The Severe Form of Hypertension Caused by the Activating S810L Mutation in the Mineralocorticoid Receptor Is Cortisone Related

http://www.100md.com 《内分泌学杂志》2003年第2期

     Abstract

    A gain of function mutation resulting in the substitution of leucine for serine at codon 810 (S810L) in the human mineralocorticoid receptor (MR) is responsible for early-onset hypertension that is exacerbated in pregnancy. All steroids, including progesterone, that display antagonist properties when bound to the wild-type MR are able to activate the mutant receptor (MR_L810). These findings suggest that progesterone may contribute to the dramatic aggravation of hypertension in MR_L810 carriers during pregnancy. However, the steroid(s) responsible for hypertension in MR_L810 carriers (men and nonpregnant women) has not yet been identified. Here we show that cortisone and 11-dehydrocorticosterone, the main cortisol and corticosterone metabolites produced in the distal nephron, where sodium reabsorption stimulated by aldosterone takes place, bind with high affinity to MR_L810. The potency with which cortisone and 11-dehydrocorticosterone bind to the mutant MR contrasts sharply with their low wild-type MR-binding capacity. In addition, cotransfection assays demonstrate that cortisone and 11-dehydrocorticosterone are potent activators of the MR_L810 trans-activation function. Because the plasma concentration of cortisol in humans is about 30-fold higher than that of corticosterone, these findings strongly suggest that cortisone is one of the endogenous steroids responsible for early-onset hypertension in men and nonpregnant women carrying the MR_L810 mutation.

    Introduction

    UNDER NORMAL conditions, aldosterone binds with high affinity to the mineralocorticoid receptor (MR) to stimulate renal sodium reabsorption. In response to aldosterone, MR modulates transcription, and this ultimately enhances the transport of sodium from the tubular lumen to the basolateral side of the principal cells of the collecting duct (1, 2). Screening for MR among 75 hypertensive patients, many with low plasma renin and/or low serum aldosterone levels, identified a missense mutation in exon 6 of the MR in a young boy, resulting in the substitution of leucine for serine (S810L) at codon 810 (MR_L810) (3). Examination of this patient’s family revealed that all MR_L810 carriers had developed hypertension before they were 20 yr old, a rare trait in the general population, whereas the members of the family who did not express the mutant MR had normal blood pressure (3). Rather intriguingly, the women harboring this mutation experienced a dramatic exacerbation of hypertension during pregnancy.

    In vitro experiments have revealed that aldosterone activates both wild-type MR (MR_WT) and MR_L810 (3). Furthermore, all steroids, including progesterone, that displayed antagonist properties when bound to MR_WT were also able to activate MR_L810. As the levels of plasma progesterone normally increase about 100-fold during pregnancy, Geller et al. (3) have postulated that this hormone may contribute to the dramatic exacerbation of hypertension in pregnant MR_L810 carriers. The steroid(s) responsible for early-onset hypertension in MR_L810 carriers (men and nonpregnant women) has still not been identified.

    Cortisol, which is 100- to 1000-fold more abundant than aldosterone in plasma, binds to MR with the same affinity as aldosterone (4, 5). In the collecting duct, the preferential accessibility of aldosterone to MR is ensured by 11ß- hydroxysteroid dehydrogenase type 2, an enzyme that metabolizes cortisol and corticosterone to form inactive cortisone and 11-dehydrocorticosterone (6, 7). Because the determinants for the ligand-induced activation of MR_L810 are distinct from those for MR_WT, we investigated the ability of cortisone and 11-dehydrocorticosterone to bind to MR_L810 and activate its trans-activation function. The findings of steroid binding studies and steroid-induced trans-activation measurements provide evidence of the specific binding of cortisone and 11-dehydrocorticosterone to MR_L810 and of their abilities to activate the mutant MR. Because the plasma level of cortisol in men is about 30-fold higher than the plasma level of corticosterone (8), these findings also suggested that cortisone could be involved in the development of hypertension in young men and nonpregnant women harboring the S810L mutation.

    Materials and Methods

    Plasmids and construction

    The expression plasmid pchMR contains the entire coding sequence of MR_WT (9). Plasmid pchMR_L810, encoding for MR_L810, was constructed from pchMR using the QuikChange procedure from Stratagene (Amsterdam, The Netherlands). The Bpu1102I-AflII fragment of pchMR_L810 was subcloned into pchMR, after being sequenced to confirm that there was no other mutation in the sequence. Plasmid pFC31Luc contains the mouse mammary tumor virus (MMTV) promoter that drives the luciferase gene (10), and plasmid TAT3-TATA-Luc contains a trimerized hormone response element fused to the alcohol dehydrogenase minimal promoter driving luciferase (11). The pSVß vector was from CLONTECH Laboratories, Inc. (Saint Quentin en Yvelines, France).

    In vitro hormone binding assay

    MR_WT and MR_L810 were expressed in vitro using the T7-coupled rabbit reticulocyte lysate system from Promega Corp. (Charbonnieres, France). Lysates containing MR_WT or MR_L810 were diluted 2-fold with TEGWD buffer [20 mM sodium tungstate and 1 mM dithiothreitol in 20 mM Tris-HCl, 1 mM EDTA, and 10% glycerol, pH 7.4 (TEG)], and incubated with 10 nM [³H]aldosterone (Amersham Pharmacia Biotech, Les Ulis, France) with or without unlabeled steroids for 30 min at 20 C. Bound and free steroids were separated by the dextran-charcoal method: 25 µl lysate were stirred for 5 min with 50 µl 4% Norit A and 0.4% dextran T-70 in TEG buffer and centrifuged at 4500 x g for 5 min at 4 C. Bound steroid was measured by counting the radioactivity of the supernatant.

    Kinetic experiments

    MR_WT and MR_L810 were expressed in vitro using the T7-coupled rabbit reticulocyte lysate system. The lysate was diluted 2-fold with ice-cold TEGWD buffer and then incubated with 10 nM [³H]aldosterone or [³H]cortisol for 30 min at 20 C. One half of the labeled lysate was kept at 20 C to measure the stability of the [³H]steroid-MR complexes, and the other was incubated with 10 µM of the corresponding unlabeled steroid for various periods. Bound and free steroids were separated with dextran-charcoal. Parallel incubations containing [³H]steroid plus a 1000-fold excess of unlabeled steroid were used to calculate the nonspecific binding. Results were corrected for receptor stability and expressed as a percentage of the binding measured at time zero.

    Cell culture and transfection

    COS-7 cells were cultured in DMEM (Life Technologies, Inc.-BRL, Cergy Pontoise, France) supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere with 5% CO₂. Cells were maintained in the medium supplemented with 10% charcoal-stripped fetal calf serum for 4 h before and then throughout the transfection procedure. Cells were transfected by the phosphate calcium precipitation method (Profection, Promega Corp.). The phosphate solution, prepared for six-well trays, contained 5 µg of one of the receptor expression vectors (pchMR or pchMR_L810), 10 µg pFC31Luc or TAT3-TATA-Luc, and 2 µg pSVß. The steroids to be tested were added to the cells 12 h after transfection, and the incubation was continued for an additional 24 h. Cell extracts were then prepared and assayed for luciferase (12) and ß-galactosidase activities (13). To standardize the transfection efficiency, the relative light units obtained in the luciferase assay were divided by the OD obtained in the ß-galactosidase assay.

    Statistical analysis

    Values are the mean ± SEM from at least three separate experiments. Statistical differences between groups were analyzed by t test. P < 0.05 was considered significant.

    Results

    MR_L810 and MR_WT were expressed in vitro and tested for the ability to bind cortisone and 11-dehydrocorticosterone. As these two steroids were available as unlabeled molecules only, their abilities to inhibit [³H]aldosterone binding were measured in competition experiments. MR_L810 or MR_WT was incubated with 5 nM [³H]aldosterone alone or with 1 µM aldosterone, cortisol, cortisone, corticosterone, or 11dehydrocorticosterone. About 80% of the [³H]aldosterone bound to MR_L810 or MR_WT was displaced by a 200-fold excess of aldosterone, cortisol, or corticosterone (Fig. 1A), indicating that these hormones bind to MR_WT and to MR_L810. Cortisone did not displace the [³H]aldosterone bound to MR_WT, but did inhibit the binding of [³H]aldosterone to MR_L810. 11-Dehydrocorticosterone displaced by 40% and 80% the [³H]aldosterone bound to MR_WT and MR_L810, respectively (Fig. 1A). The range of concentrations of the corticosteroids tested widely differs in humans. Table 1 summarizes the plasma levels of aldosterone, cortisol, cortisone, and corticosterone in men. It appears that cortisol is about 1000-fold more abundant than aldosterone in plasma. The prevalence of cortisol over corticosterone in plasma led us to further analyze the abilities of various concentrations of cortisol and cortisone to inhibit [³H]aldosterone to both MR_WT and MR_L810. The dose-response curve depicted in Fig. 1B shows that cortisone was almost efficient as aldosterone and cortisol in displacing [³H]aldosterone from MR_L810. This finding demonstrates the high affinity with which cortisone binds to MR_L810.

    fig.ommitteed

    Figure 1. Effects of steroids on the binding of tritiated aldosterone to MR_WT and mutant MR_L810. A, MR_WT and MR_L810 were synthesized in vitro in rabbit reticulocyte lysate and incubated with 5 nM [³H]aldosterone with or without 1 µM unlabeled aldosterone, cortisol, cortisone, corticosterone, and 11-dehydrocortisone for 2 h at 4 C. B, MR_L810 expressed in rabbit reticulocyte lysate was incubated with 5 nM [³H]aldosterone in the absence (100%) or presence of various concentrations of aldosterone, cortisol, or cortisone for 2 h at 4 C. Bound and unbound steroids were separated using the charcoal-dextran method. Results are expressed as a percentage of the specific [³H]aldosterone binding measured in the absence of any competitor. Each pointis the mean ± SEM of three separate experiments. ***, P < 0.001

    fig.ommitteed

    Table 1. Plasma concentration of corticosteroids in humans

    It has been reported that the stability of the steroid-receptor complexes is highly dependent upon the nature of the steroid (5). We therefore investigated whether the stability of the steroid-receptor complexes could be altered by the nature of amino acid residue at the 810 position of the MR. The half-life times (t_1/2) of steroid-MR_WT and steroid-MR_L810 complexes were calculated from dissociation kinetics studies (Fig. 2). Aldosterone dissociated more slowly from MR_L810 (t_1/2 = 420 min) than from MR_WT (t_1/2 = 90 min; Fig. 2). Cortisol also dissociated more slowly from MR_L810 (t_1/2 = 120 min) than from MR_WT (t_1/2 = 10 min; Fig. 2). Figure 2 also shows that the dissociation kinetics were slower for the aldosterone-MR_L810 complex (t_1/2 = 420 min) than for the cortisol-MR_L810 complex (t_1/2 = 120 min). Similar differences in the dissociation kinetics and t_1/2 were observed with aldosterone- and cortisol-MR_WT complexes (Fig. 2). Despite the fact that S810L mutation has improved the stability of the human MR-steroid complexes, which suggests a change in receptor conformation, aldosterone-MR complexes still remain more stable than cortisol-MR complexes (Fig. 2).

    fig.ommitteed

    Figure 2. Dissociation of aldosterone from MR_WT and MR_L810. MR_WT (black symbols) and MR_L810 (open symbols) were produced by translation in vitro and incubated with 10 nM [³H]aldosterone (circles) or [³H]cortisol (triangles) for 30 min at 20 C. The end of this incubation period was taken as time zero for kinetic analysis. An aliquot was kept at 20 C to measure the stability of steroid-receptor complexes, and another aliquot was incubated with unlabeled aldosterone or cortisol (10^-6 M). Bound and free steroids were separated by the dextran-charcoal method described in Materials and Methods. Nonspecific binding was measured in parallel for each time tested. Results were corrected for receptor stability and expressed as a percentage of the binding measured at time zero. Values are the mean ± SEM of three experiments.

    The trans-activation activity of MR_L810 and MR_WT in response to steroids was then examined in cis-trans cotransfection assays. COS-7 cells were transiently transfected with pchMR_L810 or pchMR_WT plus a reporter gene containing luciferase under the control of the MR-sensitive MMTV promoter (pFC31Luc). The transfected cells were incubated with 10 nM of the steroid to be tested. Aldosterone, cortisol, and corticosterone produced maximum activation of both MR_WT and MR_L810 (Fig. 3A). Progesterone had low MR_WT-activating efficiency, but, like aldosterone, acted as a potent activator of MR_L810 (Fig. 3A). Cortisone was unable to activate MR_WT, but activated MR_L810 to about 75% the level of aldosterone-induced MR_L810 activity (Fig. 3A). 11-Dehydrocorticosterone had a low MR_WT-activating efficiency, but maximally activated MR_L810 (Fig. 3A). Additional transfection assays were performed using the TAT3-TATA-Luc promoter. In this case, 10^-9 M cortisone, which slightly activated MR_WT (25% of the aldosterone-induced MR_WT activity), activated MR_L810 to almost the same extent as aldosterone (Fig. 3A). These results indicated that the agonist activity of cortisone is related to the S810L mutation independently of the promoter used. The dose-response curves of MR_WT and MR_L810 trans-activation activity performed with the MMTV promoter also showed that cortisone had no effect on MR_WT activity, and that the concentration of cortisone required to produce half-maximum MR_L810 trans-activation activity was higher (5 x 10^-9 M) than that of aldosterone (5 x 10^-11 M; Fig. 3B).

    fig.ommitteed

    Figure 3. Trans-activation activity of MR_WT and MR_L810 in response to steroids. A and B, COS-7 cells were transfected with pchMR_WT or pchMR_L810 and pFC31Luc or TAT3-TATA-Luc as reporter plasmids, and a ß-galactosidase internal reporter was used to correct for transfection efficiency. A, Before harvesting, the cells were treated for 24 h with 10^-8 M aldosterone, progesterone, cortisol, cortisone, corticosterone, or 11-dehydrocorticosterone (using pFC31Luc as reporter plasmid) or with 10^-9 M aldosterone or cortisone (using TAT3-TATA-Luc as reporter plasmid). B, Before harvesting, the cells transfected with pFC31Luc were treated for 24 h with increasing concentrations of aldosterone or cortisone. Trans-activation was determined by luciferase activity, normalized vs. the internal ß-galactosidase control, and expressed as a percentage of MR_WT activity measured in the presence 10^-8 M aldosterone. Each point is the mean ± SEM of three separate experiments. ***, P < 0.001.

    Discussion

    The findings show that cortisone, the main metabolite of cortisol in the kidney, activates mutant MR_L810, suggesting that cortisone is primarily involved in the early development of hypertension in patients harboring this mutation.

    Using cotransfection assays, Geller et al. (3) have shown that MR_L810 displays a constitutive activity not detected for MR_WT, and these researchers point out that this activity, which they observed without adding any steroid, may play a role in the onset of hypertension in patients harboring mutant MR_L810. However, such constitutive activity accounts for only 20% of the maximum aldosterone-induced activity, suggesting that an endogenous steroid ligand is necessary for maximum MR_L810 activation to promote hypertension. In their search for the endogenous steroid ligand activator, Geller et al. (3) tested the abilities of various steroids to activate MR_L810. Neither estradiol nor testosterone, two steroids with a 17ß-hydroxyl group, activated MR_L810 or MR_WT (3). In contrast, steroids with a 21-hydroxyl group, such as aldosterone and cortisol, activated MR_WT and MR_L810 to the same extent (Ref. 3 and the present study). Steroids without the 21-hydroxyl group, which display antagonist properties when bound to the MR_WT, are also able to activate MR_L810 (3). This is the case for the antihypertensive drug spironolactone (a synthetic steroid with 17-lactone) and progesterone (Ref. 3 and the present study). The facts that 17-hydroxyprogesterone is able to activate MR_L810 and the plasma level of this progesterone derivative is the same as that of aldosterone raised the possibility that 17{alpha} -hydroxyprogesterone may contribute to hypertension in men (3). In the present study we investigated the possibility that one of the steroid metabolites produced in the principal cells of the collecting duct could produce agonist effects at MR_L810, but not at MR_WT. We found that this was indeed the case for cortisone and 11-dehydrocorticosterone. The 11-dehydrometabolites of cortisol and corticosterone have very low agonist and antagonist activities at high concentrations when bound to MR_WT (14, 15). In sharp contrast, we found that cortisone and 11dehydrocorticosterone bind and activate MR_L810. Under normal conditions, cortisol and corticosterone are metabolized by 11ß-hydroxysteroid dehydrogenase type 2 to form the almost inactive substance, cortisone and 11-dehydrocorticosterone, respectively, allowing aldosterone to occupy the MR, and this is responsible for the fine hormonal regulation of sodium absorption in the distal nephron (6, 7, 15). Because plasma levels of corticosterone are much lower than those of cortisol, it seems unlikely that 11-dehydrocortisosterone will have any effect on MR_L810 activation in vivo. Cortisol, the main plasma corticosteroid in humans, is metabolized into cortisone in collecting duct cells (15). Cortisone, inactive on MR_WT, becomes active on MR_L810. The dose-response curve of MR_L810 trans-activation activity in response to cortisone suggests that the concentration of circulating cortisone is sufficient (70 nM) to activate the mutant MR. Thus, in MR_L810 carriers, the occupancy of MR by cortisone should lead to permanent activation of the mutant MR.

    The agonist activity of aldosterone at the hMR has been attributed to the capacity of its 21-hydroxyl group to form a strong hydrogen bond with Asn⁷⁷⁰, a residue of the H3 helix (9). The presence of a hydroxyl group in the C₁₁ position facing alanine 773 (A773) of the H3 helix interferes with the positioning of cortisol within the ligand-binding pocket of the hMR (9, 15, 16). As a result, the contact between the 21-hydroxyl of cortisol and Asn⁷⁷⁰ is weakened, and the MR_WT-activating potential of cortisol is lower than that of aldosterone (9). The accommodation of cortisone in the ligand binding pocket is even more difficult, making contact between Asn⁷⁷⁰ and the 21-hydroxyl group almost impossible. This hypothesis is illustrated in Fig. 4. The lack of contact between Asn⁷⁷⁰ and the 21-hydroxyl group is compatible with the antagonist activity of cortisone, as has been observed for progesterone and spirolactone (9).

    fig.ommitteed

    Figure 4. Schematic representation of the amino acid residues of MR_WT involved in the anchoring of cortisol and cortisone. The black arrows represent the hydrogen bonds between steroids and amino acid residues of the ligand-binding domain of MR_WT. The Ala⁷⁷³ facing the 11-ketone of cortisone impairs its positioning within the ligand binding pocket of MR_WT, leading to a weak contact between the 21-hydroxyl group of cortisone and Asn⁷⁷⁰ (dashed arrow).

    The exact mechanism of MR_L810 activation remains unknown. It has been proposed that the agonist activity of MR_L810 when bound to steroids with no 21-hydroxyl group, such as progesterone, depends on contacts between the H3 and H5 helixes via the Leu⁸¹⁰ and Ala⁷⁷³ residues (3). Cortisone can also activate MR_L810 through similar contacts. Another possible explanation, and the two are not mutually exclusive, is that the S810L mutation could modify the conformation of the mutant receptor, making it possible to accommodate cortisone within the ligand binding pocket of the mutant MR. In this case, the contact between the 21-hydroxyl group of cortisone and Asn⁷⁷⁰, which is responsible for the activation of MR_WT by aldosterone (9), may make the cortisone-dependent agonist activity of mutant MR_L810 possible. The question also arises of whether the high stability of the steroid-MR_L810 complex contributes to permanent activation of the mutant receptor. Although this possibility cannot be completely ruled out, Geller et al. (3) showed that MR_WT and MR_L810 displayed the same sensitivity to aldosterone.

    The activating S810L mutation that has been found to date in only one family (3) has brought new insights to the understanding of MR agonism mechanisms. In this study we show that cortisone, which is normally an inefficient activator of MR_WT, is able to activate MR_L810. Thus, it is reasonable to infer that the early onset of hypertension in MR_L810 carriers could be attributable to a permanent increase in renal sodium reabsorption due to activation of MR_L810 by cortisone.

    Acknowledgments

    We thank H. Richard-Foy and F. Gouilleux for providing the plasmid pFC31Luc. We are grateful to D. Pearce for providing the plasmid TAT3-TATA-Luc.

    Received July 11, 2002.

    Accepted for publication October 31, 2002.

    References

    Rossier BC, Palmer LG 1992 Mechanism of aldosterone action on sodium and potassium transport in the kidney. In: Seldin DW, Giebisch G, eds. Physiology and pathology. 2nd ed. New York: Raven Press; 1373–409

    Bonvalet JP 1998 Regulation of sodium transport by steroid hormones. Kidney Int 65:49–56

    Geller DS, Farhi A, Pinkerton N, Fradley M, Moritz M, Spitzer A, Meinke G, Tsai FTF, Sigler PB, Lifton RP 2000 Activating mineralocorticoid receptor mutation in hypertension exacerbated by pregnancy. Science 289:119–123

    Arriza JL, Simerly RB, Swanson LW, Evans RM 1988 The neuronal mineralocorticoid receptor as a mediator of glucocorticoid response. Neuron 1: 887–900

    Hellal-Levy C, Couette B, Fagart J, Souque A, Gomez-Sanchez C, Rafestin-Oblin ME 1999 Specific hydroxylations determine selective corticosteroid recognition by human glucocorticoid and mineralocorticoid receptors. FEBS Lett 464:9–13

    Edwards CR, Stewart PM, Burt D, Brett L, McIntyre MA, Sutano WS, de Kloet ER 1988 Localization of 11ß-hydroxysteroid dehydrogenase-tissue specific protector of the mineralocorticoid receptor. Lancet 2:986–989

    Funder JW, Pearce PT, Smith R, Smith AI 1988 Mineralocorticoid action: target tissue specificity is enzyme, not receptor, mediated. Science 242:583–585

    Orth DN, Kovacs JK, De Bold R 1987 The adrenal cortex. In: Wilson JD, Foster W, eds. Textbook of endocrinology. 8th ed. Philadelphia: Saunders; 509

    Fagart J, Wurtz JM, Souque A, Hellallevy C, Moras D, Rafestin-Oblin ME 1998 Antagonism in the human mineralocorticoid receptor. EMBO J 17:3317–3325

    Gouilleux F, Sola B, Couette, Richard-Foy H 1991 Cooperation between structural elements in hormono-regulated transcription from the mouse mammary tumor virus promoter. Nucleic Acids Res 19:1563–1569

    Liu W, Wang J, Sauter NK, Pearce D 1995 Steroid receptor heterodimerzation in vitro and in vivo. Proc Natl Acad Sci USA 92:12480–12484

    De Wet JR, Wood KV, Deluca M, Helsinki DR, Subramani S 1987 Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7:725–737

    Herbomel P, Bourachot B, Yanif M 1984 Two distinct enhancers with different cell specificities coexist in the regulatory region of polyoma. Cell 39:653–662

    Morris DJ, Souness GW, Bren AS, Oblin ME 2000 Interactions of mineralocorticoids and glucocorticoids in epithelial target tissues. Kidney Int 57:1370–1373

    Farman N, Rafestin-Oblin ME 2001 Multiple aspects of mineralocorticoid selectivity. Am J Phyiol 280:181–192

    Auzou G, Fagart J, Souque A, Hellal-Levy C, Wurtz JM, Moras D, Rafestin-Oblin ME 2000 A single amino acid mutation of Ala773 in the mineralocorticoid receptor confers agonist properties to 11ß-substituted spirolactones. Mol Pharmacol 58:684–691(Marie-Edith Rafestin-Oblin Anny Souque Brigitte Bocchi Gregory Pinon Jerome Fagart and Alain Vandewa)

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