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Am J Physiol Heart Circ Physiol 284: H193–H203, 2003.
First published September 5, 2002; 10.1152/ajpheart.00161.2002.
Regulation of the S100B gene by ␣1-adrenergic
stimulation in cardiac myocytes
JAMES N. TSOPORIS,1 ALEXANDER MARKS,2 LINDA J. VAN ELDIK,3
DAVID O’HANLON,2 AND THOMAS G. PARKER1
1
Division of Cardiology, Department of Medicine, The Toronto General Hospital Research Institute,
University of Toronto, Toronto M5G 2C4; 2Banting and Best Department of Medical Research,
University of Toronto, Toronto, Ontario M5G 1L6, Canada; and 3Drug Discovery Program
and Department of Cell and Molecular Biology, Northwestern University Medical School,
Chicago, Illinois 60611
Tsoporis, James N., Alexander Marks, Linda J. Van
Eldik, David O’Hanlon, and Thomas G. Parker. Regulation of the S100B gene by ␣1-adrenergic stimulation in cardiac myocytes. Am J Physiol Heart Circ Physiol 284:
H193–H203, 2003. First published September 5, 2002;
10.1152/ajpheart.00161.2002.—We previously reported that
S100B, a 20-kDa Ca2⫹-binding homodimer, inhibited the
postinfarct myocardial hypertrophic response mediated by
␣1-adrenergic stimulation through the protein kinase C
(PKC) signaling pathway. In the present study, we examined
whether the same pathway induced the S100B gene, supporting the hypothesis that S100B is a feedback negative regulator of this pathway. We transfected cultured neonatal rat
cardiac myocytes with a luciferase reporter gene driven by
the maximal human S100B promoter and progressively
shorter segments of this promoter sequentially deleted from
the 5⬘ end. We identified a basic promoter essential for
transcription spanning 162 bp upstream of the transcription
initiation site and positive (at ⫺782/⫺162 and ⫺6,689/
⫺4,463) and negative (at ⫺4,463/⫺782) myocyte-selective
regulatory elements. We showed that the basic and maximal
S100B promoters were activated specifically by ␣1-adrenergic
agonists through the ␣1A-adrenergic receptor, but not by any
other trophic hormonal stimuli. The activation of the S100B
promoter was mediated through the PKC signaling pathway.
Transcription enhancer factor-1 (TEF-1) and related to
TEF-1 (RTEF-1) influenced transcription from the maximal,
but not the basic, promoter implicating active MCAT elements upstream from the basic promoter. Acting in opposing
fashions, TEF-1 transrepressed the S100B promoter and
RTEF-1 transactivated the promoter. Our results suggest
that ␣1-adrenergic stimulation induces the S100B gene after
myocardial infarction through the PKC signaling pathway
and that this induction is modulated by TEF-1 and RTEF-1.
norepinephrine; TEF-1
MYOCARDIAL INFARCTION,
the leading cause of death in the
developed world, results in an acute loss of functional
myocardium. Among survivors, postinfarct ventricular
remodeling is an adaptive response of the heart to this
loss in an attempt to preserve cardiac performance
Address for reprint requests and other correspondence: T. G. Parker,
The Toronto General Hospital, 200 Elizabeth St., EN12-208, Toronto,
Ontario M5G 2C4, Canada (E-mail: [email protected]).
http://www.ajpheart.org
(25). Myocardial hypertrophy is an integral component
of this adaptive response. Because adult cardiac myocytes are terminally differentiated and have lost the
ability to divide, this increase in mass is due to enlargement of individual myocytes. The hypertrophic
response can be reproduced in rat and mouse experimental models and cultured neonatal cardiac myocytes
from these species (5, 25, 26). Experimental work in
these models has implicated hormonal stimulation,
including ␣1-adrenergic agonists, angiotensin (ANG)
II, peptide growth factors, and mechanical stretch as
instigators of the hypertrophic response (4, 30, 35).
Some of these, including ␣1-adrenergic agonists, activate the protein kinase C (PKC) signaling pathway (15,
17). Furthermore, in addition to growth, myocytes have
been shown to respond to trophic stimuli in these
models by reexpression of several fetal genes, including
the embryonic ␤-myosin heavy chain (␤-MHC), the
skeletal isoform of ␣-actin (skACT), and atrial natriuretic factor (4, 15, 17, 35).
The relative contributions of the various trophic
stimuli that initiate and sustain the hypertrophic response after myocardial infarction have not been defined. Moreover, some form of negative regulation may
be involved to limit unchecked hypertrophy. We have
previously proposed that S100B, a 20-kDa Ca2⫹-binding homodimer normally expressed in brain astrocytes,
was a candidate for an intrinsic negative regulator of
the myocardial hypertrophic response on the basis of
several experimental observations. First, whereas
S100B is normally not expressed in the myocardium,
S100B protein was identified immunohistochemically
in the peri-infarct region of the human heart after
myocardial infarction, and S100B mRNA was detected
in the rat heart after coronary artery ligation in an
experimental model of myocardial infarction (32, 33).
Second, in cotransfection experiments in cultured neonatal rat cardiac myocytes, S100B inhibited the ␣1-
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0363-6135/03 $5.00 Copyright © 2003 the American Physiological Society
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Submitted 27 February 2002; accepted in final form 29 August 2002
H194
␣1-ADRENERGIC INDUCTION OF S100B IN HEART
MATERIALS AND METHODS
Human S100B promoter-driven reporter plasmids. A luciferase reporter gene system (Promega; Madison, WI) was
used to study the relative ability of different 5⬘ DNA regions
of the human S100B gene to promote transcription in transient transfection assays, as previously described (3). In this
system, a maximal human S100B promoter and progressively shorter fragments sequentially deleted from the 5⬘ end
of the maximal promoter by restriction enzyme digestion
were cloned upstream of the luciferase gene into the plasmid
pGL3, which lacks eukaryotic enhancer and promoter sequences. Initially, a 9.8-kb HindIII fragment of human
genomic DNA (1) spanning exons 1 and 2 (and the intervening intron) of the human S100B gene and 6,689 bp of upstream and 320 bp of downstream DNA sequence, which was
numbered in accordance with the human S100B genomic
sequence in the National Center for Biotechnology Information database, was subcloned into a BlueScript plasmid (pBS;
Stratagene; La Jolla, CA) to generate pBS100 9.1. A HindIII/
BamHI fragment spanning the DNA nucleotide sequence
⫺6,689/⫹698 relative to the transcription initiation site was
excised from pBS100 9.1 and designated as the maximal
S100B promoter. This fragment includes 6,689 bp of DNA
sequence upstream of exon 1, exon 1 (71 bp), and 627 bp of
downstream sequence and was subcloned into pGL3 to generate pGBS ⫺6,689/⫹698. The reporter plasmids containing
progressively shorter segments of the maximal promoter
were generated by subcloning the fragments KpnI/BamHI,
PvuII/BamHI, and SmaI/BamHI, excised from pBS100 9.1,
into pGL3 to generate pGBS ⫺4,463/⫹698, pGBS ⫺782/
⫹698, and pGBS ⫺162/⫹698, respectively (3). The plasmid
pGBS ⌬⫺162/⫹698 was generated similarly from a fragment
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created by deleting the region ⫺162/⫹698 from pGBS
⫺4,463/⫹698 (see Fig. 3).
Expression plasmids. The expression plasmids were obtained from the following sources: SR␣-⌬PKC-␤ [containing a
partially deleted constitutively active PKC-␤ cDNA driven by
a sarcoma virus (SV)40 early promoter] was from Dr. P. C.
Simpson (14); pXJ40-TEF-1A and pXJ40-RTEF-1 (containing
human TEF-1 and RTEF-1 cDNA, respectively, driven by a
cytomegalovirus enhancer) were from Dr. A. F. R. Stewart
(31); pBK28-c-Fos (containing a human c-Fos cDNA driven
by a murine SV long-terminal repeat promoter) and pSV-cJun (containing a human c-Jun cDNA driven by a SV40 early
promoter) were from Dr. I. Verma; and Rous sarcoma virus
(RSV)-CAT (containing a CAT cDNA driven by a RSV longterminal repeat promoter) was from Stratagene (La Jolla,
CA). Plasmids were purified using midi or maxi kits (Qiagen;
Valencia, CA).
Left coronary artery ligation. Left coronary artery ligation
was performed in the rat as previously described and as
approved by the animal care committee of the Toronto General Research Institute (24). Briefly, 8-wk-old rats were anesthetized intraperitoneally with 90 mg/kg ketamine and 10
mg/kg xylazine. A left thoracotomy was performed, and a 6-0
silk suture was placed through the myocardium into the
anterolateral left ventricular wall. This area corresponds to
the course of the left anterior descending artery in the rat.
The suture was positioned approximately midway between
the apex and base. The suture was then tied and the chest
cavity closed.
Tissue catecholamine measurements. For tissue norepinephrine (NE) measurements, left ventricular tissue was
homogenized in 0.4 N perchloric acid containing 5 mM reduced glutathione and then centrifuged to produce a proteinfree supernatant. Tissue NE was assayed using the Biotrak
catecholamines 3H-labeled radioenzymatic assay system
(Amersham Pharmacia Biotech; Baie d’Urfe, Quebec, Canada) according to the instructions of the manufacturer. In
brief, the assay utilized the enzyme catechol-O-methyltransferase to catalyze the transfer of a 3H labeled methyl group
from S-adenosyl-L-[methyl-3H]methionine to NE. The resulting product, [3H]normetanephrine, was isolated with the use
of thin-layer chromatography. [3H]normetanephrine was
converted by periodate oxidation to [3H]vanillin and extracted. The radioactivity attributable to NE was determined
by liquid scintillation counting.
Cell transfections. Low-density (⬃150 cells/mm2) noncontractile neonatal cardiac myocytes and fibroblasts were isolated from ventricles of 2-day-old Sprague-Dawley rats and
established in culture as previously described (21, 30) and
as approved by the animal care committee of the Toronto
General Research Institute. Transfection was carried out
by calcium phosphate precipitation (14) with the use of specified quantities of the following plasmids: pW9Fc (9 ␮g);
pGBS ⫺162/⫹698, pGBS ⌬⫺162/⫹698, pGBS ⫺782/⫹698,
pGBS ⫺4,463/⫹698, and pGBS ⫺6,689/⫹698 (5 ␮g); and SR
␣-⌬PKC-␤, pXJ40-TEF-1A, pXJ40-RTEF-1, pBK28-c-Fos,
and pSV-c-Jun (0.1 ␮g), as indicated in the legends. RSVCAT (0.1 ␮g) was included in all plates as an internal control
for transfection efficiency. Myocyte cultures were maintained
in medium supplemented with 5% fetal bovine serum for 18 h
after transfection before transfer to serum-free medium and
treatment with 20 ␮M NE, 20 ␮M phenylephrine (PE), 20 ␮M
isoproterenol (Iso), 20 ng/ml 3,5,3⬘-triiodothyronine (T3), 100
nM ANG II, the phorbol ester phorbol 12-myristate 13-acetate (PMA; 10 nM), the ␣1A-adrenergic receptor antagonist
WB-4101 (2 ␮M), the ␣1B-adrenergic receptor antagonist
chloroethylclonidine (CEC) (2 ␮M), the PKC inhibitor stau-
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adrenergic-mediated induction of ␤-MHC and skACT
through the PKC signaling pathway (32). Third, in
S100B-overexpressing transgenic mice, S100B protein
and mRNA were detected in the heart after ␣1-adrenergic agonist infusion, and the hypertrophic response
normally evoked in the heart and cultured myocytes in
response to ␣1-adrenergic stimulation in wild-type
mice was abrogated (33).
The above data suggest that S100B functions as a
negative feedback regulator of the ␣1-adrenergic-mediated component of the hypertrophic response after
myocardial infarction. A corollary of this suggestion is
that the S100B gene is induced after myocardial infarction by the same ␣1-adrenergic pathway. To formally
examine this possibility, we used luciferase reporter
constructs to study the activation of the human S100B
promoter by components of the ␣1-adrenergic signaling
pathway in cultured rat myocytes. These components
included PKC, and the transcription factors transcription enhancer factor-1 (TEF-1) and related TEF-1
(RTEF-1), which bind to MCAT elements. These elements have previously been reported (14, 16, 17, 19,
31) to be involved in the induction of the fetal genes
␤-MHC and skACT in cultured rat neonatal cardiac
myocytes and in the intact rat heart. We report the
specific induction of the S100B promoter by ␣1-adrenergic stimulation in rat myocytes through the PKC
signaling pathway and the selective modulation of this
induction by TEF-1 and RTEF-1 acting through MCAT
elements in the S100B promoter.
␣1-ADRENERGIC INDUCTION OF S100B IN HEART
thesis of a 210-bp fragment from human RNA (32). The rat
S100B primers 5⬘-TTGCCCTCATTGATGTCTTCCA-3⬘ and
5⬘-TCTGCCTTGATTCTTACAGGTGAC-3⬘ direct the synthesis of a 500-bp fragment from rat RNA. GAPDH primers
5⬘-ATCACCATCTTCCAGGAGCG-3⬘ and 5⬘-TTGTCATACCAGGAAATGAG-3⬘ were selected based on conserved sequences in the human, rat, and mouse and direct the synthesis of a 720-bp fragment. PCR was performed for 30 cycles,
with denaturation at 94°C for 1 min, annealing at 50°C for 1
min, and extension at 72°C for 1 min, with an extra 5-min
extension for the last cycle. Aliquots of PCR products (10 ␮l)
were separated by electrophoresis on 1.5% agarose gels in 40
mM Tris acetate-1 mM EDTA (pH 8.0). Gels were visualized
after ethidium bromide staining under UV transillumination.
Gel mobility shift assay. Oligonucleotides to the sense and
antisense strands containing putative TEF-1 binding sites
were synthesized by an automated solid-phase synthesizer
(Gene Assembler Plus, Amersham Pharmacia Biotech) by
␤-cyanoethyl phosphoramidite chemistry. The radiolabeled
probe for the gel mobility shift assays was the (⫺84/⫺61)
mouse skACT promoter fragment containing the canonical
MCAT sequence (17). Cold competitor probes from the
S100B promoter were designed encoding MCAT1, a mutant
MCAT1mut, MCAT2, and MCAT2mut. These oligonucleotide
sequences are shown in Fig. 7B. MCAT1 and MCAT2 sites
are mapped on the S100B promoter in Fig. 7A. Nuclear
extracts from cardiac myocytes were prepared as described
by Kariya et al. (14). Binding reactions were performed as
described previously (14, 16, 17).
Statistical analysis. Treated-to-control ratios were tested
for deviation from unity by calculation of confidence limits.
Mean values were compared by analysis of variance by the
Fig. 1. Absence of basal S100B mRNA expression in
myocardium in vivo and myocardial cells in culture.
Adult rats were killed at 35 days after coronary artery
ligation (cor. art. lig.). Steady-state levels of S100B and
GAPDH mRNAs were determined by RNase protection.
The protected fragments specific for rat S100B (264
bp) and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH; 413 bp) mRNAs are shown. The results of a
representative experiment include RNA from cultured neonatal (cult. neo.) myocytes and fibroblasts,
fetal, neonatal, and adult hearts and brains, and
hearts from experimental animals as indicated. Rat
tRNA was used as a negative control.
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rosporine (20 nM), the Ca2⫹ channel blocker nifedipine (1
␮M), the Ca2⫹ ionophore A-23187 (10 nM), or vehicle diluent
(100 ␮M ascorbic acid for NE, PE, Iso, T3, ANG II, CEC, and
WB-4101 or 0.01% DMSO for staurosporine, nifedipine,
A-23187, and PMA) for 48 h. The cell lysates were assayed for
luciferase and CAT activity by following published techniques (7, 11). Cotransfection with any of the plasmids or
treatment of cultures as described above did not affect CAT
activity. The differences of the respective luciferase and CAT
activities between duplicate dishes were ⬍10% of their mean.
Luciferase activity was normalized for transfection efficiency
on the basis of CAT activity in the same dish. Treated-tocontrol ratios were tested from deviation from unity by calculation of confidence limits.
RNase protection assay. RNA was isolated from 1) cultured
rat neonatal myocytes and fibroblasts, 2) rat tissues, including the fetal, neonatal, and adult heart and brain, and 3)
residual noninfarcted rat left ventricular myocardium outside the territory supplied by the ligated coronary artery (32),
with the use of a one-step guanidine thiocyanate-phenol
method (6). RNase protection assays to determine steadystate levels of S100B and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNase were performed as previously
described (32).
Detection of human S100B mRNA by RT-PCR. RNA was
isolated from myocyte cultures and human and rat brains by
a one-step guanidine thiocyanate/phenol method (6) and pretreated with RNase-free DNase 1 (Boehringer-Mannheim;
Laval, Quebec, Canada) before first-strand DNA synthesis
with random hexamer primers. Human brain RNA was derived from archival specimens without patient identifiers.
The human S100B primers 5⬘-TGGACAATGATGGAGACGG-3⬘ and 5⬘-ATTAGCACAACACGGCTGG-3⬘ direct the syn-
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␣1-ADRENERGIC INDUCTION OF S100B IN HEART
Student-Newman-Keuls test, with significance defined as
P ⬍ 0.05.
RESULTS
Fig. 2. Induction of the human and rat S100B gene by ␣1-adrenergic stimulation in neonatal rat myocyte cultures
transfected with the human S100B gene. Rat (A) and human (B) S100B mRNAs were assayed by RT-PCR in
human and rat brain, respectively, and in rat neonatal myocyte cultures transfected with the human S100B gene
either untreated (vehicle only) or stimulated with norepinephrine (NE), isoproterenol (Iso), phenylephrine (PE), or
3,5,3⬘-triiodothyronine (T3), as described in MATERIALS AND METHODS. The results of a representative experiment are
presented. GAPDH mRNA was used as an internal control for the RT-PCR reaction. The sizes of the RT-PCR
products for rat S100B (500 bp), human S100B (210 bp), and GAPDH (720 bp) are indicated.
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S100B exhibits no basal expression in cultured neonatal rat cardiac myocytes or fibroblasts but is present
in residual rat myocardium adjacent to myocardial
infarction. Steady-state levels of S100B and GAPDH
mRNAs were determined with the use of an RNase
protection assay in cultured rat neonatal cardiac myocytes and fibroblasts and in the fetal, neonatal, and
adult rat heart and brain as well as in noninfarcted
adult myocardium adjacent to an infarct resulting from
coronary artery ligation (Fig. 1). S100B mRNA was not
detected in cultured neonatal myocytes or fibroblasts
or in the fetal, neonatal, and adult heart and not in the
fetal or neonatal brain, but it was highly expressed in
the adult brain. After coronary artery ligation, S100B
mRNA was expressed in the peri-infarct region of the
adult myocardium (35 days postinfarction) at levels
equivalent to those seen in the adult brain. The constant amounts of GAPDH mRNA in all samples served
as controls for the quality of the RNA and confirmed
equal loading in all lanes.
Coronary artery ligation is associated with increased
levels of NE. Thirty-five days after coronary artery
ligation, increased NE levels were seen in the periinfarct region of the adult myocardium (35 days postin-
farction) compared with sham-operated animals
(696 ⫾ 34 vs. 468 ⫾ 25 ng/g left ventricular tissue,
respectively, P ⬍ 0.05, n ⫽ 6).
Expression of exogenous human and endogenous rat
S100B gene in transfected rat cardiac myocytes. Cultured neonatal rat cardiac myocytes were transfected
with plasmid pW9Fc carrying 17.3 kb of human
genomic DNA that includes a full-length S100B gene.
Human and rat S100B mRNAs were assayed by RTPCR. The endogenous rat and human S100B mRNAs
were expressed in rat and human brains, respectively
(Fig. 2, A and B). The endogenous rat S100B mRNA
was absent from untreated rat myocyte cultures and
present after treatment with the ␣1-adrenergic agonists NE and PE but not with the ␤1-agonist Iso or T3
(Fig. 2A). Similarly, the level of the human S100B
mRNA derived from the transfected human S100B
gene was markedly increased in cultures treated with
␣1-adrenergic agonists NE and PE from a low basal
level in untreated cultures (Fig. 2B). Treatment with
the ␤1-adrenergic agonist Iso or T3 did not increase the
level of human S100B mRNA above the basal level.
␣1-Adrenergic stimulation specifically activates human S100B promoter through PKC signaling pathways. To study the activation of the human S100B
promoter by hormonal stimulation, we initially cloned
progressively shorter segments of the maximal human
S100B promoter sequentially deleted from the 5⬘ end in
␣1-ADRENERGIC INDUCTION OF S100B IN HEART
construct pGBS ⌬⫺162/⫹698 abolished transcription
(Fig. 3).
In response to hormonal stimulation, transcription
from the basic (cloned in pGBS ⫺162/⫹698) and maximal (cloned in pGBS ⫺6,689/⫹698) promoters was
stimulated by the ␣1-agonists NE and PE. Treatment
with PE stimulated transcription fourfold, equally
from the basic and maximal promoters (Fig. 4A). The
PE response was blocked with the use of the ␣1Aadrenergic receptor antagonist WB-401 but not the
␣1B-adrenergic receptor antagonist CEC (Fig. 4A).
Treatment with Iso, T3, or ANG II did not stimulate
transcription from the basic or maximal S100B promoter (Fig. 4B). Transcription from the basic and maximal S100B promoters was also stimulated by PMA, an
activator of PKC, and the stimulation of transcription
by PE and PMA was blocked by the PKC antagonist
staurosporine (Fig. 5A). Neither the calcium ionophore
A-23187 nor the calcium channel antagonist nifedipine
had any effect on basal or PE-stimulated transcription
(Fig. 5A). By using a cotransfection strategy, we found
that transcription was stimulated twofold, equally
from both promoters by ⌬PKC-␤, a constitutively active mutant of PKC-␤ (Fig. 5B). This stimulation was
blocked by staurosporine. Furthermore, stimulation
Fig. 3. Basal activity of human S100B promoter-luciferase (Luc) reporter constructs in neonatal rat cardiac
myocytes. The maximal S100B promoter (⫺6,689/⫹698) and progressively shorter fragments sequentially deleted
from the 5⬘ end of the maximal promoter were cloned into the promoterless plasmid pGL3 in front of the luciferase
reporter gene to generate plasmids pGBS ⫺6,689/⫹698, pGBS ⫺4,463/⫹698, pGBS ⫺782/⫹698 and pGBS
⫺162/⫹698, which are depicted schematically. The plasmid pGBS ⌬⫺162/⫹698 was generated similarly from a
fragment created by deleting the region ⫺162/⫹698 from pGBS ⫺4,463/⫹698. The relative luciferase activity of
each plasmid after transfection into neonatal rat myocyte cultures is shown on the right of each plasmid. Values
are the mean of six independent experiments, with duplicate dishes in each experiment. The base sequence
numbering is expressed relative to the transcription initiation site (⫹1), in accordance with the human S100B
genomic sequence in the National Center for Biotechnology Information database. Exon 1 is illustrated as an open
bar and the luciferase gene as an arrow. The restriction enzyme sites are indicated as follows: H, HindIII; B,
BamH I; K, KpnI; P, PvuII; S, SmaI.
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front of the luciferase reporter gene. These constructs
were transfected into cultured neonatal rat cardiac
myocytes. We showed that basal transcription from the
various S100B promoter constructs was selective for
cardiac myocytes because it was absent in similarly
transfected cultured neonatal rat fibroblasts (data not
shown). We also identified a basic promoter cloned in
pGBS ⫺162/⫹698 and defined as the shortest S100B
promoter fragment assayed, which spanned 162 bp
upstream of the transcription initiation site, 71 bp of
exon 1, and 627 bp of intron 1 (Fig. 3). The basic
promoter contains several general transcription elements, including a TATA box, a reverse CCAAT box,
and two GC boxes (1). The relative luciferase activities
of the various cloned constructs that comprised additional sequences upstream of the basic promoter
were 2-fold for pGBS ⫺782/⫹698, 0.5-fold for pGBS
⫺4,463/⫹698, and 4-fold for the maximal promoter
⫺6,689/⫹698, all relative to the basic promoter (Fig. 3).
This indicates the presence of positive regulatory
elements in the sequences ⫺782/⫺162 and ⫺6,689/
⫺4,463 and of a negative regulatory element in the
sequence ⫺4,463/⫺782. There were no additional transcription initiation sites upstream of the basic promoter, as absence of the basic promoter from the cloned
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DISCUSSION
Fig. 4. ␣1-Adrenergic stimulation specifically activates the basic and
maximal human S100B promoters through the ␣1A-adrenergic receptor. Neonatal rat cardiac myocytes were transfected with pGBS
⫺162/⫹698 (basic S100B promoter) or pGBS ⫺6,689/⫹698 (maximal
S100B promoter). A: transfected myocytes were treated with ␣1adrenergic agonists NE or PE, the ␣1A-adrenergic receptor antagonist WB-4101 (WB) or the ␣1B-adrenergic receptor antagonist chloroethyl clonidine (CEC), or with PE in the presence of WB or CEC
and assayed for luciferase activity, which is expressed relative to
myocytes treated with vehicle as described in MATERIALS AND METHODS. B: transfected myocytes were treated with Iso, T3, or ANG II and
assayed for luciferase activity, as described above. Bars are the
means ⫾ SE of luciferase expression from 6 independent experiments, with duplicate dishes in each experiment. * P ⬍ 0.05 vs.
myocytes treated with vehicle and assigned a value of 1.
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The postinfarct hypertrophic response in the myocardium is initiated by multiple trophic signals. The
state of local and systemic sympathetic hyperactivity
that accompanies myocardial infarction contributes
one of these signals through ␣1-adrenergic stimulation
of the ␣1A-adrenergic receptor (12, 18, 29). Intracellular second messengers for signal transduction triggered by ␣1-adrenergic stimulation include Ca2⫹ and
PKC (23). The latter signaling pathway involves
PKC-␤ and possibly other isoforms of the enzyme (␣, ␦,
⑀, and ␨) that have been shown to be present in the rat
heart (2, 28). Distal components of the PKC signaling
pathway include TEF-1, RTEF-1, Fos, and Jun (15, 17,
19, 31). Phosphorylation of these proteins by PKC is
one of the control mechanisms that influences their
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with ⌬PKC-␤, when combined with PE treatment, did
not further increase the stimulation obtained with PE
alone (Fig. 5B).
TEF-1 and RTEF-1 modulate transcription from human S100B promoter through elements located upstream of basic promoter. Cotransfection with TEF-1 or
RTEF-1, which are distal components of the PKC signal transduction pathway, did not influence transcription from the basic promoter (⫺162/⫹698) or the sequence ⫺782/⫹698, but influenced transcription from
the upstream sequences ⫺4,463/⫹698, and ⫺6,689/
⫹698 implicating the presence of MCAT elements located in the upstream sequences. The effects of TEF-1
and RTEF-1 on transcription from the upstream sequences were different. Whereas TEF-1 inhibited basal
transcription and reversed the transcriptional stimulation mediated by PE (Fig. 6A), RTEF-1-stimulated
basal transcription 1.5- to 2-fold and had no effect on
the PE-induced transcriptional stimulation (Fig. 6B).
Cotransfection with the transcription factors Fos or
Jun, which are also distal components of the PKC
signaling pathway, did not influence transcription
from either the basic or maximal promoter (data not
shown).
S100B MCAT elements exhibit specific binding to
cardiac myocyte nuclear extracts. Two MCAT motifs
were identified in the maximal S100B promoter,
MCAT1 (⫺1,984/⫺1,978) and MCAT2 (⫺1,333/⫺1,327)
(Fig. 7A). To determine whether the S100B MCAT
sequences bound members of the TEF-1 family, gel
mobility shift assay was performed with the use of
cardiac myocyte nuclear extract and the radiolabeled
⫺84/⫺61 skACT promoter containing the canonical
MCAT sequence (Fig. 7B). The interaction of TEF-1
with the skACT MCAT produced a band pattern (lane
1) that was competed out by excess unlabeled MCAT
sequences of the skACT promoter (lane 2) and MCAT1
(lane 3) and MCAT2 (lane 5) of the S100B promoter.
Demonstrating specificity, mutated MCAT1 (lane 4) or
MCAT2 (lane 4) did not compete out this binding.
These results suggest that the MCAT1 and MCAT2
elements within the S100B promoter can interact with
members of the TEF-1 family in cardiac myocytes.
␣1-ADRENERGIC INDUCTION OF S100B IN HEART
transactivation or transrepression of specific promoters (13, 34). TEF-1 and RTEF-1 belong to a family of
eukaryotic transcription factors that share sequence
homology in the tetraethylammonium (or ATTS) DNA
binding domain (13). In mammalian promoters, TEF-1
and RTEF-1 bind to M-CAT elements (9, 19). It is
possible that these two transcription factors play selective opposing roles in the regulation of fetal gene induction during the hypertrophic response induced by
␣1-adrenergic stimulation in cardiac myocytes. This
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Fig. 5. ␣1-Adrenergic stimulation activates the basic and maximal
human S100B promoters through the protein kinase C (PKC) signaling pathway. A: neonatal rat cardiac myocytes were transfected
with pGBS ⫺162/⫹698 (basic S100B promoter) or pGBS ⫺6,689/
⫹698 (maximal S100B promoter). Transfected myocytes were
treated with PE, the Ca2⫹ ionophore A-23187 (Ion), the Ca2⫹ channel
blocker nifedipine (Nif), the PKC antagonist staurosporine (Stau), or
phorbol 12-myristate 13-acetate (PMA), or with NE in the presence
of Ion, Nif, or Stau, and PMA ⫹ Stau, and assayed for luciferase
activity, which is expressed relative to myocytes treated with vehicle,
as described in MATERIALS AND METHODS. B: neonatal rat cardiac
myocytes were transfected with pGBS ⫺162/⫹698 (basic S100B
promoter) or pGBS ⫺6,689/⫹698 (maximal S100B promoter), or
cotransfected with the above promoters and ⌬PKC-␤ (a constitutively active mutant of PKC-␤). Control myocytes were cotransfected
with the plasmid vector SR␣. The transfected or cotransfected plasmids were treated with PE or Stau and assayed for luciferase
activity, which is expressed relative to myocytes transfected with the
plasmid vector SR␣ and treated with vehicle as described in MATERIALS AND METHODS. Bars represent the means ⫾ SE of luciferase
expression from six independent experiments, with duplicate dishes
in each experiment. * P ⬍ 0.05 vs. control myocytes assigned a value
of 1.
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possibility is based on the observations that RTEF-1
transactivates the promoters of the fetal genes ␤-MHC
and skACT, whereas TEF-1 downregulates transcription from the skACT promoter in these cells (31).
The results of our previous studies implicated S100B
as a negative modulator of the hypertrophic response
contributed by ␣1-adrenergic stimulation (32, 33). We
were able to demonstrate that S100B inhibited the
induction of ␤-MHC and skACT through the PKC pathway in the context of the hypertrophic response induced by ␣1-adrenergic stimulation in cultured rat
myocytes (32). The present study was undertaken to
examine whether the S100B gene induction in cardiac
muscle was mediated by the ␣1-adrenergic pathway.
This would support the hypothesis that S100B functions as a feedback regulator of this component of the
hypertrophic response.
We (33) reported a de novo presence of S100B protein
and mRNA in the peri-infarct region of human and rat
hearts. In the present study, we found that the presence of S100B mRNA in the peri-infarct region of the
rat heart after an experimentally provoked myocardial
infarction consequent to coronary artery ligation coincided with increased left ventricular NE levels. We
planned to demonstrate that the induction of steadystate S100B mRNA levels from undetectable in normal
heart to a level equivalent to that seen in the rat brain
after myocardial infarction was due to an induction of
the S100B gene by increased NE levels. For this purpose, we first transfected cultured neonatal rat cardiac
myocytes with a full-length human S100B gene and
showed that both the endogenous rat S100B gene and
the transfected human S100B gene were specifically
induced by the ␣1-adrenergic agonists NE and PE but
not the ␤1-adrenergic agonist Iso or T3.
To begin to dissect the functional activation of the
human S100B promoter in the heart, we transfected
cultured rat myocytes with constructs incorporating a
luciferase reporter gene driven by the human S100B
promoter. In the first instance, we used a maximal
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␣1-ADRENERGIC INDUCTION OF S100B IN HEART
S100B promoter, spanning 6,689 bp upstream of the
transcription initiation site, and progressively shorter
fragments sequentially deleted from the 5⬘ end of this
promoter, to define a basic promoter essential for transcription (spanning 162 bp upstream of the transcrip-
Fig. 6. Transcription enhancer factor-1 (TEF-1) or related to TEF-1
(RTEF-1) modulate transcription from the human S100B promoter
through elements located upstream of the basic promoter. Neonatal
rat cardiac myocytes were transfected with pGBS ⫺162/⫹698, pGBS
⫺782/⫹698, pGBS ⫺4,463/⫹698, or pGBS ⫺6,689/⫹698 or cotransfected with the above promoters and TEF-1 (A) or RTEF-1 (B).
Control myocytes were cotransfected with the plasmid vector pXJ40.
The transfected or cotransfected myocytes were treated with PE and
assayed for luciferase activity, which is expressed relative to myocytes transfected with the plasmid vector pXJ40 and treated with
vehicle as described in MATERIALS AND METHODS. Bars represent
means ⫾ SE of luciferase expression from six independent experiments, with duplicate dishes in each experiment. * P ⬍ 0.05 vs.
control myocytes assigned a value of 1.
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tion initiation site), two positive regulatory elements
(located in the regions ⫺782/⫺162 and ⫺6,689/
⫺4,463), and one negative regulatory element (located
in the region ⫺4,463/⫺782). There was no transcription from any of the S100B promoter fragments in
cultured neonatal rat cardiac fibroblasts. In our previous studies, in cultured neonatal rat astrocytes, we
used a similar strategy starting with a maximal promoter that spanned 4,463 bp upstream of the transcription initiation site (3). In these studies, we identified the same basic promoter, the positive regulatory
element at ⫺782/⫺162, and the negative regulatory
element at ⫺4,463/⫺782. Presently, by using a longer
maximal promoter as our starting point for generating
the 5⬘ deletion fragments, we extended these results by
identifying an additional positive regulatory element
at ⫺6,689/⫺4,463. The similarity in the basic human
S100B promoter and upstream regulatory elements
identified in rat astrocytes and myocytes, and the absence of transcription in rat fibroblasts, indicate a
tissue-selective distribution of specific transcription
factors that activate the S100B promoter and are conserved among mammalian species. This is consistent
with the experimental evidence suggesting highly specialized functional roles for S100B in the mammalian
brain and heart (8).
We then used the basic and maximal S100B promoters to study the induction of S100B in cardiac myocytes
by ␣1-adrenergic stimulation. Both promoters were activated by the ␣1-adrenergic agonists NE and PE
through the ␣1A-adrenergic receptor, but not by any
other hormonal myocardial trophic signals, including
T3, ANG II, or the ␤1-adrenergic agonist Iso. The activation by PE was blocked by the PKC antagonist staurosporine suggesting involvement of the PKC signaling
pathway. This suggestion was supported by showing
activation of the S100B promoter by treatment with
PMA (which activates PKC) and by cotransfection with
⌬PKC-␤, a constitutively active mutant of PKC-␤. The
activation by PMA and ⌬PKC-␤ was also blocked by
staurosporine again implicating the PKC signaling
pathway. The twofold activation seen with ⌬PKC␤ was
approximately one-half of that seen with PE and the
two together were not additive, suggesting that both
PKC-␤ and other isoforms of PKC are involved in this
activation consistent with previously published results
(2, 28). There were no significant differences in the
␣1-ADRENERGIC INDUCTION OF S100B IN HEART
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activation of the S100B promoter by PE in the presence
or absence of either the Ca2⫹ ionophore A-23187 or the
Ca2⫹ channel blocker nifedipine, suggesting that Ca2⫹
was not a likely second messenger of the ␣1-adrenergic
signal leading to the activation of the S100B promoter.
Taken together, the above data indicate that the same
␣1-adrenergic pathway that initiates and sustains the
hypertrophic response in cardiac myocytes by activating PKC and is subject to negative modulation by
S100B also induces the S100B gene (32). This supports
the hypothesis that S100B is a negative feedback regulator of this pathway.
Whereas ␣1-adrenergic activation of the S100B promoter proceeded equally well with the basic and maximal promoters, the same was not the case for the
transcription factors TEF-1 and RTEF-1. Both factors
required the maximal promoter for their action, consistent with the presence of MCAT sequences (CATTCCT at ⫺1,333 to ⫺1,327 and CATTCCA at ⫺1,984 to
⫺1,978) upstream of the basic promoter that exhibited
specific binding activity. TEF-1 transrepressed the
maximal full-length S100B promoter under basal conditions and with PE, thereby preventing ␣1-adrenergic
AJP-Heart Circ Physiol • VOL
activation of the S100B promoter that was previously
documented. The inhibition of the S100B promoter was
not seen with 10-fold and 100-fold lower concentrations
of TEF-1 (data not shown). The inhibitory effect of
TEF-1 is comparable to the response of the skACT
promoter and may result from TEF-1 overabundance
sequestering or “squelching” a limiting cofactor (31).
Conversely, RTEF-1 transactivated the same S100B
promoter twofold versus the fourfold activation seen
with PE, and the two together were not additive. This
suggests that a component of the ␣1-adrenergic induction of the S100B promoter is mediated in part through
RTEF-1. It is conceivable that this two-tiered mechanism of induction of S100B confers the following advantages. First, the RTEF-1 component provides a
means to regulate the induction of the S100B gene
coordinately with the induction of fetal genes such as
␤-MHC and skACT, and, second, the non-RTEF-1 component ensures an induction of the S100B gene in
conditions where the amount of RTEF-1 might be limiting. Moreover, because PE alone activated the basic
and maximal S100B promoters equally well, the above
results also suggest that in the presence of the maxi-
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Fig. 7. The S100B MCAT binds myocyte TEF-1.
A: schematic representation of the ⫺6,698/⫹698
S100B promoter fragment containing the first
exon and a portion of the first intron. The location of the restriction sites used to generate the
series of 5⬘ promoter deletions and of the MCAT,
GATA4, MEF-2, and TATA sequences is indicated. See Fig. 3 for restriction enzyme site designations. B: endogenous TEF-1 in cardiac myocyte nuclear extracts binds to the S100B MCAT
elements. A double-stranded oligonucleotide of
the mouse ⫺84/⫺61 skACT promoter sequence,
containing the canonical MCAT sequence, was
end labeled with 32P and incubated with 10 ␮g of
cardiac myocyte nuclear extract. Free and complex probes were separated on a 6% native gel
(bottom). Arrows indicate the position of free and
the shifted TEF-1-DNA complex. Gel mobility
shift assay was done with no competitor (lane 1),
with 100-fold molar excess of unlabeled ⫺84/⫺61
skACT promoter fragment (lane 2), and with
100-fold molar excess of unlabeled S100B,
MCAT1, and MCAT2 fragments (lanes 3 and 5)
and their mutated versions (lanes 4 and 6). Sequences of the oligonucleotides are shown at the
top. Binding sites are underlined and boldface.
Lowercase letters identify mutated bases.
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␣1-ADRENERGIC INDUCTION OF S100B IN HEART
We thank C. McMahon, A. Haddad, and J. Culbert for excellent
technical assistance and Drs. L. R. Karns, P. C. Simpson, A. F. R.
Stewart, and I. Verma for the gift of the essential expression plasmids and for helpful discussions.
This work was supported in part by the Canadian Institutes of
Health Research.
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