(Received for publication, June 30, 1994; and in revised form, October 21, 1994)
From the
Induction of the fetal isogenes skeletal -actin (skACT) and
-myosin heavy chain (
-MHC) is characteristic of cardiac
growth in many models, suggesting a conserved signaling pathway.
However, divergent regulation has also been observed.
-Protein
kinase C (PKC) and transcriptional enhancer factor-1 (TEF-1) are
involved in induction of
-MHC in
-adrenergic-stimulated hypertrophy of cultured cardiac
myocytes (Kariya, K., Farrance, I. K. G., and Simpson, P. C.(1993) J. Biol. Chem. 268, 26658-26662; Kariya, K., Karns, L.
R., and Simpson, P. C.(1994) J. Biol. Chem. 269,
3775-3782). In the present study, we asked whether the skACT
promoter used the same mechanism. A mouse skACT promoter fragment
(-113/-46) was induced by both
-adrenergic
stimulation and co-transfection of activated
-PKC, and contained
three required DNA sequence elements: M-CAT, CArG, and Sp1. The skACT
M-CAT element bound TEF-1 in cardiac myocytes. Thus the skACT and
-MHC promoters both require a TEF-1 binding site for activation by
-adrenergic stimulation, but differ in that skACT also
requires a CArG box. These results provide a potential molecular basis
for divergent regulation of the fetal program, and also imply that PKC
and TEF-1 are conserved transducers for this program during cardiac
growth.
A characteristic feature of hypertrophic growth in adult cardiac
muscle is qualitative changes in gene expression, changes that have
come to be known as the ``fetal program'' (for reviews, see (1, 2, 3, 4) ). The fetal program,
most evident in the rat heart subjected to pressure overload, but seen
also in man(1) , consists of the recapitulation of a pattern of
cardiac-specific gene expression that characterizes the pre-natal
developing heart. For example, among the contractile protein genes, the
skeletal isoform of -actin (skACT) (
)and the
-isoform of myosin heavy chain (
-MHC) are expressed in the
fetal ventricle and are replaced by the respective adult isoforms,
cardiac
-actin and
-MHC, during rat post-natal development.
Induction of pressure overload, as by aortic banding, up-regulates
skACT and
-MHC, and this up-regulation is clearly
pre-translational and probably transcriptional(5) . The fetal
program includes up-regulation of other gene products, including atrial
natriuretic factor (ANF), smooth muscle actin, ventricular myosin light
chain 1,
-tropomyosin, the
3 subunit of the Na,K-ATPase, B
creatine kinase, and the T-type calcium current, as well as
down-regulation of others, such as the sarco(endo)plasmic reticulum
calcium ATPase, which is expressed at very low levels in the fetal
heart. The similarity of gene expression during these major cardiac
growth transitions, the fetal period, and pressure overload, has raised
the possibility of a conserved signaling pathway for transcriptional
regulation, one that is particularly important in cardiac growth.
Additional evidence for a conserved pathway is provided by studies
with cultured cardiac myocytes. Cultured myocytes from both the
neonatal and the adult rat heart recapitulate the fetal program when
challenged with a variety of growth stimuli, including
-adrenergic agonists, endothelin 1, angiotensin II,
TGF-
and bFGF, myotrophin, and mechanical stretch (for references,
see (6) ). Thus if there is a conserved signaling pathway for
the fetal program, this pathway might be activated by a variety of
different extracellular stimuli.
Despite this evidence for a
conserved pathway, it is also clear that the cardiac fetal genes are
not regulated identically. Divergent regulation within the fetal
program is well exemplified by -MHC and skACT (for a review, see (3) ). With pressure overload of the rat heart, skACT mRNA is
detected in the ventricle much earlier than is
-MHC
mRNA(7) ; and the skACT mRNA elevation is transient despite
continued load, whereas that of
-MHC is
sustained(8, 9) . Furthermore, skACT mRNA is found
throughout the ventricle after pressure overload, whereas
-MHC
mRNA tends to be localized to the inner wall of the ventricle and
around large coronary arteries(7) . In the aging heart,
-MHC mRNA reaccumulates, whereas skACT mRNA remains at a low
level(10, 11) . When the aged heart is subjected to
pressure overload,
-MHC mRNA is further up-regulated, but skACT
mRNA is not induced(10) . Thus, it is evident that the signals
for induction of skACT and
-MHC during cardiac growth in vivo must diverge in some way, despite the fact that they also might
share some conserved pathway for transcriptional regulation.
We have
been using a model of cultured neonatal rat cardiac myocytes to define
conserved and divergent pathways for transcriptional signaling during
cardiac growth(12) . In this model, both skACT and -MHC
are up-regulated at the transcriptional level during hypertrophic
growth stimulated by an
-adrenergic
agonist(13, 14, 15) . Therefore, this system
provides the opportunity to compare the induction of skACT and
-MHC by the same growth stimulus in the same population of cardiac
myocytes, asking whether they share a common, conserved pathway for
induction or whether they are activated by divergent pathways.
Our
recent studies have outlined a pathway that includes transcriptional
enhancer factor-1 (TEF-1) and -protein kinase C (PKC) for
-adrenergic induction of the
-MHC promoter in the
cultured cardiac myocytes. An
-adrenergic agonist and
activated
-PKC both stimulate the
-MHC promoter through an
enhancer core/muscle CAT (M-CAT) element(6, 16) . This
-adrenergic and
-PKC response element binds
cardiac myocyte TEF-1 in vitro(17) , and a mutation
that disrupts TEF-1 binding abolishes both
-adrenergic
and
-PKC induction of the
-MHC promoter(6) . Thus
TEF-1 is involved somehow in
-MHC promoter induction by
-adrenergic agonists in cardiac myocytes, and
-PKC appears to be a transducer for
-adrenergic
signaling at TEF-1.
TEF-1 was cloned as a HeLa cell transcription
factor for a Simian virus 40 (SV40) enhancer core motif (18) (for reviews, see (19) and (20) ).
Recently, TEF-1 has been found to have a role in cardiac-specific
transcription, even though TEF-1 mRNA (20, 21) and
binding activity (M-CAT binding factor, MCBF) (6, 22, 23) are not restricted to myocardium.
An M-CAT element, 5`-CATNC(C/T)(T/A)-3`(22, 24) , is
present in the promoters of several cardiac- and skeletal
muscle-specific genes(25) ; mediates activity in cultured
cardiac myocytes of the promoters of cardiac troponin T(26) ,
-MHC(6, 27, 28) , and
skACT(29) ; and binds TEF-1 in cardiac muscle (20, 23, 24) and cardiac
myocytes(17, 29) . As assayed by promoter-reporter
injection in the adult heart in vivo, TEF-1 binding sites are
required for expression of
-MHC (30) (see sequence in (6) ) and
-MHC (23) . In transgenic mice,
-MHC promoter fragments of 600 bp (31) and 354
bp(32) , which contain at least two TEF-1 binding
sites(6, 17) , are sufficient for developmentally
regulated cardiac-specific expression. Finally, disruption of the TEF-1
gene in mice causes impaired cardiac growth with embryonic
lethality(33) . Thus a target for
-adrenergic
stimulation of the
-MHC promoter is critical for cardiac growth
and gene transcription.
In the present study, we asked whether skACT
induction during -adrenergic-stimulated cardiac
myocyte hypertrophy also involved
-PKC and TEF-1. We present three
main findings. First, activation of the skACT promoter requires at
least three DNA sequence elements: M-CAT, CArG, and Sp1. Second, the
skACT promoter fragment activated by
-adrenergic
stimulation is also activated by
-PKC. Third, the skACT M-CAT
element binds TEF-1 in cardiac myocytes. We interpret these data to
support two main conclusions. First, there is divergence in
transcriptional activation of skACT and
-MHC, even by the same
agonist in the same population of cardiac myocytes. Induction of the
skACT promoter requires a CArG box, and, by inference, the serum
response factor (SRF), whereas this is not the case for
-MHC.
These results therefore provide a potential molecular basis for
differential regulation within the fetal program, as exemplified by
skACT and
-MHC. Second, TEF-1 is a common factor in activation of
both the skACT and
-MHC promoters during cardiac myocyte growth
stimulated through an
-adrenergic receptor, and PKC
might transduce a signal from the receptor to TEF-1. Since TGF-
induces a skACT promoter in cardiac myocytes through the same response
elements as an
-adrenergic agonist(29) , PKC
and TEF-1 might be conserved elements for activation of the fetal
program by diverse stimuli for cardiac growth.
The mouse skACT gene
(pMACT-) was provided by M. Hu and N. Davidson (35) . The
pMACT SacI-HinfI fragment (-46 to +47) was
inserted into SP64-CAT cut with SacI and SmaI, to
generate -46-skACT-CAT. The plasmid -46-skACT-CAT contained
47 of the 57 bp of the skACT first (untranslated) exon fused to the CAT
gene (see Fig. 1). The deletions between -1400 and
-113 were constructed by using convenient restriction sites, as
shown in Fig. 1A, and converting the sites at the
5`-deletion end point to a SacI site by linker addition. These SacI fragments, from the new SacI site at the
5`-deletion end point to the SacI site at -46, were
inserted into the SacI site of -46-skACT-CAT. SkACT-CAT
plasmids containing the first intron were made by fusing the skACT
promoter to CAT within the untranslated portion of exon 2, utilizing
the DraI site at +1031 (1 bp 5` of the initiator ATG)
(see Fig. 1A). Site-directed mutagenesis was performed
on the -113/-46 skACT promoter fragment subcloned in
pGEM-3Z, using the polymerase chain reaction(6) . The resulting
polymerase chain reaction-generated fragments were inserted into the SacI site of -46 skACT-CAT. The thymidine kinase-LUX
plasmids were constructed by inserting the -113/-46 and the
-155/-76 skACT fragments into the SacI site of
pT81-LUX(36) , containing the -81/+52 herpes simplex
virus thymidine kinase promoter 5` of the LUX gene. Mutant sequences
and the end points and orientation of the promoter fragments in the
reporter plasmids were verified by DNA sequencing.
Figure 1:
The -113/-46 sequence of
the skACT promoter is required for activation by
-adrenergic stimulation. A, the upper
diagram represents the -1400-skACT-CAT reporter plasmid.
Shown are the restriction sites used to generate the series of 5`
promoter deletions, and the locations of the CArG, M-CAT, Sp1, and TATA
elements. The lower diagram illustrates the structure of the
skACT-CAT plasmids containing the first intron. Arrows indicate the start site of transcription at +1; and dark
boxes, untranslated skACT exons. B, cardiac myocytes were
transfected with the series of skACT-CAT plasmids illustrated in A. Cells were treated for 24 h with 20 µM phenylephrine (
1) or its vehicle (Basal).
All dishes were co-tranfected with a constant amount of RSV-LUX as a
control for transfection efficiency. CAT activity was assayed and
calculated relative to the LUX activity measured in the same sample.
The basal activity of -1400-skACT-CAT was set at 100. Values are
the mean ± S.E. from at least three experiments, with duplicate
dishes for each plasmid in each experiment.
Twenty
h after plating, neonatal myocytes or 3T3 cells were refed with minimal
essential medium containing 5% calf serum and 30 mM HEPES, pH
7.5. Equimolar amounts of the CAT plasmids (1.8 pmol, 5.0-6.8
µg) were transfected into duplicate dishes using the CaPO method(16) . RSV-LUX (0.04 pmol, 0.2 µg) was included
in all plates as an internal control for variability of transfection
efficiency. In transfections with pT81-LUX plasmids, RSV-CAT (0.06
pmol, 0.2 µg) was included as the internal control. This amount of
co-transfected RSV-LUX or RSV-CAT has no inhibitory effect on
expression from reporter plasmids(16) . For the PKC
co-transfection experiments, 0.2 or 0.8 pmol (0.5 or 2.0 µg) of the
expression plasmids were included per plate. Total DNA per plate was
maintained at 25 µg by the addition of variable amounts of pSP64 or
pUC18. The CaPO
precipitate was removed from the cells
after 2 h, and the medium was changed to serum-free minimal essential
medium with porcine insulin (10 µg/ml) (Lilly), bovine transferrin
(10 µg/ml) (HyClone), and bovine serum albumin (1 mg/ml) (Intergen
no. 3130-00, Armour). After 16 h, the cells were refed with the same
medium and treated for 24 h with various agents or their vehicle (100
µM ascorbic acid): 20 µML-phenylephrine HCl (Sigma); 0.2 µM WB4101
(Research Biochemicals, Natick, MA); 0.2 µML-isoproterenol HCl (Sigma); or 5% calf serum. Harvesting
of transfected cells, preparation of cell extracts, and CAT and LUX
assays were performed as described elsewhere(16) . Data are
expressed as the relative activity, determined by normalizing the
reporter plasmid CAT or LUX activity to the activity of the
co-transfected RSV control plasmid in the same cell extract.
Figure 4:
Three elements in the -113/-46
sequence, M-CAT, CArG, and Sp1, are required for activation. The lower part of the figure gives the sequence of the
-113/-46 wild-type (wt) mouse skACT promoter, with
the CArG, M-CAT, and Sp1 elements underlined. The
-108/-43 chicken skACT promoter (58) is shown
also, indicating the conservation of these motifs across species. Dots indicate identity with mouse wild-type sequence. Region I and Region II of the chicken skACT promoter
are protected in DNase I footprinting by embryonic muscle nuclear
extract(22) , and the footprint over Region II is competed
completely by an oligonucleotide that binds
TEF-1(22, 24) . Mutations (mut) of the mouse
-113/-46 sequence were generated by polymerase chain
reaction. As shown in the upper part of the figure, the
wild-type and mutant mouse -113/-46 fragments, inserted
into -46-skACT-CAT, were transfected into duplicate dishes of
cardiac myocytes, along with the internal control RSV-LUX; and the
cells were treated with vehicle (Basal), 20 µM phenylephrine (1), or 5% calf serum (Serum)
for 24 h. The basal CAT activity of -46-skACT-CAT was set at 1.
The basal and serum values are the mean ± S.E. of at least three
experiments, and the
values are the mean ±
variation of two experiments. The 5` (-111/-108) and 3`
(-54/-51) mutations had no effect on promoter activity
(data not shown). Also, a different mutation of the chicken skACT
promoter just 5` to the CArG/SRE1 (M -100/-95) has no
effect on basal or TGF-
-induced activity in rat cardiac
myocytes(29) .
Treatment of transfected
cardiac myocytes with the -adrenergic agonist
phenylephrine (20 µM, 24 h) increased the activity of
-1400 skACT-CAT by 2-fold (phenylephrine/vehicle = 2.00
± 0.09-fold, n = 21, p < 0.01) (and
see Fig. 1B). The
-adrenergic
antagonist, WB4101 (0.2 µM), blocked the
phenylephrine-stimulated increase in CAT activity (0.94 ±
0.05-fold, n = 6, p = NS); and the
-adrenergic agonist, isoproterenol (0.2 µM), did not
increase CAT activity (isoproterenol/vehicle = 1.08 ±
0.06-fold, n = 13, p = NS). The
promoterless vector, SP64-CAT (0-skACT-CAT) was not activated by
phenylephrine (Fig. 1B) or isoproterenol (data not
shown). These data confirmed that the mouse skACT promoter was
activated through an
-adrenergic receptor, and a
similar finding has been reported previously for the human skACT
promoter(38) .
The magnitude of skACT promoter activation by
-adrenergic stimulation (2-fold) was less than that
found previously for
-adrenergic induction of skACT
mRNA (11-fold) (13) or skACT gene transcription
(6-fold)(14) . We attribute this lesser degree of promoter
activation to differences in experimental protocols. In the prior
studies of endogenous skACT mRNA abundance and transcription, myocytes
were maintained in serum-free culture for at least 72 h prior to
treatment with an
-adrenergic agonist, and basal skACT
transcripts and transcription were absent or very
low(13, 14) . In the present studies, myocytes were
serum-depleted for only 16 h prior to
-adrenergic
stimulation, and basal skACT promoter activity was detectable easily.
Further, serum (5% v/v) activated -1400-skACT-CAT potently (3.86
± 0.36-fold, n = 12, p < 0.01; and
see Fig. 4). Thus it seemed likely that the ``basal''
activity of -1400-skACT-CAT was higher than might have been
expected from our prior studies of endogenous skACT transcription, due
to the residual effects of serum on the promoter, and that this
elevation of ``basal'' activity reduced apparent induction by
-adrenergic stimulation. An additional factor reducing
apparent induction of the skACT promoter was that
-adrenergic stimulation increased expression from the
co-transfected control plasmid, RSV-LUX, slightly but significantly
(1.28 ± 0.04-fold, n = 87, p <
0.01). Since
-adrenergic activation of skACT-CAT was
calculated relative to LUX activity, phenylephrine stimulation of the
skACT promoter was relatively underestimated. We cannot exclude that
basal activity was spuriously high due to the absence of negative
regulatory elements outside the 1400-bp skACT promoter fragment, or
that induction was less due to absence of positive elements. However,
730 bp of rat skACT 5`-flanking sequence is sufficient for appropriate
expression during development in transgenic mice(39) . The
magnitude of promoter induction with
-adrenergic
stimulation was not a function of the particular reporter gene, since
2-fold induction was found also with a LUX reporter (see below and Fig. 2).
Figure 2:
The -113/-46 skACT promoter
sequence is sufficient for activation by -adrenergic
stimulation. Overlapping fragments of the skACT promoter were inserted
into the enhancer test plasmid, pT81-LUX, 5` of the 81-bp herpes
simplex virus thymidine kinase promoter, as illustrated in the lower portion of the figure. Cardiac myocytes were transfected
with the skACT-thymidine kinase-LUX plasmids or pT81-LUX, as in Fig. 1B except that RSV-CAT was the internal control,
and treated for 24 h with 20 µM phenylephrine (
1) or its vehicle (Basal). The relative LUX
activity of the vector with no skeletal actin promoter fragment (pTK81-LUX) was set at 1. Values are the mean ± S.E.
from three experiments.
Figure 3:
Activated -PKC stimulates the skACT
promoter. The -1400-skACT-CAT and -113-skACT-CAT plasmids
were co-transfected with activated mutants of
-PKC (act
-PKC) or
-PKC (act
-PKC), or with the
expression vector without the PKC cDNA (vector). RSV-LUX,
which is not affected by the PKC mutants(16) , was also
co-transfected as an internal control. CAT activity was measured after
24 h and normalized to the LUX activity in each sample. The relative
CAT activity for each skACT plasmid co-transfected with the vector was
set at 1. Values are the mean ± S.E. of five to six experiments.
The autoradiograph is a representative assay showing the increase in
CAT activity from the -113-skACT-CAT plasmid co-transfected with
activated
-PKC.
Figure 5:
The skACT M-CAT motif binds myocyte TEF-1.
A double-stranded oligonucleotide of the rat -215/-196
-MHC promoter sequence was end-labeled with
P and
incubated with 10 µg of cardiac myocyte nuclear extract. Free and
complexed probe were separated on a 4% native gel. Arrows indicate the position of protein-DNA complexes (C1, C2, and C3), identified after autoradiography. GMSA
was done with no competitor (none, lane 1), with 100-fold
molar excess of unlabeled wild-type -113/-46 skACT promoter
fragment (-113/-46 skACT, lane 2), or with the
-113/-46 fragment with the M-CAT mutations shown in Fig. 4(-113/-46 M-CAT mutant, lane 3).
Complex C2 is specific (6) and is produced by binding of
TEF-1(17) .
There are three main results of the present study. First,
activation of the skACT promoter during
-adrenergic-stimulated cardiac myocyte hypertrophy
requires at least three DNA sequence elements: M-CAT, CArG, and Sp1;
second, the skACT promoter fragment activated by
-adrenergic-stimulation is also activated by
-PKC; and third, the skACT M-CAT element binds TEF-1 in cardiac
myocytes. Because we have examined skACT promoter activation by the
same agonist and in the same population of myocytes used for recent
studies of the
-MHC
promoter(6, 16, 17) , we can draw inferences
about conserved and divergent signaling pathways for
-adrenergic-stimulated transcription during cardiac
myocyte growth. Taken with those recent studies of the
-MHC
promoter, the present results have two main implications. First, they
suggest that TEF-1 is a common factor in activation of the skACT and
-MHC promoters during cardiac myocyte growth stimulated through an
-adrenergic receptor, and that PKC might transduce the
signal from the receptor to TEF-1. Since TGF-
induces a skACT
promoter in cardiac myocytes through the same response elements as an
-adrenergic agonist(29) , PKC and TEF-1 might
be conserved elements for activation of the fetal program by diverse
stimuli for cardiac growth. Second, the results show that there is
divergence in transcriptional activation of skACT and
-MHC, even
by the same agonist in the same population of myocytes. Induction of
the skACT promoter requires a CArG box, and, by inference, the SRF,
whereas this is not the case for
-MHC. Thus, these results provide
a potential molecular basis for differential regulation of the fetal
program. It would now be interesting to test whether differences in the
amount or activity of TEF-1 or the SRF contribute to the spatial and
temporal differences in skACT and
-MHC expression known to occur
during cardiac hypertrophy in vivo (see Introduction).
Other observations are consistent with a role for TEF-1
in gene expression during cardiac growth. An M-CAT element binding
TEF-1 is required for induction of the chicken skACT promoter by a
different growth agonist in cultured rat cardiac myocytes,
TGF-(29) . In vivo, TEF-1 is up-regulated in the
rat heart by pressure overload hypertrophy(23) . TEF-1 knockout
mice have impaired cardiac growth (33) .
Critical to testing
the involvement of TEF-1 in cardiac growth is understanding the
mechanism of TEF-1 regulation by -adrenergic agonists
or other stimuli. Evidence suggests that PKC might transduce a signal
from the
-adrenergic receptor to TEF-1 (see below),
and PKC could regulate TEF-1 directly or indirectly (for a review, see (17) ). The mechanism is likely to be complex, since multiple
TEF-1 isoforms, produced by alternate splicing, are present in the
chick (20) and in the rat(45) , and TEF-1-related MCBFs
have also been proposed (21) .
In the present study, a
CArG mutation inactivated the 113-bp skACT promoter, suggesting that
the SRF is required for basal expression of the mouse skACT promoter in
rat cardiac myocytes, in agreement with prior studies of the chicken
skACT promoter(29, 48) . In contrast, there is no CArG
box in the active 215-bp -MHC promoter(6, 27) ,
nor has the SRF been implicated in expression of the
-MHC promoter
in cardiac myocytes(6, 27, 28) . Our prior
results on skACT and
-MHC mRNA levels in the cultured cardiac
myocytes are also consistent with a differential dependence on the SRF
for basal expression. Specifically, skACT mRNA (13) and
transcription (14) are very low or undetectable after prolonged
culture in the absence of serum, whereas
-MHC mRNA is well
expressed in serum-free cultures(15) . This difference might be
explained by serum regulation of SRF abundance and
activity(49) , and skACT, but not
-MHC, dependence on the
SRF. Activation by the SRF could also explain why the basal activity of
the skACT promoter was higher in this study than had been expected from
prior study of endogenous skACT transcription, as mentioned under
``Results.'' Thus skACT and
-MHC appear to differ in
that the SRF, or another CArG-binding factor, is required for
expression of skACT but not
-MHC.
It is not clear whether the
SRF is a target for -adrenergic signaling at the skACT
promoter, or whether the SRF is simply a part of a basal
transcriptional complex required for detection of skACT induction. In
this study, an skACT promoter fragment (-155/-76)
containing a CArG and a putative Sp1 site did not confer basal or
-adrenergic-stimulated expression on the 81-bp
thymidine kinase promoter. In contrast, the chicken skACT SRE1/CArG
confers both basal expression in cardiac myocytes and responsiveness to
bFGF (48) and TGF-
(29) on the 56-bp c-fos promoter. Although it is likely that bFGF and TGF-
signal
differently in some respects from an
-adrenergic
agonist, the different heterologous promoters might also contribute to
these results(50) .
The apparent requirement of the
skACT promoter for Sp1 or a related protein might not be different from
the -MHC promoter. The 20-bp
-MHC promoter element
(-215/-196) that confers
-adrenergic
inducibility on the 109-bp thymidine kinase promoter is bound by TEF-1,
but not by Sp1(17) . However, the 109-bp thymidine kinase
promoter does contain a high affinity Sp1 binding site, centered at
about -100 bp(51) . The 215-bp
-MHC promoter, which
requires an intact TEF-1 binding site for induction(6) ,
contains two GAG-like motifs, in the
e4 and
e5
footprints(27) ; and a GAG motif in an
-adrenergic-inducible rat ANF promoter fragment binds
an Sp1-related protein in cardiac myocytes(52) . Thus Sp1 or an
Sp1-related protein might also contribute to expression in cardiac
myocytes of the
-MHC promoter, as appears to be the case with the
skACT, cardiac actin, and ANF promoters.
In the present study, the 1400-bp skACT
promoter was stimulated by both an -adrenergic agonist
and co-transfection of activated
-PKC, and the element or elements
required for these responses were contained within the
-113/-46 skACT sequence. We did not attempt to map the PKC
response element or elements in the -113/-46 skACT
sequence, because the M-CAT and CArG mutations each had a major effect
on basal and induced expression. Nevertheless, there is indirect
evidence that the M-CAT motif binding TEF-1 is also important for
-PKC activation of the skACT promoter. Specifically, in this
study, activated
-PKC was a much more potent stimulus for the
1400- and 113-bp skACT promoters than was activated
-PKC. The same
preference for
-PKC over
-PKC is found with a 3300-bp
-MHC promoter (16) and the 20-bp PKC-inducible element
that binds TEF-1(6) , but not with the AP-1
element(16) , which binds Fos and Jun. Thus, the
-MHC and
skACT promoters are both preferentially activated by
-PKC,
implying that TEF-1 might be a better substrate for
-PKC than for
-PKC, a possibility that can now be tested.
The skACT CArG
might participate in the response to PKC, and hence in the response to
-adrenergic stimulation. Indeed, the c-fos SRE is stimulated by an
-adrenergic agonist in
cardiac myocytes (
)and by co-transfection of activated
-PKC or
-PKC in cell lines (see (6) ) and in cardiac
myocytes. (
)PKC stimulation of the SRE appears to require
ternary complex factors (TCFs), for example SAP-1 and Elk-1, which
interact with SRF bound to the SRE (for a review, see (56) ).
The pressure overload response element of the c-fos promoter,
assayed by DNA injection in the isolated perfused rat heart, maps to
the SRE; and a mutation that disrupts TCF binding abolishes
induction(57) . These results are consistent with a role for
PKC in pressure overload in vivo, and suggest that TCFs are
present in cardiac muscle(57) , as reported in a preliminary
fashion by others(29) . However, the skACT CArG/SRE does not
form a detectable complex with TCF(47) . Therefore the CArG/SRE
might not be a target for PKC on the skACT promoter, in contrast with
the c-fos promoter.