ABSTRACT. We have studied the effects of progesterone on the transcription of the mineralocorticoid receptor. (MR) gene in neurons in vitro and in uiuo.
001372271951$03.0010 Endocrmology Copynght 0 1995 by The Endocrine
Regulation Expression
Vol. 136, No. 9 Prmted in U.S.A. Society
of Rat Mineralocorticoid Receptor in Neurons by Progesterone
MAIJA CASTRtiN”, VLADIMIR K. PATCHEV, FLORIAN HOLSBOER, THORSTEN TRAPP,
OSBORNE F. X. ALMEIDA, EERO CASTRkN
AND
Department of Neuroendocrinology (M.C., V.K.P., O.F.X.A., F.H., T.T.), Max-Planck Institute of Psychiatry, Clinical Institute, 80804 Munich, Germany; A. I. Virtanen Institute (E.C.), University Kuopio, FIN-7021 1 Kuopio, Finland
ABSTRACT We have studied the effects of progesterone on the transcription of the mineralocorticoid receptor (MR) gene in neurons in vitro and in uiuo. Progesterone treatment caused a 2.5fold increase in activity of the MR promoter in transiently transfected N2A neuroblastoma cells. Similarly, MR promoter activity in GH, pituitary cells was increased 2-fold after treatment with the specific progesterone receptor agonist R5020, with an even greater induction after priming with 17P-estradiol. Progesterone treatment also produced a dose-dependent increase in MR messenger RNA (mRNA) levels in primary hippocampal neuron cultures. In uiuo, chronic administration of progesterone to estrogen-primed adrenalectomized/ovariectomized rats significantly increased MR mRNA levels in all hippocampal subfields, as
T
WO DISTINCT types of corticosteroid receptors, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR), mediate the effects of adrenal steroids in the central nervous system (1,2). The MR, which regulates electrolyte balance in response to mineralocorticoids in kidney, binds the glucocorticoid corticosterone with a IO-fold higher affinity than GR in rat hippocampus (3). In the rat brain, MR is expressed predominantly in neurons of limbic structures, most notably in the hippocampus. GR, which is ubiquitous, is also expressed at high levels in the hippocampus (4). The hippocampal corticosteroid receptors are involved in the regulation of several neuroendocrine, behavioral, and cognitive functions (2,5). MR and GR also mediate the negative feedback of adrenal corticosteroids on the limbit-hypothalamo-pituitary-adrenal-(LHPA) axis (1,6). Decreased levels of corticosteroid receptors in the hippocampus have been associated with impaired glucocorticoid feedback, resulting in hyperactivation of the LHPA-axis during chronic stress and aging (1,7,8). Corticosteroid and progesterone receptors (PR) share extensive homology in their amino acid sequences and represent a distinct subfamily within the steroid receptor family (9). After binding of the cognate ligand, these receptors produce their genomic effects by interacting with the same, or similar, DNA sequences in the promoter regions of hormone responsive genes (10,ll). In contrast to MR and GR, only low-to-moderate levels of PR have been Received January 30, 1995. Address all correspondence and requests for reprints to: Dr. Maija Castrkn, Department of Pediatrics, Kuopio University Hospital, P.O. Box 1777, FIN-70211 Kuopio, Finland. * Supported by a fellowship from the Academy of Finland.
of
determined by semiquantitative in situ hybridization histochemistry. Whereas chronic estradiol treatment decreased MR mRNA levels in the hippocampus, progesterone administration in the absence of estradiol priming was without any effect. These results indicate that 1) progesterone increases MR mRNA levels in uitro and in uivo; 2) the stimulatory effects of progesterone are at least partially mediated by induction of MR promoter activity; and 3) estrogen priming is essential for the effect of progesterone upon MR mRNA in viuo. Further, they suggest the possibility of heterologous regulation of corticosteroid receptors in the brain, whereby the responsiveness of the limbichypothaiamo-pituitary-adrenal system to- corticosteroids may be modulated. (Endocrinology 136: 3800-3806, 1995)
found in the rat hippocampus (12). Nevertheless, the likelihood of interactions between corticosteroids and progesterone at the receptor and/or promoter level, warrants a closer examination of the effects of progesterone on corticosteroid receptor expression. The expression of the rat MR (rMR) gene is controlled by at least three promoters, and at least three messenger RNA (mRNA) subtypes differing in their 5’-untranslated regions have been demonstrated (13,14). We have previously characterized a functional promoter for the rat MR (13). This promoter controls the expression of the a-form of MR transcript, which appears to be the only MR subtype that is increased after adrenalectomy (ADX) and down-regulated by corticosteroid replacement in viva (14). We have shown that the activity of this promoter is up-regulated by corticosteroids in vitro, with the involvement of several weak elements (15). Heterologous regulation of MR levels in the brain by hormones other than adrenal steroids is less well understood. MR concentrations have been shown to vary during development, in aging, and after pharmacological modifications of transmitter systems in the brain (4). In addition, sex differences in glucocorticoid binding, as well as in MR and GR mRNA levels, have been reported in the rat brain (16-18). Estrogen administration to gonadectomized rats decreased both MR and GR mRNA expression in the hippocampus and was shown to interfere with glucocorticoid receptor function (19). Progesterone has been shown to have no effect on the regulation of GR mRNA expression (20); however, a recent report suggested that progesterone may influence binding of the ligand to MR (21).
3800
REGULATION
OF MR GENE
In the present study, we investigated the effects of progesterone on the MR gene expression in neurons and pituitary cells. We show that progestins induce MR promoter activity in N2A and GH, cells and that this induction is enhanced by estrogen priming. The transcriptional activation of the MR gene by progesterone was demonstrated as a dose-dependent increase in steady-state MR mRNA levels in primary cultures of hippocampal neurons. Finally, we document that progesterone treatment increases MR mRNA levels in the hippocampus of adrenalectomized/ovariectomized (ADX/OVX) female rats when estradiol treatment precedes progesterone administration. Materials
and Methods
Plasmids The reporter plasmids pMR5’-1500 and pMR5’-520 were created by subcloning HirrdIII/NhrI fragments from reporter constructs pMRLUC5’-1500 and pMRLUC5’-635 (13) into pGL-Basic vector (Promega Corp., Madison, WI). The SocI site of the vector and the Hind111 ends of the inserts were blunt-ended with T, DNA polymerase, and the subcloning was performed after N/ICI digestion of the vector and inserts. The size of the promoter sequence in the plasmid pMR5’-1500 is estimated by gel electrophoresis, whereas the sequence of the insert in pMR5’-520 has been characterized by sequencing. The PstI cloning site in the plasmid pMR5’-520 is included to the promoter sequence. The construction of an expression vector for cPR,, has previously been described (22). The control plasmid pMR/St!/I for PCR was created by deleting a St!/1 fragment of 207 base pairs (bp) from the rMR complementary DNA1 (cDNA1) containing plasmid (13).
Cell cultures
and transient
expression
analysis
Mouse neuroblastoma (Neuro 2A) and rat pituitary (GHJ cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) under an atmosphere of 95% 0, and 5% CO,. Estrogen priming of GH, cells was performed by addition of 10 nM 17a-estradiol (Sigma Chemical Co., St. Louis, MO) to the medium for 48 h before transfections. N2A cells were transiently transfected by the CaPO, precipitation method as described previously (13). Ten micrograms of reporter plasmid, 2.5 wg of cPR, expression plasmid, pGEM4 as a carrier DNA (Promega Corp., Madison, WI), and 5 pg pCHll0, a plasmid containing P-galactosidase gene downstream of a Simian virus 40 early promoter (Pharmacia, LKB, Freiburg, Germany), were coprecipitated with calcium phosphate in a total volume of 1 ml. The precipitate was added to the medium of four subconfluent (-30%) plates. After 16 h the cells were washed with 1X PBS, and the medium was replaced with phenol red-free DMEM supplemented with 10% charcoalstripped steroid-free FCS (9). Progesterone (100 nM) (Sigma) was added, and cells were grown for additional 24 h before harvesting. Transfections into GH, cells were performed using electroporation (Biotechnologies and Experimental Research Inc., San Diego, CA), after determination of the electric field strength (23); 5 pg of reporter plasmid and 5 pg of carrier plasmid were cotransfected with 5 Kg of pCHll0. Electroporated cells were replated in phenol red-free DMEM supplemented with 10% charcoal-stripped steroid-free FCS and incubated with R5020 (24) and /or ZK 98,299 (Onapristone; Ref. 25). After 24 h, cells were harvested, extracts in 0.1 M potassium phosphate and 1 mM dithiothreitol (DTT) (50 ~1) were prepared by freeze-thawing, and luciferase activity assays were performed (26). All results were corrected for f3-galactosidase activity (271, which was used to monitor transfection efficiency.
Hippocampal
primary
cultures
and RNA preparation
Neurons were prepared from hippocampi of 17.day-old rat embryos and cultured in a serum-free medium as described previously (28). After 7 days in culture, the medium was replaced by DMEM supplemented with 10%) charcoal-stripped steroid-free FCS, and the incubation was continued
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for an additional 24 h in the presence of different concentrations of progesterone. Total cellular RNA from hippocampal neurons (0.5 X 10h cells) was isolated by a modification of the method of Chomzcynski and Sacchi (28).
cDNA
synthesis
and PCR amplification
A quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to measure the levels of MR mRNA in neurons (28). PCR primers (5’.TTC AGG CTG CTC AGA GGA AG-3’ and 5’-CTC AAG CTT CCT TGT TGG TTC-3’) were targeted to the 5’-region t-198 bp to +386 bp, Fig. 1) of the rMR cDNA1 (13). Total RNA was reversetranscribed together with an irz vitro transcribed RNA from a shortened MR control plasmid (pMR/StyI; see Plnmids). The reverse transcription was performed in a volume of 10 ~1 in a reaction buffer containing 60 mM KCI, 15 mM Tris, 3 mM MgCl,, 0.3% Tween, 1 mM dNTP, 0.01 M DTT, 1.25 U RNase inhibitor, 50 pmol random hexanucleotides (Promega) and 100 U Moloney Murine Leukemia Virus reverse transcriptase (Pharmacia LKB). The reaction mix was subjected to PCR in a volume of 100 ~1 containing 10 ~1 Tq DNA polymerase 10X buffer (Promega) [9.5 M KCl, 100 mM Tris-HCl (pH 9.0 at 25 C) and 1% Triton X-1001,1.25 mM MgCl,, 0.375 mM of each deoxynucleotide triphosphate, 1.2 FM of each forward and reverse primer, and 2.5 U of Tnq polymerase (Promega). PCR conditions were: 1 min denaturation at 94 C, 1 min annealing at 58 C and 2 nun extension at 72C. Twenty-five cycles were used based on the preliminary studies, which demonstrated that 5 fg of MR RNA standard is amplified within the linear part of the concentration curve when 25-30 cycles were used in these conditions (see the concentration curve in Ref. 15). Negative control without template was included to each PCRexperiment. The specificity of the amplification products was confirmed by Southern blotting and hybridization with rMR cDNA probe. The amplification products (15 ~1 samples) were run on a 1.5% agarose gel and blotted onto Hybond N plus membranes (Amersham); the filters were hybridized with a rMR cRNA probe complementary to the N-terminal EcoRI fragment of the rMR cDNA. The filters were prehybridized for 4 h at 42 C in hybridization buffer [SO% formamide, 50 mM NaH,PO,, 0.5% sodium dodecyl sulfate (SDS), 5 mM EDTA, 3 X SSC (1 X SSC is 0.15 M NaCl, 0.015 M sodium citrate), 5 X Denhardt’s solution (0.5% Ficoll, 0.5% polyvinylpyrrolidone, 0.5% BSA) and 250 Fg/ml salmon sperm DNA] and hybridized for 20 h at 42 C. The membranes were washed at 42 C in 2 X SSC, 0.1% SDS for 10 min, and at 68 C in 0.2 X SSC, 0.1% SDS for 30 min, before exposure to Kodak x-ray film in cassettes with intensifying screens. The resulting autoradiograms were analyzed by a laser scanning device (LKB, Bromma, Sweden). Results were normalized to the internal control.
Animal
surgery,
treatment
and tissue collection
Adult female Wistar rats (Max-Planck Institute; Martinsried, Germany) weighing 220-250 g were housed in groups of five under controlled illumination (12-h light, 12-h dark) and had free access to food and water. After 1 week of acclimation, the rats were bilaterally ADX and/or OVX under barbiturate anesthesia (Brevimytal; Lilly, Bad Hom-
5’ I promoter
cj---I
n
\/
584
w
377
b bp
FIG. 1. Schematic illustration of the rMR promoter region upstream of the first untranslated exon of the rMR gene and the primer sets used for RT-PCR experiments. The first untranslated exon is represented as a white box, coding exons as shadowed boxes, and the primers as dark boxes. The arrow indicates the translational start site. The amplification product of 584 bp represents amplification of the specific MR mRNA product; the product of 377 bp is the internal standard amplified from the shortened MR RNA.
REGULATION OF MR GENE BY PROGESTERONE
3802
burg, Germany; 30 mg/kg BW). Sham-operated controls received lumbar skin incisions. Five days after surgery, ADX/OVX rats were treated for 5 consecutive days with either estradiol benzoate (E,; 20 pg/rat daily), or progesterone (I’, 1 mg/rat daily). These steroids were also applied in a sequential fashion to a separate group of ADX/OVX rats (E, + I’): E, was injected for 3 consecutive days followed by 2 days of progesterone treatment in the doses mentioned above. Steroids were purchased from Sigma and dissolved in corn oil. Groups of vehicle-treated sham-operated (0, ADX, OVX and ADX/OVX rats were used for comparisons. The ovarian cycles in sham-operated and ADX rats were monitored by daily vaginal smears; these rats were killed at the first documented diestrus, but not earlier than 5 days after surgery. Rats were killed by decapitation between 0900 and 1000 hours. Brains were rapidly removed from the skull, snap-frozen in prechilled isopentane, and stored at -70 C until cryosectioning. All animal procedures were carried out in compliance with local animal welfare laws and NIH Guidelines on the Care and Use of Laboratory Animals.
In situ hybridization In situ hybridization was performed on 12. pm-thick frozen coronal sections. The sections were thaw-mounted on Probe-coated glass slides (Fisher Scientific, Pittsburgh, PA) so that one section from each experimental group was mounted on each slide to reduce variation. The sections were postfixed in 4% buffered paraformaldehyde and permealized with acetic anhydride (0.25% in 0.1 M triethanolamine buffer, pH 8.0). Dehydrated sections were hybridized overnight at 60 C in a buffer containing 50% formamide, 0.3 M NaCl, 2 mM Tris (pH 8.0), 1 mM EDTA sodium salt, 1 X Denhardt’s solution, 500 kg/ml yeast transfer RNA, 10% dextran sulfate, and 10 mM DTT. Antisense and sense (control) probes were transcribed in vitro in the presence of 35S-UTP using T7 and Sp6 RNA polymerases (Boehringer, Mannheim, Germany), respectively, to a specific activity of approximately 10’ cpm/pg and were used at a concentration of lo4 cpm/pl. The sections were washed four times for 15 min each in 1 X SSC at room temperature, treated with RNase A (20 yg/ml) and then washed four times for 15 min in 0.1 X SSC at 60 C. Dehydrated sections were dried and exposed to Hyperfilm /3-max (Amersham) for 6 days together with a set of 14C-labeled radioactive standards (American Radioactive Chemicals, St. Louis, MO). Selected sections were exposed to NTB-2 autoradiographic emulsion (Kodak, Rochester, NY) for 4 weeks. X-ray films were scanned (Hewlett-Packard, Palo Alto, CA) and analyzed by the NIH Image 1.52 program (Research Services Branch, NIMH, Bethesda, MD). Effects of steroid manipulations on MR mRNA levels were analyzed in the following hippocampal subfields: CAl-2, CA3 proximal (CA3prox), CA3 distal (CA3dist) and dentate gyrus. Optical densities were converted to nCi/mg of tissue using a curve fitting program based on readings from coexposed radioactive standards.
Statistical
Endo. Vol 136.
1995 No 9
pared with that of the promoterless construct (Fig. 2). A 5’-deletion to -297 bp did not alter the stimulation by progesterone. The induction of the MR promoter activity by progesterone in Neuro 2A cells was detected only when the expression vector of cPR, (22) was cotransfected. No induction of the promoter activity by progesterone was detected when expressionvectors of MR or GR were cotransfected (data not shown). When GH, cells,which expressPR (291,were used in transient transfection experiments, a 2-fold induction of promoter activity by the synthetic specific PR agonist R5020(24) was detected without additional cotransfection of PR. As shown in Fig. 3, the induction of MR promoter activity by 10nM progesteronewas enhancedwhen the cells were pretreated for 48 h with estradiol; the latter isknown to increasethe expression of PR in these cells (29). In addition, the increasein promoter activity by progesterone treatment could be prevented by ZK 98,299 (100 nM), a progesterone antagonist considered to be devoid of antiglucocorticoid activity (25) (Fig. 3). Increase cultures
in MR mRNA of hippocampal
levels by progesterone neurons
in primary
Total RNA was prepared from primary hippocampal neurons after incubation for 24 h in the absenceor presence of progesterone in different concentrations. The regulation of MR mRNA levels by progesterone was investigated using a quantitative polymerase chain reaction method (RT-PCR). A shortened MR RNA standard (Fig. 1) was used to control the amplification efficiency and to verify that the amplification product derived from hippocampal MR mRNA corresponds
analysis
Statistical analysis of the effects of progesterone on MR promoter activity was performed using Student’s t test for unpaired samples. One-way analysis of variance, combined with the Tukey test, was used to evaluate the effects of hormone treatments on MR mRNA levels. The level of significance was set at P < 0.05.
100 -1500
% of control 200
300
ILUC I -520 I
ILUCl -297 ILUCl
Results Regulation
of MR promoter
by progesterone
Our previous studies have shown that the sequence upstream of the first untranslated exon of the rMR gene (Fig. 1) exerts promoter activity in transient transfection assays(13). The basal activity of this promoter was induced by progesterone (100 nM) in mouse neuroblastoma (Neuro 2A) cells. The activities of constructs pMR5’-1500 and pMR5’-520 containing promoter fragments of 1500 bp and 520 bp, respectively, were both increased approximately 2.5-fold as com-
IRSV
ILUCl
FIG. 2. MR promoter activity and its responsiveness to progesterone treatment in N2A cells. The N2A neuroblastoma cells were cotransfected with reporter plasmids pMR5’-1500, pMR5’-520 or pMR5’-297 containing promoter sequences in front of the luciferase gene as described in Materials and Methods together with expression plasmid for cPR,. After transfection, cells were incubated with progesterone (100 nM) or vehicle for 24 h. Promoter activity was expressed as light units per unit of galactosidase activity, and the stimulation by progesterone is shown as percentual change in promoter activity following progesterone treatment us. promoter activity without treatment. The values show the mean Z SD of three independent experiments.
REGULATION
3803
OF MR GENE BY PROGESTERONE
amino terminal part of the common protein coding region in different types of MR transcripts were used (data not shown). Regulation of hippocampal progesterone in vivo
q q -I-
control +R5020 +E2/R5020 +R5020/ ZK 98299
FIG. 3. Effects of progestins on the rMR promoter activity in GH, cells. The rMR reporter plasmid pMR5’-520 was transfected in GH, cells by electroporation, and the luciferase activity was measured in cell extracts after treatment with synthetic progestin R5020 without estrogen priming (+R5020), R5020 with estrogen priming (+Ed R.5020) and R5020 together with antagonist ZK98.299 or vehicle. Promoter activity was expressed as light units per unit of galactosidase activity. Treatment-induced changes are presented as percent of control promoter activity. *, Significant increase U’ < 0.05) with respect to control. #, Significant decrease (P < 0.05) with respect to treatment with R.5020 without estrogen priming. 250 -
MR mRNA
levels by
A cDNA clone (13) containing the amino terminal part of the rat MR cDNA was used to synthesize 35S-labeledcRNA probes for hybridization analysis of MR mRNA expression in the brain. The distribution of MR expression in the hippocampus appeared to be identical to that obtained with probes containing the 3’ untranslated region described previously (Fig. 5; Ref. 4,30,31). MR were abundantly expressed in all hippocampal subfields, the hybridization signal being weaker in the distal CA3 area, as compared to other subfields. In situ hybridization with the sense probe did not reveal any signal above background (data not shown). Removal of the endogenous gonadal steroids by OVX did not significantly affect MR mRNA levels in any hippocampal subfield studied (Fig. 6). The combination of ADX and OVX significantly increased MR mRNA levels in all hippocampal subfields (Figs. 5 and 6). Estradiol replacement in ADX/OVX animals significantly decreased MR transcripts in subfields CAl-2 and CA3dist (Fig. 6). Progesterone treatment alone for 5 days after ADX/OVX did not produce any significant changesin MR mRNA levels, although a slight increasein the CA3prox region was observed (Fig. 6). However, when ADX/OVX animals were treated with estradiol for 3 consecutive days before progesterone administration, a sig-
200 z s g0
150
% *
100
50
I
0
I
-10
#
-9
I
-8
I
-7
I
-6
LOG [STEROID] FIG. 4. Dose-dependent regulation of MR mRNA levels in primary hippocampal neurons. MR mRNA levels were determined by RTPCR after incubation of cultures for 24 h in the absence or presence of different concentrations of progesterone. The figure shows results from a representative experiment that was repeated three times.
to the (Y form of MR mRNA (Fig. 1). As shown in Fig. 4, progesterone treatment caused a dose-dependent increasein MR mRNA levels. The effect of progesterone was clearly distinguishable at a concentration of 1 nM, reaching a plateau between 0.1 and 1 PM concentrations (Fig. 4). Similar increase in MR mRNA levels was seenwhen primers amplifying the
FIG. 5. Localization of MR mRNA in hippocampal subfields. A, Photomicrograph illustrating the distribution of MR mRNA in the dorsal hippocampus of sham-operated female rats; B, ADx/OVX caused a significant increase in MR mRNA levels in all hippocampal subfields; C, progesterone administration to ADx/OVX animals after estrogen priming significantly increased the MR mRNA levels in all hippocampal subfields above those seen in ADX/OVX vehicle-treated rats. CA1-3, Hippocampal fields CA13; DG, dentate gyrus. Magnification 12x.
REGULATION
OF MR GENE
’ () qn/iL)x/ovx ADX/OVX
o,x
I
q
+E2
ADX/OVX H ADX/OVX
+PROG +E? +PROG
CA l-2
CA3prox
CA?dist
Hippocampal
subfield
DC
FIG. 6. Quantification of MR mRNA levels in hippocampal sections from sham-operated (n = 51, ADX (n = 41, OVX (n = 5), ADX/OVX (n = 4), ADx/OVX + E,-treated (n = 4), ADx/OVX + P-treated (n = 51, and ADX/OVX + E, + P-treated (n = 5) rats. Data are expressed as group means -t SEM. *‘, Significant differences between ADx/OVX and sham-operated animals; #, significant changes induced by steroid hormone supplementation as compared with ADX/OVX vehicletreated rats.
nificant increase in MR mRNA levels above those in vehicletreated rats was detected in all hippocampal subfields (Figs. 5 and 6). Discussion The present study demonstrates the transcriptional induction of MR gene expression by progesterone. Progesterone itself, as well as the synthetic PR agonist R5020, was shown to induce the activity of a MR promoter in neuroblastoma and pituitary cells, and this induction was further enhanced by estrogen priming. Progesterone increased the levels of the (Y form of MR mRNA controlled by this promoter in primary cultures of hippocampal neurons in vitro; when estradiol treatment preceded the progesterone administration in viva an increase in MR mRNA levels in the hippocampus of ADX/OVX female rats was also demonstrated. Progesterone directly activated the MR promoter, indicating that the progesterone-induced increase in MR mRNA expression is transcriptional, although an additional effect of progesterone on MR mRNA stability is possible. The 2.5-fold induction of the MR gene activity by progesterone is in the same range as the 2-fold induction of the uteroferrin gene in human placental cell line (33). The effects of progesterone were shown to require PR expression and were not mediated via MR or GR in transient transfection assays. Similarly, progesterone has been shown to induce only low transcriptional response via MR and GR on the mouse mammary tumor virus promoter compared with the response via PR, although binding of progesterone to corticosteroid receptors was demonstrated (34). It has been proposed that the structure of the ligand-binding domain of MR and GR in com-
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Endo . 1995 V
