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J. Biol. Chem., Vol. 278, Issue 28, 25542-25547, July 11, 2003
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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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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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 (P1–P5) operate in the mouse
Bcl-x gene, each of which is associated with a different first exon (exons
A–E) (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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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Immunoblot Analysis—Myocytes 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 Synthesis—Myocytes 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 DNA—Rat 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 5–20 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 Analysis—Ratiometric 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 24–31 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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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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 2–4 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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H2O2 Induces Bcl-xL mRNA Expression—To 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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Up-regulation of Bcl-xL mRNA by H2O2 Is Mediated by Selective Promoter Activation—As 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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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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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2 h (Fig.
3A). The subsequent increase in Bcl-xL mRNA presumably
accounts for the re-expression of Bcl-xL protein from 4–6 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 Myocytes—Although 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.
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FOOTNOTES |
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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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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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