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The Journal of Neuroscience, September 1, 1998, 18(17):6672-6680
Transcriptional Effects of Estrogen on Neuronal Neurotensin
Gene Expression Involve cAMP/Protein Kinase A-Dependent Signaling
Mechanisms
Jyoti J.
Watters1 and
Daniel M.
Dorsa1, 2
Departments of 1 Pharmacology and
2 Psychiatry and Behavioral Sciences, University of
Washington, Seattle, Washington 98195
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ABSTRACT |
Steroid hormones exert dramatic effects on neuronal expression of
genes that encode neuropeptides. Expression of the
neurotensin/neuromedin (NT/N) gene in preoptic area neurons is
dramatically enhanced by estrogen in vivo, even though
its promoter lacks palindromic estrogen response elements. We report
here that estrogen promotes transcription of this gene by interactions
with the cAMP cascade in a neuronal cell line, SK-N-SH, and in a mouse
model. In neuroblastoma cells, estrogen increases cAMP and the
phosphorylation of the cAMP response element-binding protein in a time
frame that precedes induction of NT/N gene transcription. Interference
with the cAMP/protein kinase A signal transduction cascade blocks the
ability of estrogen to elicit increases in transcription of this gene.
Furthermore, in studies performed in vivo using mice
deficient in protein kinase A, estrogen fails to induce increases in
NT/N mRNA but retains its ability to promote estrogen response
element-dependent progesterone receptor gene transcription. These data
represent the first report of a nonclassical effect of estrogen on the
expression of an endogenous estrogen-regulated neuropeptide gene
through cAMP-mediated mechanisms both in a neuroblastoma cell line and
in hypothalamic neurons. More importantly, this "cross-talk" may
represent a more generalized mechanism by which steroid hormones act
through other signal transduction cascades to regulate the expression
of other genes in the brain.
Key words:
estrogen; neurotensin; cAMP; nonclassical; gene-transcription; mouse brain; signal transduction; SK-N-SH cells
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INTRODUCTION |
Estrogen exerts many of its effects
by the well characterized mechanism of transactivation, involving
nuclear receptor dimerization and binding to consensus estrogen
response elements (EREs) (for review, see Malayer and Gorski,
1993 ). However, there is accumulating evidence to suggest that estrogen
might promote gene transcription by signaling through pathways other
than those traditionally associated with steroid hormone-induced gene
transcription. This is particularly relevant to understanding how
estrogen might influence the expression of genes whose promoters do not
contain recognizable EREs. We show here that in some cases, steroid
hormone modulation of peptidergic neurotransmission involves an ability
to influence pathways leading cAMP-dependent gene transcription, both
in vitro and in vivo.
A link between estrogen exposure and accumulation of intracellular cAMP
in uterine tissue has been suggested since the early 1960s; however,
the mechanism by which the hormone elicits this effect is still
unknown. Recent reports indicate that estrogen treatment of various
cultured peripheral cell types induces the accumulation of cAMP. The
accumulation of cAMP has been noted in MCF-7 cells, a human breast
cancer cell line, in which 1 nM 17 -estradiol induced
maximal cAMP production within 1 hr of treatment (Aronica et al.,
1994). Similar effects have been observed in human prostate cells
(Nakhla et al., 1994). Most recently, the involvement of cAMP and
protein kinase A (PKA) have been noted in estradiol-induced dendritic
spine outgrowth in cultured hippocampal neurons (Murphy and Segal,
1997). Transcriptional effects of estrogen involving cAMP-dependent
signaling, however, have not been linked previously to the expression
of a neurotransmitter gene in brain neurons.
Neurotensin/neuromedin (NT/N) is one of several neuropeptide genes
dramatically regulated by estrogen exposure in vivo
(Alexander et al., 1989a,b, 1991; Brot et al., 1993; Alexander and
Leeman, 1994; Szot and Dorsa, 1994). Neurotensin is a 13-amino acid
peptide, thought to act as a neurotransmitter or neuromodulator in the CNS (Leeman et al., 1982). It is involved in stimulation of
prolactin release and may participate in the preovulatory leutenizing
hormone surge (Alexander et al., 1989a, 1991; Alexander and Leeman,
1994). NT/N is expressed in an estrogen-dependent manner in the medial preoptic nucleus (MPON) of the rodent hypothalamus (Axelson et al.,
1992) and also in the bed nucleus of the stria terminalis (our
unpublished observations), two nuclei that are rich in nuclear estrogen
receptor protein. In the female, NT/N mRNA levels in the MPON reflect
the changes in circulating estrogen during the estrous cycle (Alexander
et al., 1989a, 1991). We have shown recently that a single dose of
estrogen administered to rats rapidly induces the persistent
phosphorylation of the cAMP response element-binding protein (CREB) in
these brain regions (Zhou et al., 1996). The promoter of the
neurotensin gene contains CREB recognition sites, in addition to other
potentially important transcription factor binding sites such as those
for AP-1 complexes and glucocorticoid receptors (Kislauskis et al.,
1988). In light of the relationship between estrogen and cAMP
generation in peripheral cells, and the fact that estrogen can induce
the phosphorylation of the CREB protein in brain regions in which the
neurotensin gene is regulated by estrogen, we have examined the
possibility that estrogen might modulate the transcription of this gene
through activation of the cAMP/PKA pathway.
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MATERIALS AND METHODS |
Animals. Female C57/BL6 mice at 6 weeks of age were
obtained from Simonsen Laboratories (Gilroy, CA), maintained on a 12 hr light/dark cycle, and housed at an American Association for the Accreditation of Laboratory Animal Care-accredited research facility. RII knock-out (Adams et al., 1997), C1
knock-out (Qi et al., 1996), and wild-type mice were used between 6 and
8 weeks of age and were treated similarly to the C57/BL6 mice. Animals
were bilaterally ovariectomized (OVX) and allowed to recover for 1 week
before hormone replacement. Mice weighed ~20-25 gm and were
intraperitoneally injected with either 1 µg of estradiol benzoate
(EB) or 100 µg of tamoxifen (T) in 100 µl of sesame oil vehicle.
Animals treated with both EB and T were given T 30 min before estrogen
exposure, and animals receiving EB or T or vehicle alone received
vehicle at the 30 min time point to ensure that all animals were
injected with equal amounts of solution at equal time periods and to
control for effects caused by the injection. Intact mice received 100 µl of sesame oil vehicle at both times. One group of RII and C
knock-out animals received 10 µg of EB daily for 3 d before killing to evaluate a very large, chronic dose of estrogen treatment. Animals were killed at the indicated times after injection by cervical dislocation. Brains were removed and immediately frozen on dry
ice. They were then sectioned by cryostat in 20-µm-thick sections and
thaw-mounted onto RNase-free positively charged slides (Fisher
Scientific, Santa Clara, CA) and frozen at  80°C before being
assayed.
In situ hybridization. In situ hybridization
was performed on slide-mounted brain sections following a processing
procedure described previously by Miller et al. (1988). Tissue slices
were hybridized with a 35S-UTP-labeled riboprobe
complementary to the coding region of the mouse NT/N gene (generously
provided by Dr. Gene Erwin, University of Colorado, Denver, CO) or with
a riboprobe complementary to the ligand-binding domain of the rat
progesterone receptor (PR; kindly provided by Dr. OK-Kyong Parke-Sarge,
Northwestern University). NT/N antisense riboprobe was synthesized as
described previously (Adams et al., 1997). Rat PR riboprobe was
synthesized as described previously (Park and Mayo, 1991). Optical
density was determined using the MicroComputer Imaging Device (MCID;
Imaging Research Inc., St. Catherine's, Ontario, Canada). Sense
riboprobe revealed no specific labeling of any brain region
(data not shown). For optical density measurements, tissue background
was subtracted from each reading, and both left and right MPON readings
from two consecutive sections from each animal were measured (bregma, 0.1 mm) (Franklin and Paxinos, 1997).
Reporter gene constructs. The NT/N promoter-reporter
construct used in our experiments is identical to that used previously (Harrison et al., 1995). The construct contains the first 216 nucleotides of the rat NT/N gene promoter, previously shown to be the
minimal fragment necessary to induce NT/N gene expression by various
agents, including cAMP (Kislauskis and Dobner, 1990). pCH110 was
purchased from Pharmacia (Uppsala, Sweden) and used as a control
against which to normalize for transfection efficiency. Dominant
negative CREB (KCREB) was provided by Dr. Richard Goodman (Vollum
Institute, Portland, OR). PKI was provided by Dr. Richard Maurer
(Vollum Institute).
Cell culture. SK-N-SH cells were obtained from the American
Type Culture Collection (Rockville, MD). Cells were grown as reported previously (Watters et al., 1997). Passages 3-9 were used for these
experiments because the use of early passages was noted to be essential
for estrogen effects. Data shown are representative experiments. Each
experiment was repeated at least five times. Procedures used for
transfection are as reported previously (Watters et al., 1997), except
N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate was the method used for transfection (Boehringer Mannheim, Indianapolis, IN). SK-N-SH cells were transfected in six-well
plates at ~80-90% confluency. Luciferase assays were done according
to the manufacturer's protocol for cell lysis and luciferase
measurement using a kit from Promega (Madison, WI). Luciferase
measurements were done in a luminometer. -Galactosidase assays were
performed in duplicate using the cell lysates used for luciferase
measurement in 96-well titer plates. The substrate o-nitrophenyl
-D-galactopyranoside (Sigma, St. Louis, MO) was in
accordance with the kit from Promega. Average normalized luciferase activity units for a representative experiment would be ~17,000 for
vehicle treatment of the NT-luciferase reporter construct, and
estrogen induction would be ~35,000 luciferase units. One microgram
each of PKI, REVAB, and KCREB constructs was added
to existing NT/pCH110 concentrations, and 1 µg of pGEM7Z was added to
wells not receiving these DNA constructs to ensure equal amounts of
DNA. Cells were treated with the various doses of water-soluble 17-estradiol (Sigma), 10 µM forskolin (Sigma), 1 µM tamoxifen (Research Biochemicals, Natick, MA), or 1 µM ICI 182,780 (Zeneca Ltd., London, England) for 8 hr.
Protein kinase inhibitors H89, bisindolylmaleimide II (BIM; Calbiochem,
San Diego, CA), and KN-62 (Research Biochemicals) were used at a final
concentration of 5 µM, and cells were pretreated for 1 hr
before stimulation. The doses of inhibitor used were the same as those
determined to block the effects of forskolin and phorbol ester on NT/N
gene transcription.
cAMP measurement. Cells for cAMP determination were grown as
for transfection and labeled for 3 hr in phenol red-free MEM (with
supplements) and 10% charcoal-stripped calf serum with
[3H]adenine (NEN, Natick, MA). After labeling,
cells were washed twice with PBS and replaced with fresh medium for 2 hr and then treated in triplicate for various times with 3 nM 17-estradiol and 200 µM
isobutylmethylxanthine (IBMX; Sigma), a phosphodiesterase inhibitor.
After estrogen treatment, medium was removed, 5% TCA containing 1 µM cold cAMP was added, and cells were allowed to precipitate overnight at 4°C. cAMP assays were performed by the method described previously (Wong et al., 1991). Actinomycin D experiments were performed as above, except cells were pretreated for
30 min with 1 µM actinomycin D before stimulation of cAMP with estrogen.
Protein kinase A activity assay. PKA kinase activity was
assayed on cell homogenates as described (Clegg et al., 1987) using Kemptide (Peninsula Laboratories, Belmont, CA) as a substrate for the enzyme. Assays were done in the presence or absence of 5 µM cAMP. Residual kinase activity measured in the
presence of 4 µg/ml PKI peptide (Sigma) was subtracted. Hypothalami
from wild-type controls, C knock-out, and RII knock-out mice were
dissected (n  3), immediately frozen on dry ice, and
stored at 80°C until the day of assay.
Western blotting. SK-N-SH cells grown as above were treated
with 200 µM IBMX and 3 nM water-soluble
17-estradiol for the times indicated or with water vehicle in the
presence of IBMX. Mice were treated intraperitoneally with 100 µg of
tamoxifen for 15 or 75 min. Nuclear extracts from MPON and SK-N-SH
cells were obtained as reported previously (Watters et al., 1996),
except 5 µM Microcystin L-R (Sigma) was added to buffers
A and B just before use. Western blots were performed as described by
Sambrook et al. (1989). A total of 10 or 15 µg of nuclear extract
protein were loaded per well, and gels were transferred to
nitrocellulose membranes (Amersham, Arlington Heights, IL). Anti-PCREB
antisera 8466 kindly provided by Dr. David Ginty (Johns Hopkins,
Baltimore, MD) and the anti-CREB antibodies (Upstate Biotechnology,
Lake Placid, NY) were used at a final dilution of 1:5000. Methods used have been reported elsewhere (Ginty et al., 1993). Bands were visualized using the ECL reagent (Amersham). PCREB immunoreactivity was
normalized to CREB immunoreactivity to control for unequal protein
loading. Bands for tamoxifen induction of PCREB in vivo were
quantitated using the MCID system, as above. Quantitation of estrogen
induction of PCREB in SK-N-SH cells was performed using the NIH Image
software program (National Institutes of Health, Bethesda, MD).
Statistics. Statistical analyses were performed using the
ANOVA pre hoc test and the Scheffe F test or
Fisher PLSD tests for post hoc significance. Significance
levels were set at 95% confidence limits. Data are represented as
means ± SEM.
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RESULTS |
Rapid effects of estrogen on NT/N expression in the MPON of mouse
brain are unresponsive to estrogen receptor antagonism
Tamoxifen is one of the few antagonists that have been previously
used to antagonize the effects of estrogen in the brain after
peripheral administration (Wade et al., 1993a,b). NT/N mRNA levels were
significantly reduced (p < 0.05) in
ovariectomized vehicle-replaced mice when compared with all other
treatments, including intact controls (Fig.
1A,B). EB rapidly
increased (within 2 hr) NT/N mRNA levels above those of ovariectomized
animals, which remained elevated when measured 8 hr after injection.
Tamoxifen alone acted agonistically to increase NT/N mRNA levels at
both the 3 and 8 hr time points. Furthermore, when administered before EB, it failed to block the effects of estrogen on NT/N gene expression. These data imply that the in vivo effects of estrogen on the
NT/N gene are not mediated by the classical mechanism of estrogen
action. Tamoxifen interferes with estrogen binding to the
ligand-binding domain of the estrogen receptor protein. Although
estrogenic activity of tamoxifen has been noted in other tissues (for
review, see Kuo and Runowicz, 1995), these effects have been attributed
to effects on estrogen receptor function that involve actions other than those mediated by estrogen receptor-ERE (Webb et al., 1995). The
inability of tamoxifen to block the effects of estrogen on this gene,
and its apparent potent agonistic activity, suggest that
cross-talk with other signaling pathways could be involved. An
alternate explanation may involve the recent finding that estrogen receptor antagonists such as tamoxifen interacting with the estrogen receptor (ER) cause transactivation at AP-1 sites (Paech et al.,
1997). This is most likely not the mechanism used here, because cotransfection of ER along with NT in our cell model system failed to augment the estrogen responsiveness of the NT reporter construct (data not shown).
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Figure 1.
A, Effect of estrogen and estrogen
antagonists on NT/N mRNA measured by in situ
hybridization in the MPON of the mouse brain. EB (1 µg) was
administered intraperitoneally to bilaterally ovariectomized
(OVX) females. Mice were pretreated with T (100 µg) for 30 min before EB exposure. Intact animals received injections
of sesame oil vehicle and were killed 8 hr after treatment. EB- and
T-treated animals were killed at the times indicated after agonist or
antagonist exposure. A, Intact; B, OVX
and vehicle-replaced; C, OVX + EB treatment for 2 hr;
D, OVX + EB treatment for 8 hr; E, OVX + T treatment for 3 hr; F, OVX + T treatment for 8 hr;
G, OVX + EB + T treatment for 8 hr. B,
Graphical depiction of NT/N mRNA autoradiograms. The
y-axis denotes optical density of MPON after subtraction
of background. n 3 animals per treatment group.
*p < 0.05 from all other treatments.
C, Western blot indicating PCREB immunoreactivity in the
MPON of mouse brain after tamoxifen exposure for 15 and 75 min. Each
lane represents 15 µg of protein from a pool of MPON
nuclear extract from three or more mice per group. Top
panel, PCREB immunoreactivity level; bottom
panel, CREB immunoreactivity level. D, Graphical
representation of the optical density of the ratio of PCREB to CREB-IR
after tamoxifen treatment of mice.
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The estrogen receptor antagonist tamoxifen induces phosphorylation
of the CREB in mouse brain
Because tamoxifen treatment was unable to block the effects of
estrogen on NT/N gene transcription and was in fact an agonist, we
investigated the ability of this estrogen receptor antagonist to induce
the phosphorylation of CREB in the MPON of mice. Tamoxifen increased
the phosphorylation of CREB within 15 min, and the increase persisted
at the 75 min time point (Fig. 1C,D). Levels of CREB protein
itself were not altered over this time course. The antisera recognizes
the phosphorylation of Ser-133 that is essential for transcriptional
activation by the CREB protein.
In vitro modeling of estrogen-induced transcription of
NT/N- and CRE-containing constructs in the SK-N-SH neural cell line
Figure 2A depicts
a dose-response curve for the effects of estrogen on an NT/N
promoter-luciferase construct transiently transfected into SK-N-SH
cells. The lowest dose to elicit a maximal effect on NT/N reporter gene
expression was 3 nM 17-estradiol. Also shown is the
effect of forskolin, a direct activator of adenylate cyclase, on the
expression of the NT/N reporter construct. It elicited a threefold to
fourfold induction of the NT/N reporter, whereas estrogen elicited
a twofold to threefold stimulation. Figure 2B
illustrates the effect of E2 on a luciferase reporter construct containing the promoter of the  subunit of the
glycoprotein hormones, 168, a gene well documented to be regulated
by cAMP-dependent mechanisms (Delegeane et al., 1987). The 168
reporter construct contains 168 nucleotides of the promoter region of
the subunit gene, which includes the cAMP-responsive elements. As
was noted with the NT/N promoter, estradiol induces the expression of
this gene at a similar concentration.
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Figure 2.
Dose-response curves depicting the effect of
17-estradiol (E2) treatment on cAMP-responsive
reporter gene constructs transiently transfected into SK-N-SH cells.
A, NT/N promoter luciferase construct; B,
the 168 luciferase construct. Forskolin was administered at a dose
of 10 µM, and vehicle-treated wells received water
vehicle.
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The effects of estrogen on cAMP accumulation are independent of
gene transcription in SK-N-SH cells
The accumulation of intracellular cAMP that was maximal at 60 min
and returned to baseline within 90 min of treatment was induced by 3 nM 17-estradiol in the presence of IBMX (Fig.
3A). Given the protracted
period of this effect, it seemed possible that estrogen could be acting
on nuclear estrogen receptors to enhance the expression of
estrogen-inducible genes that promote cAMP accumulation at 60 min.
Therefore, we performed identical experiments in the presence of the
transcription inhibitor actinomycin D. A dose of actinomycin D (1 µM), previously determined to block ERE-mediated
transcription in this cell line, was added to cells 30 min before
estrogen exposure and failed to block the increases in cAMP observed
with estrogen treatment.
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Figure 3.
A, Effect of estrogen and
actinomycin D on cAMP accumulation in SK-N-SH cells. Cells were treated
with 3 nM estrogen alone, estrogen and 1 µM
actinomycin D together, or water vehicle in the presence of 200 µM IBMX for the times indicated. Data are graphed as
percent vehicle-induced cAMP accumulation for estrogen treatment alone
or as vehicle-induced cAMP accumulation in the presence of actinomycin
D. *p < 0.05 from vehicle-treated cAMP levels.
B, Western blot depicting estrogen induction of PCREB
immunoreactivity in SK-N-SH cells. Ten micrograms of nuclear extract
protein were loaded per lane. PCREB immunoreactivity is denoted in the
top panel, and the bottom panel indicates
CREB immunoreactivity in the same cells. C, Quantitative
ratio of optical densities of PCREB to CREB immunoreactivities of
Western blot bands.
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Estrogen induces the phosphorylation of the cAMP response
element-binding protein in SK-N-SH cells
Because the peak of estrogen-induced cAMP accumulation occurred at
60 min after estrogen treatment of these cells, we reasoned that the
phosphorylation of the CREB protein should occur within 5-15 min after
activation of PKA (Fig. 3B, top panel).
Time points earlier and later were also evaluated, with no noticeable
change in phosphorylation status of the CREB protein. In contrast,
using an antibody to monitor total CREB nuclear protein, no change was evident in the amount of CREB itself (Fig. 3B, middle
panel). Figure 3B, bottom panel, illustrates the
quantitative depiction of the PCREB-to-CREB ratio of immunoreactivity
as measured by optical density. A pronounced increase in PCREB-IR was
noted between 65 and 75 min after estrogen treatment. The ratio of
phosphorylated CREB to total CREB-IR increased dramatically over this
period. This response waned by 90 min.
Effect of estrogen receptor antagonists and various protein kinase
inhibitors on NT/N gene expression in SK-N-SH cells
The estrogen receptor antagonists tamoxifen and ICI 182,780 failed to block estrogen-induced NT/N gene transcription (Fig. 4A). In fact, as
observed in vivo with tamoxifen, it and ICI 182,780 acted as
agonists of the NT/N gene response, and when coadministered elicited
the same response as estradiol itself. Figure 4B
illustrates the ability of H89, a potent and selective PKA inhibitor,
to block the transcription of the NT/N reporter gene construct induced by estrogen. H89 was also able to inhibit the induction of the NT/N
gene induced by tamoxifen. Other protein kinase inhibitors, BIM
selective for several protein kinase C isoforms, and KN-62, an
inhibitor of calmodulin kinase II, were incapable of blocking the
effect of estrogen on transcription of the NT/N construct. Together
these data suggest a selective role for PKA.
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Figure 4.
A, Effect of estrogen receptor
antagonists on estrogen-induced NT/N gene transcription in SK-N-SH
cells. NT/N gene expression is induced by 3 nM estrogen.
Pretreatment with 1 µM tamoxifen
(T) or 1 µM ICI 182,780 (I) fails to block the effects of
estrogen. One micromolar tamoxifen and I alone and both together have
agonistic activity on the NT/N gene. *p < 0.05 from all other treatments. B, Blockade of 3 nM estrogen and 1 µM T induced NT/N gene
transcription by 5 µM H89 but not by 5 µM
BIM or KN-62. *p < 0.05 from vehicle and
E2 + H89 treatments. C, Effect of dominant
negative PKA and CREB and overexpression of PKI on estrogen induction
of NT/N gene transcription in SK-N-SH cells. KCREB,
REVAB, and PKI were cotransfected into SK-N-SH cells
with the wild-type NT/N luciferase reporter construct. Vehicle-treated
cells received water, and estradiol-treated cells received 10 nM E2. Coexpression of the dominant negative
PKA subunit REVAB or of the nonactivatable form of CREB
(KCREB) inhibited the effects of estrogen on the NT/N gene.
Overexpression of the PKA peptide inhibitor also blocked the effects of
estrogen on NT/N transcription. *p < 0.05 from
vehicle treatment of NT reporter construct alone.
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Effect of dominant negative PKA and CREB and overexpression of PKI
in SK-N-SH cells on NT/N gene transcription
A dominant negative PKA construct, REVAB (Clegg et
al., 1987), a regulatory subunit that is unable to bind cAMP and
therefore is incapable of releasing free catalytic subunit, was
cotransfected along with the NT/N reporter construct into SK-N-SH
cells. The presence of REVAB inhibited the ability of
estrogen to increase NT/N gene transcription (Fig. 4C).
Additionally, a dominant negative form of CREB, KCREB (Walton et al.,
1992), in which a mutation in the DNA binding domain allows CREB
dimerization but not transactivation, also abolished estrogenic
induction of the NT/N gene. Last, overexpression of the native peptide
inhibitor of PKA, PKI (Day et al., 1989), resulted in blockade of the
effects of estrogen on the NT/N gene, further indicating the
involvement of cAMP/PKA-mediated mechanisms in the effects of estrogen
on NT/N gene expression.
Mice bearing a targeted disruption in PKA are unable to elicit
estrogen-induced increases in NT/N mRNA in the MPON
Mice bearing targeted disrupted genes for either
the regulatory type II (Brandon et al., 1998) or catalytic
1 (Qi et al., 1996) subunit of the PKA holoenzyme were
studied to assess the involvement of the PKA-dependent signaling
pathway in mediating both the chronic and acute effects of estrogen on
NT/N gene transcription in the brain. RII knock-out mice exhibit
region-specific reductions in PKA activity. The cAMP-stimulated PKA
activity in the MPON of these knock-out mice was reduced to 50% of
wild-type animals (Fig. 5A).
This was not true of C1 knock-out mice (Fig.
5A). In the chronic paradigm, wild-type,
C1 /, and RII/
mice were ovariectomized for 7 d and replaced with 10 µg of EB intraperitoneally for 3 d. In both wild-type and
C1/ mice, significant increases
in NT/N mRNA were observed after estrogen treatment, whereas in RII
knock-out mice, this response was completely absent (Fig.
5B,C). However, detectable levels of NT/N mRNA were evident
in the MPON of some intact RII knock-out intact animals.
Because it was possible that disruption of the RII gene had in some
way impaired ERE-dependent transcriptional effects involving the
estrogen receptor, we performed experiments on the RII mice to test
the ability of estrogen to induce PR mRNA expression in the MPON. PR
gene expression was examined because the role of ERE-dependent effects
of estrogen on transcription of this gene have been well documented
(Kraus et al., 1994). We did this to assure that the lack of effect we
observed on NT/N mRNA was not a result of an improperly phosphorylated
and functioning estrogen receptor. Mice were treated with 1 µg of EB,
100 µg of tamoxifen, or both for 3 hr, with tamoxifen being
administered 30 min before EB. Previous studies had shown that maximal
effects of estrogen on PR mRNA in wild-type animals are evident within 6 hr. We observed a significant increase in PR mRNA in response to EB
treatment of the RII/ mice (Fig. 5E).
Tamoxifen alone was without effect and when given together with EB
reduced the induction elicited by EB alone by ~50%. In these same
animals, NT/N mRNA was unaltered by any of these treatments (Fig.
5D), again indicating that the animals are capable of
exhibiting an ERE-mediated transcriptional response in preoptic neurons
but not one that appears to be PKA-dependent.
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Figure 5.
A, Graph depicting decreased PKA
activity levels in MPON of mouse brain. cAMP-stimulated PKA activity is
decreased by >50% in RII knock-out mice when compared with
C1 knock-out and wild-type controls. Basal PKA activity
levels are similar in both wild-type and knock-out animals.
B, Composite of coronally sliced mouse brain sections
depicting NT/N mRNA expression in the MPON of wild-type (top
panel), C1 knock-out (middle
panel), and RII knock-out (bottom
panel) mouse brains in ovariectomized
(OVX; sesame oil vehicle) and ovariectomized and
estrogen (10 µg)-replaced animals (OVX + E) for 3 d. C, Graphical
representation of NT/N mRNA optical density in the MPON of wild-type,
RII, and C1 knock-out animals. *p < 0.05 from OVX animals. D, Graphical representation of
NT/N mRNA optical density measured in the MPON of RII knock-out
animals ovariectomized and acutely treated with 1 µg of EB, 100 µg
of T, or both together for 3 hr. E, Graphical
representation of PR mRNA optical density measured in the MPON. EB
elicited a significant increase in PR mRNA in the MPON
(*p < 0.05), which was significantly reduced 50%
by pretreatment of animals with T.
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DISCUSSION |
We have used a human neuroblastoma cell line, SK-N-SH, as a model
in which to study the molecular events through which estrogen promotes
expression of the neuropeptidergic gene NT/N. In vivo, estrogen alters the expression of neurotensin as it varies throughout the estrous cycle in various estrogen receptor-rich regions of the rat
brain. The time course of estrogen-dependent induction of neurotensin
gene expression in the MPON is very rapid and occurs within 2-3 hr
after peripheral estrogen treatment.
In cultured neuroblastoma cells, estrogen drives the expression of a
luciferase NT/N promoter-luciferase construct maximally at a
concentration of 3 nM, well within the physiological range of estrogen concentrations encountered by the rodent hypothalamus, because the hormone varies throughout the estrous cycle (Bixo et al.,
1986). Consensus ERE-like elements are not present in the promoter
region fused to the luciferase reporter. We have shown previously that
tamoxifen behaves as an antagonist of the effects of estrogen on
ERE-mediated gene transcription in SK-N-SH cells both in the wild-type
state and when the human estrogen receptor (ER) is overexpressed in
these cells (Watters et al., 1997). However, tamoxifen acts as a full
agonist to induce NT/N-luciferase, which strongly suggests that the
effects of both estrogen and tamoxifen on NT/N transcription in this
cell line do not involve ERE-mediated mechanisms.
Estrogen increases NT/N-luciferase activity in these cells
approximately twofold to threefold, an effect comparable with that of
forskolin, a direct activator of adenylate cyclase. Estrogen also
elicits an increase in intracellular cAMP in these neural cells, which
peaks within 1 hr after treatment. It appears that this increase in
cAMP allows estrogen to drive the expression of the CRE-containing
promoter 168 and the NT/N promoter. The increase in cAMP induced by
estrogen was not blocked by actinomycin D, a transcription inhibitor,
indicating that although the period of estrogen activation of cAMP is
delayed, gene transcription is likely not required for the effects of
estrogen on cAMP. In MCF-7 cells, estrogen promotes an increase in cAMP
that is maximal at 1 hr. Aronica et al. (1994) have reported that these
increases in cAMP were caused not by inhibition of a phosphodiesterase
but by the activation of adenylate cyclase. Additionally, we found that
estrogen promotes the phosphorylation of the cAMP response element-binding protein in SK-N-SH cells, an event that is necessary for transcriptional activation of the CREB protein and subsequently of
promoters containing CREs. The phosphorylation of CREB can be performed
on the Ser-133 residue by several enzymes, including PKA, which appears
to be activated in these cells, by virtue of the increases in cAMP
elicited by estrogen. The time course of CREB phosphorylation we have
observed is consistent with this hypothesis.
In our study, estrogen and tamoxifen action was blocked by addition of
H89, a potent and selective PKA inhibitor, and by cotransfection of
dominant negative CREB and PKA regulatory subunit, implying a central
role for PKA and the phosphorylated form of CREB in the effects of
estrogen on the NT/N promoter. Additionally, overexpression of the
endogenous PKA peptide inhibitor PKI also blocked the effects of
estrogen on NT/N transcription.
The estrogen receptor antagonists tamoxifen and ICI 182,780 failed to
block the effects of estrogen on the NT/N reporter gene construct. In
fact, both mimic the effects of estrogen itself. These antagonists have
also been reported to increase cAMP in MCF-7 cells (Aronica et al.,
1994). In vivo, tamoxifen was unable to reverse the effects
of estrogen on NT/N gene transcription and, in addition, promoted both
the phosphorylation of the CREB protein and subsequent activation of
NT/N expression in the MPON. Tamoxifen is a partial estrogen receptor
antagonist, and in certain tissues such as the uterus, it exerts
agonistic effects (for review, see Kuo and Runowicz, 1995). The
estrogenic effects of tamoxifen that have been noted in other tissues
have been attributed to effects on estrogen receptor function that
involve actions other than those mediated by estrogen receptor-ERE
interactions (Webb et al., 1995) possibly via AP-1 sites. Tamoxifen has
been shown to block the actions of estrogen in brain, both on
estrogen-induced sexual behavior and induction of progesterone receptor
mRNA (McKenna et al., 1992). ICI 182,780 interferes with activated
hormone-receptor complex binding to DNA by preventing dimerization and
nuclear translocation. Because ICI 182,780 was unable to block the
effects of estrogen on NT/N transcription in this cell line, DNA
binding of the activated estrogen receptor also appears not to be
involved. Interestingly, overexpression of ER in these cells along
with the neurotensin-luciferase reporter construct failed to further augment the effects of estrogen on the NT/N gene (our unpublished observation). Taken together, these data suggest that ER and classic
estrogen receptor-ERE transactivation is not involved in the
transcription of the NT/N gene.
An alternative explanation is suggested by studies in MCF-7 cells
(Fujimoto and Katzenellenbogen, 1994). In these breast cancer cells,
activation of PKA reduces the antagonist activity of tamoxifen as
measured using ERE-containing reporter constructs, reducing the
antagonist effects of tamoxifen in cells in proportion to the degree to
which PKA was activated. Conversely, the agonist activity of tamoxifen
was enhanced. This effect was also shown to be promoter-specific and
was not noted in all ERE reporter constructs tested. Thus, it is
possible that by virtue of the cAMP increases induced by estrogen in
SK-N-SH cells (and perhaps in MPON neurons), PKA activation modulates
the agonist and antagonist potency of tamoxifen on transcription of the
NT/N gene. Alternatively, the estrogen receptor present in these cells
may differ from the classical estrogen receptor. This potentially novel
estrogen receptor appears to have the ability to transactivate an ERE
to a small degree, and it appears to be capable of eliciting increases
in intracellular levels of cAMP. Recently, a novel estrogen receptor, termed ER, has been cloned from the rat prostate, having significant homology in the DNA binding domain but not in the ligand binding domain
of the estrogen receptor protein (Kuiper et al., 1996). Thus, there is
evidence suggesting the existence of at least one alternate receptor
for estrogen, potentially possessing different ligand specificities. A
further possibility is that tamoxifen may be acting as an agonist
through its previously documented ability to augment transactivation
via AP-1 sites present in the promoters of certain genetic origin (Webb
et al., 1995). This interaction has been described in uterine cells but
does not appear to occur in cells of breast. In HeLa cells, Webb et al.
(1995) showed that the estrogen receptor forms a protein complex with c-Jun and c-Fos proteins and acts to facilitate
transcription of AP-1-containing reporter constructs but not of
ERE-containing constructs. A raloxifene response element (RRE) has been
elucidated recently that differs in nucleotide sequence from the ERE
and appears to support transactivation induced by metabolites of
estrogen and by the estrogen receptor antagonist raloxifene (Yang et
al., 1996). Sequence analysis of the NT/N reporter used in our studies revealed that a segment of DNA is present that exhibits 75% identity to the RRE. It is therefore possible that some of the agonist effects
of the estrogen receptor antagonists and of estrogen itself we observe
on the NT/N gene might partly be mediated through this homologous site.
Another potential explanation for how the estrogen receptor antagonists
function as agonists of the NT/N response is given by a recent report
that transactivation by ER at AP-1 sites occurs when the receptor is
complexed with ER antagonists (Paech et al, 1997). Thus, there are
several potential explanations for the agonist effect of the estrogen
receptor antagonists used in our experiments. It will be necessary to
further characterize the estrogen receptor present in SK-N-SH cells to
determine which of these mechanisms is involved in the effects noted
here. It is conceivable that the estrogen receptor present in these
cells also exists in vivo and may be responsible for the
estrogen-dependent modulation of cAMP reported in the literature for
several decades.
A role for PKA in mediating the effects of estrogen in vivo
is implied by the fact that the knock-out mice deficient in the RII
subunit of the PKA holoenzyme, but not those for the C1 subunit, lack the ability to induce NT/N gene transcription.
RII/ mice show a 50% decrease in cAMP-stimulated
PKA activity in the hypothalamus, whereas PKA activity in the same
region of CB1/ animals are similar
to the wild-type controls. Estrogen does, however, elicit increases in
PR mRNA in the RII/ animals, an effect that is
believed to be via ERE-mediated events (Savouret et al., 1991). This
strongly suggests that although the knock-out animals retain the
ability to mount a "classical" estrogen response, they are
deficient in transcriptional responses involving PKA-dependent
signaling.
Given these results, it is also possible that estrogen, or
antiestrogens such as tamoxifen, may have an effect on NT/N mRNA stability in addition to the transcriptional effects we have observed in vivo. This mechanism has been shown to be involved in the
pulsatility of luteinizing hormone-releasing hormone secretion in the
rat hypothalamus (Maurer and Wray, 1997). Although post-transcriptional mechanisms appear not to be involved in the modulation of NT/N promoter-luciferase constructs in SK-N-SH cells, this mechanism might
very well be used in addition to transcriptional mechanisms in the
intact brain. It is also possible that PKA activation by estrogen in
the brain might indirectly increase the stability of NT/N mRNA by
activating another protein that might bind to and stabilize the mRNA.
More experiments directed at determining the processes induced by
estrogen in vivo, in relation to the mRNA for NT/N, would be
needed to answer this question specifically. In a more general sense,
estrogen might affect the expression of many neuropeptide genes in the
brain using post-transcriptional modifications of their mRNAs either
solely or in addition to other mechanisms such as transcription.
Our data represent the first report of transcriptional effects of
estrogen involving cross-talk with another signal transduction pathway
in neurons both in vitro and in vivo. They
provide a possible mechanism by which estrogen is able to regulate the
expression of several neuropeptidergic genes with promoters that are
devoid of classical estrogen response elements. Both our in
vitro findings on cAMP-dependent gene transcription and the lack
of effect of estrogen in a mouse model deficient in PKA activity
provide evidence that estrogen may exert effects on the expression of
numerous target genes containing cAMP response elements. More
importantly, these data illustrate the potential importance of
cross-talk signaling as a relevant feature of steroid hormone action in
the brain.
| |
FOOTNOTES |
Received March 11, 1998; revised June 8, 1998; accepted June 12, 1998.
This work was supported by Pharmacological Sciences Training Grant
GM670489 (J.J.W.) and United States Public Health Service Grants
NS20311 and AG05136 (D.M.D.). We thank Cong Xu and Monique Adams for
their laboratory help and assistance and helpful scientific discussions. We also thank Elena Chartoff and Sherry Neher for their
help with preparation of this manuscript. Also, we are grateful to the
following individuals for providing reagents: Dr. Paul Dobner for the
NT-luciferase construct, Dr. Richard Goodman for the KCREB construct,
Dr. Richard Maurer for the PKI construct, Dr. Gene Erwin for the mouse
NT/N riboprobe plasmid, and Dr. David Ginty for the 8466 anti-PCREB
antibody. Additionally, we thank Zeneca Ltd. for donating the ICI
182,780 compound used in our experiments and Dr. Daniel Storm for
generously allowing us to perform the cAMP assays in his laboratory.
Last, we thank Dr. Stanley McKnight, Rejean Idzerda, and Eugene Brandon
for so generously providing the PKA knock-out mice that were used in
our studies and for performing the PKA assay on the hypothalami of
these mice.
Correspondence should be addressed to Dr. Daniel M. Dorsa, Department
of Pharmacology and Psychiatry, University of Washington, Box 356560, Seattle, WA 98195.
| |
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C. N. Rudick and C. S. Woolley
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Top
Abstract
Introduction
