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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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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mammary tumor virus-GRE (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. 1
). 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 MTV-GRE-CAT vector without the
hMR expression vector (Fig. 1). 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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MTV
promoter, yielding the plasmids GRE-, (GRE)2-, and (GRE)4-MTV-CAT,
respectively. As shown in Fig. 2A, aldosterone activated these promoters 10-, 35-, and 66-fold
respectively, but had no effect on the MTV promoter itself (Fig. 2A). 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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), 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. 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 MTV-GRE-CAT plasmid and the hMR
expression vector (Fig. 3A). 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. 3B). These cells were transiently
transfected by a 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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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. 4A). 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. 2C).
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). 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 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 5 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. 5 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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). The GRE-hMR complexes and free
probes were quantified on a PhosphorImager (Molecular Dynamics,
Sunnyvale, CA) (Fig. 6B). As shown in Fig. 6A, 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. 7, 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. 7B). 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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). 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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-32P ATP and PKA (100 U) as described in Materials
and Methods. The data presented in Fig. 9 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 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
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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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M.-C. Zennaro, A. Souque, S. Viengchareun, E. Poisson, and M. Lombes A New Human MR Splice Variant Is a Ligand-Independent Transactivator Modulating Corticosteroid Action Mol. Endocrinol., September 1, 2001; 15(9): 1586 - 1598. [Abstract] [Full Text] [PDF]
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M. Nishi, H. Ogawa, T. Ito, K.-I. Matsuda, and M. Kawata Dynamic Changes in Subcellular Localization of Mineralocorticoid Receptor in Living Cells: In Comparison with Glucocorticoid Receptor using Dual-Color Labeling with Green Fluorescent Protein Spectral Variants Mol. Endocrinol., July 1, 2001; 15(7): 1077 - 1092. [Abstract] [Full Text] [PDF]
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 B. G. Rowan, N. Garrison, N. L. Weigel, and B. W. O'Malley 8-Bromo-Cyclic AMP Induces Phosphorylation of Two Sites in SRC-1 That Facilitate Ligand-Independent Activation of the Chicken Progesterone Receptor and Are Critical for Functional Cooperation between SRC-1 and CREB Binding Protein Mol. Cell. Biol., December 1, 2000; 20(23): 8720 - 8730. [Abstract] [Full Text]
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G. Lazennec, L. Canaple, D. Saugy, and W. Wahli Activation of Peroxisome Proliferator-Activated Receptors (PPARs) by Their Ligands and Protein Kinase A Activators Mol. Endocrinol., December 1, 2000; 14(12): 1962 - 1975. [Abstract] [Full Text]
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 E. Falkenstein, H.-C. Tillmann, M. Christ, M. Feuring, and M. Wehling Multiple Actions of Steroid Hormones---A Focus on Rapid, Nongenomic Effects Pharmacol. Rev., December 1, 2000; 52(4): 513 - 556. [Abstract] [Full Text] [PDF]
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 M. D. Sadar and M. E. Gleave Ligand-independent Activation of the Androgen Receptor by the Differentiation Agent Butyrate in Human Prostate Cancer Cells Cancer Res., October 1, 2000; 60(20): 5825 - 5831. [Abstract] [Full Text]
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K. J. Greenland, I. Jantke, S. Jenatschke, K. E. Bracken, C. Vinson, and B. Gellersen The Human NAD+-Dependent 15-Hydroxyprostaglandin Dehydrogenase Gene Promoter Is Controlled by Ets and Activating Protein-1 Transcription Factors and Progesterone Endocrinology, February 1, 2000; 141(2): 581 - 597. [Abstract] [Full Text] [PDF]
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) 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.
, mol wt = 22,000; casein ß, mol wt = 24,000;
and casein-
, mol wt = 19,000).