© 1995 American Heart Association, Inc.
Articles |
Involvement of Multiple cis Elements in Basal- and 
-Adrenergic Agonist–Inducible Atrial Natriuretic Factor Transcription
Roles for Serum Response Elements and an SP-1–Like Element
From the Department of Biology and Molecular Biology Institute, San Diego (Calif) State University.
Correspondence to Dr Christopher C. Glembotski, Department of Biology, San Diego State University, San Diego, CA 92182.E-mail cglembotski@sunstroke.sdsu.edu.
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Abstract |
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Abstract In the present study, cis elements in the 5'-flanking sequence (FS) of the rat atrial natriuretic factor (ANF) gene involved in regulating basal and
1-adrenergic–inducible transcription were
investigated. Truncation analyses using ANF-luciferase reporter
constructs transfected into primary neonatal rat cardiac myocytes
showed that an A/T-rich serum response element (SRE) at -114 bp
of the ANF 5'-FS, which bound serum response factor (SRF), was required
for basal and inducible transcription. In constructs composed of 134 bp
of rat ANF 5'-FS driving luciferase (ANF-134Luc), mutations in the SRE
at -114 bp disrupted SRF binding and ANF promoter activity.
However, the same mutations in ANF-638Luc had little effect, suggesting
a collaborating role for more distal sequences, such as the other SRE
in ANF-638 at -406 bp. In ANF-638Luc, mutations in the SRE at
-406 bp that disrupted SRF binding to that site decreased ANF
reporter activity by only 25%; however, mutating both of the SREs
completely blocked 1-adrenergic–inducible activity.
Mutation analyses showed that an 
(SP-1)–like site at
-69 bp, shown previously to confer inducibility in reporters with
134 bp of ANF 5'-FS, was not required in ANF-638Luc. However, double
mutants in the SP-1–like region and either SRE completely blocked
1-adrenergic–inducible ANF promoter activity. These
findings emphasize that no single element is responsible for
1-adrenergic agonist–regulated ANF transcription
but that the SREs at -114 and -406 bp and the SP-1–like
sequence at -69 bp mediate the effect in collaboration.
Key Words: atrial natriuretic factor • 1-adrenergic agonist inducibility • transcription • serum response element • serum response factor
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A great deal of interest has focused on the intracellular signals linking cell surface receptor activation with gene expression and the related hypertrophic cell growth of cardiac myocytes.1 A characteristic feature of treating primary neonatal ventricular myocytes with a variety of growth stimuli (eg,
1-adrenergic agonists,
endothelin, transforming growth factor-ß, stretch, and electrical
pacing of contractions) is the reactivation of certain cardiac genes,
such as -skeletal actin, ß-myosin heavy chain, myosin
light chain-2, and ANF, which are normally expressed primarily during
embryonic development.2 3 4 5 6 Recent studies have indicated
that the induction of these genes is at least partly transcriptional
and that their coordinate induction suggests the involvement of
conserved signaling pathways.7 8 9
The present study was undertaken to gain a better understanding of
the mechanisms by which 1-adrenergic agonists induce ANF
transcription in primary neonatal ventricular myocytes. In
cardiac myocytes, 1-adrenergic agonists, which
activate ANF transcription (eg, see References 8 and 108 10 ),
promote the conversion of membrane phosphoinositides to
diacylglycerol and inositol 1,4,5-tris-phosphate, which results in
the activation of PKC.11 12 13 14 In some cells, such PKC
activation leads to the formation of fos/jun heterodimers,
which can induce the expression of genes possessing
cis-acting elements specific for AP-1
binding.15 16 17 Although 1-adrenergic
agonists can activate myocardial cell PKC18 and
although the resulting AP-1 may be required for some aspects of the
hypertrophic growth response, it is unclear whether ANF transcription
is stimulated via AP-1 binding at the ANF 5'-FS. For example, although
the transfection of primary myocardial cells with fos and/or
jun expression constructs or constitutively active PKC
constructs can activate cotransfected ANF
reporters,19 20 there has been no functional
identification of AP-1–binding cis sequences or TREs in the
ANF gene, and in the rat ANF 5'-FS, there are no true consensus TREs.
Furthermore, there is also evidence that fos overexpression
can repress ANF transcription.21 Also, one recent study
has implicated the involvement of an SP-1–like protein binding at a
promoter-proximal site of the ANF promoter in
1-adrenergic agonist–stimulated
transcription.22 Therefore, it is possible that although
1-adrenergic agonist–activated ANF
transcription may involve PKC, as previously suggested,21
it may not involve AP-1 as a transcription factor.
One case of transcriptional activation that conditionally involves PKC,
but not AP-1, is the induction of the human c-fos gene
through SRF binding to an SRE in the 5'-FS. In several cell lines, it
has been shown that some growth factors (eg, phorbol esters) augment
c-fos expression through the activation of
MAPK23 ; this is followed by the MAPK-mediated
phosphorylation of an accessory factor (eg,
p62TCF, also known as Elk-1 or Sap-1) that binds to
SRF/SRE to form the ternary complex that enhances transcription (for
recent review, see Reference 2424 ). Alternatively, some growth factors
(eg, serum) augment c-fos transcription through the MAPK-
and pp90rsk-mediated phosphorylation of SRF
itself,25 apparently independently of ternary complex
formation.26 The recent demonstration that
1-adrenergic agonists activate myocardial cell
MAPK,27 along with the finding that the blockade of MAPK
inhibits 1-adrenergic agonist–induced ANF
transcription,28 supports the hypothesis that SRF could
participate in the PE-inducible transcription of at least some of the
myocardial cell genes activated during the hypertrophic
program.
To evaluate the possible involvement of SRF in ANF induction, the FSs
of the rat ANF gene most proximal to the transcriptional start site
were tested for the ability to mediate basal and
1-adrenergic agonist–inducible transcription. In
the present study, we report on the identification of several
SRE-like elements in the ANF 5'-FS that bind a cardiac-derived
protein that is antigenically indistinguishable from SRF and that
participate together in PE-inducible ANF transcription.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plasmid Constructs and Oligonucleotide Probes
An EcoRI–Spe I fragment of the plasmid pANF 30038 was cloned into pGeneLight2-Promoter (pGL2-P, Promega), replacing the simian virus 40 promoter of the vector with rat ANF 5'-FSs from -638 to +65, to produce ANF-638Luc. Similar truncations were produced with HindIII–Spe I (ANF-134Luc) and Nla IV–Spe I (ANF-65Luc) fragments. Other truncations between -134 and -65 were created by using the Promega Erase-a-base kit according to the manufacturer's instructions. Site-directed mutagenesis of ANF-134Luc and ANF-638Luc was performed by using the Promega Altered Sites in vitro mutagenesis kit according to the manufacturer's instructions and the appropriate oligonucleotides shown in Fig 1
. The ANF sequences in all of the expression
constructs used in the present study terminate on the 3' end at ANF
+65. All plasmid constructions were verified by dideoxy sequencing.
Plasmids used for electroporation were purified according to
manufacturer's instructions on a pZ523 column (5-Prime 3-Prime) and
transfected at a concentration of 10 µg luciferase vector per 3 µg
ß-gal vector (CMV-ß-gal, Clontech) per 35-mm well.
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Synthetic oligonucleotides (Fig 1
),
which were used as probes and/or competitors in EMSAs, were prepared by
the CSUPERB Microchemical Core Facility, San Diego (Calif) State
University. Each pair of oligonucleotides was prepared
with the addition of Psp A1 sequences to the 5' of the positive (+)
strand and Sal1 sequences to the 5' of the negative (-) strand to
facilitate labeling.
Cell Culture and Transfections
Myocardial cells were prepared as described
previously.5 Briefly, 1- to 4-day neonatal rat hearts were
dissected, the atrial and ventricular portions were
subdissected, and the apical one third of the ventricle was removed for
trypsin dissociation. Cultures prepared by this method contain >98%
cardiac myocytes. For transfections, freshly dissociated
ventricular cells at a density of 10 million cells per
milliliter minimal medium (DMEM/F-12 [GIBCO/BRL] containing 1 mg/mL
BSA) were mixed with DNA, electroporated in a BioRad (Hercules) gene
pulser (600 V, 25 µF, 100 
, 0.2-cm gap cuvette), and plated on
fibronectin-coated wells. Nonmyocardial cells obtained by
preplating dissociated tissue, when electroporated under the conditions
described above, demonstrated no luciferase or ß-gal activity
after 48 hours in serum-containing medium, suggesting that any
nonmyocardial cells present in the cultures do not directly
contribute to reporter enzyme activity. Viability of electroporated
ventricular myocytes 12 to 14 hours after plating in
DMEM/F-12 (1:1) plus 10% FBS (Gemini Bioproducts) was
empirically determined to be 
30%; therefore, final density was
750 000 to 1 million cells per 35-mm well (78 000 to 100 000 cells
per square centimeter). After 12 to 14 hours after electroporation in
10% FBS, cells were washed thoroughly, and the medium was replaced
with minimal medium. Twenty-four hours later, the medium was again
replaced with minimal medium with or without the additions as described
in the figure legends. Luciferase and ß-gal assays were performed
as described previously.33 34 Luciferase activity was
measured for 30 seconds on a Bio Orbit 1251 Luminometer
(LKB/Pharmacia). Data are expressed as "relative luciferase"
(arbitrary integrated luciferase units per ß-gal unit),
representative of at least three independent
experiments performed with two different plasmid preparations, and
represent the mean±SEM of triplicate 35-mm wells.
Preparation of Nuclear Extracts
Nuclei from ventricular tissue were obtained as
described previously.35 Nuclei were extracted essentially
as described previously36 in high salt buffer C (20 mmol/L
HEPES [pH 7.9], 25% glycerol, 1.5 mmol/L MgCl2,
420 mmol/L NaCl, 0.2 mmol/L EDTA, 0.5 mmol/L dithiothreitol, 1 mmol/L
phenylmethylsulfonyl fluoride, 2 µg/mL leupeptin, and 2
µg/mL aprotinin) and then dialyzed (12 000– to 14 000–molecular
weight cutoff) against buffer D (20 mmol/L HEPES [pH 7.9], 20%
glycerol, 100 mmol/L KCl, 0.2 mmol/L EDTA, 1 mmol/L
phenylmethylsulfonyl fluoride, and 0.5 mmol/L dithiothreitol).
Extracts were stored at -70°C.
EMSA
EMSA was performed as described previously,37 with
minor modifications. Probes were prepared by Klenow
fragment–mediated filling of the sticky ends of
double-stranded oligonucleotides. A typical binding
assay contained 20 000 cpm double-stranded probe and 5 to 10 µg
protein in 1x binding buffer (30 mmol/L NaCl, 0.1 mmol/L EDTA, 8
mmol/L Tris-HCl [pH 8.2], 8% glycerol, 1 nmol/L dithiothreitol, and
0.2 mmol/L ZnCl2). After a 10-minute preincubation of
extract and 0.1 µg nonspecific competitor (poly dI-dC, Pharmacia),
the probe was added. Binding was allowed to proceed at room temperature
for 30 minutes before separation of bound and free probe on a 4%
native polyacrylamide gel (29:1 bis/acrylamide) in
0.25x Tris-borate-EDTA buffer at room temperature. For supershift
experiments, 1 µL of the appropriate antiserum was preincubated for
30 minutes with 5 to 10 µg of extract before the addition of
nonspecific competitor, followed by probe. The antisera, which were
raised against human SRF and rat SRF, have been previously
described.39 DNA-protein complexes were detected by
autoradiography. The autoradiograms
of some gels in this manuscript were scanned with a Molecular Dynamics
Personal Densitometer, and the resulting image was imported to Adobe
Photoshop and Claris MacDraw Pro II for final figure preparation.
Interference Analyses
Methylation interference analysis was performed as
described previously,40 as was carbethoxylation
interference,41 42 with minor modifications. Glycogen was
used in place of tRNA as a precipitation carrier. DMS was used to treat
singly end-labeled double-stranded probe, whereas DEPC
modification was performed on labeled single-stranded
oligonucleotide before annealing to produce
double-stranded probe. For all probes, labeled, modified, and
double-stranded product was purified from a 15% nondenaturing
polyacrylamide gel by electroblotting to NA-45 paper
(Schleicher & Schuell) and eluting according to the manufacturer's
instruction before use in EMSA. The EMSA was scaled up five times with
respect to nuclear extract and nonspecific competitor (dI-dC), with
300 000 cpm of probe. Specific complexes 1 and 2 and free probe were
isolated from the EMSA gel and also electroblotted to and eluted from
NA-45 paper. Piperidine-cleaved probe was fractionated on a 10%
denaturing polyacrylamide gel and visualized by
autoradiography.
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Results |
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Truncation Analysis of SREs and ANF Induction
In the proximal 638 bp of the rat ANF 5'-FS, consensus SREs, or near-consensus (9 of 10 bases match) SREs (ie, SRE=CC[A/T]6GG; eg, see Reference 4343 ), are located at -406 and -114 bp relative to the transcription start site.3 To gain a better understanding of the potential roles for these SREs in basal and
1-adrenergic
agonist–inducible ANF transcription, a group of reporter
constructs was prepared by fusing various lengths of the ANF 5'-FS to a
luciferase reporter gene, transfecting primary myocardial cells, and
determining basal and PE-inducible levels of reporter enzyme
activity.
Reporter activity was similar with plasmids containing 638 and 134 bp
of ANF 5'-FS (Fig 2), suggesting that
sequences proximal to -134 bp were sufficient for maximal
PE-inducible transcription. Whereas truncation to 122 bp of 5'-FS
resulted in an 2-fold loss of both basal and PE-inducible reporter
activity, further truncation to -109 bp resulted in decreases of
10-fold, suggesting that sequences between -134 and -109
bp were required for optimal basal and PE-inducible transcription.
PE-inducible reporter activities were between 5- and 7-fold for
ANF-638GL, ANF-134GL, and ANF-122GL and then decreased to between
3.5-fold and 2-fold for the shorter constructs. This suggested that
although sequences between -638 and -122 bp were required
for maximal PE inducibility, more proximal sequences residing between
-122 and -65 bp remained capable of conferring some degree
of PE-inducible reporter activity. Since there was a dramatic decrease
of reporter activity observed between -122 and -109 bp and
since there is an A/T-rich SRE-like sequence centered at -114 bp
(Fig 3), further studies were focused on
determining whether and how this SRE-like region could participate in
ANF transcription.
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P<.01 vs other control values using ANOVA followed by Newman-Keuls
post hoc ANOVA.
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Nuclear Proteins That Bind to the A/T-Rich Region of the ANF
5'-FS
The nature of factors that might bind to the A/T-rich sequence in
the ANF 5'-FS was studied by EMSAs using a double-stranded
oligonucleotide containing ANF SRE/114 (ANF
[-134 to -95]; see Fig 1) as a probe and
neonatal ventricular tissue nuclear extracts. A single
major complex was obtained that could be inhibited by the addition of
100-fold molar excess of the unlabeled probe, c-fos SRE, or
YY-1 double-stranded oligonucleotides, whereas
TRE,15 MCAT, PERE,22 and GATA
double-stranded oligonucleotides were not effective
competitors (Fig 4). Competition by the
c-fos SRE and YY-1 oligonucleotides, both of
which have canonical SREs and bind SRF, is consistent with the
putative identification of an SRE-like site within ANF SRE/114.
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To map more precisely the nature of the DNA-protein interactions,
carbethoxylation and methylation interference analyses were
carried out. DEPC-mediated modification of adenines at positions
-113, -112, -111, -110, and -105 on the +
strand of the ANF 5'-FS interfered with complex formation (Fig 5A, DEPC +), as did modification of adenines -114
and -116 on the - strand (Fig 5A, DEPC
-). DMS-mediated modification of guanines at -109,
-108, and -107 of the + strand of the ANF 5'-FS interfered
with complex formation (Fig 5A, DMS +), as did
modification of the guanines at -117 and -106 of the
- strand (Fig 5A, DMS -). The contact
sites made between the protein responsible for the band shift in Fig 4 and the ANF promoter sequences are diagrammed in Fig 5B. The importance of the A/T-rich core and the
adjacent pyrimidines in this region suggests a binding event similar to
the binding of SRF to canonical SREs (eg, see Reference 4343 ).
Accordingly, the characteristics of the protein(s) that binds to ANF
SRE/114 to form the major complex on the EMSA were analyzed
further by using several antisera raised against transcription factors
that bind to A/T-rich elements.
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).
Among the nuclear proteins known to bind to A/T-rich elements are MEFs
2A through 2D,44 45 some of which are also known as
related to rat SRF46 and recognize the consensus
CTA[A/T]4TAG and CArG binding factor,47 now
known to be SRF.39 To characterize the protein(s) binding
to the SRE-like region in ANF SRE/114, antisera specific for MEF 2 and
SRF were used in the EMSAs. Using either ANF SRE/114 or the
c-fos SRE probe (see Fig 1),
ventricular nuclear extracts produced a major band with the
same mobility (Fig 6). Only the SRF
antiserum produced a supershifted complex with either probe,
demonstrating that this complex is the result of the binding of an
SRF-related protein to this region of the ANF 5'-FS. Indeed, the
A/T-rich sequence in the ANF promoter (CT[TTAAAA]GG) is similar to
the canonical SRE core sequence (CC[A/T]6GG) but differs
significantly from the consensus MEF 2 binding site
(CTA[A/T]4TAG).
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Mutations that were predicted to disrupt the binding of an SRF-like
protein to ANF SRE/114 were prepared. Whereas the native ANF SRE/114,
c-fos SRE, and YY-1 oligonucleotides each
blocked band formation in the EMSA, as expected, unlabeled
oligonucleotides containing cluster mutations made at
the putative SRE-like site (eg, ANF/C114 or ANF/C121; see Figs 1 and 3)
were ineffective competitors of complex formation (Fig 7), consistent with the need for
an intact SRE for efficient complex formation. An unlabeled
oligonucleotide harboring a similar-sized cluster
mutation to the 3' region of the putative SRE-like site in ANF (ANF
SRE/C101; see Figs 1 and 3) served as an effective competitor, as
expected, since the region affected lies outside the SRE-like area. As
expected, the TRE, MCAT, PERE, and GATA
oligonucleotides were ineffective competitors. These
data support the interference and supershift analyses
indicating that the complex represents the binding of an
SRF-like protein to ANF SRE/114.
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Effects of Mutations on PE Inducibility
To evaluate further the importance of the SRE at -114 bp in
ANF transcription, the effects of the cluster mutations were determined
in transfection experiments. Beginning with ANF-134Luc, which consists
of 134 bp of ANF 5'-FS driving luciferase expression, three mutations
were prepared, two of which spanned the putative SRE (C114 and C121;
see Fig 3) and one of which was located outside this
region (C101). Compared with the native sequence, both of the
SRE-directed mutants possessed 5-fold lower basal transcription and
8-fold lower PE-inducible transcription (Fig 8A). The C101 mutation, which has no
effect on SRF binding, also had no effect on transcription.
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While these studies were in progress, an article was published
demonstrating that a GC-rich region at -69 bp, which binds an
SP-1–like protein, was important for PE-inducible ANF
transcription.22 Since this element, which was named PERE,
was intact in the ANF SRE/C114 and ANF SRE/C121 mutants, we sought to
determine whether the SRE-like sequence at -114 bp and the
SP-1–like sequence at -69 bp both participated in regulating ANF
transcription. Accordingly, a mutation was prepared in the PERE on
ANF-134Luc (see Fig 3); this was identical to the
cluster mutation used previously to identify the
element.22 Like the C114 and C121 mutations, the C69
mutation within ANF-134Luc resulted in a coordinate 6- to 8-fold
decrease in both basal and PE-inducible transcription (Fig 8A). Thus, it was apparent that in the context of
ANF-134Luc, both ANF SRE/114 and the previously identified ANF SP-1/69
sites were required for optimal basal and PE-inducible transcription;
however, neither one alone was sufficient to confer induction.
Given the dramatic impact of the SRE/114 and SP-1/69 mutations in
ANF-134Luc, we wished to confirm the importance of these elements in a
larger context of the ANF promoter. Accordingly, both the C114 and C69
mutations were prepared in ANF-638Luc. Although the C114 mutation in
ANF-638Luc resulted in a drop in both basal and PE-inducible
transcription, the decrease was modest, amounting to only a 50%
reduction (Fig 8B). Thus, it was clear that although
the SRE at -114 bp was important for optimal induction using
ANF-638Luc, regions of the ANF 5'-FS distal to -134 bp could
serve as enhancers and in part they could "rescue" mutations in
more promoter-proximal regions of the 5'-FS. We also investigated
the effects of the PERE cluster mutation in ANF-638Luc. The PERE
mutation resulted in a small but significant decline in basal and
PE-inducible transcription of 8% to 9%; this was unexpected given
the dramatic effects of this mutation in the -134 context in the
present study (Fig 8A) as well as the previous
report.22 Continuing with the hypothetical involvement of
SRF, we next evaluated whether mutating the SRE at -406 bp, which
is the only other SRE in ANF-638Luc, effected ANF transcription.
Although basal transcription was not affected significantly in the ANF
SRE/C406 mutant, PE-inducible transcription decreased, but by only 25%
(Fig 8B). This result was consistent with the
possibility that SRF binding to both ANF SRE/406 and ANF SRE/114 might
be required for maximal basal and PE-inducible transcription.
To evaluate whether an SRF-like protein could bind to ANF SRE/406, EMSA
was carried out. Using a probe that included the ANF SRE/406 and some
FS (see Fig 1), a nuclear protein/DNA complex was
observed that possessed the same mobility as that obtained with the ANF
SRE/114 probe (Fig 9). Indeed, both
complexes were efficiently supershifted by using the SRF antiserum.
Moreover, the competition profile for the ANF SRE/406 complex was
consistent with SRF binding, since it could be competed by ANF
SRE/114, c-fos SRE, and YY-1; however, it could not be
competed by ANF SRE/C406 or ANF SRE/C114 (which are SRE-directed
cluster mutations; see Fig 1), the native PERE, or an
MCAT consensus sequence.
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Effects of Double Mutations on PE Inducibility
Since either the ANF SRE/C114 or the ANF SP-1/C69 mutants alone
blocked transcription in ANF-134Luc but had minimal effects in
ANF-638Luc, as did ANF SRE/C406, we determined whether these potential
cis elements might participate combinatorially to regulate
ANF transcription. Accordingly, double mutations were prepared in
ANF-638Luc; interestingly, mutating any two of the three elements
severely inhibited transcription, both basal and PE inducible (Fig 10). When any two of the SRE/C114,
SRE/C406, and/or SP-1/C69 mutations were present in ANF-638Luc,
basal transcription decreased by 2-fold, whereas PE-inducible
transcription decreased by 5-fold. This amounted to a decrease in
PE-inducible transcription from 3.5-fold in ANF-638Luc to 1.5-fold
in any of the three double mutants.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Taken together, these results demonstrate that multiple regions of the ANF 5'-FS are involved in regulating both basal and PE-inducible transcription and that two of these are A/T-rich SRE-like sequences at -406 and -114 bp, which likely bind an SRF-related protein. Although a GC-rich region at -69 bp, previously identified as a PERE, is also important for both basal and inducible transcription in promoter/reporter constructs possessing 134 bp of ANF 5'-FS, as shown previously,22 when tested in the context of larger stretches of the ANF 5'-FS and promoter, the SP-1–like region appears to be less important, much like the ANF SRE at -114 bp. It is possible that this SP-1–like element accounts for the partial PE-inducible reporter activity obtained by using constructs possessing -109 bp or less of the ANF 5'-FS (see Fig 2
).
The results of the present study emphasize the value of carrying
out mutational analyses of putative cis elements in
both small and large promoter contexts; this approach revealed that
elements distal to -134 bp can apparently contribute to basal and
PE-inducible ANF transcription such as to complement or duplicate the
functions of proximal elements. Indeed, a previous study identified the
potential importance of sequences residing between -134 and
-638 bp of the ANF 5'-FS in PE inducibility,8 and a
very recent study has indicated the probable involvement of a
non–SRF-binding A/T-rich sequence at -530 bp of the ANF 5'-FS in
ANF transcription.1 Thus, it is very probable that an
array of cis elements residing in the ANF 5'-FS, and perhaps
elsewhere in the gene, contribute to the regulation of basal and
inducible transcription.
Although this is the first report indicating that
1-adrenergic receptor activation can enhance ANF
transcription through an SRF-related molecule(s), several previous
studies have identified SREs in other genes as potential enhancers of
cardiac gene expression. For example, an SRE in the cardiac -SkA
gene was shown to confer basic FGF– but not acidic FGF–inducible
transcription to reporter expression.48 More recently, the
characterization of the transcription factors that interact with this
SRE has shown that in addition to SRF, TEF-1 and SP-1 also participate
in basal and inducible transcription,49 and
1-adrenergic induction of -SkA in cardiac myocytes
also requires all three elements.50 In another example,
transfection experiments showed that stretch-induced
c-fos transcription in cardiac myocytes maps to the SRE in
that gene51 ; however, no direct evidence was
presented to address the nature of the protein(s) binding to
that element. Also, a report from Argentin et al52
indicated that the SRE at -406 bp of the rat ANF 5'-FS
contributed to basal ANF transcription in ventricular
myocytes; however, the protein(s) that bound to this element, which did
not apparently bind to the c-fos SRE, was not positively
identified.
What is the mechanism through which SRF mediates cardiac gene
induction? In part, the answer to this question may depend on the
identity of the gene under study as well as the stimulus. Most previous
studies relating to the mechanism of SRF action have investigated the
induction of the c-fos gene in non–muscle cell lines.
In general, stimuli that induce c-fos transcription via SRF
fall into two categories: (1) the PKC/ternary complex–dependent
stimuli (eg, phorbol ester), which require MAPK-mediated
p62TCF phosphorylation,53 and
(2) the PKC/ternary complex–independent stimuli (eg,
serum),26 which require pp90RSK-mediated SRF
phosphorylation.25 Although a variety of
stimuli can mediate myocardial cell hypertrophy and
transcriptional activation, a potential role for SRF has been indicated
for stretch-induced c-fos, basic FGF–induced -SkA
expression, and (from the present study) ANF induction. Myocardial
cell stretch is known to activate PKC and MAPK,49
suggesting that it may fall into category 1. Indeed, mutations of the
c-fos SRE, which are known to block ternary complex
formation,26 also block stretch-activated
c-fos transcription.51 It is also clear that
the 5'-FS of the chicken -SkA gene possesses several SRE-like
sequences, one of which confers basic FGF induction of
promoter/reporter constructs in rat myocardial cells.48 49
Interestingly, the -SkA SRE-1 possesses no flanking Ets-domain
sequence, and coupled with the belief that FGF operates through
flg-mediated activation of tyrosine kinase and not PKC, it
would appear that SRF may mediate induction through the PKC/ternary
complex–independent mechanism. We hypothesize that SRF-regulated
ANF transcription falls into the PKC/ternary complex–independent
category. Our preliminary studies have shown that no ternary complex is
ever formed when either the ANF SRE/114 or ANF SRE/406 is used as a
probe. This finding would indicate that in myocardial cells,
PE-activated MAPK may lead to pp90rsk activation and that the
subsequent phosphorylation of SRF at Ser-103 would lead
to transcriptional activation, as described for the c-fos
gene in a different cell type.25 We have preliminarily
demonstrated that SRF is phosphorylated at Ser-103 in
myocardial cells, lending credibility to this hypothesis.
In summary, the present study provides direct evidence that SRF
participates in both basal and PE-inducible ANF transcription,
providing the framework for a complete signaling pathway from the cell
surface 1-adrenergic receptor to transcriptional
activation of the ANF gene. Interestingly, recent studies on
ß-myosin heavy chain induction in primary myocardial cells have
demonstrated that the binding of a TEF-1–like protein to an MCAT
sequence in the 5'-regulatory region of that gene confers PE-inducible
transcription.54 Thus, it appears that in cardiac myocytes
there are multiple enhancers through which 1-adrenergic
receptor activation can lead to apparently coordinated transcriptional
activation of a collection of genes that are typically expressed
primarily during early development. These factors include but are not
limited to TEF-1,54 a cardiac SP-1–like
protein,22 and an SRF-like protein (present study).
Future studies focusing on the molecular mechanisms by which these
proteins are ultimately activated as enhancers by
1-adrenergic receptor activation will be of interest, as
will an analysis of how they collaborate to confer not only
hormone-inducible transcription but also stage- and
tissue-specific expression.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by National Institutes of Health grants NS-25073 and HL-46345. A.B. Sprenkle was supported by a predoctoral fellowship from the American Heart Association, California Affiliate, Inc (grant 92-418). We gratefully acknowledge the expert technical assistance of Donna Thuerauf. We thank R. Prywes (Columbia University, New York, NY) for the SRF and MEF 2 antisera.
Received August 16, 1995; accepted September 25, 1995.
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References |
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H. E. Hoover, D. J. Thuerauf, J. J. Martindale, and C. C. Glembotski alpha B-crystallin Gene Induction and Phosphorylation by MKK6-activated p38. A POTENTIAL ROLE FOR alpha B-CRYSTALLIN AS A TARGET OF THE p38 BRANCH OF THE CARDIAC STRESS RESPONSE J. Biol. Chem., July 28, 2000; 275(31): 23825 - 23833. [Abstract] [Full Text] [PDF]
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C. P. Mack, A. V. Somlyo, M. Hautmann, A. P. Somlyo, and G. K. Owens Smooth Muscle Differentiation Marker Gene Expression Is Regulated by RhoA-mediated Actin Polymerization J. Biol. Chem., January 5, 2001; 276(1): 341 - 347. [Abstract] [Full Text] [PDF]
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Q. Liang, L. J. De Windt, S. A. Witt, T. R. Kimball, B. E. Markham, and J. D. Molkentin The Transcription Factors GATA4 and GATA6 Regulate Cardiomyocyte Hypertrophy in Vitro and in Vivo J. Biol. Chem., August 3, 2001; 276(32): 30245 - 30253. [Abstract] [Full Text] [PDF]
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C. Morisco, K. Seta, S. E. Hardt, Y. Lee, S. F. Vatner, and J. Sadoshima Glycogen Synthase Kinase 3beta Regulates GATA4 in Cardiac Myocytes J. Biol. Chem., July 20, 2001; 276(30): 28586 - 28597. [Abstract] [Full Text] [PDF]
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R. Craig, M. Wagner, T. McCardle, A. G. Craig, and C. C. Glembotski The Cytoprotective Effects of the Glycoprotein 130 Receptor-coupled Cytokine, Cardiotrophin-1, Require Activation of NF-kappa B J. Biol. Chem., September 28, 2001; 276(40): 37621 - 37629. [Abstract] [Full Text] [PDF]
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R. Craig, A. Larkin, A. M. Mingo, D. J. Thuerauf, C. Andrews, P. M. McDonough, and C. C. Glembotski p38 MAPK and NF-kappa B Collaborate to Induce Interleukin-6 Gene Expression and Release. EVIDENCE FOR A CYTOPROTECTIVE AUTOCRINE SIGNALING PATHWAY IN A CARDIAC MYOCYTE MODEL SYSTEM J. Biol. Chem., July 28, 2000; 275(31): 23814 - 23824. [Abstract] [Full Text] [PDF]
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