(Received for publication, January 16, 1996; and in revised form, February 23, 1996)
From the
``Fetal'' gene transcription, including activation of
the skeletal -actin (SkA) promoter, is provoked in cardiac
myocytes by mechanical stress and trophic ligands. Induction of the
promoter by transforming growth factor
or norepinephrine requires
serum response factor (SRF) and TEF-1; expression is inhibited by YY1.
We and others postulated that immediate-early transcription factors
might couple trophic signals to this fetal program. However, multiple
Fos/Jun proteins exist, and the exact relationship between control by
Fos/Jun versus SRF, TEF-1, and YY1 is unexplained. We
therefore co-transfected ventricular myocytes with Fos, Jun, or JunB,
and SkA reporter genes. SkA transcription was augmented by Jun,
Fos/Jun, Fos/JunB, and Jun/JunB; Fos and JunB alone were neutral or
inhibitory. Mutation of the SRF site, SRE1, impaired activation by Jun;
YY1, TEF-1, and Sp1 sites were dispensable. SRE1 conferred Jun
activation to a heterologous promoter, as did the c-fos SRE.
Deletions of DNA binding, dimerization, or trans-activation domains of
Jun and SRF abolished activation by Jun and synergy with SRF. Neither
direct binding of Fos/Jun to SREs, nor physical interaction between
Fos/Jun and SRF, was detected in mobility-shift assays. Thus, AP-1
factors activate a hypertrophy-associated gene via SRF, without
detectable binding to the promoter or to SRF.
Mechanical load induces adaptive growth of cardiac muscle by
cell enlargement, with little or no capacity for proliferative growth
in terminally differentiated ventricular muscle cells (hypertrophy, not
hyperplasia). Characteristically, this increase in myocyte mass is
associated with qualitative and quantitative changes in
cardiac-specific gene expression, including up-regulation of an
ensemble of genes (including skeletal -actin (SkA), (
)
myosin heavy chain, and atrial natriuretic factor
(ANF)) that are highly expressed in embryonic but not normal adult
ventricular myocardium, a phenomenon referred to as reinduction of a
``fetal'' phenotype(1, 2, 3) . SkA
expression has been associated with increased contractility in the
rodent heart (4) and with impaired contractility in patients
with heart failure(5) . In addition to this set of genes, two
others are induced, and to varying degrees have been implicated, in
this response to mechanical load. First, ``immediate-early''
transcription factors including Fos and Jun potentially might couple
trophic signals to long-term changes in growth and gene expression.
Second, myocardial growth factors including angiotensin II and
transforming growth factor
(TGF-
) evoke most aspects of the
fetal/hypertrophic program in cultured cardiac muscle
cells(1, 2, 3) . Induction of TGF-
by
load, passive stretch,
-adrenergic agonists, and
angiotensin II suggests that TGF-
might participate in the onset,
maintenance, or inhibition of cardiac hypertrophy, as an autocrine or
paracrine factor(1) . We recently demonstrated that induction
of the SkA promoter by TGF-
requires the MADS box protein serum
response factor (SRF) and the SV40 enhancer-binding protein,
TEF-1(6) ; both also mediate activation of this promoter by
-adrenergic agonists(7) . Dominant-negative
mutations of the type II and type I TGF-
receptor, which share
related serine/threonine kinase domains, suffice to disrupt
TGF-
-dependent transcription(8, 9) ; however, the
molecular circuit that confers signal from the TGF-
receptor
complex to SkA promoter-binding proteins is unknown.
Among the
secondary or tertiary messengers that might be involved in this
signaling cascade, it is noteworthy that activation of nuclear oncogene
transcription factors Fos, Jun, and JunB precedes growth and the
up-regulation of ``fetal'' cardiac genes, in cultured
myocytes (10, 11) and intact
animals(12, 13, 14) . Fos and Jun proteins
(Fos, FosB, Fra-1, Fra-2, Jun, JunB, and JunD) each possess a basic
domain for DNA binding and a leucine heptad repeat (leucine zipper) as
an interface for homo- or heterodimerization(15) . Each member
of this AP-1 transcription factor family recognizes the
12-O-tetradecanoylphorbol-13-acetate response element (TRE:
TGA(G/C)TCA), although noncanonical sites also are
reported(16, 17, 18) . Three lines of
evidence support the inference that Fos/Jun proteins might mediate
TGF- signal transduction: TGF-
up-regulates the expression of junB and c-fos in skeletal myocytes (19) ,
cardiac myocytes, (
)and other cell types, and AP-1 sites
mediate autoinduction of TGF-
1 itself(20) . Moreover, in
skeletal muscle, forced expression of either Fos or Jun reproduces the
suppressive effect of TGF-
on myogenic differentiation, although
the issue of physical association between Fos/Jun with myogenic
helix-loop-helix proteins is unresolved (21, 22, 23) .
Related co-transfection
studies likewise support the premise that Fos/Jun mediates the
induction of fetal cardiac genes by TGF-, as shown for
ANF(24, 25) ,
myosin heavy chain, (
)and SkA(18) . In the latter study, forced
expression of Jun (or Fos plus Jun) up-regulated transcription of the
human SkA promoter in cardiac myocytes from neonatal rats and in P19
teratocarcinoma cells. Deletion analysis of the SkA promoter indicated
that nucleotides -153 to -36 were required for maximal
trans-activation by Fos/Jun. Although no consensus AP-1 site was found
within this region, sequence-specific binding to a noncanonical motif
was believed to occur. Despite this suggestive information, the
conclusion that Fos/Jun proteins augment the transcription of SkA and
other fetal cardiac genes would be premature. Dichotomous results,
repression (24) as well as activation by Fos/Jun(25) ,
have been reported for ANF. Mechanical load, ischemia, and
isoproterenol each highly induce JunB in myocardium in
vivo(13, 26, 27) , but few functional
comparisons among Jun proteins are known for cardiac
myocytes(24) . Although prior results pointed to direct binding
of Fos/Jun near the first SRE of the human SkA promoter, it has not
been demonstrated whether point mutations of this construct that
abolish AP-1 binding remain susceptible, or not, to induction by AP-1
factors. Finally, given the emerging importance of SRF in concert with
TEF-1, and given the displacement of SRF at the first SRE by a
GLI-Krüppel protein, YY1, the relationship between
control by these three factors and the induction by Fos/Jun merits
study.
In the present report, we demonstrate that Jun, Fos plus Jun, Fos plus JunB, and Jun plus JunB all transactivate the SkA gene in cardiac myocytes, whereas JunB, like Fos, is ineffective individually. Notably, a SRE is necessary for AP-1 responsiveness of the SkA promoter, and suffices to confer induction by AP-1 to a heterologous promoter. Induction required full-length Jun protein, and did not involve measurable binding of AP-1 factors to the SkA SRE1, physical association of AP-1 and SRF, augmentation of SRF binding by AP-1, or potentiation of the SRF trans-activation domain, residues 266-508 (SRF(266-508)). Together, these results indicate that AP-1 factors can act through SRF to induce a hypertrophy associated gene, SkA, but trans-activate SRE reporter genes in the absence of direct binding to the promoter or DNA-bound SRF.
Rat c-fos cDNA, provided by T.
Curran(31) , was subcloned as an EcoRI-XhoI
fragment into the SV40-driven eukaryotic expression vector, pSV-SPORT1
(Life Technologies, Inc.). Mouse c-jun and junB
cDNAs, provided by E. Olson(19) , were subcloned as EcoRI fragments into pSV-SPORT1. Mouse JunRK and
Jun
LZ, a gift from I. M. Verma(32) , were subcloned as XhoI-SacI and PstI DNA fragments,
respectively, into pSV-SPORT1; Jun
RK and Jun
LZ are mutations
of Jun in which the DNA binding and dimerization domains have been
deleted, respectively (amino acids 251-276 and 281-313).
TAM67 (a deletion mutant of human Jun lacking amino acids 3-122
in the trans-activation domain, driven by the CMV promoter) and an
insertless CMV vector derived from pCMV-
-gal (Clontech, Palo Alto,
CA) were kindly donated by M. J. Birrer(33) . Human SRF
plasmids were kindly provided by R. Prywes. SRF (residues 1-508,
wild type), SRF (residues 1-338), and SRFpm1 (34) were,
respectively, subcloned as XbaI-BamHI, XbaI-PvuII, and XbaI-BamHI DNA
fragments into pSV-SPORT1. SRF(1-338) harbors a deletion of the
transcriptional activation domain, and SRFpm1 contains three point
mutations in the basic region that abolish DNA binding.
GAL4-SRF(266-508) comprises the DNA-binding domain of yeast GAL4
(amino acids 1-147), fused to residues 266-508 in the SRF
trans-activation domain(35) , and was subcloned into the
CMV-driven expression vector pCGN. Plasmid DNAs were purified using
Quiagen Maxiprep columns (Chatsworth, CA).
Cells were transfected by
a modified DEAE-dextran method. DNA (2.5 µg/ml reporter and
0-10 µg/ml expression vectors) was mixed with 150 µg/ml
DEAE-dextran (average molecular weight, 500,000; Sigma) in DF
supplemented with 2.5% Cosmic calf serum (Hyclone). For all
comparisons, DNA and promoter content were kept constant using
equivalent amounts of vector. Cells were washed once, were incubated
with 1 ml/35-mm dish of DNA-DEAE-dextran complex for 1 h, and were then
shocked for 30 s with 10% dimethyl sulfoxide in DF. Cells were cultured
overnight in DF supplemented with 5% horse serum, after which the
medium was replaced by DF supplemented with 5 µg/ml transferrin, 1
nM NaSeO
, 1 nM LiCl, 25
µg/mL ascorbic acid, and 100 µg/ml bovine serum albumin (fatty
acid free). Twenty-four h later, cells were harvested and assayed for
luciferase activity (6) and for protein content using the
Bradford assay (Pierce). Luciferase activity for each promoter was
corrected for protein content of each extract and was normalized to the
activity of each promoter in parallel cultures of control,
vector-transfected cells. Co-transfection with a constitutive lacZ gene
was omitted for three reasons. The brief half-life of luciferase
compared to other reporter proteins can lead to misleading
interpretation, especially for transcriptional repression(36) ;
commonly used viral promoters contain functional AP-1 or SRF sites (37, 38, 39) and are up-regulated by
expression vectors used here; (
)neutral core promoters that
are unaffected by Fos/Jun, such as the c-fos -57/+109 core promoter, are insufficiently active in
cardiac myocytes for accurate quantitation of lacZ activity,
but can be utilized to drive luciferase in parallel cultures, as
an ostensibly constitutive control. Protein content was not
significantly altered by any of the inteventions, compared to the
corresponding vehicle-treated, vector-transfected cells. Where
indicated, serum-free medium was supplemented with 1 ng/ml of
TGF-
1 purified from porcine platelets (R& Systems,
Minneapolis, MN) or the vehicle (4 µM HCl, 1 µg/ml
bovine serum albumin, fatty acid free).
Figure 1: Fos/Jun proteins transactivate the skeletal actin promoter in ventricular muscle cells. Cardiac myocytes were transfected with 2.5 µg of the luciferase reporter genes and 0-10 µg of the Fos, Jun, or JunB vectors. Control cultures were transfected with 2.5 µg of reporter plasmid and 10 µg of the empty SV40-driven vector. For each reporter plasmid, luciferase activity is expressed relative to that in vector-transfected cells. Results are the mean ± S.E. for at least four transfections with two exceptions; n = 3 for the SkA reporter co-transfected with Fos plus JunB, and for the TRE reporter co-transfected with Jun plus JunB. *p < 0.05, versus vector-transfected control cells; p < 0.05, versus parallel cultures co-transfected with 5 µg of Jun.
To verify that induction of the SkA promoter by forced
expression of Fos, Jun, and JunB is specific, a canonical
AP1-responsive element and a constitutive neutral promoter were
examined, as positive and negative controls, respectively. Control of
the SkA promoter by the various permutations of AP-1 factors accurately
resembled activation of the human collagenase TRE1, although the TRE1
was more highly induced. By contrast, little or no activation was seen
using the c-fos neutral core promoter, 56Fos. Jun,
Fos/Jun, and Fos/JunB up-regulated the
56Fos reporter by no more
than 40, 70, and 60%, respectively (p
0.01). Indeed,
slight inhibition was observed at high concentrations of Fos or JunB (p
0.01).
Figure 2: Trans-activation of the SkA promoter by Jun requires SRF binding and the TATA box. A, schematic representation of the proximal SkA promoter, from SRE1 to the TATA box. B, cardiac myocytes were co-transfected with 10 µg of the Jun expression vector versus the empty vector control, and 2.5 µg of the luciferase reporter genes. Nucleotides altered by the linker-scanning BglII mutations and the binding site affected are indicated at the left. For each reporter plasmid, luciferase activity (mean ± S.E.) is shown for Jun-transfected cells relative to vector-transfected control cells; n = 6 for each condition tested. *p < 0.05, versus induction of the wild-type SkA promoter.
Figure 3:
SRF binding sites confer AP-1
responsiveness to a heterologous promoter. A, cardiac myocytes
were co-transfected with the luciferase reporter genes, 0 or 10 µg
of Jun, and 0-100 ng of the SRF vector. Mutations altering the
DNA binding domain of Jun (JunRK) and SRF (SRFpm1), Jun dimerization domain (Jun
LZ) and
trans-activation domain of Jun (TAM67) and SRF (SRF91-338) also were tested, as indicated. Luciferase
activity (mean ± S.E.) is expressed relative to the activity of
each reporter, respectively, in parallel cultures of vector-transfected
cells. n
6 for each condition tested, except n = 3 for the c-fos SRE co-transfected with SRF or
SRF plus Jun. *p < 0.05, versus vector-transfected
control cells; p < 0.05, versus cells
co-transfected with 10 µg of Jun; **p < p0.05, versus cells co-transfected with 10 µg of Jun plus 25 ng
of SRF. B, cardiac fibroblasts were co-transfected with the
luciferase reporter genes and 10 µg of Jun versus the
empty vector control. Luciferase activity (mean ± S.E.) is
expressed relative to the activity of each reporter, respectively, in
parallel cultures of vector-transfected cells. n
6 for
each condition tested. *p < 0.005 versus vector-transfected control cells. C, schematic diagram of
wild-type and mutant Jun and SRF.
To test what domains of Jun might be necessary or
sufficient for these effects, we co-transfected cardiac myocytes with
10 µg of the Jun expression vector, SRE reporter genes, and
0-100 ng of an SRF expression vector (Fig. 3, A and C). This concentration of SRF by itself did not
significantly activate any of the three reporter genes. However,
exogenous SRF was synergistic with Jun, up-regulating both the SkA
SRE1- and c-fos SRE-56Fos constructs, up to 14-fold,
nearly three times the level of induction produced by Jun alone. When
wild-type SRF was co-transfected with Jun mutants, no trans-activation
was observed with a deletion of the DNA-binding domain (Jun
RK),
deletion of the dimerization domain (Jun
LZ), or partial deletion
of the trans-activation domain (TAM67). Each of the three Jun mutants
also failed to transactivate, by themselves, the full-length SkA
reporter gene.
Conversely, in cells cotransfected with Jun,
the synergistic effect of wild-type SRF was abolished with SRFpm1, a
point mutation of SRF which cannot bind DNA, and was markedly impaired
with a mutation that deletes a trans-activation domain,
SRF(1-338); the remaining cooperative effect is consistent with
the residual activation domain of this mutation (43) . As the
high levels of endogenous Jun and SRF in
myocardium(6, 44) , together with the limited
efficiency for transfection of ventricular myocytes, would confound
efforts to compare the levels of each protein in vivo, we
synthesized each Jun and SRF mutation in vitro in the presence
of [
S]methionine and verified that the
translational efficiency and inherent stability of each protein were
equivalent to that of wild-type Jun and SRF.
Thus,
functional DNA-binding, dimerization, and trans-activation domains were
each necessary for up-regulation of the SkA promoter by Jun. The
necessity for full-length Jun contrasts with effects shown for the
isolated Jun trans-activation domain in repression of myogenin
activity(22) .
To test whether Jun could augment gene expression via the trans-activation domain of SRF, we employed a GAL4 fusion protein comprising the DNA-binding domain of yeast GAL4 fused to SRF amino acids 266-508, GAL4SRF(266-508). Five µg of GAL4SRF(266-508) were co-transfected with or without the Jun expression vector, using a GAL4-dependent luciferase reporter (Fig. 4). In the absence of exogenous Jun, the GAL4/SRF fusion protein increased transcription of the reporter 8.39 ± 0.42 (p < 0.01); transcription via the SRF activation domain was not augmented significantly by the addition of exogenous Jun. Thus, Jun did not increase transcription, when the SRF activation domain was tethered to DNA by a heterologous DNA-binding domain. This also argues against a generalized activation of transcription by Jun.
Figure 4: Jun does not potentiate the SRF trans-activation domain. Cardiac myocytes transfected with the GAL4-dependent luciferase reporter gene, pUAS(GAL4)EpLuc3, were co-transfected as shown with 5 µg of GAL4-SRF(266-508), 5 µg of the Jun expression vector, or both. Luciferase activity (mean ± S.E.) is expressed relative to that in vehicle-treated, vector-transfected cells. n = 6 for each condition tested. *p < 0.01, versus the control cells.
To address
two further possibilities, that Jun might activate the SRE by physical
association with native SRF, or facilitate DNA binding by SRF, gel
mobility-shift assays were performed using SRF, Fos, and Jun, produced
by in vitro transcription and translation (Fig. 5A). A HindIII-PvuII DNA
fragment of the SkA SRE1-56Fos luciferase reporter gene was used
as probe, encompassing the SkA SRE1 plus all sequences of the core
c-fos promoter including the TATA box. A double-stranded
oligonucleotide containing the consensus AP-1 binding site was used,
for comparison. Recombinant SRF bound the SRE1-
56 Fos probe, was
displaced by the competitor that binds SRF but not YY1
(M-81/-79), and was displaced poorly by the reciprocal
mutation, which disrupts SRF binding (M-94/-89). Jun, Fos,
and co-translated Fos/Jun showed no direct binding to the probe, did
not form a higher order complex with DNA-bound SRF, and did not alter
binding of SRF to DNA in any fashion. To ensure that the lack of
protein-protein interaction was not due to inadequate expression of Jun
or Fos, parallel experiments were performed using the AP-1 consensus
probe. Jun and co-translated Fos/Jun bound the TRE, were displaced by
excess unlabeled TRE, and were unaffected by the mutated TRE. Thus,
both AP-1 factors were expressed as stable proteins with the expected
DNA-binding and dimerization properties. To exclude direct association
between Fos/Jun proteins and SRF, we demonstrated no binding of
recombinant SRF to Jun homodimers or Fos/Jun heterodimers, and no
effect of SRF on the binding of AP-1 factors to the TRE probe (Fig. 5B).
Figure 5:
Fos/Jun proteins do not bind the SkA SRE1
or DNA-bound SRF in vitro. SRE1-56 Fos (A) and
TRE (B) probes were end-labeled with
P, incubated
with Fos, Jun, and SRF proteins produced in vitro, and
analyzed by the electrophoretic mobility shift assay in the presence or
absence of a 100-fold excess of the indicated unlabeled competitor. The
amount of Fos, Jun, and SRF designates microliters of the programmed
reticulocyte lysate. The total quantity of reticulocyte lysate per lane
was maintained at 6 µl using lysate programmed with the empty SV40
vector. ns, nonspecific binding produced by the control
reticulocyte lysate.
The present investigations show that transcription of SkA, a
``fetal'' cardiac gene associated with myocardial
hypertrophy, can be augmented by AP-1 transcription factors (Jun, Fos
plus Jun, Fos plus JunB, or Jun plus JunB) in ventricular myocytes from
neonatal rats. Despite marked differences in the constructs and
procedures used, this corroborates and extends the report of Bishopric et al.(18) , that forced expression of Jun or Fos
plus Jun up-regulates the human SkA promoter in cardiac cells. Because
dichotomous results, both induction and repression, were reported for
the ANF gene(27, 28) , consensus regarding the
functional role of Fos/Jun factors in hypertrophy has been lacking.
Although JunB, like Fos and Jun, is highly induced by both mechanical
load (13, 14) and trophic factors that up-regulate the
endogenous SkA gene (TGF-, catecholamines, and angiotensin
II
(18, 45) ), even less functional
evidence has been available for JunB in cardiac muscle(24) .
Whereas Fos proteins do not form homodimers, Jun and JunB form both
homodimers and heterodimers with Fos/Jun proteins and more distantly
related factors, via the leucine zipper. Our results demonstrate
functional synergy between JunB and Fos in cardiac myocytes; either
alone had no effect or was inhibitory, while Fos plus JunB activate the
SkA promoter. Permutations of Fos, Jun, and JunB that activate the SkA
gene correspond to those that induced the human collagenase TRE, a
canonical AP-1-responsive element.
Our results diverge from previous
findings, however, on mechanisms to explain trans-activation of SkA by
AP-1. Here, SRE1 was both necessary and sufficient. A molecular basis
for activation of the SkA promoter by SRF in concert with TEF-1 has
been proposed(6, 7) , with virtually identical
conclusions for the avian and rat promoters. Cooperation of SRF and
TEF-1 also was implicated in two distinct transduction pathways for
hypertrophy, TGF- and
-adrenergic agonists. By
contrast, the TEF-1 site is dispensable for full augmentation of the
SkA promoter by Jun. In agreement, nested deletions of the human SkA
promoter suggested that nucleotides encompassing the first SRE
(-153 to -87) were required for maximal trans-activation by
Fos plus Jun, whereas more proximal sequences including the TEF-1 site
at nucleotides -71 to -65 do not mediate AP-1
responsiveness(18) . While direct binding of Jun and Fos/Jun to
a noncanonical site near SRE1 was reported for the human SkA promoter,
our results point instead toward an indirect mechanism, mapped to SRE1
itself. First, Jun can induce the avian SkA promoter in the absence of
TEF-1, Sp1, or YY1 binding, yet the SRF binding site and TATA box are
indispensable. This is intriguing, given that SRF contacts the RAP74
subunit of transcription factor IIF(39, 41) , while
Fos and Jun contact other basal factors, transcription factor IIB and
TATA box-binding protein(42, 46) . Second, the
isolated SkA SRE1 is sufficient for AP-1-dependent expression and is
interchangeable, in this respect, with the c-fos SRE. Third,
using recombinant Fos and Jun produced in reticulocyte lysates, we
detected no binding to the SkA SRE1. This discrepancy with direct
binding of AP-1 factors reported for the human SkA promoter (18) may be explained by technical differences: in the earlier
study, proteins were produced in E. coli, truncated Fos and
Jun were used, and five times more protein was used for the SkA probe versus the authentic TRE. Alternatively, sequence
dissimilarities may be germane. Among the characterized vertebrate SkA
genes, only the human promoter matches the TGACTCA consensus TRE at
five positions that include both cytosine residues.
Control of the
SkA SRE1 by Jun and synergy with SRF both required full-length Jun
protein with intact DNA binding, dimerization, and trans-activation
domains. As no binding of AP-1 factors was detected to SRE1, mechanisms
alternative to direct association must be considered. Conceivably, AP-1
factors might increase transcriptional activity of an SRF binding site
through protein-protein interactions with SRF, increasing SRF
abundance, or affecting transcriptional activity of SRF. Our results
provide no support for a ternary or quarternary complex of Fos/Jun with
DNA-bound SRF. No physical interaction was seen between SRF and AP-1 in
gel mobility-shift assays, nor did AP-1 factors augment DNA binding by
SRF. However, measurements of DNA-protein interaction might overlook
low affinity binding or interactions that require a co-activator. Using
GAL4 fusion proteins to overcome limitations of both gel retardation
assays and endogenous SRF, we found that Jun could not potentiate the
C-terminal activation domain of SRF. It is a formal possibility that
Jun might interact (with affinity too low to be stable in
vitro) only with native SRF or the native SRFSRE complex.
Increased SRF abundance is unlikely to explain AP-1 dependent SkA
transcription, since SRF was not limiting in cardiac myocytes. Our
findings are more consistent with the alternative, that Fos/Jun
transcription factors increase, instead, the transcriptional activity
of SRF. In principle, this could be contingent on altered expression of
an autocrine or paracrine factor(23) , a protein kinase
modulating SRF activity(47) , or a co-activator. Whereas SRF
accessory proteins include most obviously the ternary complex factors
Elk-1/TCF and SAP-1 (48) , association and synergy with SRF
both were demonstrated (49) for the cardiac-restricted
homeodomain protein, Nkx-2.5, vertebrate homologue of the Drosophila tinman gene(50, 51) .
In
summary, our studies reveal a novel AP-1-dependent pathway for gene
induction in cardiac myocytes, via indirect activation of SREs. Hence,
AP-1 factors might plausibly be involved in the up-regulation of genes
containing SREs, including skeletal and smooth muscle -actin and
the immediate-early gene c-fos, in the setting of cardiac
hypertrophy. It is unknown whether the greater induction of
``fetal''
-actin transcripts relative to cardiac
-actin reflects inherent differences among SREs, contextual
sequences, or elements elsewhere in the respective promoters. The SRE
has been implicated in numerous settings as a pivotal regulatory
element for cardiac gene expression during hypertrophy, for activation
of the SkA promoter by TGF-
(6) , basic FGF(52) ,
and
-adrenergic agonists(7) , up-regulation of
ANF by
-adrenergic signals(53) , and induction
of c-fos by load(54) , passive stretch(55) ,
or angiotensin II (56) . Control of SRE-dependent transcription
by AP-1 factors was selective, as the neutral core promoter was
unaffected by Jun, contrasting with the global increase in
transcription provoked by Ras under similar conditions(36) .
Conversely, TGF-
selectively up-regulates fetal cardiac genes,
with little change in RNA or protein content(57) . Hence, the
overall increase in cell RNA and protein can be dissociated from the
``fetal/hypertrophic'' program. Analogously, rapamycin blocks
the increase in total cell protein and p70 S6 kinase activity in
angiotensin II-treated cardiac myocytes, while not affecting induction
of c-fos or fetal cardiac genes(58) . The latter
report highlights other data pointing to the possibility, with which
the present study and our Ras results concur(36) , of
distinguishable signaling cascades for these components of the
hypertrophic phenotype, that the global increase of cell protein is
mediated by p70 S6 kinase, while the fetal program might be mediated,
at least in part, by mitogen-activated protein kinase induction of Fos,
and Jun N-terminal kinase acting though Jun(58) .