Cyclic AMP (cAMP) Effects on
Chorionic Gonadotropin Gene
Transcription and mRNA Stability:
Labile Proteins Mediate Basal
Expression Whereas Stable
Proteins Mediate cAMP
Stimulation
Vivian L. Fuh, Jacky M. Burrin, and J. Larry Jameson
Thyroid Unit
Massachusetts General Hospital
Harvard Medical School
Boston, Massachusetts 02114
modification of preexisting proteins. However, other
labile proteins are required for gene transcription
and mRNA stability and the effects of cAMP on a
and CG# mRNA accumulation occur in part via selective mRNA stabilization. (Molecular Endocrinology 3: 1148-1156, 1989)
Cyclic AMP stimulates a marked accumulation of
CG« and CG/? mRNAs that reflects, in part, increased
rates of gene transcription. We find that a major
component of cAMP stimulation of a and CG/9
mRNAs is independent of new protein synthesis.
After treatment of JEG-3 choriocarcinoma cells with
cycloheximide, basal levels of a and CG0 mRNAs
decreased over 12 h to 27% and 13% of control
values, respectively. However, cycloheximide treatment did not affect the degree of cAMP-stimulation
of a and CG/3 mRNA levels which increased 20- and
26-fold, respectively. Similarly, cycloheximide did
not block cAMP-stimulated transcription of the a and
CG/? genes. The effect of cAMP treatment on a and
CG/? mRNA stability was assessed by decay after
removal of cAMP, pulse-chase analyses, and decay
after inhibition of RNA synthesis by actinomycin D.
The half-lives of a and CG/? mRNAs determined by
decay rates after removal of cAMP were 6.0 h and
7.2 h, respectively. Consistent with these measurements of mRNA stability, a and CG/9 mRNA half-lives
determined by pulse-chase analyses were 8.8 h and
8.6 h, respectively. Cyclic AMP treatment increased
the half-lives of a and CG£ mRNAs 1.8- and 3.4-fold,
respectively. Thus, the effects of cAMP on a and
CG/? gene expression are predominantly transcriptional, but cAMP also increases mRNA levels via a
posttranscriptional mechanism. Inhibition of RNA
synthesis by treatment with actinomycin D caused a
rapid decay of both a (tV2 = 2.8h) amd CG/5 (t1/2 =
3.1 h) mRNAs with no apparent stabilization by
cAMP, providing evidence that labile proteins also
stabilize basal a and 0 mRNA levels. We conclude
that cAMP stimulates a and CG/? transcription via
INTRODUCTION
CG is a placental hormone that stimulates steroidogenesis after implantation of the fertilized ovum (1). CG is
a heterodimer composed of a- and /?-subunits that are
encoded by separate genes (2). Cyclic AMP stimulates
the biosynthesis and secretion of CG in placental explants as well as in cell lines of placental origin (1, 3).
The effects of cAMP on CG biosynthesis are largely
pretranslational. For example, a and CG/3 mRNA levels
increase 10- to 30-fold after treatment of choriocarcinoma cell lines with 8-bromo-cAMP for 24 h (4-7). Two
lines of evidence indicate that cAMP acts at a transcriptional level. First, nuclear run-on assays demonstrate a
4- to 10-fold increase in the transcription rates of the a
and CG/? genes after treatment with 8-bromo-cAMP (5,
6). Second, transient expression of transfected fusion
genes that are linked to the a or CG/? promoters is
increased markedly after treatment with 8-bromo-cAMP
(5, 8-12). Based upon these studies, it is clear that the
effects of cAMP on a and CG/3 gene expression occur
in large part due to increased gene transcription rates.
However, it is not known whether the effects of cAMP
on CG gene expression occur directly via preexisting
proteins as opposed to indirectly via an intermediate
step involving new protein synthesis. Differences in the
kinetics of cAMP-stimulation of a and CG/? transcription
rates (6), mRNA accumulation (4, 5), and activation of
0888-8809/89/1148-1156502.00/0
Molecular Endocrinology
Copyright © 1989 by The Endocrine Society
1148
CGa and /3 mRNAs
transiently expressed fusion genes (12) have led to the
hypothesis that the a-gene may be activated directly,
whereas activation of the CG/3 gene may occur indirectly. Moreover, it is also important to assess potential
effects of cAMP on mRNA stability which could also
influence the rate and degree of mRNA accumulation
(13).
To further characterize the cellular mechanisms that
regulate basal and cAMP-stimulated a and CG/3 gene
expression, we determined the effects of blocking new
protein synthesis on transcription rates, mRNA stability,
and steady state mRNA levels. We find that blocking
protein synthesis reduces basal a and CG/3 mRNA
levels, but has little effect on the degree of cAMP
stimulation, suggesting that cyclic AMP effects are
mediated via relatively stable, preexisting protein(s).
1149
A
-Cycloheximide
- CAMP
+ CAMP
+Cycloheximide
-cAMP
+ cAMP
a
CGB
Actin
B
a mRNA
CGBmRNA
20
CC
1 0
(9x)
E
RESULTS
Effects of Cycloheximide on cAMP-Stimulated
Accumulation of a and CG/3 mRNAs
Cycloheximide was used to inhibit protein synthesis in
JEG-3 cells. Treatment with cycloheximide (25 ng/m\)
was limited to 4 h because longer treatments were
toxic for JEG-3 cells when the treated cells were assessed for subsequent viability. A 4-h exposure to
cycloheximide inhibited protein synthesis by greater
than 95%. Inhibition of protein synthesis persisted for
at least 20 h after removal of cycloheximide (data not
shown).
An experimental paradigm was employed in which
JEG-3 cells were preincubated with cycloheximide for
1 h before addition of 8-bromo-cAMP. After 4 h of
treatment with cycloheximide, the media was replaced
without or with 8-bromo-cAMP and cells were incubated for an additional 8 h (Fig. 1). Cycloheximide
decreased the basal levels of a and CG/3 mRNAs by
73% and 87%, respectively (Fig. 1), indicating that
relatively labile proteins are involved either in transcription of these genes or in stabilization of the expressed
mRNAs. Because basal actin mRNA levels were minimally affected by treatment with cycloheximide, the role
of the labile proteins appears to be relatively specific
for the a and CG/3 genes and/or mRNA transcripts.
Treatment of JEG-3 cells with 8-bromo-cAMP caused
a marked stimulation of both a and CG0 mRNAs (Fig.
1) (4-7). Pretreatment with cycloheximide decreased
the absolute levels of both a and CG/3 mRNAs after
stimulation with 8-bromo-cAMP. However, when compared with basal levels after cycloheximide treatment,
cAMP-stimulation of a (20-fold) and CG/3 (26-fold)
mRNA levels was unaffected or even enhanced by
treatment with cycloheximide. These results indicate
that new protein synthesis is not required for cAMPstimulation of a or CG/3 mRNA levels by 8-bromocAMP, suggesting that cAMP-stimulation occurs via
relatively stable preexisting protein(s).
The effects of cycloheximide treatment on the kinet-
-CYCLOHEX +CYCLOHEX -CYCLOHEX +CYCLOHEX
Fig. 1. Effect of Cycloheximide on Basal and cAMP-Stimulated
a and CG/3 mRNA Levels
JEG-3 cells were incubated in the absence or presence of
1 ITIM 8-bromo-cAMP for 12 h. Some groups of cells were
preincubated for 1 h with cycloheximide (25 ng/n\\) which was
removed 4 h later. Total RNA (20 /xg) from triplicate plates of
cells was analyzed by Northern blots for a, CG/3, and actin
mRNAs. A, Autoradiograms of a (6-h exposure), CG/3 (6-day
exposure), and actin (16-h exposure) mRNAs. The autoradiograms shown were overexposed to allow visualization of
basal mRNA levels. B, The mRNA levels shown in A were
quantified using scanning densitometry. a and CG/3 mRNA
levels were corrected for hybridization to actin. The values
(means ± SEM) shown are relative to the basal mRNA level in
the absence of cycloheximide or 8-bromo-cAMP. The fold
increase by treatment with 8-bromo-cAMP is shown in parentheses.
ics of a and CG/3 mRNA stimulation by 8-bromo-cAMP
were assessed to determine the rapidity of onset of
cycloheximide and to ensure that effects on cAMP
stimulation did not change over time (Fig. 2). In the
absence of cycloheximide, treatment with 8-bromocAMP increased a mRNA within 2 h and caused a 15fold stimulation over 26 h. In contrast, stimulation of
CG0 mRNA was not detected until 6 h of treatment,
but the degree of stimulation exceeded 100-fold after
26 h. Cycloheximide treatment inhibited basal a and
CG/3 mRNA levels throughout the measured time
course (data not shown). After cycloheximide treatment, cAMP-stimulation of a mRNA was reduced by
30-40% as early as 3 h after treatment and inhibition
persisted throughout 26 h of stimulation (Fig. 2A).
cAMP-Stimulated CG/3 mRNA levels were inhibited by
cycloheximide treatment within 7 h and were consistently reduced thereafter by 70-80%. Effects on CG/3
mRNA may have occurred at earlier time points, but
the levels of expression were too low for accurate
MOL ENDO-1989
1150
Vol 3 No. 7
B
a mRNA
CGB mRNA
1 50
-Cyclohex. + cAMP
-Cyclohex.+cAMP
100
+ Cyclohex,+cAMP
50
+Cyclohex,+cAMP
-Cyclohex,-cAMP
0
5
-Cyclohex,-cAMP
5
1 0 1 5 2 0 2 5 3 0
10 1 5 2 0
25
30
TIME (Hrs)
TIME (Hrs)
Fig. 2. Kinetics of a and CG/3 mRNA Accumulation after Treatment with Cycloheximide
JEG-3 cells were incubated in the absence or presence of 1 mM 8-bromo-cAMP for various lengths of time. Some groups Of
cells were preincubated for 1 h with cycloheximide (25 ng/m\) which was removed after 4 h. Treatment groups are indicated in the
figure. Total RNA (20 ^g) was analyzed for a mRNA (A) or CG/3 mRNA (B) levels by Northern blot hybridization. Actin mRNA was
used to correct for variations in applied RNA. Results are the mean of duplicate treatment groups and the values are relative to
basal RNA levels.
quantification. Thus, the effects of cycloheximide on a
and CG/3 gene expression occur rapidly and the kinetics
for cAMP stimulation of a and CG/3 mRNAs are not
substantially altered by inhibition of protein synthesis.
There was no evidence over this time course for superinduction by cycloheximide as might occur if the synthesis of labile repressors were blocked by inhibition of
protein synthesis.
Effects of Cycloheximide on 8-Bromo-cAMP
Stimulation of a and CG/3 Gene Transcription
Nuclear run-on experiments were performed to assess
the effect of inhibition of protein synthesis on cAMPstimulated a and CG/3 gene transcription. JEG-3 cells
were treated with 8-bromo-cAMP for 12 h in the absence or presence of pretreatment with cycloheximide
(Table 1). In the basal state, the transcription rate for
the a-gene was low (0.3 ppm) and CG/3 gene transcription was below detection. As shown previously (5, 6),
Table 1. Effects of Cycloheximide on cAMP Stimulation of a
and CGjS Gene Transcription Rates
Transcription Rate (ppm)a
+CAMP
Basal
-CHX
a
CG/3
a
0.3
—
+CHX
-CHX
+CHX
3.2
2.3
0.9
0.5
JEG-3 cells were incubated in the absence (basal) or presence of 1 mM 8-bromo-cAMP (+cAMP) for 12 h. Cycloheximide
(25 Mg/ml) (CHX) was added 1 h before addition of 8-bromocAMP and it was removed after 4 h. Nuclei were isolated and
used for transcription run-on assays as described in Materials
and Methods. Data are mean values from duplicate treatment
groups. - , Below detection.
treatment with 8-bromo-cAMP for 12 h stimulated the
transcription rates for both the a and CG/3 genes by at
least 10-fold. After pretreatment with cycloheximide,
basal a-gene expression was reduced, but cAMP-stimulated transcription was not blocked. The effects of
cycloheximide on basal CG/3 gene transcription could
not be assessed due to the low transcription rates.
However, cAMP treatment increased CG/3 gene transcription despite pretreatment with cycloheximide.
Taken together with the effects of cycloheximide on
mRNA levels, these results suggest that inhibition of
protein synthesis reduces basal transcription, but does
not prevent the cAMP-stimulated component of transcription.
Decay of a and CG/f? mRNAs after Removal of
8-Bromo-cAMP
Although cAMP is known to mediate most, if not all, of
its effects on a and CG/3 mRNAs at the transcriptional
level, it is important to assess whether some of these
effects occur posttranscriptionally. We used three independent techniques to estimate the half-lives of a
and CG/3 mRNAs. Withdrawal of 8-bromo-cAMP was
used to determine the decay rates of a and CG/3
mRNAs from the cAMP-stimulated state. JEG-3 cells
were treated with 8-bromo-cAMP for 16 h, washed
extensively, and incubated in the presence or absence
of 8-bromo-cAMP for various lengths of time before
isolation of RNA for Northern blot analyses (Fig. 3). In
the presence of 8-bromo-cAMP, a and CG/3 mRNA
levels were maintained at the stimulated level throughout the course of the experiment. However, after removal of 8-bromo-cAMP, a and CG/3 mRNA levels
decayed with half-lives of 6.0 h and 7.2 h, respectively.
CGa and /3 mRNAs
1151
B
CGR mRNA
a mRNA
+ cAMP
1 00
80
60
LLJ
DC
CE
2=6.0h
20
0
5
10
15
TIME AFTER WITHDRAWAL (Hrs)
E
o
o
+ CAMP
40
20
t1/2=7.2h
0
5
10
15
TIME AFTER W I T H D R A W A L
2 0
(Hrs)
Fig. 3. Rates of Decay of a and CG/3 mRNAs after Removal of 8-Bromo-cAMP
JEG-3 cells were treated with 1 mM 8-bromo-cAMP for 16 h after which media were removed and cells were washed and
replenished with fresh media. The cells were subsequently incubated in the absence or presence of 1 mM 8-bromo-cAMP for
various lengths of time before harvest for isolation of RNA. a (A) and CG/3 (B) mRNA levels were determined from duplicate plates
of cells as described in the previous figures. The values shown are relative to the mRNA level 16 h after initiation of treatment with
8-bromo-cAMP. Note that ordinate is plotted on a logarithmic scale and a least squares fit of the data was used to derive the
mRNA decay rates.
Pulse-Chase Analyses of a and CG0 mRNA
Turnover Rates
Pulse-chase analyses were used to estimate a and
CG/3 mRNA half-lives in the absence or presence of 8bromo-cAMP. JEG-3 cells were treated with 8-bromocAMP for 16 h to allow incorporation of the 3H-labeled
uridine pulse after near-maximal induction of a and CG/3
mRNAs (Figs. 1, 3) (5). After a 4-h pulse, the cells were
chased with unlabeled uridine and cytidine for various
lengths of time either in the absence or the presence of
8-bromo-cAMP (Fig. 4). 3H-Labeled RNA was hybridized to filters containing excess a or CG/3 cDNAs to
measure the levels of labeled a and CG/3 mRNAs remaining after initiation of the chase. In the absence of
cAMP, the half-lives of the a and CG/3 mRNAs were
8.8 h and 8.6 h, respectively. Addition of 8-bromocAMP caused a transient increase in labeled a and CG/3
mRNAs, likely reflecting transcriptional activation during
the intial phase of the chase (14). However, after 4 h of
the chase, the 3H-labeled UTP pooled was depleted by
greater than 90%. The half-lives o f « and CG/3 mRNAs
in the presence of cAMP were 16.6 h and 29.0 h,
respectively. Thus, addition of 8-bromo-cAMP increases the half-life of a mRNA 1.8-fold and CG0 mRNA
3.4-fold.
Effects of Actinomycin D on the Decay Rates of a
and CG/? mRNAs
The effects of 8-bromo-cAMP on the stability of a and
CG/3 mRNAs was also examined after treatment with
an inhibitor of RNA synthesis, actinomycin D. In the
absence of prior stimulation by cAMP, treatment with
actinomycin D markedly decreased (>70%) the basal
levels of a and CG/3 mRNAs and the addition of cAMP
had little, if any, effect on the rapid decline in mRNA
levels (Fig. 5A).
After stimulation of a and CG/3 mRNA levels with 8bromo-cAMP for 16 h, the mRNA decay rates after
treatment with actinomycin D were measured more
readily. In the absence or presence of 8-bromo-cAMP,
a mRNA levels decayed with a half-life of 2.8 h. CG/3
mRNA also decayed rapidly (t/2 = 3.1 h) after treatment
with actinomycin and its half-life was unaffected by
addition of 8-bromo-cAMP. Therefore, both the a and
CG/3 mRNAs are less stable after treatment with actinomycin D, suggesting that ongoing synthesis of relatively labile protein(s) is required to maintain RNA stability. Moreover, no effect of cAMP on mRNA stability
was found after treatment with actinomycin D suggesting that labile factors may be necessary for cAMPmediated stabilization of mRNA.
DISCUSSION
A fundamental feature of CG biosynthesis is that distinct a- and /3-peptides, that are the product of separate
genes (2, 15), must be combined to form biologically
active hormone. In most instances, the rates of a and
CG/3 subunit biosynthesis rise and fall in parallel to meet
the demands for formation of intact hormone (1). For
example, when secretion of CG is maximal during the
first trimester of pregnancy, both the a and CG/3 genes
are highly expressed, but their activity declines in the
latter stages of gestation (16, 17). Similarly, treatment
with agents such as 8-bromo-cAMP increases CG secretion and stimulates expression of both the a and
CG/3 genes (1, 4, 6). These examples of coordinant
expression of the a and CG/3 genes raise the possibility
Vol 3 No. 7
MOL ENDO-1989
1152
B
a mRNA
CGB mRNA
+CAMP
+CAMP
t1/2=29.0h
t 1 / 2 = 16.6h
t-j 12=
5
10
15
20
25
8.8h
30
CHASE (Hrs)
2
5
10
15
20
= 8.6h
25
CHASE(Hrs)
Fig. 4. Pulse-Chase Analysis of the Effects of 8-Bromo-cAMP on the Turnover Rates of a and CG/3 mRNAs
JEG-3 cells were treated with 1 rriM 8-bromo-cAMP for 16 h and then pulsed with [3H]uridine for 4 h in the presence of 8-bromocAMP. After removal of the media, cells were washed twice and replenished with chase media containing 5 mM uridine and 2.5 rriM
cytidine. At the indicated intervals after the chase, cells were harvested for isolation of RNA. Specific 3H-labeled RNA was measured
by hybridization to filters containing excess a or CG/3 cDNAs as described in Materials and Methods. The data points are the mean
of duplicate plates of cells and are plotted relative to the mRNA level at the end of the 4-h pulse. Specific hybridization of « and
CG/3 mRNAs were 757 cpm and 572 cpm/106 cells at the end of the pulse. A least squares fit of the data was used to derive the
mRNA half-lives.
that the cellular mechanisms that regulate these genes
may be similar or identical. On the other hand, there
are examples of unbalanced expression of the a and
CG/3 genes. Although both a and CG/3 mRNA levels
decline after the first trimester of pregnancy, there is a
striking increase in the ratio of a/CG(3 mRNAs by late
gestation (16, 17). In addition, the a-subunit is expressed alone or in great excess relative to the CG/3subunit in certain neoplasms (18, 19). These studies
taken together with evidence that the kinetics of cAMPinduction of a and CG/3 gene expression are different,
imply that the cellular mechanisms that regulate a and
CG/3 gene expression may be fundamentally different.
In this report, we demonstrate that cAMP stimulation
of a and CG/3 gene expression does not require new
protein synthesis, eliminating mechanisms in which
cAMP-stimulated expression of one gene is regulated
directly, whereas the other gene is induced indirectly
by synthesis of a new regulatory protein or transcription
factor. Furthermore, we find that labile factors dramatically influence basal expression levels and mRNA stability, and that inhibition of protein synthesis affects
basal CG/3 gene expression to a greater degree than a
gene expression.
The mechanisms for cAMP regulation of a-gene transcription have been well characterized. Cyclic AMP
increases the transcription rate of the a gene as assessed either by nuclear run-on assays (5, 6) or in
transient expression assays (8,11). We now show that
cAMP also stabilizes a mRNA, however, to only a small
degree. Thus, effects of cAMP on the steady state level
of a mRNA predominantly reflect increased transcription of the a-gene. The cAMP responsive regions of the
a-gene have been delineated by extensive mutagenesis
of the promoter. A repeated 18 base-pair (bp) element
that contains the palindrome, TGACGTCA, functions
both as a basal enhancer and as a cAMP-responsive
element (CRE) (9-11). A related DNA sequence is found
in several other cAMP-responsive genes (20) and a 3843 kilodalton protein(s) termed CREB, interacts specifically with the CRE (21, 22). CREB is a phosphoprotein
and it functions as a transcription factor as indicated by
its ability to mediate transcription in vitro (23). Although
treatment of cells with cAMP stimulates transcriptional
activity, it does not increase the amount of CRE-binding
activity in cellular extracts (24). The fact that new
protein synthesis is not required for cAMP-stimulation
of a gene expression is consistent with a mechanism
in which the effects of cAMP are mediated at a posttranscriptional level, likely via phosphorylation of CREB.
These results also suggest that CREB is relatively
stable or that it is present in great excess relative to
the amounts required for maximal stimulation of gene
expression. Additional evidence that CREB is relatively
stable is provided by the fact that it is heat stable (25)
and that binding activity is retained after denaturation
and renaturation (22).
Cyclic AMP responsive sequences in the CG/? gene
have been localized to a 450-bp region of the 5'-flanking
sequence, but the precise sequence determinants for
cAMP responsiveness have not been defined (12). Interestingly, there are no sequences within this 450 bp
region of the CG/3 gene that resemble the a CRE, even
allowing for several sequence mismatches. Given the
relatively strict sequence determinants for CREB binding to the a CRE (26, 27), it is likely that distinct proteins
will be found to mediate cAMP stimulation of a and
CG0 gene transcription. Because cycloheximide does
not prevent cAMP-stimulation of CG/? gene expression,
a posttranslational modification is also likely to mediate
cAMP activation of CG/3 gene expression.
CGa and /3 mRNAs
1153
•
BASAL
UJ
1.0
UJ
_J
0.8
ACTINOMYCIN
-cAMP
0.6
ACTINOMYCIN
+CAMP
z
DC
E
RELATI
UJ
0.4
0.2
0
a mRNA
CGB mRNA
a mRNA
CGB mRNA
CAMP
= 2.8h
cAMP
t1/2=3.1h
t1/2=3.1h
0
5
10
15
20
TIME AFTER ACTINOMYCIN (Hrs)
0
5
10
15
20
TIME AFTER ACTINOMYCIN (Hrs)
Fig. 5. Effects of 8-Bromo-cAMP on the Rates of Decay of a and CG/3 mRNAs after Treatment with Actinomycin D
A, Effects of actinomycin D and 8-bromo-cAMP on basal a and CG/3 mRNA levels. JEG-3 cells were incubated in the absence
or presence of 1 mui 8-bromo-cAMP for 12 h. For plates in which actinomycin D was added, JEG-3 cells were preincubated for 1
h with actinomycin D (5 (IM) which was removed after 4 h. Total RNA from triplicate plates of cells was analyzed for a and CG/3
mRNAs as described in previous figures. B, Effects of actinomycin D and 8-bromo-cAMP on the decay of cAMP-stimulated a and
CG/3 mRNA levels. After treatment of JEG-3 cells with 1 ITIM 8-bromo-cAMP for 16 h, media were removed and the cells were
washed twice and replenished with fresh media containing actinomycin D (5 HM) with or without 1 rtiM 8-bromo-cAMP. At the
indicated intervals after addition of actinomycin D, duplicate plates of cells were harvested for isolation of RNA. a and CG/3 mRNA
levels were corrected for hybridization to actin as described in previous figures. A least squares fit of the data beginning with the
2-h time point was used to derive the mRNA decay rates, a mRNA levels at 16 h were below detection.
Potential effects of cAMP on a and CG/3 mRNA
stability have not been addressed in previous studies.
In the basal state, a and CG/3 mRNA half-lives are
similar, whether assessed by rates of mRNA decay
after removal of 8-bromo-cAMP or by pulse-chase kinetics. In contrast, after inhibition of transcription with
actinomycin D, the decay rates for both « and CG/3
mRNAs are markedly increased, suggesting that labile
proteins are involved in mRNA stabilization. Treatment
with 8-bromo-cAMP increased the half-lives of a, and
to a greater degree, CG/3 mRNA. As with any treatment
that affects both transcription and mRNA stability, the
apparent mRNA half-life can be altered to the extent
that the chase is incomplete or that the treatment alters
nucleotide pools (13). We found that the chase depleted
the UTP pool by greater than 90% within 4 h. Thus,
cAMP-stimulated transcription should not account for
the alterations in mRNA half-lives, particularly for the
data points after 4 h. Moreover, cAMP did not affect
[3H]incorporation into total RNA and it increased the
half-life CGjS mRNA to a greater degree than a mRNA,
consistent with specific effects rather than an influence
on nucleotide pools. Cyclic AMP has been reported to
stabilize other mRNAs including lactate dehydrogenase
A (28) and osteocalcin mRNAs (29). The mechanisms
for mRNA stabilization by cAMP are not known. However, for certain transcripts, such as the transferrin
receptor mRNA, specific sequences that interact with
cellular proteins have been identified (30, 31).
Several mechanisms could account for more rapid
accumulation of a mRNA relative to CG|8 mRNA after
treatment with cAMP. For experiments in which the
approach to steady state is assessed, alterations in
mRNA half-life by treatment with cAMP could affect the
MOL ENDO-1989
1154
rate of approach to steady state (13). Our results demonstrate, however, that the changes in half-life induced
by cAMP are relatively small and because CG/3 mRNA
is stabilized to a greater degree than a mRNA, the
alterations in half-life should not account for the delay
in CGjS gene expression. In addition, measurements of
transcription rates and activation of promoters linked
to a common reporter gene should not be influenced
by differences in mRNA stability. Thus, the alterations
in kinetics appear to arise at the level of transcriptional
activation.
Differences in the kinetics for activation of a and CG/?
gene transcription whether assessed by nuclear run-on
assays or by transient expression of reporter genes,
may be due in part to the inability to accurately measure
changes in CG/3 promoter activity below a certain
threshold level (6, 12). More sensitive techniques for
measuring promoter activity, such as the luciferase
assay, may allow this issue to be readdressed. In
contrast to the cAMP-stimulated component of expression, inhibition of protein synthesis profoundly affects
the basal level of expression, particularly for the CG/3
gene. Thus, inhibition of protein synthesis causes an
absolute reduction in mRNA levels that can be attributed to the loss of factors involved in basal expression
rather than cAMP stimulation. A similar situation is
found for the «1-acid glycoprotein gene in which the
apparent requirement for ongoing protein synthesis for
glucocorticoid induction is due to the interaction of a
labile factor with a proximal promoter element rather
than an indirect mechanism for glucocorticoid action
(32).
Given that new protein synthesis is not required for
the cAMP activated component of expression of either
the a or CGj8 genes, the delay in CG/3 gene activation
may be due to a requirement for rate-limiting steps in
basal transcriptional activation that do not occur for the
a-gene. In this regard, it is noteworthy that the CG/3
gene does not contain characteristic promoter elements
such as a TATA box (33). Transcriptional activation of
the CG/3 gene may therefore involve proteins and mechanisms that are distinct from those that initiate transcription from TATA box recognition elements.
It is apparent from these studies and from earlier
work that multiple factors are involved in transcription
of the a and CG/3 genes. This report provides evidence
that factors such as the cAMP-responsive proteins are
preexisting and relatively stable. These proteins can
rapidly modulate transcription after posttranslational
modification. Other factors, such as those involved in
basal transcription and mRNA stability, are relatively
labile, providing a mechanism by which the activity of
other genes can rapidly influence expression of the a
and CGj8 genes. Identification of the multiple DNA regulatory elements in the a and CG/3 promoters will allow
isolation and characterization of the cellular factors that
mediate basal and regulated transcription.
Vol.3 No. 7
60-mm plates and grown in Dulbecco's modified Eagle's medium containing 5% fetal calf serum, 5% calf serum, penicillin
(100 U/ml), and streptomycin (100 Mg/ml).
RNA Isolation and Hybridization
RNA was isolated by extraction in guanidinium thiocyanate
and centrifugation through a cesium chloride cushion (34).
RNA concentration was determined by measuring the absorbance at 260 nm. Total RNA was treated with glyoxal (35),
fractionated by electrophoresis through 1.4% agarose gels,
and transferred to Nytran (Schleicher & Schuell, Keene, NH)
membranes. 32P-Labeled oligonucleotides were hybridized to
RNA as described previously (5). Oligonucleotides were synthesized on an Applied Biosystems 380A DNA synthesizer
(Foster City, CA) and are complementary to the sense strand
at the following positions relative to the translational start
codon: a-subunit (codons 63-69), CG/3 (codons 108-112),
actin (codons 15-25). Oligonucleotides were 5'-end labeled to
a specific activity of approximately 5 x 106 cpm/pmol using T4
polynucleotide kinase (36). Alternatively, CG/3 hybridization
probes were prepared using oligonucleotide-primed cDNA synthesis corresponding to a sense strand template that spans
codons 56 to 73 of the human CG/35 gene (15). After autoradiography, membranes were stripped completely of hybridized
oligonucleotide by heating to 90 C for 10 min in 10 mM sodium
phosphate, pH 7, and then rehybridized with a different labeled
oligonucleotide. RNA levels were quantified by scanning appropriately exposed film images with an LKB 2202 laser densitometer (LKB Instruments, Rockville, MD). Autoradiograms
were exposed to allow quantification of peak areas within the
linear response range of the film.
Transcription Run-on Assays
JEG-3 cells were treated as described in Results and nuclei (1
x 107) were isolated and resuspended in 150 ti\ transcription
buffer (20% glycerol, 50 mM HEPES, 5 mM MgCI2, 5 mM
MgOAC, 150 mM NH4CI, 2 mM dithiothreitol, 7.5 mM creatine
phosphate, 20 U creatine phosphokinase, pH 8.0) containing
0.5 mM each of ATP, GTP, and CTP, and 500 MCi [a-32P]UTP
(600 Ci/mmol, New England Nuclear, Boston, MA). After incubation at 37 C for 10 min, labeled RNA (8 x 106 cpm) was
isolated by digestion with DNase I (35 U) and proteinase K
(100 jig) (5, 37) followed by extraction in guanidinium thiocyanate and centrifugation through a CsCI cushion as described
above using 50 ng rat liver mRNA as carrier. Specific a and
CG/3 transcripts were hybridized to membranes containing
excess plasmid cDNAs (5 ^9) and quantified as described
previously (5).
Pulse-Chase Procedure
JEG-3 cells (1 x 106) were treated with 1 mM 8-bromo-cAMP
for 16 h and then incubated with 1.5 ml media containing 330
/tCi/ml 5,6[3H] uridine (Amersham Searle, Arlington Heights,
IL) and 1 mM 8-bromo-cAMP for 4 h. The cells were then
washed twice and incubated with 5 ml media containing 5 mM
uridine and 2.5 mM cytidine with or without 8-bromo-cAMP for
up to 24 h. The [3H]UTP pool determined by polyethyleniminecellulose TLC (38) was decreased by 9 1 % after 4 h of the
chase. At various intervals, cells were harvested for isolation
of RNA as described above. 3H-Labeled RNA (10 M9) was
hybridized to immobilized a and CG/3 cDNAs as described
previously (5). Control filters containing pGEM-7 plasmid DNA
were included in each hybridization to allow determination of
nonspecific binding (10-15% of specific binding at the start of
the chase).
MATERIALS AND METHODS
Cell Culture
Acknowledgments
JEG-3 choriocarcinoma cells (HTB 36) were obtained from the
American Type Culture Collection. Cells were subcultured into
We thank J. Fiddes and H. Goodman for providing the a and
CG/3 cDNAs and P. Behn for oligonucleotides. We are grateful
CG« and /3 mRNAs
to Chris Lindell for contributions to initial experiments and R.
DiBlasi and R. Mooradian for typing the manuscript.
Received March 13,1989. Revision received April 18,1989.
Accepted April 18, 1989.
Address requests for reprints to: Dr. J. Larry Jameson,
Massachusetts General Hospital, Thyroid Unit, Boston, Massachusetts 02114.
This work was supported P.H.S. Grant HD-23519, and by
the Upjohn Scholar and Chugai Faculty awards (to J.L.J.).
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