Differential Regulation and Transcriptional Control of Immediate Early Gene Expression in Forskolin-Treated WEHI7.2 Thymoma Cells
Dailing Mao,
Elizabeth A. Warner,
Susan A. Gurwitch and
Diane R. Dowd
E. A. Doisy Department of Biochemistry and Molecular
Biology Saint Louis University Health Sciences Center St.
Louis, Missouri 63104
 |
ABSTRACT
|
---|
Agents that increase intracellular cAMP are
frequently growth inhibitory for lymphocytes and induce apoptosis in
cortical thymocytes by regulating gene expression. In the present
study, immediate early gene expression was examined in WEHI7.2 thymoma
cells undergoing cAMP-mediated apoptosis. Temporal differences in
c-fos, junB, and inducible cAMP early repressor
(ICER) steady-state mRNA levels were observed after forskolin exposure.
Maximal induction of c-fos and junB occurred
within 1 h, returning to basal levels by 3.5 h. In contrast,
a 1.5-h time lag was observed before ICER transcript levels increased,
reaching maximal levels after 3.5 h. This rise in expression,
correlating with the decrease in c-fos and junB
levels, preceded apoptotic DNA fragmentation by 1.5 h. Transient
expression of ICER promoter constructs demonstrated that cAMP
responsiveness occurred through cAMP-autoregulatory response element
(CARE)3/4, two of the four proposed response elements in the ICER
promoter. In contrast to the cAMP-responsive cell line JEG-3, CARE1/2
was not functional for cAMP-activated transcription in WEHI7.2 cells.
An observed differential binding pattern of WEHI and JEG nuclear
extracts to these elements may account for the cell-specific
differences in expression patterns. To determine the role of endogenous
ICER in regulating gene expression, cells were treated with two
sequential doses of forskolin after which ICER and c-fos
mRNA levels were measured. The high levels of cAMP-induced ICER
expression dramatically reduced a second induction of
c-fos. These data suggest that ICER expression may function
as an antioncogene to attenuate the expression of certain
protooncogenes, thereby preventing transformation and oncogenesis due
to continuous overexpression. Moreover, inhibition of
growth-stimulatory genes may be required for the activation of the cell
death machinery in specific cells.
 |
INTRODUCTION
|
---|
The balance between cellular differentiation, proliferation, and
cell death is controlled by complex signaling pathways. These pathways
include such diverse molecules as receptors, kinases, and transcription
factors interacting in concert to exert an effect on growth and
viability. A variety of pathways act in the differentiating thymocyte
as the cell undergoes positive and negative immunological selection.
Mitogenic agents trigger maturation and differentiation, whereas
exposure to glucocorticoids and the E series prostaglandins lead to
apoptotic cell death in certain populations of thymocytes.
PGE1 and PGE2 signal through a receptor/G
protein/adenylate cyclase-coupled pathway. As cAMP levels in the cell
rise, the subsequent activation of protein kinase A (PKA) results in
the phosphorylation of cytoplasmic and nuclear proteins. One of the
nuclear targets is the transcription factor cAMP response
element-binding protein (CREB). In the thymocyte cell line WEHI7.2, we
have shown that forskolin treatment results in the phosphorylation of
the CREB homodimer and subsequent cAMP response element
(CRE)-dependent transcription (1). While the activation of
cAMP-mediated transcription requires the phosphorylation of CREB,
transcriptional attenuation may be due in part to its dephosphorylation
(2, 3, 4). A refractory period begins 68 h after stimulation of
adenylate cyclase, lasts for 35 days, and represents a time during
which additional stimulation of adenylate cyclase does not result in
the activation of transcription. The down-regulation of the c subunit
of PKA appears to be responsible for the observed refractory period
(5). Thus, three phases have been observed for cAMP-mediated
transcription: the activation phase, the attenuation phase, and the
refractory phase.
The recent discovery of the cAMP response element modulator (CREM)
proteins suggests that the activity of the functional CREB dimer is
regulated not only by its phosphorylation state, but also by
protein/protein interactions (6, 7). CREM
, CREMß, CREM
, and
inducible cAMP early repressor (ICER) are alternatively spliced
isoforms of the CREM gene, whose protein products function to repress
CREB activity either by the formation of a nonfunctional CREB:CREM
heterodimer or by competition between CREM and CREB homodimers for
binding to a CRE. The ICER isoform utilizes a distinct promoter located
in an intron and is itself induced by cAMP (6). The induction of ICER
was originally identified in neuroendocrine cells but more recently has
been shown to occur during some stages of lymphocyte development (8).
The cAMP inducibility of the ICER promoter may be due to the presence
of four cAMP-autoregulatory response elements (CAREs) located in
two clusters of two elements each (6). Each cluster was shown to exert
cAMP inducibility in JEG-3 choriocarcinoma cells and was able to bind
the ICER protein in vitro. Moreover, ICER is proposed to
down-regulate its own expression and to down-regulate the expression of
cAMP-inducible genes. Thus, CREM/ICER may function to attenuate the
transcriptional response to cAMP.
The cellular effects of agents that increase cAMP are varied. For
example, cAMP stimulates proliferative pathways in some endocrine cells
and antiproliferative pathways in many immune cells. Increases in cAMP
levels in developing thymocytes block differentiation by blocking cell
cycle progression and by inducing apoptosis in a subset of cells (9, 10). Furthermore, cAMP-induced apoptosis in thymocytes is preceded by a
G1 block in the cell cycle (11) and proceeds through a mechanism
requiring activated gene expression (12, 13). The molecular bases of
cAMP cell cycle arrest and apoptosis are poorly understood.
In this study, we examined the effect of forskolin, an activator of
adenylate cyclase, on the expression of the immediate early genes
c-fos, junB, and ICER in the apoptotic thymocyte.
The c-fos and junB protooncogenes have previously
been shown to be cAMP-responsive, and each have been implicated in
apoptosis induced by a variety of stimuli (14, 15, 16, 17, 18). Moreover,
junB expression has been implicated as a negative regulator
of cell growth in neuronal cells, fibroblasts, and mammary carcinoma
cells (19, 20). ICER regulation by cAMP was detected recently in
developing and mature human T lymphocytes, and it was suggested that
ICER may function in the circadian control of lymphocyte development
and activity (8) similar to its proposed role in the pineal gland (21, 22). Because of the importance of cAMP signaling in the developing
thymocyte, it was of considerable interest to determine whether these
immediate early genes were induced during cAMP-induced apoptosis. We
observed a rapid and transient induction of the c-fos and
junB protooncogenes and a delayed induction of ICER
expression. The kinetics of ICER expression was similar for the thymoma
cells and a neuroendocrine cell line; however, there were qualitative
differences that may be explained by thymoma-specific complexes bound
to the ICER promoter. Moreover, ICER induction precedes DNA
fragmentation in the WEHI7.2 cells and thus may represent an early step
in the apoptotic cascade. The induced endogenous ICER expression
correlated with the attenuated expression of c-fos and a
reduced responsiveness to a second treatment of forskolin. Thus, ICER
may function in the attenuation of the cAMP response, in an early
transcriptional refractory period, as well as in apoptosis of
thymocytes.
 |
RESULTS
|
---|
Rapid Induction of c-fos, junB, and
ICER in Forskolin-Treated WEHI7.2 Precedes DNA Fragmentation
The WEHI7.2 murine thymoma cell line was used in this study as a
model system for cAMP-mediated activation of gene expression and
apoptosis (1, 23, 24). To determine whether the expression of
c-fos, junB, and CREM genes is induced by cAMP in
the WEHI7.2 cell line, cells were treated with forskolin for various
times, and mRNA was analyzed by Northern blot protocols (Fig. 1A
). An increase in steady-state CREM
mRNA levels was detected within 1.5 h. A maximal 10-fold increase
in CREM RNA levels was observed at 3.5 h, after which time
expression was reduced essentially to basal levels by 24 h. This
gradual decline after CREM induction is consistent with negative
autoregulation by CREM/ICER on ICER gene transcription (6).
c-fos and junB were also induced by forskolin,
although the kinetics of induction are significantly different than
CREM. c-fos and junB steady-state mRNA levels
increased within 20 min and were maximal at 1.5 h. Transcript
levels were attenuated to basal levels by 3.5 h and were reduced
to below basal levels by 6 h. Thus, the increase in CREM
expression correlated with the decrease in c-fos and
junB.

View larger version (34K):
[in this window]
[in a new window]
|
Figure 1. Effect of Forskolin on Expression of Immediate
Early Genes and Apoptosis
A, WEHI7.2 cells were incubated with 10 µM forskolin for
the indicated time periods. mRNA was isolated and 3 µg were subjected
to Northern blot analysis. The resulting bloti was sequentially hybridized with probes for CREM,
c-fos, junB, and -actin (for
normalization). B, WEHI7.2 cells were preincubated with 10 µg/ml
cycloheximide for 20 min to block protein synthesis. This was effective
at blocking 96% of protein synthesis as analyzed by incorporation of
[35S]methionine in newly synthesized proteins (40).
Forskolin (10 µM) then was added to the cells and
incubated for 20 min or 3 h. mRNA was isolated and analyzed as in
panel A. f, Forskolin; c, cycloheximide; c/f, cycloheximide
pretreatment followed by forskolin treatment. C, WEHI7.2 cells were
incubated with 10 µM forskolin for the indicated time
periods. Soluble DNA fragments were extracted and separated by
electrophoresis on a 1.5% agarose gel (see Materials and
Methods for details).
|
|
The cAMP-mediated induction of Fos and Jun family members has been
shown to be a primary response, independent of protein synthesis.
Indeed, the rapid induction of c-fos and junB in
the WEHI7.2 cells was not blocked by cycloheximide at levels that
inhibited protein synthesis by 96% (Fig. 1B
). Moreover, in the
cycloheximide-treated cells, the return of c-fos and
junB mRNA to basal levels was inhibited. Similarly,
cycloheximide was ineffective at blocking the induction of CREM
transcripts. Three hours of cycloheximide and forskolin cotreatment led
to a 26-fold induction of CREM whereas forskolin alone induced CREM
expression 10-fold. These data are consistent with the hypothesis that
the ICER protein represses its own expression in a negative
autoregulatory loop and suggests that CREM/ICER may also function to
attenuate the cAMP-induced expression of c-fos and
junB.
The relative kinetics of induced gene expression and cell death were
compared. Because WEHI7.2 cells excluded vital dyes for up to 16 h
after treatment with forskolin (D. Dowd, unpublished observations), we
used DNA fragmentation as a marker of apoptosis. During apoptosis, the
DNA is cleaved into double-stranded fragments whose lengths are
multiples of 180 bp, the length associated with the nucleosome (25, 26). DNA cleavage was observed after 3 h of forskolin treatment,
and the concentration of fragments increased over time (Fig. 1C
). Thus,
the induction of CREM/ICER gene expression precedes DNA fragmentation
by approximately 1.5 h.
ICER Is the CREM Isoform Induced by Forskolin
The CREM gene is spliced to yield multiple
isoforms of the protein, which can differ by the presence or absence of
the N terminus and by the DNA-binding domain used (6, 7). Using a
full-length CREM
cDNA probe, we detected induction of four
transcripts: 2.3, 1.6, 1.4, and 1.1 kb. To specifically identify which
of the CREM products were regulated, we used probes specific for the N
terminus and C terminus of CREM (Fig. 2A
). The 3'-CREM probe, which encodes
C-terminal sequences common to all CREM and ICER isoforms, demonstrated
an induction pattern virtually identical to the full- length probe with
an approximately 10-fold induction in each of the four transcripts. In
contrast, the 5'-probe, specific for CREM
, -ß, -
, and -
but
absent in the ICER isoforms, demonstrated multiple transcripts with no
induction of expression of the 2.3-kb band and a 1.6-fold induction of
the smaller 1.4- and 1.1-kb species. JEG-3 choriocarcinoma cells
treated with forskolin demonstrated only a single transcript
hybridizing to the 5'-sequence that was not regulated by cAMP. A
pattern of induced transcripts similar to WEHI7.2 was observed when the
3'-ICER-specific probe was used (Fig. 2B
), consistent with that
previously reported (6). It is interesting to note that although the
kinetics of induction appear similar in WEHI7.2 and JEG-3, the pattern
of transcripts was slightly different. Whereas the level of the 2.3-kb
transcript increased 10-fold in WEHI7.2 cells, it represented only a
small fraction of the regulated transcripts in the JEG-3 cells.

View larger version (25K):
[in this window]
[in a new window]
|
Figure 2. The ICER Isoform Is Regulated in WEHI7.2 and JEG-3
Cells
A, WEHI7.2 cells were incubated with 10 µM forskolin for
the indicated time periods, mRNA was isolated, and 3 µg were
subjected to Northern blot analysis. The blot was probed sequentially
with full-length CREM, CREM (3'), and CREM (5'). CREM (3') encodes
sequences of CREM corresponding to nucleotides 389745 of CREM cDNA
and common to CREM/ICER; the CREM (5') probe corresponds to
nucleotides -1 to 353 of CREM cDNA, which do not
hybridize to ICER isoforms; -actin was used for normalization. B,
JEG-3 cells were incubated with 10 µM forskolin for the
indicated time periods, and mRNA was isolated and analyzed as in panel
A. C, RNase protection to determine which DNA-binding domain is encoded
by the induced ICER isoform. RNA from WEHI7.2 cells treated with
ethanol vehicle (-f) or 10 µM forskolin (+f) for 3
h was hybridized to probe 1 (specific for DBD I, lanes 15), probe 2
(to detect DBD II of ICER II or intron/DBD II encoded by ICER I, lanes
611), and probe 3 (actin control, lanes 1215). Lanes 3 and 8 ( )
contain sense RNA produced using CREM cDNA and is a control for the
presence of DBD I and intron/DBD II. Lane 9 (ß) contains sense RNA
produced using CREMß cDNA as a control for DBD II (ICER II) in the
absence of DBD I and intronic sequences. Lanes 2, 7, and 13 (tRNA)
represent complete digestion of probe in the absence of WEHI7.2 RNA and
presence of tRNA. The lengths of the probes and protected fragments are
given in nucleotides (nt). The presence of multiple bands with each of
the CREM probes corresponds to partial RNase cleavage of protected
probe fragments or incomplete digestion in the presence of RNA. Shown
are representative experiments.
|
|
The 3'-probe used for Northern blot analysis encoded
both DNA-binding domain (DBD) I and DBD II and thus is capable of
detecting isoforms that utilize either of the DBDs. We employed
ribonuclease (RNase) protection assays to determine whether the ICER
isoform(s) induced in WEHI7.2 cells used DBD I or II, (ICER I and II,
respectively; Fig. 2C
). Probe I was used to detect DBD I, specifically.
A DBD I-specific fragment was protected in ethanol-treated cells (lane
4), which comigrated with the protected band resulting from
hybridization with CREM
. This band was induced in cells treated with
forskolin (lane 5). These observations were confirmed using probe II, a
probe that contained a portion of DBD II and noncoding sequence between
DBD I and DBD II. This noncoding sequence is spliced from ICER II, and
thus this probe generates two products of different sizes when
hybridized to CREM/ICER isoforms encoding DBD I, DBD II, and the
intervening sequence (e.g. ICER I, CREM
; lane 8) or
those encoding only DBD II (e.g. ICER II, CREM ß; lane 9).
We observed a forskolin-induced increase in the protected band
comigrating with DBD I, and no detectable band corresponding to the
exclusive use of DBD II (compare lanes 10 and 11). Thus, these data
indicate that the ICER isoform induced by forskolin in WEHI7.2 cells
utilizes DBD I.
CARE3/4 Directs cAMP-Induced Expression in WEHI7.2 Cells
Because of the observed differences in transcript pattern between
WEHI7.2 thymocytes and JEG-3 choriocarcinoma cells (Fig. 2
, A and B),
ICER promoter activity was examined in transient expression studies in
these two cell lines. The ICER native promoter, containing
approximately 400 bp of 5'- untranslated sequence, was isolated by PCR
amplification of genomic DNA and cloned into a CAT reporter vector.
This ICER-CAT construct conveyed cAMP- inducible CAT activity in
WEHI7.2 and JEG-3 cells (Fig. 3
). The
ICER promoter encodes a cluster of four CRE-like elements (termed
cAMP-autoregulatory elements, CAREs) that direct cAMP inducibility in
JEG-3 choriocarcinoma cells (6). The 62-bp cluster of four CAREs
conferred cAMP inducibility to the thymidine kinase (tk) promoter (27),
as did two copies of the CREs from the
-glycoprotein hormone
promoter (1, 27, 28). This CARE14 construct was positively responsive
to forskolin in both cell lines. The CAREs were further divided into
two segments, CARE1/2 and CARE3/4, and tested for cAMP inducibility in
WEHI7.2 and JEG-3 cells. CARE1/2 demonstrated cAMP inducibility in
JEG-3 cells, in agreement with the observations of Molina et
al. (6), but it was ineffective in WEHI7.2 cells. In contrast,
CARE3/4 was responsive to forskolin in both JEG-3 and WEHI7.2 cells.
However in WEHI7.2 cells, the level of CAT activity measured utilizing
the CARE3/4tkCAT construct was 3-fold less than that obtained with
CARE14tkCAT. This suggests that although CARE1/2 was unresponsive to
cAMP, it augments the activation of CARE3/4. CARE3 and CARE4 were
examined separately for the ability to induce expression from a
CAREtkCAT expression vector. Both CARE3 and CARE4 were capable of
sustaining forskolin-activated transcription in WEHI7.2 and JEG-3 cells
(data not shown).

View larger version (19K):
[in this window]
[in a new window]
|
Figure 3. Transcriptional Activation by cAMP in WEHI7.2 and
JEG-3 Cells
WEHI7.2 cells or JEG-3 cells were transfected by lipofection with the
pCRE(2)tkCAT control plasmid or with ICER-CAT, CARE14tkCAT,
CARE1/2tkCAT, or CARE3/4tkCAT reporter constructs. (Plasmid
construction is detailed in Materials and Methods).
Cells were treated with 5 µM forskolin for 16 h and
extracts analyzed for CAT activity. Fold induction is reported relative
to vehicle-treated cells ± SEM, n 2.
|
|
The sequences of CARE1 (TGAGCTGCA) and CARE2 (TGATGGCA) each are 62%
identical to the consensus palindromic CRE, TGACGTCA. In comparison,
CARE4 (TGATGTCA) differs by only 1 bp from the palindrome, and CARE3 is
a perfect CRE. Thus, we reasoned that the inability of forskolin to
activate the CARE1/2tkCAT construct in WEHI7.2 cells may be due to a
reduced affinity for binding to CREBs. Gel mobility shift analyses were
employed to determine the relative affinity of CREM and CREB for the
CARE1/2 and CARE3/4 DNA fragments (Fig. 4
). Whereas CREB
and CREM
bound
avidly to the CARE3/4 oligonucleotide (Fig. 4A
, lanes 914), binding
of equivalent amounts of extracts to CARE1/2 was greatly reduced (lanes
27). Note that at the highest levels of CREB or CREM protein
extracts, virtually all of the CARE3/4 probe is bound, whereas only a
small fraction of the CARE1/2 probe is bound by these proteins. When
CARE3/4 was used as the probe, both the CREB and CREM extracts resulted
in two major bands of retarded mobility. These complexes may correspond
to the occupancy of one CARE (band I) and two CAREs (band II). CARE1/2
demonstrated only a single major band with the CREB and CREM extracts
comigrating with CREB band I and CREM band I, respectively. These data
suggest that only one of the two CARE sites is occupied at these
protein concentrations.

View larger version (61K):
[in this window]
[in a new window]
|
Figure 4. Mobility Shift Analysis of ICER Fragments Bound to
CREB or CREM
A, CREB and CREM have higher affinity for binding to CARE3/4 than to
CARE1/2. Lanes 17, Extracts were incubated with
32P-labeled CARE1/2 probe. Lanes 814, Extracts were
incubated with 32P-labeled CARE3/4 probe. Lanes 1 and 8,
Free probe; lanes 24 and 911, increasing amounts of CREB extract
(0.16, 0.32, and 0.64 µg); lanes 57 and 1214, increasing amounts
of CREM extract (0.2, 0.6, and 1.2 µg). The protein-DNA complexes
were resolved from free probe with a nondenaturing polyacrylamide gel
and visualized by autoradiography of the dried gel. The filled
arrows labeled I and II represent CREB occupancy of one or two
CARE sites, respectively; the open arrows labeled I and
II represent CREM occupancy of one or two CARE sites, respectively. B,
DNA competition for binding to CREB and CREM. CREB extract (0.06 µg;
lanes 17), or 0.16 µg CREM extracts (lanes 915) were preincubated
in the absence of DNA-radiolabled probe, with increasing amounts of
unlabeled CARE1/2 oligonucleotide (5-, 20-, and 100-fold excess; lanes
24 and 1012) or with increasing amounts of unlabeled CARE3/4
oligonucleotide (5, 20, and 100-fold excess; lanes 57 and 1315).
32P-labeled CARE3/4 probe was then added and incubated for
20 min, and the complexes were analyzed as in panel A. Filled
arrows labeled I and II represent CREB occupancy of one or two
CARE sites, respectively; open arrow labeled I
represents CREM occupancy of one CARE site. At this concentration of
extract we observed minimal occupancy of both sites. Lane 8 contains
0.16 µg control COS7 extract. Note that there are no protein-DNA
complexes at this concentration of extract when CREM is not expressed.
|
|
The lower affinity of CARE1/2 compared with CARE3/4 was confirmed in
competition assays (Fig. 4B
). As before, the level of CREB protein used
resulted in the appearance of two slowly migrating bands (lane 1),
presumably due to the occupancy of one or both CAREs. The amount of
CREM extract used in this experiment produced primarily one band,
corresponding to the occupancy of one CARE (lane 9, Fig. 4B
; and lane
12, Fig. 4A
). A 20-fold excess of unlabeled CARE3/4 was effective at
eliminating CREB-specific band II (lane 6) and reducing the
CREM-specific band by 50% (lane 14), whereas a 100-fold excess
completely eliminated binding of labeled CARE3/4 to extracts containing
recombinant CREB or CREM (lanes 7 and 15). A 100-fold excess of CARE1/2
was an ineffective competitor when CREB or CREM protein was bound to
CARE3/4 (lanes 4 and 12). When CREB was bound to the CARE3/4 probe,
there did appear to be slight reduction in band II with 100-fold excess
of CARE1/2. This suggests that there was a small competition effect of
the CARE1/2 oligonucleotide reducing the amount of probe in which both
CARE sites were occupied.
Both CARE3 and CARE4 were efficient at binding CREB and CREM. Indeed,
the binding affinities of CREB and CREM to each of these sequences were
similar. Gel mobility shift DNA-binding assays demonstrated that both
CARE3 and CARE4 were effective competitors for CREB and CREM binding to
a 32P-labeled CARE3 probe (data not shown). The ability of
CREB to bind to both CARE3 and CARE4 is consistent with the ability of
both of these elements to confer cAMP responsiveness on a heterologous
reporter system (Ref. 6 and data not shown).
To further address the question of differential behavior of
CARE-driven transcription in WEHI7.2 and JEG-3 cells, we examined the
binding of nuclear proteins to the CARE1/2 and CARE3/4 oligonucleotides
(Fig. 5
). Increasing concentrations of
nuclear extracts were incubated with the CARE3/4 oligonucleotide and
examined by gel mobility shift analysis (Fig. 5A
). Extracts from both
cell lines formed a protein/DNA complex that comigrated with the
baculoviral-expressed CREB protein. Its identity as CREB was confirmed
with the use of an anti-CREB antibody, which resulted in a supershifted
protein/antibody/DNA complex. The WEHI7.2 extract also formed a complex
of slower mobility (denoted by the open arrow, lanes 35),
which was absent in the JEG-3 extract (lanes 79). This complex was
not supershifted by the anti-CREB antibody. However, the supershifted
CREB band comigrated with this unique band, making it difficult to
determine whether the antibody adversely affected it.

View larger version (28K):
[in this window]
[in a new window]
|
Figure 5. Mobility Shift Analysis of CAREs Bound to WEHI7.2
and JEG-3 Nuclear Extracts
A, Differential binding to CARE3/4. Lane 1, 0.03 µg
baculoviral-expressed CREB ; lane 2, 0.03 µg CREB incubated with
an anti-CREB antibody. Note the supershifted complex formed in the
presence of antibody. Lanes 35, Increasing concentrations of WEHI7.2 nuclear extract (0.2, 0.6, and 1.0
µg protein, respectively); lane 6, 1.0 µg WEHI7.2 nuclear extract
incubated with an anti-CREB antibody; lanes 79, increasing
concentrations of JEG-3 nuclear extract (0.2, 0.6, and 1.0 µg
protein, respectively); lane 6, 1.0 µg JEG-3 nuclear extract
incubated with an anti-CREB antibody. The WEHI7.2-specific complex is
denoted by an open arrow; CREB is denoted by a
filled arrow. B, Differential binding to CARE1/2. Lanes
are as in panel A with the exception that lanes 1 and 2 contain 0.15
µg baculoviral-expressed CREB . C, DNA competition for binding to
nuclear protein complexes. WEHI7.2 nuclear extract (1.0 µg) was
preincubated in the absence of DNA-radiolabeled probe, with increasing
amounts of unlabeled CARE1/2 oligonucleotide (2.5, 10, and 50-fold
excess; lanes 13) or with increasing amounts of unlabeled CARE3/4
oligonucleotide (2.5, 10, and 50-fold excess; lanes 57). Lane 4
contains WEHI7.2 extract in the absence of competitor DNA.
32P-Labeled CARE1/2 probe was then added and incubated for
20 min and the complexes were separated. The filled
arrow represents the complex common between WEHI7.2 and JEG-3
extracts; open arrows designate complexes specific for
WEHI7.2, and the asterisk denotes the only complex with
which CARE3/4 competes.
|
|
In addition, there were observed differences in the pattern of
complex formation when CARE1/2 was used as the probe (Fig. 5B
). Both
WEHI7.2 and JEG-3 extracts formed complexes that comigrated with
baculoviral-expressed CREB (filled arrow), however, these
complexes did not interact with the anti-CREB antibody, suggesting that
the complexes did not contain CREB. As with the CARE3/4 probe, WEHI7.2
extracts formed two additional protein/DNA complexes, which were absent
in the JEG-3 extracts (open arrows). The three complexes
formed by the WEHI extracts were competed with unlabeled CARE1/2
oligonucleotide (Fig. 5C
), but only the fastest migrating complex
(denoted by the asterisk) was competed by the CARE3/4
oligonucleotide, suggesting that this protein complex can bind to both
CARE1/2 and CARE3/4. In contrast, the remaining two slower migrating
bands were unaffected by CARE3/4, suggesting that they are specific for
the CARE1/2 oligonucleotide and are not capable of binding to a typical
CRE. These data support the conclusion that the protein/CARE1/2 complex
that is seen in both WEHI and JEG extracts is not CREB, as CREB binding
the CARE1/2 would be competed by the CARE3/4 oligonucleotide.
Induction of c-fos Is Reduced after a Second Treatment
of Forskolin
The overexpression of ICER in transient expression studies blocks
the cAMP-induced expression of a number of cAMP-responsive promoter
constructs (8, 29, 30, 31). Moreover, ectopic expression of the CREM
transcriptional repressor in WEHI7.2 cells significantly inhibits the
responsiveness of a c-fos promoter to cAMP in agreement with
data reported earlier by Foulkes et al. (Ref. 32; and data
not shown). Although these experiments provide functional information
on CREM and ICER bioactivity, they do not address whether such an
inhibitory activity occurs in the context of native cellular levels of
endogenous ICER. We addressed this question by examining whether the
cAMP-induced expression of native ICER altered gene regulation after a
second stimulation of adenylate cyclase. Because activation of
adenylate cyclase by forskolin does not desensitize the enzyme with
respect to subsequent treatments (33, 34), we were
able to reactivate adenylate cyclase by removing the first treatment
and stimulating a second time with forskolin (see Materials and
Methods for details). Forskolin was added to WEHI7.2 cells for
3.5 h to achieve maximal levels of ICER expression. Then, cells
were harvested and resuspended in fresh media, and a second treatment
of forskolin was added. Gene induction in the presence of increased
ICER was then monitored (Fig. 6
;
quantification in Table 1
). As shown
previously, c-fos expression was induced approximately
3-fold at 0.5 h after the initial incubation with forskolin, and
the levels of c-fos dropped below basal levels by 3.5
h. In contrast, maximal levels of ICER message were observed at
3.5 h. At 3.5 h, cells were harvested and resuspended in
fresh media, and a second treatment with forskolin was immediately
initiated. After 0.5 h of additional incubation, c-fos
levels were only modestly induced and were not significantly higher
than basal levels. This observation could not be explained by a
reduction in CREB activation as the CREB protein was phosphorylated
after the second treatment to levels similar to the initial treatment
(Fig. 6
and Table 1
). Thus, induction of endogenous
ICER severely impaired c-fos expression.

View larger version (120K):
[in this window]
[in a new window]
|
Figure 6. Secondary Induction of cAMP-Mediated Gene
Expression
WEHI7.2 cells were treated with 10 µM forskolin. After
3.5 h incubation, media was replaced with fresh media and 10
µM forskolin was immediately added. Cells were incubated
for an additional 0.5 h. When indicated, a sample of cells was
harvested and divided for mRNA isolation or Western blot analysis. An
antiphospho-CREB antibody was used to examine CREB phosphorylation.
Lane 1, Control; lane 2, forskolin, 0.5 h; lane 3, forskolin
3.5 h; lane 4, forskolin 4 h; lane 5, forskolin 3.5 h +
forskolin 0.5 h. Experimental detail is described in
Materials and Methods. Quantification of the data is
included in Table 1 . Shown is a representative experiment.
|
|
It is worth noting that the first treatment of forskolin had to be
removed before the second incubation. If the second addition of
forskolin was placed directly in the initial forskolin-containing
media, an effect was not seen, i.e. CREB was not
phosphorylated and c-fos mRNA levels were unaffected (data
not shown). This is in agreement with earlier observations whereby
forskolin-treated membranes were washed before subsequent reactivation
of adenylate cyclase by this agent (33, 34). Thus, removal of the
initial forskolin treatment was absolutely required to reactivate
adenylate cyclase and subsequently rephosphorylate CREB.
 |
DISCUSSION
|
---|
We and others have shown that cAMP induces gene expression in
thymocytes, and the regulation of gene expression is critical for
induction of the apoptotic response. In the present study, we examine
the expression of immediate early genes in WEHI7.2 thymoma cells in
response to adenylate cyclase stimulation. Forskolin led to the rapid,
transient induction of c-fos and junB gene
expression. Expression of both genes was maximal within 20 min of
stimulation. In contrast, induction of ICER was not apparent for
1.5 h and was maximal at 3.5 h. Although slightly delayed in
comparison to c-fos and junB, additional protein
synthesis was not required, suggesting a primary transcriptional
response typical of immediate early genes. The kinetics of
cAMP-mediated induction of ICER were similar for an unrelated cell
line, JEG-3, suggesting similar pathways to up-regulation of
transcription of the ICER gene.
The ICER protein exists as multiple isoforms. RNase protection assays
revealed that the ICER protein(s) induced in WEHI7.2 cells encoded DBD
I. Isoforms that lacked DBD I and that used DBD II exclusively were not
detected. However, the Northern blot analysis suggests the presence of
multiple transcripts; therefore we cannot rule out the possibility that
there are additional isoforms that we were unable to distinguish by
these experiments.
Although ICER was induced in both WEHI7.2 and JEG-3 cells, there were
three differences in the expression and regulation of CREM/ICER between
the cell lines. First, 5'-sequences of CREM
hybridized to multiple
transcripts in WEHI7.2 cells whereas only a single transcript was
observed for JEG-3 cells. These transcripts were not regulated by
forskolin and may represent cell-specific differences in the basal
expression of various CREM isoforms. The significance of this finding
remains to be elucidated. Second, the levels of the 2.3-kb transcript
increased more in the WEHI7.2 cells relative to the JEG-3 cells. The
2.3-kb transcript represented only a minor species in JEG-3 cells
treated with forskolin, whereas it was a major isoform in the WEHI
cells. Third, regulation in JEG-3 cells occurred through both CARE1/2
and CARE3/4. Moreover, CARE3/4 was sufficient in JEG-3 cells for
maximal induction equivalent to CARE14. In WEHI7.2 cells, CARE3/4 was
able to support cAMP-induced transcription whereas CARE1/2 was
nonfunctional in transient transfection assays. Although CARE3/4
conferred cAMP sensitivity to the tk promoter, it was not sufficient
for full activity. Other sequences appear to be required for maximal
induction. In this regard, CARE1/2 augmented the activity of CARE3/4 to
produce the maximal effect. One possible explanation for this
observation is that these sequences serve as binding sites for other
proteins that are required for maximal activation. Gel mobility shift
experiments using nuclear extracts demonstrate the presence of multiple
complexes associated with CARE1/2 that are unique to WEHI7.2. Although
CARE1/2 does not support cAMP-mediated gene expression in WEHI7.2
cells, it may recruit specific proteins to the area, which leads to the
synergistic effect on transcription observed with CARE14 in WEHI7.2
cells. The absence of these complexes in JEG-3 cells may explain the
lack of synergy of CARE1/2 and CARE3/4 in these cells. This would
suggest the presence of cell type-specific factors in thymocytes that
function in the regulation of ICER transcription in a manner different
from choriocarcinoma cells.
How CARE1/2 functions in cAMP-mediated transcription in JEG-3 cells is
currently unclear. CREB from nuclear extracts did not bind to these
elements, in agreement with our results demonstrating a low affinity of
CREB for these sequences. However, a nuclear protein/DNA complex was
formed that was common to both JEG-3 and WEHI extracts. This protein(s)
may be responsible for the transcriptional activation observed. The
absence of a response in WEHI7.2 cells may be due to the presence of
other complexes not observed in JEG-3 extracts that may be inhibiting
the response to cAMP. There are more than 12 different CREB/activating
transcription factor (ATF) family members that bind to similar CRE-like
sequences and may have a higher affinity for these sequences than does
CREB. Thus the presence or absence of these may represent an additional
level of transcriptional control and specificity. We are currently
attempting to identify these factors to determine their potential role
in the response to cAMP.
The kinetics of cAMP-mediated induction and attenuation of
c-fos transcription in WEHI7.2 cells are typical of
immediate early genes: rapid induction followed by transcriptional
attenuation and down-regulation. Subsequent to the rapid induction of
c-fos, we detected a reduction in mRNA levels to basal
levels within 3 h, decreasing below basal levels within 6 h
after forskolin treatment. It is proposed that transcriptional
attenuation after cAMP induction of the somatostatin gene requires
phosphatase activation and dephosphorylation of CREB (2, 3, 4).
Although this mechanism is certainly of importance in the attenuation
of expression of genes such as somatostatin, its role in the
attenuation of c-Fos expression in WEHI7.2 cells is unclear.
Significant CREB phosphorylation is detected after 3 h of
forskolin treatment (1), a time when c-fos and
junB mRNA levels have returned to basal. CREM transcript
levels also are increased at 3.5 h after forskolin treatment,
suggesting that CREB remains functional at this time point. Thus, it is
unlikely that dephosphorylation of CREB plays a predominant role in the
attenuation of c-fos expression in WEHI7.2 cells. It has
been suggested that c-Fos may act to feedback and negatively regulate
its own expression after stimulation with growth factors (35, 36, 37). This
attenuation occurs through the palindromic sequence known as the dyad
symmetry element, which is also the element that confers phorbol ester
and serum responsiveness. Thus, the activation and repression of
fos expression by phorbol ester and serum occur through the
same DNA element. In comparison, the dyad symmetry element is not
responsive to cAMP, whereas the CREs located in the promoter are
responsive. Although it is possible that Fos may negatively regulate
cAMP-induced transcription, it is unlikely, since these transcription
factors act through separate response elements.
Another potential candidate for the transcriptional attenuation is
ICER. It is suggested that ICER functions to attenuate the
transcriptional response to cAMP (6, 8, 22, 29, 30, 31). In the WEHI7.2
cells, the induction of ICER correlates with the return of
c-fos and junB expression to basal levels,
suggesting that ICER may be involved in the attenuation phase of the
response. Cotreatment of cells with forskolin and cycloheximide
prevents the transcriptional attenuation of c-fos and
junB and indicates that new protein synthesis is required
for the rapid return of these oncogene transcripts to basal levels
(reviewed in Ref.38). This is consistent with the idea that one
component of the superinduction is the lack of ICER repression.
In addition to transcriptional attenuation, overexpression of ICER in
transient reporter assays prevents the induction of a variety of
cAMP-responsive genes (8, 29, 30, 31), and expression of the CREM
, ß
and
transcriptional repressors can inhibit cAMP induction of
c-fos in transient transfection assays (Ref. 32 and data not
shown). While these studies are paramount in understanding the role for
this family of transcriptional repressors, the levels of CREM/ICER in
the cells are superphysiological and thus do not represent levels of
protein actually occurring in the cell under normal physiological
conditions. Thus, we asked whether induced levels of native ICER were
sufficient to prevent the regulation of cAMP-induced gene expression at
time periods subsequent to transcriptional attenuation of the
c-fos gene. WEHI cells were treated with forskolin for
3.5 h, a time period required for induction of ICER and
attenuation of c-fos expression. Cells were then treated a
second time with forskolin and c-fos levels were measured.
Although c-fos levels increased, the relative levels
increased only to slightly above basal levels. The reduced induction of
c-fos could not be explained by the down-regulation of the
cAMP signaling machinery because the CREB protein was phosphorylated
after the second treatment to levels similar to the initial treatment.
These data indicate that: 1) adenylate cyclase was activated; 2) the
phosphatases responsible for attenuating the effects of CREB activation
did not prevent its reactivation; and 3) PKA was active; thus the
refractory period due to PKA down-regulation had not begun. Thus, other
factors appear to be responsible for preventing the relative levels of
the c-fos protooncogene from rising above the basal levels
upon subsequent activation.
In this regard, the production of transcriptional
attenuators/repressors such as ICER may be required for early stages in
the rapid down-regulation of cAMP-induced genes and the reduced
responsiveness to subsequent activation. The dephosphorylation of CREB
may prevent further activation of gene expression when cAMP levels
remain elevated. At later time periods, down-regulation of the
catalytic subunit of PKA functions to make the cell refractory to
stimulation by the same agent. This concerted effort to reduce Fos in
the cells after stimulation and keep levels low may be required to
prevent oncogenesis. Deregulated expression of c-fos results
in neoplastic transformation of rat fibroblasts (reviewed in Ref.38).
Thus, ICER may function as an antioncogene by preventing protooncogenes
such as c-fos from being expressed at high levels for an
extended period of time, a condition supporting neoplasia.
In WEHI7.2 thymoma cells, the induction of ICER immediately precedes
the detection of apoptosis-associated DNA cleavage. Thus, ICER may
function in the early steps of apoptosis in cells that have an intact
death cascade. The growth-promoting effects of protooncogenes might be
dominant to death factors and block apoptosis. By down-regulating
protooncogenes such as c-fos, ICER may be allowing for the
death program to proceed. Moreover, ICER may function in part by
inhibiting cell growth by down-regulating cell cycle genes. For
example, cyclin A expression and cell cycle progression are induced by
cAMP. ICER may function to return the level of cyclin A to its
noninduced state and return the cells to the
G0/G1 stage of the cell cycle (39). Clearly,
the stable expression of ICER sense and antisense cDNA in WEHI7.2 cells
will help to address the issue surrounding the possible involvement of
ICER in both transcriptional attenuation and the apoptotic cascade, and
such studies will be instrumental in enhancing our understanding of
cAMP-induced gene expression leading to apoptosis.
 |
MATERIALS AND METHODS
|
---|
Materials
Forskolin was purchased from Sigma (St. Louis, MO), dissolved in
ethanol, and stored at -20 C in the dark.
Cell Culture
WEHI7.2 thymoma cells were grown in DMEM containing 10% calf
bovine serum, 0.063 g/liter penicillin, 0.1 g/liter streptomycin. JEG-3
cells were grown in MEM containing 10% calf bovine serum, 0.063
g/liter penicillin, 0.1 g/liter streptomycin. All cells were grown at
37 C in an atmosphere of 6% CO2 and 90% humidity.
Northern Blot Analysis
Northern analysis was performed as described previously (40)
with the following modification: mRNA was purified using a PolyATract
mRNA isolation system (Promega, Madison, WI), according to
manufacturers instructions, and 3 µg mRNA were subjected to
electrophoresis. Quantification of Northern blots was performed using a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
The analysis of gene expression after two treatments with forskolin was
performed as follows. Five aliquots of WEHI7.2 cells were pooled and
one aliquot (control, 0 h) was removed for Northern and Western
analyses. The remaining cells were treated with 10 µM
forskolin and aliquots removed for analysis after 0.5 h
(+forskolin, 0.5 h) and 3.5 h (+forskolin, 3.5 h). The
final two samples were divided, and one sample continued to incubate
for an additional 0.5 h (+forskolin, 4 h). The other sample
was harvested, and the cells were resuspended in fresh media, 10
µM forskolin was added, and the cells were incubated
0.5 h (+forskolin, 3.5 h; +forskolin, 0.5 h). Individual
pools were divided for the analysis of phospho-CREB by Western blot and
for mRNA isolation and Northern blot analysis.
DNA Fragmentation
DNA fragmentation was assessed by the methods of Wyllie et
al. (26). Briefly, cells were treated with 10 µM
forskolin for the indicated time points, harvested by centrifugation at
900 g, and lysed on ice in an excess volume of hypotonic
buffer consisting of 5 mM Tris-HCl, pH 7.5, 5
mM EDTA, and 0.5% Triton X-100. The lysates were
centrifuged at 13,000 x g, and the supernatant
fraction containing soluble DNA fragments were extracted two times with
phenol-chloroform-isoamyl alcohol (25:24:1) and once with
chloroform-isoamyl alcohol (24:1). The DNA in the supernatants was
precipitated in 66% ethanol and 300 mM sodium acetate, pH
5.3. Precipitates were resuspended in 10 mM Tris-HCl, pH
8.0, 1 mM EDTA and incubated for 30 min at 25 C with 25
µg/ml RNase. Soluble DNA fragments from equivalent number of cells
were separated by electrophoresis on a 1.5% agarose gel and visualized
by ethidium bromide staining.
RNase Protection
RNA was analyzed according to manufacturers directions using
the RPA II Ribonuclease Protection Assay Kit (Ambion, Austin, TX). The
cDNAs corresponding to CREM probe I [nucleotides 554653 of the CREM
cDNA sequence (7)] and probe II (nucleotides 841-1158) were
constructed in pBluescriptKS+ and 32P-labeled
probes were made using MAXIscript (Ambion). The mouse ß-actin
probe was supplied by Ambion and was used as a control for
normalization of RNA concentration. CREM
and CREMß cDNAs were
cloned into pSG5 and unlabeled RNA made using MAXIscript. Briefly 2
µg total RNA were hybridized to a 32P-labeled RNA probe,
single-stranded RNA was digested, and the product was separated on a
standard urea gel. The gel was dried and products were visualized by
autoradiography.
Gel Mobility Shift Analysis
Gel mobility shift analysis used procedures and conditions
described previously (41). The CARE1/2 probe consists of the following
sense-strand sequence corresponding to positions -154 to -121 in the
ICER promoter (6): TTTCAGTGAGCTGCACATTGATGGCAGTGATAGG. The CARE3/4
probe consists of the following sense strand sequence corresponding to
positions -120 to -92: CTGGTGACGTCACTGTGATGTCAGTGCTC.
CREM
-containing extracts were obtained by transfecting COS-7 cells
with pSG5-CREM
(7) and preparing nuclear extracts by the method of
Shapiro et al. (42). Control COS-7 extracts were obtained in
a similar manner from cells transfected with the pSG5 plasmid lacking
the cDNA insert. Murine CREB
cDNA was subcloned into the pVL1392
polyhedrin transfer plasmid for expression in a baculovirus system.
Recombinant baculovirus was isolated and plaque purified by standard
procedures (43). Sf-9 cells were infected for 48 h with CREB
recombinant baculovirus. Whole cell extracts were prepared by
sonication of infected cell pellets in 10 mM Tris-HCl (pH
7.8), 1 mM EDTA, 0.3 mM zinc acetate, 5
mM dithiothreitol, 0.3 M KCl, followed by
centrifugation at 200,000 x g for 30 min. WEHI7.2 and
JEG-3 nuclear extracts were prepared by the method of Shapiro et
al. (42). Supershift experiments were performed using an anti-CREB
antibody (New England Biolabs, Beverly, MA).
Construction of Reporter Plasmids
The ICER promoter from -378 to +1 from the translational start
site was isolated by PCR amplification of genomic DNA. DNA primers were
designed corresponding to position -378 to -365 and +1 to -13, and
standard PCR conditions were employed for amplification of genomic DNA
(44). The amplified sequence was subcloned into the ptkCAT plasmid (27)
in which the tk promoter was removed. CARE1/2tkCAT and CARE3/4tkCAT
were constructed using complimentary DNA oligonucleotides corresponding
to base pairs -154 to -121 and -120 to -92, respectively, and
subcloned into ptkCAT. The sequence of all constructs was confirmed
using a Sequenase kit (USB Corp., Cleveland, OH) according to
manufacturers recommendation.
Transient Transfection Assay
Logarithmically growing cells were harvested and resuspended in
DMEM, 0.5% calf bovine serum at a concentration of 3 x
106 cells/ml. Cells were transfected with the indicated
reporter plasmid (2 µg) for 5 h using Lipofectamine reagent
(Life Technologies, Gaithersburg, MD), according to manufacturers
recommendations. Then cells were diluted with DMEM, 10% calf bovine
serum to 6 x 105 cells/ml and treated with forskolin
or ethanol vehicle for 16 h. Cell extracts were prepared and
assayed for CAT activity as described (45).
Western Blot (Immunoblot) Analysis
Western blot analysis with antiphosphorylated CREB was performed
as previously described (1). Phosphorylated CREB was quantitated by
densitometric analysis of the resulting autoradiogram.
 |
ACKNOWLEDGMENTS
|
---|
We gratefully acknowledge the generosity of Pamela Mellon for
the tkCAT and pCRE(2)tk CAT plasmids, Günther Schütz for
the CREB
cDNA, Paolo Sassone-Corsi for the CREM
and CREMß cDNA
vectors, and M. Greenberg for the antiphosphorylated CREB antibody. We
would like to thank Paul MacDonald for help with baculovirus-mediated
expression of CREB and for helpful discussions. Special thanks go to
Darcy Krueger for critical reading of the manuscript and to Monique
Heitmeier and Michelle Benoit for technical assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Diane R. Dowd, Ph.D., E.A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University Health Sciences Center, 1402 South Grand Boulevard, St. Louis, Missouri 63104. E-mail: dowddr{at}wpogate.slu.edu
This work was supported by NIH Grant AI-35910 (to D.R.D.).
Received for publication September 9, 1997.
Revision received December 12, 1997.
Accepted for publication December 30, 1997.
 |
REFERENCES
|
---|
-
Dowd DR, Ryerse JS, MacDonald PN, Miesfeld RL,
Kamradt MC 1997 Crosstalk during Ca2+-, cAMP-, and
glucocorticoid-induced gene expression in lymphocytes. Mol Cell
Endocrinol 128:2937[CrossRef][Medline]
-
Hagiwara M, Alberts A, Brindle P, Meinkoth J, Feramisco J,
Deng T, Karin M, Shenolikar S, Montminy M 1992 Transcriptional
attenuation following cAMP induction requires PP-1 mediated
dephosphorylation of CREB. Cell 70:105113[Medline]
-
Wadzinski BE, Wheat WH, Jaspers S, Peruski Jr LF, Lickteig
RL, Johnson GL, Klemm DJ 1993 Nuclear protein phosphatase 2A
dephosphorylates protein kinase A-phosphorylated CREB and regulates
CREB transcriptional stimulation. Mol Cell Biol 13:28222834[Abstract]
-
Alberts AS, Montminy M, Shenolikar S, Feramisco JR 1994 Expression of a peptide inhibitor of protein phosphatase 1 increases
phosphorylation and activity of CREB in NIH 3T3 fibroblasts. Mol Cell
Biol 14:4398407[Abstract]
-
Armstrong R, Wen W, Meinkoth J, Taylor S, Montminy M 1995 A
refractory phase in cyclic AMP-responsive transcription requires down
regulation of protein kinase A. Mol Cell Biol 15:182632[Abstract]
-
Molina CA, Foulkes NS, Lalli E, Sassone-Corsi P 1993 Inducibility and negative autoregulation of CREM: an alternative
promoter directs the expression of ICER, an early response repressor.
Cell 75:875886[Medline]
-
Foulkes NS, Borrelli E, Sassone-Corsi P 1991 CREM gene: use
of alternative DNA-binding domains generates multiple antagonists of
cAMP-induced transcription. Cell 64:739749[Medline]
-
Bodor J, Spetz AL, Strominger JL, Habener JF 1996 cAMP
inducibility of transcriptional repressor ICER in developing and mature
human T lymphocytes. Proc Natl Acad Sci USA 93:35363541[Abstract/Free Full Text]
-
McConkey DJ, Orrenius S, Jondal M 1990 Agents that elevate
cAMP stimulate DNA fragmentation in thymocytes. J Immunol 145:12271230[Abstract/Free Full Text]
-
Lalli E, Sassone-Corsi P, Ceredig R 1996 Block of T lymphocyte
differentiation by activation of the cAMP-dependent signal transduction
pathway. EMBO J 15:528537[Abstract]
-
Lemaire I, Coffino P 1977 Cyclic Amp-induced cytolysis in S49
cells: selection of an unresponsive "deathless" mutant. Cell 11:149155[Medline]
-
Durant S, Homo-Delarche F 1983 Cytolytic effects of
dexamethasone and of agents stimulating cyclic AMP content in isolated
mouse thymocytes. Mol Cell Endocrinol 31:215225[CrossRef][Medline]
-
Anderson KL, Anderson G, Michell RH, Jenkinson EJ, Owen JJT 1996 Intracellular signaling pathways involved in the induction of
apoptosis in immature thymic T lymphocytes. J Immunol 156:40834091[Abstract]
-
Marti A, Jehn B, Costello E, Keon N, Ke G, Martin F, Jaggi R 1994 Protein kinase A and AP-1 (c-Fos/JunD) are induced during
apoptosis of mouse mammary epithelial cells. Oncogene 9:121323[Medline]
-
Gillardon F, Eschenfelder C, Uhlmann E, Hartschuh W,
Zimmermann M 1994 Differential regulation of c-fos, fosB, c-jun, junB,
bcl-2 and bax expression in rat skin following single or chronic
ultraviolet irradiation and in vivo modulation by antisense
oligodeoxynucleotide superfusion. Oncogene 9:321925[Medline]
-
Preston GA, Lyon TT, Yin Y, Lang JE, Solomon G, Annab L,
Srinivasan DG, Alcorta DA, Barrett JC 1996 Induction of apoptosis by
c-Fos protein. Mol Cell Biol 16:211218[Abstract]
-
Smeyne RJ, Vendrell M, Hayward M, Baker SJ, Miao GG, Schilling
K, Robertson LM, Curran T, Morgan JI 1993 Continuous c-fos
expression precedes programmed cell death in vivo. Nature 363:166169[CrossRef][Medline]
-
Grassilli E, de Prati AC, Monti D, Troiano L, Menegazzi M,
Barbieri D, Franceschi C, Suzuki H 1992 Studies of the relationship
between cell proliferation and cell death. II. Early gene expression
during concanavalin A-induced proliferation or dexamethasone-induced
apoptosis of rat thymocytes. Biochem Biophys Res Commun 188:12611266[Medline]
-
Schlingensiepen KH, Schlingensiepen R, Kunst M, Klinger I,
Gerdes W, Seifert W, Brysch W 1993 Opposite functions of jun-B and
c-jun in growth regulation and neuronal differentiation. Dev Genet 14:30512[Medline]
-
Schlingensiepen KH, Wollnik F, Kunst M, Schlingensiepen R,
Herdegen T, Brysch W 1994 The role of Jun transcription factor
expression and phosphorylation in neuronal differentiation, neuronal
cell death, and plastic adaptations in vivo. Cell Mol
Neurobiol 14:487505[Medline]
-
Stehle JH, Foulkes NS, Molina CA, Simonneaux V, Pévet P,
Sassone-Corsi P 1993 Adrenergic signals direct rhythmic expression of
transcriptional repressor CREM in the pineal gland. Nature 365:314320[CrossRef][Medline]
-
Foulkes NS, Duval G, Sassone-Corsi P 1996 Adaptive
inducibility of CREM as transcriptional memory of circadian rhythms.
Nature 381:8385[CrossRef][Medline]
-
Harris AW, Bankhurst AD, Mason S, Warner NL 1973 Differentiated functions expressed by cultured mouse lymphoma cells.
J Immunol 110:431438[Medline]
-
Dowd DR, Miesfeld RL 1992 Evidence that glucocorticoid- and
cyclic AMP-induced apoptotic pathways in lymphocytes share distal
events. Mol Cell Biol 12:36003608[Abstract]
-
Wyllie AH 1980 Glucocorticoid-induced thymocyte apoptosis is
associated with endogenous endonuclease activation. Nature 284:555556[Medline]
-
Wyllie AH, Morris RG, Smith AL, Dunlop D 1984 Chromatin
cleavage in apoptosis: association with condensed chromatin morphology
and dependence on macromolecular synthesis. J Pathol 142:6777[Medline]
-
Steger DJ, Altschmied J, Buscher M, Mellon PL 1991 Evolution
of placenta-specific gene expression: comparison of the equine and
human gonadotropin
-subunit genes. Mol Endocrinol 5:243255[Abstract]
-
Delegeane AM, Ferland LH, Mellon PL 1987 Tissue-specific
enhancer of the human glycoprotein hormone
-subunit gene: dependence
on cyclic AMP-inducible elements. Mol Cell Biol 7:39944002[Medline]
-
Monaco L, Foulkes NS, Sassone-Corsi P 1995 Pituitary
follicle-stimulating hormone (FSH) induces CREM gene expression in
Sertoli cells: involvement in long-term desensitization of the FSH
receptor. Proc Natl Acad Sci USA 92:1067310677[Abstract]
-
Lalli E, Sassone-Corsi P 1995 Thyroid-stimulating hormone
(TSH)-directed induction of the CREM gene in the thyroid gland
participates in the long-term desensitization of the TSH receptor. Proc
Natl Acad Sci USA 92:96339637[Abstract]
-
Tinti C, Conti B, Cubells JF, Kim K-S, Baker H, Joh TH 1996 Inducible cAMP early repressor can modulate tyrosine hydroxylase gene
expression after stimulation of cAMP synthesis. J Biol Chem 271:2537525381[Abstract/Free Full Text]
-
Foulkes NS, Laoide BM, Schlotter F, Sassone-Corsi P 1991 Transcriptional antagonist cAMP-responsive element modulator (CREM)
down-regulates c-fos cAMP-induced expression. Proc Natl Acad
Sci USA 88:54485452[Abstract]
-
Seamon KB, Daly JW 1981 Forskolin: a unique diterpene
activator of cyclic AMP-generating systems. J Cyclic Nucleic Res 7:201224
-
Seamon KB, Padgett W, Daly JW 1981 Forskolin: unique diterpene
activator of adenylate cyclase in membranes and in intact cells. Proc
Natl Acad Sci USA 78:33633367[Abstract]
-
Hartig E, Loncarevic IF, Buscher M, Herrlich P, Rahmsdorf HJ 1991 A new cAMP response element in the transcribed region of the human
c-fos gene. Nucleic Acids Res 19:41539[Abstract]
-
Konig H, Ponta H, Rahmsdorf U, Buscher M, Schonthal A,
Rahmsdorf HJ, Herrlich P 1989 Autoregulation of fos: the
dyad symmetry element as the major target of repression. EMBO J 8:255966[Abstract]
-
Schonthal A, Buscher M, Angel P, Rahmsdorf HJ, Ponta H,
Hattori K, Chiu R, Karin M, Herrlich P 1989 The Fos and Jun/AP-1
proteins are involved in the downregulation of Fos transcription.
Oncogene 4:62936[Medline]
-
Angel P, Karin M 1991 The role of Jun, Fos and the AP-1
complex in cell-proliferation and transformation. Biochim Biophys Acta 1072:12957[CrossRef][Medline]
-
Desdouets C, Matesic G, Molina CA, Foulkes NS,
Sassone-Corsi P, Breshot C, Sobczak-Thepot J 1995 Cell cycle
regulation of cyclin A gene expression by the cyclic AMP-responsive
transcription factors CREB and CREM. Mol Cell Biol 15:33013309[Abstract]
-
Dowd DR, MacDonald PN, Komm BS, Haussler MR, Miesfeld R 1991 Evidence for early induction of calmodulin gene expression in
lymphocytes undergoing glucocorticoid-mediated apoptosis. J
Biol Chem 266:1842318426[Abstract/Free Full Text]
-
MacDonald PN, Dowd DR, Nakajima S, Galligan MA, Reeder MC,
Haussler CA, Ozato K, Haussler MR 1993 Retinoid x receptors
stimulate and 9-cis retinoic acid inhibits
1,25-dihydroxyvitamin D3-activated expression of the rat
osteocalcin gene. Mol Cell Biol 13:59075917[Abstract]
-
Shapiro DJ, Sharp PA, Wahli WW, Keller MJ 1988 A
high-efficiency HeLa cell nuclear transcription extract. DNA 7:4755[Medline]
-
Summers M, Smith GE (eds) 1987 A Manual of Methods for
Baculovirus Vector and Insect Cell Culture Procedures. Texas
Agriculture Experimental Station and Texas A & M University, College
Station, TX, pp 157
-
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JD, Smith
JA, Struhl K 1988 Current Protocols in Molecular Biology. John Wiley &
Sons, Inc., New York
-
Rouet P, Raguenez G, Salier J-P 1992 Optimized assays for
quantifying transient expressions of co-transfected ß-galactosidase
and CAT reporter genes. BioTechniques 13:700701[Medline]