From the Neuroscience Research Institute, University
of Ottawa, Ottawa, Ontario K1H-8M5, Canada and the
Department of
Pharmacology and Therapeutics, McGill University, Montreal,
Canada, H3G-1Y6
Received for publication, June 20, 2000, and in revised form, December 20, 2000
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ABSTRACT |
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Negative regulation of neuronal serotonin (5-HT1A)
receptor levels by glucocorticoids in vivo may contribute
to depression. Both types I (mineralocorticoid) and II (glucocorticoid)
receptors (MR and GR, respectively) participate in
corticosteroid-induced transcriptional repression of the 5-HT1A gene;
however, the precise mechanism is unclear. A direct repeat 6-base pair
glucocorticoid response element (GRE) half-site 5'-TGTCCT separated by
6 nucleotides was conserved in human, mouse, and rat 5-HT1A receptor
promoters. In SN-48 neuronal cells that express MR, GR, and 5-HT1A
receptors, deletion or inactivation of the nGRE (negative GRE)
eliminated negative regulation of the rat 5-HT1A or heterologous
promoters by corticosteroids, whereas its inclusion conferred
corticosteroid-induced inhibition to a heterologous promoter.
Bacterially expressed recombinant MR and GR preferentially bound to the
nGRE as a heterodimer, as identified in nuclear extracts of
MR/GR-transfected COS-7 cells, and with higher affinity than MR or GR
homodimers. In SN48 and COS-7 cells,
concentration-dependent coactivation of MR and GR was
required for maximal inhibitory action by corticosteroids and was
abrogated in the L501P-GR mutant lacking DNA binding activity. Corticosteroid-mediated transcriptional inhibition was greater for
MR/GR in combination than for MR or GR alone. These data represent the
first identification of an nMRE/GRE and indicate that
heterodimerization of MR and GR mediates direct corticosteroid-induced
transrepression of the 5-HT1A receptor promoter.
Adrenal corticosteroids readily enter the central nervous
system to regulate a diversity of processes that involve the
serotonergic system, including mood and emotion. For example, gene
knockout of neuronal glucocorticoid type II
(GR)1 or corticotrophin-releasing
hormone receptor genes results in decreased anxiety behaviors in mice
(1, 2). In contrast, knockout of the 5-HT1A receptor gene results in
increased anxiety (3-5). Limbic brain regions involved in mood
control, such as the hippocampus and septum, are richly
innervated by serotonergic neurons (6). Glucocorticoids exert
strong negative regulation on the serotonergic system (7-9), and the
limbic system is particularly sensitive to glucocorticoids because both
high and low affinity glucocorticoid receptor types I and II
(i.e. MR and GR) are present (10-12). The 5-HT1A receptor
plays an important dual role in the serotonergic system, because it is
expressed both postsynaptically in the limbic system and
presynaptically as the primary somatodendritic autoreceptor of
serotonergic raphe neurons (13, 14). The level of expression of
somatodendritic 5-HT1A receptors determines serotonergic tone, in part,
by inhibiting raphe firing activity and is thus implicated in mood
disorders, such as depression and generalized anxiety, that involve the
serotonergic system (11, 15, 16). Major depression has also been
associated with disruption of the hypothalamic-pituitary-adrenal axis;
as many as 50% of depressed patients fail to suppress
corticotrophin and cortisol levels upon dexamethasone challenge.
Interactions between glucocorticoids and the serotonin system may be
critical in the origin or maintenance of the depressed state.
The 5-HT1A receptor is strongly expressed in the hippocampus, septum,
and other limbic areas (17, 18) and functions as a key regulator of the
limbic system. Transcription of the 5-HT1A receptor gene is negatively
regulated by corticosteroids especially in the limbic system. In the
rat, adrenalectomy is followed by a rapid increase (within hours) in
de novo 5-HT1A RNA synthesis (as measured by nuclear run-on
assay (19)), total 5-HT1A mRNA, and binding sites. These changes
are completely suppressed by low concentrations of corticosterone that
preferentially activate MR (20-23). The extent of increase in 5-HT1A
receptor gene transcription upon adrenalectomy was most robust in the
hippocampus and septum, with smaller changes in the raphe nuclei. Using
MR- and GR-selective ligands (in rat) and gene knockout approaches (in
mouse) (24-26), MR (primarily) and GR have been implicated in negative
regulation of 5-HT1A gene expression, but the precise mechanism remains unclear.
Positive gene regulation by glucocorticoids results in an 8-12-fold
induction of transcription and is mediated by binding of GR/MR homo- or
heterodimers to a canonical GRE composed of an inverted repeat of GRE
half-sites (TGTTCT) separated by 3 nucleotides (27, 28). However,
negative regulation of gene transcription by glucocorticoids results in
40-70% inhibition of transcription and involves diverse mechanisms
(29). Direct steroid-induced repression of target gene transcription
occurs at an nGRE (e.g. proopiomelanocortin (30)). Indirect
mechanisms are mediated via interactions of glucocorticoid receptors
with a variety of other transcription factors such as Pbx and Oct-1
(31, 32), XTF (33), or c-Jun and c-Fos (34). The mechanism of
glucocorticoid action on the 5-HT1A gene was examined using deletion
constructs of the promoter to localize the site of gene repression by
corticosteroids in SN-48 cells, a septal cell line that expresses
5-HT1A receptors. Because interactions between MR and GR appear to play
an important role in glucocorticoid-mediated regulation of 5-HT1A
receptor transcription in vivo, gel shift and cotransfection
experiments were designed to assess functional interactions at the
5-HT1A promoter. Our findings indicate that a novel nGRE confers direct negative regulation by corticosteroids to the rat 5-HT1A receptor or
heterologous promoters and suggest that MR and GR form a novel head-to-tail heterodimeric complex with the nGRE to mediate
glucocorticoid-induced gene repression.
Plasmid Construction--
Previously reported DNA
fragments or constructs of the rat 5-HT1A receptor promoter (35)
(GenBankTM accession number AF217200) were cloned
into the pGL3-Basic vector (Promega, Madison, WI) to generate
luciferase reporter constructs (1519-luc, 1186-luc, 1145-luc, 1098-luc,
995-luc, 800-luc, 618-luc, and 426-luc). Point mutations were
incorporated in the 1519- and 1186-luciferase constructs to generate
1519 m and 1186 m constructs by site-specific mutagenesis
using U.S.E. Mutagenesis Kit (Amersham Pharmacia Biotech). The
six point mutations within the nGRE sequence ( Cell Culture and Transfections--
SN-48 and COS-7 cells were
maintained in Dulbecco's modified Eagle's medium (DMEM, Life
Technologies, Inc.) supplemented with 10% fetal calf serum at
37 °C in 5% CO2. SN-48 and COS-7 cells were grown to
50-60% confluence, and the medium in each plate were replaced
12 h before transfection. Calcium phosphate coprecipitation was
used for both cell lines (35). Cells were transfected with the
following amounts of normal or mutant constructs: 5-HT1A gene deletion
constructs (10 µg); 3xnGRE/SV40 (1 µg); 1xnGRE/SV40 (3 µg); 1.2 pM p6RMR (5.4 µg); 1.2 pM p6RGR (5 µg); 0.6 pM MR/GR; or as indicated. Cells were incubated with DNA
precipitates for about 10 h and then were supplemented with
charcoal-stripped, steroid-free fetal calf serum for 36 h. SN-48
cells were differentiated by a reduction of fetal calf serum
(charcoal-treated serum) to 1% v/v and the addition of 1 µM retinoic acid (39). Cells were resuspended in reporter
lysis buffer (Promega) for detection of luciferase or Recombinant Protein Expression--
pGEX-2T-X568 encoding GST-GR
fusion protein containing amino acids 407-568 of the rat GR (DNA
binding domain) was a gift from Dr. Robert Haché (41).
pGEX-2TKS-X922 encoding GST-MR fusion protein containing amino acids
105-922 of rat MR (GST-MR) was created by cloning the
NotI-HindIII fragment of p6RMR in frame into the
NotI-HindIII sites of pGEX-2TKS, modified from
pGEX-2TK (Amersham Pharmacia Biotech) by Dr. M. Ghahremani (University of Ottawa). GST-GR- or GST-MR-transformed Eschrichia coli
cells were grown at 37 °C overnight followed by induction with 1 mM isopropy-1-thio- DNase I Footprint Assay--
The 1186-luc construct was digested
with MluI, end-labeled using Klenow DNA polymerase with
[ Electrophoretic Mobility Shift Assay (EMSA)--
Complementary
39-bp oligonucleotides ( Western Blot Analysis--
Nuclear protein was extracted from
SN48 cells as described above. Western blot was performed as described
previously (42). Proteins were electrophoresed on sodium lauryl sulfate
containing 10% polyacrylamide gel. The MR or GR antibody (1/1000) was
rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.). The
secondary antibody (1/3000) and chemiluminescence procedures were
conducted according to the manufacturer (Amersham Pharmacia Biotech).
Statistical Analysis--
The statistical significance was
evaluated using a Student's t test for two-group
comparisons or by one-way analysis of variance with the Tukey test for
multiple comparisons among more than two groups.
Identification of nGRE-like Sequence in the Rat 5-HT1A Receptor
Promoter--
We have previously identified a major TATA-driven
transcriptional initiation site at MR and GR Associate with the 5-HT1A nGRE--
To determine whether
nuclear proteins interact with the putative nGRE, the DNase I
protection assay was conducted using SN-48 nuclear extracts or purified
recombinant glucocorticoid receptors (Fig.
2). DNase I digestion using nuclear extracts
revealed a pair of strongly protected segments between
To examine further the interaction of GR or MR with the nGRE, an EMSA
was conducted using double-stranded 39-bp oligonucleotides incorporating the nGRE sequence incubated with nuclear protein extracts
from SN-48 cells (Fig. 3) or with recombinant
GR and MR proteins (Fig. 4). A specific
protein complex from SN-48 nuclei associated with the nGRE and was
competed by excess unlabeled nGRE oligonucleotide but not by the
unrelated E2F sequence (Fig. 3A, lane 4) or by
the mutated nGRE sequence (data not shown). The inclusion of either
anti-GR or anti-MR antibody displaced this complex (Fig. 3A,
lanes 5 and 6). As observed by others (32, 43),
the interaction of antibody with either rat GR or MR prevented high
affinity binding to labeled nMRE/GRE (39-oligo), displacing the
DNA-protein complex rather than inducing a supershift of the complex.
By contrast, inclusion of either nonspecific antibody or preimmune
serum did not displace the complex (Fig. 3A, lanes 7 and 8). Using GR antibody against MR or MR antibody
against GR in Fig. 4, A (lane 6) and B
(lane 6), we did not find a cross-reaction of MR and GR
antibodies to each other, arguing against nonspecific actions of the
antisera. To determine directly whether MR and GR are expressed in SN48
cells, Western blots were conducted using nuclear extract from SN48
cells (Fig. 3B). In parallel samples single MR- and
GR-reactive species of 120- and 95-kDa, respectively, were detected
with comparable levels of expression as estimated by densitometry. In
Fig. 3C, the density of the protein-DNA complex was
increased in a concentration-dependent manner by prior
treatment of SN-48 cells with dexamethasone, suggesting
glucocorticoid-mediated translocation of GR/MR to the nucleus.
MR and GR Form a Heterodimeric Complex with the 5-HT1A
nGRE--
To examine whether GR or MR binds directly to the nGRE
sequence, EMSA was performed using purified recombinant GR or MR
proteins (Fig. 4). In the presence of GR or MR, single protein-DNA
complexes were detected, in agreement with evidence from DNase I
protection analysis (Fig. 2). These complexes were not present in the
absence of protein and were displaced by unlabeled nGRE (Fig. 4,
A and B) or by the addition of the appropriate
antibody to GR (Fig. 4A, lane 5) or MR (Fig.
4B, lane 5). In contrast, noncorresponding NF1
antibody or preimmune serum did not compete (Fig. 4, A and B, lanes 6 and 7). Thus GR or MR alone
can bind directly to the nGRE sequence, which is displaced by anti-GR
or anti-MR, respectively. The interaction between GR and MR at the nGRE
was examined directly by EMSA using recombinant GR and MR (Fig.
4C). Incubation of labeled nGRE with GR, MR, or MR/GR in an
equal plasmid molar ratio resulted in distinct complexes with readily
distinguishable electrophoretic mobilities, presumably representing GR
and MR receptor homodimers (predicted molecular mass = 98 and
196 kDa, respectively) and the MR/GR heterodimer (147 kDa).
Thus GR, and MR preferentially associate as a heterodimer at the nGRE,
consistent with observations that MR and GR heterodimerize with a
positive GRE (44, 45).
To assess GR/MR receptor interactions in vivo, COS-7 cells
were transfected with expression plasmids for Flag-GR alone, GST-MR alone, or both, and receptor expression was assessed by EMSA performed on nuclear extracts using the nGRE oligonucleotide (Fig.
4D). The COS-7 cells are derived from CV-1 cells, which lack
endogenous MR and GR and therefore provide a null background for
expression of transfected receptors (38). Consistent with an absence of detectable MR/GR, no gel-retarded bands were present in extracts from COS-7 cells not transfected with MR or GR plasmids (lane 9). Nuclear extracts from COS-7 cells transfected with 2 pM GST-MR, 2 pM Flag-GR, or 1 pM
each GST-MR and Flag-GR plasmids together resulted in specific
protein-DNA complexes that were supershifted with the appropriate
antibodies (anti-GST or anti-Flag). The predicted molecular masses of
these full-length recombinant receptor constructs were 140 kDa for
GST-MR and 95 kDa for Flag-GR; hence the electrophoretic mobilities of
homodimers or heterodimers were distinguishable (MR, MR/GR, and GR were
indicated by arrows 1, 2, and 3, respectively, Fig. 4D) in 4% nondenatured gel. These results
indicate that the transfected MR and GR in COS-7 cells were functional
and present at a similar density as detected by Western blot (data not
shown) and that both associated with the nGRE of the 5-HT1A receptor gene. Furthermore, anti-GST or -Flag antibody independently
supershifted binding of the nGRE-protein complex in cells transfected
with GR+MR (lanes 8, 9), indicating that the GR/MR
heterodimer forms the major species of the complex.
The binding affinity of recombinant MR, GR or MR/GR heterodimer to the
nMRE/GRE was evaluated by EMSA using increasing concentrations of
radiolabeled 39-oligo followed by quantification of bound and free oligonucleotide (Fig. 5). Scatchard
analysis revealed that the dissociation constant
(Kd) value was 0.13 × 10 MR and GR Coactivation for Glucocorticoid Transrepression at the
5-HT1A nGRE--
The relative importance of MR, GR, or both receptors
in transcriptional repression was assessed by cotransfection of MR, GR, or both expression plasmids with the 1186-luciferase 5-HT1A construct in COS-7 cells (Fig. 6A). A MR/GR
molar ratio of 1:1 was found to be optimal for glucocorticoid
transrepression (data not shown) and was used in these experiments.
Dexamethasone and aldosterone had no effect on luciferase activity of
the 1186-luc construct in the absence of MR or GR. Transrepression was
greatest upon coactivation of both MR and GR (53% inhibition), less
for MR alone (33% inhibition), and least for GR alone (26%
inhibition). The mutated nGRE (1186 m-luc) was not responsive to
corticosteroids in the presence of both MR and GR, confirming the
results obtained in SN-48 cells (Fig. 1). The greater activity (53 versus 41% inhibition) of the GR/MR combination in COS-7
(Fig. 6A) versus SN-48 cells (Fig. 1A)
may be due to higher levels of receptor expression in transfected COS-7
cells.
The activity of the nGRE to mediate glucocorticoid transrepression at a
heterologous promoter was examined by placing one to three copies of
the nGRE sequence upstream of the SV40 promoter in the pGL3 luciferase
vector (Fig. 6, B and C). The SV40 promoter was
unresponsive to corticosteroids, even in the presence of cotransfected MR and GR (data not shown). For each construct, no effect of
corticosteroids was observed in the absence of cotransfected MR or GR,
and the mutated nGRE was unresponsive to corticosteroids in the
presence of both MR and GR, as observed for the 5-HT1A promoter. In
each case, cotransfection with both MR/GR conferred significantly more corticosteroid responsiveness on the nGRE-containing constructs than
that of transfection with MR or GR alone as seen for the 5-HT1A
promoter (Fig. 6A). The presence of three copies of the nGRE
did not enhance the corticosteroid responsiveness of the SV40 promoter
(Fig. 6C), which is consistent with previous findings (30).
Interestingly, the corticosteroid responsiveness of the 5-HT1A promoter
(53% inhibition in COS-7 cells) was greater than for the SV40 promoter
(41% inhibition). These results indicate that the nGRE independently
confers glucocorticoid transrepression on a heterologous promoter, with
preferential repression activity for coactivation of MR and GR compared
with independent activation of either receptor.
To assess the importance of DNA binding in glucocorticoid action, the
mutant L501P-GR, which incorporates a mutation in the zinc finger
domain to inactivate DNA binding (37), was cotransfected with each of
the reporter constructs (Fig. 6, A-C). This mutant GR was
completely inactive in all cases, indicating that direct receptor-DNA interaction is required for glucocorticoid-induced repression at the nGRE of the 5-HT1A receptor gene.
Pharmacology and Receptor Dependence of the nGRE Response--
The
concentration dependence of Dex- or Ald-mediated dexamethasone- or
aldosterone-mediated repression of the 3xnGRE/SV40 construct in COS-7
cells cotransfected with GR (Fig.
7A) or MR (Fig. 7B)
alone, respectively. Both Dex (EC50 = 2.5 nM)
or Ald (EC50 = 0.08 nM) mediated saturable
responses at concentrations similar to those required for ligand
binding to their respective receptors. The functional importance of
coactivation of MR and GR in SN-48 and COS-7 cells was addressed
further using a pharmacological approach (Fig. 7, C and
D). Three treatment conditions were used to selectively
activate, respectively, GR alone, MR alone, or both: dexamethasone + MR
antagonist spironolactone (Dex + Spi); aldosterone (MR agonist) + GR
antagonist RU38486 (Ald + RU); or Dex + Ald. In SN-48 cells
cotransfected with 1186-luc (Fig. 7C), treatment with Dex
and Ald resulted in the greatest level of transrepression (40% of
control), whereas the other treatments produced a smaller but
significant repression. COS-7 cells cotransfected with MR and GR
plasmids in an equal molar ratio, and 3xnGRE/SV40 displayed glucocorticoid receptor selectivity similar to that in SN-48 cells (Fig. 7D). Corticosteroid-induced repression was greatest
(40% of control) upon activation of both MR and GR, whereas activation of either receptor alone displayed significant less repression activity. Thus, coactivation of GR and MR is required for optimal transrepression at the nGRE in both neuronal SN-48 cells and COS-7 cells.
A Novel nGRE Regulates the 5-HT1A Receptor Gene--
The level of
expression of the 5-HT1A receptor has been implicated in mental
illnesses such as major depression, anxiety, and related disorders. For
example, gene knockout of the 5-HT1A receptor gene results in mice with
increased anxiety-related behaviors (3-5). On the other hand,
depressed suicides show elevated levels of 5-HT1A autoreceptors
compared with nondepressed suicides (46). Dysregulation of
glucocorticoids is observed in a large proportion of depressed
patients, suggesting a link between regulation of the 5-HT1A receptor
by glucocorticoids and the etiology or maintenance of depression.
One of the most powerful regulators of 5-HT1A receptor expression in
the rat brain is corticosterone, which rapidly and completely inhibits
adrenalectomy-induced expression of the 5-HT1A receptor gene.
Transcriptional repression of the 5-HT1A receptor gene by glucocorticoids has been demonstrated in the rat hippocampus, septum,
and frontal cortex (20, 21, 23). However, the precise site of
glucocorticoid regulation at the 5-HT1A receptor gene has yet to be
clarified. We have identified a novel nGRE that mediates direct
transcriptional repression of the rat 5-HT1A receptor gene by
corticosteroids in 5-HT1A-expressing septal SN-48 or raphe RN46A
neuronal cells. The 5-HT1A promoter activity was reduced by 40-60%,
consistent with the extent of inhibition observed at other nGRE
elements (29, 31). Importantly, we demonstrate that transrepression of
the 5-HT1A promoter or heterologous promoters is mediated by the nGRE
and is transduced preferentially by MR over GR, but transrepression is
strongest upon coactivation of both receptors. Thus the nGRE of the
5-HT1A receptor gene represents the first nMRE/GRE to be identified.
The ligand- and receptor-dependent properties of this novel
nGRE correspond well with the known properties of regulation of the
5-HT1A receptor transcription in limbic areas of the brain in
vivo. In particular, both pharmacologic and gene knockout studies
have provided evidence that MR is involved in corticosteroid-induced
repression of 5-HT1A transcription. For example, low concentrations of
corticosteroids or synthetic ligands that selectively activate MR are
sufficient to reverse the adrenalectomy-induced increase in 5-HT1A
receptor level in the hippocampus (24, 25, 47). Similarly, knockout of
the murine GR gene did not greatly impair corticosterone-mediated
repression of hippocampal 5-HT1A RNA, suggesting that MR alone is
sufficient for transrepression of the 5-HT1A gene by glucocorticoids
(26).
Glucocorticoid-induced repression of the rat 5-HT1A receptor gene at
the nGRE proceeded via direct protein-DNA interaction as opposed to
indirect mechanisms involving protein-protein interactions (29). We
demonstrate that both GR and MR bind to the nGRE and that mutations in
the nGRE or the receptor that disrupt this interaction block
repression. In particular, the point mutant L501P-GR, which lacks DNA
binding capability, failed to mediate or alter GR/MR-induced transrepression. This is in contrast to an indirect mechanism proposed
for corticosterone-mediated inhibition of NF
The NF Heterodimerization of MR and GR at the nGRE--
Both MR and GR
bound directly to the 5-HT1A nGRE, and when both were present formation
of a heterodimeric complex was favored. Consistent with this
finding, the affinity for the nMRE/GRE of the MR+GR combination
was significant higher than that of GR or MR alone. Corticosteroid
repression of transcriptional activity of the rat 5-HT1A receptor gene
proceeded through a mechanism involving preferential coactivation of
both MR and GR. Cooperative interactions between MR and GR at a
positive GRE have been reported in the nervous system (54), in which MR
and GR activate transcription synergistically through heterodimer
formation. It has been proposed that glucocorticoid receptor
heterodimerization may play a crucial role in glucocorticoid action in
the brain, particularly in tissues (e.g. hippocampus) that
express MR and GR (45). The high affinity of MR for corticosterone
would allow for high sensitivity to the actions of glucocorticoids in
regions that express this receptor (55). The strong expression of
5-HT1A receptors in MR-enriched hippocampal CA1 pyramidal cells and the
cooperative interactions between MR and GR at the 5-HT1A nGRE strongly
suggest that heterodimerization is the key mechanism for inhibitory
regulation by glucocorticoids of the 5-HT1A receptor gene in the brain.
Structure and Conservation of the nGRE--
Negative
regulation of gene transcription by glucocorticoids involves repression
via an nGRE composed of one GRE half-site (56, 57) or three half-sites
(30, 58). An nGRE-like element has been found in several genes
including the following: the pro-opiomelanocortin gene (30, 59, 60),
the corticotrophin-releasing hormone gene (58), the
gonadotropin-releasing hormone gene (32), the human interleukin-1
In conclusion, we have identified a novel nMRE/GRE that demonstrates
the crucial role of MR/GR heterodimers in mediating responses to
corticosteroids, not only for transcriptional activation but also for
transcriptional repression. This is consistent with the general
importance of heterodimerization of the steroid receptor family of
nuclear receptors (44, 63). Furthermore, our results provide a
plausible mechanism for the sensitivity of the rat 5-HT1A receptor gene
to corticosteroid-induced transrepression, particularly in the
hippocampus where MR and GR are both expressed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1169 to
1148
bp) were generated using a mutagenic primer:
5'-CAGCTCCTGgaaTTcAACTTaTaCTTTATAACACGCGGC-3' (upper strand sequence
between
1176 and
1137 bp; the lowercase letters indicate base
substitutions). For construction of normal or mutant nGRE-luciferase plasmids three tandem 39-bp-containing nGREs (3xnGRE/SV40), one 39-bp-containing nGRE (1xnGRE/SV40), or the mutated nGRE were subcloned into the SmaI or MluI/XhoI
sites, respectively, of the pGL3 promoter (Promega). DNA sequencing was
used to confirm the identity and orientation of the subcloned
fragments. The p6RGR (36), p6RL501P-GR (37), and p6RMR (38)
expression plasmids, which contain full-length rat GR, L501P-GR, and
MR, respectively, were gifts from Dr. Robert Haché, Ottawa
Hospital Research Institute, Ottawa, Canada.
-galactosidase
activity as described (40) or collected for extraction of nuclear protein.
-D-galactopyranoside for
4-6 h at 23 °C. Induced bacteria were resuspended in lysis buffer
(25 mM Hepes, pH 7.9, 100 mM KCl, 20%
glycerol, 2 mM EDTA, 0.2 mM phenymethylsulfonyl fluoride) supplemented with 100 µg/ml lysozyme and incubated for 10 min at 4 °C. Cells and DNA were sheared by passage through a series
of 18-, 20-, and 25-gauge needles followed by sonication in the
presence of 0.1% Nonidet P-40 at 4 °C. Lysates were centrifuged for
10 min at 10,000 × g and the supernatants incubated
with 3 ml of glutathione-Sepharose 4B beads (Amersham Pharmacia
Biotech) for 90 min. The beads were subsequently washed four times with 10 bed volumes of lysis buffer and three times with 20 bed volumes of
60% lysis buffer containing 0.1% Nonidet P-40, eluted in glutathione elution buffer (Amersham Pharmacia Biotech), and stored at
80 °C.
For transfections, GST-MR was subcloned into the SmaI site of pcDNA3 vector, and Flag-GR was created by cloning the polymerase chain reaction product of GR in frame into the EcoRI site of
pcDNA3-Flag vector downstream from the Flag epitope.
-32P]dCTP (3000 Ci/mmol) (40), and digested with
HindIII to generate a 1186-bp fragment, which was purified
on an agarose gel. The 60-µl reaction contained: 20,000 cpm of
labeled probe, 3 µg of poly(dI-dC) (Roche Molecular
Biochemicals), nuclear extract or recombinant GST, GR, or MR in
binding buffer (20 mM Hepes, 0.2 mM EDTA, 0.2 mM EGTA, 100 mM KCl, 5 mM
MgCl2, 5% glycerol, and 2 mM dithiothreitol,
pH 7.9) (40). Following incubation samples were digested with DNase I
(Amersham Pharmacia Biotech) for 2 min at room temperature with the
addition of 15 µg of yeast tRNA as carrier. After phenol/chloroform
extraction, samples were resuspended in formamide dye solution and
electrophoresed through 8% polyacrylamide/urea gel. The total nuclear
protein for all extracts was quantitated according to the Bradford
method with bovine albumin as standard.
1180 to
1141 bp) containing the
dexamethasone-responsive element were end-labeled with
[
-32P]dATP and used in EMSA (40). Nuclear extracts or
recombinant GR and MR were preincubated at room temperature for 20 min
with or without competitor DNA or antibodies in a 20-µl
reaction containing EMSA buffer (20 mM Hepes, 0.2 mM EDTA, 0.2 mM EGTA, 100 mM KCl, 5% glycerol, and 2 mM dithiothreitol, pH 7.9), and 2 µg
of poly(dI-dC). 32P end-labeled probe (50,000 cpm) was
added and incubated for an additional 20 min at room temperature. The
reaction was separated on a 5% polyacrylamide gel at 4 °C, which
was dried and exposed to film overnight at
80 °C with an
intensifying screen.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
967 bp from the translational
initiation ATG of the rat 5-HT1A receptor gene, which is regulated by
proximal GC-rich enhancer elements and by upstream dual repressor
elements that confer neuron-specific expression (35, 40). To identify DNA elements that confer corticosteroid-mediated transcriptional repression, a series of six 5'-deletion fragments fused to a luciferase reporter gene was transfected in septal SN-48 cells differentiated to a
neuronal phenotype, and luciferase activity in the absence or presence
of 1 µM dexamethasone was measured (Fig.
1). Dexamethasone induced a 40% reduction in
luciferase activity for the 1519- or 1186-luciferase constructs, but no
significant effect was observed for any of the shorter luciferase
constructs (Fig. 1A), including 995-, 800-, and
618-luciferase (not shown). Within the dexamethasone-responsive region between positions
1186 and
1145 bp, a GRE-like sequence (
1169 to
1148 bp)
TGTCCT-nnnnnn-TGTCCT was
identified, and the 6-bp spacing between repeated 6-bp GRE half-sites
(TGTCCT) was conserved in human and mouse 5-HT1A promoters.
Mutations were incorporated to disrupt the GRE half-sites (1519m-
and 1186m-luciferase, see "Materials and Methods"), resulting in
a complete loss of corticosteroid transrepression for both constructs
(Fig. 1B). Similarly, in rat raphe RN46A cells (35), another
5-HT1A-expressing neuronal cell line, 1 µM dexamethasone
induced a 32% reduction in transcriptional activity of the
1186-luciferase; and this was absent in the mutant 1186m construct
(data not shown). Thus, the nGRE was critical for
dexamethasone-mediated negative regulation of 5-HT1A receptor gene
transcription in neuronal cells.
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Fig. 1.
Activity of the 5-HT1A receptor promoter
expressed in SN-48 cells is suppressed by dexamethasone.
A, SN-48 cells treated with dexamethasone (1 µM, 48 h) showed inhibition of transcriptional
activity (plotted as percent of vehicle control) of the rat 5-HT1A gene
5'-flanking reporter constructs. A schematic diagram of the rat 5-HT1A
receptor gene is shown on the left. The percent inhibition
by dexamethasone is shown on the right. B, loss
of glucocorticoid transrepression by mutation of the nGRE. Mutated nGRE
constructs were generated in the context of the 1519- and
1186-luciferase gene by site-specific mutagenesis (1519m and
1186m; see "Materials and Methods"). The pGL3-Basic vector was
measured to determine background activity. All measurements were
performed in triplicate in four independent experiments. Shown is the
average ± S.E. expressed as percent inhibition compared with
vehicle control. *, p < 0.02 compared with
control.
1173 and
1145 bp and corresponding to the two GRE-like half-sites (Fig.
2B, lanes 2 and 3). The interaction
between GR and MR at the nGRE was examined directly using recombinant
GR and MR (Fig. 2, A and B, lanes
4-9). Purified GR or MR or GR/MR heterodimerization protected the
same nGRE sequence observed using SN-48 nuclear extracts (Fig.
2B, lanes 2 and 3), but GST protein
did not protected this region (Fig. 2A, lanes 2 and 3), suggesting that the DNA-protein complex observed
involves a direct interaction of GR or MR at two half-sites on the nGRE
sequence.
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Fig. 2.
Association of MR and GR with an nGRE-like
element of the 5-HT1A receptor gene. DNase I protection analysis
of nGRE-like region of the rat 5-HT1A receptor gene was performed. GST
protein (A) and nuclear extracts from SN-48 cells
(B) or GST-MR or GST-GR (A and B) were
incubated with a DNA probe generated from the 1186-luc construct,
digested with DNase I (5 units/reaction. (lanes 2,
4, 6, and 8) or 10 units/reaction. (lanes
1, 3, 5, 7, and 9)), and electrophoresed
(see "Materials and Methods"). Lanes: 1, no
protein; 2 and 3, GST protein (1 µg/lane,
A) or SN-48 nuclear extract (30 µg/lane, B);
4 and 5, recombinant MR (100 ng/lane);
6 and 7, recombinant MR + GR (50/25 ng/lane);
8 and 9, recombinant GR (50 ng/lane). A
protein-DNA interaction site (vertical lines on the
right) spanned from 1173 to
1145 bp upstream
from the translation initiation ATG (the corresponding 5-HT1A gene
sequence is shown on the right in B). The
nucleotides in the box indicate consensus GRE
half-sites.
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Fig. 3.
Direct interaction of MR and GR present in
SN-48 nuclear extracts at the 5-HT1A nGRE. DNA-protein
interactions with labeled 5-HT1A nGRE were detected by EMSA.
A, detection of MR and GR in the protein-nGRE complex.
Labeled 39-bp complementary oligonucleotides corresponding to the
protected region ( 1180 to
1141; 39 oligo) were incubated
(50,000 cpm/lane) without (lane 1) or with 30 µg of
nuclear extract from SN-48 cells (lanes 2-6). A 100-fold
excess of unlabeled oligonucleotide was included as indicated: 39-mer
(cold 39, lane 3) or E2F (lane 4).
Co-incubation was with 0.4 µg of anti-MR or anti-GR or anti-NF1
antibodies (lanes 5, 6, and 7) or with
preimmune rabbit serum (lane 8), as indicated. B,
the expression of MR and GR in SN48 cells were detected by Western blot
(upper panel). Nuclear protein was extracted from SN48 cells
and electrophoresed (50 µg/lane) on sodium lauryl sulfate-containing
10% polyacrylamide gel, and MR or GR were detected in parallel with
separate rabbit polyclonal antibodies (1/1000 dilution each). The
intensity of bands was quantified by Microcomputer Imaging
Device (Imaging Research Inc., St. Catherines, Ontario, Canada). Data
represent the mean ± S.E. of three independent experiments.
C, dexamethasone enhances protein-nGRE complex formation.
Nuclear extracts (10 µg) were made from SN-48 cells pretreated with
different concentrations of dexamethasone as indicated and analyzed by
EMSA (inset). Quantification for nGRE binding data
(inset) is presented as a semi-logarithmic plot of relative
optical density of the nGRE-protein complex versus
dexamethasone concentration. Each point represents the mean ± S.E. of three independent experiments. The specific nGRE-protein
complex increased upon pretreatment with dexamethasone.
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Fig. 4.
Heterodimerization of recombinant MR and GR
with the 5-HT1A nMRE/GRE. Labeled 39-oligo ( 1180 to
1141
bp) was incubated (50,000 cpm/lane) with 2 µg of recombinant
GST-GR (A) or 4 µg of recombinant GST-MR (B) as
indicated. No bands were detected in the absence of nuclear extract
(lane 1). A single specific complex (indicated by an
arrow) was observed with either GR or MR (lane 2)
that was competed by unlabeled 39-oligo (Cold 39,
lanes 3 and 4). Recombinant proteins were
preincubated with 0.4 µg of anti-GR antibody (A, lane 5),
anti-MR antibody (B, lane 5), or anti-NF1 antibody or normal
rabbit serum (lanes 6 and 7). C,
heterodimerization of recombinant MR and GR at the 5-HT1A nGRE. Labeled
39-oligo (-1180 to
1141 bp) was incubated with equimolar GR (2 µg,
lane 2), MR (4 µg, lane 3), or both (1 µg of
GR + 2 µg of MR; lane 4) as indicated. Lane 1 was loaded with only free probe. Note that distinct shifted bands are
readily identified as indicated by arrows labeled 1,
2, and 3. D, heterodimerization of MR
and GR from nuclear extracts at the 5-HT1A nGRE. COS-7 cells, which
lack endogenous GR and MR, were transfected with 8 µg of expression
plasmids Flag-GR for GR alone (2 pmol, lanes 2 and
3), 12 µg of GST-MR for MR alone (2 pmol, lanes
4 and 5), or both (1 pmol each of MR and GR plasmids,
lanes 6-8). Nuclear proteins were extracted, incubated with
labeled 39-bp nGRE oligonucleotide, and analyzed on 4% polyacrylamide
gel. No bands were detected in the absence of nuclear extract
(lane 1) or in nuclear extracts from cells not transfected
with MR or GR (lane 9). The nGRE-protein complexes (MR,
MR/GR, and GR), indicated by arrows labeled 1, 2, and
3, respectively, were supershifted by the addition of 0.4 µg of anti-GST or anti-Flag antibody (lanes 2, 4, 7, and 8) as indicated.
9 M for GR, 0.12 × 10
9 M for MR, and 0.08 × 10
9 M for GR + MR (Fig.
5B). These Kd values are in the same
range as for GR binding to the nGRE (0.25 × 10
9 M) in the
pro-opiomelanocortin gene (30). The Kd value of GR + MR was significantly lower than that of MR or GR alone (p < 0.05). These results indicate that the binding
affinity of MR/GR heterodimer to nMRE/GRE is significantly enhanced
compared with homodimers, suggesting that MR/GR heterodimerization may play an important role in down-regulation of the 5-HT1A receptor gene.
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Fig. 5.
Saturation binding of nMRE/GRE to recombinant
GR, MR, or GR + MR combination. A, increasing amounts
of 32P-labeled 39 oligo (1-25 × 104
cpm/lane) were incubated with equimolar amounts of recombinant GST-GR
(2 µg), GST-MR (4 µg), or GST-GR + GST-MR (1 µg/2 µg) and
analyzed by EMSA. The arrows indicate the migration of the
protein-DNA complexes. B, bound and free 39-oligo
(nMRE/GRE) in panel A were quantitated by
Microcomputer Imaging Device (Imaging Research Inc.), and the
Kd of nMRE/GRE binding to GR + MR, GR, or MR was
determined by Scatchard analysis. Scatchard plot is displayed together
with the Kd values calculated from four independent
experiments.
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Fig. 6.
Coactivation of MR and GR for maximal
transrepression at the nGRE in the 5-HT1A promoter or a heterologous
promoter. The indicated reporter constructs were cotransfected
with expression plasmids MR or GR alone or MR/GR together as indicated.
The reporter constructs used were: A, 1186-luc or 1186 m-luc, constructs of the 5-HT1A gene, as indicated; B,
1 × nGRE/SV40, containing the 5-HT1A nGRE or mutant nGRE located
upstream of the SV40 promoter; and C, 3 × nGRE/SV40,
containing three tandem copies of the 5-HT1A nGRE or mutant nGRE
located upstream of the SV40 promoter (see "Materials and
Methods"). Cells were transfected with equimolar concentrations of
MR, GR, both (or neither), or DNA binding mutant L501P-GR and treated
for 48 h with 20 nM Ald, 100 nM Dex, or
both Ald and Dex, respectively. The activity of SV40 vectors
cotransfected with MR/GR was not altered by treatment with Ald and Dex
(not shown). Note that mutated nGRE or receptor (L501P-GR) failed to
mediate corticosteroid repression. Data represent percent inhibition
compared with vehicle control and are plotted as mean ± S.E. of
at least six independent transfections done with triplicate samples. *,
p < 0.05 and **, p < 0.02 compared
with control.
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Fig. 7.
Pharmacology of corticosteroid-induced
repression at the 5-HT1A nGRE indicates cooperative interactions.
A and B, concentration-dependent
repression by Ald or Dex. COS-7 cells were transfected with 3 × nGRE/SV40 and GR or MR expression plasmids and then treated with
increasing concentrations of Dex (1-1000 nM) or Ald (10 pM-10 nM) for 48 h as indicated in
A and B, respectively. The induced inhibition is
expressed as a percentage of the maximal inhibition. Each point is the
mean (±S.E.) of triplicate samples from three independent experiments.
C, pharmacology of corticosteroid-induced repression in
SN-48 cells. SN-48 cells were transfected with the 1186-luciferase
construct and treated for 48 h with vehicle (1186-luc), 100 nM dexamethasone and 100 nM spiroxatrine
(Dex + Spi), 10 nM aldosterone and 100 nM RU-486 (Ald + RU), or Dex + Ald.
D, pharmacology of corticosteroid-induced repression in
COS-7 cells. COS-7 cells were transfected with 3 × nGRE/SV40 and
both MR- and GR-expression plasmids in equal ratio. Cells were treated
for 48 h as described in C. All measurements were done
in triplicate samples and represent the average ± S.E. of three
independent experiments. Luciferase activity was normalized to
-galactosidase activity and is expressed as relative light units. *,
p < 0.05 and **, p < 0.02, in
comparison with control.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B-induced, but not
basal, transcription of the 5-HT1A receptor gene (48). In COS-1 cells,
cotransfection of 5-HT1A reporter constructs with the p50 or p65
subunit of NF
B activated transcription by over 5-fold and
was mediated by two NF
B elements located at
365 and
64 bp of the
5-HT1A receptor gene. Transcription induced by NF
B was inhibited
50% by glucocorticoids upon cotransfection of GR, but not MR, which is
different from the dependence on MR and GR observed in the hippocampus.
The NF
B-dependent indirect mechanism differed from direct transrepression at the nGRE, which suppressed basal transcription of the 5-HT1A receptor promoter in neuronal and
non-neuronal cells via activation of both MR and GR. The importance of
the NF
B-dependent mechanism was not assessed under basal
conditions or in neuronal cells (48). In the present studies,
constructs that contained both NF
B sites but lacked the nGRE element
(e.g. 1.145-, 1.098-, 995-, 800- and 618-luc constructs,
Fig. 1A) did not mediate dexamethasone-induced inhibition of
basal transcription of the 5-HT1A promoter. The results obtained with
nGRE mutants 1519 m and 1186 m also rule out participation in
corticosteroid action of an adjacent nGRE-like sequence of the 5-HT1A
promoter (at
1149 to
1134 bp). Furthermore, inclusion of
the nGRE element upstream of heterologous promoters conferred
corticosteroid-induced inhibition by both GR and MR receptors, the same
receptor preference observed for 5-HT1A gene regulation in hippocampal
neurons. Thus, direct glucocorticoid-induced transrepression at the
nGRE is likely to represent the dominant mechanism of basal regulation
of the 5-HT1A gene.
B-dependent indirect pathway of glucocorticoid
regulation may provide a complementary mechanism to regulate 5-HT1A receptor expression, especially in fibroblast or immune cells where
NF
B is implicated in the stimulation of 5-HT1A receptor expression
(49). For example, in non-neuronal Chinese hamster ovary cells, NF
B
mediates 5-HT1A agonist-induced expression of the 5-HT1A receptor (50),
regulation that is opposite to the negative regulation of the 5-HT1A
receptor gene by agonists in hippocampal neurons (51). In B and T
lymphocytes, where mitogens induce NF
B-dependent
transcriptional activation of the 5-HT1A promoter (49, 52, 53),
antagonism of NF
B action by glucocorticoids could inhibit mitogen action.
gene (61), and the bovine prolactin gene (33, 56). In the present
study, the GRE-like sequence has a repeated 6-bp GRE half-site
(TGTCCT) separated by 6 nucleotides, which is conserved
(uppercase) in both human (
1249
TGTCCT-TTgnnn-TGTCCTTTA) and
mouse (
1158 TGTCCT-ccAnnn-TGTCCTTTc)
5-HT1A receptor genes. Note that in all cases the direct repeat GRE
half-site (TGTCCT) and 6-bp gap are absolutely conserved.
Although the nGRE contains two GRE-like half-sites, suggesting the
formation of receptor dimers, the exact number of receptors involved in the present interaction needs to be elucidated by x-ray
crystallography. Nevertheless, a theoretical consideration of the
topology of GR homodimer binding to the nGRE, based on the crystal
structure of GR homodimer binding to a consensus GRE (62), suggests
that two receptors would bind in a head-to-tail conformation on the same face of the double helix. The critical parameter for this arrangement is the 6-nucleotide spacing between the two conserved GRE
half-sites, which is sufficient for a complete 360° rotation of the
two sites into alignment. The absolute conservation among 5-HT1A gene
homologues of the 6-bp spacing between two GRE half-sites suggests that
this is a crucial element of the nGRE. Indeed, elimination of 3 bp of
the 6-bp gap in the 5-HT1A nGRE to rotate the receptor dimer 180°
converts the negative GRE into a positive GRE that mediates
glucocorticoid-induced gene transcription of 3-4-fold the basal
level in SN48 or GR-transfected COS-7 cells (data not shown).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Robert Haché, University of Ottawa, for providing plasmids and experimental compounds, and Dr. M. H. Ghahremani University of Ottawa for assistance.
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Note Added in Proof |
---|
Recent evidence that MR and GR interact to form heterodimers in solution (Savory, J. G., Préfontaine, G. G., Lamprecht, C., Liao, M., Walther, R. F., Lefébvre, Y. A., and Haché, R. J. (2001) Mol. Cell Biol. 21, 781-793) may account in part for the preferred formation of heterodimers at the nGRE of the 5-HT1A receptor gene.
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FOOTNOTES |
---|
* This research was supported by the Canadian Institute of Health Research (CINR).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Fellow of the Ontario Mental Health Foundation.
¶ A CIHR M.D./Ph.D. trainee.
** Recipient of the Novartis/CIHR Michael Smith Chair in Neurosciences. To whom correspondence should be addressed. Tel.: 613-562-5800, ext. 8307; Fax: 613-562-5403; E-mail: palbert@uottawa.ca.
Published, JBC Papers in Press, February 2, 2001, DOI 10.1074/jbc.M005363200
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ABBREVIATIONS |
---|
The abbreviations used are: GR, glucocorticoid receptor; GRE, glucocorticoid response element; nGRE, negative GRE; MR, mineralocorticoid receptor; 5-HT, serotonin; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; bp, base pairs; Dex, dexamethasone; Ald, aldosterone.
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