The Severe Form of Hypertension Caused by the Activating S810L Mutation in the Mineralocorticoid Receptor Is Cortisone Related
Abstractx{)z:2, 百拇医药
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 (MRL810). These findings suggest that progesterone may contribute to the dramatic aggravation of hypertension in MRL810 carriers during pregnancy. However, the steroid(s) responsible for hypertension in MRL810 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 MRL810. 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 MRL810 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 MRL810 mutation.
Introductionc*9o, 百拇医药
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 (MRL810) (3). Examination of this patient’s family revealed that all MRL810 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 (MRWT) and MRL810 (3). Furthermore, all steroids, including progesterone, that displayed antagonist properties when bound to MRWT were also able to activate MRL810. 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 MRL810 carriers. The steroid(s) responsible for early-onset hypertension in MRL810 carriers (men and nonpregnant women) has still not been identified.nr}*%, 百拇医药
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 MRL810 are distinct from those for MRWT, we investigated the ability of cortisone and 11-dehydrocorticosterone to bind to MRL810 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 MRL810 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 Methodsi*aier, 百拇医药
Plasmids and constructioni*aier, 百拇医药
The expression plasmid pchMR contains the entire coding sequence of MRWT (9). Plasmid pchMRL810, encoding for MRL810, was constructed from pchMR using the QuikChange procedure from Stratagene (Amsterdam, The Netherlands). The Bpu1102I-AflII fragment of pchMRL810 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).i*aier, 百拇医药
In vitro hormone binding assayi*aier, 百拇医药
MRWT and MRL810 were expressed in vitro using the T7-coupled rabbit reticulocyte lysate system from Promega Corp. (Charbonnieres, France). Lysates containing MRWT or MRL810 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 experimentsep, 百拇医药
MRWT and MRL810 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.ep, 百拇医药
Cell culture and transfectionep, 百拇医药
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 pchMRL810), 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 analysisn, 百拇医药
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.n, 百拇医药
Resultsn, 百拇医药
MRL810 and MRWT 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. MRL810 or MRWT 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 MRL810 or MRWT was displaced by a 200-fold excess of aldosterone, cortisol, or corticosterone (Fig. 1A), indicating that these hormones bind to MRWT and to MRL810. Cortisone did not displace the [3H]aldosterone bound to MRWT, but did inhibit the binding of [3H]aldosterone to MRL810. 11-Dehydrocorticosterone displaced by 40% and 80% the [3H]aldosterone bound to MRWT and MRL810, 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 MRWT and MRL810. The dose-response curve depicted in Fig. 1B shows that cortisone was almost efficient as aldosterone and cortisol in displacing [3H]aldosterone from MRL810. This finding demonstrates the high affinity with which cortisone binds to MRL810.
fig.ommitteedna+}, 百拇医药
Figure 1. Effects of steroids on the binding of tritiated aldosterone to MRWT and mutant MRL810. A, MRWT and MRL810 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, MRL810 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 < 0.001na+}, 百拇医药
fig.ommitteedna+}, 百拇医药
Table 1. Plasma concentration of corticosteroids in humansna+}, 百拇医药
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 (t1/2) of steroid-MRWT and steroid-MRL810 complexes were calculated from dissociation kinetics studies (Fig. 2). Aldosterone dissociated more slowly from MRL810 (t1/2 = 420 min) than from MRWT (t1/2 = 90 min; Fig. 2). Cortisol also dissociated more slowly from MRL810 (t1/2 = 120 min) than from MRWT (t1/2 = 10 min; Fig. 2). Figure 2 also shows that the dissociation kinetics were slower for the aldosterone-MRL810 complex (t1/2 = 420 min) than for the cortisol-MRL810 complex (t1/2 = 120 min). Similar differences in the dissociation kinetics and t1/2 were observed with aldosterone- and cortisol-MRWT 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), 百拇医药
Figure 2. Dissociation of aldosterone from MRWT and MRL810. MRWT (black symbols) and MRL810 (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.), 百拇医药
The trans-activation activity of MRL810 and MRWT in response to steroids was then examined in cis-trans cotransfection assays. COS-7 cells were transiently transfected with pchMRL810 or pchMRWT 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 MRWT and MRL810 (Fig. 3A). Progesterone had low MRWT-activating efficiency, but, like aldosterone, acted as a potent activator of MRL810 (Fig. 3A). Cortisone was unable to activate MRWT, but activated MRL810 to about 75% the level of aldosterone-induced MRL810 activity (Fig. 3A). 11-Dehydrocorticosterone had a low MRWT-activating efficiency, but maximally activated MRL810 (Fig. 3A). Additional transfection assays were performed using the TAT3-TATA-Luc promoter. In this case, 10-9 M cortisone, which slightly activated MRWT (25% of the aldosterone-induced MRWT activity), activated MRL810 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 MRWT and MRL810 trans-activation activity performed with the MMTV promoter also showed that cortisone had no effect on MRWT activity, and that the concentration of cortisone required to produce half-maximum MRL810 trans-activation activity was higher (5 x 10-9 M) than that of aldosterone (5 x 10-11 M; Fig. 3B).
fig.ommitteedzy;, 百拇医药
Figure 3. Trans-activation activity of MRWT and MRL810 in response to steroids. A and B, COS-7 cells were transfected with pchMRWT or pchMRL810 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 MRWT activity measured in the presence 10-8 M aldosterone. Each point is the mean ± SEM of three separate experiments. ***, P < 0.001.zy;, 百拇医药
Discussion
The findings show that cortisone, the main metabolite of cortisol in the kidney, activates mutant MRL810, suggesting that cortisone is primarily involved in the early development of hypertension in patients harboring this mutation.k@s9/;, http://www.100md.com
Using cotransfection assays, Geller et al. (3) have shown that MRL810 displays a constitutive activity not detected for MRWT, 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 MRL810. However, such constitutive activity accounts for only 20% of the maximum aldosterone-induced activity, suggesting that an endogenous steroid ligand is necessary for maximum MRL810 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 MRL810. Neither estradiol nor testosterone, two steroids with a 17ß-hydroxyl group, activated MRL810 or MRWT (3). In contrast, steroids with a 21-hydroxyl group, such as aldosterone and cortisol, activated MRWT and MRL810 to the same extent (Ref. 3 and the present study). Steroids without the 21-hydroxyl group, which display antagonist properties when bound to the MRWT, are also able to activate MRL810 (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 MRL810 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 MRL810, but not at MRWT. 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 MRWT (14, 15). In sharp contrast, we found that cortisone and 11dehydrocorticosterone bind and activate MRL810. 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 MRL810 activation in vivo. Cortisol, the main plasma corticosteroid in humans, is metabolized into cortisone in collecting duct cells (15). Cortisone, inactive on MRWT, becomes active on MRL810. The dose-response curve of MRL810 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 MRL810 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 MRWT-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).9m-1+v2, 百拇医药
fig.ommitteed
Figure 4. Schematic representation of the amino acid residues of MRWT 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 MRWT. The Ala773 facing the 11-ketone of cortisone impairs its positioning within the ligand binding pocket of MRWT, leading to a weak contact between the 21-hydroxyl group of cortisone and Asn770 (dashed arrow).#}^-h-], 百拇医药
The exact mechanism of MRL810 activation remains unknown. It has been proposed that the agonist activity of MRL810 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 MRL810 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 MRWT by aldosterone (9), may make the cortisone-dependent agonist activity of mutant MRL810 possible. The question also arises of whether the high stability of the steroid-MRL810 complex contributes to permanent activation of the mutant receptor. Although this possibility cannot be completely ruled out, Geller et al. (3) showed that MRWT and MRL810 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 MRWT, is able to activate MRL810. Thus, it is reasonable to infer that the early onset of hypertension in MRL810 carriers could be attributable to a permanent increase in renal sodium reabsorption due to activation of MRL810 by cortisone.!1, 百拇医药
Acknowledgments!1, 百拇医药
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.!1, 百拇医药
Received July 11, 2002.!1, 百拇医药
Accepted for publication October 31, 2002.!1, 百拇医药
References!1, 百拇医药
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Farman N, Rafestin-Oblin ME 2001 Multiple aspects of mineralocorticoid selectivity. Am J Phyiol 280:181–192s'/, 百拇医药
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)
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 (MRL810). These findings suggest that progesterone may contribute to the dramatic aggravation of hypertension in MRL810 carriers during pregnancy. However, the steroid(s) responsible for hypertension in MRL810 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 MRL810. 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 MRL810 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 MRL810 mutation.
Introductionc*9o, 百拇医药
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 (MRL810) (3). Examination of this patient’s family revealed that all MRL810 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 (MRWT) and MRL810 (3). Furthermore, all steroids, including progesterone, that displayed antagonist properties when bound to MRWT were also able to activate MRL810. 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 MRL810 carriers. The steroid(s) responsible for early-onset hypertension in MRL810 carriers (men and nonpregnant women) has still not been identified.nr}*%, 百拇医药
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 MRL810 are distinct from those for MRWT, we investigated the ability of cortisone and 11-dehydrocorticosterone to bind to MRL810 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 MRL810 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 Methodsi*aier, 百拇医药
Plasmids and constructioni*aier, 百拇医药
The expression plasmid pchMR contains the entire coding sequence of MRWT (9). Plasmid pchMRL810, encoding for MRL810, was constructed from pchMR using the QuikChange procedure from Stratagene (Amsterdam, The Netherlands). The Bpu1102I-AflII fragment of pchMRL810 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).i*aier, 百拇医药
In vitro hormone binding assayi*aier, 百拇医药
MRWT and MRL810 were expressed in vitro using the T7-coupled rabbit reticulocyte lysate system from Promega Corp. (Charbonnieres, France). Lysates containing MRWT or MRL810 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 experimentsep, 百拇医药
MRWT and MRL810 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.ep, 百拇医药
Cell culture and transfectionep, 百拇医药
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 pchMRL810), 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 analysisn, 百拇医药
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.n, 百拇医药
Resultsn, 百拇医药
MRL810 and MRWT 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. MRL810 or MRWT 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 MRL810 or MRWT was displaced by a 200-fold excess of aldosterone, cortisol, or corticosterone (Fig. 1A), indicating that these hormones bind to MRWT and to MRL810. Cortisone did not displace the [3H]aldosterone bound to MRWT, but did inhibit the binding of [3H]aldosterone to MRL810. 11-Dehydrocorticosterone displaced by 40% and 80% the [3H]aldosterone bound to MRWT and MRL810, 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 MRWT and MRL810. The dose-response curve depicted in Fig. 1B shows that cortisone was almost efficient as aldosterone and cortisol in displacing [3H]aldosterone from MRL810. This finding demonstrates the high affinity with which cortisone binds to MRL810.
fig.ommitteedna+}, 百拇医药
Figure 1. Effects of steroids on the binding of tritiated aldosterone to MRWT and mutant MRL810. A, MRWT and MRL810 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, MRL810 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 < 0.001na+}, 百拇医药
fig.ommitteedna+}, 百拇医药
Table 1. Plasma concentration of corticosteroids in humansna+}, 百拇医药
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 (t1/2) of steroid-MRWT and steroid-MRL810 complexes were calculated from dissociation kinetics studies (Fig. 2). Aldosterone dissociated more slowly from MRL810 (t1/2 = 420 min) than from MRWT (t1/2 = 90 min; Fig. 2). Cortisol also dissociated more slowly from MRL810 (t1/2 = 120 min) than from MRWT (t1/2 = 10 min; Fig. 2). Figure 2 also shows that the dissociation kinetics were slower for the aldosterone-MRL810 complex (t1/2 = 420 min) than for the cortisol-MRL810 complex (t1/2 = 120 min). Similar differences in the dissociation kinetics and t1/2 were observed with aldosterone- and cortisol-MRWT 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), 百拇医药
Figure 2. Dissociation of aldosterone from MRWT and MRL810. MRWT (black symbols) and MRL810 (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.), 百拇医药
The trans-activation activity of MRL810 and MRWT in response to steroids was then examined in cis-trans cotransfection assays. COS-7 cells were transiently transfected with pchMRL810 or pchMRWT 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 MRWT and MRL810 (Fig. 3A). Progesterone had low MRWT-activating efficiency, but, like aldosterone, acted as a potent activator of MRL810 (Fig. 3A). Cortisone was unable to activate MRWT, but activated MRL810 to about 75% the level of aldosterone-induced MRL810 activity (Fig. 3A). 11-Dehydrocorticosterone had a low MRWT-activating efficiency, but maximally activated MRL810 (Fig. 3A). Additional transfection assays were performed using the TAT3-TATA-Luc promoter. In this case, 10-9 M cortisone, which slightly activated MRWT (25% of the aldosterone-induced MRWT activity), activated MRL810 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 MRWT and MRL810 trans-activation activity performed with the MMTV promoter also showed that cortisone had no effect on MRWT activity, and that the concentration of cortisone required to produce half-maximum MRL810 trans-activation activity was higher (5 x 10-9 M) than that of aldosterone (5 x 10-11 M; Fig. 3B).
fig.ommitteedzy;, 百拇医药
Figure 3. Trans-activation activity of MRWT and MRL810 in response to steroids. A and B, COS-7 cells were transfected with pchMRWT or pchMRL810 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 MRWT activity measured in the presence 10-8 M aldosterone. Each point is the mean ± SEM of three separate experiments. ***, P < 0.001.zy;, 百拇医药
Discussion
The findings show that cortisone, the main metabolite of cortisol in the kidney, activates mutant MRL810, suggesting that cortisone is primarily involved in the early development of hypertension in patients harboring this mutation.k@s9/;, http://www.100md.com
Using cotransfection assays, Geller et al. (3) have shown that MRL810 displays a constitutive activity not detected for MRWT, 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 MRL810. However, such constitutive activity accounts for only 20% of the maximum aldosterone-induced activity, suggesting that an endogenous steroid ligand is necessary for maximum MRL810 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 MRL810. Neither estradiol nor testosterone, two steroids with a 17ß-hydroxyl group, activated MRL810 or MRWT (3). In contrast, steroids with a 21-hydroxyl group, such as aldosterone and cortisol, activated MRWT and MRL810 to the same extent (Ref. 3 and the present study). Steroids without the 21-hydroxyl group, which display antagonist properties when bound to the MRWT, are also able to activate MRL810 (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 MRL810 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 MRL810, but not at MRWT. 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 MRWT (14, 15). In sharp contrast, we found that cortisone and 11dehydrocorticosterone bind and activate MRL810. 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 MRL810 activation in vivo. Cortisol, the main plasma corticosteroid in humans, is metabolized into cortisone in collecting duct cells (15). Cortisone, inactive on MRWT, becomes active on MRL810. The dose-response curve of MRL810 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 MRL810 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 MRWT-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).9m-1+v2, 百拇医药
fig.ommitteed
Figure 4. Schematic representation of the amino acid residues of MRWT 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 MRWT. The Ala773 facing the 11-ketone of cortisone impairs its positioning within the ligand binding pocket of MRWT, leading to a weak contact between the 21-hydroxyl group of cortisone and Asn770 (dashed arrow).#}^-h-], 百拇医药
The exact mechanism of MRL810 activation remains unknown. It has been proposed that the agonist activity of MRL810 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 MRL810 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 MRWT by aldosterone (9), may make the cortisone-dependent agonist activity of mutant MRL810 possible. The question also arises of whether the high stability of the steroid-MRL810 complex contributes to permanent activation of the mutant receptor. Although this possibility cannot be completely ruled out, Geller et al. (3) showed that MRWT and MRL810 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 MRWT, is able to activate MRL810. Thus, it is reasonable to infer that the early onset of hypertension in MRL810 carriers could be attributable to a permanent increase in renal sodium reabsorption due to activation of MRL810 by cortisone.!1, 百拇医药
Acknowledgments!1, 百拇医药
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.!1, 百拇医药
Received July 11, 2002.!1, 百拇医药
Accepted for publication October 31, 2002.!1, 百拇医药
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