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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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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).
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). The results show that ectopic ICER
expression is strongly induced upon cadmium treatment in At-ICER(S)
cells.
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). 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. 1B). 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. 1C; 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. 1C; see
accompanying paper).
Both the overexpression and blockage of ICER function in AtT20 cells
significantly affect their morphology (Fig. 2). 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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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. 3). 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. 1C; 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. 3, left panel).
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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. 4A). 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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). 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. 4B). 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. 1C; 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. 3) 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. 4C). 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. 4C) 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. 5). 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. 6A). 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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). Thus, the
cAMP-signaling pathway regulates ACTH synthesis at multiple levels. We
have analyzed PC1 expression in forskolin-stimulated AtT20 cells (Fig. 6A). 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. 1C 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. 6, 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 6D 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 |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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. 3). Furthermore, we have shown that cyclin A
expression is down-regulated upon induction of ectopic ICER (Fig. 4).
However, induced ICER ectopic expression alone, although associated
with a significant repression of cyclin A mRNA (Fig. 5), is not
sufficient to affect passage through the cell cycle (Fig. 3). 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
activator isoform has no effect upon AtT20
cell cycle progression (our unpublished results). Given that CREM
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. 2). 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. 5). 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. 5). 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. 6). 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. 6B). 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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. 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
