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Bcl-2 Rescues Ceramide- and Etoposide-induced
MitochondrialApoptosis through Blockage of Caspase-2
Activation*S
Received for publication, October 29, 2004, and in revised form,
February 17, 2005Published, JBC Papers in Press, April 6, 2005, DOI
10.1074/jbc.M412292200
Chiou-Feng Lin, Chia-Ling Chen, Wen-Tsan Chang, Ming-Shiou
Jan**, Li-Jin Hsu,Ren-Huang Wu, Yi-Ting Fang, Ming-Jer Tang,
Wen-Chang Chang, and Yee-Shin Lin
From the Departments of Microbiology and Immunology,
Biochemistry, Physiology, and Pharmacology and theInstitute of
Basic Medical Sciences, National Cheng Kung University Medical
College, Tainan 701, Taiwan and the**Department of Microbiology and
Immunology, Chung-Shan Medical University, Taichung 402, Taiwan
Recent studies indicate that caspase-2 is involved inthe early
stage of apoptosis before mitochondrial dam-age. Although the
activation of caspase-2 has beenshown to occur in a large protein
complex, the mecha-nisms of caspase-2 activation remain unclear.
Here wereport a regulatory role of Bcl-2 on caspase-2 upstreamof
mitochondria. Stress stimuli, including ceramide andetoposide,
caused caspase-2 activation, mitochondrialdamage followed by
downstream caspase-9 and -3 acti-vation, and cell apoptosis in
human lung epithelial cellline A549. When A549 cells were
pretreated with thecaspase-2 inhibitor
benzyloxycarbonyl-Val-Asp(-OMe)-Val-Ala-Asp(-OMe)-fluoromethyl
ketone or transfectedwith caspase-2 short interfering RNA, both
ceramide-and etoposide-induced mitochondrial damage and apo-ptosis
were blocked. Overexpression of Bcl-2 preventedceramide- and
etoposide-induced caspase-2 activationand mitochondrial apoptosis.
Furthermore, caspase-2was activated when A549 cells were introduced
withBcl-2 short interfering RNA or were treated with
Bcl-2inhibitor, which provided direct evidence of a
negativeregulatory effect of Bcl-2 on caspase-2. Cell survival
wasobserved when caspase-2 was inhibited in Bcl-2-silenc-ing cells.
Blockage of the mitochondrial permeabilitytransition pore and
caspase-9 demonstrated that Bcl-2-modulated caspase-2 activity
occurred upstream of mi-tochondria. Further studies showed that
Bcl-2 was de-phosphorylated at serine 70 after ceramide
andetoposide treatment. A protein phosphatase inhibitor,okadaic
acid, rescued Bcl-2 dephosphorylation andblocked caspase-2
activation, mitochondrial damage,and cell death. Taken together,
ceramide and etoposideinduced mitochondria-mediated apoptosis by
initiatingcaspase-2 activation, which was, at least in part,
regu-lated by Bcl-2.
During the process of apoptosis, there is, in general, a
reduc-tion of mitochondrial transmembrane potential (m) followedby
the release of cytochrome c, which binds to Apaf-1 and
promotes caspase-9 and -3 activation (13). Bcl-2 family
pro-teins serve as critical regulators of mitochondrial
apoptosis,functioning as either inhibitors or promoters of cell
death (4).Bcl-2 inhibits apoptosis by blocking cytochrome c release
frommitochondria (5) through prevention of channel formation,which
is mediated by proapoptotic Bax and Bid (68). A recentstudy (9)
indicated that, in healthy cells, Bcl-2 adopts a
typicaltail-anchored topology. Induction of apoptosis by ceramide
oretoposide triggered a change of Bcl-2 to the
multispanningtransmembrane topology. In addition to membrane
topology,Bcl-2 phosphorylation is required for its full
anti-apoptoticfunction (10, 11).Caspase-2 acts upstream of
mitochondria to promote cyto-
chrome c release and apoptosis (1218), although caspase-2may
also act downstream of mitochondria (19, 20). One study inthe
mechanisms of caspase-2 activation showed that caspase-2complex
formation occurs independently of an Apaf-1/apopto-some pathway and
that the recruitment of caspase-2 into thiscomplex is sufficient to
mediate its activation upstream ofmitochondria (21). A recent
report (22) demonstrated that ac-tivation of caspase-2 occurs in
the complex that contains thep53-induced death-domain-containing
protein and the adapterprotein RAIDD (RIP (ribosome-inactivating
protein)-associatedICH-1/CED-homologous protein with death domain).
Increasedp53-induced death-domain-containing protein may result
incaspase-2 activation to regulate apoptosis induced by
genotoxicstress. Interestingly, Bcl-2 suppresses p53-dependent
apopto-sis that requires Bax and caspase-2 as essential
apoptoticmediators (23).We previously demonstrated sequential
caspase-2 and -8
activation upstream of mitochondria during ceramide- and
eto-poside-induced apoptosis (24). In the present study, the
rela-tionship between Bcl-2 and caspase-2 in mitochondrial
apopto-sis induced by ceramide and etoposide was investigated.
Bcl-2overexpression rescued ceramide- and etoposide-induced
apo-ptosis (2532). Furthermore, ceramide caused Bcl-2 dysfunc-tion
through its dephosphorylation at serine 70 mediated byprotein
phosphatase 2A (33). Chemotherapeutic etoposidecaused Bcl-2
cleavage, which led to cell apoptosis (34). In ad-dition, protein
phosphatase facilitated cells undergoing etopo-side-induced
apoptosis (35, 36). Using Bcl-2 short interferingRNA or Bcl-2
inhibitor ethyl
2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate
(HA14-1),1 we
* This work was supported by Grant 91-B-FA09-1-4 from the
Minis-try of Education (MOE) Program for Promoting Academic
Excellence ofUniversity (Taiwan). The costs of publication of this
article were de-frayed in part by the payment of page charges. This
article musttherefore be hereby marked advertisement in accordance
with 18U.S.C. Section 1734 solely to indicate this fact.S The
on-line version of this article (available at
http://www.jbc.org)
contains supplemental Figs. S1S3. A postdoctoral fellow
supported by the National Health Research
Institutes, Taiwan, ROC (PD9403). To whom correspondence should
be addressed. Tel.: 886-6-235-3535
(ext. 5646); Fax: 886-6-208-2705; E-mail:
[email protected].
1 The abbreviations used are: HA14-1, ethyl
2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate;
MDCK,Madin-Darby canine kidney; z, benzyloxycarbonyl; fmk,
fluoromethylketone; PI, propidium iodide; GFP, green fluorescent
protein; EGFP,enhanced GFP; siRNA, short interfering RNA; OA,
okadaic acid; DAPI,4,6-diamidino-2-phenylindole; OD, optical
density.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 25, Issue of
June 24, pp. 2375823765, 2005 2005 by The American Society for
Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org23758
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showed directly that Bcl-2 negatively regulated caspase-2.Upon
ceramide and etoposide stimulation, protein phospha-tase-mediated
Bcl-2 dephosphorylation led to activation ofcaspase-2,
mitochondrial damage, and apoptosis.
EXPERIMENTAL PROCEDURES
Cell Cultures and ReagentsThe human lung epithelial cell
lineA549, Madin-Darby canine kidney (MDCK) cells, and their bcl-2
trans-fectants (A549-B2 and MDCK-B6) and vector controls (A549-P2
andMDCK-C1) were provided by Dr. M. T. Lin, Department of
Biochemis-try, and Dr. M. J. Tang, Department of Physiology,
National ChengKung University, Taiwan. A549 and MDCK cells were
cultured inDulbeccos modified Eagles medium supplemented with 10%
fetal bo-vine serum, 50 units/ml penicillin, and 0.05 mg/ml
streptomycin. Theywere maintained at 37 C in 5% CO2. Human prostate
cancer DU145cells were cultured in minimum essential medium
supplemented with10% fetal bovine serum. A549-B2, A549-P2, MDCK-B6,
and MDCK-C1cells were constructed using lipofection with a
transfection reagent(Lipofectamine 2000, Invitrogen) and then
cultured in Dulbeccos mod-ified Eagles medium supplemented with 10%
fetal bovine serum and500 g/ml G418 (Calbiochem). Bcl-2 cDNA
constructed in expressionvector, pUSEamp, was purchased from
Upstate Biotechnology. Cera-mide analogue C2-ceramide (BioMol) and
etoposide (BioVision) weredissolved in Me2SO. The broad-spectrum
caspase inhibitor benzyloxy-carbonyl-Val-Ala-Asp(-OMe)-fluoromethyl
ketone (z-VAD-fmk) andcaspase-9, -3, and -2 inhibitors
benzyloxycarbonyl-Leu-Glu(-OMe)-His-Asp(-OMe)-fluoromethyl ketone
(z-LEHD-fmk),
benzyloxycarbonyl-Asp(-OMe)-Glu(-OMe)-Val-Asp(-OMe)-fluoromethyl
ketone (z-DEVD-fmk), and
benzyloxycarbonyl-Val-Asp(-OMe)-Val-Ala-Asp(-OMe)-fluoromethyl
ketone (z-VDVAD-fmk), respectively, were purchasedfrom Sigma and
dissolved in Me2SO. Bcl-2 inhibitor HA14-1 (Tocris),bongkrekic acid
(Calbiochem), and okadaic acid (OA, Sigma) were dis-solved in
Me2SO.
Analysis of Cell ApoptosisFor apoptosis detection characterized
byDNA fragmentation, cells were fixed with 70% ethanol in
phosphate-buffered saline for propidium iodide (PI, Sigma) staining
and then wereanalyzed using flow cytometry (FACSCalibur; BD
Biosciences). DAPI(Sigma) was also used for apoptotic cell staining
in 5 g/ml for 30 minat room temperature and was followed by
microscopy detection. Apo-
ptotic cell membrane disruption characterized by the presence of
phos-phatidylserine was performed using the annexin V-phycoerythrin
de-tection kit (BioVision).
Detection of Caspase ActivationCellular caspase activation
wasdetermined using the ApoAlert caspase colorimetric assay kit
(Clon-tech) for caspase-3 and an ApoAlert caspase fluorescent assay
kit forcaspase-9 according to the manufacturers instructions.
Caspase-2 ac-tivity was detected using a caspase-2 assay kit
(Calbiochem). Opticaldensity (OD) measurements were performed using
a microplate reader,and the substrate activities shown as
p-nitroanilide (nmol) were calcu-lated for caspase-3 and -9. For
caspase-2, the relative substrate activitywas shown by the OD
values. Caspase-3 activation monitored in cellswas performed using
PhiPhiLux-G2D2 staining (OncoImmunol) anddetected with fluorescent
microscopy. The activation of caspases wasalso detected using
Western blot analysis as described below.
Western Blot AnalysisTo detect cytochrome c release, cytosolic
ex-tract without the mitochondrial fraction was separated using an
ApoA-lert cell fractionation kit (Clontech) according to the
manufacturersinstructions. To detect other proteins, total cell
lysate was used. West-ern blotting was then performed (BD
Biosciences). Briefly, cell extractwas separated by SDS-PAGE and
then transferred to a polyvinylidenedifluoride membrane
(Millipore). After blocking, blots were developedwith a series of
antibodies as indicated. Rabbit antibodies specific forhuman
caspase-9 and -3 (Cell Signaling Technology), cytochrome c(Santa
Cruz Biotechnology), green fluorescent protein (GFP) (SantaCruz
Biotechnology), Bcl-2, and phospho-Bcl-2 serine 70 (R&D)
wereused. Monoclonal antibodies against human caspase-2 (R&D)
and -ac-tin (Sigma) were used. Finally, blots were hybridized with
horseradishperoxidase-conjugated goat anti-rabbit IgG or anti-mouse
IgG (Calbio-chem) and developed using an AEC substrate kit (Zymed
LaboratoriesInc.).
Mitochondrial Functional AssayThe loss of mitochondrial
trans-membrane potential (m) value was determined using rhodamine
123(Sigma). Cells were incubated with rhodamine 123 (50 M) for 30
min incultured medium. After being washed with phosphate-buffered
saline,cells were resuspended in cold phosphate-buffered saline and
immedi-ately underwent flow cytometric analysis. Mitochondrial
dehydrogen-ase activity was determined using a WST-8 assay kit
(Dojindo Labora-tories, Kumamoto, Japan).
FIG. 1. Ceramide and etoposide induce mitochondria-mediated
apoptosis in A549 cells. A, human A549 epithelial cells were
treatedwith or without 50 M C2-ceramide or 25 M etoposide for 48 h.
The changes in cell morphology (characterized by cell rounding up)
and in nuclearmorphology (characterized by nuclear fragmentation)
are shown by phase-contrast microscopic observation and DAPI
staining, respectively. B,time- and dose-dependent (as indicated)
ceramide- or etoposide-induced A549 cell apoptosis was detected
using PI staining followed by flowcytometric analysis. The
percentages of apoptotic cells are shown (means S.D. of triplicate
cultures). C, using rhodamine 123 followed by flowcytometric
analysis, the percentages of ceramide- or etoposide-treated cells
with m (MTP) reduction at different times are shown (means S.D.of
triplicate cultures). D, the activation of caspase-9 and -3 induced
by ceramide or etoposide time dependently as determined by caspase
activityassay kits are shown (means S.D. of triplicate cultures).
pNA, p-nitroanilide. E, with or without caspase inhibitors as
indicated, ceramide- oretoposide-induced cell apoptosis at 48 h was
detected using PI staining. The percentages of apoptotic cells are
shown (means S.D. of triplicatecultures).
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ImmunostainingFor intracellular immunostaining, cells werefixed
with 1% formaldehyde in phosphate-buffered saline and
perme-abilized with 0.01% saponin in phosphate-buffered saline. A
series ofantibodies was used as indicated, followed by fluorescein
isothiocya-nate-conjugated goat anti-rabbit IgG (Calbiochem)
staining. Rabbitanti-Bcl-2 and anti-phospho-Bcl-2 serine 70
(R&D) antibodies wereused for flow cytometric analysis. For
confocal microscopy, rabbitanti-truncated Bid (tBid) (Calbiochem)
antibodies were used. MitoTracker Red CMXRos (Molecular Probes) was
used for mitochondrialstaining.
Short Interfering RNA (siRNA) PreparationPlasmids
expressingshort hairpin RNA were constructed using standard
techniques. ThepSUPER/enhanced GFP (EGFP) contained the GFP gene
frompEGFP-N1 (Clontech) inserted into pSUPER vector (37) (kindly
pro-vided by Dr. R. Agami, The Netherlands Cancer Institute,
Amsterdam,The Netherlands). To generate pSUPER-Casp2/EGFP and
pSUPER-Bcl2/EGFP, pSUPER/EGFP was digested with BglII and HindIII,
andthe annealed targeting oligonucleotides ACAGCTGTTGTTGAGCGAAfor
caspase-2 (15) and GCTGCACCTGACGCCCTTC for Bcl-2 (23) were
ligated into the vector. To generate the double knockdown
constructpSUPER-Bcl2/Casp2/EGFP, the Casp2 short hairpin RNA
expressioncassette from pSUPER-Casp2/EGFP was inserted into
pSUPER-Bcl2/EGFP. The pSUPER-Casp2/EGFP/Neo, pSUPER-Bcl2/EGFP/Neo,
andpSUPER-Bcl2/Casp2/EGFP/Neo were generated by inserting the
Neor
gene from the pIRESneo2 (Clontech) into the
pSUPER-Casp2/EGFP,pSUPER-Bcl2/EGFP, and pSUPER-Bcl2/Casp2/EGFP
vectors, respec-tively (supplemental Fig. S1).
A549 and DU145 cells were cultured in 6-well plastic plates
inDulbeccos modified Eagles medium and minimum essential
medium,respectively, supplemented with 10% fetal bovine serum (5
105/well). Before short hairpin RNA-expression vector transfection,
cellswere washed with serum-free medium and cultured with 2 l
ofLipofectamine 2000 and 1 g of DNA. After 6 h of incubation,
cellswere maintained in cultured medium containing 10% fetal
bovineserum for an additional 24 h before experiments. A FACSAria
cellsorter (BD Biosciences) was used to sort EGFP-positive cells
forvector control and siRNA-expressing cells in some experiments
asindicated.
FIG. 2. Requirement of caspase-2 activation during ceramide- or
etoposide-induced mitochondrial apoptosis. A, A549 cells
weretreated with 50 M C2-ceramide or 25 M etoposide for various
time periods followed by a caspase-2 activity assay; the substrate
relative activities(OD) are shown (top, means S.D. of triplicate
cultures). Also, ceramide (C)- or etoposide (E)-induced caspase-2
processing was determined usingWestern blot analysis (bottom).
Protein expression of -actin was used as an internal control. B,
ceramide or etoposide induced caspase-2-mediatedmitochondrial
disruption. With or without caspase-2 inhibitor z-VDVAD-fmk or
broad spectrum caspase inhibitor z-VAD-fmk, m (MTP)reduction and
cell apoptosis induced by 50 M C2-ceramide or 25 M etoposide for 48
h were determined using rhodamine 123 and PI staining,respectively,
followed by flow cytometric analysis (means S.D. of triplicate
cultures). The activities of caspase-9 and -3 (means S.D. of
triplicatecultures) were determined using caspase activity assay
kits (top). Ceramide- or etoposide-induced cytosolic cytochrome c
expression with or withoutcaspase inhibitors was determined using
Western blot analysis (bottom). Protein expression of -actin was
used as an internal control. pNA,p-nitroanilide. C, blockage of
ceramide- or etoposide-induced cell apoptosis by caspase-2 siRNA.
A549 cells were transfected with siRNA tocaspase-2 as described
under Experimental Procedures. The expression of procaspase-2 (45
kDa) in vector-transfected cells (Vector) and
caspase-2siRNA-transfected cells (Casp-2 siRNA) was detected using
Western blot analysis (top). Protein expression of EGFP was used as
an internalcontrol. The changes in morphology of transfected cells
with 50 M C2-ceramide or 25 M etoposide treatment for 48 h were
determined usingfluorescent microscopy (EGFP) plus phase-contrast
microscopy (Merge). The apoptotic cells are shown in transfected
cells (filled arrowheads) oruntransfected cells (open arrowheads).
D, ceramide- or etoposide-treated transfected cells were stained
with annexin V and caspase-3-specificfluorescence substrate
PhiPhiLux-G2D2 as described under Experimental Procedures. The
percentages of annexin V-positive and active-caspase-3-positive
cells are shown (means S.D. of triplicate cultures).
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RESULTS AND DISCUSSION
Ceramide and Etoposide Induce Mitochondria-mediated Apo-ptosis
in A549 CellsApoptotic cell death induced by ceramideand etoposide
has been widely reported (2832, 38, 39). Toinvestigate the
involvement of mitochondrial dysfunction andcaspase activation,
C2-ceramide and etoposide were used toinduce apoptosis in A549
human lung epithelial cells. First, cellgrowth inhibition after
ceramide and etoposide treatment wasdemonstrated using a WST-8
assay to detect mitochondrialdehydrogenase activity (data not
shown). A549 cells that hadbeen exposed to ceramide and etoposide
for 48 h were fixed andstained with DAPI. In ceramide- and
etoposide-treated cul-tures, but not in untreated cultures, cells
exhibited apoptoticmorphology (Fig. 1A). The dose and time kinetics
of ceramide-and etoposide-induced cell apoptosis were shown using
PIstaining and then flow cytometric analysis (Fig. 1B). To
furtherinvestigate the involvement of mitochondrial damage, the
lossof mitochondrial transmembrane potential (m) was deter-mined.
Using lipophilic cationic fluorochrome rhodamine 123staining, we
found that ceramide and etoposide time depend-ently induced m
reduction in A549 cells (Fig. 1C). Using acaspase activity assay
andWestern blot analysis, the activity ofcaspase-9 and -3 (Fig. 1D)
and the processing of caspase-3 fromproform (35 kDa) to active form
(17 kDa) (data not shown) wereobserved time dependently after
exposure to ceramide andetoposide. An increase in cytosolic
cytochrome c expression wasalso observed (data not shown).
Furthermore, pretreatmentwith the irreversible caspase-9 and -3
inhibitors z-LEHD-fmkand z-DEVD-fmk, respectively, blocked cell
death detected us-ing PI staining (Fig. 1E). We therefore
ascertained the involve-ment of mitochondrial damage, cytochrome c
release, and
caspase-9 and -3 activation in ceramide- and
etoposide-inducedA549 cell apoptosis.Requirement of Caspase-2
Activation before Mitochondrial
DamageOur previous study (24) demonstrated that activa-tion of
caspase-2 was required for ceramide- and etoposide-induced T cell
apoptosis before mitochondrial damage. We thusconfirmed the
involvement of caspase-2 in response to ceramideand etoposide
stimulation in A549 cells. Time-dependent acti-vation of caspase-2
was shown by the activity assay (Fig. 2A,top). The caspase-2
cleavage was also detected using Westernblot analysis (Fig. 2A,
bottom). To examine whether caspase-2was required for the
mitochondrial intrinsic pathway of apo-ptosis, we inactivated
caspase-2 in A549 cells by pretreatmentwith the inhibitor
z-VDVAD-fmk. The results showed the inhi-bition of cell apoptosis,
m reduction, and caspase-9 and -3activation after z-VDVAD-fmk
pretreatment in response toceramide and etoposide stimulation (Fig.
2B, top). Accordingly,cytosolic cytochrome c (Fig. 2B, bottom) and
cleavage fragmentsof active caspase-9 and -3 (data not shown) were
shown in theceramide- and etoposide-treated cultures, but not in
the cul-tures pretreated with z-VDVAD-fmk. The
broad-spectrumcaspase inhibitor z-VAD-fmk caused an effect similar
to that ofz-VDVAD-fmk (Fig. 2B). We further introduced the short
hair-pin RNA specific for caspase-2 into A549 cells for
interferencewith caspase-2 expression. The mean transfection
efficiency ofcaspase-2 siRNA was 52.3% and of vector control was
60.8%according to a flow cytometric analysis of EGFP-positive
cellsin a representative experiment. An 50% inhibition ofcaspase-2
expression was observed in the caspase-2 siRNA-transfected cells
compared with the vector control (Fig. 2C,top). After ceramide or
etoposide treatment, apoptotic cells
FIG. 3. Overexpression of Bcl-2 inactivates caspase-2, which
rescues ceramide- or etoposide-induced apoptosis. A, A549 cells
withBcl-2 overexpression were established as described under
Experimental Procedures. The protein expression of Bcl-2 in
wild-type (WT),vector-transfected (P2), and Bcl-2-transfected (B2)
was detected using Western blot analysis (top) and immunostaining
followed by flow cytometricanalysis (bottom). The relative mean
fluorescence intensity (MFI) is shown. For Western blotting,
protein expression of -actin was used as aninternal control. B,
cell apoptosis induced by 50 M C2-ceramide or 25 M etoposide at 48
h was detected using DAPI staining (left) or PI staining(middle)
followed by fluorescent microscopy or flow cytometric analysis,
respectively. The percentages of apoptotic cells are shown. For
mreduction detection, ceramide- or etoposide-treated cells were
detected using rhodamine 123 followed by flow cytometric analysis
(right). Thepercentages of cells with m reduction are shown. C,
caspase activation in A549-P2 and A549-B2 cells induced by 50 M
C2-ceramide (top) or 25M etoposide (bottom) for various time
periods was determined using activity assay kits. The substrate
activities for caspase-9 and -3 (pNA,p-nitroanilide) (left, means
S.D. of triplicate cultures) and the relative activity for
caspase-2 using OD (right) are shown. D, the processing ofcaspase-2
in these cells after ceramide (top) or etoposide (bottom) treatment
was detected using Western blot analysis.
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characterized by destructive morphology (Fig. 2C, all
arrow-heads) were seen in both EGFP-positive vector control
(filledarrowheads) and EGFP-negative cells (open arrowheads). Inthe
caspase-2 siRNA-transfected group, however, only EGFP-negative
cells underwent apoptosis (open arrowheads),whereas EGFP-positive
caspase-2 siRNA-expressing cellsshowed resistance to ceramide and
etoposide stimulation withintact morphology. Similarly, ceramide-
and etoposide-inducedapoptosis was blocked in caspase-2
siRNA-expressing cells,detected using annexin V staining and
caspase-3 activity inEGFP-positive cells (Fig. 2D). These results
showed that inter-ference with the expression of caspase-2 blocked
the mitochon-dria-dependent pathway of apoptosis induced by
ceramide andetoposide.Bcl-2 Blocks Caspase-2-mediated Ceramide- and
Etoposide-
induced ApoptosisBcl-2 acts as an anti-apoptotic factor toblock
death signals, including those from ceramide and etopo-side (2532).
To investigate the relation between Bcl-2 andcaspase-2 activity
during ceramide- and etoposide-induced apo-ptosis, we used
Bcl-2-overexpressing cells. First, an elevatedexpression of Bcl-2
in A549 cells transfected with bcl-2 (A549-B2), compared with the
wild-type (WT) and vector control(A549-P2), was confirmed using
Western blot analysis (Fig. 3A,top), intracellular immunostaining,
and then flow cytometricanalysis (Fig. 3A, bottom). Cell apoptosis,
characterized byDNA fragmentation, was detected using DAPI staining
(Fig.3B, left) or PI staining followed by flow cytometric
analysis(Fig. 3B, middle). Compared with A549-P2 cells, which
had45.1 and 35.7% apoptotic cells after ceramide and
etoposidestimulation, respectively, apoptotic cells were reduced to
15.1and 8.3% in A549-B2 cells. Using rhodamine 123 staining,
the
m reductions in A549-P2 and A549-B2 cells were 39.2 and7.1%,
respectively, after ceramide treatment and 45.6 and 4.8%after
etoposide treatment (Fig. 3B, right). Ceramide-inducedtBid
expression was detected using tBid-specific antibody incombination
with Mito Tracker Red dye. Results showed Bidcleavage and
translocation to mitochondria in ceramide-treated A549-P2 but not
in A549-B2 cells (data not shown). Acaspase substrate activity
assay revealed a time-dependentincrease in caspase-9, -3, and -2
activities in A549-P2 but not inA549-B2 cells (Fig. 3C). Also,
cleavage of procaspase-2 was seenin A549-P2 but not in A549-B2
cells after ceramide and etopo-side treatment (Fig. 3D). MDCK cells
with or without Bcl-2overexpression after ceramide and etoposide
stimulationshowed similar results (supplemental Fig. S2).Role of
Bcl-2 in Regulating Caspase-2 ActivationBcl-2 has
blocked caspase-3 and -2 sequential activation after
mitochon-drial damage (19). In that case, caspase-2 acted
downstream ofmitochondria and caspase-3, and Bcl-2 acted at a point
down-stream from the release of mitochondrial cytochrome c.
Bcl-2had also acted upstream or downstream of caspase-8 in Fas-
orstaurosporine-induced apoptosis (4, 40, 41). Our previous
studydemonstrated caspase-2 and -8 sequential activation in
ceram-ide- and etoposide-induced mitochondrial damage in
concomi-tant with the production of tBid and the release of
cytochromec (24). To directly investigate the regulation of Bcl-2
oncaspase-2, we used the gene-silencing technique and Bcl-2
in-hibitor. siRNA transfection in A549 cells induced Bcl-2
orcaspase-2 knockdown, or both. After cell sorting, flow
cytomet-ric analysis showed, and Western blot analysis confirmed,
thatthe EGFP-positive cells with Bcl-2 siRNA, caspase-2 siRNA,Bcl-2
plus caspase-2 siRNA, and vector control were 82.2, 84,
FIG. 4. Down-regulation of Bcl-2 results in caspase-2
activation. A, A549 cells were transfected with Bcl-2 siRNA,
caspase-2 siRNA, orBcl-2 plus caspase-2 siRNA as described under
Experimental Procedures. After cell sorting, the expression of
Bcl-2 (28 kDa) and caspase-2 (45kDa) in vector-transfected cells
(Vector) and siRNA-transfected cells was detected using Western
blotting (top). Protein expression of EGFP wasused as an internal
control. In the bottom panel, EGFP-positive cells are indicated as
successfully transfected cells (all arrowheads). With orwithout
z-VDVAD-fmk, the morphology changes of 48-h posttransfected cells
were detected using phase-contrast plus fluorescent microscopy.
Theapoptotic cells (open arrowheads) are shown. B, for apoptosis
and caspase-activation analysis, 24 and 48 h posttransfection,
cells were stained withannexin V or caspase-3-specific fluorescence
substrate using PhiPhiLux-G2D2 staining, respectively. The
percentages of positive cells in vector-transfected and Bcl-2
siRNA-transfected groups are shown (means of duplicate cultures).
After cell sorting, the caspase-2 activity was detectedusing a
caspase-2 activity assay kit. The substrate relative activity is
shown using OD (means of duplicate cultures). C, A549 cells were
treatedwith 100 M Bcl-2 inhibitor HA14-1 for 18 and 36 h, and cell
apoptosis was detected by PI staining followed by flow cytometric
analysis. Also,activation of caspase-3 and -2 was determined using
caspase activity assay kits. The substrate activity for caspase-3
(pNA, p-nitroanilide) and therelative activity for caspase-2 using
OD are shown (means of duplicate cultures).
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90, and 89.8%, respectively, in one representative
experiment(Fig. 4A, top). We next showed that the hallmarks of
apoptosiswere present in EGFP-positive cells with Bcl-2 siRNA but
notin those with vector control. Cells with Bcl-2 siRNA
transfec-tion showed apoptotic morphology compared with
untrans-fected cells or vector control cells (Fig. 4A, open
arrowheads).Bcl-2-silencing cells co-transfected with caspase-2
siRNA orpretreated with z-VDVAD-fmk rescued apoptosis caused
byBcl-2 inhibition. Bcl-2 siRNA-transfected cells showed a
time-dependent increase of apoptotic cell death with annexin
Vbinding and caspase-3 activation compared with vector controlcells
(Fig. 4B). To further investigate caspase-2 activation afterBcl-2
knockdown, we sorted EGFP-positive cells and deter-mined the
activation of caspase-2. A substrate activity detec-tion assay
suggested that caspase-2 was activated in Bcl-2siRNA-transfected
cells but not in vector control cells (Fig. 4B).HA14-1, a small
molecule inhibitor of Bcl-2, induced cell apo-ptosis via the
mitochondrial intrinsic pathway through Bcl-2structural dysfunction
(42, 43). HA14-1 caused Bax transloca-tion to mitochondria and
cytochrome c release (43). Our resultsshowed that treatment with
HA14-1 induced A549 cells toundergo apoptosis. The m reduction
(data not shown) andcaspase-3 and -2 activation (Fig. 4C) were also
observed. HA14-1-induced cell apoptosis and caspase activation were
detectedin Bcl-2-overexpressing cells (data not shown). To verify
thespecificity of HA14-1 on Bcl-2 dysfunction, we tested a
Bcl-2-independent apoptotic pathway in DU145 cells, human pros-
tate cancer cells. A previous study (44) showed that
Bcl-2down-regulation by antisense RNA and siRNA strategies
wasunable to facilitate cell apoptosis in DU145 cells. Our
resultsconfirmed that DU145 cells resisted a serial dose of
Bcl-2siRNA (supplemental Fig. S3A). DU145 cells treated with 100M
HA14-1 did not undergo apoptosis, m reduction, orcaspase-3 and -2
activation (supplemental Fig. S3B). However,HA14-1 may have caused
DU145 cell death at higher concen-trations (data not shown). These
results corresponded with aprevious report (23) demonstrating the
involvement ofcaspase-2 in Bcl-2-silencing cell apoptosis.Based on
our previous findings (24), ceramide- and etopo-
side-induced caspase-2 activation acted upstream of
caspase-8activation, Bid cleavage, and m reduction. To further
con-firm that Bcl-2-modulated caspase-2 activation acted upstreamof
mitochondrial damage, we used the mitochondrial perme-ability
transition pore inhibitor bongkrekic acid and caspase-9inhibitor
z-LEHD-fmk. After pretreatment with bongkrekicacid or z-LEHD-fmk,
apoptosis (Fig. 5, top) and caspase-3 ac-tivation (middle) caused
by Bcl-2 knockdown or HA14-1 wascompletely blocked. Most
importantly, caspase-2 activation in-duced by Bcl-2 knockdown or
HA14-1 was not inhibited in thepresence of bongkrekic acid or
z-LEHD-fmk (bottom). We con-cluded that Bcl-2-modulated caspase-2
activation functionedupstream of mitochondria.Bcl-2 Dysfunction
Caused by Phosphatase during Ceramide-
and Etoposide-induced ApoptosisIt had been reported thatceramide
caused Bcl-2 dephosphorylation at serine 70 and re-sulted in Bcl-2
functional destruction through protein phospha-
FIG. 5. Caspase-2 activation acts upstream of
mitochondrialdamage after Bcl-2 dysfunction. With or without
bongkrekic acid(BA) or caspase-9 inhibitor z-LEHD-fmk, A549 cells
were treated with50 M C2-ceramide, 25 M etoposide, or 100 M HA14-1,
or transfectedwith Bcl-2 siRNA. Cell apoptosis and caspase
activation were deter-mined at 48 h. Cell apoptosis was detected
using PI (filled bar) orannexin V (open bar) staining followed by
flow cytometric analysis orfluorescent microscopy. The percentages
of apoptotic cells and annexinV-positive cells are shown (means of
duplicate cultures). The activationof caspase-3 and -2 was detected
by substrate cleavage using PhiPhi-Lux-G2D2 staining or an activity
assay kit. The quantified concentra-tions for caspase-3 (filled
bar) and the percentages of active caspase-3-positive cells (open
bar) are shown. Bcl-2 siRNA-transfected cells weresorted and used
for caspase-2 activity detection. The substrate-relativeactivity is
shown using OD (means of duplicate cultures).
FIG. 6. Phosphatase causes Bcl-2 dysfunction, which facili-tates
ceramide- and etoposide-induced mitochondrial apopto-sis. A,
Western blot analysis of phospho-Bcl-2 serine 70 (pBcl-2) andBcl-2
protein expression (28 kDa) in total A549 cell extracts at
varioustime points after treatment with 50 M C2-ceramide (top) or
25 Metoposide (bottom). Protein expression of -actin was used as an
inter-nal control. B, using immunofluorescence staining followed by
flowcytometric analysis, the expression of pBcl-2 in A549 cells
after cera-mide treatment with or without 100 nM protein
phosphatase inhibitorOA was detected at 24 h. The histogram of
protein expression is shownas a representative of two individual
experiments. The mean fluores-cence intensity (MFI) of each protein
expression is shown. C, A549 cellswere treated with ceramide (solid
line) or etoposide (dotted line) with orwithout 100 nM OA for 24
and 48 h. Cell apoptosis and m (MTP)reduction were detected using
PI and rhodamine 123 staining, respec-tively, followed by flow
cytometric analysis. The percentages of apo-ptotic cells and
positive cells with m reduction are shown (means ofduplicate
cultures). Also, activation of caspase-3 and -2 were deter-mined
using caspase activity assay kits. The activity as relative OD
isshown (means of duplicate cultures).
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tase 2A (33). We therefore investigated whether the
dephos-phorylation of Bcl-2 at serine 70 also occurred in A549
cells andrelated to caspase-2 activation. Indeed, Bcl-2 was
dephospho-rylated at serine 70 by 12 and 24 h after ceramide and
etopo-side treatment, respectively, and the expression level of
Bcl-2decreased at 48 and 24 h posttreatment (Fig. 6A).
Similarresults with Bcl-2 serine 70 dephosphorylation were seen
usingintracellular immunostaining followed by flow cytometric
anal-ysis. Moreover, dephosphorylation of Bcl-2 caused by
ceramidewas rescued by pretreatment with the phosphatase
inhibitorOA (Fig. 6B). Furthermore, ceramide- and
etoposide-inducedcell apoptosis, m reduction, and caspase-3 and -2
activationwere all repressed by OA (Fig. 6C). OA caused maximal
inhi-bition at a dose of 100 nM, but a higher dose was cytotoxic
(datanot shown). Based on these results, ceramide- and
etoposide-induced mitochondrial damage was initiated by caspase-2
ac-tivation, caspase-2 was regulated by Bcl-2, and Bcl-2 was,
atleast in part, regulated by protein phosphatase.
Nevertheless,although the phosphorylated status of Bcl-2 appears
involved,the modulatory role of Bcl-2 on caspase-2 activation
remains tobe defined.Concluding RemarksIn the present study, the
interrela-
tionship between Bcl-2 and caspase-2 was revealed.
Bcl-2knockdown by siRNA or Bcl-2 inhibition by an inhibitor
re-sulted in an autonomic activation of caspase-2, providing
directevidence that caspase-2 is negatively regulated by Bcl-2.
Uponapoptotic stimulation by ceramide and etoposide, Bcl-2
wasdephosphorylated by protein phosphatase and lost its regula-tory
control on caspase-2. This study, therefore, addresses anovel
anti-apoptotic mechanism of Bcl-2. In its phosphorylatedstate, it
blocks caspase-2 activation, although the underlyingmechanism
remains unclear. However, Bcl-2-modulatedcaspase-2 activation and
cell apoptosis were not involved inBcl-2-insensitive cells such as
DU145. Recently, the silencing ofBcl-2 by siRNA or inhibitor has
been used in tumor therapy(23, 42, 43). Cell apoptosis occurred in
Bcl-2-silencing cellsthrough caspase-2- and p53-regulated pathways
(23). In addi-tion, Bcl-2 had been shown to act upstream of
caspase-2 acti-vation in PC12 cell apoptosis induced by growth
factor depri-vation (45). Based on our results, ceramide- and
etoposide-induced caspase-2 activation before mitochondrial damage
wasinitiated because of Bcl-2 dysfunction. In ceramide- and
etopo-side-induced apoptosis, Bcl-2 dysfunction occurred because
ofits dephosphorylation at serine 70. Furthermore, there was
adecrease in Bcl-2 expression after ceramide and
etoposidetreatment. Involvement of Bcl-2 cleavage in the
accelerationof etoposide-induced U937 cell apoptosis had previously
beenreported (34).Apoptotic signaling mediated by ceramide has
provided new
insights into the mechanism of action of chemotherapy
andradiotherapy in antitumor activity (4648). The up-regulationof
the endogenous ceramide level induced by etoposide wasdemonstrated
(30, 49, 50). Ceramide-induced apoptosis hasbeen associated with
dephosphorylation of various kinasessuch as Akt, Bcl-2-associated
death promoter (BAD), forkheadtranscription factor (FKHR), and
GSK-3 (5154). Whetherthese kinases are involved in the regulation
of protein phos-phatase on Bcl-2 and in the regulation of Bcl-2 on
caspase-2remains to be investigated. Our preliminary results show
thatBcl-2 and caspase-2 can be co-precipitated. Whether Bcl-2
andcaspase-2 can directly interact with each other or
whetheradaptor proteins are necessary for Bcl-2 and caspase-2
bindingrequires further investigation. The possible regulatory
mecha-nisms between protein phosphatase, protein kinase, Bcl-2,
andcaspase-2 need to be further explored. Taken together, these
findings shed light on the role of Bcl-2 in the inhibition
ofcaspase-2 before mitochondrial damage.
AcknowledgmentsWe thank Wan-Hua Tsai from the Institute ofBasic
Medical Sciences (NCKU, Taiwan) for conducting the Bcl-2
over-expression system and Ming-Chen Yang and Wen-Wei Chang from
theDepartment of Microbiology and Immunology (NCKU, Taiwan) for
as-sistance with cell sorting. We also thank Dr. C. W. Chiang for
commentson the manuscript and Bill Franke for editorial
assistance.
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Tang, Wen-Chang Chang and Yee-Shin LinRen-Huang Wu, Yi-Ting
Fang, Ming-JerChang, Ming-Shiou Jan, Li-Jin Hsu, Chiou-Feng Lin,
Chia-Ling Chen, Wen-Tsan
ActivationApoptosis through Blockage of
Caspase-2Etoposide-induced Mitochondrial Bcl-2 Rescues Ceramide-
andMechanisms of Signal Transduction:
doi: 10.1074/jbc.M412292200 originally published online April 6,
20052005, 280:23758-23765.J. Biol. Chem.
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