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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{gamma} (20) coding sequence under the control of the human cadmium-inducible metallothionein IIA promoter (29), in either the sense or the antisense orientation (Fig. 1AGo). 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{gamma} 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 {gamma} 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{gamma} 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{gamma}. 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).

 
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. 1BGo 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. 1BGo). 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. 2AGo). 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. 2AGo). 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. 2BGo). 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{gamma} 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.

 
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{tau} 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. 2CGo). 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 3AGo 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. 3Go). 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. 7Go). 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.

 
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. 3BGo). Importantly, a second stimulus failed to trigger a novel cycle of CREB phosphorylation even 5 days after the first cycle of inducibility (Fig. 3BGo). 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 4Go 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. 4Go) 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. 2AGo). We also performed the analysis of the kinetics of ICER protein induction after a 5-min stimulation by forskolin (Fig. 4Go). 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.

 
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 5AGo 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 5BGo 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. 5CGo).



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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 [{gamma}-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).

 
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. 6Go). Our results demonstrate that CREB phosphorylation peaks with similar speed and magnitude in both chronic and short treatments (compare Figs. 3BGo and 6Go). 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. 3BGo and 6Go).



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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).

 
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. 7Go) 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.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{tau}, 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. 2AGo). Furthermore, expression of an ICER antisense RNA influences the attenuation process, resulting in an extended transcriptional response (Fig. 2BGo). Importantly, the ICER-mediated block of cAMP-induced transcription takes place in the presence of normal CREB phosphorylation (Fig. 2CGo). 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. 3AGo). 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. 3BGo). CREB is rapidly phosphorylated upon stimulation and subsequently dephosphorylated (Fig. 3BGo). 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. 3BGo). 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. 4Go). Under these conditions the kinetics of CREB/CREM phosphorylation and ICER induction are identical to those observed under chronic treatment of the cells (Fig. 4Go). 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. 5Go and 6Go). Importantly, the length of the stimulation determines the length of the transcriptional refractory period (Fig. 7Go). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{gamma} under the control of the human metallothionein IIA (hMTIIA) (29) promoter. In these constructs, the ICERII{gamma} 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 14–17 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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Willoughby JO, Terry LC, Brazeau P, Martin JB 1977 Pulsatile GH, prolactin and TSH secretion in rats with hypothalamic deafferentation. Brain Res 127:137–152[CrossRef][Medline]
  2. Greenspan SL, Kibanski A, Schoenfeld D, Ridgway EC 1986 Pulsatile secretion of thyrotropin in man. J Clin Endocrinol Metab 63:661–668[Abstract]
  3. Lopez FJ, Dominguez JR, Sanchez-Criado JE, Negro-Vilar A 1989 Distinct pulsatile prolactin secretory patterns during the estrous cycle: possible encoding for diverse physiological responses. Endocrinology 124:536–542[Abstract]
  4. Haisenleder DJ, Yasin M, Marshall JC 1992 Enhanced effectiveness of pulsatile 3',5' -cyclic adenosine monophosphate in stimulating prolactin and {alpha}-subunit gene expression. Endocrinology 131:3027–3033[Abstract]
  5. Haisenleder DJ, Yasin M, Marshall JC 1995 Regulation of gonadotropin, thyrotropin subunit and prolactin messenger ribonucleic acid expression by pulsatile or continuous protein kinase C stimulation. Endocrinology 136:13–19[Abstract]
  6. McKnight SG, Clegg CH, Uhler MD, Chrivia JC, Cadd GG, Correll LA, Otten AD 1988 Analysis of the cAMP-dependent protein kinase system using molecular genetic approaches. Recent Prog Horm Res 44:307–335[Medline]
  7. Roesler WJ, Vanderbark GR, Hanson RW 1988 Cyclic AMP and the induction of eukaryotic gene expression. J Biol Chem 263:9063–9066[Free Full Text]
  8. Lalli E, Sassone-Corsi P 1994 Signal transduction and gene regulation: the nuclear response to cAMP. J Biol Chem 269:17359–17362[Free Full Text]
  9. Ziff EB 1990 Transcription factors: a new family gathers at the cAMP response site. Trends Genet 6:69–72[CrossRef][Medline]
  10. Sassone-Corsi P 1995 Transcription factors responsive to cAMP. Annu Rev Cell Dev Biol 11:355–377[CrossRef][Medline]
  11. Hoeffler JP, Meyer TE, Yun Y, Jameson JL, Habener JF 1988 Cyclic AMP-responsive DNA-binding protein: structure based on a cloned placental cDNA. Science 242:1430–1433[Medline]
  12. Gonzalez GA, Montminy MR 1989 Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59:675–680[Medline]
  13. Hai TW, Liu F, Coukos WJ, Green MR 1989 Transcription factor ATF cDNA clones: an extensive family of leucine zipper proteins able to selectively form DNA-binding heterodimers. Genes Dev 3:2083–2090[Abstract]
  14. Foulkes NS, Borrelli E, Sassone-Corsi P 1991 CREM gene: use of alternative DNA-binding domains generates multiple antagonists of cAMP induced transcription. Cell 64:732–749
  15. de Groot RP, den Hertog J, Vandenheede JR, Goris J, Sassone-Corsi P 1993 Multiple and cooperative phosphorylation events regulate the CREM activator function. EMBO J 12:3903–3911[Abstract]
  16. Rehfuss RP, Walton KM, Loriaux MM, Goodman RH 1991 The cAMP-regulated enhancer-binding protein ATF-1 activates transcription in response to cAMP-dependent protein kinase A. J Biol Chem 266:18431–18434[Abstract/Free Full Text]
  17. Foulkes NS, Mellström B, Benusiglio E, Sassone-Corsi P 1992 Developmental switch of CREM function during spermatogenesis: from antagonist to activator. Nature 355:80–84[CrossRef][Medline]
  18. Foulkes NS, Sassone-Corsi P 1992 More is better: activators and repressors form the same gene. Cell 68:411–414[Medline]
  19. Mellström B, Naranjo JR, Foulkes NS, Lafarga M, Sassone-Corsi P 1993 Transcriptional response to cAMP in brain: specific distribution and induction of CREM antagonists. Neuron 10:655–665[Medline]
  20. Molina CA, Foulkes NS, Lalli E, Sassone-Corsi P 1993 Inducibility and negative autoregulation of CREM: an alternative promoter directs the expression of ICER, an early response promoter. Cell 75:875–886[Medline]
  21. Stehle JH, Foulkes NS, Molina CA, Simonneaux V, Pévet P, Sassone-Corsi P 1993 Adrenergic signals direct rhythmic expression of transcriptional repressor CREM in the pineal gland. Nature 365:314–320[CrossRef][Medline]
  22. Lamas M, Sassone-Corsi P 1996 CREM and the transcriptional response to cAMP. Curr Opin Endocrinol Diabetes 3:403–407
  23. Lalli E, Sassone-Corsi P 1995 Thyroid -Stimulating Hormone (TSH)-directed induction of the CREM gene in the thyroid gland participates in the long-term desensitization of the TSH-receptor. Proc Natl Acad Sci USA 92:9633–9637[Abstract]
  24. Foulkes NS, Duval G, Sassone-Corsi P 1996 Adaptative inducibility of CREM as transcriptional memory of circadian rhythms. Nature 381:83–85[CrossRef][Medline]
  25. Lohse MJ 1993 Molecular mechanisms of membrane receptor desensitization. Biochim Biophys Acta 1179:171–188[Medline]
  26. Conti M, Nemoz G, Sette C, Vicini E 1995 Recent progress in understanding the hormonal regulation of phosphodiesterases. Endocr Rev 16:370–389[Medline]
  27. Alberts AR, Montminy M, Shenolikar S, Feramisco JR 1994 Expression of a peptide inhibitor of protein phosphatase 1 increases phosphorylation and activity of CREB in NIH 3T3 fibroblasts. Mol Cell Biol 14:4398–4407[Abstract]
  28. Armstrong R, Wen W, Meinkoth J, Taylor S, Montminy M 1995 A refractory phase in cyclic AMP-responsive transcription requires down-regulation of protein kinase A. Mol Cell Biol 15:1826–1832[Abstract]
  29. Yu SF, VonRuden T, Kantoff PW, Garber C, Seiberg M, Ruther U, Anderson WF, Wagner EF, Gilboa E 1986 Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells. Proc Natl Acad Sci USA 83:3194–3198[Abstract]
  30. Lamas M, Molina C, Foulkes NS, Jansen E, Sassone-Corsi P 1997 Ectopic ICER expression in pituitary corticotroph AtT20 cells: effects on morphology, cell cycle and hormonal production. Mol Endocrinol 11:1425–1434[Abstract/Free Full Text]
  31. Monaco L, Foulkes NS, Sassone-Corsi P 1995 Pituitary follicle-stimulating hormone (FSH) induces CREM gene expression in Sertoli cells: involvement in long-term desensitization of the FSH receptor. Proc Natl Acad Sci USA 92:10673–10677[Abstract]
  32. Ginty DD, Kornhauser JM, Thompson MA, Bading H, Mayo KE, Takahashi JS, Greenberg ME 1993 Regulation of CREB phosphorylation in the suprachiasmatic nucleus by light and a circadian clock. Science 260:238–241[Medline]
  33. Sassone-Corsi P 1994 Rhythmic transcription and autoregulatory loops: winding up the biological clock. Cell 78:361–364[Medline]
  34. Lamas M, Lalli E, Foulkes NS, Sassone-Corsi P 1996 Rhythmic transcription and autoregulatory loops: nuclear pacemaker CREM. Cold Spring Harbor Symp Quant Biol 61:285–294[Medline]
  35. Tasken K, Anderson KB, Skalhegg BS, Tasken KA, Hansson V, Jahnsen T, Blomhoff HK 1993 Reciprocal regulation of mRNA and protein for subunits of cAMP-dependent protein kinase (RI and C{alpha}) by cAMP in a neoplastic B cell line (Reh). J Biol Chem 31:23483–23489
  36. Sasaki K, Cripe TP, Koch SR, Andreone TL, Peterson DD, Beale EB, Granner KK 1984 Multihormonal regulation of phosphoenolpyruvate carboxykinase gene transcription. J Biol Chem 259:15242–15251[Abstract/Free Full Text]
  37. Buscher M, Rahmsdorf H, Liftin M, Karin M, Herrlich P 1988 Activation of the c-fos gene by UV and phorbol ester: different signal transduction pathways converge to the same enhancer element. Oncogene 3:301–311[Medline]
  38. Hagiwara M, Alberts A, Brindle P, Meinkoth J, Feramisco J, Deng T, Karin M, Shenolikar S, Montminy M 1992 Transcriptional attenuation following cAMP induction requires PP-1-mediated dephosphorylation of CREB. Cell 70:105–113[Medline]
  39. Wadzinski B, Wheat W, Jaspers S, Peruski L, Lickteig R, Johnson G, Klemm D 1993 Nuclear protein phosphatase 2A dephosphorylates protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation. Mol Cell Biol 13:2822–2834[Abstract]
  40. Morgan JI, Cohen DR, Hempstead JL, Curran T 1987 Mapping patterns of c-fos expression in the central nervous system after seizure. Science 237:192–197[Medline]
  41. Gorman C, Padmanabhan R, Howard BH 1983 High efficiency DNA transformation of primate cells. Science 221:551–553[Medline]
  42. Chomczynski P, Sacchi N, 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[CrossRef][Medline]
  43. Clegg CH, Correll LA, Cadd GA, McKnight GS 1987 Inhibition of intracellular cAMP-dependent protein kinase using mutant genes of the regulatory type I subunit. J Biol Chem 262:13111–13119[Abstract/Free Full Text]