Modulation of Human Mineralocorticoid Receptor Function by Protein Kinase A

Charbel Massaad, Nathalie Houard, Marc Lombès and Robert Barouki

INSERM Unité 490 (C.M., R.B.) Centre universitaire des Saints-Pères 75270 Paris Cedex 06, France
INSERM Unité 478 (N.H., M.L.) Faculté de Médecine Xavier Bichat 75018 Paris France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The mineralocorticoid receptor (MR) acts as a ligand-dependent transcription factor modulating specific gene expression in sodium-transporting epithelia. Physiological evidence suggest a cross-talk between the cAMP- and aldosterone-signaling pathways. We provide evidence that protein kinase A (PKA), a major mediator of signal transduction pathways, modulates transcriptional activity of the human MR (hMR). Using transient transfection assays in HepG2 cells, we show that 8-bromo-cAMP, a protein kinase A activator, stimulates glucocorticoid response element (GRE)-containing promoters in a ligand-independent manner. This effect was strictly MR dependent since no activation of the reporter gene was observed in the absence of cotransfected hMR expression plasmid. Furthermore, a synergistic activation was achieved when cells were treated with both aldosterone and cAMP. This synergistic effect was also observed in the CV1 and the stable hMR-expressing M cells but was dependent on the promoter used. In particular, synergism was less pronounced in promoters containing several GREs. We show that (protein kinase-inhibiting peptide (PKI), the peptide inhibitor of PKA, prevented both cAMP and aldosterone induction, which indicates that a functional cAMP pathway is required for stimulation of transcription by aldosterone. Using MR-enriched baculovirus extracts in gel shift assays, we have shown that the binding of the MR to a GRE-containing oligonucleotide was enhanced by PKA. Increased DNA binding of hMR is likely to reflect an increase in the number of active receptors, as measured by Scatchard analysis. Using a truncated MR, we show that the N-terminal domain is required for the effect. Finally, the N-terminal truncated MR was not directly phosphorylated by PKA in vitro. We conclude that PKA acts indirectly, probably by relieving the effect of an MR repressor.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Aldosterone, the endogenous steroid hormone affecting the homeostasis of sodium, potassium, and hydrogen ions, acts through its intracellular receptor, the mineralocorticoid receptor (MR). After its binding to the MR, aldosterone triggers the translocation of the receptor into the nucleus and promotes its binding to cognate responsive elements that are similar to those of the glucocorticoid, progesterone, and androgen hormones (1, 2, 3). After binding to DNA, the hormone-receptor complex stimulates the transcription of target genes. This mechanism is very similar to that of several other steroid hormones.

In the kidney, both arginine vasopressin (AVP) and aldosterone contribute to salt and water homeostasis. AVP acts via two different receptors, V1 and V2, which are coupled to two distinct second messengers, Ca2+ and cAMP, respectively. Aldosterone and AVP (through its V2 receptor) exert synergistic actions on sodium reabsorption in the distal nephron (4), most notably via activation of the Na+/K+-ATPase in the collecting duct (5, 6, 7, 8), the pump enhancing the sodium reabsorption. Moreover, Alfaidy et al. (9) recently reported a synergy between aldosterone and V2, but not V1 receptor activation, in controlling the activity of 11ß-hydroxysteroid dehydrogenase, the MR protecting enzyme which plays a pivotal role in the mineralocorticoid selectivity in aldosterone-sensitive cells (9). These studies have raised the question of a physiologically relevant cross-talk between these two hormones, particularly, the role of cAMP in aldosterone signaling.

Recent studies have suggested that binding of steroid hormones to their receptors is not sufficient to trigger a potent response; in some cases, cAMP has been shown to play an important role. In the presence of hormone, protein kinase A activators (e.g. 8-bromo-(Br)-cAMP, isobutylmethylxanthine) elicit a synergistic activation of the transcription mediated by the estrogen receptor (ER) (10), the glucocorticoid receptor (GR) (11, 12), and the progesterone receptor (PR) (13, 14, 15). In these cases, protein kinase A (PKA) was shown to enhance the DNA-binding activity of these receptors. Surprisingly, treatment with 8-Br-cAMP alone was sufficient to activate the human AR (hAR) (16) and the chicken PR (cPR) (14) but not human PR (hPR) (16, 17). Thus, activation of the hAR and the cPR could be achieved through PKA signaling in the absence of the hormone. This is not the case for GR and ER signaling. Thus, while cAMP appears to interfere with steroid hormone action, the mechanisms involved appear to differ according to the receptor (18). Furthermore, it is not clear whether basal amounts of cAMP or PKA levels are required for these receptors’ action.

The interaction between mineralocorticoid effects and other signal transduction pathways remains unclear. In this study we demonstrate that 8-Br-cAMP potentiates the aldosterone induction of a glucocorticoid response element (GRE)-containing promoter. PKA treatment of MR-containing extracts enhances the binding of MR to GRE, probably due to an increase of active MR levels. Finally, the amino-terminal domain of the MR is essential in mediating PKA action, although this domain is not phosphorylated by PKA.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activation of Human hMR (hMR) by 8-Br-cAMP in HepG2 Cell Line
The mineralocorticoid hormone aldosterone activates 9-fold the {Delta}mammary tumor virus-GRE ({Delta}MTV-GRE) promoter in HepG2 cells when an hMR expression vector is cotransfected into these cells. 8-Br-cAMP was added in the absence or presence of aldosterone (Fig. 1Go). cAMP alone elicited a 3- to 4-fold activation of transcription (40% of the activation elicited by aldosterone). When added together, aldosterone and cAMP activated the promoter activity 26-fold, evidence for a synergistic effect. To confirm that the effect of cAMP required the presence of MR, HepG2 cells were transiently transfected by {Delta}MTV-GRE-CAT vector without the hMR expression vector (Fig. 1Go). In this case, neither aldosterone nor cAMP activated transcription, which indicates that the ligand-independent as well as the ligand-dependent activation of gene expression was MR specific.



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Figure 1. MR-Dependent Effect of cAMP

HepG2 cells were transiently transfected with {Delta}MTV-GRE-CAT plasmid, with (+hMR) or without (-hMR) hMR expression vector. Cells were treated with aldosterone (0.1 nM), 8-Br cAMP (0.5 mM), or both. Results are expressed as the fold induction over basal level. Results are the mean ± SEM of 15 independent experiments.

 
Influence of the Number of GREs
Promoters that are regulated by steroid hormones contain either one or several hormone response elements. To test the effect of the number of GREs on the MR-dependent cAMP activation, we subcloned one, two, or four GREs into the HindIII site of the {Delta}MTV promoter, yielding the plasmids GRE-, (GRE)2-, and (GRE)4-{Delta}MTV-CAT, respectively. As shown in Fig. 2AGo, aldosterone activated these promoters 10-, 35-, and 66-fold respectively, but had no effect on the {Delta}MTV promoter itself (Fig. 2AGo). In the absence of aldosterone, cAMP alone caused a 4-fold increase in transcription elicited by these constructs. Thus, as expected, the effect of aldosterone was dependent on the number of GREs present in the regulatory region of the target promoter while the effect of cAMP was not. The effect of both cAMP and aldosterone was synergistic for the (GRE)1 and (GRE)2 plasmids but less so in the case of (GRE)4 plasmid.



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Figure 2. Promoter-Dependent Effect of cAMP

HepG2 cells were transiently transfected with hMR expression vector and with one of the following constructions: {Delta}MTV-[(GRE)0]-CAT, {Delta}MTV-[(GRE)1]-CAT, {Delta}MTV-[(GRE)2]-CAT, or {Delta}MTV-[(GRE)4]-CAT plasmids (A), MMTV-CAT plasmid (B), or TK-GRE-CAT plasmid (C). Cells were treated with aldosterone (0.1 nM), 8-Br-cAMP (0.5 mM), or both for 24 h. Results are expressed as the fold induction over basal level. Results are the mean ± SEM of 5 to 8 independent experiments. All differences between cAMP and control were significant (P < 0.05). Differences between aldosterone and aldosterone + cAMP were significant (P < 0.05) except in the case of (GRE)4 and MMTV promoters.

 
Other promoters were also tested in the HepG2 cells. cAMP activated the transcription of the mouse MTV (MMTV) promoter 4-fold and modestly potentiated the effect of aldosterone (Fig. 2BGo), although the difference between aldosterone and aldo-sterone+cAMP effects in this particular case was not significant. Using a different promoter context, cAMP was as efficient as aldosterone in activating the recombinant GRE-TK-CAT promoter (2-fold), while the addition of both drugs elicited a 3.5-fold induction. Thus, the efficiency of both cAMP and aldosterone depends on the basal promoter used (TK vs. {Delta}MTV), an observation that has also been made in the case of other nuclear receptors (13).

Effect of cAMP on hMR in Different Cell Lines
The ability of cAMP to enhance the hMR-mediated transcription was also tested in another cell line, CV-1 (Simian kidney fibroblast). CV-1 cells were transfected with {Delta}MTV-GRE-CAT plasmid and the hMR expression vector (Fig. 3AGo). Cells were then treated with aldosterone, cAMP, or both effectors. cAMP activated the transcription of this plasmid, to an extent approximately 20% of that elicited by aldosterone alone. cAMP also potentiated the effect of aldosterone (400% the effect of aldosterone). To determine whether cAMP-mediated activation of the hMR was not a function of the transient transfection procedure, we tested the effect of cAMP on a clone M, stably expressing MR, derived from RC.SV3 cells, which are isolated from rabbit kidney tubules (Fig. 3BGo). These cells were transiently transfected by a {Delta}MTV-(GRE)2-CAT plasmid. As we have observed in HepG2 cells, cAMP activated transcription of the reporter gene approximately 4-fold (20% of the activation elicited by aldosterone). Added together, cAMP and aldosterone displayed a synergistic effect on transcription, to levels almost double those with aldosterone alone.



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Figure 3. Effect of cAMP in Different Cells

A, CV1 cells were transiently transfected with {Delta}MTV-GRE-CAT plasmid and the hMR expression vector. Cells were treated with aldosterone (0.1 nM), 8-Br-cAMP (0.5 mM), or both; 100% is the maximal activation elicited by aldosterone corresponding to 401 ± 10 arbitrary units. Results have been expressed as percent aldosterone effect because basal activity was very close to blank values, making calculations of fold activation imprecise. B, M cells were transiently transfected with the {Delta}MTV-(GRE)2-CAT plasmid and the hMR expression vector. Cells were treated with aldosterone (0.1 nM), 8-Br-cAMP (0.5 mM), or both; 100% is the maximal activation elicited by aldosterone, corresponding to 24,088 ± 3,854 arbitrary units. Results are the mean ± SEM of three and six independent experiments, respectively.

 
Taken together, these results indicate that cAMP activates the transcription of GRE-containing promoters. This cAMP-mediated activation was dependent on the presence of the hMR and was observed in three cell lines with different efficiencies.

Effect of PKA and PKI
Since the increase in intracellular levels of cAMP results in the activation of PKA, we examined the effect of the PKA on hMR activity. HepG2 cells were cotransfected with the RSV-hMR expression vector, GRE-TK-CAT, and in conjunction with various amounts of a PKA expression vector (Fig. 4AGo). The basal activity of the GRE construct increased in the presence of PKA, which also potentiated the effect of aldosterone. The effect of PKA was not observed in the absence of an hMR expression vector (not shown). PKA mimics the effect of cAMP confirming the results obtained with 8-Br-cAMP (Fig. 2CGo).



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Figure 4. Effect of PKA and PKI

A, HepG2 cells were transiently transfected with the hMR expression vector, the TK-GRE-CAT plasmid, and with increasing amounts of the PKA expression vector. Cells were treated or not with aldosterone (0.1 nM). Results are expressed as fold induction over basal level. B, HepG2 cells were transiently transfected with hMR expression vector, with the TK-GRE-CAT plasmid, and with increasing amounts of the PKI expression vector. Cells were treated with aldosterone (0.1 nM) or 8-Br-cAMP. Results are expressed as the fold induction over basal level and represent the mean ± SEM of six independent experiments.

 
To assess the effect of PKI , we cotransfected in HepG2 cells the RSV-MR and TK-GRE-CAT as well as various amounts of a PKI expression vector. Transfection of the PKI plasmid dramatically inhibited induction elicited by both cAMP and aldosterone (Fig. 4BGo). Indeed, in the presence of PKI, neither cAMP nor aldosterone has any effect. These results show that the action of cAMP is repressed by PKI and, more importantly, that the activation elicited by aldosterone requires an active PKA. Neither PKA nor PKI elicited any effect on the TK promoter (not shown).

We then examined whether 8-Br-cAMP could alter the dose-response curve of aldosterone. HepG2 cells were transfected with the {Delta}MTV-GRE-CAT plasmid and the hMR expression vector. Increasing concentrations of aldosterone were added (1 pM to 10 nM) with or without 8-Br-cAMP (0.5 mM). RU486 was added to block the possible aldosterone binding to the endogenous glucocorticoid receptor, thus avoiding the activation of GR at high concentrations of aldosterone. Figure 5Go shows that 8-Br-cAMP enhances the transactivation elicited by aldosterone over a wide dose range. Dose-response curves were redrawn from the data presented in Fig. 5Go as a percent of maximal activation (inset). 8-Br-cAMP enhances the activation without causing any significant shift in the dose-response curve, indicating that 8-Br-cAMP does not modify the apparent affinity of the receptor for the hormone.



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Figure 5. Aldosterone Dose-Response Curves

HepG2 cells were transiently transfected by hMR expression vector and {Delta}MTV-GRE-CAT plasmid. Cells were than treated by increasing concentrations of aldosterone (1 pM to 10 nM) with ({blacktriangleup}) or without (•) cAMP. Results are expressed as the fold induction over basal level. Inset, 100% is the maximal activation elicited by aldosterone or aldosterone + cAMP. Results are the mean of four independent experiments.

 
PKA Increases hMR Binding to GRE
We evaluated the effect of PKA on the binding of MR to a GRE sequence using electrophoretic mobility shift assay (EMSA) with radiolabeled GRE and baculovirus MR-enriched extracts preincubated or not with increasing amounts of PKA (50 U or 100 U) (Fig. 6AGo). The GRE-hMR complexes and free probes were quantified on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) (Fig. 6BGo). As shown in Fig. 6AGo, PKA enhanced the binding of MR to the GRE. After PKA treatment, the GRE-MR complexes were 2- to 3-fold more abundant than that of untreated complexes. To determine whether PKA treatment modifies the number of active MR species or the affinity of the receptor for a GRE-containing oligonucleotide, we performed EMSA using recombinant MR preincubated or not with PKA (100 U) and incubated with increasing concentrations of labeled GRE. As shown in Fig. 7Go, the GRE-MR treated by PKA were 2- to 3-fold more abundant than that of untreated complexes. The amount of bound and unbound oligonucleotides was quantified on a PhosphorImager. Scatchard analysis revealed that the dissociation constant (Kd) value was unaffected by PKA treatment (Kd = 6 x 10-9 M) (Fig. 7BGo). Interestingly, the number of DNA- binding hMR molecules was twice as high after treatment with PKA. These results indicate that the enhanced transcriptional activation of hMR by PKA is due to an increased amount of active MR protein without affecting the apparent affinity of hMR for the response element.



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Figure 6. Effect of PKA on the Binding of MR to GRE

A, Radiolabeled GRE was incubated with MR-enriched baculovirus extracts. These extracts were preincubated or not during 45 min with increasing amounts of PKA (50 U and 100). The arrow indicates the migration of the MR-GRE complex. B, The complexes were quantified by PhosphorImager (ImageQuant software); 100% corresponds to the binding of the MR without preincubation with PKA. These results are the mean ± SEM of eight independent experiments.

 


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Figure 7. Scatchard Plots

A, MR extracts were preincubated (PKA = 100 U) or not (PKA = 0 U) with PKA. EMSA was performed using these extracts and increasing amounts of radiolabeled GRE (104 cpm to 2.5 106 cpm). The arrow indicates the migration of the MR-GRE complex. B, Complexes and free probes were evaluated by PhosphorImager (ImageQuant software). The figure shows a representative experiment that was repeated six times.

 
The N-Terminal Domain of hMR Is Required for PKA Activation
To localize more precisely the domain of the MR that is important for PKA regulation, the properties of an N-terminal deleted MR were tested (MR-352). This domain contains a transcription activation function (TAF-2). MR- or MR352-enriched baculovirus extracts were incubated or not with PKA. EMSA showed that the MR-GRE complex is twice as abundant after treatment with PKA (Fig. 8Go). In contrast, the complex formed by MR352-GRE was not affected by PKA treatment. These results indicate that the amino-terminal domain of the MR is required for the activation by PKA and is the likely direct or indirect target of this kinase.



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Figure 8. Effect of PKA on N-Terminal Deleted MR

Wild-type MR (wt) and N-terminal deleted MR (del352) baculovirus-enriched extracts were preincubated (+) or not (-) with PKA (100 U). EMSAs were performed using these extracts and radiolabeled GRE. The arrow indicates the migration of the MR-GRE complex. B, Complex abundance was quantitated on a PhosphorImager (100% corresponds to the binding of the MR without preincubation with PKA). These results are the mean ± SEM of 10 independent experiments.

 
PKA could activate the hMR either by directly phosphorylating the N-terminal domain of this receptor, or by phosphorylating another protein that interacts with this domain. To test the first hypothesis, the N-terminal domain of the hMR (N516) was expressed in Escherichia coli. The molecular mass of the purified truncated protein was 54 kDa as revealed by Coomassie gel staining (not shown). The purified N516 fragment was then incubated with radiolabeled {gamma}-32P ATP and PKA (100 U) as described in Materials and Methods. The data presented in Fig. 9Go show that PKA did not trigger the incorporation of 32P in the N516 fragment. Thus, the N516 fragment is not phosphorylated by PKA. As expected, a similar amount of recombinant estrogen receptor (ER) and casein were both phosphorylated by PKA under the same experimental procedures.



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Figure 9. Phosphorylation of the N516 Fragment

Either N516 (2 µg) purified fragment, recombinant purified ER (2 µg), or pure dephosphorylated casein (2 µg) were incubated with PKA (100 U) and radiolabeled ATP. The reaction mixture was loaded on an SDS-polyacrylamide gel as described in Materiald and Methods. Casein isoforms have different molecular weights (casein-{alpha}, mol wt = 22,000; casein ß, mol wt = 24,000; and casein-{kappa}, mol wt = 19,000).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Previous studies have shown that steroid receptors could be activated in a ligand-independent manner. cPR (13) and hAR (16) are activated by cAMP even in the absence of progesterone or testosterone, respectively. In contrast, hGR (11) and hPR (19) are not activated by the PKA-signaling pathway alone; in this case, cAMP potentiates the activation by the corresponding hormones. In this report, we demonstrate that hMR can be activated in the absence of aldosterone through an alternative signaling pathway involving cAMP, a stimulator of PKA. In addition, cAMP potentiates the activation of transcription by aldosterone of a GRE-containing promoter. These results were observed in three different cell lines that indicate that the effect of PKA is not cell specific.

One interesting observation made here is that the effect of PKA was dependent on the promoter structure. Using promoters containing one, two, or four GREs in tandem, we have shown that the effect of 8-Br-cAMP alone was constant in all cases (4-fold), while the effect of aldosterone was additive. Interestingly, cAMP strongly potentiated the aldosterone effect in the GRE or (GRE)2-containing promoter (3-fold) and to a lesser extent in the case of (GRE)4 or MMTV promoters. These results suggest that the activation by cAMP or PKA-signaling pathways is critical for promoters containing a small number of regulatory sequences like GRE or (GRE)2. In this case, PKA activation results in an optimal induction by aldosterone. In contrast, for promoters like MMTV in which the activation by aldosterone is very potent, the synergistic effect of cAMP is relatively weak. This could be due to a saturation in the ability of various effectors to stimulate promoter activity. The molecular mechanisms involved remain to be determined.

As expected, PKI, a peptide inhibitor of PKA, abolished the activation by 8-Br-cAMP. Surprisingly, PKI also dramatically inhibited gene regulation by aldosterone. These data suggest that the effects of aldosterone alone depend on the presence of a basal intracellular activity of PKA. We were not able to use an MR-antagonist (e.g. spironolactone, RU26752) to inhibit cAMP action. These molecules exhibit a partial agonist effect on MR that was potentiated by 8-Br-cAMP (Ref. 20 and data not shown).

How could PKA affect MR function? Several lines of evidence suggest that it is the number of active MR species that is increased by PKA. Indeed, the apparent affinity of MR for aldosterone is unchanged. In addition, Scatchard analysis suggested that the affinity of the receptor for DNA is not modified while the number of functional, DNA-binding species of receptor is increased. It should also be noted that the migration of the receptor is apparently not altered by PKA even in very low acrylamide gels (not shown). However, since the MR dimer-DNA complex is very large (>200 kDa), it is difficult to exclude a minor modification.

Another important finding is that the deletion of the N-terminal domain prevents the effect of PKA. The lack of a consensus PKA phosphorylation site within the hMR and the absence of phosphorylation of the N516 deletion fragment of the hMR indicate that the MR itself is unlikely to be the direct target for phosphorylation by PKA. One possible model accounting for all these data is that the MR could be maintained in an inactive state through an interaction with a protein or a complex of proteins. Through a phosphorylation step that remains to be determined, PKA could release the MR from the complex and thus allows it to interact with DNA and activate transcription. Although other models could also be suggested, the one presented here is compatible with our observations and with several features of the biology of steroid hormone receptors. Indeed, these receptors interact with various proteins either in the cytosol or in the nucleus. Some of these proteins are known to maintain the receptors in a inactive form that provides a possible mechanism for cross-talk between various signaling pathways. A similar model was recently demonstrated in the case of the progesterone receptor. cAMP, via PKA, phosphorylates nuclear corepressors, NCoR and SMRT. When phosphorylated, these corepressors are released from the PR and allow it to interact with the transcription machinery (21). One possible function of the PKA effects is to provide a differential regulation of steroid receptors that otherwise share several similar properties. While many functions of the MR and the GR are similar, cAMP displays different effects on these two receptors. Indeed, deletion of the N-terminal domain of the GR did not alter the action of PKA on this receptor (11) while it dramatically abolished the action of PKA on MR. Thus, different specific proteins could interact with the amino-terminal domains of these receptors. These proteins, which could be phosphorylated via the PKA pathway, could confer specific regulation elicited by these receptors as also suggested by Lim-Tio and Fuller (22).

The interaction between the cAMP and the aldosterone signaling could have physiological consequences. As mentioned, the synergistic effects observed between vasopressin and aldosterone could, at least partially, be accounted for by the cross-talk described in this study (7, 9). Furthermore, our data provide an explanation for the interactions between ß-adrenergic receptor blockers and MR action. Indeed propanolol, which results in a decrease in cAMP levels, has been shown to alter MR signaling in kidney cell tubules (23). These observations highlight the contribution of both induced and basal levels of cAMP in aldosterone effects and provide a framework to explain some aspects of physiological and pharmacological regulation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
The human hepatoma cell line HepG2 (24) was maintained in DMEM supplemented with 10% FCS (GIBCO, Grand Island, NY), 100 U/ml penicillin, 100 µl/ml streptomycin (Diamant), and 0.5 µg/ml fungizone (Squibb). CV-1 monkey kidney cells were grown in DMEM supplemented with 10% FCS. The rabbit kidney tubule cells (RC.SV3) were isolated as described by Vandewalle et al. (25) and were grown in a medium composed of DMEM-Ham F12 (1:1) supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, 2 mM glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, 20 mM HEPES, 0.5 µg/ml fungizone, and 2% charcoal-stripped FCS.

Plasmids
The hMR expression vectors (RSV-MR) were a generous gift from Dr. R. Evans (San Diego, CA) (26). The PKA plasmid was a gift from Dr. S. McKnight (Seattle, WA) (27), and the PKI expression vector was a gift from Dr. R. Maurer (Portland, OR) (28) .The plasmid {Delta}MTV-CAT was derived from the plasmid MMTV-CAT by deletion of the sequence from position -190 to -88 of the MMTV long terminal repeat. It was a gift from R. Evans (San Diego, CA), and its construction was described by Umesono et al. (29). A HindIII site, created at the deletion site, was used as a cloning site for all the oligonucleotides used in this study. The double-stranded oligomers [GRE, (GRE)2 and (GRE)4] have 5'-extensions that are compatible with a HindIII site. However, the restriction site is lost in the recombinant plasmid. The (GRE)4 sequence was obtained by the ligation of two (GRE)2 oligonucleotides into the HindIII site of the {Delta}MTV-CAT plasmid. Sequence of GRE: strand A: 5'-AGCTGCTCAGCT GGTACA CTC CGTCCT CTACT-3', strand B: 5'-AGCTAGTAG AGGACG GAG TGTACC AGCTGAGC.3' –Sequence of (GRE)2: strand A: 5'-AGCTGCTCAGCT GGTACA CTC CGTCCT ATTATC GGTACA CTC CGTCCT ATTATCTACT-3', strand B: 5'-AGCTAGTAGATAAT AGGACG GAG TGTACC GATAAT AGGACG GAG TGTACC AGCTGAGC-3' (GRE half-sites are underlined). The GRE sequence that we used was derived from the promoter of the aspartate aminotransferase gene (30). It had the same efficiency in transcription as a consensus GRE sequence. The luciferase plasmid (SV40-Luc) was purchased from Promega (Madison, WI).

Cellular Transfection
Transfection experiments were performed as previously described (31). Briefly, 1 day before the transfection, HepG2 cells (106 cells per 10-cm dish) were seeded into the usual culture medium containing 10% FCS. Ten milliliters of fresh medium with 10% charcoal-treated serum were added to the cells 2–3 h before the transfection. The chloramphenicol acetyltransferase (CAT) plasmids (5 µg of DNA), the hMR expression vectors (1 µg and 10 ng, respectively), and the luciferase expression vector (1 µg) were introduced into the cells by the calcium phosphate coprecipitation technique followed by a glycerol shock. After the glycerol shock, 10 ml of fresh medium containing 5% charcoal-treated serum were added to the cells. Sixteen hours later, serum-free medium was added, and cells were then treated with the various hormones or 8 Br-cAMP. After an additional 24-h incubation, cells were homogenized for chloramphenicol acetyltransferase (CAT) and luciferase assays.

A similar transfection protocol was used for CV1 cells (6.105 cells per 10-cm dish) using different amounts of transfected DNA: 10 µg of CAT plasmid, 2 µg of hMR expression vector, and 10 µg of luciferase expression vector. In this case, no glycerol shock was performed. Furthermore, during the treatment with the various drugs, serum was not removed from the culture medium because it is essential for the survival of these cells.

Generation of Stable hMR-Expressing M Cells
The RC.SV3 cells originating from the rabbit kidney distal tubules immortalized by SV40 infection (25) were kindly provided by Dr. P. Ronco (Hôpital Tenon, Paris). Cells were grown in a defined medium composed of DMEM-Ham F12 (GIBCO-BRL, Gaithersburg, MD) supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, 2 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin, 20 mM HEPES, 50 nM sodium selenate, and 2% charcoal-stripped FCS. To obtain stable hMR expressing clones, pCDNA3-hMR was constructed using the XmaIII-AflII fragment (2995 bp) encompassing the full-length coding sequence of hMR inserted into the SmaI site of pcDNA3 vector after all ends were made blunt by T4 DNA polymerase treatment. Ten micrograms of expression hMR vector were transfected into RC.SV3 cells by the calcium phosphate method. The day after transfection, cells were rinsed with PBS and fed with fresh medium. The next day, the cells were divided and treated with 200 µg/ml genetecin G418 (GIBCO-BRL). Individual clones were isolated and expanded. The presence of functional hMR was subsequently tested on each clone by transiently transfecting the pFC31-Luc, which contains the MMTV promoter driving the luciferase gene together with pSVßgal (CLONTECH, Palo Alto, CA), a plasmid encoding for ß-galactosidase, used as internal transfection control. After aldosterone treatment, enzymatic activities for luciferase and ß-galactosidase were assayed as previously described (2). Among the 23 geneticin-resistant clones, 6 clones displayed an approximately 10-fold induction of luciferase activity upon aldosterone. The M clone was subsequently used for further studies. Results were standardized for transfection efficiency and expressed as the ratio of luciferase activity over ß-galactosidase activity in arbitrary units.

Luciferase Assay
Luciferase was assayed with a kit from Promega according to the manufacturer’s instructions (32). Briefly, the transfected cells were washed twice with 5 ml of calcium and magnesium-free PBS and lysed in 500 µl of Reporter Lysis Buffer 1X (Promega) for 15 min. After a 5-min centrifugation, 20 µl of the supernatant were mixed with 100 µl of luciferase assay reagent (Promega) at room temperature. The luciferase activity was measured using a luminometer 30 sec after addition of the assay reagent.

CAT Assay
CAT activity was determined by the two-phase assay developed by Neumann et al. (33). Briefly, 60 µl of cellular extract, heated at 65 C for 10 min, were incubated with 1 mM chloramphenicol, 0.5 mM acetyl CoA, and 0.5 µCi [3H]-acetyl CoA (New England Nuclear, Boston, MA; product no. NET-290 L) at 37 C for 30 min. The solution was then transferred to a minivial and layered with 4 ml of Econofluor (New England Nuclear product no. NEF 969). After vigorous mixing, the two phases were allowed to separate for at least 15 min, and the radioactivity was then counted in a scintillation counter. Under these conditions, the product of the reaction, acetylated chloramphenicol, but not unreacted acetyl-CoA, can diffuse into the Econofluor phase. For these experiments, blanks were obtained by assaying CAT activity in cells that have undergone the same treatment in the absence of a CAT plasmid.

Recombinant hMR Baculovirus Nuclear Extracts
The recombinant baculovirus AcNPV-hMR was originally described in Ref. 3 . The phMR3750 plasmid (kindly provided by Dr. Jeff Arriza), which contains the entire hMR coding sequence, was cleaved by BamHI and HindIII. The resulting 2289-bp fragment, which encodes for a N-terminal truncated hMR (Ser 352-Lys 984) was inserted into the BamHI-HindIII site of pBlueBac His A vector (InVitrogen, San Diego, CA), and the recombinant baculovirus AcNPV-NH352hMR was produced by standard procedures in Spodoptera frugiperda (Sf9) cells as previously described (3). The functional properties of recombinant full-length or 6HisNH352-truncated hMR were indistinsguishable in terms of aldosterone- binding characteristics and hetero-oligomeric structure (data not shown). Whole-cell extracts from baculovirus-infected Sf9 cells were prepared as previously described (2). Briefly, cells were rinsed twice with cold PBS and homogenized with a glass-glass Potter apparatus at 4 C in 20 mM Tris-HCl, pH 7.4, 0.6 M KCl, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 20% glycerol. The homogenates were incubated for 30 min on ice and centrifuged at 25,000 x g at 4 C. Supernatants considered as whole-cell nuclear extracts were frozen in liquid nitrogen until further use.

Expression and Purification of the N516 hMR Protein
The complementary DNA sequence encoding for the N-terminal domain of hMR was synthetized by PCR with the p3750 plasmid as template and primers as follows (sense oligonucleotide: 5'-GATCGAAGATCTATGGAGACCAAAGGCTACC-ACAGT-3' and reverse oligonucleotide BR 184§ 5'-GGTC-TCTAGCCGATCGTGATAAAG-3'. Thirty cycles were carried out with an annealing temperature of 54 C. After salt precipitation, the fragment was cleaved by BglII and EcoRI and inserted into the BglII EcoRI sites of the pTrcHis B vector (Invitrogen). The plasmid was sequenced to confirm the correct open reading frame that encodes for the N-terminal domain of hMR tagged by six histidine residues (1–516 amino acid residues). The recombinant protein was induced in transformed E. coli strain TOP10 after 4-h 1 mM isopropyl-ß-D-thiogalactoside stimulation and purified with X press system (Invitrogen) according to the manufacturer’s recommendations onto Probond resin colums after 200 mM imidazole elution. A 50–80% homogeneous approximately 54 kDa protein was observed by the purification procedure as revealed by SDS-PAGE analysis and Coomassie blue staining. This recombinant protein was further used for phosphorylation assays.

In Vitro Treatment with PKA
MR enriched-baculovirus extracts were incubated at 30 C during 30 min with various amounts of the catalytic subunit of the PKA (Sigma Product Ref: P 8289) (50 U and 100 U). The assay was performed in 50 µl of phosphorylation buffer: 1 mM EGTA, 10 mM MgCl2, 20 mM Tris-HCl, pH 7.8, and 100 µM ATP (34). The reaction was stopped by freezing, and the samples were prepared for EMSA or SDS gel electrophoresis.

EMSA
Oligonucleotides were hybridized and labeled using the Klenow fragment of DNA polymerase I. The assay was done essentially as described by Cao et al. (35). Binding reactions were carried out in 20 µl buffer containing 20 mM Tris-HCl (pH 7.8), 1 mM dithiothreitol, 1 mM EDTA, 10% (vol/vol) glycerol, 3 µg of BSA per µl, 100 mM NaCl, 0.3 ng of radiolabeled purified DNA probe, and 1 µg of dIdC. MR at the amounts indicated in the figures legends was added last. After incubation at 4 C for 30 min, the reaction mixtures were loaded on a preelectrophoresed (100 V/12 cm, 30 min) 4.5% polyacrylamide gel (acrylamide/bisacrylamide, 29:1) containing 0.25x Tris-borate-EDTA, and electrophoresis was continued for 90 min (200 V/12 cm). Gels were then dried and autoradiographed. In supershift experiments, the FD4 monoclonal antibody was incubated with the receptor during the binding reaction. The complexes and free probes were quantitiated on a Phosphorimager (Molecular Dynamics, Storm 860).

SDS Gel Electrophoresis
After phosphorylation, N516 hMR protein, hER, and casein proteins were mixed with Laemmli buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 2% glycerol, 0.006% bromophenol blue) and heated at 90 C for 10 min. The reaction mixtures were loaded on an SDS-polyacrylamide gel (0.375 M Tris-HCl, 0.1% SDS, 12% acrylamide) for 5 h (250 V/12 cm). The gel was dried and autoradiographed.


    ACKNOWLEDGMENTS
 
We would like to thank Drs. J. Hanoune, F. Pecker, S. Lotersztajn, and C. Pavoine for their helpful comments.


    FOOTNOTES
 
Address requests for reprints to: Dr. Robert Barouki, INSERM Unité 490, Centre Universitaire des Saints-Pères, 45, rue des Saints-Pères, 75270 Paris Cedex 06, France. E-mail: robert.barouki{at}biomedicale.univ-paris5.fr

C.M. is a recipient of "La Ligue Contre le Cancer" Ph.D. fellowship. This work was supported by the INSERM and the Université René Descartes (Paris V).

Received for publication June 23, 1998. Revision received October 2, 1998. Accepted for publication October 8, 1998.


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