Ectopic ICER Expression in Pituitary Corticotroph AtT20 Cells: Effects on Morphology, Cell Cycle, and Hormonal Production

Monica Lamas, Carlos Molina, Nicholas S. Foulkes, Erik Jansen and Paolo Sassone-Corsi

Institut de Génétique et de Biologie Moléculaire et Cellulaire,(M.L., C.M., N.S.F., P.S.-C.), B. P. 163, 67404 Illkirch, Strasbourg, France,
Laboratory for Molecular Oncology (E.J.), Center for Human Genetics, University of Leuven and Flanders Interuniversity Institute,for Biotechnology, 3000 Leuven, Belgium


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The products of the cAMP response element modulator (CREM) gene play an important role in the transcriptional response to cAMP in endocrine cells. By virtue of an alternative, intronic promoter within the gene, the inducible cAMP early repressor (ICER) isoform is generated. ICER was shown to act as a dominant negative regulator and to be cAMP-inducible in various neuroendocrine cells and tissues. ICER negatively autoregulates its own expression and has been postulated to participate in the molecular events governing oscillatory hormonal regulations. To elucidate ICER function in pituitary physiology, we have generated AtT20 corticotroph cell lines expressing the sense or antisense ICER transcript under the control of the cadmium-inducible human methallothionein IIA pro-moter. Here we demonstrate that changes in the regulated levels of ICER have drastic consequences on the physiology of the corticotrophs. Ectopic ICER expression induces remarkable modifications in AtT20 morphology. Cells with persistent, nonregulated high levels of ICER are blocked in the G2/M phase of the cell cycle, while the opposite effect is obtained in cells expressing an antisense ICER transcript. We show that the effect of ICER on the AtT20 cell cycle is correlated to a direct down-regulation of the cyclin A gene promoter by ICER. Finally, we show that ACTH hormonal secretion from the corticotrophs is completely blocked by ICER ectopic expression. Interestingly, this effect is not due to a direct regulation of the POMC gene, but is mediated by a transcriptional control of the prohormone convertase 1 gene. These results point to a key regulatory function of CREM in pituitary physiology.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Extracellular signals such as hormones and growth factors control the proliferation and function of their target cells by binding to membrane receptors and activating specific signaling cascades. The second messenger cAMP plays a crucial role in the regulation of receptor-mediated cell proliferation in the mammalian neuroendocrine system, exerting both positive and negative effects (1, 2). The effects elicited by cAMP in the progression of the cell cycle are complex and not fully understood. The inhibition of cAMP-dependent protein kinase (PKA) has been implicated in the induction of mitosis in mammalian cells (3). In addition, cell cycle progression in Xenopus egg extracts is accompanied by fluctuations in cAMP and in the activity of PKA (4). Other studies have correlated the antiproliferative effect of cAMP with the inhibition of protein phosphatase activity (5). Finally, the involvement of cAMP-responsive nuclear factors, such as cAMP response element binding protein (CREB), cAMP response element modulator (CREM), and activation transcription factor 1, in the regulation of neuroendocrine cell proliferation has been suggested (6, 7, 8, 9).

Transcription factors responsive to cAMP belong to the bZip family and bind as dimers to cAMP-response promoter elements (CRE) (10). They are activated upon phosphorylation by PKA at a serine residue located in the activation domain (Ser-133 in CREB and Ser-117 in CREM) (11, 12). These proteins are modular in structure and present two major functionally independent activation domains (13, 14). The CREM gene encodes both activators and repressors of cAMP-responsive transcription (10). The isoform ICER (inducible cAMP early repressor) (15) appears to play a central role in the physiology of the neuroendocrine system. ICER is a cAMP-inducible small protein of 14 kDa generated from an alternative, intronic promoter in the CREM gene. It functions as a powerful repressor of cAMP-induced transcription (15, 16) and, by binding to its own promoter, represses its own transcription, thus constituting a negative autoregulatory feedback loop (15, 17). ICER lacks the activation domain and thus escapes from PKA-dependent phosphorylation. Thus, in contrast to the other CRE-binding proteins, the principal determinant of ICER activity is its intracellular concentration and not its degree of phosphorylation (15).

ICER is expressed at high levels predominantly in tissues of neuroendocrine origin, namely the pineal, pituitary, adrenal, and thyroid glands (15, 16, 17, 18, 19). We have previously shown that ICER is strongly induced at night in the pineal gland by clock-derived adrenergic signals (16) and that it is involved in the establishment and entrainment of circadian rhythms (20). In the rat thyroid gland, induction of ICER represses expression of the TSH receptor gene and is thereby involved in the homologous long-term desensitization of the receptor (18).

Coupling of gene expression to the cAMP signaling pathway has great importance in the physiology of the pituitary gland. An example is given by transgenic mice expressing a CREB mutant that cannot be phosphorylated by PKA (6). Since cAMP serves as a mitogenic signal for the somatotroph cells of the anterior pituitary, the mutant CREB-coding sequence was placed under the control of the somatotroph-specific promoter of the GH gene. The pituitary glands of transgenic mice expressing this construct appeared atrophied and were deficient in somatotroph cells. Moreover, the transgenic mice exhibited a dwarf phenotype. No other cell type in the pituitary was influenced by expression of the transgene. It is noteworthy that the block of CREB function by the dominant repressor generated a transgenic phenotype equivalent to the one obtained by targeted cell death of the somatomammotrophs (21). This indicated that CRE-binding proteins are likely to have pivotal functions in normal pituitary development.

Here we have investigated the role of the cAMP-inducible repressor ICER in the physiology of pituitary-derived corticotroph cells. We have used the mouse anterior pituitary cell line AtT20 to generate a series of stably transfected clones ectopically expressing the sense or antisense ICER transcript (17). AtT20 cells recapitulate the hormonal response of the corticotroph cells in the pituitary gland and have been widely studied (22, 23, 24). We describe the antiproliferative effect of cAMP in these cells and show that ectopic ICER expression enhances the cAMP-dependent arrest at the G2/M stage of the cell cycle. The molecular mechanism responsible for the G2/M arrest appears to involve a direct transcriptional deregulation of cyclin A gene expression by ICER. It has been previously described that, in addition to the regulation of cell proliferation, the cAMP signal transduction pathway is involved in the regulation of hormonal secretion (25). Furthermore, changes in the proliferation characteristics of corticotroph cells are often associated with disturbed hormonal secretion (22). In AtT20 cells, various hormones and growth factor rapidly stimulate secretion of the POMC-derived peptide, ACTH (26). Here we have analyzed the cAMP-mediated induction of ACTH levels in AtT20 mutant cells, and we demonstrate that ACTH secretion is severely impaired by ICER overexpression. Furthermore, we demonstrate that the molecular mechanism by which ICER affects ACTH secretion involves transcriptional down-regulation of the gene encoding the endoproteolytic enzyme prohormone convertase 1 (PC1).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ectopic ICER Expression Affects the Morphology of AtT20 Cells
To investigate the effect of ectopic ICER expression in endocrine cells, we generated clones of AtT20 pituitary corticotrophs expressing either the sense [At-ICER(S)] or the antisense ICER [At-ICER(AS)] under the control of the human cadmium-inducible metallothionein IIA promoter (27). As a control, we used cells expressing only the neomycin resistance gene (At-Neo). We have analyzed the effect of cadmium treatment on ICER expression in these cells. At-ICER(S) cells were treated with increasing amounts of CdCl2, and ICER protein expression was determined by Western blot analysis (Fig. 1AGo). The results show that ectopic ICER expression is strongly induced upon cadmium treatment in At-ICER(S) cells.



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Figure 1. Effect of Cadmium Treatment on ICER Expression in AtT20 Stably Transfected Cell Lines

A, Western blot analysis of ICER protein from At-ICER(S) treated during 6 h with increasing concentrations of CdCl2, which induces the expression of the ectopically transfected ICER II{gamma}. C indicates control protein sample from cells treated with 10-6 M forskolin during 6 h. Equivalent amounts of protein were loaded. B, RNase protection analysis of CREM RNA using the p6/N1 probe (15) in the At-Neo and At-ICER(S). Clones were treated with 50 µM CdCl2 for 6 h to induce the expression of the stably transfected ICER II{gamma} cDNA. The size of the bands corresponding to the specific endogenous CREM-protected fragments is indicated [DBDI and DBDII correspond to the two DNA-binding domains of the CREM gene (43)]. Note the decreased expression of the endogenous CREM gene in the presence of ectopic ICER protein. C, Western blot analysis of At-ICER(AS) cells treated with various concentration of CdCl2 and 10-6 forskolin as indicated. Cells were pretreated for 6 h by cadmium and then stimulated for 6 h by forskolin. The cAMP-induced synthesis of endogenous ICER proteins is inhibited by the cadmium-stimuled ICER antisense transcript.

 
The increase in ICER protein levels correlates with a significant cadmium-mediated induction of the ectopic ICER gene transcription (Fig. 1BGo). RNA samples from At-Neo and At-ICER(S), treated or not with CdCl2, were analyzed by RNase protection with a probe designed to score for transcripts containing both DBD I and DBD II of the CREM endogenous gene (Fig. 1BGo). The increase in ectopic ICER levels results in the down-regulation of the endogenous CREM gene.

In At-ICER(AS) cells, endogenous ICER protein is not detectable in nonstimulated cells and is induced after activation of the cAMP signal transduction pathway by forskolin (Fig. 1CGo; see accompanying paper and 15 . Pretreatment of the cells with 50 µM CdCl2 drastically reduces the cAMP-induced levels of the endogenous ICER, although a certain amount of protein is still detected, indicating that the antisense ICER transcript is not able to block completely endogenous cAMP-induced ICER synthesis (Fig. 1CGo; see accompanying paper).

Both the overexpression and blockage of ICER function in AtT20 cells significantly affect their morphology (Fig. 2Go). The At-ICER(S) cells present an elongated and fusiform morphology, show increased adhesion to the substrate, and reduce formation of aggregates. These cells are very different from the original AtT20. In contrast, the At-ICER(AS) cells show a round cell body and tend to aggregate during growth. The morphology of the At-Neo cells is comparable to that of the parental AtT20 cells. These observations are consistent with previous reports showing that the cAMP pathway is involved in changes in cell shape associated with a marked reorganization of microtubule network (3).



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Figure 2. Ectopic ICER Expression Affects the Morphology of AtT20 Cells

Photomicrographs of the neomycin-resistant AtT20 clone, expressing only the resistance gene (At-Neo) and clones expressing the ICERII{gamma} cDNA either in the sense [At-ICER(S)] or in the antisense [At-ICER (AS)] orientation.

 
Persistent ICER Levels Induce AtT20 Cell Cycle Arrest in G2/M
cAMP is an inhibitory growth factor in a number of endocrine cells (2). We have observed that prolonged treatment of normally growing corticotroph AtT20 cells with forskolin results in a significant decrease in cell proliferation (M. Lamas and P. Sassone-Corsi, unpublished observations). These effects of cAMP on cell proliferation prompted us to investigate whether altering the inducible pattern of ICER may influence the AtT20 cell cycle.

At-Neo, At-ICER(S), and At-ICER(AS) cells were treated with 50 µM CdCl2 and/or 10-6 M forskolin for 12 h and then examined by flow-cytometric analysis (Fig. 3Go). The results show that ectopic ICER expression significantly influences the cell cycle of AtT20 cells. In At-Neo cells, treatment with forskolin induces a significant increase in the number of cells blocked in G2/M. This result is in accordance with the antiproliferative effect of cAMP on AtT20 cells mentioned above. The same increase is observed in the At-ICER(S) cells, whereas forskolin appears to have only a marginal effect on the At-ICER(AS) cells. This is possibly due to a basal level of ICER antisense transcript present in these cells even without cadmium treatment [Fig. 1CGo; see also accompanying paper (17)]. In general, these results indicate that ICER plays a significant role in mediating the antiproliferative effect of cAMP. This conclusion appears to be confirmed by the results obtained by treating the cells with CdCl2 (Fig. 3Go, left panel).



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Figure 3. Flow-Cytometric Analysis of AtT20 Stable Cell Lines

The first peak consists of cells in G1 and the second peak of cells in G2 and mitosis (G2/M). Between the two peaks are S phase cells. The indicated three different stable cell lines were treated (+) or not (-) with 50 µM CdCl2 without (-) or with (+) forskolin (FSK) for 12 h before flow-cytometric analysis.

 
As expected, no difference is observed by treating the At-Neo cells with cadmium. In contrast, CdCl2-mediated induction of the ICER levels in the At-ICER(S) cells causes a remarkable increase in the number of cells blocked in G2/M. This effect, however, is observed only in the presence of forskolin, indicating that ICER alone is not sufficient to bring the cells to arrest in G2/M. Importantly, the reverse effect is observed upon forskolin stimulation in the At-ICER(AS) cells treated with cadmium. Thus, although ICER cannot be considered as the only determinant of the cAMP-induced effect on AtT20 cell cycle, these results establish that its deregulated expression has a dramatic effect on the control of the cell cycle.

Transcriptional Down-Regulation of Cyclin A by ICER
It has been extensively established that the progression of cells through the cell cycle is controlled by a group of proteins known as cyclins (28, 29). Cyclin A has been implicated in both S phase and the G2/M transition in mammalian cells (30, 31), whereas cyclins B and D1 are expressed during the G2/M transition and early G1, respectively (32, 33). Interestingly, recent studies have established a direct link between members of the CREB/CREM family and the regulators of the cell cycle and specifically, with the transcriptional regulation of cyclin A (8, 34, 35, 36).

The cyclin A promoter contains a CRE that is required for the cell cycle-regulated inducibility of the gene by cAMP and is thought to contribute to the precise timing of cyclin A expression in fibroblasts (8). In vascular endothelial cells, the CRE cooperates with downstream regulatory factors to modulate cell cycle progression (35). This prompted us to investigate whether cyclin A gene expression may be directly regulated by ICER in the AtT20 cells. We first wanted to analyze the pattern of expression of cyclin A in AtT20 cells at different stages of the cell cycle to determine whether it would oscillate as happens in fibroblasts (Fig. 4AGo). AtT20 cells were synchronized in M phase by nocodazole or in G1 phase by aphidicolin treatment, and samples were collected at the indicated hours after release from the drug. We found that cyclin A transcript levels are cell cycle regulated in pituitary AtT20 cells, exhibiting an expression pattern analogous to fibroblasts (8).



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Figure 4. Regulation of Cyclin A Expression by CREM

a, Cyclin A expression during the AtT20 cell cycle. RNase protection analysis of cyclin A expression in AtT20 cells, at various times (hr) after release from aphidicolin or nocodazole block as indicated. b, Effect of ICER on cyclin expression. RNase protection assay of cyclin transcripts from At-Neo or At-ICER(S) clones not treated (-) or treated (+) with CdCl2. The size and position of the specific transcripts are indicated. c, Gel-mobility shift analysis using an oligonucleotide containing the CRE present between positions -80 to -73 of the cyclin A promoter (40). Nuclear extracts were prepared from At-Neo, At-ICER(S) and At-ICER(AS) cells not treated (-) or treated (+) with CdCl2. Binding to a control site (Sp1) was equivalent in all extracts (not shown).

 
We then compared the pattern of expression of cyclin A in the At-Neo and At-ICER(S) cells. When ICER is induced ectopically by cadmium in these cells, cyclin A basal expression is significantly down-regulated (Fig. 4BGo). In addition, the forskolin-induced expression of the cyclin A gene is also blocked. This effect appears to be specific for cyclin A as the expression of cyclin D1 and cyclin B genes is not affected (Fig. 4BGo). Cyclin A expression is not significantly affected in the At-ICER(AS) cells (not shown). The levels of ICER protein in these experimental conditions have been analyzed [Fig. 1CGo; see also accompanying paper (17)]. A reduced amount of ICER protein is still present in the cadmium-treated At-ICER(AS) cells and is likely to blunt the effect of the antisense approach. However, these results suggest that the observed effect of ICER in deregulating the AtT20 cell cycle (Fig. 3Go) involves ICER-mediated transcriptional repression of cyclin A expression. Our results are consistent with previous transfection studies in fibroblasts showing that cyclin A transcription can be blocked by overexpression of CREM repressors (8).

To substantiate this thesis, we decided to analyze whether induced ICER levels and cyclin A deregulation may coincide with an increased occupation of the cyclin A CRE site by ICER. Indeed, this would establish a direct link to the consequent down-regulation of cyclin A expression. We prepared nuclear extracts from cadmium-induced and uninduced At-Neo, At-ICER(S), and At-ICER(AS) cells and used them in a binding assay with an oligonucleotide bearing the cyclin A CRE sequence. We observed a strong, inducible binding of the ectopic ICER to the site, exclusively in the At-ICER(S) cells (Fig. 4CGo). Binding activity among the various extracts was normalized using a control Sp1 oligonucleotide (not shown). This result is in accordance with the specific down-regulation of cyclin A in the same cells (Fig. 4CGo) and with previous transfection experiments showing the repression of cAMP-induced cyclin A transcription by CREM (8).

Impairment of ACTH Secretion by ICER
Changes in both morphology and proliferation rate of secretory cells are often associated with disturbed hormonal secretion (22, 37). In AtT20 cells, various hormones and growth factors stimulate secretion of the POMC peptide, ACTH (26). ACTH secretion in the pituitary gland is physiologically induced by CRF and can be mimicked in AtT20 cells by treatment with forskolin to activate the adenylate cyclase pathway. We have analyzed by RIA the ACTH levels in cells stimulated with 10-6 M forskolin. Cells were treated during various times (1–6 h), and the medium was removed and analyzed. As expected, treatment of the At-Neo cells results in an increase of ACTH synthesis and release. In striking contrast, ACTH secretion is severely impaired in ICER-overexpressing cells (Fig. 5Go). On the contrary, At-ICER(AS) cells responded similarly to control cells (not shown). To determine the molecular mechanism of the decreased ACTH levels in the At-ICER(S) cells, we analyzed the levels of precursor POMC transcripts in these cells. In remarkable contrast with the ACTH levels, the pattern of POMC expression upon forskolin stimulation is equivalent in both AtT20 clones (Fig. 6AGo). Thus, the effect of ICER on ACTH synthesis is independent of POMC gene transcription. A CRE-like sequence is present in the human POMC promoter and has been involved in the regulation of POMC gene expression by cAMP (38). More recently, however, it has been shown that at least part of POMC transcriptional response to cAMP is mediated by the nurr1 and nurr77 nuclear receptors (39). Our data are supportive of these latter results since they indicate that ICER is not directly involved in the regulation of POMC transcription.



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Figure 5. ACTH Secretion Is Impaired in ICER Overexpressing Cells

Time course of ACTH release from AtT20 clones incubated with forskolin. Each point represents the mean value from three determinations from three different experiments.

 


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Figure 6. Regulation of PC1 Expression by ICER

A, Northern blot analysis of POMC and PC1 expression in At-Neo and At-ICER(S) cells treated (+) or not (-) with forskolin for 4 h. B, DNA sequence of the human PC1 gene 5'-flanking region. Boxes indicate the putative CRE motifs. Numeration indicates position from the translation initiation site as +1 as in Jansen et al. (41). C, Gel-mobility shift analysis using oligonucleotides containing the CRE present between positions -283 to -276 (CRE1) and a CRE-like motif (CRE2) located 20 bp downstream from CRE1 in the human PC1 promoter (39). Nuclear extracts were prepared from At-Neo, At-ICER(S), and At-ICER(AS) cells treated with CdCl2 and forskolin for 6 h. D, At-Neo and At-ICER (S) cells were transiently transfected with a human PC1-promoter-luciferase reporter construct containing sequences up to -288 bp (44). Twenty four hours after transfection cells were treated or not with CdCl2 and FSK for 6 h, after which luciferase activity was measured.

 
ICER Regulates the PC1 Gene
How is ICER able to regulate ACTH synthesis without affecting POMC gene expression? It is well known that the release of mature ACTH from the precursor POMC in AtT20 cells involves posttranslational processing by PC1 (40). Interestingly, PC1 transcription has been shown to be hormonally regulated through two CREs (41) (Fig. 6BGo). Thus, the cAMP-signaling pathway regulates ACTH synthesis at multiple levels. We have analyzed PC1 expression in forskolin-stimulated AtT20 cells (Fig. 6AGo). There is a decrease in PC1 expression in the At-ICER(S) cells as compared with the control At-Neo cells. No significant effect was observed in At-ICER(AS) cells (not shown) probably due to a residual amount of ICER protein present in these cells [see Fig. 1CGo and accompanying paper (17)]. We have prepared nuclear extracts from the At-Neo, At-ICER(S), and At-ICER(AS) cells to perform binding assays using oligonucleotides containing the sequence corresponding to two CREs in the PC1 promoter (Fig. 6Go, B and C). This experiment demonstrates that the two CREs are readily recognized by ICER and establish a direct link with the down-regulation of the transcript. We finally tested whether ICER overexpression could affect cAMP-induced transcription elicited by a promoter containing the PC1 5'-flanking region fused to the reporter luciferase gene. This construct was assayed for promoter activity by transient transfection into both At-Neo and At-ICER(S) cells. Figure 6DGo shows that while in control cells stimulation with forskolin leads to a 7.5-fold induction of promoter activity, in At-ICER(S) cells this induction is drastically reduced and absent in cells treated with cadmium.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The cAMP-dependent signaling pathway plays a crucial role in the specific regulation of gene expression within the neuroendocrine system. Indeed, cAMP is a key second messenger in the regulation of both differentiated function (e.g. hormonal secretion) and cell number, acting as a mitogenic or an antimitogenic stimulus. These effects are the consequence of the cAMP-mediated regulation of gene expression in the nucleus via the modulation of transcription factor activity (10). Our results describe for the first time the molecular mechanism by which the cAMP-responsive nuclear repressor ICER affects cell growth, proliferation, and hormonal secretion in a neuroendocrine cell line.

We have described that chronic treatment of pituitary corticotroph AtT20 cells with forskolin causes cell cycle arrest at the G2/M boundary and that ICER ectopic expression dramatically enhances cell cycle arrest by cAMP (Fig. 3Go). Furthermore, we have shown that cyclin A expression is down-regulated upon induction of ectopic ICER (Fig. 4Go). However, induced ICER ectopic expression alone, although associated with a significant repression of cyclin A mRNA (Fig. 5Go), is not sufficient to affect passage through the cell cycle (Fig. 3Go). Thus, the function of ICER in cell cycle regulation seems to be tightly linked with that of cAMP. Importantly, it has been recently demonstrated that the control of activation-inactivation cycles of the cAMP signal transduction pathway plays a critical role in regulating transition through the cell cycle (3, 4).

Direct links between cAMP-responsive nuclear factors and the regulators of cell cycle have been established (8, 34). While activation transcription factor 1 mediates down-regulation of the cyclin A gene in endothelial cells (34), CREB and CREM have been shown to modulate cyclin A expression in fibroblast cells (8). In addition, a powerful induction of ICER has been observed during liver regeneration, a model of cellular proliferation (42). Our results provide new insights into the molecular mechanisms that could account for the effects elicited by cAMP in the cell cycle. Since ICER appears to be predominantly distributed in neuroendocrine tissues, where it is also powerfully inducible by cAMP (15, 16), it is likely that its function in the cell cycle is specific for such systems. Importantly, cyclin A expression is down-regulated in fibroblasts at the same points of the cell cycle as in neuroendocrine cells, clearly demonstrating the existence of an array of regulatory mechanisms operating in different cell types to achieve the same control. It is particularly striking that ectopic expression of the CREM{tau} activator isoform has no effect upon AtT20 cell cycle progression (our unpublished results). Given that CREM{tau} and ICER both share the same C-terminal DNA-binding domain (15, 16, 43), it is evident that specificity of ICER function resides in its repressor function and/or in the lack of the transcriptional activation domain. Taken together, our results suggest that ICER functions at transcriptional checkpoints in concert with cAMP, maybe through control of cyclin A gene expression. Interestingly, a link between cyclin A expression and adhesion-dependent cell cycle progression has been described (44). We show that ectopic expression of ICER dramatically alters the morphology of AtT20 cells (Fig. 2Go). It is thus tempting to speculate about a possible relationship between the effects of ectopic ICER expression on cell morphology and its regulation of cyclin A expression.

In addition to its effect on cell proliferation, ICER-deregulated expression impairs hormonal secretion in AtT20 cells (Fig. 5Go). It is well established that activation of the cAMP signal transduction pathway mediates hormonal secretion in these cells (25). Thus, various hormones and growth factors rapidly stimulate secretion of the POMC-derived peptide, ACTH (26). We have shown that in ICER-overexpressing cells, ACTH secretion cannot be induced by cAMP stimulation (Fig. 5Go). The molecular mechanism by which ICER affects ACTH secretion in these cells does not involve direct transcriptional deregulation of the POMC gene. Instead, we demonstrate that ICER influences POMC processing by down-regulating the expression of the gene encoding for the endoproteolytic enzyme PC1 (Fig. 6Go). It has been previously described that PC1 is involved in the tissue-specific processing of POMC, resulting in the release of mature products from the inactive precursor molecule (45). The PC1 promoter has been recently cloned, and it was demonstrated that PC1 tissue-specific expression and hormonal regulation require two distinct CREs within the proximal promoter region (41) (Fig. 6BGo). Recent analysis of protein-DNA interactions at the PC1 CRE regulatory elements has demonstrated that different members of the bZip superfamily or transcription factors bind specifically to these sequences (46). Here we have shown that the CREs in the PC1 promoter are direct targets of ICER. Therefore, we establish a direct link between cAMP-responsive nuclear factors and the expression of neuroendocrine-specific processing enzymes. Our results indicate that ICER is a major effector of the cAMP signal transduction pathway in neuroendocrine cells and reinforces its role in the regulation of hormonal physiology.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture and Transfections
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 as described (17). At least three independent clones of each stably transfected cell line were used to perform the experiments described. Photomicrographs were taken using a Wild MPS 51S microscope (Leitz, Zürich, Switzerland). Cells were transfected by the calcium phosphate coprecipitation technique (47) with 10 µg total DNA. Depending on the experiment, 24 h after transfection cells were treated with 10-6 M forskolin (Sigma, St. Louis, MO) as 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. The results shown are representative of at least three independent experiments. PC1 constructs have been previously described (46).

Cell Cycle Analysis
The synchrony of the cells was monitored by flow cytometric analysis of cellular DNA content, after staining ethanol-fixed cells in a solution of 40 µg/ml ethidium bromide and 40 µg/ml of RNase A (Sigma). DNA content was determined by flow cytometry using an ATC 3000 cell sorter (Odam-Brocker, Wissembourg, France). When required, cells were synchronized at the G1/S boundary by aphidicolin (Sigma) or in metaphase by addition of nocodazole (Sigma) as described (48, 49).

RNA Analysis
Total RNA was extracted by the guanidinium thiocianate procedure as described previously (50). Typically, aliquots of 20 µg of total RNA were analyzed either by RNase protection as described (51) or blotted onto Hybond-N membranes (Amersham Corp, Arlington Heights, IL) and hybridized following instructions of the manufacturer. To score for cyclin A expression, a 236-bp internal HindIII fragment from murine cyclin A cDNA (positions 844–1080) was subcloned into pBluescript SK-. The plasmid DNA was then linearized at a unique NheI site (position 876) to prepare an RNA probe. Similarly, for cyclin B a 151-bp SphI fragment was excised from a human cyclin B cDNA (position 1467–1618). The resulting plasmid was linearized at a unique XmnI site (position 1363). For cyclin D1, a 281-bp HindIII fragment was excised from a human cyclin D1 cDNA (position 1031–1312), and the resulting plasmid was linearized at a unique StyI site (position 837). All three antisense probes were generated using the T7 RNA polymerase promoter. 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.

Protein Analysis
AtT20 cell cultures were harvested in PBS, and cell pellets were 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 (ECL) detection system (Amersham) in combination with a peroxidase-conjugated antibody.

Gel Mobility Shift Analysis
Nuclear extracts and mobility shift assays were done as previously described (49). A 32-bp double-stranded oligonucleotide (5'-TCGATCGCCTTGAATGACGTCAAGGCCGCG-AC-3') containing the cyclin A CRE (50) and two 20-bp double-stranded oligonucleotides containing the two PC1 CREs (5'-GGGATCTGACGTCAAGAGAT-3' and 5'-GGGA-TTTGACGTGTAAACAC-3') (41) were used as probes in the binding reactions.

Measurements of ACTH Levels
Cells were plated on six-well culture dishes and incubated for 12 h. After incubation, the medium was removed, cells were washed with PBS, and fresh medium including 10-6 forskolin was added. For the time course experiments, 300 µl-aliquots were removed from each well. ACTH was measured using the rat ACTH RIA Kit (Peninsula Laboratories, Belmont, CA) following the manufacturer’s instructions. Each experiment was performed in triplicate, and the results shown here are representative of at least three different experiments.


    ACKNOWLEDGMENTS
 
We would like to thank E. Borrelli, J. Pines, and J. Sobczak-Thepot for discussions; C. Brechot (Paris) for the cyclin A promoter; R. Müller (Marburg) for various cyclins probes; and all the members of the Sassone-Corsi laboratory for discussions and helpful support. The excellent technical assistance of E. Heitz and C. Waltzinger is greatly appreciated.


    FOOTNOTES
 
Address requests for reprints to: Paolo Sassone-Corsi, Biologie Moleculaire et Cellulaire, Institut de Genetique, 1 Rue Laurent Fries, Strasbourg, France.

This work was funded by grants from Centre National de la Recherche Scientifique, Institut National de la Santé et pour la Recherche Médicale, Centre Hospitalier Universitaire Régional, Rhône-Poulenc Rorer (Bioavenir), Fondation de la Recherche Médicale and Association pour Recherche sur le Cancer. M. L. was supported by the Ministerio Educacion y Ciencia (Spain) and 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
 

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