(Received for publication, February 10, 1995; and in revised form, July 13, 1995)
From the
The acetyl-CoA carboxylase (ACC) gene contains two distinct
promoters, denoted PI and PII. PI is responsible for the generation of
class I ACC mRNAs which are induced in a tissue-specific manner under
lipogenic conditions. PII generates class II ACC mRNAs which are
expressed constitutively. During 30A5 preadipocyte differentiation,
both promoters are activated; the preadipocytes must be pretreated with
cAMP for this activation to occur. In this report, we present evidence
that CAAT enhancer-binding protein- (C/EBP-
) is induced and
involved in the PI activation by cAMP. Expression of the reporter gene
under the control of the PI promoter is activated within 3 h after
treatment of 30A5 cells with a cyclic AMP analogue,
8-(4-chlorophenylthio)-adenosine 3`,5`-cyclic monophosphate, and
3-isobutyl-1-methylxanthine, in association with the accumulation of
C/EBP-
mRNA and protein. These accumulations were inhibited in the
presence of H8, a protein kinase inhibitor; H8 also inhibited
activation of PI by cAMP. However, the induction of reporter gene
expression and the increase of C/EBP-
mRNA by cAMP were not
affected by treatment with tumor necrosis factor
, which
completely inhibited the accumulation of C/EBP-
mRNA.
Overexpression of C/EBP-
by transfection with the C/EBP-
gene
led to increased binding of C/EBP-
to DNA and partial PI
activation. cAMP did not affect the amount of C/EBP-
binding to
the DNA but did promote phosphorylation of C/EBP-
and PI
activation. As in the case of C/EBP-
, C/EBP-
bound to the
CCAAT box of the PI promoter. These results indicate that cAMP not only
induces, but also activates, bound C/EBP-
through phosphorylation
for PI activation. Our studies also indicate that cAMP induces
C/EBP-
. C/EBP-
induction, however, precedes that of
C/EBP-
.
30A5 preadipocytes are derived from C3H 10T1/2 mouse fibroblasts
that can be differentiated into adipocytes(1, 2) . The
induction conditions for differentiation are pretreatment of the cells
at confluence with dexamethasone and insulin for 3 days, followed by
incubation in basal medium containing insulin alone. Under these
conditions, about 80% of the cells become laden with fat droplets by
day 7 or 8(1) . The differentiation can be accelerated by at
least 2-4 days when cells are pretreated with cAMP and
3-isobutyl-1-methylxanthine (IBMX) ()at confluence for as
little as 1 h instead of the dexamethasone and insulin
pretreatment(3) . Pretreatment of the cells can be omitted if
the cells are kept in the same medium for 5 days, so that some
nutrients become limited; these conditions also allow the cells to
differentiate upon incubation in medium containing insulin. During the
nutrient limitation of 30A5 cells, the intracellular level of cAMP
increases to a level comparable to cAMP and IBMX concentrations used in
the pretreatment(3) . These studies indicated that cAMP is
essential to induce differentiation of preadipocytes(3) .
Activation of acetyl-CoA carboxylase (ACC) gene expression, which is
closely associated with the differentiation, also requires cAMP
pretreatment of the cells(3, 4) . ACC gene transcripts
consist of multiple forms which are generated as a result of
differential splicing of two primary transcripts from two distinct
promoters, PI and PII(5, 6) . Previously, we
classified those mRNA species transcribed from PI as class I ACC mRNAs,
whereas those transcribed from PII were designated class II mRNAs.
Stimulated lipogenic conditions lead to induction of the class I ACC
mRNAs in the liver(5) . Likewise, induction of PI promoter
expression occurs during differentiation of 30A5 preadipocytes into
adipocytes(4) . Based on these observations, together with
others(5) , we suggested that PI is an inducible promoter under
stimulated lipogenic conditions, whereas PII is constitutively
expressed(5) . In a recent report we showed that accumulation
of CCAAT/enhancer-binding protein
(C/EBP-
) mRNA occurred in
close association with the PI gene products during the differentiation
of 30A5 preadipocytes(4) . Furthermore, we showed that
C/EBP-
binds to a specific sequence, GCAAT, in the PI promoter and
that binding allowed the expression of PI which was otherwise
repressed(4) . In this paper, we demonstrate that cAMP
treatment of 30A5 cells activates both C/EBP-
and C/EBP-
gene
expression, which lead to PI activation. However, in the case of
C/EBP-
, increased binding of C/EBP-
to the CAAT box alone is
not sufficient to activate PI unless the bound C/EBP-
is
phosphorylated and activated by cAMP.
Figure 1: cAMP action on various subdeleted PI-CAT constructs. The numbers in the diagram designate the subdeleted position of the promoter. pPI-997/CAT(mut) contains the PI promoter fragment (from -1008 to -12) from which the sequence GCAAT (the CCAAT box at -63/-67) was deleted. The construction of these plasmids is described under ``Experimental Procedures.'' Stable clones containing these plasmids were cultured for 24 h with and without cAMP and IBMX before analysis of CAT activity. The basal promoter activities and those in the presence of cAMP were analyzed. The standard deviations were obtained from three independent experiments.
The induction of CAT activity by cAMP occurred only with those cells containing the plasmids with the CCAAT box sequence. For example, cAMP routinely induced CAT activities 20-30-fold over those in the absence of cAMP in the case of pPI-CAT0, pPI-CAT2, pPI-CAT5; and 40-80-fold in the case of pPI-997/CAT and pPI-D/CAT. The CAT expressions of cells with pPI-TATA (PI sequence from -34 to -12), which contain no CCAAT box, were hardly affected by the addition of 8-CPT cAMP and IBMX. The CAT expression of pPI-997/CAT(mut), in which a 5-bp sequence of the CCAAT box from pPI-997/CAT was deleted, also showed no effect of cAMP on the induction of CAT activity, although the basal promoter activity increased about 3-fold as observed previously(4) . Finally, cAMP had no effect on cells containing the plasmid pPII-CAT22. This plasmid contains a small piece of the PII sequence, which shows promoter activity by itself but contains no CCAAT box. These results suggested that the CCAAT box sequence of the PI promoter is involved in the stimulation of PI promoter expression by cAMP.
Figure 2:
DNase I footprinting of PI with nuclear
extracts from 30A5 cells. The following DNAs were used: A, PI
fragment (-303 and -12 bases) prepared from pPI-997/CAT; B, PI fragment (-303 and -12) from which the 5-bp
GCAAT of the CCAAT box were deleted. This DNA fragment was prepared
from pPI-997/CAT(mut). The plasmids were digested with BamHI
and labeled using [-
P]ATP and T4 DNA
kinase. One end-labeled fragment was prepared by digestion with NspI. 20 µg of nuclear extract from 30A5 cells that had
been incubated with (lane4) or without (lane3) 100 µM cAMP and 500 µM IBMX
for 48 h or 20 µg of bovine serum albumin (lanes1 and 2), were incubated with 1.5
10
cpm of the
P-labeled DNA probe. The reaction
mixtures were then subjected to DNase I (0.2-1 unit) treatment at
room temperature for 1 min. After terminating the reaction, samples
were extracted and analyzed in 8% denaturing polyacrylamide gels by
electrophoresis and autoradiography. The protected region is shown at
the left side of ladders with the sequences. The 5-bp deleted
region is indicated by a smallbox between the
ladders of the A and B digestion patterns. Dottedlines note corresponding regions between the two
probes.
In order to identify the binding factor(s), 30 base pair oligonucleotides, spanning this protected region, were synthesized and mobility shift DNA-binding assays were performed. The 30-base pair fragment generated 3 bands (Fig. 3, lane1). All these bands were competed out in the presence of excess amounts of non-labeled probes, but not by either the mutated sequence, in which the AA of the GCAAT sequence in the CCAAT box was substituted for by GG, or an unrelated DNA sequence, a 30-base pair fragment from PII (data not shown; see also (4) ). Therefore, these three bands (B1, B2, and B3) appear to be specific to this 30-bp DNA fragment containing the GCAAT sequence.
Figure 3:
Supershift assay. 10 µg of nuclear
extract from confluent 30A5 cells that had been incubated without (lanes 1-4) or with (lanes 5-8) 100
µM 8-CPT cAMP and 500 µM IBMX for 48 h were
then incubated with anti-C/EBP- (lanes2 and 6), anti-C/EBP-
(lanes3, and 7), anti-AP2 (lanes4 and 8), or
without antibody (lanes1 and 5) prior to
addition of the labeled 30-bp DNA fragment (-76 to -47) of
PI. Then the mixtures were incubated further with the labeled 30-bp DNA
fragment (1.5
10
cpm). B1, B2,
and B3 indicate shifted bands of the 30-bp DNA fragment with
nuclear extracts. Star indicates the supershifted band with
anti-C/EBP-
or anti-C/EBP-
.
Figure 4:
Northern blot analysis of C/EBP- and
mRNAs. 15 µg of total RNA from 30A5 cells (lanes1 and 4) or 30A5 cells incubated without (lanes2 and 5) or with (lanes3 and 6) 100 µM 8-CPT cAMP and 500
µM IBMX for 48 h were fractionated in formaldehyde, 1%
agarose gels and transferred onto Hybond nylon membranes. The membranes
were hybridized separately with a nick-translated rat C/EBP-
cDNA
probe (lanes 1-3) or a rat C/EBP-
cDNA probe (lanes 4-6).
Figure 5:
Time dependence of cAMP action on the
activation of PI and the expression of C/EBP- and C/EBP-
. A, Northern blot analysis. Total RNAs were prepared from
confluent 30A5 cells at various time points during incubation of the
cells with cAMP and IBMX as described under ``Experimental
Procedures.'' The accumulation of mRNAs for C/EBP-
and
C/EBP-
was analyzed at various time points by Northern blot
analysis. B, Western analysis of C/EBP-
was carried out
using the extracts (30 µg) prepared from the cytosolic (1 and 3) and nuclear fractions (2 and 4)
of the control cells (lanes1 and 2) and
cells that had been treated with a cAMP analogue (lanes3 and 4) for 3 h. The two panels (a and b) show the results of two independent experiments. C, CAT activity from the stable clone transfected with
pPI-997/CAT (150 µg of proteins) was assayed at various time points
during incubation of the cells with cAMP and
IBMX.
The effect of TNF-
further supports our thesis that C/EBP-
is responsible for cAMP
activation of the PI promoter. When confluent 30A5 cells were treated
with TNF-
together with 8-CPT cAMP and IBMX for 2 days, TNF almost
completely repressed the induction of mRNAs for C/EBP-
, while
C/EBP-
induction was not affected at all, as shown by Northern
blot analysis (Fig. 6A). However, as shown in Fig. 6B, the same concentration of TNF-
had no
effect on the induction of CAT activity, in spite of complete
suppression of C/EBP-
induction. Therefore, cAMP can activate the
PI promoter in 30A5 cells through the transcriptional activation of the
C/EBP-
gene without cAMP activation of the C/EBP-
gene.
Figure 6:
Effect
of TNF- on the expression of C/EBP-
and C/EBP-
, and cAMP
activation of the PI promoter. A, confluent 30A5 cells were
incubated in medium without (lane1) or with 0.1
mM 8-CPT cAMP and 0.5 mM IBMX, with (lane3) or without (lane2) 200 units of
TNF-
/10 ml. Total RNAs were prepared as described under
``Experimental Procedures,'' and the accumulation of mRNAs
for C/EBP-
and C/EBP-
was analyzed by Northern blot analysis. B, cellular extracts were prepared from stable clones with
pPI-997/CAT, and CAT activity was assayed as described under
``Experimental Procedures.''
Figure 7:
Transactivation of PI-CAT by C/EBP-.
Five µg of pPI-997/CAT was co-transfected with increasing amounts
of MSV-C/EBP-
as indicated. The total amount of DNA was adjusted
to 15 µg in each case using pMSV. CAT activities were measured
after 48 h as described under ``Experimental Procedures.''
Each value represents the mean ± S.E. of three
experiments.
One of our most interesting observations is that the activation of
PI expression by overexpressed C/EBP- was not fully achieved
unless the overexpressed C/EBP-
was exposed to cAMP (Fig. 8). As shown in Fig. 8, cAMP treatment of 30A5
cells for 2 h increased CAT activity about 2-fold. The same levels of
CAT activities were observed when pPI-997/CAT plasmids were
co-transfected with either of the plasmids containing C/EBP-
or
C/EBP-
genes. On the other hand, cAMP treatment of 30A5 cells that
were co-transfected with both plasmids increased CAT activity between
8- and 10-fold. However, there was no effect of cAMP on CAT expression
in cells with the C/EBP-
gene. This suggests that, with respect to
the activation of CAT gene expression, cAMP acts on C/EBP-
, and
not in a manner independent of C/EBP-
.
Figure 8:
Effect of transient transfection with
C/EBP and cAMP on PI expression. 30A5 preadipocytes were co-transfected
with 15 µg of pMSV/EBP- or pMSV/EBP-
, and the cells were
maintained for 48 h until 2 h before the CAT assay when cAMP was added
to some of the samples, as follows: column1, cells
were transfected with 15 µg of PMSV vector; column2, control cells were treated as described for column1, except for a 2-h treatment with cAMP; column3, pMSV/EBP-
-transfected cells; column4, pMSV/EBP-
-transfected cells were treated with
cAMP; column5, cells were transfected with
pMSV/EBP-
; column6,
pMSV-EBP-
-transfected cells from column5 were
then treated with cAMP. The values represent the mean ± S.E. of
6 experiments.
In order to examine
whether or not cAMP treatment of C/EBP- expressing cells affects
the binding efficiency of C/EBP-
, nuclear extracts were prepared
from cells that had been treated with or without cAMP and DNA-band
shift analysis was performed (Fig. 9). There were no significant
differences between the binding activities of nuclear extracts from
cell preparations that had been treated with cAMP and cells that were
not treated (Fig. 9).
Figure 9:
Effect of transient transfection of
C/EBP- and cAMP treatment on C/EBP-
binding activity. The
DNA-mobility shift assay was performed as described under
``Experimental Procedures'' using nuclear extract from cells
that had been transfected with pMSV/EBP-
for 48 h before the
treatments indicated. Lane1, control; lane2, treated with 8-CPT cAMP (100 µM, 30 min); lane3, C/EBP-
transient-transfected (48 h); lane4, C/EBP-
transient-transfected cells
treated with 8-CPT cAMP (100 µM) for 30 min before nuclear
extract was prepared; lane5, free
probe.
Furthermore, the amounts of nuclear
C/EBP- (Fig. 10, panelA) are not
affected by short term cAMP treatment, i.e. up to 2 h, in the
control cells (Fig. 10, lane1versus2), or in cells transfected with the C/EBP-
gene (Fig. 10, lane3versus4).
However, the short term cAMP treatment did induce phosphorylation of
C/EBP-
(Fig. 10A, panelB).
Figure 10:
Effect of cAMP on C/EBP-
phosphorylation. A, Western blot analysis for the amounts of
C/EBP-
(panelA) and the extent of
phosphorylation of C/EBP-
(panelB) were carried
out as described under ``Experimental Procedures.'' cAMP
treatments were carried out for 30 min (lanes2 and 4), and C/EBP-
expressions were for 48 h (lanes3 and 4) as described under ``Experimental
Procedures.'' Lane1, control; lane2, control + cAMP; lane3,
C/EBP-
-transfected; lane4,
C/EBP-
-transfected and cAMP-treated. B, in order to
examine the specificity of antibodies against phosphoserine, the effect
of dephosphorylation of the phosphorylated C/EBP-
was examined.
Nuclear extracts (80 µg) from cells expressing C/EBP-
that had
been treated with cAMP were incubated with and without intestinal
alkaline phosphatase (20 units) for 30 min. The reactivity of the
samples to the antibodies was examined as described under
``Experimental Procedures'': lanes1 and 3, control nuclear extracts; lanes2 and 4, phosphatase-treated samples. Lanes1 and 2 were probed with anti-phosphoserine and lanes3 and 4 with anti-C/EBP-
. Each lane contained 40
µg of nuclear proteins.
As shown in Fig. 10B, the antibodies against
phosphoserine are specific to phosphorylated C/EBP- (Fig. 11, lane1versus2),
and thus discriminate between phosphorylated C/EBP-
and
nonphosphorylated C/EBP-
. The total amount of C/EBP-
did not
change as a result of phosphatase treatment (lanes3 and 4).
Figure 11:
Phosphorylated status of C/EBP- and
its DNA binding ability. Nuclear extracts were prepared from cells
expressing C/EBP-
and were treated with cAMP as described in Fig. 10. Nuclear protein preparations (10 µg/sample) were
then incubated with intestinal alkaline phosphatase-agarose conjugate
(20 units of phosphatase) (lane2) or with an
equivalent amount of agarose (lane1) for 30 min.
Following these treatments, agarose was removed by centrifugation for 2
min at 14,000
g. Supernatants were used for
DNA-binding assay (A) and for Western analysis using
anti-phosphoserine (B) as described under ``Experimental
Procedures.''
Finally, in order to clearly establish that
the phosphorylation of C/EBP- does not affect its binding ability,
nuclear extract from the cAMP-treated cells was treated with or without
intestinal alkaline phosphatase-Sepharose, and the effect of
phosphatase on the status of phosphorylation and DNA binding activity
was examined. As shown in Fig. 11A, the binding
activity of the phosphatase treated nuclear extract was not affected,
while C/EBP-
was almost completely dephosphorylated, as shown in Fig. 11B.
These observations support the thesis that
phosphorylation of C/EBP- by cAMP-dependent protein kinase causes
transcriptional activation as in the case of
Ca
-dependent phosphorylation of
C/EBP-
(23) , rather than a covalent modification leading
to facilitated translocation from the cytosol to the nucleus and thus
DNA binding(22) .
Previously, we reported that in 30A5 cell cultures both
differentiation and activation of PI of the ACC gene require a short
period of cAMP treatment before the cells are exposed to
insulin(3, 4) . While analyzing the role(s) played by
cAMP in making the cells competent to respond to insulin, we observed
that PI of the ACC gene was in a state of repression by virtue of the
presence of a negative cis-element, and that C/EBP-
binding to the CCAAT box led to the activation of PI
expression(4) . We also presented evidence that the repressed
state of PI involved interaction between the negative cis-element and the CCAAT box. Deletion of the CCAAT box
(GCAAT sequence in PI) from PI, or masking the CCAAT box with
C/EBP-
, resulted in the activation of PI. These results suggested
that the interaction between the negative cis-element and the
CCAAT box could control PI expression in either a negative or positive
direction depending on the presence or absence of C/EBP-
. C/EBP is
a family of proteins that belongs to a class of the basic
region-leucine zipper proteins (bZIP class). C/EBP-
is an isoform
of C/EBP-
. These proteins are capable of forming a complex within
or without the family, but the relationships among these proteins in
controlling specific genes has been difficult to assess.
In this
report, we have demonstrated that the expression of the C/EBP-
gene is activated by cAMP and the increased level of C/EBP-
and
modification of the increased level of C/EBP-
may be responsible
for the activation of PI when 30A5 cells are treated with cAMP. This
conclusion is based on the observation that overexpression of
C/EBP-
alone, without cAMP treatment, caused only minimal
activation of PI. In the latter case, cAMP treatment presumably
modified the preexisting C/EBP-
. Although cAMP treatment led to
the accumulation of both mRNAs of C/EBP-
and C/EBP-
,
activation of PI could occur when cAMP only stimulated C/EBP-
accumulation. Furthermore, when C/EBP-
accumulation was inhibited
by the use of an inhibitor of protein kinase, H8, there was no PI
activation, and thus the C/EBP-
accumulation can be causally
related to PI activation. Although H8 can inhibit other protein kinases
as well as cAMP-dependent protein kinase, other kinases would not
contribute to the effects of cAMP observed in the experimental setting
being used here, since they are not stimulated by cAMP(21) .
TNF-
, which inhibited the accumulation of C/EBP-
, had no
effect on C/EBP-
accumulation, and this accumulation activated PI.
Finally, we have shown that C/EBP- gene transfection alone can
activate CAT gene expression in the presence of cAMP. Although we have
shown that cAMP treatment of the cells induces C/EBP-
expression
and C/EBP-
phosphorylation, one could still argue that the site of
cAMP action involves something other than C/EBP, and that such action
of cAMP together with cAMP action on C/EBP-
leads to the
activation of the promoter. If that were the case, the full activation
of CAT gene expression would still require C/EBP-
. At this point,
we cannot completely exclude such a possibility.
It had been
reported that C/EBP- was phosphorylated by
Ca
-calmodulin-dependent protein kinase II. The
phosphorylated C/EBP-
transactivated the gene containing the
Ca
-calmodulin-dependent protein kinase II-responsive
element in a pituitary cell line (G/C)(23) . The
phosphorylation of the serine at position 276 of C/EBP-
did not
alter its binding affinity for the
Ca
-calmodulin-dependent protein kinase II-responsive
element or its ability to form a homodimer. On the other hand, it has
been shown that when C/EBP-
was phosphorylated, translocation of
the phosphorylated C/EBP-
into the nucleus was
stimulated(22) . The mechanism of cAMP activation of c-fos gene transcription seems to have a similar explanation, i.e. C/EBP-
is translocated to the nucleus, where it activates
transcription by binding to the serum-responsive element of the
c-fos promoter. NF-IL6, a homolog of C/EBP-
, is
phosphorylated by mitogen-activated protein kinase at threonine 235,
which is essential for its transcriptional activity, and it has been
suggested that differential phosphorylation at different sites may play
a role under various physiological conditions(24) . In this
report, we established that cAMP induced C/EBP-
expression in the
process of PI activation in 30A5 preadipocytes. Lack of C/EBP-
mRNA induction by cAMP in the presence of H8 (data not shown) and in
the 30A5 cells expressing mutant regulatory subunit of cAMP-dependent
protein kinase (data not shown) further support the thesis that cAMP
induces C/EBP-
gene expression. Our present experimental results,
however, contribute further to the controversial subject of how cAMP
affects C/EBP-
action. Our results in this regard support the view
that in the 30A5 system cAMP-mediated phosphorylation of C/EBP-
activates transcription, and not binding of C/EBP-
to DNA or
translocation from the cytosol to the nucleus.
The C/EBP binding
site appears to mediate cAMP control of certain promoters(25) .
For example, it has been reported that C/EBP- activation of the
phosphoenolpyruvate carboxylase gene involves an interaction between
CRE and C/EBP-
(26) . Transcription of the
phosphoenolpyruvate carboxylase gene is rapidly stimulated by cAMP as
in the case of the c-fos gene(27, 28) . It
appears that C/EBPs are capable of facilitating communication between
enhancers and promoters based on their ability to interact with other
factors, e.g. CRE(26) . Since PI does not contain CRE,
it is unlikely that this is how cAMP activates PI. Also, we did not
detect any CRE-binding protein in the C/EBP-
containing complex.
Whether or not the occurrence of the two C/EBP-
complexes is due
to two forms of C/EBP-
(22) is not clear at this time.
In our present studies, using chimeric gene constructs, we
demonstrated that an increased level of C/EBP- is a factor in the
activation of PI of ACC in response to cAMP. It would be simplistic to
suggest that the observed effect of C/EBP-
on PI is the only cause
of the activation of the endogenous gene. The nuclear extract from
cAMP-treated cells showed increased amounts of both C/EBP-
and
C/EBP-
. It is particularly interesting to note that there are two
shifted bands of the DNA probe containing C/EBP-
(Fig. 3).
How these two complexes are formed is not known, but these observations
suggest the occurrence of heterodimeric protein complexes between
C/EBP-
and other proteins(18) . Even such putative
heterodimeric protein complexes appear to be independent of cAMP
treatment in this case. The expression of genes involved in
differentiation and development of higher eukaryotes is presumably
regulated by coordinated interaction of various cis-acting DNA
regulatory sequences that interact with specific trans-acting
factors(29) . How the binding of a regulatory protein at a
distant cis-element affects the binding and activity of RNA
polymerase is still a subject of intensive investigation and
speculation(30, 31, 32) .
Finally, we also
presented evidence that cAMP activates C/EBP- gene expression as
well. Such an action of cAMP had been suggested previously under
unpublished results(25) .