polymorphism influences the initial sensitivity of the alpha4beta2 nAChR to ethanol.     

Activation of the rat kidney mineralocorticoid receptor

Activation of the rat kidney mineralocorticoid receptor was investigated using diethylaminoethyl (DEAE)-cellulose, DNA-cellulose, and gel permeation chromatography. Specific labeling of the mineralocorticoid receptor was achieved by labeling with [3H]aldosterone in the presence of the pure glucocorticoid RU28362. The specificity of labeling was confirmed by the lack of immunoreactivity of [3H]aldosterone-labeled material with the monoclonal antiglucocorticoid receptor antibody BURG-1. The unactivated aldosterone-mineralocorticoid receptor complex did not bind to DNA-cellulose, was eluted from DEAE-cellulose at relatively high salt (195 mM KCl) concentration, and had an apparent Stokes radius when chromatographed on Sephacryl S300 of 6.3 nm. After activation, the aldosterone-mineralocorticoid receptor complex had increased affinity for DNA-cellulose and decreased affinity for DEAE-cellulose and appeared as a smaller complex when chromatographed on Sephacryl S300. These changes were blocked by sodium molybdate. Our results indicate that activation of the rat kidney mineralocorticoid receptor is analogous to activation of the glucocorticoid receptor and suggest that activation of the mineralocorticoid receptor involves dissociation of a multimeric receptor form.     

Ca2+ but not H2O2 modulates GRE-element activation by the human mineralocorticoid receptor in HEK cells

The mineralocorticcoid receptor (MR) plays an important role in salt and water homeostasis as well as during cardiovascular and renal fibrosis but little is known regarding its modulation by other signaling pathways. To investigate a possible modulation under controlled conditions we used human embryonic kidney (HEK) cells (devoid of endogenous MR) transfected with the human MR and measured transactivation with a GRE-SEAP-reporter construct. MR was compared to the glucocorticoid receptor (GR) as well as to MR lacking the N-terminal domains AB (MR(CDEF)). Chelation of cytosolic Ca2+ enhanced MR activity and SGK1-expression, whereas elevation of cytosolic Ca2+ with ionomycin or thapsigargin reduced MR activity. GR activity was not affected by ionomycin or thapsigargin. MR(CDEF) activity was not affected by chelation or elevation of cytosolic Ca2+. Inhibition of ERK1/2 activation by U0126 or activation of PKA by cAMP, previously shown to modulate MR and GR activity, did not affect MR(CDEF) activity either. H2O2<500micromol/l did not affect basal nor hormone-induced reporter activity. Higher concentrations exerted the same relative inhibitory effect on GRE-SEAP-activity under basal conditions as in the presence of aldosterone-stimulated MR and elicited cytotoxic effects. Our data indicate that the genomic function of MR can be modulated by cytosolic Ca2+, PKA and ERK1/2 via an interaction with the AB-domain. H2O2 seems not to affect relative MR activity directly under our experimental conditions.     

A common intron 2 polymorphism of the glucocorticoid receptor gene is associated with insulin resistance in men

Objective  Clinical similarities between the metabolic syndrome and Cushing's syndrome have led to speculation of genetic association between them. The Bcl1 polymorphism in intron 2 of the glucocorticoid receptor (GR) gene has been associated with insulin resistance/hyperinsulinaemia. Our objective was to test the association of rs2918419, a T→C single nucleotide change in intron 2 downstream of the Bcl1 locus, with components of the metabolic syndrome and its interaction with the Bcl1 locus.
Design and methods  We genotyped a subsample of 325 White subjects (116 men) in the Newcastle Heart Project (NHP), a population-based study in north-east England. Gender-specific statistical analysis by stepwise backward multiple regression was performed to test the association of allele status with adiposity, glucose and insulin responses to oral glucose tolerance test (OGTT), fasting lipids and blood pressure.
Results  Minor allele frequency was 0·14 for rs2918419 and 0·39 for the Bcl1 polymorphism. rs2918419 was associated with higher fasting insulin concentration and insulin resistance in men but not in women. Contrary to earlier studies, the Bcl1 polymorphism on its own was not associated with insulin resistance/hyperinsulinaemia in either gender. Subjects carrying variant rs2918419 alleles also had variant alleles at the Bcl1 locus. In men, but not women, Bcl1 variant alleles on a background of rs2918419 wild-type alleles associated with lower fasting insulin compared to wild-type alleles at both loci or variant alleles at both loci.
Conclusions  We report that rs2918419 was linked with hyperinsulinaemia and insulin resistance in men. Carrying Bcl1 variant alleles without rs2918419 was not associated with hyperinsulinaemia/insulin resistance. Previous reports of the association of Bcl1 polymorphism with obesity-related characteristics may reflect linkage disequilibrium with rs2918419.     

Determinants of spironolactone binding specificity in the mineralocorticoid receptor

Spironolactone is a mineralocorticoid receptor (MR) antagonist in clinical use. The compound has a very low affinity for the glucocorticoid receptor (GR). Determinants of binding specificity of spironolactone to the MR were investigated using chimeras created between the ligand-binding domains (LBDs) of the MR and the GR. These chimeras had previously been used to investigate aldosterone binding specificity to the MR. Spironolactone was able to compete strongly for [(3)H]-aldosterone and [(3)H]-dexamethasone binding to a chimera containing amino acids 804-874 of the MR, and weakly for [(3)H]-dexamethasone binding to a chimera containing amino acids 672-803 of the MR. Amino acids 804-874 were also critical for aldosterone binding specificity. Models of the MR LBD bound to aldosterone and spironolactone were created based on the crystal structure of the progesterone receptor LBD. The ligand-binding pocket of the MR LBD model consisted of 23 amino acids and was predominantly hydrophobic in nature. Analysis of this model in light of the experimental data suggested that spironolactone binding specificity is not governed by amino acids in the ligand-binding pocket.     

Paradoxical mineralocorticoid receptor activation and left ventricular diastolic dysfunction under high oxidative stress conditions

BACKGROUND: Salt status plays a pivotal role in angiotensin-II-induced organ damage by regulating reactive oxygen species status, and it is reported that reactive oxygen species activate mineralocorticoid receptors. METHOD: To clarify the role of reactive oxygen species-related mineralocorticoid receptor activation in angiotensin-II-induced cardiac dysfunction, we examined the effect of the following: salt status; an MR antagonist, eplerenone; and an antioxidant, tempol in angiotensin-II-loaded Sprague-Dawley rats. RESULTS: Angiotensin-II/salt-loading elevated blood pressure, and neither eplerenone nor tempol antagonized the rise in blood pressure significantly. Left ventricular diastolic function was monitored by measuring peak velocity of a mitral early inflow (E), the ratio of mitral early inflow to atrial contraction related flow (E/A), deceleration time of mitral early inflow and -dP/dt, the time constant (T), and filling pressure (left ventricular end-diastolic pressure) by echocardiography or cardiac catheterization. Despite the suppressed serum aldosterone, left ventricular diastolic function was deteriorated with angiotensin II/high salt, but not affected by angiotensin II/low salt. However, angiotensin-II/salt-induced cardiac dysfunction was restored by eplerenone and tempol. Nicotinamide adenine dinucleotide phosphateoxidase-derived superoxide formation was greater in the hearts of the angiotensin II/high-salt rats than of the angiotensin II/low-salt rats. The expression of the Na(+) -H(+) exchanger isoform 1, a target of mineralocorticoid receptor activation, was significantly increased in the angiotensin II/high-salt group. Both tempol and eplerenone inhibited the angiotensin-II/salt-induced upregulation of Na(+) -H(+) exchanger isoform 1. CONCLUSION: These findings demonstrate that mineralocorticoid receptor activation by oxidative stress can cause left ventricular diastolic dysfunction in a rat model of mild hypertension.     

Mechanisms of ligand specificity of the mineralocorticoid receptor

The mineralocorticoid receptor (MR) differs from the other steroid receptors in that it responds to two physiological ligands, aldosterone and cortisol. In epithelial tissues, aldosterone selectivity is determined by the activity of 11β-hydroxysteroid dehydrogenase type 2, while in other tissues, including the heart and regions of the central nervous system, cortisol is the primary ligand for the MR where it may act as an antagonist. Clinical trials have demonstrated the potential of MR antagonists in the treatment of cardiovascular disease, though their use has been limited by concurrent hyperkalaemia. In order to better target the MR, an understanding of the structural determinants of tissue- and ligand-specific MR activation is needed. Interactions of the MR have been identified, which exhibit ligand discrimination and/or specificity. These interactions include those of the ligand-binding domain with ligand, with the N-terminal domain and with putative co-regulatory molecules. Agonist and antagonist binding have been characterised using chimeras between the human MR and the glucocorticoid receptor or the zebra fish MR together with molecular modelling. The interaction between the N-terminus and the C-terminus is aldosterone dependent but is unexpectedly antagonised by cortisol and deoxycorticosterone in the human MR. Nuclear receptor-mediated transactivation is critically dependent on, and modulated by, co-regulatory molecules. Proteins that interact with the MR in the presence of either aldosterone or cortisol, but not both, have been identified. The successful identification of ligand-specific interactions of the MR may provide the basis for the development of novel MR ligands with tissue specificity.     

A polymorphism in the 3' untranslated region of the gene for tumor necrosis factor receptor 2 modulates reporter gene expression

The gene encoding the human TNF alpha receptor (TNFR) 2 contains polymorphisms in the 3' untranslated region (UTR). Previous studies have shown that some variant alleles in this region are associated with obesity and insulin resistance. However, the effect of these polymorphisms on the expression of TNFR2 has not been studied to date. To examine the role played by different haplotypes in the control of TNFR2 expression (haplotypes A1-A5, referring to nucleotides 1663 G/A, 1668 T/G, and 1690 T/C), we introduced these sequences into the 3'-UTR of a heterologous reporter gene and expressed the corresponding constructs in a human T-cell line. We demonstrate that a 485-nt fragment of the TNFR2 3'-UTR that contains a U-rich region decreases reporter expression and that haplotypes A1-A4 exert a stronger effect than A5. Furthermore, time-course assays of mRNA stability using actinomycin D revealed that haplotypes A1-A4 destabilize the mRNA. The proximal TNFR2 3'-UTR, independently of haplotype differences, responded to T-cell activation by increasing mRNA decay. Electromobility shift analysis demonstrated that protein(s) found in T-cell extracts bind to the 485-nt fragment. We suggest that an increased rate of TNFR2 mRNA decay protects cells from unrestrained TNF alpha effects and that this protection is weakened in A5 subjects. These findings may explain the association of this haplotype with obesity and increased leptin levels.     

Inactivating mutations of the mineralocorticoid receptor in Type I pseudohypoaldosteronism

Type I pseudohypoaldosteronism (PHA1) is a rare form of mineralocorticoid resistance characterized by neonatal renal salt wasting and failure to thrive. Typical biochemical features include high levels of plasma aldosterone and renin, hyponatremia and hyperkalemia. Different mutations of the human mineralocorticoid receptor (hMR) gene have been identified in subjects affected by the autosomal dominant or sporadic form of the disease. Our laboratory has investigated a large number of subjects with familial and sporadic PHA1. Several different mutations have been detected, which are localized in different coding exons of the hMR gene. These mutations either create truncated proteins, either affect specific amino acids involved in receptor function. In this paper, we review hMR mutations described to date in PHA1 and their functional characterization. We discuss the absence of mutations in some kindreds and the role of precise phenotypic and biological examination of patients to allow for identification of other genes potentially involved in the disease.