From the Department of Pharmacology, University of Washington, Seattle, Washington 98195-7280
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ABSTRACT |
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A program of stringently-regulated gene
expression is thought to be a fundamental component of the circadian
clock. Although recent work has implicated a role for
E-box-dependent transcription in circadian rhythmicity, the
contribution of other enhancer elements has yet to be assessed. Here,
we report that cells of the suprachiasmatic nuclei (SCN) exhibit a
prominent circadian oscillation in cAMP response element (CRE)-mediated
gene expression. Maximal reporter gene expression occurred from
late-subjective night to mid-subjective day. Cycling of
CRE-dependent transcription was not observed in other brain
regions, including the supraoptic nucleus and piriform cortex. Levels
of the phospho-active form of the transcription factor CREB (P-CREB)
varied as a function of circadian time. Peak P-CREB levels occurred
during the mid- to late-subjective night. Furthermore, photic
stimulation during the subjective night, but not during the subjective
day, triggered a marked increase in CRE-mediated gene expression in the
SCN. Reporter gene experiments showed that activation of the p44/42
mitogen-activated protein kinase signaling cascade is required for
Ca2+-dependent stimulation of CRE-mediated
transcription in the SCN. These findings reveal the CREB/CRE
transcriptional pathway to be circadian-regulated within the SCN, and
raise the possibility that this pathway provides signaling information
essential for normal clock function.
In mammals, the suprachiasmatic nuclei
(SCN)1 of the hypothalamus
contain a circadian oscillator that functions as the major biological
clock (1-3). The biorhythm generated by the SCN allows an organism to
predict and coordinate its daily physiological processes to an
approximate 24-h period. If the SCN are lesioned, there is a loss of
physiological and behavioral circadian rhythms (4, 5). The most
effective regulator of the endogenous clock is light; endogenous clock
rhythmicity is entrained to the environmental light cycle by photic
cues conveyed from the eyes to the SCN via the retinohypothalamic tract
(RHT) (6). For example, if an animal receives a light flash during the
dark phase of the day/night light cycle, the circadian rhythm is reset,
or phase-shifted (7). Light-induced phase-shifting results from the
synaptic release of glutamate from the RHT onto the SCN (8-10).
Entrainment of the clock by light is thought to involve changes in gene
expression. In support of this, several studies have shown that
immediate early gene induction is triggered by light (11-13).
Recent work has revealed important information about specific proteins
and transcriptional events essential for circadian rhythmicity. For
example, CLOCK and BMAL1 proteins heterodimerize to form a
transcription factor that binds the E-box enhancer element, resulting
in mper1 gene expression (14). In Drosophila,
PER/TIM dimers negatively regulate CLOCKdependent
transcription (15), thus forming a negative feedback loop. Mutations of
any of these genes disrupts circadian rhythmicity (16-21), suggesting
that this transcriptional loop is essential for normal clock function.
Given these results, it is likely that the E-box enhancer element
regulates the rhythmic expression of other genes within the SCN.
However, the transcriptional activation of several circadian-regulated genes (vasopressin, brain-derived neurotrophic factor, Fos; Refs. 22-24) requires complex interactions of several different classes of
enhancer elements (25-27), suggesting the involvement of different transcription pathways in circadian gene regulation within the SCN. The
elucidation of these pathways will provide valuable insight into the
series of coordinated transcriptional events underlying circadian rhythmicity.
Ostensibly, transcriptional pathways that contribute to SCN rhythmicity
should have the capacity to integrate signaling information from a
variety of stimuli, as well as possess properties that allow for its
stringent regulation. One candidate is the CREB/CRE transcriptional
pathway. This pathway has been shown to be activated by multiple
kinases, including protein kinase A (PKA),
Ca2+/calmodulin-dependent kinase, and
mitogen-activated protein kinase (MAPK) (28-31). The CREB/CRE
transcriptional pathway also has the capacity to integrate the
activation of multiple signaling pathways and the strength of signal
into striking variations in downstream gene transcription (32-35).
Furthermore, CRE-mediated transcription can be rapidly repressed
through a myriad of mechanisms, including inducible early cAMP
repressor induction, phosphatase activation, or as a result of CREB
heterodimerization with inhibitory transcription factors (32, 36).
These unique functional properties led us to explore whether this
transcriptional pathway plays a role in circadian rhythmicity. Toward
this end, we used a mouse CRE- Treatments--
A description of the generation of the
CRE-
For immunohistochemical analysis of CRE/ Tissue Collection--
Cervical dislocation followed by
decapitation allowed for the rapid removal of the brain, which was
immediately placed in ice-cold oxygenated Dulbecco's modified Eagle's
medium (pH 7.4) and cut into 400-µm coronal sections with a
vibratome. For P-CREB experiments, animals were sacrificed and their
brains were removed under dim red illumination < 10 lux. For
immunohistochemistry, sections were placed in a 6%
formaldehyde/phosphate-buffered saline (PBS) solution for 4-6 h at
room temperature. Sections were then cryoprotected with 30% sucrose
for at least 12 h. Thin (35-40 µm) sections were cut through
the SCN using a sliding microtome. For Western analysis, 400-µm
coronal brain sections were quick-frozen onto glass coverslips. The SCN
and lateral hypothalamus were then excised with the use of a dissecting
microscope. Tissue was stored at Immunohistochemistry--
For
For immunolabeling against the Ser-133 phosphorylated form of CREB,
free-floating sections were initially blocked as described above,
except that PBST also contained NaF (1 mM). Tissue was then
incubated overnight with phospho-specific CREB antibody (1:500, New
England Biolabs). On Western blots, this affinity-purified antibody
specifically recognizes the phosphorylated form of CREB and the
phosphorylated forms of CREB-related proteins ATF-1 and CREM. The
tissue was then incubated for 6 h with a fluorescein-conjugated secondary antibody raised in goat and directed against rabbit IgG (4 µg/ml final dilution, Cappel), then stained with an
Alexa-488-conjugated anti-fluorescein antibody raised in rabbit (2 µg/ml final dilution, Molecular Probes). Data obtained with this
technique were confirmed by an alternative technique that employed a
biotin-linked secondary antibody directed against the IgG domain of the
P-CREB antibody, followed by the administration of a
streptavidin-conjugated lissamine-rhodamine fluorophore. Although
results with this technique were consistent with those obtained using
the Alexa-488 tertiary antibody, we noticed a high level of nonspecific
labeling. Trial experiments showed that the extraordinary sensitivity
of the ABC immunostaining technique (Vector Laboratories) did not allow
for subtle differences in the levels of P-CREB immunoreactivity to be
observed. All samples were processed concurrently.
For immunolabeling against map-2, tissue was incubated with a mouse
monoclonal anti-map-2 antibody (1:1000 final dilution, Sigma), then
labeled with Cy-2-conjugated goat anti-mouse IgG secondary antibody (1 µg/ml final dilution, Jackson Laboratories). After each antibody
treatment, the tissue was washed six times (>5 min/wash) in PBST.
Tissue was mounted using Gelmount (Biomedia). For nuclear staining,
tissue was labeled with the DNA-specific dye syto-13 (1 nM
final dilution, Molecular Probes) for 15 min, then washed six times in
PBST.
Data Collection and Analysis--
Images of the sections were
collected using a Bio-Rad MRC-600 scanning laser confocal microscope
equipped with a krypton-argon laser. SCN images were collected at a
1.2× amplification using a 10× Nikon objective. Data analysis was
performed using Metamorph software (Universal Imaging).
Semiquantitative densitometric analysis of immunoreactivity was used to
quantify endogenous rhythmicity of CRE-regulated reporter gene
expression and P-CREB rhythmicity. A 140 (x axis) × 200 (y axis) pixel oval was placed over the digitized SCN image
to determine mean fluorescent intensity. A basal level of background
fluorescence was determined for each section by acquiring a mean
fluorescent signal from the lateral hypothalamic area. This value was
then subtracted from the value for the corresponding SCN. Imaging and
data analysis were performed "blind." The amplitude of the signal
was determined by dividing the peak CT fluorescent intensity signal
from the trough CT fluorescent intensity signal. An analogous procedure
was used for analysis of other nuclei. For light flash experiments,
conventional counting was used to determine the number of
immunoreactive cells. The sum of immunopositive cells from three
central SCN sections was averaged and expressed as the total number of
immunoreactive cells per nuclei. To identify maximal variations in
signal intensity, the sensitivity of the confocal microscope was set
differently for image collection from endogenous rhythm experiments as
compared with light stimulation experiments.
Tissue Culture--
Initially, whole neonatal rat brain was cut
into 400-500-µm coronal slices using a tissue chopper. A polished
22-gauge needle was then used to punch out the SCN. The tissue was
immediately placed in sterile dissociation medium (DM: 90 mM Na2SO4, 30 mM K2SO4, 16 mM MgCl2,
0.25 mM CaCl2, 32 mM HEPES, 0.01%
phenol red, 1 mM kynurenic acid (Sigma), pH 7.7), washed
three times, and then finely minced. Next, the tissue was incubated for
30 min in DM containing 100 units of papain latex (Worthington) and 4.5 mg of cysteine (Sigma) at 37 °C. After removal of the proteolytic solution, the tissue was washed in standard tissue culture medium (minimal essential medium (Life Technologies, Inc.), 5% fetal bovine
serum, 100 units/ml penicillin/streptomycin, and 6 g/liter glucose),
then triturated into a single cell suspension. Cells were then
pelleted, washed twice, then plated in 48-well dishes (Costar) coated
with high molecular mass (>540 kDa, Sigma) poly-D-lysine. Cells cultures were maintained at 37 °C and 5% CO2 in a
Napco 6100 incubator. Cytosine arabinofuranoside (8 µM)
was added to the tissue culture medium on day 2. For Western analysis,
the region containing the SCN was excised from postnatal day 1 rat brain. Using the optic chiasm for orientation, an ~1-mm cube of tissue was removed. Tissue was washed, triturated, and plated as
described above.
Transfections--
SCN cells (1 × 104
cells/well) were transfected with DOSPER (Roche Molecular
Biochemicals). On day 5 in culture, cells were treated with a complex
of 0.6 µg of DNA and 6 µl of DOSPER, as described by the
manufacturer, in 100 µl of minimal essential medium. After 6 h,
the DNA-DOSPER complex was replaced with conditioned tissue culture
medium. In some experiments pcDNA3.1-LacZ (Invitrogen) was added (4 ng/well) for transfection efficiency normalization. Transfection
efficiency typically did not vary by more than 10% for quadruplicate
determinations. The following plasmids have been described previously:
dominant-negative MEK S222A (37), dominant-negative PKA, and the
CRE-luciferase construct (38).
Reporter Assays--
After removal of media, 150 µl of cell
lysis buffer (0.2% Triton X-100, 4 mM ATP, 6 mM MgCl2, 100 mM potassium
phosphate, pH 7.8) was added to each well. Following one freeze/thaw
cycle, luciferase activity was measured, as described in Ref. 39, and Western Blots--
Excised brain tissue was resuspended in 100 µl of buffer H (50 mM Circadian Oscillation in CRE-mediated Gene Expression--
If the
CRE enhancer element plays a role in endogenous clock rhythmicity, then
its regulation must fulfill several criteria. In the absence of photic
cues, gene expression mediated by activation at the CRE must by
regulated in a rhythmic manner. Furthermore, this rhythmicity should be
observed within the SCN, and have a period close to 24 h. To
address whether CRE-mediated gene expression shows circadian
oscillations in the SCN, animals transgenic for the
CRE-
Circadian regulation of CRE-mediated transcription was also analyzed by
Western blot. As above, transgenic mice were sacrificed over the
circadian cycle. Within the SCN, the expression of the CRE-driven
One necessary step proximal to the induction of CRE-mediated gene
expression is the phosphorylation of CREB at Ser-133. Given the
circadian oscillation in CRE-mediated transcription, one might expect
circadian variations in the phosphorylation state of CREB in the SCN.
To address this question, coronal SCN sections from mice sacrificed at
different circadian times were labeled for P-CREB immunoreactivity
(Fig. 3A). As with
CRE-mediated gene expression, levels of P-CREB immunoreactivity
exhibited significant (analysis of variance: p < 0.005, F = 4.33) circadian oscillations under free-running conditions (Fig. 3B). Maximal P-CREB signal was
observed from CT 18-22. Levels of P-CREB declined during subjective
day, then began to rise with onset of subjective night. As with
CRE-mediated reporter gene expression, P-CREB rhythmicity occurred
throughout the dorso-ventral axis of the SCN.
Photic Activation of CRE-dependent
Transcription--
The ability of light to phase-delay the clock and
overt rhythmicity during early-subjective night, to phase-advance the
clock during late-subjective night, and to have no effect during
subjective day has been well characterized (7). These phase-shifting
effects of light result from the synaptic release of glutamate from the RHT and the subsequent activation of ionotropic glutamate receptors in
the SCN (8-10). Within the SCN, photic stimulation during subjective night also induces the expression of a number of immediate early genes
(11-13). Although these data suggest a connection between photic
stimulation, glutamate release, gene induction, and phase regulation,
the inducible transcriptional pathways activated have not been
determined. A clue to the transcriptional pathways that may be critical
for phase-shifting was provided by the finding that photic stimulation
triggers phosphorylation of CREB at Ser-133 (40). Although suggestive,
this observation does not show that CRE-induced transcription occurred,
since CREB phosphorylation at Ser-133 is necessary, but not sufficient
to trigger CRE-mediated transcription (32, 41-45). To assess the
inducibility of CRE-dependent transcription, reporter mice
were entrained to a 12-h L/D cycle, then allowed to free-run under D/D
conditions for 5 days. Animals were then exposed to light (400 lux) of
varying durations during either the subjective day or the subjective
night, and sacrificed 8 h later for analysis of CRE-mediated
transcription. A 60-min light exposure at CT 16.5, a time at which
light phase-delays activity rhythms (7), elicited a robust increase in
CRE-mediated gene expression relative to control animals not exposed to
light (Fig. 4, A and
B). The induction of CRE-mediated transcription was
primarily localized to the retinoreceptive, ventrolateral region of the
SCN. A 60-min light exposure at CT 22.5 (a time point shown to
phase-advance activity rhythms; Ref. 7), also triggered CRE-mediated
gene expression (Fig. 4, A and B). A
densitometric comparison of the ventrolateral SCN revealed that light
induced an ~4-fold increase in CRE-mediated gene expression relative
to peak levels driven by the endogenous clock under D/D conditions. To
assess whether light-mediated gene expression was regulated in a
phase-restricted manner, animals were exposed to light during the
subjective day. A 60-min light treatment during mid-subjective day (CT
6) did not significantly increase CRE-mediated gene expression (Fig. 4,
A and B).
If CRE-dependent transcription plays a critical role in the
phase-shifting effects of light, then very brief light treatments known
to phase-shift the clock should also trigger CRE-mediated gene
expression. To address this question, mice were exposed to light for
only 5 min during subjective night. Compared with control transgenic
mice, light treatment increased CRE-mediated gene expression in a
highly localized band of cells within the retinoreceptive region of the
SCN (Fig. 4C).
Signaling to the CRE--
To assess the contribution of different
signaling pathways to activation of CRE-mediated transcription, we
cultured SCN neurons from P1 rat and transfected them with a
CRE-regulated reporter construct. Neurons obtained from a coronal punch
of the SCN (Fig. 5A) stained
positive for the neuron-specific antigen MAP-2 (Fig. 5B) and
were readily transfected (Fig. 5C). Treatment with the adenylyl cyclase activator forskolin (5 µM) triggered an
~6-8-fold increase in CRE-mediated transcription (Fig.
5D). K+ (50 mM) or NMDA (50 µM, plus glycine: 2 µM) elicited an ~ 2-4-fold increase in CRE-mediated transcription (Fig.
5D). Ca2+ influx resulting from
depolarization-mediated opening of voltage-activated ion channels is
the likely mechanism for K+ stimulation of CRE-mediated
transcription since administration of the voltage-activated
Ca2+ channel blocker isradipine (2 µM)
blocked CRE-dependent transcription evoked by high
K+ (data not shown). Likewise, NMDA stimulation of
CRE-mediated transcription is very likely initiated by Ca2+
influx.
Identification of signaling pathways that couple to
CRE-dependent transcription in the SCN should provide
mechanistic insights as to how phase-regulated transcriptional
activation is conferred. The MAPK (28), the
calmodulin-dependent protein kinase (30), and PKA (29)
signaling pathways couple receptor stimulation to
CRE-dependent transcription. To assess the contribution of the MAPK cascade, SCN neurons were cotransfected with the CRE-reporter and a dominant-negative interfering form of MEK, an upstream activator of MAPK. This completely inhibited K+- and NMDA-stimulated
gene expression (Fig. 5E). Similar results were obtained
with PD 98059, a specific inhibitor of MEK (data not shown).
Forskolin-activation of CRE-dependent transcription was
slightly reduced by disruption of the MAPK signaling pathway. Co-expression of the dominant-negative regulatory subunit of PKA (D/N
PKA) blocked Ca2+ as well as cAMP-dependent
transcription (Fig. 5E). These results suggest that MAPK and
PKA signaling are both required for
Ca2+-dependent CRE-regulated transcription in
SCN cells.
Immunoblotting cultured SCN cell extracts showed that potassium (40 mM) depolarization as well as forskolin (5 µM) triggered CREB phosphorylation at Ser-133 (Fig.
6A: lane
K, potassium; lane F, forskolin;
lane C, control). In addition, glutamate (20 µM) administration elicited CREB phosphorylation
(lane G, glutamate). This suggests that either
Ca2+ influx or cAMP can trigger CREB phosphorylation in the
SCN. Pretreatment with the specific MEK inhibitor, PD 98059 (75 µM), markedly attenuated potassium stimulation of P-CREB,
as well as reducing forskolin- and glutamate-evoked CREB
phosphorylation. Equal amounts of extract were also probed for CREB
expression. Cell extracts were also probed with an antibody that
detects the activated (dually phosphorylated Thr-202 and Tyr-204) form
of the extracellular signal-regulated kinases erk-1 and erk-2 (here,
collectively referred to as ERK). All three agonists triggered robust
ERK phosphorylation (Fig. 6B). Pretreatment with PD 98059 blocked agonist-induced ERK phosphorylation.
Pairing of Ca2+ and cAMP signals resulted in robust,
synergistic, activation of CRE-mediated transcription in the SCN.
Specifically, the coadministration of forskolin (5 µM)
and K+ (50 mM) triggered an ~3-fold
potentiation of reporter expression (Fig. 5F). To determine
whether circadian-regulated genes are modulated in a similar manner, we
assessed transcriptional activation driven by the CRE-containing
vasoactive intestinal peptide promoter. Vasoactive intestinal peptide
is found in high levels in SCN (46) and has been shown to regulate
clock rhythmicity (47). Stimulation with either forskolin (5 µM) or K+ (50 mM) elicited
similar levels of reporter expression as the CRE construct (Fig.
5F). Pairing of forskolin and K+ synergistically
stimulated reporter expression to a level that was similar to that
observed with the CRE-driven reporter (Fig. 5F). Together,
these results reveal that Ca2+- and
cAMP-dependent signaling pathways act with robust synergy to potentiate CRE-dependent transcription in SCN cells.
Since Ca2+ can activate the MAPK and PKA pathways, it may
function as a signal integrator for regulation of SCN function.
The results presented here show that the SCN exhibit a prominent
circadian oscillation in CRE-mediated gene expression in dark-adapted
animals. Furthermore, photic stimulation during the subjective night
triggered CRE-dependent transcription, whereas light
treatment during the subjective day was not effective. Transient transfection experiment revealed that the ERK/MAPK pathway activity is
essential for Ca2+-dependent stimulation of
CRE-mediated transcription in SCN cells. Together, these results
provide the first evidence linking the CREB/CRE transcriptional pathway
to endogenous timing mechanisms.
Photic Stimulation of CRE-mediated Transcription--
There is a
large body of evidence suggesting that the phase-shifting effects of
light result from new protein synthesis. However, the enhancer elements
mediating transcriptional activation have not been thoroughly
characterized. Our data suggest that the CRE may play a central role in
the ability of light to activate gene expression and, in turn,
phase-shift the clock. In support of this, we show that light induces
CRE-mediated gene expression. Additionally, the finding that a brief
5-min light treatment triggers a highly localized induction of
CRE-mediated gene expression correlates with a duration of light shown
to phase-shift overt activity rhythms (11). The general pattern of
CRE-mediated gene expression, both temporally and anatomically,
parallels the induction of several immediate early genes thought to be
involved in phase-shifting the clock (11-13). Given that the promoters
for these immediate early genes (including c-fos,
junB, and NGFI-B) contain at least one CRE
(48-50), it is reasonable to hypothesize that the CRE may play a
central role in mediating the ability of light to trigger immediate
early gene induction. Our results showing that light-induces CRE-regulated gene expression in a phase-restricted manner are consistent with work showing that light-induced CREB phosphorylation is
restricted to the subjective night (40). These results suggest that
stringent regulation of the CREB/CRE-transcriptional pathway during the
day may be a critical element that confers the phase-restricted phase-shifting effects of light.
Signaling Pathways--
A role for Ca2+ in photic
entrainment of the clock has been suggested by the finding that
light-induced phase-shifts require NMDA receptor activation (8). Given
the evidence identifying a transcriptional component to light-induced
phase shifts, we assessed the signaling mechanisms that couple
Ca2+ to gene expression in the SCN. In primary cultures of
SCN neurons, we found that increasing cytosolic Ca2+,
either through high K+ or NMDA administration, resulted in
enhanced CRE-dependent transcription. In addition, the
co-activation of Ca2+ and cAMP pathways resulted in a
robust synergistic activation of CRE-dependent
transcription. Interestingly, besides glutamate, RHT nerve terminals
also express PACAP (51), a peptide capable of stimulating cAMP
production. Light-induced release of transmitters capable of
stimulating Ca2+ and cAMP pathways may be important for
robust activation of the CREB/CRE transcriptional pathway in the SCN.
Along these lines, we have observed that modest stimulation of cAMP
signaling pathways that, alone, was unable to increase CRE-mediated
transcription, potently augmented
Ca2+-dependent CRE-mediated transcription in
the SCN.2
CRE-mediated gene expression is regulated by a variety of cellular
stimuli acting through a number of different kinase cascades (28-31).
Our work shows that Ca2+ stimulation of CRE-mediated
transcription was dependent upon activation of the MAPK signaling
pathway. Cotransfection with a dominant-negative interfering form of
MEK or treatment with the MEK inhibitor PD 98059 blocked
Ca2+-stimulated gene expression. Coupling of
Ca2+ to activation of the MAPK cascade has been shown to be
dependent upon an enhancement of Ras-GTPase catalytic activity (52).
Ca2+-dependent Ras activation is triggered by a
variety of signaling intermediates, including calmodulin kinases (42,
53), Src (54), Ras-GRF (55), and the epidermal growth factor receptor (56). The MAPK pathway gains access to the nucleus via the
activation-dependent nuclear translocation of ERK. ERK has
been shown to triggered CREB phosphorylation through activation of RSK
1, 2, and 3, all of which are CREB kinases (57). A requirement for MAPK
activity was also revealed by the observation that
Ca2+-induced CREB phosphorylation was attenuated by the MEK
inhibitor PD 98059. In addition, forskolin-stimulated CREB
phosphorylation was reduced by PD 98059, indicating that the
cAMP-dependent signaling pathway acts, in part, via
activation of the MAPK cascade. Along these lines, cAMP has been shown
to activate the MAPK cascade in hippocampal and cortical neurons
(31).
Within the SCN, elevated cytosolic Ca2+ has been shown to
trigger CREB phosphorylation through a mechanism requiring the
production of nitric oxide (58). Recently,
Ca2+-dependent nitric oxide activation was
shown to elicit ERK phosphorylation in neuronal cultures (59), thus
providing a pathway by which light-induced nitric oxide production
could trigger sequential MAPK activation. Although other transcription
factors may be activated by increased cytosolic Ca2+, it is
intriguing to note that nitric oxide synthetase antagonism blocks both
glutamate-induced CREB-phosphorylation and glutamate-induced phase-shifts (58, 60). This suggests a strong correlation between clock
phase-shifting and the activation of a transcriptional pathway involved
in triggering CRE-mediated gene expression. Taken together, the results
presented here provide a mechanism by which glutamate receptor
stimulation leads to CRE-dependent transcription.
Endogenous Rhythmicity--
Rhythmic transcription appears to be
central to maintaining circadian timekeeping. For example, a mutated
form of the putative transcription factor CLOCK abolishes circadian
activity rhythms under D/D conditions (16). Our data show that
CRE-mediated gene expression is regulated in a circadian manner under
free-running conditions. Levels of reporter protein began to rise
during the late subjective night and peaked during mid-subjective day.
Given the approximately 6 h between transcription and maximal
reporter expression, one may deduce that induction of CRE-mediated gene expression is restricted to the subjective night, and possibly the
early subjective morning. This result indicates that the
phase-dependent regulation of endogenous CRE-mediated gene
rhythmicity overlaps with the phase dependence of light inducible
CRE-mediated gene expression, suggesting that a similar signaling
mechanism may govern both processes.
It is unclear why the light-evoked stimulation of CRE-mediated gene
expression was greater than the peak in reporter expression resulting
from endogenous pacemaker activity. Possible explanations may include
more robust activation of signaling pathways by light or synergism
between signaling pathways that are activated by light and the
endogenous clock. Interestingly, we recently reported that light
triggers MAPK activation in the SCN, and that MAPK activity is
regulated in a circadian manner in the SCN under D/D conditions
(61).
Recent work performed in Drosophila has revealed a robust
circadian oscillation in CRE-mediated gene expression and an
interdependence between rhythmic CRE-dependent
transcription and period oscillations, indicating that the
CRE transcriptional pathway is a component of the circadian clock (74).
Further work in mammalian systems may reveal a similar interaction
between the CRE transcriptional pathway and period homolog rhythmicity.
Circadian variations in the phosphorylation state of the transcription
factor CREB at Ser-133 were also observed. The peak in the P-CREB
rhythm preceded the reporter gene peak by approximately 6 h, an
expected time lag for transcriptional activation and maximal protein
expression. The circadian P-CREB rhythm does not result from CREB
oscillations, since levels of CREB in the SCN were stable at subjective
day versus night time points. This result suggests that
circadian oscillations in P-CREB result from circadian fluctuations in
the activation state of CREB kinases or phosphatases.
It is unclear how rhythmic CRE-mediated gene expression is maintained
under free-running conditions. However there are several plausible
explanations. Given that extracellular membrane receptor-mediated signaling events regulate CRE-mediated gene expression, one may expect
to observe circadian changes in the level of extracellular transmitters
capable of eliciting CRE-mediated gene expression. In support of this
idea, circadian variations in the concentrations of excitatory amino
acids have been observed within the region of the SCN under
free-running conditions, and in slice preparations (62-64). Circadian
oscillations in CRE-dependent transcription also may be a
result of an inherent rhythmic transcriptional program of SCN pacemaker
cells. Another possibility is that the amount or ratio of CREB
heterodimerization partners within the SCN varies over the circadian
cycle. In support of this idea, CREM-deficient mice do not express
circadian locomotor
activity.3
The circadian expression of a variety of genes within the SCN may
result from circadian CRE-dependent transcription. For
example, the promoters for several peptides that show circadian
oscillations at the mRNA or protein level in the SCN, including
vasopressin, somatostatin, and, during development, vasoactive
intestinal peptide (22, 65, 66), contain one or more CREs (67- 69).
Interestingly, these peptides have the capacity to both modulate
CRE-dependent transcription, either positively or
negatively (70-72), and to alter rhythmicity when added to the SCN
(47, 73). Conceivably, the circadian expression of these proteins could
be generated by temporally overlapping feedback loops that either
activate or inhibit CRE-mediated transcription. Based on the results
presented here, we propose that the CRE transcriptional pathway plays
an important role in orchestrating the series of transcriptional events
essential for both endogenous clock rhythmicity and the ability of
light to phase-shift the clock.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase transgenic reporter
strain to monitor CRE-mediated transcription in vivo.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase reporter mouse strain is found in Ref. 33. For
all experiments, 6-8-week-old mice were housed individually in clear
polycarbonate cages in a well ventilated room. Mice were entrained to a
12-h light/dark (L/D) cycle for at least 7 days before being
transferred to constant darkness (D/D). Light intensity during L was
~400 lux. Food and water were available ad libitum
throughout the experiment. Circadian activity rhythms were monitored
using motion sensors connected to VitalView software (Mini-Mitter Co.,
Sunriver, OR).
-galactosidase rhythmicity,
mice (n = 3-6/time point) kept in D/D for 6 days were sacrificed at 4-h intervals over a 24-h period. For the Western analysis of CRE/
-galactosidase rhythmicity, mice (4/time point) kept
in D/D for 6 days were sacrificed at 6-h intervals over a 24-h period.
For photic stimulation experiments, animals were exposed to light (400 lux) at CT 16.5, CT 22.5, or CT 6. Mice were then returned to constant
darkness. Eight hours after light treatment, animals were sacrificed
and brain slices were processed for
-galactosidase immunoreactivity.
70 °C.
-galactosidase immunolabeling,
free-floating sections were blocked for 2 h in 1% normal goat
serum and 10% bovine serum albumin in PBS with 0.1% Triton X-100
(PBST). After blocking, sections were incubated overnight at 4 °C
with an affinity-purified polyclonal
-galactosidase antibody raised
in rabbit (1:1000 final dilution, Cappel) in PBST, and 2.5% bovine
serum albumin. The tissue was then incubated for 6 h with a
lissamine-rhodamine-conjugated secondary antibody raised in goat and
directed against rabbit IgG (2 µg/ml final dilution, Jackson
Laboratories) in PBST containing 2.5% bovine serum albumin. To ensure
minimal immunolabeling variability for endogenous rhythm experiment,
all samples were processed concurrently.
-galactosidase activity was assayed, as described in Ref. 30, using
a luminometer (Berthold).
-glycerophosphate, 1.5 mM EGTA, 0.1 mM Na3VO4,
1 mM dithiothreitol, 10 µg/ml aprotinin, 2 µg/ml
pepstatin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl
fluoride, pH 7.4) and sonicated for 15 s. 100 µl of 5× sample
buffer was then added, and the samples were heated to 90 °C for 10 min. Extracts were then vortexed (20 s) and centrifuged (8 min at
13,000 × g). 40 µl of extract was loaded onto an 8%
SDS-PAGE gel and electrophoresed using standard procedures. For
cultured SCN neurons, agonist-treated cells were lysed in hot
(90 °C) 2.5× sample buffer (80 µl/dish). After vortexing and centrifugation (7 min at 13,000 × g), 30 µl of
extract was loaded onto a 12% SDS-PAGE gel and electrophoresed using
standard procedures. Once transblotted, membranes (Immobilon P:
Millipore) were blocked with 10% powdered milk in PBS. Membrane were
then incubated overnight at 4 °C in PBST with primary rabbit
antibody against
-galactosidase (1:500, 5Prime
3Prime Inc.) or
P-CREB (1:1000, New England Biolabs), or phospho-p44/42-(P-ERK)
specific antibody (1:1000 final dilution, New England Biolabs).
Membranes were then treated with a goat anti-rabbit IgG alkaline
phosphatase-conjugated secondary antibody (1:2000, Cappel).
Immunoreactivity was developed using the Western-star alkaline
phosphatase detection system (Tropix). A second cultured SCN cell
membrane and the same
-galactosidase membrane were then probed with
a monoclonal anti-CREB antibody (1:1000, Santa Cruz), and labeled with
a rabbit anti-mouse IgG antibody conjugated to horseradish peroxidase.
The Renaissance chemiluminescence detection reagent (NEN Life Science
Products) was used to visualize immunoreactivity. Membranes were washed
six times with a 5% milk/PBST solution after each antibody treatment.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase reporter (33) were entrained to a 12-h L/D cycle,
then placed in total darkness (D/D). Under this condition, circadian
rhythmicity is controlled by the endogenous pacemaker. After 6 days in
D/D, animals were sacrificed every 4 h over a 24-h cycle and
coronal sections containing the SCN were labeled immunohistochemically
for the expression of the reporter gene. Quantitation revealed a
significant (analysis of variance: p < 0.001, F = 7.16) circadian variation in CRE-mediated gene
expression in the SCN (Fig.
1A). Maximal gene expression
was observed from early- to mid-subjective day (Fig. 1B).
Levels of the reporter gene dropped markedly from mid-subjective day to
mid-subjective night, then rose during late-subjective night.
Significant variations in CRE-mediated gene expression were not
observed in other hypothalamic nuclei (supraoptic, Fig. 1C)
or in other brain regions (piriform cortex and primary motor cortex,
data not shown).
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Fig. 1.
Circadian oscillations in CRE-mediated gene
expression under free-running conditions. C57/BL6 mice transgenic
for the CRE- -galactosidase construct were initially entrained to a
12-h L/D cycle, then placed in constant darkness (D/D). After 6 days in
D/D, animals were sacrificed every 4 h, and SCN-containing tissue
was immunolabeled for the expression of
-galactosidase.
A, color-coded confocal images of coronal sections through
the central SCN at different CT during the subjective day and night.
CT 0 defines the beginning of subjective day; CT
12 defines the beginning of subjective night. Dark
red fluorescent hues correspond to weak reporter gene
expression; yellow fluorescent hues correspond to strong
reporter gene expression. The peak in CRE-mediated gene expression was
observed at CT 6; minimal levels of reporter gene were observed at CT
18. B, graphical representation of the relative level of
reporter gene in the SCN is shown for each time point. A minimum of
three animals were analyzed for each time point. C,
circadian fluctuations in the level of CRE-mediated gene expression
were not observed in the supraoptic nucleus (SON). A minimum
of three animals were analyzed for each time point. Error
bars in B and C denote S.E.
OC, optic chiasm; 3V, third ventricle.
-galactosidase reporter construct oscillated over the circadian
cycle. Relatively high levels of reporter were observed during
late-subjective night and early-subjective day (Fig.
2A). Reporter expression
within the lateral hypothalamus did not significantly fluctuate over a
24-h period (Fig. 2B). Probing the same membranes revealed
that levels of the transcription factor CREB did not significantly vary
as a function of circadian time in either the SCN or the lateral
hypothalamus. Together, these data reveal the presence of an
endogenous, circadian, oscillation of CRE-dependent transcription localized to the SCN.
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Fig. 2.
Western analysis of CRE-mediated gene
expression. After CRE- -galactosidase transgenic mice were
maintained for 6 days in D/D, tissue was isolated at different
circadian times and analyzed for
-galactosidase expression.
A, SCN tissue showed a marked variation in
-galactosidase
levels as a function of circadian time. CREB levels probed from the
same membrane were consistent over circadian time. B,
significant circadian variations in
-galactosidase or CREB were not
observed in tissue from the lateral hypothalamus.
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Fig. 3.
Circadian oscillation in the Ser-133
phosphorylated form of the transcription factor CREB (P-CREB).
Mice were initially entrained to a 12-h L/D cycle, then placed in
constant darkness (D/D). After 6 days in D/D, animals were sacrificed
every 4 h, and SCN-containing tissue was immunolabeled for the
expression of P-CREB. A, color-coded confocal images of
representative P-CREB expression in the SCN are shown for each CT.
Blue/green fluorescent hues correspond to
relatively weak P-CREB expression; yellow fluorescent hues
correspond to strong P-CREB expression. B, graphical
representation of the relative level of P-CREB in the SCN is shown for
each time point. Peak P-CREB expression was at CT 18, minimal
expression was at CT 10. A minimum of three animals were analyzed for
each time point. Error bars in B
denote S.E.
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Fig. 4.
Light induces phase-dependent
stimulation of CRE-mediated gene expression in the SCN. Initially,
mice transgenic for the CRE-regulated reporter gene construct were
placed on a 12-h L/D cycle, then transferred to D/D. After 5 days in
total darkness, animals were exposed to white light (60 min, 400 lux)
at different circadian times: early subjective night (CT 16.5), late
subjective night (CT 22.5), or mid-subjective day (CT 6). Eight hours
after light exposure, animals were sacrificed and their brains were
removed and immunocytochemically processed for reporter expression.
A, photic stimulation during the subjective night, but not
during the subjective day, triggered CRE-mediated gene expression. Of
note, the late subjective night group (CT 22.5) was sacrificed at CT
6.5 and the mid-subjective day group (CT 6) was sacrificed at CT 14. Given this, the observed CT variation in immunoreactive cells in
control groups (shown as black bars in
B) corresponds to the variations shown in Fig. 1. Also, note
that different confocal settings were used to maximize variations in
light flash versus endogenous rhythm experiments.
B, quantitation of CRE-mediated gene expression for the
three circadian times assayed. Error bars denote
S.E. The number above each time point refers to the number
of animals assayed. ** = p < 0.0001, two-tailed
Student's t test. C, relative to a control
animals, a 5-min light treatment at CT 16.5 induced a highly localized
expression of the CRE-regulated reporter gene.
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Fig. 5.
Multiple signaling pathways activate
CRE-dependent transcription in the SCN. A,
coronal punch-excision of the SCN from postnatal day 1 rat. Following
SCN removal, section was labeled with the DNA stain syto-13.
Asterisk (*) identifies excised region. B,
primary SCN culture immunolabeled for the neuro-specific antigen MAP-2.
C, the same field of cells was also immunolabeled for the
expression of a transiently transfected -galactosidase reporter
construct. Arrows identify transfected neuron. D,
SCN neurons transiently transfected with a CRE-luciferase construct
were stimulated with forskolin (5 µM), K+ (50 mM), or NMDA (50 µM + glycine: 2 µM) for 6 h, and then assayed for luciferase
activity. E, SCN cells were co-transfected with a
CRE-luciferase construct and a 6-fold excess of vector control
(PCDNA3), dominant negative MEK (D/N MEK), or
dominant negative PKA (D/N PKA). D/N MEK blocked
Ca2+-dependent transcription, whereas D/N PKA
blocked both Ca2+- and cAMP-dependent
transcription. F, simultaneous stimulation of
Ca2+ and cAMP signaling pathways triggered synergistic
stimulation of both CRE-dependent (CRE-LUC)
transcription and transcription driven by the vasoactive intestinal
peptide promoter (VIP-LUC). Experiments are averages of
quadruplicate determinations. Bars denote standard error of
the mean.
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Fig. 6.
The MAPK signaling cascade couples
cAMP-dependent and Ca2+ signaling pathways to CREB
phosphorylation. A, 20-min treatment of cultured SCN
neurons with K+ (40 mM; K,
K+), forskolin (5 µM; F,
forskolin), or glutamate (20 µM; G, glutamate)
triggers an increase in the Ser-133 phosphorylated form of the
transcription factor CREB relative to mock-treated cultures
(C, control). A 90-min pretreatment with the MEK inhibitor
PD 98059 (75 µM) attenuated agonist-induced CREB
phosphorylation. Cell extracts were also probed for CREB. B,
agonist treatment triggers the phosphorylation of erk-1 and erk-2 at
Thr-202 and Tyr-204. PD 98059 blocked agonist-induced erk-1 and erk-2
phosphorylation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. Silvio Gutkind for the dominant-negative Ras plasmid, Neil Nathanson for the vip-luciferase construct, Stanley McKnight for the CRE-luciferase and dominant-negative PKA constructs, and Lauren Baker for helpful advice during manuscript preparation. Confocal microscopy and image analysis was conducted in the W. M. Keck Center for Neural Signaling, University of Washington.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant NS 37056.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.
Recipient of National Research Service Award F32 MH 11857-01.
§ To whom correspondence should be addressed: University of Washington, D-429 Health Sciences Bldg., Box 357280, Seattle, WA 98195-7280. Tel.: 206-543-7028; Fax: 206-685-3822; E-mail: dstorm{at}u.washington.edu.
2 K. Obrietan and D. R. Storm, unpublished observation.
3 P. Sassone Corsi, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: SCN, suprachiasmatic nuclei; CRE, cAMP response element; CREB, CRE-binding protein; P-CREB, phospho-active CREB; MAPK, mitogen-activated protein kinase; PKA, protein kinase A; L, light; D, dark; CT, circadian time; PBS, phosphate-buffered saline; PBST, PBS with Triton X-100; RHT, retinohypothalamic tract; ERK, extracellular signal-regulated kinase; D/N, dominant negative; DM, dissociation medium; MEK, MAPK kinase.
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