Oncogene (1998) 17, 1903 ± 1910
 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00
http://www.stockton-press.co.uk/onc
Modulation of endoplasmic reticulum calcium pump by Bcl-2
Tuan H Kuo, Hyeong-Reh Choi Kim, Liping Zhu, Yingjie Yu, Huei-Min Lin and Wayne Tsang
Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA
Members of the bcl-2 gene family encode proteins that
function either to promote or to inhibit apoptosis.
Despite numerous e€orts, the mechanism of action of
Bcl-2, an anti-apoptotic protein, is still not clear. In
particular, the relation between Bcl-2 and the endoplasmic reticulum (ER) calcium store is not well-understood.
In the present work, we examined the e€ect of Bcl-2 on
the ER store. We demonstrate that overexpression of
Bcl-2 in breast epithelial cells modulates ER store by
upregulating calcium pump (SERCA) expression without
a€ecting the release channel (IP3R). The steady state
levels of SERCA2 mRNA and protein were both
increased in Bcl-2 expression clones. The increase in
SERCA2 protein leads to accelerated calcium uptake
and enhanced Ca2+ loading. In addition, we also show
the detection of intracellular interaction between Bcl-2
and SERCA molecules by co-immunoprecipitation. Since
high lumenal Ca2+ concentration of ER is essential for
normal cell functions, the results suggest that Bcl-2
preserves the ER Ca2+ store by upregulating SERCA
gene expression as well as by a possible interaction with
the pump.
Keywords: Bcl-2; endoplasmic reticulum calcium pump;
gene expression
Introduction
Endoplasmic reticulum (ER) is a major intracellular
Ca2+ storage site in mammalian cells (Carafoli, 1987).
Maintenance of Ca2+ homeostasis within the ER is
essential for a number of vital cellular functions,
including cell growth (Short et al., 1993; Waldron et
al., 1994; Cheng et al., 1996), signal transduction
(Berridge, 1993; Calpham, 1995), protein translation
(Brostrom and Brostrom, 1990), processing and
transport (Lodish et al., 1992; Sambrook, 1990). Ca2+
movement through the ER membrane are regulated by
two major Ca2+ transporters, namely the ER Ca2+ATPase pump (SERCA) and the IP3 receptor channel
(Berridge, 1993; Calpham, 1995). During cell signaling,
the IP3R channel is responsible for the release of Ca2+
from the ER, while SERCA functions to pump
cytosolic Ca2+ back into the ER against a steep
concentration gradient. Together, in conjunction with
the ER calcium binding proteins, they are responsible
for the maintenance of high lumenal Ca2+ concentration of ER which is essential for normal ER function.
Recently, a connection between alteration of ER Ca2+
and apoptosis was suggested in studies involving the
Correspondence: TH Kuo
Received 24 February 1998; revised 6 May 1998; accepted 6 May
1998
use of Ca2+ ionophores and thapsigargin (Tg), a
selective inhibitor of SERCA (Jiang et al., 1994; Lam
et al., 1994; Zhu and Loh, 1995; Furuya et al., 1994;
Preston et al., 1997; Bian et al., 1997). It has been
shown that inhibition of SERCA by Tg directly leads
to depletion of the intracellular Ca2+ store and
induction of apoptosis. In addition, a decrease in ER
calcium also precedes apoptosis induced by other
agents such as hydrogen peroxide, okadaic acid or
growth factor withdrawal (Preston et al., 1997;
Distelhorst et al., 1996; Ba€y et al., 1993). The
importance of the ER calcium is further supported
by studies showing that overexpression of the Ca2+
binding protein calbindin, which resides in the lumen
of the ER, or a decrease in Ca2+ e‚ux from the ER
can delay the onset of apoptosis in glucocorticoidtreated lymphoid cell line (Dowd et al., 1992).
Members of the bcl-2 gene family encode proteins
that function either to promote or to inhibit apoptosis
(reviewed by Kroemer, 1997). Anti-apoptotic members
such as Bcl-2 and Bcl-XL prevent cell death in response
to a wide variety of stimuli including thapsigargin
(Kroemer, 1997). Conversely, pro-apoptotic proteins
such as Bax and Bak can accelerate death (Yang and
Korsmeyer, 1996). One common feature of Bcl-2related proteins is that these proteins are localized to
the outer mitochondrial, outer nuclear and ER
membranes as a result of a carboxy-terminal membrane anchor (Yang and Korsmeyer 1996). Three
dimensional structural analysis has suggested that
Bcl-2 related proteins can potentially form a membrane pore, thus allowing the passage of ions or
proteins through the membrane (Minn et al., 1997;
Antonsson et al., 1997; Schendel et al., 1997). Despite
numerous e€orts, the mechanism of action of Bcl-2 is
still not clear. Partly this is due to the pleiotropic
e€ects of Bcl-2 involving several organelles including
mitochondria (Zamzami et al., 1996; Kluck et al., 1997;
Vander Heiden et al., 1997), ER (Lam et al., 1994;
Ba€y et al., 1993) and nucleus (Marin et al., 1996). It is
not known whether these e€ects are independent of
each other or may be linked. While recent evidence has
provided some clues to the action of Bcl-2 at the
mitochondria level, other study has suggested that Bcl2 also protects against apoptosis when speci®cally
targeted to the ER using an ER retention sequence
(Zhu et al., 1996). It is therefore important to
understand the e€ects of Bcl-2 in various subcellular
sites which may all contribute to the anti-apoptosis
process. In the present work, we focus our study on the
e€ect of Bcl-2 on ER organelle. We demonstrate that
overexpression of Bcl-2 in breast epithelial cells
modulates ER Ca2+ store by upregulation of SERCA-2 expression with little e€ect on IP3R-type 3
expression. In addition, we show the in vitro
interaction between Bcl-2 and SERCA by co-immunoprecipitation.
Endoplasmic reticulum calcium pump and Bcl-2
TH Kuo et al
1904
Results
Upregulation of SERCA2 expression in Bcl-2 transfected
cell lines
Our previous study has indicated that alterations in
calcium homeostasis are often associated with changes
in the mRNA and protein levels of the major calcium
transporters that include pumps and channels (Liu et
al., 1996). Based on this consideration, we reasoned
that if Bcl-2 action involves changes in Ca2+ homeostasis, then these changes may be re¯ected in the
expression of calcium pumps and channels. We
therefore tested the e€ect of Bcl-2 overexpression on
SERCA and IP3R expression. Breast epithileal cell lines
(MCF10A) transfected with Bcl-2 cDNA were selected
and individual clones were compared with control cell
line transfected with the vector alone (termed Neo).
Protein lysate were collected and loaded on a 12.5%
polyacrylamide gel for monitoring Bcl-2 expression by
Western blotting (Figure 1a). Densitometric analysis
showed that the relative level of Bcl-2 expressing clones
varied from 1.3-fold for clone 40 to 3.5-fold for clone
30 as compared to Neo control (Figure 1a). The same
samples were also loaded on a 7.5% gel for analysing
SERCA isoform 2 (the major isoform) expression
(Figure 1b). Proper control experiments were carried
out (using actin probe) to con®rm equal loading of the
protein samples (not shown). Comparison of Figure 1a
with Figure 1b indicated a close correlation between
Figure 1 Upregulation of SERCA2 protein and mRNA expression
in Bcl-2 transfected cell lines. Cell lines of MCF10A stably
transfected with Bcl-2 cDNA were analysed. Samples prepared
from Bcl-2 clones 40, 30, 6 and 8 were compared with control (Neo)
transfected with vector only. (a) Protein lysates for Bcl-2 detection
were run on a 12% gel followed by Western blotting. (b) Protein
lysates for SERCA2 detection were run on a 7.5% gel followed by
Western blotting. (c) RNA samples from clone 30 and Neo control
were compared by Northern blotting. Although not shown, control
for sample loading was carried out using actin probe. The Western
and Northern blotting experiments have been repeated two times.
Using di€erent exposure time, it has been determined that the image
values were within the linear range
the Bcl-2 expression level and the SERCA2 protein
levels; higher Bcl-2 expression was accompanied by
higher SERCA expression. The results suggest that Bcl2 overexpression leads to increased SERCA2 protein
levels. Northern analysis (Figure 1c) also showed that
Bcl-2 expressing clone 30 had a twofold increase in the
SERCA2 mRNA level as compared to Neo control.
Thus Bcl-2 expression upregulates SERCA2 expression
at both the mRNA and protein levels. Increased
SERCA2 mRNA levels are at least partly responsible
for the observed increase in SERCA2 protein.
Upregulation of SERCA2 expression leads to increased
SERCA activity
To determine if enhanced SERCA2 expression
produces enzymatically active protein, microsomes
were prepared from the Bcl-2 expressing clones 30
and 40 and compared with the preparation from Neo
control in the calcium uptake assay (Figure 2a). This
assay measures the ATP-mediated, oxalate-dependent
45
Ca uptake into microsomes which is speci®c for
SERCA pump (see Materials and methods). Under the
assay condition, in the presence of sodium azide
(mitochondrial inhibitor) and oxalate (that traps the
Ca2+ in the ER by forming insoluble calcium oxalate),
Figure 2 Increased SERCA activity in Bcl-2 transfected cell
lines. Microsomal membranes were prepared from the Bcl-2
clones 30 and 40, and compared with Neo control. (a) SERCA
activity was determined by the ATP-mediated, oxalate-dependent
Ca2+ uptake assay. (b) The formation of the SERCA phosphoenzyme intermediate (E-P) as indicated by a 105-kDa band after
SDS gel electophoresis and autoradiography
Endoplasmic reticulum calcium pump and Bcl-2
TH Kuo et al
the majority (97%) of the Ca2+ uptake was due to
SERCA activity, with less than 3% of the uptake being
oxalate-independent (data not shown). The results
suggest that non-speci®c Ca2+ uptake due to contaminating mitochondrial membranes was negligible. The
45
Ca uptake was also completely inhibited by 100 nM
thapsigargin, supporting the notion that the activity
assay is speci®c for SERCA. Figure 2a shows that the
Bcl-2 overexpressing clones 30 and 40 have enhanced
45
Ca uptake activity (approximately 1.5 ± 2-fold higher
than Neo) and the increases are proportional to their
SERCA protein levels as determined by the Western
analysis (Figure 1b). Therefore, increased SERCA2
protein led to enhanced SERCA activity. This
conclusion is further supported by the assay of
phospho-enzyme intermediate (E-P) formation (Figure
2b), which directly measures the amount of 32P-labeled
active enzyme present in the calcium transport reaction
(Cheng et al., 1996). The autoradiogram from the E-P
reaction (Figure 2b), detects a radioactive band at 100
kDa representing the active SERCA. The intensity of
this E-P band is proportionally increased in clones 30
and 40 according to their Bcl-2 levels (Figure 2b). Thus
Bcl-2 overexpression led to increased Ca2+ uptake as
well as increased E-P formation, indicative of increased
active SERCA expression.
Increased loading of the ER calcium stores in Bcl-2
transfected cell line
Previous evidence from our laboratory has shown that
the loading state of the agonist-sensitive intracellular
Ca2+ stores correlates with the SERCA expression level
(Kuo et al., 1997). Based on this information, it was
expected that higher expression of SERCA in Bcl-2
overexpressing cell lines would result in higher Ca2+
load in ER stores. As a ®rst approximation to estimate
the loading state of the ER stores, Bcl-2 overexpressing
cells and Neo control cells were treated with
thapsigargin (Tg), a speci®c inhibitor of the SERCA.
The addition of Tg (1 mM) resulted in the release of ER
calcium into the cytosol, where it was measured by
Indo-1 as an increase in intracellular Ca2+ (Liu et al.,
1996). To block Ca2+ in¯ux due to the release of ER
calcium, also called capacitative Ca2+ entry (Putney,
1986), these studies were done in the absence of
extracellular Ca2+ (see Materials and methods). Under
this condition, the increase of cytosolic Ca2+ after the
addition of Tg (together with 0.1 mM EGTA) is largely
due to the release from the ER store. After reaching a
peak within 20 ± 60 s, the [Ca2+]c starts to decline
slowly, returning to baseline after 5 min (Figure 3a).
This slow decline of cytosolic Ca2+ is due to the action
of the plasma membrane Ca2+ ATPase pump (PMCA)
which removes Ca2+ out of the cell. Since PMCA is a
low capacity pump (Carafoli, 1987), it takes longer
time to reduce [Ca2+]c to basal level. Figure 3a shows
that while the resting levels of [Ca2+]c in Bcl-2 clone 30
cell line and Neo control were similar (ranging from
80 ± 90 nM), the Tg-induced release of Ca2+ was
signi®cantly higher in clone 30 as compared to Neo
control. ER calcium release was estimated by
measuring the di€erence between basal and peak
Ca2+ (Ba€y et al., 1993). Results from ®ve experiments
indicated that the Tg-releasable ER Ca2+ for clone 30
was 315+53 nM versus 140+15 nM for Neo control
(P50.004). The Tg-induced depletion of the ER store
was complete for both clone 30 and Neo control as
evidenced by the absence of further Ca2+ release by the
second addition of Tg (data not shown). Thus the Tgreleasable ER Ca2+ was approximately two times
higher in Bcl-2-transfected cell line (clone 30) as
compared to Neo control.
Similar experiments were carried out using ionomycin
(5 mM) to release the ER Ca2+. In contrast to
thapsigargin, ionomycin is a non-speci®c calcium
ionophore which can release ER Ca2+ as well as
mitochondria Ca2+ (Liu and Hermann, 1978). However, addition of ionomycin after the depletion of ER
Ca2+ by Tg did not cause signi®cant further release of
Ca2+, indicating that mitochondrial accumulation of
Ca2+ in these cells was minimal (data not shown). For
this reason, the ionomycin-releasable Ca2+ was also
used as an estimate of the ER Ca2+ release. Figure 3b
shows a typical trace for the ionomycin-mobilizable
Ca2+ pool from the Bcl-2 clone 30 as compared to Neo
control. Again the basal Ca2+ levels were similar for
both cell types (80 ± 90 nM). However, the peak Ca2+
was much higher for Bcl-2 clone 30 than control.
Results from six experiments indicated that the
ionomycin-releasable ER Ca2+ for clone 30 and for
Neo control were 703+147 nM and 230+101 nM
respectively (P50.003). Further studies were carried
out using ATP (0.1 mM) as agonist to measure the
agonist-sensitive Ca2+ pool (Figure 3c). ATP binds to
purinergic receptor which activates the phospholipase C
and generates IP3 which in turn activates the IP3R and
causes the release of ER Ca2+. Results from ®ve
experiments indicated that ATP-releasable ER Ca2+
for clone 30 and for Neo control were 680+100 nM
and 320+27 nM respectively (P50.003). The results
again suggest that the ER loading state of the Bcl-2
clone 30 was approximately two times higher than Neo
control.
It is important to point out that the Tg-induced
Ca2+ signal had a lower peak Ca2+ and slower kinetics
than ionomycin- or ATP-induced Ca2+ signals
(compare Figure 3a with b and c). The lower peak
Ca2+ observed with Tg treatment is explained on the
basis of slower release of Ca2+ by the non-speci®c
channels of the ER, reaching peak only after the
activation of plasma membrane Ca2+ pump (PMCA)
which by removing Ca2+ out of the cell can reduce the
maximal Ca2+ concentration detected at the peak of
the signal. In the case of ATP-mobilized Ca2+ signaling
(Figure 3c), peak Ca2+ was higher due to the faster
release of Ca2+ by IP3-activated channel which precedes
the activation of both PMCA and SERCA pumps. The
combined action of PMCA and SERCA then results in
a more rapid decay of the Ca2+ signal in both ATP-and
ionomycin-treated cells (Figure 3b and c) as compared
to Tg-treated cells (Figure 3a). Despite their di€erent
mechanism of action, all three agents (Tg, ionomycin,
and ATP) caused a larger ER Ca2+ release (twofold or
more) in Bcl-2 clone 30 as compared to Neo control,
suggesting a higher loading state of the ER store in
Bcl-2 expressing cells.
Physical interaction between Bcl-2 and SERCA pump
Because SERCA and Bcl-2 are both localized to the
ER membrane, the possibility that SERCA and Bcl-2
1905
Endoplasmic reticulum calcium pump and Bcl-2
TH Kuo et al
1906
may physically associate and participate in the
regulation of Ca2+ homeostasis was explored. To test
for interaction between SERCA and Bcl-2, cells from
Bcl-2 overexpressing clone 30 and also from human
lymphoma cell line DHL-4 were used for this study.
Because of the fact that Bcl-2 localizes to several
organelles and interacts with multiple proteins in the
cell, the immunoprecipitation experiment was carried
out by using SERCA antibody instead of Bcl-2
antibody. Cells were metabolically labeled with
S-methionine and then processed for immunoprecipitation experiment using the anti-SERCA2 antibody.
Figure 4a shows the autoradiography of the 35Slabeled immunoprecipitate after electrophoresis on 5 ±
20% gradient gel followed by blotting. Three major
radioactive bands at 105 kDa, 52 kDa and 28 kDa
were detected. While the 105 kDa and 28 kDa bands
were identi®ed by immuno-detection as SERCA2 and
Figure 3 Increased loading of the ER calcium stores in Bcl-2
transfected cell line. Ca2+ response in cells overexpressing Bcl-2
(clone 30) was compared with the vector-transfected control
(Neo). Ca2+ response induced by various agents in single cells
were recorded using the ACAS 570 laser cytometer as described in
Materials and methods. Cells incubated in Ca2+ free medium for
40 ± 60 s, were treated with 1 mM thapsigargin (a), 5 mM ionomycin
(b), or 0.1 mM ATP (c) and time-resolved measurements were
performed to follow the changes in [Ca2+]c during the course of
treatment (see Materials and methods). Note that Bcl-2 clone 30
(lighter trace) has increased loading in the ER store compared
with Neo control cells (darker trace). Experiments typify results
from ®ve or more independent experiments
Figure 4 Co-precipitation of SERCA with Bcl-2. 35S-labeled cells
(clone 30) were treated with anti-SERCA (a) or anti-Bcl-2
(c, lane 2). After binding to protein G-Sepharose, the immune
precipitate was washed ®ve times and samples loaded on a 5 ± 12%
gradient gel. The gel was blotted to nitrocellulose membrane and
processed for autoradiography (a, c) and also for Western blot
detection of SERCA and Bcl-2 separately (b). To show the
speci®city of the immunoprecipitation, the whole cell lysate before
precipitation with anti-Bcl-2 was included in c, lane 1. Numerous
protein bands were present in the lysate (lane 1) while the Bcl-2
precipitate (lane 2) contained only a limited number of bands
35
Endoplasmic reticulum calcium pump and Bcl-2
TH Kuo et al
Bcl-2 respectively (Figure 4b), the identity of the
52 kDa band was unknown. Control experiment using
normal rabbit serum for immunoprecipitation indicated no radioactive bands in the precipitate
(data not shown). Because of the short duration of
metabolic labeling (2 h, see Materials and methods),
the radioactive bands were faint and required 10 days
to develop. Using lymphoma cell line DHL-4 which
expresses high levels of endogenous Bcl-2, it was
found that anti-SERCA2 antibody also coprecipitated SERCA, Bcl-2 and the 52-kDa protein
(data not shown). The co-precipitation of these three
proteins was further vari®ed by using anti-Bcl-2
antibody with DHL-4 cell lysate. Figure 4c shows
numerous cellular protein bands in the DHL-4
detergent solubilized cell lysate before precipitation
(lane 1), and a distinct pattern of the immune
complex after precipitation with anti-Bcl-2 (lane 2).
Because of the required 10 day exposure (for lane 2),
protein bands in Lane 1 became unvoidably
dark. It is interesting that the Bcl-2 precipitate
(Figure 4c lane 2) appeared to contain additional
bands than the SERCA precipitate (Figure 4a) and
this is consistent with the fact that Bcl-2 is known to
interact with proteins of mitochondria as well as ER.
Despite this di€erence, it is clear that both precipitates
contain SERCA, Bcl-2 and 52-kDa as common
proteins. To avoid redundancy with Figure 4b, the
Western analysis for the anti-Bcl-2 immune complex
was omitted here. Taken together, the results suggest
that there is possible interaction between Bcl-2 and
SERCA in the breast epithelial cell line as well as
DHL-4 cell line, and that the SERCA immune
complex appears to be a multi-protein complex.
The e€ect of Bcl-2 on IP3R protein expression and
calcium release
Since both SERCA pump and IP3R channel participate
in the regulation of ER Ca2+ homeostasis, it was
important to determine if Bcl-2 expression might also
in¯uence the steady state level of the IP3R protein and its
activity. Whole cell lysates were prepared from the Bcl-2
expressing clone 30 and compared with that from the
Neo control by Western blot analysis. The results
(Figure 5a) indicated no signi®cant change in the IP3Rtype3 protein levels in clone 30 as compared to Neo
control. However, the possibility that Bcl-2 may a€ect
the other two isoforms can not be excluded. To ®nd out if
there is functional alteration of the IP3R by Bcl-2
transfection, we have carried out studies to determine
the IP3-mediated Ca2+ release in permeabilized cells. For
this purpose, both 45Ca2+ uptake and release in saponinpermeabilized cells were followed (Figure 5b). The
uptake of 45Ca2+ in the ER of saponin-permeabilized
cells after 10 min incubation was 1.5-fold higher for clone
30 as compared to Neo control (Figure 5b left panel,
5000 versus 3000 c.p.m.). This result con®rmed Figure 2
data obtained by microsomal assay of the 45Ca2+ uptake.
The IP3-induced release of 45Ca2+ was initiated after
10 min when the uptake reached plateau (Figure 5b right
panel). Addition of 5 mM IP3 (indicated by arrow) caused
an immediate release of Ca2+ such that greater than 90%
of the accumulated radioactivity was released in 20 s.
The speci®city of the IP3-induced release was con®rmed
when control experiment carried out in the absence of IP3
1907
Figure 5 The e€ect of Bcl-2 on IP3R expression and IP3-induced
Ca2+ release. (a) Lysate prepared from Bcl-2 clone 30 was
compared with control (Neo); samples were run on a 7.5% gel,
followed by Western blotting for IP3R-3 expression. (b) Assay of
45
Ca2+ uptake and 45Ca2+ release in saponin-permeabilized cells
(see Materials and methods). Neo control (®lled square) and
Bcl- 2 clone 30 (circle) were permeabilized and incubated for
45
Ca2+ uptake assay at various time intervals (0 ± 10 min). After
10 min of uptake, the medium was switched to e‚ux medium and
IP3- mediated 45Ca2+ release was assayed from 5 ± 60 s. The
results indicated no signi®cant change in IP3R protein levels or
calcium release by Bcl-2
indicated less than 5% of release (data not shown).
Calculation of the initial rate of IP3-induced Ca2+ release
from three experiments (Figure 5b) indicated no
signi®cant di€erence between Bcl-2 clone 30 cells versus
Neo control. Thus Bcl-2 does not a€ect IP3-induced
calcium release. However, other subtler e€ects of Bcl-2
on the IP3R, such as regulation of phosphorylation can
not be ruled out.
Discussion
The present results show for the ®rst time that
overexpression of Bcl-2 a€ects the ER Ca2+ store by
upregulating the SERCA2 expression with little e€ect
on the IP3R-3 expression. The 2 ± 3-fold increase in
SERCA2 pump expression is largely responsible for the
2 ± 3-fold increase in microsomal Ca2+ uptake and ER
Ca2+ loading in these cells (Figures 2, 3 and 5). Thus
we have provided clear evidence for a role of Bcl-2 in
the maintenance of ER Ca2+ pump expression as well
as Ca2+ uptake. As this work was in progress, a report
appeared (He et al., 1997) showing similar maintenance
of Ca2+ homeostasis in the ER by Bcl-2. By studying
Endoplasmic reticulum calcium pump and Bcl-2
TH Kuo et al
1908
WEH17.2 lymphoma cells and the corresponding Bcl-2
stable-transfectant, W.Hb12 cells, these authors found
large increases of SERCA-mediated calcium uptake in
Bcl-2 overexpressing cells (He et al., 1997). Thus both
studies are in agreement regarding the enhancement of
SERCA-mediated Ca2+ uptake by Bcl-2. However, the
two studies di€er in the suggested mechanism for this
Bcl-2 e€ect. While our study suggests that the twofold
increase in SERCA pump density is the major reason
for the twofold increased Ca2+ uptake, the other study
(He et al., 1997) suggests a direct mediation of Ca2+
uptake by Bcl-2. In addition to the SERCA-mediated
uptake, a direct mediation of Ca2+ movement across
ER membrane by Bcl-2 seems to be an intriguing
possibility, especially in view of X-ray and NMR
studies showing that Bcl-2 family proteins can
potentially form ion channels (Minn et al., 1997;
Antonsson et al., 1997; Schendel et al., 1997). Indeed,
this Tg-resistant Ca2+ uptake was detected in W.Hb12
cells overexpressing Bcl-2 (He et al., 1997). However,
this SERCA-independent Ca2+ uptake (presumably
mediated by Bcl-2) in W.Hb12 cells was very low
which amounts to only 1% of the SERCA-dependent
uptake (see Figure 1 of He et al., 1997). In MCF10A
clone 30 cells overexpressing Bcl-2, we failed to detect
such Tg-resistant Ca2+ uptake. It is possible that our
assay may not be sensitive enough to detect such small
di€erences. Although our study does not rule out a
novel function of Bcl-2 as Ca2+ transporter, it is clear
that this small uptake mediated by Bcl-2 can not
quantitatively account for the 2 ± 3-fold increase of
Ca2+ uptake detected in MCF10A cells as well as in
W.Hb12 cells. We would rather propose that Bcl-2
mediated upregulation of SERCA gene expression is
responsible for the large increase of Ca2+ uptake in
Bcl-2 overexpressing cells.
The Bcl-2 e€ect on ER Ca2+ load is likely related to
its anti-apoptotic function. Previously, we and others
have demonstrated in various cell types that SERCA
expression is required for cell cycle progression whereas
low SERCA expression leads to cell cycle arrest (Short
et al., 1993; Cheng et al., 1996). Furthermore, we have
evidence that treatment of MCF10A cell culture with
calcium ionophore A23187 (1 mM) caused a depletion
of ER Ca2+ store and subsequent cell death. After 48 h
in the presence of A23187, approximately 70% of
MCF10A cells had died and only 30% survived. In
contrast, Bcl-2 overexpressing clone 30 cells were
resistant to this treatment, with only 22% death and
78% survival. Thus Bcl-2 can inhibit cell death caused
by disruption of Ca2+ homeostasis with ionophore
treatment. Other laboratories also showed that
depletion of ER Ca2+ leads to apoptosis and Bcl-2
can inhibit both store depletion and associated cell
death (Lam et al., 1994; Bian et al., 1997; Distelhorst et
al., 1996). Thus the ®lling state of the ER store which
is mainly determined by SERCA expression exerts a
profound control over cell growth and cell death. It is
also worth mentioning that the high Ca2+ loading state
is not observed in cells that overexpress the Bax, a proapoptotic protein (our unpublished results). In contrast
to the Bcl-2 overexpressing cells, the Bax-overexpressing cells are associated with either no change or a
decrease of the Ca2+ ®lling state when compared to the
Neo control. Thus Bcl-2 could preserve the normal ER
function by maintaining ER Ca2+ homeostasis.
The mechanism of SERCA-upregulation by Bcl-2 is
not clear. Previously, during the study of intracellular
Ca2+ homeostasis (Liu et al., 1996; Kuo et al., 1997), we
have shown that while Ca2+ pumps and channels
cooperate in their action to maintain [Ca2+]c, their own
gene expression is subjected to regulation by the Ca2+
signal. Speci®cally, we have shown that the ®lling state of
the ER store (as determined by the size of the agonistinduced Ca2+ signal) is an important factor for
regulating SERCA gene expression (Kuo et al., 1997).
Low ®lling state is associated with low SERCA
expression and high ®lling state with high SERCA
expression (Kuo et al., 1997). Furthermore, Ca2+ signal
stimulates SERCA gene transcription as demonstrated
by nuclear run-on assay (Kuo et al., 1997). This is
consistent with the presence of a putative Ca2+ response
element (Li et al., 1993) in the SERCA promoter
sequence (Lytton et al., 1989). Taken together, it is
plausible that a Bcl-2-mediated Ca2+ uptake (by way of
the slow ion channel) could accelerate the transcription
of SERCA gene, resulting in increased SERCA
expression. During apoptosis-condition, when SERCA
function is depressed and the depletion of the ER pool
occurs, this ability of Bcl-2 to mediate Ca2+ uptake
becomes critical to maintain a threshold level of ER Ca2+
that is essential for cell survival. While we favor this
explanation, the possible involvement of other Bcl-2
a€ected signal pathways in SERCA expression can not
be ruled out.
In addition to the Bcl-2 e€ect on SERCA expression,
we have also shown a possible interaction between Bcl-2
and SERCA by immunoprecipitation. This physical
association may be a direct one or may be bridged by
the 52 kDa protein (Figure 4). In either case, the results
suggest that Bcl-2 might modulate SERCA function
through protein-protein interaction. In summary, we
have demonstrated that Bcl-2 modulates ER store by
regulating SERCA gene expression and perhaps by
physical association with SERCA protein. Further study
on the functional interaction between Bcl-2 and SERCA
is now being investigated.
Materials and methods
Cell lines and culture conditions
Breast epithelial cell line (MCF10A) was stably transfected
with pcDNAI expression vector with or without Bcl-2
cDNA insert (provided by Dr S Korsmeyer) as described
previously (Upadhyay et al., 1995). The resulting Bcl-2
expressing clones were selected and compared to cell line
that was transfected with the vector without the insert.
These cell lines are termed Bcl-2 clones and Neo-control
respectively. Human DHL-4 lymphoma cell line expressing
high endogenous level of Bcl-2 was provided by Dr L
Boxer (Wilson et al., 1996). Bcl-2 or Neo control cell lines
were routinely cultured in DMEM medium supplemented
with appropriate serum and factors at 378C in a 5% CO2
atmosphere as described (Upadhyay et al., 1995). DHL-4
cell lines were cultured in RPMI medium as described
previously (Wilson et al., 1996).
RNA preparation and Northern analysis
For detecting SERCA mRNA, 70 ± 80% con¯uent cells were
used for RNA preparation and Northern analysis (Liu et al.,
1996). The blots were hybridized with speci®c cDNA probes.
Endoplasmic reticulum calcium pump and Bcl-2
TH Kuo et al
The SERCA cDNA probe was a 330 bp fragment (nt 892 ±
1222) of the SERCA2 gene (Lytton et al., 1989).
Western analysis
For detecting SERCA or IP3R protein, 70 ± 80% con¯uent
cells were lysed and protein samples were separated on
7.5% polyacrylamide gel for Western blotting (Liu et al.,
1996). The SERCA2 speci®c antibody was obtained from
Anity BioReagent. The IP3R-type 3 speci®c antibody was
obtained from Transduction Laboratory.
Intracellular Ca2+ measurements
Intracellular calcium was measured using a Meridian
Ultima laser confocal microscope and the calcium-sensitive
¯uorescent dye Indo-1 as described before (Liu et al., 1996).
Bcl-2 clones were grown overnight on a glass coverslip, then
loaded with Indo-1 AM (2 mM) in loading bu€er containing
(in mM) 5.4, KCl; 137, NaCl; 0.44, KH2PO4; 4.2, NaHCO3;
0.34, Na2HPO4; 11.1, D-glucose; 5, HEPES, pH 7.3, 2,
CaCl2; 0.1% bovine serum albumin. Indo-1 ¯uorescence was
measured at an excitation wavelength of 360 nm and
emission wavelengths of 405 and 485 nm. To estimate the
loading state of the ER store, cells were rinsed (after dye
loading) in loading bu€er that does not contain Ca2+, and
then treated with thapsigargin (1 mM) plus 0.1 mM EGTA.
Time-resolved measurements were performed to follow the
changes in cytosolic Ca2+ during the course of treatment
(0 ± 10 min). For measurement of capacitative Ca2+ entry,
the thapsigargin-EGTA bu€er was replaced with bu€er
containing 2 mM Ca2+. For each experiment, there were
between four and nine cells per ®eld.
Microsomal membrane preparation
Cells were lysed in an ice-cold bu€er containing (in mM) 10
HEPES, pH 7, 10 KCl, 0.05 EGTA, 0.05 dithiothreitol and a
mixture of protease inhibitors (in mg/ml: 0.1 soybean trypsin
inhibitor, 0.05 aprotinin and 0.01 leupeptin). The lysates
were then homogenized and centrifuged for 10 min at 48C
and 3500 g. The supernatants were centrifuged for 60 min at
100 000 g, and the resulting pellets resuspended in 20 mM
HEPES, pH 7, 160 mM KCl and 0.1 mM dithiothreitol. The
microsomes were used immediately for SERCA assay or
phospho-enzyme (E-P) formation.
Ca2+ transport assay and measurement of phosphoenzyme
intermediate
The assay of SERCA activity was based on the measurement
of ATP-mediated, oxalate-dependent calcium uptake into
crude microsomes according to Cheng et al. (1996) and Kuo
et al. (1992). The reaction mixture contained 100 mM KCl, 50
mM K-HEPES (pH 6.8), 6 mM MgCl2, 5 mM NaN3, 7 mM
oxalate, 50 mM 45Ca2+, 15 mg of microsomal membranes and
the reaction was initiated by addition of 5 mM ATP.
Radioactive Ca2+ accumulation at each time point was
determined by ®ltration (Kuo et al., 1992). The formation of
phospho-enzyme intermediate (E-P) during the catalytical
cycle was carried out at 08C, using 50 mg of crude membrane
and 0.08 mM of [32P]ATP in the reaction as previously
described (Cheng et al., 1996).
Metabolic labeling with 35S-methionine and
immunoprecipitation of Bcl-2
MCF10A transfected with Bcl-2 (clone 30) or human
lymphoma cell line DHL-4 that expresses high level of
endogenous Bcl-2 was used for this study. Cells (3 6 106)
grown in 100 mm dish were pre-labeled for immunopre-
cipitation experiment (Boulay et al., 1997). Brie¯y, after a
starvation period of 30 min in methionine/cysteine-free
DMEM medium (ICN), the cells were incubated for 3 h at
378C with 5 ml of the same medium containing 20 mCi of
[35S]-methionine (Amersham). The cells were washed twice
and scraped from the dish in the presence of PBS and
harvested by centrifugation. The cell pellet was homogenized in 500 ml of RIPA bu€er (20 mM Tris/HCl, pH
8.0, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.5%
deoxycholic acid, 0.1% SDS, 0.1 mM PMSF, 1 mg/ml
soybean trypsin inhibitor and 0.5 mg/ml leupeptin) by
drawing the cells through syringe needles (25 gauge). The
cell extracts were clari®ed by an incubation of 30 min with
40 ml of a 50% slurry of pre-washed protein G-sepharose
(Gibco ± BRL) in RIPA bu€er. Prewashed Protein GSepharose was prepared by treatment for 1 h with 25 mg/
ml bovine serum albumin in RIPA bu€er followed by two
washes with RIPA bu€er alone. After a centrifugation of
5 min, 5 ml of Bcl-2 antibody (KLH Denmark) was added
to the supernatant and incubated for 18 h at 4 8C with
shaking. The antigen-antibody complexes were separated
from the mixture by incubating with pre-washed Protein
G-Sepharose for 2 h at 4 8C. The beads were centrifuged
and washed four times with RIPA bu€er at room
temperature and recovered each time by centrifugation.
The immune complexes were then eluted in SDS sample
bu€er and loaded on a 5-20% gradient gel for
electrophoresis and blotting. The membrane blot was
either exposed to X-ray ®lm for autoradiography or
processed for immuno-detection as described previously
(Liu et al., 1996).
Measurement of Ca2+ release in permeabilized cells
The assay of IP3 induced 45Ca2+ release was done
according to Zhao and Muallem (1990). Bcl-2 clone 30
and Neo control cells (66104 per well) were plated in
24-well culture plate for 24 h before the assay. Cells were
washed in KMH medium containing (in mM) 140 KCl,
3 MgCl2, 10 HEPES, and then permeabilized with
0.0075% saponin as described (Zhao and Muallem,
1990). Ca2+ uptake was initiated by the addition of
uptake medium containing 5 mM Ca, 5 mg/ml oligomycin,
10 mM antimycin A, 2 mM ATP and 45Ca. During the
uptake period (0 ± 10 min), samples were withdrawn for
measurement of 45Ca uptake into the microsome. After
10 min incubation, the uptake medium was replaced with
e‚ux medium which had the same composition as uptake
medium but without ATP and 45Ca2+ and contained
5 mM IP3. The IP3-induced e‚ux was stopped at various
times (from 5 ± 60 s) and cells were washed and processed
for measurement of 45Ca2+ content as described previously
(Zhao and Muallem, 1990).
Densitometry analysis and statistics
Autoradiograms and protein blots were quanti®ed by using
Ambis image analysis system (San Diego). Each experiment
presented is representative of at least two experiments
performed independently. Where applicable, data
(mean+s.d.) were analysed by Student's t-test and
signi®cance de®ned as a P-value of 5 0.05.
Acknowledgements
The authors acknowledge the support of National
Institutes of Health (HL-39481 to THK, CA-64139 to HRCK) and the American Heart Association/Michigan (to
THK). We also thank Dr Shmuel Muallem for discussion.
1909
Endoplasmic reticulum calcium pump and Bcl-2
TH Kuo et al
1910
References
Antonsson B, Conti F, Ciavatta A, Montessuit S, Lewis S,
Martinow I, Bemasconi L, Bemard A, Memod JJ, Mazzei
G, Maundrell K, Gambale F, Sadoul R and Martinou J-C.
(1997). Science, 277, 370 ± 372.
Ba€y G, Miyashita T, Williamson JR and Reed JC. (1993). J.
Biol. Chem., 268, 6511 ± 6519.
Berridge, MJ. (1993). Nature, 361, 315 ± 325.
Bian X, Hughes FM, Huang Y, Cidlowski JA and Putney
JW. (1997). Am. J. Physiol., 272, C1241 ± C1249.
Boulay G, Zhu X, Peyton M, Jiang M, Hurst R, Stefani E
and Birbaumer L. (1997). J. Biol. Chem., 272, 29672 ±
29680.
Brostrom CO and Brostrom MA. (1990). Annu. Rev.
Physiol., 52, 577 ± 590.
Calpham DE. (1995). Cell, 80, 259 ± 268.
Carafoli E. (1987). Annu. Rev. Biochem., 56, 395 ± 433.
Cheng G, Liu BF, Yu Y, Diglio C and Kuo TH. (1996). Arch.
Biochem. Biophys., 329, 65 ± 72.
Distelhorst CW, Lam M and McCormick TS. (1996).
Oncogene, 12, 2051 ± 2055.
Dowd, DR, MacDonald PN, Komm BS, Haussler MR and
Miesfeld R. (1992). Mol. Endocrinol., 6, 1843 ± 1848.
Furuya Y, Lundmo P, Short AD, Gill DL and Isaacs JT.
(1994). Cancer Res., 54, 6167 ± 6175.
He H, Lam M, McCormick TS and Distelhorst CW. (1997).
J. Cell Biol., 138, 1219 ± 1228.
Jiang S, Chow SC, Nicotera P and Orrenius S. (1994). Exp.
Cell Res., 212, 84 ± 92.
Kluck RM, Bossy-Wetzel E, Green DR and Newmeyer DD.
(1997). Science, 275, 1132 ± 1136.
Kroemer G. (1997). Nature Med., 3, 614 ± 620.
Kuo TH, Tsang W, Wang KKW and Carlock L. (1992).
Biochim. Biophys. Acta, 1138, 343 ± 349.
Kuo TH, Liu BF, Yu Y, Wuytack F, Raeymaekers L and
Tsang W. (1997). Cell Calcium, 21, 399 ± 408.
Lam M, Dubyak G, Chen L, Nunez G, Miesfeld RL and
Distelhorst CW. (1994). Proc. Natl. Acad. Sci. USA, 91,
6569 ± 6573.
Li WW, Alexandre S, Cao X and Lee AS. (1993). J. Biol.
Chem., 268, 12003 ± 12009.
Liu BF, Xu X, Fridman R, Muallem S and Kuo TH. (1996).
J. Biol. Chem., 271, 1 ± 9.
Liu C and Hermann TE. (1978). J. Biol. Chem., 253, 5892 ±
5894.
Lodish HF, Kong N and Wikstrom L. (1992). J. Biol. Chem.,
267, 12753 ± 12760.
Lytton J, Zarian-Herzberg A, Periasamy M and MacLennan
DH. (1989). J. Biol. Chem., 264, 7049 ± 7056.
Marin M, Fernandez A, Bick RJ, Brisbay S, Buja LM,
Snuggs M, McConkey DJ, von Eschenbach AC, Keating
MJ and McDonnell TJ. (1996). Oncogene, 12, 2259 ± 2266.
Minn AJ, Valez P, Schendel SL, Liang H, Muchmore SW,
Fesk SW, Fill M and Thompson CB. (1997). Nature, 385,
353 ± 357.
Preston GA, Barrett JC, Biermann JA and Murphy E.
(1997). Cancer Res., 57, 537 ± 542.
Putney JW. (1986). Cell Calcium, 7, 1 ± 12.
Sambrook JF. (1990). Cell, 61, 197 ± 199.
Schendel SL, Xie Z, Montal MO, Matsuyama S, Montal M
and Reed JC. (1997). Proc. Natl. Acad. Sci. USA, 94,
5113 ± 5118.
Short AD, Bian J, Ghosh TK, Waldron RT, Rybak SL and
Gill DL. (1993). Proc. Natl. Acad. Sci. USA, 90, 4986 ±
4990.
Upadhyay S, Li G, Liu H, Chen YQ, Sarkar FH and Kim
HR-C. (1995). Cancer Res., 55, 4520 ± 4524.
Vander Heiden MG, Chandel NS, Williamson EK,
Schumacker PT and Thompson CB. (1997). Cell, 91,
627 ± 637.
Waldron RT, Short AD, Meadows JJ, Ghosh TK and Gill
DL. (1994). J. Biol. Chem., 269, 11927 ± 11933.
Wilson BE, Mochon E and Boxer LM. (1996). Mol. Cell.
Biol., 16, 5546 ± 5556.
Yang E and Korsmeyer SJ. (1996). Blood, 88, 386 ± 401.
Zamzami N, Susin SA, Marchetti P, Hirsch T, GomezMonterrey I, Castedo M and Kroemer G. (1996). J. Exp.
Med., 183, 1533 ± 1544.
Zhao H and Muallem S. (1990). J. Biol. Chem., 265, 21419 ±
21422.
Zhu W, Cowie A, Wasfy GW, Penn LZ, Leber B and
Andrews DW. (1996). EMBO J., 15, 4130 ± 4141.
Zhu WH and Loh TT. (1995). Life Sci., 57, 2091 ± 2099.