Regulation of Bcl-xL Expression by H2O2 in Cardiac Myocytes*,
Donna M. Valks,
Timothy J. Kemp and
Angela Clerk
From the
National Heart and Lung Institute Division (Cardiac Medicine Section),
Faculty of Medicine, Imperial College London, Flowers Building, Armstrong
Road, London SW7 2AZ, United Kingdom
Received for publication, April 10, 2003
, and in revised form, April 23, 2003.
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ABSTRACT
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Oxidative stress promotes cardiac myocyte apoptosis through the
mitochondrial death pathway. Since Bcl-2 family proteins are key regulators of
apoptosis, we examined the effects of H2O2 on the
expression of principal Bcl-2 family proteins (Bcl-2, Bcl-xL, Bax, Bad) in
neonatal rat cardiac myocytes. Protein expression was assessed by
immunoblotting. Bcl-2, Bax, and Bad were all down-regulated in myocytes
exposed to 0.2 mM H2O2, a concentration that
induces apoptosis. In contrast, although Bcl-xL levels initially declined, the
protein was re-expressed from 46 h. Bcl-xL mRNA was up-regulated from 2
to 4 h in neonatal rat or mouse cardiac myocytes exposed to
H2O2, consistent with the re-expression of protein. Four
different untranslated first exons have been identified for the Bcl-x gene
(exons 1, 1B, 1C, and 1D, where exon 1 is the most proximal and exon 1D the
most distal to the coding region). All were detected in mouse or rat neonatal
cardiac myocytes, but exon 1D was not expressed in adult mouse hearts. In
neonatal mouse or rat cardiac myocytes, H2O2 induced the
expression of exons 1B, 1C, and 1D, but not exon 1. These data demonstrate
that the Bcl-x gene is selectively responsive to oxidative stress, and the
response is mediated through distal promoter regions.
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INTRODUCTION
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Cardiac myocytes, the contractile cells of the heart, are terminally
differentiated cells, which withdraw from the cell cycle in the perinatal
period. Consequently, myocyte cell death, as occurs (for example) following
ischemia and/or reperfusion, significantly affects cardiac function. One of
the principal insults that may promote cardiac myocyte death is oxidative
stress, and the levels of oxidative stress are increased in ischemic hearts
(1,
2) with higher levels being
produced following reperfusion
(3). H2O2
(0.10.5 mM), as an example of oxidative stress, promotes
cardiac myocyte apoptosis through the mitochondrial death pathway
(4). However, lower
concentrations are non-toxic and may have cytoprotective and/or growth
promoting effects
(58).
Bcl-2 family proteins are key regulatory components of the mitochondrial
cell death pathway (9,
10). Some family members are
cytoprotective (e.g. Bcl-2, Bcl-xL, Bcl-w, Mcl-1), whereas others
promote apoptosis (e.g. Bad, Bak, Bax, Bid, Bim, Bmf). These proteins
act at the mitochondria to regulate cytochrome c release. Bcl-2
family proteins act either as heterodimers or as oligomers and the dynamic
equilibrium between such complexes appears to determine the predisposition to
apoptosis. Translocation of Bax to the mitochondria and oligomerization of Bax
and/or Bak promotes permeabilization of the outer mitochondrial membrane and
release of cytochrome c and other apoptosis-inducing factors,
possibly by forming a pore in the membrane. Heterodimerization with either
Bcl-2 or Bcl-xL prevents oligomerization and protects from apoptosis. Other
pro-apoptotic proteins, such as Bad, Bim, or Bmf compete for binding to
Bcl-2/Bcl-xL causing release of Bax/Bak, which can then form oligomers and
induce apoptosis. Thus, the balance between pro- and anti-apoptotic Bcl-2
family proteins influences the rate of apoptosis.
Bcl-2 and Bcl-xL are the principal Bcl-2 family proteins that protect cells
from apoptosis. The regulation of the Bcl-x gene appears particularly complex.
Initial studies of the mouse Bcl-x gene demonstrated that, like Bcl-2, the
Bcl-x gene consisted of three separate exons
(11). Exon 1 is untranslated
and is separated from the first coding exon, exon 2, by a short facultative
intron. Exon 3 codes for the C terminus of Bcl-xL and is separated from exon 2
by a large intron of at least 9 kb. At least four proteins potentially derive
from alternatively spliced Bcl-x mRNAs. Exons 1, 2, and 3 are spliced together
to produce Bcl-xL. Splicing from an alternative donor site within exon 2 to
exon 3 results in a shorter form of the protein (Bcl-xS), which lacks a
central region present in Bcl-xL, but the reading frame is maintained and the
C terminus is identical (11).
In contrast to Bcl-xL, Bcl-xS induces apoptosis. Read-through of the donor
site at the end of exon 2 produces Bcl-x
(12), and splicing of exon 2
to a novel exon 4 generates Bcl-x
(13). Apart from alternative
splicing of the coding exons of the Bcl-x gene, there is additional complexity
in the regulation of the 5' non-coding region. Grillot et al.
(11) reported the presence of
at least two principal transcription initiation sites for exon 1 at 655
and 727, in addition to a cluster of initiation sites at the start of
exon 2. Subsequently, an alternative first exon upstream of exon 1 (exon 1B)
was identified in human lymphoma cells
(14). A third study suggested
that at least five different promoters (P1P5) operate in the mouse
Bcl-x gene, each of which is associated with a different first exon (exons
AE) (15). This
terminology is confusing, since exons A and B correspond, respectively, to
exons 2 and 1 identified by Grillot et al.
(11), and exon C corresponds
to the region identified as exon 1B
(14). The different exons of
the Bcl-x gene are summarized in Fig.
1. Despite the identification of potential promoters and
transcriptional start sites further upstream in the Bcl-x gene (i.e.
exons D and E (15)),
expression of these exons was not detected in erythroid progenitor cells or
differentiating erythroblasts in which Bcl-xL is up-regulated
(16), and their significance
in a biological system remains to be established.

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FIG. 1. Exon structure of the mouse Bcl-x gene. The exon numbering system
has been adapted from the schemes by Grillot et al.
(11) and MacCarthy-Morrogh
et al. (14), with the
system (exons AE) used by Pecci et al.
(15) shown below. Bcl-x primer
pairs that were used are also shown (for further details see
Table I). bp numbering extends
from the first coding ATG. Exons and introns are not shown to scale.
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TABLE I Primers used for RT-PCR
Nucleotide positions for each primer are shown in parentheses. Different
primers were used for analysis of the Bcl-x promoters for mouse (M) and rat
(R).
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A number of Bcl-2 family proteins are expressed in cardiac myocytes
including Bcl-2, Bcl-xL, Bad, Bax, and Bid
(4,
17,
18). As in other cells,
overexpression of Bcl-2 in cardiac myocytes is cytoprotective
(1921).
H2O2-induced apoptosis in neonatal myocytes is
associated with rapid translocation of Bad and Bax to the mitochondria (<5
min), an event which precedes cytochrome c release
(4,
17), suggesting that, in the
context of oxidative stress, Bad/Bax translocation may be one of the
initiating events. Bcl-2 family proteins are therefore key regulators of
apoptosis in cardiac myocytes, as in other cells, and the expression of the
various family members is likely to influence the rate of myocyte death. In
this study, we examined the effects of H2O2 on the
expression of four key family members which are readily detected in cardiac
myocytes (Bcl-2, Bcl-xL, Bax, and Bad).
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EXPERIMENTAL PROCEDURES
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Preparation of Neonatal Rat or Mouse Cardiac
MyocytesMyocytes were dissociated from the ventricles of
13-day Sprague-Dawley rat hearts as described previously
(22) and plated at a density
of 1.4 x 103 cells/mm2 in 35-mm Primaria tissue
culture dishes precoated with 1% (w/v) gelatin. Neonatal mouse myocytes were
prepared from the ventricles of C57Bl6 mice using essentially the same
procedure as for neonatal rat myocytes, and cells were plated at a density of
2.0 x 103 cells/mm2. Myocytes were plated in
Dulbecco's modified Eagle's medium/M199 (4:1) containing 5% (v/v) fetal calf
serum and 10% (v/v) horse serum (16 h, 37 °C), and then incubated in
serum-free medium for 24 h prior to experimentation. Myocytes were exposed to
H2O2 (0.02 mM, 0.2 mM, 2
mM) for the times indicated.
Immunoblot AnalysisMyocytes were washed in
Ca2+/Mg2+-free Dulbeccos
phosphate-buffered saline (Invitrogen), extracted in Buffer A (20
mM glycerophosphate, pH 7.5, 50 mM NaF, 2
µM microcystin LR, 2 mM EDTA, 0.2 mM
Na3VO4, 10 mM benzamidine, 200
µM leupeptin, 10 µM
trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, 5
mM dithiothreitol, 300 µM phenylsulfonyl fluoride, 1%
(v/v) Triton X-100) and centrifuged (10,000 x g, 5 min). The
supernatants were boiled with 0.33-volume sample buffer (0.33 M
Tris-HCl, pH 6.8, 10% (w/v) SDS, 13% (v/v) glycerol, 133 mM
dithiothreitol, 0.2 mg/ml bromphenol blue). Proteins (15 µg) were separated
by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 15% (w/v)
polyacrylamide gels and transferred to nitrocellulose membranes as described
previously (23). Blots were
incubated in Tris-buffered saline containing 0.05% (v/v) Tween 20 (TBST) and
5% (w/v) nonfat milk powder to block nonspecific binding. Blots were washed
(TBST, 3 x 5 min) and incubated with primary antibodies in TBST
containing 5% (w/v) bovine serum albumin (16 h, 4 °C). Primary antibodies
to Bcl-xL (H-5), Bcl-2 (C-2), or Bax (B-9) were from Santa Cruz Biotechnology
Inc. and used at 1/400 dilution; antibodies to total Bad (B36420
[GenBank]
) were from BD
Transduction Laboratories and used at 1/1000 dilution. Blots were washed
(TBST, 3 x 5 min) and incubated with secondary antibodies conjugated to
horse-radish peroxidase diluted in TBST containing 1% (w/v) nonfat milk powder
(1 h, 20 °C). Bands were detected by enhanced chemiluminescence (Santa
Cruz Biotechnology Inc.) and were quantified by scanning densitometry.
Total RNA Isolation and cDNA SynthesisMyocytes were
homogenized in 0.5 ml of RNAzol B (AMS Biotech) and total RNA prepared
according to the manufacturer's instructions. RNA pellets were dissolved in
diethyl pyrocarbonate-treated water, and the concentrations were calculated
from the A260. First strand cDNA synthesis was performed
from total RNA (2 µg) in a reaction mixture containing 500 oligo(dT) and 1
mM dNTP mix. Tubes were heated (65 °C, 5 min). 5 x
first-strand buffer, 0.1 M dithiothreitol, recombinant ribonuclease
inhibitor (40 units) and Superscript II RNase H reverse transcriptase (200 U)
(Invitrogen) were added, and cDNA synthesis was performed (50 min, 42
°C).
Preparation of Genomic DNARat spleen (stored at 80
°C) was powdered under liquid N2, homogenized in Buffer ATL
(Qiagen DNeasy tissue DNA extraction kit), and passed through a 21-gauge
needle 520 times. DNA was extracted according to the manufacturer's
instructions, eluted in 100 µl, and the concentration was calculated from
the A260.
Ratiometric
RT1-PCR
AnalysisRatiometric RT-PCR was performed as previously described,
and in our hands, results from this method are comparable with those obtained
by quantitative "real-time" PCR
(24). Primers were designed
for the coding regions of rat Bcl-2, Bad, and Bax and for mouse or rat Bcl-xL
coding region. Primers for the Bcl-xL coding region (exon 2) were designed not
to detect Bcl-xS. Primers were also designed for each of the potential first
exons upstream of exon 2 of the Bcl-x gene and to amplify the region across
exons 1 and 2 (Table I and
Fig. 1). To avoid confusion, we
have used the Grillot/MacCarthy-Morrogh
(11,
14) terminology referring to
exons D and E of the Pecci et al. study
(15) as exons 1C and 1D
(Fig. 1). RT-PCR reactions were
carried out in a 50-µl volume containing 100 ng of cDNA or 20 ng of genomic
DNA template, 100 µM concentration of each primer, 50
mM KCl, 20 mM Tris-HCl (pH 8.4 at 25 °C), 1.5
mM MgCl2, 0.01% (v/v) Tween 20, and a 0.2 mM
concentration each of dATP, dCTP, dGTP, dTTP, using 1 unit of Taq
polymerase (Invitrogen). The following conditions were used: 95 °C, 3 min
followed by 2431 cycles of denaturation (95 °C, 50 s), annealing
(61 °C, 50 s), and extension (72 °C, 50 s). Amplification products
were analyzed by ethidium bromide-agarose gel electrophoresis using 2% (w/v)
agarose gels and the bands captured under UV illumination. All primer sets
generated clean products of the predicted sizes
(Table I). Products were
analyzed by scanning densitometry and normalized to GAPDH. Results are
expressed relative to unstimulated controls.
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RESULTS
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H2O2 Promotes
Down-regulation of Bcl-2 Family Proteins in Cardiac MyocytesWe
examined the effects of different concentrations of H2O2
(2, 0.2, and 0.02 mM) on the expression of four principal Bcl-2
family proteins, which are expressed in cardiac myocytes: Bcl-2, Bcl-xL, Bax,
and Bad. 0.2 mM H2O2 results in myocyte death
associated with features of apoptosis
(4), whereas higher
concentrations induce non-apoptotic cell death. 0.02 mM
H2O2 is non-toxic, but has effects on gene
expression.2 Neonatal
rat cardiac myocytes were exposed to H2O2 for up to 24
h, and the levels of Bcl-2, Bcl-xL, Bax, and Bad were compared by
immunoblotting. High concentrations of H2O2 (2
mM) induced profound down-regulation of all four proteins
(Fig. 2, AD).
Down-regulation of pro-apoptotic Bax and Bad was apparent within 12 h
(Fig. 2, A and
B), whereas down-regulation of anti-apoptotic Bcl-2 and
Bcl-xL was not significant before
4 h
(Fig. 2, C and
D). The levels of Bax and Bad were also decreased in
myocytes exposed to 0.2 mM H2O2
(Fig. 2, E and
F), albeit to a lesser extent than in myocytes exposed to
2 mM H2O2
(Fig. 2, A and
B). Bcl-2 was down-regulated to a similar degree and over
a similar time course in response to both concentrations
(Fig. 2, C and
G). In contrast, although Bcl-xL protein initially
decreased in myocytes exposed to 0.2 mM H2O2
(
70% over 24 h), it was re-expressed, and after 24 h the levels of
Bcl-xL were similar to those of unstimulated control cells
(Fig. 2H). The
expression of all four Bcl-2 family proteins did not change in myocytes
exposed to 0.02 mM H2O2 (results not shown).
These data indicate that, of the four Bcl-2 family proteins, only Bcl-xL is
re-expressed following stimulation by oxidative stress at a concentration that
promotes apoptosis.

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FIG. 2. H2O2 induces down-regulation of Bax, Bad, Bcl-2,
and Bcl-xL in rat cardiac myocytes. Rat neonatal cardiac myocytes were
exposed to 2 mM (AD) or 0.2 mM
(EH) H2O2 for the times indicated.
Extracts were immunoblotted for Bax (A and E), Bad
(B and F), Bcl-2 (C and G), or Bcl-xL
(D and H). Representative blots are shown in the upper
panels; densitometric analysis of the data is provided in the lower
panels expressed relative to controls. Results are means ± S.E.
for 3 (B and D), 4 (G and H), 5 (A,
C and F), or 7 (E) independent experiments.
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H2O2 Induces
Bcl-xL mRNA ExpressionTo determine whether re-expression of Bcl-xL
protein in response to 0.2 mM H2O2 reflected
an increase in mRNA expression (rather than an effect on protein synthesis or
stability) and that this was a selective effect, neonatal rat cardiac myocytes
were exposed to 0.2 mM H2O2, and ratiometric
RT-PCR analysis of Bcl-xL, Bcl-2, Bax, and Bad was performed. Primers were
designed to detect expression of the coding regions of the mRNAs for each gene
(Table I). Consistent with the
protein data (Fig.
2H), Bcl-xL (exon 2) mRNA was up-regulated from 2 to 4 h,
and this was sustained over the 8-h period studied
(Fig. 3A). In
contrast, there was no change in expression of Bcl-2 or Bax mRNA
(Fig. 3, B and
C), and Bad was marginally down-regulated at the mRNA
level (Fig. 3D). Since
more detailed analysis of the regulation of Bcl-x has been performed for the
mouse gene than for the rat gene
(15), we confirmed that Bcl-xL
mRNA was up-regulated in neonatal mouse cardiac myocytes in response to 0.2
mM H2O2.As in rat myocytes, Bcl-xL mRNA
expression was increased at 2 and 4 h (Fig.
3F). Thus, 0.2 mM H2O2
increases the expression of Bcl-xL mRNA in cardiac myocytes, and this probably
accounts for the selective re-expression of Bcl-xL protein.

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FIG. 3. H2O2 (0.2 mM) promotes selective
up-regulation of Bcl-xL mRNA in rat or mouse cardiac myocytes. Rat
neonatal cardiac myocytes were exposed to 0.2 mM
H2O2 for the times indicated. The expression of Bcl-xL
(exon 2) (A), Bcl-2 (B), Bax (C), or Bad
(D) mRNA was analyzed by ratiometric RT-PCR. Representative images
from a single experiment are shown in the upper panels. The
expression of GAPDH in the samples from the same experiment is also shown
(E). Densitometric analysis of the data is provided in the lower
panels expressed relative to controls. Results are means ± S.E.
for 4 (B) or 5 (A, C, and D) independent
experiments. F, mouse neonatal cardiac myocytes were exposed to 0.2
mM H2O2 for the times indicated, and the
expression of Bcl-xL mRNA was analyzed by ratiometric RT-PCR. A representative
image of Bcl-xL (exon 2) expression and the corresponding GAPDH is shown in
the upper panel. Densitometric analysis of the data is provided in
the lower panel expressed relative to controls. Results are means
± S.E. for three independent experiments.
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Up-regulation of Bcl-xL mRNA by
H2O2 Is Mediated by
Selective Promoter ActivationAs illustrated in
Fig. 1, four putative
untranslated first exons have been identified for the mouse Bcl-x gene
(15). To avoid confusion we
have extended the terminology used by MacCarthy-Morrogh et al.
(14) and refer to exons 1 (the
original first exon identified by Grillot et al.
(11)), 1B, 1C, and 1D (which
correlate to exons C, D, and E described by Pecci et al.
(15)). Transcripts containing
exons 1 and 1B had been previously detected in heart extracts, but whether
exons 1C and 1D are also expressed had not been studied. Using specific
primers to each of the untranslated exons of the mouse Bcl-x gene for
ratiometric RT-PCR analysis, we confirmed that exons 1 and 1B were expressed
in adult mouse heart and all other tissues studied (spleen, kidney, liver, and
brain) (Fig. 4A). Exon
1C, but not exon 1D, was also detected in all adult mouse tissues
(Fig. 4A and results
not shown). Bcl-x exons 1, 1B, and 1C were all readily detected in neonatal
mouse cardiac myocyte cultures confirming expression in the myocytes
themselves. However, although it was not detected in adult hearts, exon 1D was
expressed in neonatal cardiac myocytes
(Fig. 4B), suggesting
that expression of this region is down-regulated during postnatal development
of the heart. Since all primers were designed to amplify regions within
individual predicted exons, we considered it necessary to confirm that there
was no contamination of samples with genomic DNA and primers were designed for
RT-PCR to amplify the region across the intron between exons 1 and 2. RT-PCR
analysis of cDNA samples resulted in a single product of 433 bp, consistent
with splicing of exon 1 to exon 2 generating a single mRNA species
(Fig. 4C). This
contrasts with a previous study in which differential splicing across this
region produced three different mRNAs
(15). Amplification of genomic
DNA resulted in a product of 615 bp (Fig.
4C) encompassing the intron and confirming that there was
no significant contamination of samples with genomic DNA.

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FIG. 4. Expression of Bcl-x non-coding exons 1, 1B, 1C, and 1D in mouse tissues,
and cardiac myocytes. RNA was extracted from adult mouse heart, spleen,
kidney, liver, and brain (A and B) or from mouse neonatal
cardiac myocytes (B and C), or genomic DNA was prepared
(A and C). The expression of Bcl-x exons 1, 1B, 1C, and 1D,
and the region encompassing the intron between exon 1 and exon 2 was examined
by ratiometric RT-PCR analysis using the primers detailed in
Table I.
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To determine which of the untranslated exons are expressed in response to
H2O2, neonatal mouse cardiac myocytes were exposed to
0.2 mM H2O2, and the different exons were
analyzed by ratiometric RT-PCR. The expression of exon 1 did not change in
response H2O2, whereas exons 1B, 1C, and 1D were all
significantly increased (Fig.
5A). The sequence for the region 5' to the rat
Bcl-x gene has recently become available and comparison of the region
encompassing all potential untranslated first exons with the mouse sequence
indicates a high degree of conservation from some distance upstream of exon 1D
through to the first coding ATG of the Bcl-x gene (see on-line Supplemental
Material). Using primers specific for the rat sequence
(Table I), we demonstrated
that, consistent with the data for mouse myocytes
(Fig. 5A), 0.2
mM H2O2 selectively increased the expression
of exons 1B, 1C, and 1D in neonatal rat cardiac myocytes with no change in
expression of exon 1 (Fig. 5, B
and C). These data suggest that oxidative stress results
in specific expression of the distal untranslated exons of the Bcl-x gene in
cardiac myocytes rather than the most proximal exon 1.

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FIG. 5. H2O2 selectively up-regulates the expression of
Bcl-x exons 1B, 1C, and 1D, but not exon 1. Mouse (A) or rat
(B and C) neonatal cardiac myocytes were exposed to 0.2
mM H2O2 for the times indicated. RNA was
extracted, and the expression of Bcl-x exons 1, 1B, 1C, and 1D was examined by
ratiometric RT-PCR analysis using the primers detailed in
Table I. Representative images
from a single experiment are shown in the upper panels, in addition
to the expression of GAPDH in the same samples. Densitometric analysis of the
data is provided in the lower panels expressed relative to controls.
Results are means ± S.E. for 3 (C), 4 (A), or 5
(B) independent experiments.
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DISCUSSION
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Down-regulation of Bcl-2 Family Proteins during Cardiac Myocyte
DeathH2O2, as an example of oxidative
stress, induces cardiac myocyte death. In response to 2 mM
H2O2, which should induce a more necrotic phenotype, all
four Bcl-2 family proteins studied were profoundly down-regulated
(Fig. 2, AD).
In contrast, in response to 0.2 mM H2O2,
which induces apoptosis, although the down-regulation of Bcl-2 was similar to
that seen in response to 2 mM H2O2, the
effect on Bax and Bad was less pronounced
(Fig. 2,
EG). Furthermore, although Bcl-xL
decreased initially, the protein was re-expressed
(Fig. 2H). The effects
of the two concentrations on Bcl-2 family proteins are therefore distinct and
may be a reflection of the type of cell death. The mechanisms involved in the
down-regulation of these Bcl-2 family proteins were not investigated in this
study. However, the down-regulation of Bcl-2, Bax, and Bad in response to 0.2
mM H2O2 is likely to be primarily due to
increased protein degradation and/or reduced protein synthesis, since the mRNA
levels for Bcl-2 and Bax did not change and, although there was a decrease in
Bad mRNA, this was relatively modest (Fig.
3, BD). The initial decline in Bcl-xL protein also
probably reflects increased degradation, since Bcl-xL mRNA expression did not
change significantly before
2 h (Fig.
3A). The subsequent increase in Bcl-xL mRNA presumably
accounts for the re-expression of Bcl-xL protein from
46 h
(Fig. 2H). There was
some variability in the time at which Bcl-xL protein was re-expressed, which
may be due to a conflict between the rate of protein degradation and synthesis
of new protein. The significance of Bcl-xL re-expression in response to 0.2
mM H2O2 is not clear. However, this
concentration does not induce apoptosis in all myocytes over 24 h
(4), and re-expression of
Bcl-xL in surviving cells may be a significant component of the survival
program.
Regulation of Bcl-xL mRNA Expression in Cardiac
MyocytesAlthough Bcl-x transcripts have been reported to derive
from at least five different promoters (i.e. the region between exons
1 and 2 and regions upstream of exons 1, 1B, 1C, and 1D), only the three most
proximal (regions upstream of exons 2, 1, and 1B) were previously shown to be
operative in the heart (15).
In this study, we demonstrated that exon 1C was expressed in adult mouse heart
and neonatal cardiac myocytes, and although it was not detected in adult
hearts, exon 1D was expressed in neonatal cardiac myocytes
(Fig. 4A). These data
suggest that there is developmental regulation of the upstream promoters such
that exon 1D is no longer expressed in adult hearts. Our preliminary data
suggest that, in neonatal cardiac myocytes, exons 1D and 1C are expressed as a
single exon that incorporates the intervening sequence (results not shown).
This suggests that during development there is a switch in the transcriptional
initiation site that is used. Exons 1C/1D do not appear to be continuous with
exon 1B (results not shown). Consistent with this, comparison of the mouse and
rat sequences indicates that there is a region of low homology between exon 1C
and exon 1B (see on-line Supplemental Material).
Although all four untranslated exons were detected in neonatal cardiac
myocytes, only the expression of exons 1B, 1C, and 1D was significantly
increased by 0.2 mM H2O2, with no increase in
exon 1 (Fig. 5). The reasons
for multiple promoter usage and differential expression of untranslated exons
are unclear, although previous studies suggest that the promoter may influence
the isoform of Bcl-x which is produced
(15). In cardiac myocytes,
Bcl-xL (Fig. 2) and Bcl-xS
(results not shown) were up-regulated concomitantly, and it is possible that,
for example, exon 1B may preferentially induce Bcl-xL whereas exons 1C/1D may
induce Bcl-xS. However, there is minimal evidence in support of such a
scenario. Alternatively, switching of non-coding exons to produce different
5'-untranslated regions may influence the mode and rate of translation.
In cardiac myocytes, H2O2 (>0.1 mM)
suppresses global protein synthesis by
95% over at least 4 h, probably
because of repression of cap-dependent initiation of translation
(25). However, a small group
of proteins continues to be synthesized, including p21CIP1/WAF1
(26), presumably through
cap-independent mechanisms. An increasing number of eukaryotic mRNAs have been
identified which contain internal ribosome entry sites required for
cap-independent protein synthesis (see
ifr31w3.toulouse.inserm.fr/IRESdatabase),
and many of these are associated with apoptosis
(27). In the case of
insulin-like growth factor 2, mRNAs are produced with three different
5'-untranslated regions, only one of which confers cap-independent
protein synthesis (28). The
specific up-regulation of exons 1B, 1C, and 1D, but not exon 1, by
H2O2 in cardiac myocytes may therefore be required for
translation to occur in the context of global inhibition of protein synthesis.
Most studies so far have focused on the regulation of expression of Bcl-xL
from the promoter regions upstream of exons 1 and 2, and binding sites for a
number of transcription factors have been identified in this region
(11). It is now clear that
further analysis of sequences upstream from exons 1B, 1C, and 1D is required
to identify the regions that are responsive to H2O2 and
establish which transcription factors are involved.
It is of note that the homology between rat, mouse and human Bcl-x gene is
extremely high across the region encompassing exons 1B through to exon 3.
However, although the homology between the rat and mouse sequences across
exons 1C and 1D is high (see on-line Supplemental Material), we have had
problems aligning this region with the human sequence (42% identity at best).
It is possible exons 1C and 1D represent a different gene from Bcl-x, but the
homology between rat and mouse extends almost continuously from some distance
5' to exon 1D through the first coding ATG of Bcl-x, and the regulation
of exons 1C and 1D in response to H2O2 is similar to
that of exons 1B and Bcl-xL exon 2. Furthermore, the homology between the rat
and mouse sequences is lost abruptly some distance 5' to exon 1D (see
on-line Supplemental Material), suggesting that this may be the extreme
5' end of the gene. The reasons for the lack of homology with the human
sequence are not clear. If exons 1C and 1D are part of another gene, it is
possible that this gene is elsewhere on the human genome, but we have been
unable to find any human sequences with significant homology to the rat/mouse
sequences. It seems more probable that evolutionary constraints on the primary
sequence were not high. Motifs for internal ribosome entry sites are not
conserved at the primary sequence level, and it is the secondary structure
that appears to be of prime importance
(29). If the upstream region
of the Bcl-x gene produces a mRNA capable of promoting cap-independent
translation, the constraints would therefore be expected to occur at the level
of secondary structure rather than primary sequence. Further analysis is
required to determine whether the upstream region is relevant to expression of
human Bcl-x gene products and if this region does indeed promote translation
through cap-independent mechanisms.
 |
FOOTNOTES
|
---|
* This work was supported by grants from the British Heart Foundation, the
Medical Research Council, the Wellcome Trust, and the Royal Brompton and
Harefield NHS Trust. The costs of publication of this article were defrayed in
part by the payment of page charges. This article must therefore be hereby
marked "advertisement" in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact. 
The on-line version of this article (available at
http://www.jbc.org)
contains supplemental data and a supplemental figure. 
To whom correspondence should be addressed: NHLI Division (Cardiac Medicine
Section), Faculty of Medicine, Imperial College London, Flowers Bldg.,
Armstrong Rd., London SW7 2AZ, UK. Tel.: 44-20-7594-3009; Fax:
44-20-7594-3419; E-mail:
a.clerk{at}imperial.ac.uk.
1 The abbreviations used are: RT, reverse transcriptase; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase. 
2 T. J. Kemp and A. Clerk, unpublished data. 
 |
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