The Dynamics of the Transcriptional Response to Cyclic Adenosine 3',5'-Monophosphate: Recurrent Inducibility and Refractory Phase
Monica Lamas and
Paolo Sassone-Corsi
Institut de Génétique et de Biologie Moléculaire
et Cellulaire, Centre Nationale de la Recherche Scientifique,
INSERM, B. P. 163, Strasbourg, France
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ABSTRACT
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Activation of the cAMP signal transduction pathway
results in the transcriptional induction of many genes. Several of them
are induced with kinetics characteristic of the early response. One of
these, the cAMP response element modulator (CREM) gene, is
cAMP-inducible by virtue of an intronic promoter that directs the
synthesis of the dominant negative inducible cAMP early repressor
(ICER). ICER is involved in the down-regulation of its own promoter via
an autoregulatory loop. Thus, while phosphorylation of cAMP response
element binding protein (CREB) by the cAMP-dependent protein kinase A
is the prerequisite for induction, it has been proposed that the
following attenuation involves both CREB dephosphorylation and
repression by the inducible repressor ICER. Here we show that ectopic
expression of sense or antisense ICER in corticotroph AtT20 cells
dramatically modifies the normal CREM inducibility profile. We have
investigated the kinetics of CREM inducibility by recurrent stimulation
of the cAMP-signaling pathway. We define the presence of a refractory
phase that follows the first induction cycle. Accumulation of cAMP,
protein kinase A activity, CREB/CREM phosphorylation, and ICER levels
contribute to the refractory period. Strikingly, the length of the
refractory period is determined by the length of the stimulation by
cAMP responsible for the first cycle of induction.
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INTRODUCTION
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A large number of endocrine functions relay on the cAMP-signaling
pathway. Through this pathway and in response to short-term signals,
long-term changes at the level of gene expression occur. Many hormones,
such as pituitary-originated TSH, FSH, LH, and ACTH, are secreted in a
pulsatile manner (1, 2, 3, 4) eliciting recurrent activation of the cAMP
pathway and thus a physiological hormonal response from the plasma
membrane to the nucleus (4, 5). Via interactions with membrane-bound G
protein-coupled receptors, hormones and neuropeptides modulate the
activity of adenylate cyclase (6). Induction of this enzyme raises
intracellular cAMP levels, which in turn activate the cAMP-dependent
protein kinase A (PKA) (7, 8). Upon translocation to the nucleus, PKA
phosphorylates a group of transcription factors that regulate the
activity of cAMP-responsive genes (9, 10). PKA-mediated phosphorylation
enhances the ability of these proteins to activate transcription
without affecting its intracellular location or DNA-binding activity
(9, 10). Thus, these factors bind to consensus cAMP-responsive elements
(CREs) present in the regulatory region of several genes. Through DNA
binding these factors elicit their function as either transcriptional
activators or repressors (reviewed in 10 .
Central to cAMP-mediated transcriptional induction and attenuation are
the properties of cAMP-responsive transcription factors. The first
cAMP-responsive factor identified was CREB [cAMP response element
(CRE) binding protein] (11, 12). Since then, several other CRE-binding
proteins have been described (13, 14). Among these, only a subset
originating from the CREM (CRE modulator) and ATF-1 (activating
transcription factor 1) genes have been shown to be cAMP-responsive
(15, 16). Phosphorylation by PKA is a major determinant of their
function, thus constituting the link with the cAMP-signaling pathway
(12).
Among cAMP-responsive factors, the products of the CREM gene appear to
play a privileged role in neuroendocrine systems. The CREM gene encodes
both transcriptional activators and repressors that have a
characteristic cell- and tissue-specific pattern of expression (14, 17, 18, 19). CREM also presents a feature unique among other cAMP-regulated
genes, namely that its transcription is cAMP inducible. The inducible
CREM isoform ICER (inducible cAMP early repressor) (20, 21) is derived
by the use of an alternative intronic promoter that contains four CREs
organized in tandem (20). The ICER protein consists of the CREM bZip
DNA-binding domain (DBD) and functions as a powerful dominant negative
regulator of cAMP-induced transcription (20, 21). Importantly, ICER
also binds to CREs present in its own promoter, constituting a
feedback-regulatory loop (20, 22). ICER is predominantly expressed in
neuroendocrine tissues, e.g. the pineal, pituitary, thyroid,
and adrenal glands (21, 23).
Analysis of CREM expression in the pineal gland has revealed that the
repressor ICER is strongly induced at night by clock-derived adrenergic
signals (21). Interestingly, we defined that during daytime the CREM
gene is refractory to cAMP transcriptional induction (21). Furthermore,
we have recently documented that the CREM transcriptional induction in
the pineal gland may be either supersensitive or subsensitive in
response to changing adrenergic inputs (24). Therefore, the molecular
machinery involved in regulating CREM-inducible expression is adaptable
to periodic stimuli and to environmental changes (24).
These observations beg the question of the molecular mechanisms
involved in the transcriptional regulation of cAMP-inducible genes and
their relationship with physiological hormonal response. It is well
known that attenuation of the cAMP response involves desensitization of
membrane receptors, cAMP degradation by specific phosphodiesterases,
and activation of protein phosphatases (25, 26, 27). Interestingly, a
refractory phase in cAMP-responsive transcription after prolonged
stimulation of the cells has been described (28). This refractory
period has been shown to involve a progressive loss in PKA activity due
to a reduced synthesis of C subunit protein (28).
The properties of ICER cAMP-inducible expression and the implications
of an autoregulatory feedback loop strongly suggest a role for ICER in
the attenuation of the neuroendocrine response. We decided to
investigate the transcriptional regulatory process governing the
response to recurrent hormonal stimuli. Here we demonstrate the
fundamental properties of the CREM negative feedback loop by showing
that ectopic ICER expression blocks the cellular cAMP-induced
transcriptional response. Furthermore, we analyze in detail the
kinetics of CREM expression in the corticotroph cell line AtT20 under
various stimuli. We establish that the duration of the stimulus
inducing ICER expression differentially modifies accumulation of cAMP,
PKA activity, and CREB/CREM phosphorylation. Importantly, we
demonstrate that the length of the stimulus determines the length of
the transcriptional refractory period.
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RESULTS
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Ectopic ICER Expression Alters the Cellular Response to cAMP
In order to test the role of ICER in the dynamics of
cAMP-dependent CREM expression, we constructed stably transfected AtT20
cell lines with plasmids containing the ICER-II
(20) coding sequence
under the control of the human cadmium-inducible metallothionein IIA
promoter (29), in either the sense or the antisense orientation (Fig. 1A
). AtT20 G-418-resistant colonies were
selected and stable lines were established, expressing either the sense
[At-ICER(S)] or the antisense ICER [At-ICER(AS)].
Neomycin-resistant control lines (At-Neo) containing only the
resistance gene were also selected as controls. The clones were
analyzed for the presence of the transfected DNA by Southern blot and
for various growth and hormonal characteristics (accompanying paper,
30 . The results reported here for each cell line are
representative of independent clones and have been confirmed in several
cycles of experiments.

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Figure 1. Ectopic Expression of Sense and Antisense ICER in
AtT20 Cells
A, Schematic representation of the ICER II transcript in relation to
the CREM gene structure as previously characterized (20). Exons
encoding the glutamine-rich domains (Q1 and Q2), the P-box, the
domain, and the two alternative DBDs (DBDI and DBDII), as well as the
positions of the P1 and P2 promoters are shown (14). Below, schematic
representation of the plasmids containing the ICER II coding
sequence in either the sense (S) or the antisense (AS) orientation,
under the control of the human cadmium-inducible metallothionein IIA
(hMT IIA) promoter are shown. In these constructs, a fragment derived
from the 3'-region of the human GH gene (hGH) provides polyadenylation
and termination signals. B, Western blot analysis of ICER protein from
AtT20 stable cell lines At-Neo, At-ICER(S), and At-ICER(AS) in the
presence or absence of 50 µM CdCl2, which
induces the expression of the stably transfected ICER II . Endogenous
ICER expression was induced by treatment of the cells with
10-6 M forskolin for the times indicated.
Equivalent amounts of protein were loaded (15 µg of cell extract)
except in the case of cadmium-induced AT-ICER(S) (3 µg cell
extract).
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The At-ICER(S) and At-ICER(AS) clones were tested for cAMP inducibility
of endogenous ICER expression upon cadmium treatment (see also
accompanying paper, 30 . Protein extracts were analyzed by Western
blot assays using a polyclonal antibody raised against the ICER protein
(31). The results in Fig. 1B
show that there is an elevated basal level
of ICER protein in At-ICER(S) cells even in the absence of cadmium.
However, ectopic ICER expression is strongly induced after cadmium
treatment (note differences in total protein loading on the gel; see
legend to Fig. 1B
). Importantly, chronic treatment of At-ICER(S)
cells with forskolin does not influence the levels of ICER
protein, indicating that ectopic ICER expression impairs the
cAMP-inducibility of cellular ICER expression.
When At-ICER(AS) cells are treated with forskolin, the levels of ICER
protein are induced with kinetics identical to those in the control
cells At-Neo. Cotreatment of the same cells with cadmium results in a
significant reduction of the ICER protein levels, even after prolonged
forskolin induction, although a certain amount of ICER protein is still
detectable. These results demonstrate that the antisense ICER
transcript induced by cadmium in the At-ICER(AS) cells can, at least
partially, block the cAMP-induced levels of the endogenous ICER.
We next analyzed the effect of the induced ectopic ICER expression upon
endogenous CREM mRNA expression in these clones (Fig. 2A
). ICER expression was scored using
RNAs from treated cells. The results confirm that induced ectopic ICER
expression in the At-ICER(S) cells completely blocks the cAMP-dependent
activation of the endogenous CREM gene (Fig. 2A
). We then compared the
kinetics of induction and attenuation of ICER transcript synthesis upon
forskolin treatment in the At-Neo and At-ICER(AS) cells (Fig. 2B
). We
have previously documented that CREM expression is transiently induced
upon forskolin treatment in AtT20 cells (20). Consistently, ICER
expression reaches a peak 4 h after treatment and then decreases
rapidly in the control At-Neo cells. However, cadmium-induced
expression of antisense ICER RNA in the AT-ICER(AS) clones causes
hyperinducibility of ICER transcription at early times and a prolonged
expression of the endogenous transcript at later time points.

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Figure 2. Ectopic ICER Expression Results in Constitutive
Transcriptional Repression of the Endogenous CREM Gene
A, RNase protection analysis of CREM RNA using the p6/N1 probe (20) in
the neomycin-resistant AtT20 clone, expressing only the resistance gene
(At-Neo) and a clone expressing sense ICER (At-ICER(S)). Clones were
treated for 6 h with 50 µM CdCl2 to
induce the expression of the stably transfected ICER II cDNA and
with 10-6 M forskolin for the times indicated.
The size of the bands corresponding to the specific endogenous
CREM-protected fragments are indicated. B, RNase protection analysis of
ICER transcripts using the RNA probe P75 (20), which scores
specifically for the 5'-end of the ICER transcript from the AT-Neo
clone and a clone expressing the antisense ICER cDNA [At-ICER (AS)].
Cells were treated with CdCl2 as in panel A and with
forskolin for the times indicated. C, Western blot analysis of the
At-Neo, At-ICER(S), and At-ICER(AS) cells using a specific antibody
that recognizes only the phosphorylated form of CREB and CREM. Cells
were treated with CdCl2 as in panel A and with forskolin
for the times indicated.
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The cAMP-induced ICER expression is directed by the CREM P2 promoter,
which contains four CREs in tandem denominated CAREs (cAMP
autoregulatory elements) (20, 21). CRE-binding activators bind to the
CAREs and must be phosphorylated to elicit transcriptional activation
(10). The principal phosphoacceptor sites in CREB and CREM
are
Ser133 and Ser117, respectively, which are principally the targets of
the cAMP-dependent PKA (12, 21). To determine whether the modified CREM
inducibility in the AtT20 clones is due to impaired PKA activity, we
analyzed CREB/CREM phosphorylation after forskolin and cadmium
treatments (Fig. 2C
). We used Western analysis and a specific antibody
recognizing only the phosphorylated forms of both CREB and CREM (32).
All the clones presented an identical pattern of CREB phosphorylation
following treatment with forskolin and cadmium. These results show that
ectopic ICER protein expression results in constitutive transcriptional
repression and firmly verify the involvement of the CREM autoregulatory
loop in the dynamics of the inducibility to cAMP.
A Refractory Phase in the CREM Response to cAMP
The specific properties of CREM inducibility and the existence of
the ICER negative feedback loop predict the existence of a refractory
inducibility period in the transcription of this early response gene
(33). To test this prediction, we analyzed the pattern of cAMP
inducibility and reinducibility of CREM transcripts in AtT20 cells. We
analyzed ICER transcription from forskolin-induced cells by
ribonuclease (RNase) protection. Figure 3A
shows that after a first challenge
with forskolin, ICER expression rises rapidly and transiently with
kinetics characteristic of an early response gene (34). However, in
cells that have been previously treated with forskolin, a novel
treatment with the same agent does not lead to transcriptional
activation. Indeed, CREM is refractory to cAMP reinduction during at
least the 15 h following the initial stimulation (Fig. 3
).
Therefore, we confirm the existence of a transcriptional refractory
period in cAMP-responsive induction of gene expression. Thus, cells
that have been treated for 24 h with forskolin respond weakly to a
second cycle of stimulation (also see Fig. 7
). In fact, maximal
induction of transcription from the P2 CREM promoter is impaired during
at least 5 days after the first cAMP stimulation (not shown).

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Figure 3. Transcriptional Refractory Period in
cAMP-Responsive Induction of the CREM Gene
A, RNase protection analysis scoring specifically for ICER expression
with probe P75 (20) of total RNA from AtT20 cells. Cells were treated
with 10-6 M forskolin for the times indicated
(1st treatment). Transfer RNA (t) was used as a control for nonspecific
protection. Maximum levels of ICER transcript are observed after
treatment for 2 h. The RNase protection of a subset of cells that
had been previously treated with forskolin for 6, 8, 10, 12, or 15
h and were reinduced with forskolin for 2 h (2nd treatment) is
shown below. No transcriptional activation is observed during at least
15 h following the initial stimulation. B, Western blot analysis
of the AtT20 cells using a specific antibody that recognizes only the
phosphorylated form of CREB and CREM (32). Cells were treated with
forskolin as in panel A for the times indicated.
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Figure 7. The Length of the Stimulus Drives the Length of the
Transcriptional Refractory Period
RNase protection analysis scoring specifically for ICER expression with
probe P75 (20) of total RNA from AtT20 cells. Cells were treated with
10-6 M forskolin either chronically or for
1 h or 5 min (1st treatment), and total RNA was prepared at the
times indicated. At 24 h, a subset of these cells was restimulated
with forskolin for 2 h (2nd treatment). The analysis of CREM
expression corresponding to these samples is shown in the
bottom panel.
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Given the direct relationship between CREB/CREM phosphorylation and
transcriptional activation (12), we analyzed whether there will also be
a refractory phase for CREB phosphorylation. Following the first
stimulus there is a burst in CREB phosphorylation that persists for
2 h and is followed by a decrease to almost undetectable levels at
6 h (Fig. 3B
). Importantly, a second stimulus failed to trigger a
novel cycle of CREB phosphorylation even 5 days after the first cycle
of inducibility (Fig. 3B
). This effect may be due to a progressive loss
in PKA activity caused by a reduced synthesis of C subunit protein, as
has been described for the follicular thyroid cell line FRTL-5 (28) and
for a neoplastic B cell line (35).
CREM Transcriptional Activation Requires Only a Short cAMP
Pulse
Sustained stimulation of the cAMP-signaling pathway leads to a
transcriptional refractory period (28, 36, 37). However, in
vivo many hormones are released in a pulsatile manner such that
cells are subjected to pulsatile, short-lasting receptor-mediated
stimuli (1, 2, 3, 4). We therefore wished to determine whether short pulses
of stimulation would be sufficient to induce the cAMP signal
transduction cascade. Consequently, we decided to study the kinetics of
CREM transcriptional induction and CREB phosphorylation following a
pulsatile cAMP induction and then compare it with that observed under
constant stimulation.
We analyzed the phosphorylation state of CREB at early time points
after stimulation of AtT20 cells with forskolin. Figure 4
shows that a short, 5-min treatment
with forskolin is sufficient to induce CREB phosphorylation to a degree
equivalent to that observed upon chronic forskolin treatment. We next
analyzed the kinetics of ICER transcript and protein inducibility after
5 min of forskolin stimulation. Our results (Fig. 4
) demonstrate that a
5-min treatment with forskolin results in an efficient transcriptional
induction of the P2 promoter of the CREM gene. Again, the kinetics of
induction are similar to those observed under constant stimulation
(Fig. 2A
). We also performed the analysis of the kinetics of ICER
protein induction after a 5-min stimulation by forskolin (Fig. 4
). The
levels of ICER protein peak 4 h after induction and persist during
approximately 24 h. Therefore, the early response pattern of the
CREM gene induction is independent of the duration of the stimulus.

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Figure 4. Kinetics of CREM Transcriptional Induction upon
Short cAMP Stimulation
Cells were treated with forskolin for 5 min (represented by the
black solid bar, time scale "hours"). After
stimulation the medium was removed, the cells washed with PBS and
incubated in fresh complete medium (white bar) for the
times indicated. A, Western blot analysis of AtT20 whole cell extracts
using the specific phospho-CREB antibody. Cells were treated with
10-6 M forskolin for the times indicated. B,
RNase protection analysis scoring specifically for ICER expression with
probe P75 (20) of total RNA from AtT20 cells upon short cAMP
stimulation. C, Western blot analysis of ICER expression in AtT20 cells
treated for 5 min with forskolin. Cells were harvested at the times
indicated, and protein extracts were prepared and resolved by
SDS-PAGE.
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The Length of the Stimulus Determines cAMP Accumulation, PKA
Activity, and the Kinetics of CREB Phosphorylation
We next addressed whether steps preceding CREB phosphorylation in
the activation of the PKA signal transduction pathway could be
modulated by changing the length of cAMP stimulation. We first compared
the kinetics of cAMP accumulation under different treatments. Figure 5A
shows that, under constant forskolin
stimulation, intracellular levels of cAMP increase significantly
reaching a peak 30 min after the beginning of the treatment to then
slowly decrease. In contrast, in cells that were subjected to a short
forskolin treatment, cAMP levels reach a peak within the first 5 min of
induction to then decay quickly to basal levels within a very short
time. Importantly, we observe that the length of the first treatment
directs the magnitude of the induced cAMP levels in response to a
second pulse of stimulation. Figure 5B
shows that a 5-min pulse of
forskolin causes a 20-fold increase in the intracellular concentration
of cAMP in unstimulated cells. However, this increase is markedly
reduced in cells that have been pretreated 6 h before with
forskolin for 5 min. In addition, cells that have been constantly
treated with forskolin for 6 h show a very low increase in cAMP
intracellular levels upon a new challenge with the same agent.
Consistently, we observe comparable variations in the induction of
protein kinase A activity in these cells (Fig. 5C
).

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Figure 5. The Time-Dependent Response of the cAMP Signal
Transduction Pathway
A, Compared kinetics of accumulation of intracellular cAMP in AtT20
cells following either chronic or short forskolin treatment. Cells were
pretreated for 30 min with 0.5 mM 3-methylisobutylxanthine
(IBMX) and then treated during either 5 min (pulse) or chronically with
10-6 M forskolin for the times indicated.
Intracellular cAMP was measured by RIA. B, cAMP accumulation in AtT20
cells depends on prior stimulation. Accumulation of intracellular cAMP
following 5 min of forskolin stimulation was measured in AtT20 cells as
in panel A. Before the 5-min forskolin stimulation, cells were either
nontreated (none), treated with 5 min with forskolin, washed with PBS,
and cultured in fresh medium for 6 h (pulse) or treated
chronically for 6 h. The relative induction of cAMP with respect
to basal levels is represented. C, Forskolin induction of PKA activity
depends on prior stimulation. Comparison of PKA activity in AtT20 cells
treated as in panel B. PKA activity was assayed by measuring the
incorporation of [ -32P]ATP in counts per minute (cpm)
using the synthetic kemptide substrate in the absence (white
bars) or presence (black bars) of cAMP as
previously described (43).
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In view of these results we wished to determine whether the pattern of
CREB phosphorylation is also dependent on the length of the stimulus.
We analyzed cells that had been subjected to 1 h treatment with
forskolin by Western blot analysis with the phospho-CREB-specific
antibody (Fig. 6
). Our results
demonstrate that CREB phosphorylation peaks with similar speed and
magnitude in both chronic and short treatments (compare Figs. 3B
and 6
). Importantly, in contrast to what we observed for constantly
stimulated cells, CREB can undergo a novel cycle of phosphorylation
following a second challenge with forskolin at very early times
(compare Figs. 3B
and 6
).

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Figure 6. Kinetics of Forskolin-Induced CREB Phosphorylation
in AtT20 Cells by Western Blot Analysis Using the
Phospho-CREB/CREM-Specific Antibody
AtT20 cells were treated with forskolin for 1 h (1st treatment).
Induction of CREB phosphorylation with respect to basal levels was
assayed (-, +). Cells were then washed with PBS and incubated in fresh
medium. Protein extracts were then prepared at the times indicated
(upper time scale) to determine the length of time
during which CREB remains phosphorylated. To determine the kinetics of
reinducibility of CREB phosphorylation, a subset of cells was
restimulated (2nd treatment) with forskolin for an additional 15 min at
the times indicated (upper time scale).
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The Length of the Stimulus Defines the Length of the
Transcriptional Refractory Period
Given the striking time-dependent variations in the response of
various components of the PKA signal transduction pathway, we reasoned
that this may correspond to changes at the level of transcriptional
activation. We therefore analyzed the pattern of CREM inducibility in
AtT20 cells prechallanged 24 h before with forskolin for 5 min,
1 h, or chronically. Our results (Fig. 7
) demonstrate that the levels of
transcriptional inducibility in response to the second challenge with
forskolin are determined by the duration of the preceding stimulation.
Indeed, AtT20 cells that have been treated constantly with forskolin
show lower levels of induced ICER transcripts than cells subjected to
treatment for only 2 h or 5 min. Therefore, we conclude that the
length of the stimulus drives the length of the transcriptional
refractory period.
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DISCUSSION
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The long-term modulation of gene expression via signal
transduction pathways is a direct consequence of hormonal regulation.
In the case of the cAMP-signaling pathway, a primary event is the
PKA-mediated phosphorylation of preexisting nuclear factors such as
CREB, CREM
, and ATF-1 (for review see 10 . As a consequence,
there is the transcriptional activation of various genes containing
CREs in their regulatory regions. We have shown that one of these
promoters is contained within the CREM gene and directs the synthesis
of the cAMP-inducible repressor ICER (20). Although dephosphorylation
of CREB has been directly implicated in the attenuation of the
transcriptional response (38, 39), here we show that ICER plays a
fundamental role in defining the dynamics of cAMP-responsive
transcription. Indeed, ectopic expression of ICER blocks transcription
from the cellular CREM P2 promoter in AtT20 cells (Fig. 2A
).
Furthermore, expression of an ICER antisense RNA influences the
attenuation process, resulting in an extended transcriptional response
(Fig. 2B
). Importantly, the ICER-mediated block of cAMP-induced
transcription takes place in the presence of normal CREB
phosphorylation (Fig. 2C
). We conclude, therefore, that several
molecular events are required for cAMP-induced transcriptional
activation.
Our results establish the presence of a transcriptional refractory
period that follows the induction of the CREM gene by cAMP. During this
period the P2 CREM promoter is unresponsive to further cAMP stimulation
(Fig. 3A
). A significantly novel aspect of these observations concerns
the length of the refractory period itself, which we have demonstrated
to be determined by the mode of the first cycle of induction. The CREM
gene appears to be either supersensitive or subsensitive to a novel
cycle of cAMP stimulation, depending on the length of the first cAMP
challenge.
These results are strikingly parallel to recent data obtained in
our laboratory on the circadian cycle of CREM inducibility in the
pineal gland in response to periodic adrenergic stimuli (24). Indeed,
we have documented that during the day, the pineal gland is refractory
to adrenergic signals coming from the suprachiasmatic nucleus (21).
Subsequently we have also demonstrated that changes in the photoperiod
have profound effects on ICER expression in the pineal gland. We
demonstrated that the duration of the refractory period is determined
by the length of the night to which the animals are adapted (24).
Changing the photoperiod corresponds to modifying the adrenergic input
to the pinealocytes, and as such it could be considered as a situation
similar to what we have described in this paper. Interestingly, we have
defined that the CREM gene may be either supersensitive or subsensitive
to induction. This differential responsiveness is controlled by the
changing balance between positive (CREB phosphorylation) and negative
(ICER levels) cAMP-responsive regulators. Thus, in the pineal gland the
transcriptional response of the CREM gene is determined by the memory
of past photoperiods (24).
Here we contribute to the understanding of the nuclear regulatory
mechanism that may underlay these physiological observations. From the
data presented in this paper, it is evident that the duration and type
of stimulation (chronic or pulsatile) modulates the cellular machinery
generating either activation or attenuation signals that affect the
secondary responses to a given stimulus. A transcriptional refractory
period in early gene expression after recurrent stimulation had been
previously described (40). Recently, it has been proposed that the
existence of a transcriptional refractory period could constitute a
mechanism by which the organism could subvert the pathological
consequences of chronic hormonal stimulation (28). Our findings extend
this notion and establish a link with physiology.
A refractory phase in cAMP responsiveness has been described in
different cell types (28, 36, 37). It has been proposed that this
phenomenon may be caused by a progressive loss in PKA activity through
a reduced synthesis of the catalytic subunit protein (28). We have
analyzed the kinetics of CREB phosphorylation/dephosphorylation in
AtT20 cells treated chronically with forskolin (Fig. 3B
). CREB is
rapidly phosphorylated upon stimulation and subsequently
dephosphorylated (Fig. 3B
). Further stimulation of previously treated
cells does not lead to a new burst of phosphorylation. Importantly,
even 5 days after cAMP stimulation, reinduction of CREB phosphorylation
is not possible (Fig. 3B
). Thus, it appears that chronic cAMP
stimulation leads to impaired phosphorylation of CREB and thus to a
refractory period in cAMP-responsive transcription. However,
importantly here we have demonstrated that the levels of ICER repressor
contribute to the control of the cellular response to cAMP.
Most pituitary hormones are released in a pulsatile manner (1, 2, 3, 4) and
consequently cells are subjected to pulsatile receptor-mediated
stimuli. Thus, in the current study we have addressed the question
whether short pulses of stimulation affect the pattern of gene
expression. We have analyzed various events in the cAMP signal
transduction pathway that lead to an increase in CREM transcription.
Strikingly, we observed that a forskolin treatment as short as 5 min is
sufficient to activate the cAMP signal transduction pathway in AtT20
cells (Fig. 4
). Under these conditions the kinetics of CREB/CREM
phosphorylation and ICER induction are identical to those observed
under chronic treatment of the cells (Fig. 4
). Therefore, we can
conclude that the specific contribution of ICER to the cAMP
transcriptional refractory period is not likely to reside in the
time-dependent kinetics of ICER expression. It is likely, however, that
the variations in ICER protein concentration modify the equilibrium
between activation and inhibition signals affecting the final
transcriptional outcome. Other steps of the signal transduction
pathway, such as the accumulation of cAMP, total PKA activity, and
therefore the phosphorylation status of CREB, show a time-dependent
response (Figs. 5
and 6
). Importantly, the length of the stimulation
determines the length of the transcriptional refractory period (Fig. 7
). As a consequence, the CREM gene may undergo variable
transcriptional refractory periods. In this respect, it would be
important to determine whether in pituitary cells this mechanism could
enable the integration of pulsatile hormonal signals and thereby ensure
an integration of signal transduction, gene control, and physiological
regulation.
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MATERIALS AND METHODS
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Cells
The AtT20 cell line was obtained from American Type Culture
Collection (Rockville, MD) and cultured as recommended. AtT20 stable
cell lines At-Neo, At-ICER(S), and At-ICER(AS) were established by
cotransfection of the plasmid pRSVNeo, which carries the
neomycin-selective marker, alone or together with a plasmid expressing
sense (S) or antisense (AS) ICER II
under the control of the human
metallothionein IIA (hMTIIA) (29) promoter. In these constructs, the
ICERII
cDNA was cloned downstream of an 800-bp XbaI
hMTIIA promoter fragment. Polyadenylation and termination signals were
provided by a 600-bp fragment derived from the 3'-region of the human
GH gene. Additional details and properties of these cell lines have
been described in an accompanying paper (30). AtT20 cells were
transfected by the calcium phosphate coprecipitation technique (41) and
were plated at a density of 106 cells/10-cm plate. Cells
were transfected the following day with 10 µg of total DNA. Plates
were treated with 600 µg/ml G418 (GIBCO, Grand Island, NY) for 1417
days and selected by their antibiotic resistance. Several colonies with
similar morphology were cloned and analyzed for ICER expression. At
least three independent clones of each stable transfected cell line
were selected and used to perform the experiments described in this
work. Depending on the experiment, cells were treated with
10-6 M forskolin (Sigma, St. Louis, MO) for
the times indicated in the figure legends and in the text. Induction of
the transfected gene was achieved by treatment of the cells with 50
µM CdCl2 for 10 h.
Protein Analysis
AtT20 cell cultures were harvested in PBS and cell pellets
resuspended in Laemmli buffer and boiled for 5 min. Western blot
analysis was performed using standard procedures, and filters were
hybridized to a polyclonal antibody raised against the ICER protein
(31) or the antiphospho-CREB antibody (32) raised against a peptide
corresponding to the P-box and phosphorylated at Ser-133 (from Upstate
Biotechnology, Lake Placid NY). This antibody recognizes both CREB- and
CREM-phosphorylated proteins. Bound antibody was visualyzed by the
enhanced chemiluminiscence detection system (Amersham, Arlington
Heights, IL) in combination with a peroxidase-conjugated antibody.
RNA Analysis
Total RNA was extracted by the guanidinium thiocianate procedure
as described previously (42). Typically aliquots of 20 µg of total
RNA were then analyzed by RNase protection as described by Foulkes
et al. (17). The RNA probes p6N/1 CREM and P75 have already
been described (20). The control histone (H4) probe is a
BamHI-EcoRI fragment from histone H4 cDNA
subcloned into BSK plasmid. In all RNase protection analysis, transfer
RNA was used as a control for nonspecific protection, and equal amounts
of RNA were included in each assay. Equivalent results were obtained in
several independent experiments.
Determination of cAMP Levels and PKA Activity
For the analysis of cAMP production, AtT20 cells were seeded in
six-well cluster plates, pretreated for 30 min with 0.5 mM
3-methylisobutylxanthine, and treated with 10-6
M forskolin as indicated in the figure legends. After
incubation the medium was aspirated and replaced by 400 µl 65%
ethanol, and the plates were allowed to shake for 30 min at room
temperature. Cells were then transferred to a test tube and the ethanol
was evaporated. Cyclic AMP was measured by RIA (cAMP RIA kit,
Immunotech, Marseille, France) following the manufacturer instructions.
The results are representative of two independent experiments in which
samples were assayed in duplicate.
For the analysis of cAMP-dependent protein kinase, AtT20 cells grown in
10-cm culture plates were rinsed with PBS, collected in 0.5 ml of
homogenization buffer containing 10 mM sodium phosphate (pH
7.4), 1 mM EDTA, 250 mM sucrose, and a
combination of protease inhibitors (0.1 mM
phenylmethylsulfonyl fluoride, 1 mM 1,10-phenanthrolene, 1
µM pepstatin A), and then centrifuged for 5 min. Cell
pellets were resuspended in 0.2 ml homogenization buffer and then
sonicated. Total cellular protein content was measured using the
Bio-Rad (Bradford) protein assay. Typically 10 µl cell extract (2
mg/ml) were used to measure cAMP-dependent protein kinase activity as
previously described (43).
 |
ACKNOWLEDGMENTS
|
---|
We wish to thank E. Borrelli and all the members of the
Sassone-Corsi laboratory for help and support. The excellent technical
assistance of E. Heitz is greatly appreciated.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Paolo Sassone-Corsi, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 rue Laurent Fries, 67404 Illkirch Cedex, C.U. Strasbourg, France. This work was supported by grants from Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Centre Hospitalier Universitaire Régional, Fondation de la Recherche Médicale, Université Louis Pasteur, Association pour la Recherche sur le Cancer, and Rhône-Poulenc Rorer. M. L. was supported by the Ministerio de Educacion y Ciencia (Spain) and by the Fondation de la Recherche Médicale (France).
Received for publication March 26, 1997.
Revision received May 23, 1997.
Accepted for publication May 30, 1997.
 |
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