(Received for publication, August 21, 1996, and in revised form, October 29, 1996)
From the Department of Biology and Molecular Biology Institute, San Diego State University, San Diego, California 92182
The cardiac genes for the A- and B-type
natriuretic peptides (ANP and BNP) are coordinately induced by growth
promoters, such as 1-adrenergic receptor agonists
(e.g. phenylephrine (PE)). Although inducible elements in
the ANP gene have been identified, responsible elements in the BNP gene
are unknown. In this study, reporter constructs transfected into
neonatal rat ventricular myocytes showed that in the context of 2.5 kilobase pairs of native BNP 5
-flanking sequences, a 2-base pair
mutation in a promoter-proximal M-CAT site (CATTCT) disrupted basal and
PE-inducible transcription by more than 98%. Expression of
constitutively active forms of Ras, Raf-1 kinase, and protein kinase C,
all of which are activated by PE in cardiac myocytes, strongly
stimulated BNP reporter expression. Isolated M-CAT elements conferred
PE, protein kinase C, and Ras inducibility to a minimal BNP promoter,
however, they did not confer Raf-1 inducibility. These results show
that M-CAT elements can serve as targets for Ras-dependent,
Raf-1-independent pathways, implying the involvement of c-Jun
N-terminal kinase and/or p38 mitogen-activated protein kinases, but not
extracellular signal-regulated protein kinase/mitogen-activated protein
kinase. Moreover, the essential M-CAT element distinguishes the BNP
gene from the ANP gene, which utilizes serum response elements and an
Sp1-like sequence.
The A-, B-, and C-type natriuretic peptides
(NPs)1 are structurally related
cardiac-derived peptides with vasorelaxant, diuretic, and natriuretic
effects (1-7). Upon treatment with stimuli that can eventually lead to
the hypertrophic growth of ventricular myocytes, several embryonic
cardiac genes are reactivated, including those for ANP, BNP, -myosin
heavy chain, and skeletal
-actin (6-18). While the precise function
of this recapitulation of embryonic cardiac gene expression is unclear,
it can be speculated that increased NP production represents a
compensatory endocrine response to stimuli that often increased blood
pressure. Accordingly, a knowledge of the mechanisms by which the NPs
are induced during the hypertrophic growth program will provide a
better framework upon which to understand how the expression levels of
the hormones are regulated under less severe, but nonetheless
hemodynamically challenging physiological conditions.
A variety of studies have addressed the mechanisms responsible for ANP
induction in primary neonatal rat cardiac myocytes (11, 12, 15, 17).
Recent studies have demonstrated the importance of serum response
elements (SREs) (18) as well as SP-1-like elements (16) in the
transcriptional activation of ANP in response to
1-adrenergic agonists. Earlier reports have also
indicated the probable involvement of AP-1-binding
cis-sequences, also known as
12-O-tetradecanoylphorbol-13-acetate response elements (TREs), in regulating transcription of human ANP (19). Accordingly, it
is believed that
1-adrenergic agonists, and perhaps
other ANP inducers, stimulate myocardial cell signaling pathways, which eventually lead to the activation of serum response factor, an Sp1-like
protein, and perhaps AP-1, each of which converge on the
transcriptional enhancement of ANP.
Relatively little is known about the regulation of BNP expression.
Phorbol esters and diacylglycerol increase BNP mRNA and peptide
levels (20, 21), suggesting the involvement of PKC. Additionally, BNP
promoter activity, measured using constructs containing approximately
2.5 kb of the rat BNP 5-FS upstream of a luciferase reporter, is
inducible by phorbol esters, serum, or the
-adrenergic agonist,
phenylephrine (PE) (22). Thus, it is possible that in part the
coordinated induction of ANP and BNP involves the convergence of
intracellular signaling mechanisms upon cis-elements that
are conserved between the genes. The present study was undertaken to
test this hypothesis by mapping and identifying regions of the BNP
5
-FS that are critical for basal and inducible transcription in rat
cardiac myocytes.
Mutagenesis
Preparation of Truncated BNP/Luciferase ConstructsA 2.5-kb
portion of the rat BNP 5-FS was inserted into a luciferase reporter
construct (pGL2; Promega, Madison, WI), as described previously (22).
Truncated versions of BNP/luciferase were created either by using
native restriction sites, by synthesizing specific PCR primers, or by
unidirectional deletion (Erase-a-Base, Promega), starting with
BNP-2501GL, as described previously (18).
A series of 6-bp cluster mutations covering the BNP
5-FS between
103 bp and
42 bp was created in full-length BNP-2501
by site-directed mutagenesis using Altered Sites (Promega), as
described previously for ANP (18). Briefly, a fragment of the BNP 5
-FS from
116 to +80 bp was inserted into the pAlter vector and used as a
template. Oligonucleotides containing the desired 6-bp mutation (mutants A-H in Fig. 1) flanked by 12 nucleotides of native
BNP sequence on either side, were synthesized and used to prepare the
mutant constructs using methods described by the manufacturer. Point
mutations in the M-CAT site (
M-CAT) were prepared beginning with
oligonucleotides containing the changed nucleotides flanked on either
side by native BNP sequences, extending on the 5
side to BNP-116,
where there is a SacI site. Using these oligonucleotides as
sense primers, and an oligonucleotide complementary to sequences in the
5
-region of the luciferase gene as a common antisense primer, PCR was
carried out using BNP-116GL as the template. PCR products were then
digested with SacI (BNP-116) and BamHI (BNP+80), and then cloned into pGL2 to create BNP-116GL possessing cluster mutations A-H and the
M-CAT mutation shown in Fig. 1.
To create multiple mutations (e.g. M-CAT/GATA(
95)), a
BNP-116GL construct possessing the appropriate GATA-directed point mutation(s), prepared previously (22), was used as the template and the
sense primer, possessing the 2-nucleotide M-CAT mutation (see above),
was coupled with the luciferase primer to prepare the appropriate
PCR-generated product. As above, PCR products were then digested with
SacI (BNP-116) and BamHI (BNP+80), and then
cloned into pGL2 to create BNP-116GL possessing various combinations of
mutations in the M-CAT and GATA sites (see Fig. 3, top, for M-CAT/GATA mutations).
To prepare the cluster and point mutations in the 2.5-kb BNP 5-FS, we
utilized the SacI sites located 5
of
2501 in the pGL2
multiple cloning site and at
116 in the BNP 5
-FS. Wild type
BNP-2501GL was digested with SacI, and the fragment from
2501 to
116 was purified. This fragment was then cloned into SacI-digested BNP-116GL constructs possessing the mutations
described above to restore the full-length BNP-2501.
Two synthetic
oligonucleotides (see below) were designed so that after hybridization
there is a tandem repeat of two canonical BNP M-CAT core elements
(boxed) separated by 5 bp (see Sequence 1).
Some flanking BNP sequences were included such that the repeats
represent BNP(112 to
97)/BNP(
109 to
97). The
lowercase nucleotides at the ends are not native to the rat
BNP 5
-FS and were added to provide PspA1 sites. The
double-stranded synthetic oligonucleotide was ligated into the
PspA1 site on the 5
boundary of the rat BNP sequences in
BNP-81GL. Positive clones were sequenced and some clones contained one
insert (i.e. 2XM-CAT/BNP-81GL), and others contained two
inserts (i.e. 4XM-CAT/BNP-81GL). All plasmid constructions
were verified by dideoxy sequencing.
Cell Culture and Transfections
Myocardial cells were prepared as described (18, 22). For
transfections, freshly dissociated cells were resuspended at a density
of 30 million cells/ml of minimal medium (Dulbecco's modified Eagle's
medium/F-12 medium (Life Technologies, Inc.) containing 1 mg/ml bovine
serum albumin). For each transfection, 300 µl, or 9 million cells,
were mixed with 15 or 30 µg of BNP/luciferase (test reporter), and 9 µg of CMV--galactosidase (normalization reporter), and in some
experiments, 45 µg of a PKC, Ras, or Raf-1 expression construct (see
below). Each 300-µl aliquot was then electroporated in a Bio-Rad Gene
Pulser at 700 V, 25 microfarads, 100 ohms in a 0.2-cm gap cuvette. This
procedure results in an approximate 30% viability (18); accordingly,
the 3 million viable cells were plated into fibronectin-coated 35-mm
wells, at 1 × 106 cells/well, or into 24-mm wells at
0.5 × 106 cells/well. Thus, the plasmid
concentrations per 106 viable cells were 5 µg of
BNP/luciferase (10 µg in some experiments), 3 µg of
CMV-
-galactosidase, and in some experiments, 15 µg of PKC, Ras, or
Raf-1 test construct.
Transfected cells were maintained in Dulbecco's modified Eagle's
medium/F-12 medium supplemented with 10% fetal bovine serum for
approximately 14 h after electroporation. The cells were then washed thoroughly, and the medium was replaced with minimal medium. Unless otherwise stated, 24 h later, the medium was again replaced with minimal medium ± 50 µM phenylephrine with 1 µM propranolol added to block -adrenergic receptors.
Luciferase and
-galactosidase assays were performed as described
(18, 22). Luciferase activity was measured for 30 s on a Bio Orbit
1251 Luminometer (Pharmacia Biotech Inc.). Data are expressed as
"relative luciferase" = arbitrary integrated luciferase
units/
-gal units, representative of at least three independent
experiments performed with two different plasmid preparations, and
represent the mean and S.E. of triplicate 35- or 24-mm wells.
To assess the effects of PKC on BNP/luciferase reporter expression,
PKCOP, which codes for the production of a catalytically inactive
form of PKC (23) was used as a control, and PKAC, which codes for the
expression of a constitutively active form of PKC-
(24), was used as
described previously (25). To assess the effects of Ha-Ras on
BNP/luciferase reporter expression, pDCR Ha-RasV12, which
codes for the production of a constitutively active form of Ha-Ras (26,
27) was used with pCEP4 as the empty vector control, as described (28).
To assess the effects of Raf-1 kinase on BNP/luciferase reporter
expression, pCEP4
Raf-1:ER, an expression plasmid coding for an
estrogen-activated form of Raf-1 kinase (29), was used and pCEP4 was
used as the empty control vector. As a second test of the effects of
Raf-1 kinase, Raf BXB, which encodes a constitutively active form of
c-Raf-1 (30, 31), was used.
In all transfection experiments, three identically treated cultures were used for each treatment. Each experiment was replicated at least three times, and the average of three experiments is shown.
Electrophoretic Mobility Shift Assay (EMSA)
EMSA was carried out using nuclei from neonatal rat ventricular tissue obtained as described (18, 32). Briefly, 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 10 µg of nuclear extract protein in 1 × binding buffer (10 mM Hepes, pH 7.9, 70 mM KCl, 5 mM MgCl2, 0.4 mM EDTA, 0.1 mM EGTA, 5% glycerol, 0.5 mM dithiothreitol). After a 10-min preincubation of extract with 0.1 µg of nonspecific competitor (poly(dI-dC), Pharmacia) ± competitor, the probe was added. Binding was allowed to proceed at room temperature for 30 min prior to separation of bound and free probe on a 4% native polyacrylamide gel (29:1 bis/acrylamide) in 0.5 × Tris borate-EDTA buffer at 4 °C at 150 V. DNA-protein complexes were detected by autoradiography. The autoradiograms of some gels in this report were scanned using a Molecular Dynamics Personal Densitometer, and the resulting image was imported to Adobe Photoshop and Claris MacDraw Pro II for final figure preparation.
In a previous study we cloned and sequenced
approximately 2.5 kilobase pairs of the rat BNP 5-FS (22). A search of
this sequence revealed the presence of various putative regulatory cis-elements in this region of the rat BNP gene (Fig.
1, top). To begin mapping areas of the gene
involved in regulating BNP transcription, a series of reporter
constructs was prepared with various lengths of the BNP 5
-FS driving
luciferase expression. When primary ventricular cardiac myocytes were
transfected with these constructs, it was apparent that the removal of
about 1.5 kb of 5
-FS, down to BNP-535GL, had very little effect on
basal or PE-inducible reporter activity (Fig.
2A). Interestingly, the removal of 137 bp
between
535 and
398 appeared to result in greater basal and
inducible promoter activity, suggesting the presence of repressor
elements in this region of the gene. While the physiological role of
such repressor elements is unknown, this result is similar to that
recently observed in similar truncation analyses of the human BNP gene
(33). Further truncation resulted in a gradual decline of promoter
activity such that BNP-140GL displayed about 75% of the basal and
inducible activities as the full-length construct. Truncation beyond
this point seemed to have a more severe effect, such that BNP-116GL
possessed only about 15% of the promoter activity observed with
BNP-2501GL, while BNP-81GL and BNP-58GL possessed only 8% and 1% of
original activity.
A series of 6-bp cluster mutations (see Fig. 1) targeted at promoter
proximal regions within the full-length, BNP-2501GL was prepared.
Mutants B, C, G, and H each decreased basal and PE-inducible promoter
activity by about 30-50%, mutant D had no effect, while mutants E and
F increased PE-inducible promoter activity by as much as 25% (Fig.
2B). Strikingly, however, mutant A, which spans the BNP
5-FS between
103 to
98, resulted in a drastic, 98% reduction of
reporter activity, implying the presence of a critical regulatory
sequence in this region.
A CATTCT, or M-CAT, consensus sequence lies between 109 and
102
nucleotides in the rat BNP gene (Fig. 1). CATTCT elements bind a family
of skeletal- and cardiac muscle-derived proteins originally named M
(muscle)-CAT-binding proteins (see Fig. 1; Ref. 34). M-CAT-binding
proteins, which are present in high levels in cardiac myocytes, are
related to a family of proteins called transcription enhancement
factors (e.g. TEF-1), also known as enhancers of SV40
transcriptional activity, GT-IIC (34). Additionally, M-CAT-binding
proteins have been implicated in the transcription of other cardiac
muscle genes, such as those for cardiac troponin C,
-skeletal actin
and
-myosin heavy chain, and
-myosin heavy chain (34-38).
Accordingly, in the context of 2.5 kb of the BNP 5
-FS, a 2-bp
double-point mutation was prepared (
M-CAT), which was predicted from
previous studies to specifically disrupt the binding of M-CAT-binding
protein (39). Basal and PE-inducible promoter activity from
M-CAT
were reduced by at least 98%, as seen for mutation A (Fig.
2C), emphasizing the absolute requirement for an intact
M-CAT element in the promoter-proximal region of the BNP gene.
The GATA family of transcription factors, which have been
implicated as regulators of myocardial cell BNP gene expression (22,
40-42), bind to DNA possessing the consensus, WGTAR sequence (43, 44).
Since the cluster mutations B and C displayed somewhat reduced basal
and PE-inducible promoter activity (Fig. 2B), and since
these mutations were located over GATA-binding protein consensus sequences (Fig. 1, lower), point mutations known to disrupt
GATA binding were prepared in the context of BNP-2501GL. In agreement with our previous study, which tested similar mutations in reporter constructs possessing only 116 bp of BNP 5-FS (i.e.
BNP-116GL) (22), the mutation at GATA(
95) was of little
consequence, while the mutation at GATA(
84) resulted in
an approximate 20% decline in PE-inducibility (Fig.
3). However, when the mutations were combined
(i.e. GATA(
95,
84)), there was a much greater
than expected, 60% reduction in basal and PE-inducible reporter
activity (Fig. 3). One explanation for this unexpected reduction is the
possibility that these two GATA sites, and perhaps the protein(s) that
bind there, are interactive, either with each other, or with other elements. For example, perhaps a GATA-binding protein (BP) must bind to
the BNP gene somewhere between about
100 and
80 to confer optimal
promoter activity. If a GATA-BP binds to the native BNP gene primarily
at GATA(
84), which would leave enough room for an M-CAT-binding
protein to bind at
106, one would expect the
95 mutation to be of
little consequence. However, the
84 mutation might be expected to
disrupt GATA-BP binding there, perhaps promoting GATA-BP binding to the
alternate, and apparently less effective GATA site at
95. Then,
mutating both
84 and
95 would completely disrupt all GATA binding,
resulting in the unexpectedly low activity observed in the
GATA(
95,
84) double mutant.
To test for possible interactions between the GATA and M-CAT elements,
the various GATA-directed mutations were combined with the M-CAT
point mutation. As expected, overall promoter activities of the
constructs harboring the
M-CAT mutation were severely decreased,
probably due to the disruption of the consensus M-CAT site.
Additionally, the GATA(
95) mutation had no additional effect when
combined with the
M-CAT mutation; however, the GATA(
84) and
GATA(
95,
84) mutations further decreased promoter activity. The
abilities of the GATA-directed mutations to decrease promoter activity
in the
M-CAT constructs, as well as those possessing the native
M-CAT site, suggests that the GATA sites behave independently of the
M-CAT site.
Due to
its absolute requirement for basal and inducible BNP promoter activity,
the properties of the M-CAT element in the BNP 5-FS were studied
further. To evaluate whether cardiac nuclear proteins could bind to the
promoter-proximal M-CAT site in a manner consistent with the functional
consequences of the mutations in the reporter genes, EMSA were carried
out. A variety of oligonucleotides were prepared either synthetically
or by restriction digestion of the appropriate BNP/luciferase reporters
(Fig. 4); the BNP(
113/(
95) oligomer was used as a
labeled probe and a competitor, while the others were used as
competitors.
In the absence of any competitor, cardiac nuclear proteins and the BNP
probe formed a single, major complex observed by EMSA (Fig.
5, lane 2). While this complex was disrupted
effectively using the unlabeled probe (Fig. 5, lane 3), or
an oligonucleotide containing a canonical M-CAT element modeled after
that in the chicken troponin gene (Fig. 5, lane 4), the
BNP(113/(
95) mutant, which mimics the double point mutation,
M-CAT, was an ineffective competitor (Fig. 5, lane 5).
Oligonucleotides mimicking larger stretches of the promoter-proximal
region of the BNP 5
-FS and containing the M-CAT site (e.g.
BNP(
116/(
52) and BNP(
116/(
71)), were also effective competitors
(Fig. 5, lanes 6 and 8). However, when these
larger oligonucleotides contained either the double-point mutation in
the putative M-CAT site (i.e. BNP(
116/(
52)
M-CAT), or
cluster mutation A (i.e. BNP(
116/(
52) mutant A) (Fig. 5, lanes 7 and 9), they served as ineffective
competitors, consistent with a requirement for the binding of an
M-CAT/TEF-1-related protein to this region of the gene.
Oligonucleotides mimicking mutants B and D, which harbored 6-bp
clustered changes in regions outside the putative M-CAT region, were
effective competitors (Fig. 5, lanes 10 and 11),
consistent with the relatively minor roles of these mutations on BNP
promoter function. As further controls, it was shown that neither the
human c-fos serum response element (SRE), the collagense
12-O-tetradecanoylphorbol-13-acetate response element, or
the promoter-proximal rat ANF SRE (18) acted as competitors of the
shifted complex (Fig. 5, lanes 12-14). Thus, the EMSA
analyses are consistent with the required binding of a protein
possessing the characteristics of M-CAT-binding protein, or TEF-1, to
the putative M-CAT element in the BNP 5
-FS for optimal PE-inducible
promoter activity.
Roles of PKC, Ras, and Raf-1 in BNP Inducibility
Further
experiments were carried out to investigate whether the
promoter-proximal sequences, such as the M-CAT element, might participate in BNP promoter activation by PE and by intracellular signals activated by PE. In cardiac myocytes,
1-adrenergic receptors couple through Gq to
the activation of PKC and to stimulation of the Ras/Raf-1
kinase/extracellular signal-regulated protein kinase
(ERK)/MAPK2 pathways (45-52).
In part, these pathways are believed to participate in mediating the
gene induction that results from myocardial cell
1-adrenergic receptor activation by PE (15, 25, 48,
53-55), although activation of ERK/MAPK alone appears to be
insufficient to activate ANP expression (56). Accordingly, we assessed
and compared the effects of PE to overexpressed PKC, Ha-Ras, or Raf-1 kinase on various forms of BNP/luciferase reporter genes.
Consistent with the earlier truncation studies, BNP-81GL was virtually
inactive, regardless of the stimulus (Fig. 6). PE
activated reporter expression from BNP-140GL about 50-60% as well
from BNP-2501GL (Fig. 6A), further supporting the view that
sequences between 140 and
81 bp could confer significant
1-adrenergic agonist inducibility. Basal expression of
luciferase was similar for BNP-140GL and BNP-2501GL, but much less for
BNP-81GL; the decrease between
140 and
81 is most likely due to the
absence of the M-CAT and GATA elements from the latter construct (see
Fig. 1). Constitutively active PKC strongly and similarly activated
both BNP-2501GL and BNP-140GL (Fig. 6B), supporting the view
that sequences lying between
140 and
81 bp could confer PKC
inducibility. Ha-Ras also served as a potent activator of BNP-2501GL;
however, it stimulated BNP-140GL only about 40% as well as BNP-2501GL
(Fig. 6C), suggesting that in contrast to PKC, sequences
residing both distal and proximal to
140 are required for optimal Ras
inducibility. In further support of a role for Ha-Ras was the finding
that Raf-1 kinase served as a potent activator of BNP-2501 (Fig.
6D); surprisingly, however, Raf-1 stimulated reporter
expression from BNP-140GL relatively poorly, by only about 25% as well
as from BNP-2501GL (Fig. 6D).
Effects of constitutively active PKC, Ha-Ras,
and Raf-1 kinase on BNP promoter activity. Myocardial cells were
transfected with BNP-2501GL, BNP-140GL, or BNP-81GL and
CMV--galactosidase. For each experiment, the relative luciferase
values obtained with BNP-2501GL were set to 100% and the other values
normalized accordingly. Values are means ± S.E.,
n = 3 cultures. Panel A, cultures were treated ± PE as in Fig. 2. Panel B, in addition to the
BNP/luciferase and CMV-
-galactosidase constructs, myocardial cells
were also transfected with 15 µg of a control plasmid, p
OP
(Con), which codes for the production of catalytically
inactive PKC, or 15 µg of pPKAC (PKC), which codes for the
production of a constitutively active form of PKC-
(24). After
48 h in minimal media, the cultures were extracted and assayed for
reporter enzyme activities. Panel C, in addition to the
BNP/luciferase and CMV-
-galactosidase constructs, myocardial cells
were also transfected with 15 µg of a control plasmid, pCEP4
(Con), which contains no insert, or 15 µg of the plasmid
Ha-RasV12 (H-Ras), which codes for the
production of a constitutively active form of Ha-Ras (26, 27). After
48 h the cultures were extracted and assayed for reporter enzyme
activities. Panel D, in addition to the BNP/luciferase and
CMV-
-galactosidase constructs, myocardial cells were also
transfected with 15 µg of pCEP4/
Raf-1:ER (Raf-1), which codes for
the production of an estrogen-inducible form of Raf-1 kinase (50). After 48 h of
incubation in minimal medium (Con) or in minimal medium
containing 1 µM estradiol (Raf), the cultures
were extracted and assayed for reporter enzyme activities. In control
experiments it was shown that estradiol (48 h) did not alter the
relative luciferase values in cultures transfected with pCEP4 (data not
shown; see also Ref. 50).
These results indicated that sequences lying proximal to 140 bp were
responsible for significant levels of inducibility, although they
displayed somewhat differential responsiveness to the stimuli,
conferring relatively strong induction in response to PE, PKC, and
Ha-Ras, but very weak induction in response to Raf-1. To explore
further the importance of the promoter-proximal M-CAT element in basal
and inducible reporter activity, the BNP-related M-CAT sequences were
cloned upstream of position
81 in BNP-81GL, a construct that normally
expresses very low reporter activity. In this context the M-CAT
sequences were found to confer a significant recovery of basal as well
as PE, PKC, and Ras inducibility to BNP-81GL, with the extent of basal
and inducible reporter expression being approximately proportional to
the number of M-CAT sites (Fig. 7).
These results supported the hypothesis that the promoter-proximal M-CAT
element can contribute to BNP inducibility in response to PE, and two
of its major effectors, PKC and Ha-Ras. This view is corroborated
further by the lack of PE inducibility of M-CAT/BNP-2501GL (Fig.
2C) and a decrease in Ha-Ras inducibility of this same
construct by over 80% (data not shown). However, since Ras is a known
activator of Raf-1 in the ERK/MAPK pathway, the low Raf-1
responsiveness of BNP-140GL was inconsistent with this hypothesis,
implying that while they were responsive to Ha-Ras, sequences in
BNP-140GL were poorly responsive to Raf-1. Accordingly, the ability of
Raf-1 kinase to activate the M-CAT/BNP-81GL constructs was evaluated using the
Raf:ER expression construct. Consistent with its poor ability to activate BNP-140GL, Raf-1 did not enhance reporter expression significantly from either 2X- or 4XM-CAT/BNP-81GL (Fig. 8A). As expected, BNP-2501GL was induced
significantly by Raf-1, while BNP-140GL was induced by less than 15%
compared to BNP-2501GL. The lack of Raf-1 responsiveness supports the
view that the PE-, PKC- and Ras-mediated increases in luciferase
expression from 2X- or 4XM-CAT/BNP-81GL, shown in Fig. 7, represent
induction above basal expression; thus, the M-CAT element is apparently required for basal transcription and it can mediate inducible transcription. To confirm the unexpected lack of responsiveness of the
M-CAT element to Raf-1, a different Raf-1 expression construct, Raf
BXB, was used in a similar experiment. Again, Raf-1 kinase was
ineffective as an enhancer of reporter expression from 4XM-CAT/BNP-81GL (Fig. 8B), but like
Raf:ER, it served as a strong inducer
of reporter expression from BNP-2501GL and a relatively weak inducer of
BNP-140GL.
The results of this study indicate that BNP transcription requires
a promoter-proximal M-CAT element that can mediate transcriptional stimulation in response to PE, as well as PKC and Ras, both of which
are activated by 1-adrenergic agonists. Interestingly, however, the M-CAT element does not appear to contribute to BNP induction in response to Raf-1 kinase, which is also activated by
1-adrenergic agonists. Instead, the Raf-1-inducible
elements reside distally, between
2501 and
140 of the BNP 5
-FS,
and are yet to be identified. Accordingly, this is the first report to
suggest that in addition to conferring inducibility in response to PKC,
which was previously shown for the
-MHC gene (37), M-CAT elements in
muscle-specific genes might also be responsive to Ras-activated
signals, but not those involving Raf-1 kinase.
Although M-CAT elements have not been previously shown to mediate Ras inducibility, a large body of information supports a role for M-CAT sequences as determinants of cardiac and skeletal muscle-specific gene expression (35, 57, 58). Cardiac and striated muscle tissues are particularly enriched in M-CAT binding factors; however, they are also found in many other cell- and tissue types (39, 59-61). Nonetheless, it is believed that in combination with nearby regulatory sequences, such as E-boxes, M-CAT elements contribute to the muscle-specific gene expression (62).
In addition to conferring tissue specificity to certain muscle genes,
the hormonal inducibility of several cardiac genes is also thought to
involve M-CAT elements. For example, a promoter-proximal (210 bp)
M-CAT element in the rat
-MHC gene is required for induction by
1-adrenergic agonists and PKC (37). A similar element,
also located near the promoter of the rat skeletal
-actin gene,
appears to mediate induction by transforming growth factor-
(36), as
well as
1-adrenergic agonists and PKC (63). Indeed, the
abilities of PKC expression constructs to activate M-CAT-containing reporter genes for
-MHC (37) and for BNP (present study) lead to the
conclusion that PKC may serve as an important regulator of
TEF-1-enhanced transcription.
The precise mechanism by which TEFs might enhance BNP transcription in response to PKC activation remains unknown. However, it has been postulated that PKC could directly regulate the transcriptional enhancement effects of TEF (64). For example, TEF-1 possesses known, PKC phosphorylation sites that could alter the ability of the protein to bind to M-CAT elements and thus confer transcriptional activation. However, it is also possible, if not probable, that in comparison to PKC's abilities to activate transcription through SRF or AP-1, it may indirectly activate TEF, perhaps by way of altering the activities of other kinases or phosphatases which would ultimately alter the phosphorylation state of TEF, or closely associated proteins. Another possibility is that PKC and Ras might converge on a single pathway to ultimately effect enhancement of transcription through TEF-1.
Since PKC and Ras can both activate Raf-1 and, thus, ERK/MAPK, it is tempting to speculate that ERK/MAPK might serve as a downstream effector through which PE can stimulate transcription via TEF-1. However, the finding in this study that the M-CAT element appears to participate in Ras- but not Raf-1-inducible BNP promoter activation suggests a role for Ras-dependent, ERK/MAPK-independent pathways. Consistent with this are recent results demonstrating that PD 098059, a specific MEK inhibitor (65), blocks PE-inducible ERK/MAPK in cardiac myocytes by 80%, but does not block PE-inducible BNP promoter activity, as measured with BNP-2501GL (66). Among the Ras-dependent, ERK/MAPK-independent pathways of possible interest are those converging on the activation of the JNK and p38 members of the MAPK family (67). It is possible, therefore, that p38 MAPK and/or JNK might ultimately affect transcriptional enhancement through TEF-1, or accessory factors. Further, it is reasonable to hypothesize that even though all three MAPK family members might be activated in cardiac myocytes in response to PE, they could differentially effect changes in transcription. Indeed, the MAPKs display some substrate selectivity amongst certain transcription factors. For example, while JNK appears to preferentially phosphorylate c-Jun, ERK/MAPK preferentially phosphorylates c-Myc, p38 MAPK preferentially phosphorylates ATF-2 and ERK/MAPK and p38 MAPK phosphorylate Elk-1 to similar extents (68). Thus, to further dissect the mechanism of Ras-induction through TEF-1, it will be of interest to evaluate the abilities of all three MAPK family members to enhance transcription through BNP promoter-proximal M-CAT element.
In summary, the results from this study add new information to our
understanding of the cis-elements and the signaling
mechanisms responsible for 1-adrenergic agonist-mediated
induction of BNP and other cardiac genes. We have found that in
comparison to the
-skeletal actin and
-MHC genes, a
promoter-proximal M-CAT element is important for
1-adrenergic inducibility of the BNP gene, and that this
induction could be mediated at least partly by PKC. The present study
has added further to our knowledge of how TEF-1 might mediate cardiac
gene expression, demonstrating that the M-CAT element confers
Ras-dependent induction, but in a Raf-independent manner;
this finding implies the involvement of p38/and/or JNK/MAPK-mediated events, and/or other Ras-activated pathways not yet clearly identified. The mechanisms by which TEF-1 responds to these, and perhaps other signaling pathways, remain unknown. And while the recent findings that
there are multiple forms of the TEFs (69, 70) might also add potential
complication to the mechanism, it is also possible that such multiple
pathways provides the potential for somewhat independent induction of
TEF-responsive genes to match a variety of physiological requirements.
Future studies of how the various forms of the TEFs interact with other
accessory proteins such as E-box-binding proteins (62) or nearby
transcription factors such as GATA-binding proteins (22, 40-42) will
be required to further our understanding of this complex gene
induction mechanism.
We thank Nichole Arnold for expert technical
assistance, and Deanna Hanford, Patrick McDonough, and Dietmar Zechner
for critical reading of the manuscript. PKCOP and PKAC were gifts
from M. Muramatsu (DNAX Research Institute, Palo Alto, CA), pDCR
Ha-RasV12 was a gift from D. Bar-Sagi (SUNY, Stony Brook,
NY), pCEP4
Raf-1:ER was a gift from A. Thorburn (University of Utah,
Salt Lake City, UT), and Raf BXB was a gift from U. Rapp
(Universität Wurzburg, Wurzburg, Germany).