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
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ABSTRACT
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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.
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INTRODUCTION
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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).
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RESULTS
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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. 1A
). 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 . 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 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.
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The increase in ICER protein levels correlates with a significant
cadmium-mediated induction of the ectopic ICER gene transcription (Fig. 1B
). 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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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 cDNA
either in the sense [At-ICER(S)] or in the antisense [At-ICER (AS)]
orientation.
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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. 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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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.
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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. 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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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).
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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. 4B
). 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 (16 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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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.
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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. 6B
). 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.
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DISCUSSION
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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. 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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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 8441080) 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 14671618). 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 10311312), 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 manufacturers 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.
 |
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