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TMP CE4
TMP CE4
DOI 10.1007/s10495-008-0219-4
ORIGINAL PAPER
Abstract Tumor Necrosis Factor (TNF)-Related Apop- we demonstrate that TRAIL-induced activation of PKC-
tosis-Inducing Ligand (TRAIL) initiate pathways of cell delta mediates regulation of the PLS3-induced changes in
death in which caspase activation is mediated either cardiolipin.
directly (without mitochondrial amplification), or indirectly
via the release of apoptogenic factors from mitochondria. Keywords Apoptosis TRAIL Mitochondria
Phospholipid scramblases (PLS) are enzymes that play a Phospholipases Scramblase Cardiolipin PKC
key role in cellular function by inducing bidirectional
movement of membrane lipids. Changes in mitochondrial
membrane lipids, cardiolipin, are critical for mediating Introduction
apoptotic response in many cell-types. PLS3 is a phos-
pholipid scramblase that is localized to mitochondria and is Apoptosis, a form of programmed cell death, is an active form
thought to be involved in the regulation of apoptotic sig- of cellular destruction which, in concert with cell division,
nals. Here we report that exogenous-expression of PLS3 controls immune response, embryonic development and tissue
enhances apoptotic death induced by TRAIL. This is homeostasis [1–6]. Apoptosis plays an important role in
acheived by potentiating the mitochondrial arm of the physiology; failure of this process can be harmful and disrupt
death pathway. Thereby, PLS3 expression facilitates the normal cellular state, thereby leading to many diseases
changes in mitochondrial membrane lipids that promote the including neoplastic transformation [1, 7–9]. Apoptosis is
release of apoptogenic factors and consequent full activa- mediated by caspases that are activated by either the death
tion and processing of the caspase-9 and effector caspase-3. receptor (extrinsic) or mitochondrial (intrinsic) pathways. One
Moreover, we show that knock-down of endogenous PLS3 death ligand is the Tumor Necrosis Factor (TNF)-Related
suppresses TRAIL-induced changes in cardiolipin. Finally, Apoptosis-Inducing Ligand (TRAIL), a type II transmembrane
protein and a member of the TNF family of ligands (Fas, TNF-
alpha, etc.) [1, 8]. TRAIL induces apoptosis in transformed or
K. Ndebele T.-G. Jin N. Benhaga A. Chalah tumor, but not in normal, cells and hence has great promise as a
R. Khosravi-Far (&)
tumor-selective anti-cancer agent [1, 7, 10–14]. Additionally,
Department of Pathology, Harvard Medical School, Beth Israel
Deaconess Medical Center, 99 Brookline Ave, Boston, MA the functional expression of TRAIL has been shown on the
02215, USA surface of antigen presenting cells such as dendritic cells and
e-mail: rkhosrav@bidmc.harvard.edu monocytes that have been stimulated by cellular transforma-
tion, suggesting a role for TRAIL in the selective ablation of
P. Gona
Statistics and Consulting Unit, Department of Mathematics and transformed cells before tumorigenesis [15–20]. The TRAIL
Statistics, Boston University, 704 Commonwealth Ave, Boston, receptors, DR4 and DR5, mediate TRAIL-induced apoptosis
MA 02215, USA by recruiting, via death domain (DD) interaction, adaptor
proteins such as Fas-Associated protein with Death Domain
M. Degli-Esposti
Faculty of Life Sciences, University of Manchester, Stopford (FADD) and apical procaspases such as procaspase-8. This
Building, Oxford Road, Manchester M13 9PT, UK resulting assembly forms the Death Inducing Signaling
123
846 Apoptosis (2008) 13:845–856
Complex (DISC) [1, 3, 7, 12, 21, 22], which promotes the auto- of PLS3 and its action on mitochondrial membrane during
proteolytic activation and release of apical caspases [23, 24]. In apoptosis is not known.
most tumor cells, which are considered to be type II cells, the In this study, we show that PLS3 sensitizes Jurkat cells
activated apical caspases then initiate two main pathways of to TRAIL-induced apoptosis by promoting changes in the
cell death [5, 25–27]. In one pathway (the extrinsic), caspase-8 composition and distribution of cardiolipin. Hence, PLS3
activation directly activates effector caspase-3, which facili- enhances TRAIL-induced apoptosis by augmenting the
tates apoptosis. Alternatively, the death receptor pathway can release of apoptogenic factors and activating caspase-9,
also activate the mitochondrial pathway (intrinsic pathway). In leading to the amplification and full activation and pro-
the mitochondrial pathway, changes in the mitochondrial cessing of caspase-3 and the downstream caspase cascade.
membranes lead to the release of apoptogenic factors such as Furthermore, we demonstrate an involvement of PKCd in
cytochrome c [5, 7, 22, 28–30]. Upon entry into the cytosol, regulating this ‘‘apoptogenic’’ action of PLS3.
cytochrome c binds the caspase-activating protein Apaf1,
stimulating the binding of Apaf1 to procaspase-9, forming
Materials and methods
apoptosome and subsequently inducing processing and acti-
vation of caspase-9. As a result, activated caspase-9 is then
Reagents
available to cooperate with caspase-8 in the processing and
activation of caspase-3, leading to the demise of the cell.
RPMI culture medium and fetal bovine serum (FBS) were
There is emerging evidence that the lipid composition of
purchased from Cellgro Mediatech Inc, (Herndon VA) and
mitochondrial membranes could modulate the membrane
Biosource (Rockville, MD), respectively. Glutamine, pen-
perturbing action of pro-apoptotic proteins like Bid [22, 31–
icillin, streptomycin and culture supplements were
34]. Membrane lipid composition is altered by lipid-modify-
purchased from Gibco/BRL (Gaithersburg, MD). Caspase-
ing, e.g. phospholipases, or lipid-transporting proteins [34].
8, caspase-9, caspase-3, Bid, HSP60, GRB2 and an anti-
Among the latter, phospholipid scramblases play an estab-
body to PKCd were obtained from BD Bioscience (Palo
lished role in the exposure of phosphatidylserine (PS) on the
Alto, California). Anti-FLAG M2 agarose and anti-FLAG
membrane surface of dying cells [32, 33]. It is now recognized
M2 were obtained from Sigma (St. Louis, MO). Anti-PLS3
that there are four different members of the phospholipid
antibody was purchased from Novus Biologicals, Inc(Lit-
scramblase family in humans and three in the mouse [26–30].
tleton, CO). Recombinant human TRAIL was obtained
Phospholipid scramblase 1 has been reported to be involved in
from R&D Systems, Inc (Minneapolis, MN). The cardio-
the externalization of phosphatidylserine from the cytosolic to
lipin-sensitive probe 10-nonyl-acridine orange (NAO) was
the outer leaflet of the plasma membrane [31]. Conversely,
obtained from Molecular Probes (Eugene, OR).
phospholipid scramblase 3 (PLS3) is localized in mitochon-
dria and may be involved in the regulation of mitochondrial Cell culture
function, especially by influencing the distribution of the
mitochondria-specific lipid, cardiolipin [32, 34, 35]. Jurkat T cells (acute T cell leukemia) were purchased from
To date, the involvement of PLS3 in regulating mito- the American Type Culture Collection (Rockville, MD),
chondrial membrane lipids during TRAIL-induced cell maintained in logarithmic growth, and were cultured in
death has not been elucidated. Death receptor induced RPMI-1640. Cells were cultured in a density between 0.1
signaling has been shown to bring alterations in cardiolipin, and 1.0 9 106/ml. Media was supplemented with 10% fetal
a lipid unique to mitochondria [36]. Moreover, cardiolipin bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and
is instrumental for the action of death receptor-induced 100 lg/ml streptomycin. The cells were cultured in sus-
apoptotic signals at the mitochondrial membrane, since the pension at 37°C and 5% CO2 in a humidified incubator and
pro-apoptotic action of tBid and Bax requires their inter- carried at 0.1–2.0 9 106 cells/ml, passaging two to three
action with this lipid and its metabolites [36–41]. The times weekly as needed. Cells were pelleted and resus-
death-receptor induced changes in cardiolipin appear to pended in fresh complete media in tissue culture plates
follow an imbalance in the cellular homeostasis of phos- 24 h before use in experiments to avoid any confounding
phatidylcholin (PC) [36]. Metabolic alterations in PC also gene expression that might occur because of handling.
cause an elevation of diacylglycerol (DAG), which in turn
activates PKCd, an isoform of phospholipids-dependent Generation of PLS3 Jurkat-expressing cells
ser/thr protein kinase [36]. Interestingly, scramblase-1 and
-3 have been shown to be phosphorylated by PKCd [34]. Full-length human PLS3 was kindly provided by Dr. Theresa
However, the involvement of PLS3 in regulating cardio- Wiedmer, The Scripps Research Institute, La Jolla, CA.
lipin homeostasis and mitochondrial apoptotic pathway and PLS3 was amplified using 30 cycles of denaturation (94°C,
consequently the role of PKCd in modulating the function 1 min), annealing (50°C, 2 min), and extension (72°C,
123
Apoptosis (2008) 13:845–856 847
3 min) using the following primers: Forward primer 24 h post-transfection, cells were replaced with 10% com-
50 TAGGATCCATGGCAGGCTACTTGCC30 ; Reverse pleted medium for overnight. Each 1 9 106 cells were then
primer 50 GACTCGAGCTAACTGGTGATGGCAG30 . The treated with TRAIL for 1 h in complete medium. Silencing of
amplified PCR product was isolated, sequenced and sub- PLS3 was assessed by western blotting and NAO shift were
cloned into the pcDNA3.1FLAG vector (Invitrogen Life measured by FACS.
Technologies, Carlsbad, CA) between Bam H1 and Xho1
restriction sites. Western blotting was performed to confirm Isolation of mitochondria
expression of PLS3 in stably transfected Jurkat cells (Fig. 1).
Mitochondria were isolated using a kit from Pierce Labo-
Generation of PLS3 mutant Jurkat-expressing cells ratories (Rockford, IL) with minor modifications. Briefly,
Jurkat cells (2 9 107) were pelleted at *850g for 2 min by
We generated mutant of PLS3 with a mutagenesis approach centrifugation. The supernatant was removed and dis-
similar to that reported by Zhou et al. [47]. Zhou et al. carded. 800 ll of Mitochondria Isolation Reagent A was
found that mutation of Phe281 of the calcium-binding motif added and the tubes were vortexed at medium speed for 5 s
in PLS1 abolished function [47]. We altered the corre- and then incubated on ice for 2 min. 10 ll of Mitochondria
sponding Phe258 in PLS3 to valine and generated stable Isolation Reagent B was added and the samples were
transfectants of the mutant PLS3(F258V) in Jurkat cells. vortexed at maximum speed for 5 s using an Eppendorf
benchtop microcentrifuge. Mitochondria Isolation Reagent
Mitochondrial staining C was added and tubes were inverted several times to mix.
The samples were centrifuged at 700g for 10 min at 4°C.
The cardiolipin-sensitive probe 10-nonyl-acridine orange The supernatant was transferred to a new, 2.0 ml tube and
(NAO) was used to monitor changes in mitochondrial centrifuged at 12,000g for 15 min at 4°C. The supernatant,
lipids in vivo. Briefly, after treatment with 10 ng/ml of containing the cytosolic fraction, was transferred to a new
recombinant human TRAIL/TNFSF10 (R&D systems, tube and the remaining mitochondria pellet was washed
Minneapolis, MN), 106 cells were collected by centrifu- with 500 ll of Mitochondria Isolation Reagent C. The
gation, washed and then resuspended in PBS containing mitochondria fraction was stored at -80°C until used.
200 nM NAO (Molecular Probes, Eugene, OR) for 30 min
at room temperature. Fluorescence was subsequently Electron microscopy analysis
measured by flow cytometry using the Fl-1 channel. Using
a similar protocol, we also measured mitochondrial mem- About 2 9 106 cells were fixed in 2% PFA/2.5% glutar-
brane potential. 106 washed cells were resuspended in 1 ml aldehyde in 0.1 M Sodium cacodylate buffer, pH 7.4 for
PBS containing 25 nM CMXRos (Molecular Probes, 1 h at room temperature, postfixed in 1% osmium tetrox-
Eugene, OR) or 40 nM TMRE (Molecular Probes, Eugene, ide/1.5% potassium ferrocyanide in water for 30 min,
OR) and after 30 min incubation at 37°C fluorescence was stained in 1% uranyl acetate in maleate buffer pH 5.2 for
measured by flow cytometry with the FL-3 channel. 30 min at room temperature. After dehydration in graded
Tricyclodecan-9-yl xanthogenate (D609) was employed to ethanols (70%, 95% and 29 100%) cells were pelleted at
pharmacologically inhibit phospholipase activity [36, 42–44]. 3,000 rpm for 3 min. Pellets were infiltrated in a 1:1
D609 was purchased from Biomol (Plymouth Meeting, PA). mixture of propylene oxide and Epon (TAAB Epon,
Cells were treated with D609 in buffered DMEM or RPMI Marivac Ltd, Nova Scotia, and Canada) for 2 h at room
medium for 30 min. prior to treatment with TRAIL. temperature, transferred to embedding molds filled with
pure TAAB Epon and polymerized for 48 h at 60°C. Ultra
PLS3 siRNA thin sections (*80–90 nm) were mounted on copper grids,
stained with 0.2% lead citrate and examined in a JEOL
Jurkat cells were transfected with PLS3 siRNA pool 1200EX transmission electron microscope.
purchased from Dharmacon Inc (Chicago, IL) using elec-
troporation. This pool contains four siRNAs against PLS3 Cell viability and Sub G1 apoptosis assay
(TAACATGTCTGCCAACAAT,GGAGAAGTCAGGCAT
AGTA,GCAAATACAGGGAAGACTA,GGACGACTGCT Flow cytometry was used to assess Sub G1 DNA content in an
CCAGCTTA, pool number = M-010225–00). The control entire cell population. 2 9 105 cells were seeded in 24-well
siRNA was also purchased from Dharmacon (SIControl plates and cultured for 24 h prior to treatment. For the inhibition
Non0Targetting SIRNA POOL #1 = D-001206-13-20). assay, cells were pretreated with D609 inhibitors for 30 min.
Briefly, 5 9 106 Jurkat cells were transfected with 100 nM/ml Cells were then treated with 10 ng/ml of TRAIL at varying time
siRNA or negative control siRNA in serum-reduced medium. periods in 0.5% FBS medium. Cells were harvested, washed
123
848 Apoptosis (2008) 13:845–856
once with PBS and suspended in 400 ll propidium iodide (PI) II error). Actual observed P-values of the Wilcoxon rank sum
solution (propidium iodide 50 lg/ml, 0.1% sodium citrate and test were presented in figures. We decided a priori not to
0.1% Triton-X 100). The Sub G1 DNA content was determined implement Boneferoni-type significance level adjustment to
using a Becton Dickinson Flow Cytometer. account for multiplicity. Firstly, we viewed the hypotheses
we tested to be independent therefore not warranting
Western blot analysis adjustment of comparison-wise significance level. Secondly,
there are different and opposing viewpoints over compari-
For Western analysis, 2 9 106–5 9 106 cells were cultured at son-wise adjustments [47].
a density of 1.0 9 106/ml and stimulated. Cells were harvested
and pelleted in an Eppendorf microcentrifuge (1,200g, 5 min,
4°C), washed in 19PBS and resuspended in a cell lysis buffer Results
containing 20 mM Tris (pH 8.0), 0.5% (w/v) Nonidet P-40,
1 mM EDTA, 1 lg/ml leupeptin, 1 lg/ml pepstatin, 1 mM Expression of PLS3 in Jurkat cells augments
dithiothreitol, 1 mM PMSF and 0.1 M NaCl. After 20 min. TRAIL-induced apoptosis
incubation at 4°C, supernatants were clarified (8,000g, 5 min,
4°C) and total protein concentration was determined by the We previously demonstrated that TRAIL induces alterations
method of Bradford and Lowry [7] using Bio-Rad Protein in mitochondrial membrane lipid, cardiolipin (CL) [36]. As
Assay reagents in a microtiter assay. 30 lg of total cellular PLS3 is localized to the mitochondrion [34, 36, 40], we
protein was electrophoresed on a polyacrylamide SDS-PAGE hypothesized that it may influence TRAIL-induced apopto-
gel and then transferred to a polyvinylidine difluoride mem- sis by regulating the mitochondrial lipid membrane. Similar
brane (Amersham, Arlington Heights, IL) by electroblotting to previous reports [19, 48], we observed that TRAIL-
overnight (25 mM Tris (pH 8.3), 192 mM glycine, 20% (v/v) induces apoptosis in a time- and dose-dependent manner
methanol, 15 V, 100 mA, 4°C). Membranes were blocked (Fig. 1a), and that a concentration of TRAIL at 10 ng/ml
with 10% (w/v) electrophoresis-grade biotin-depleted non-fat applied over a time period of 6 h causes complete proteolytic
dry milk (Bio-Rad) in 1 9 PBS, rinsed in PBS, probed with cleavage of caspase-8 in Jurkat cells (Fig. 1a). Therefore,
monoclonal mouse anti-human caspase-8, -9, -3, Bid, Cyt c or throughout the remainder of this paper, TRAIL treatment
PKCd (Pharmingen) at a 1:500 dilution and washed 39 in PBS. should be assumed to involve the aforementioned concen-
The secondary antibody was HRP-conjugated goat anti-mouse tration and time period unless if otherwise noted.
or anti-rabbit whole IgG used at 1:1,000 and 1:5,000, respec- In order to investigate the role of PLS3 in TRAIL-induced
tively (Transduction Laboratories, San Diego, CA) for 1 h at apoptosis, we generated a Jurkat T lymphocytic cell line with
room temperature. The protein bands were then visualized ectopic expression of PLS3 (F-PLS3) (Fig. 1b). We then
using an enhanced chemiluminescence (ELC) detection sys- ascertained that F-PLS3 is localized to the mitochondrion by
tem (Amersham Bioscience, Piscataway, NJ). generating cytosolic and mitochondrial fractions and
examining the intracellular distribution pattern of PLS3 by
Statistics Western blotting with an anti-FLAG antibody. These studies
demonstrated that F-PLS3, in accordance with other reports
All data were analyzed as continuous variables. Studies have for endogenous PLS3 [35, 49], was localized exclusively to
shown the t-test to be robust enough to stand considerable the mitochondrial, and not the cytosolic fraction (Fig. 1c).
departures from its theoretical assumptions, but with sample The mitochondrial and cytosolic markers, Hsp60 and GRB2
sizes N B 5 replications per treatment group in this study, respectively, were used as controls to demonstrate the purity
the large sample assumption of the two-sample t-test was not of the fractions [36].
satisfied [45]. The Wilcoxon rank sum test for two samples, a We then sought to determine whether F-PLS3 expres-
nonparametric analogue to the t-test test for two independent sion enhances TRAIL-induced apoptosis. The Sub G1
samples was applicable under the present setting and was DNA content assay demonstrated that, although there was
used to compare treatment group medians [46]. A two-sided less than 2% death in untreated parental Jurkat cells or
significance level of P \ 0.10 was considered statistically Jurkat cells with ectopic expression of F-PLS3, treatment
significant instead of the commonly used significance level of parental cells with TRAIL resulted in 15% cell death
of P \ 0.05. The less restrictive significance level was (Fig. 1d). Interestingly, treating cells expressing F-PLS3
selected to counter-balance not only the small treatment with TRAIL resulted in 32% cell death (Fig. 1d), indicat-
group sample sizes but to take into consideration that the ing that F-PLS3 sensitizes Jurkat cells to TRAIL-induced
Wilcoxon test, as a nonparametric procedure has in general, apoptosis. On the other hand, Jurkat cells transfected with a
greater probability relative to the t-test of failing to reject a non functional mutant of F-PLS3 (F258V) did not sensitize
false null hypothesis (greater probability of committing type Jurkat cells to TRAIL- induced apoptosis (Fig. 1e).
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Apoptosis (2008) 13:845–856 849
A 45 D
40 35 27.87
35 30
Apoptosis %
Apoptosis %
30 25
25 20 13.23
20 15
15 10
5 1.33 2.37 2.17
10
5 0
– – – + +
0
F-PLS3
F-PLS3
Control
Vector
Vector
TRAIL 0 1 2 3 6 h
(10ng/ml)
TRAIL
55 Casp 8 E 35 c d e
30
Apoptosis (%)
25
43
β -Actin 20
15 b
Time (h) – 1 2 3 6 10
TRAIL (10ng/ml) 5 a
– + + + +
0
CONTROL
Control + - - - - - -
VECTOR
F-PLS3
B Vector - + - - - + -
F-PLS3 - - + - + - -
35 TRAIL - - - + + - +
F-PLS3(F258V) - - - - - + +
Wilcoxon signed-rank test :
43 β -Actin a) Vector vs. F-PLS3 (p=1.000)
b) Vector vs. TRAIL (p=0.0809)
c) F-PLS3 vs. TRAIL+ F-PLS3 (p=0.0809)
d) TRAIL vs. TRAIL + F-PLS3 (p=(0.0809)
Cytosolic Mitochondrial e) TRAIL + F-PLS3 vs. F-PLS3(F258V)+ TRAIL (p=0.0809)
C
Control
F-PLS3
F-PLS3
Control
Vector
Vector
35
60 Hsp 60
24 GRB 2
Fig. 1 PLS3 is localized to mitochondria and enhances TRAIL- from Jurkat cells. Hybridizations with antibodies to Hsp60 and GRB2
induced apoptosis. (a). TRAIL activates caspase-8 and induces were used as loading controls for the mitochondrial and cytosolic
apoptosis in a time-dependent manner. Jurkat cells were treated with fractions, respectively. (d) PLS3 sensitizes Jurkat cells to TRAIL-
10 ng/ml TRAIL for 0, 1, 2, 3 and 6 h. The apoptotic, subG1, induced apoptosis. Jurkat cells stably transfected with pc DNA 3.1
population were measured by FACS analysis. Western blot analysis FLAG or pcDNA 3.1 FLAG PLS3 were treated with TRAIL for 6 h.
was carried out to measure caspase-8 activation. (b) PLS3 is Jurkat cells were then recovered and stained with propidium iodide
ectopically expressed in Jurkat cells. Western blot analysis confirming (Roche, USA). Sub G1 DNA content was analyzed by FACS using
expression of PLS3 in stably transfected Jurkat cells. b-actin was used the fluorescent channel 1 (FL-1). Expression of PLS3 significantly
as a loading control. (c) PLS3 is predominantly localized to the enhanced apoptosis by more than two folds. (e) Expression of mutant
mitochondria. Western blotting indicates that expression of PLS3 is PLS3(F258V) reduced TRAIL-mediated apoptosis to 20% as com-
limited to the mitochondrial rather than the cytosolic fraction isolated pared to 32% in PLS3 expressing Jurkat cells
PLS3 expression in Jurkat cells compromises pores in the outer mitochondrial membrane [34, 35, 41].
mitochondrial integrity following TRAIL treatment However, the signaling events responsible for the changes
in cardiolipin that prime mitochondria for the pro-apoptotic
Cardiolipin (CL) present in mitochondrial membranes is action of death ligands are not well characterized. In order
highly susceptible to peroxidation and/or degradation in to determine whether F-PLS3 activity also enhanced
response to apoptosis [37, 50] and modulates the action of TRAIL-induced modification of cardiolipin [33], we first
Bid, a BH3-only member of the Bcl-2 family which monitored the cardiolipin status within live cells using
cooperates with either Bak or Bax in forming proteolipid conventional staining with the specific dye, NAO [33].
123
850 Apoptosis (2008) 13:845–856
NAO staining (Fig. 2a, b) revealed that treatment of a greater than 30% decrease in NAO staining in F-PLS3
parental Jurkat cells with TRAIL for 1 h resulted in a 15% expressing cells (Fig. 2a). We have recently reported
decrease in NAO staining. Interestingly, TRAIL resulted in that an imbalance in the cellular homeostasis of
A D 35
Vector Vector+TRAIL 30
Apoptosis %
PLS3 PLS3+TRAIL 25
20
14.03
15
10 6.26
5 2.13 2.16 2.63 2.56
1.93
0
PLS3+TRAIL
TRAIL - - - + + + +
Vector+TRAIL PLS3+D609
Control
F-PLS3
Vector+D609
F-PLS3
F-PLS3+D609
Vector
Vector
+TRAIL 10ng/ml
Vector+D609
+TRAIL
B 35
c E
30 d
% Decrease in NAO
25
20 b
15
10 a
5
Vector Vector +TRAIL
0
- - - + + + + TRAIL
Control
F-PLS3
Vector+D609
F-PLS3
F-PLS3+D609
Vector
Vector
10ng/ml
C
PLS3 RNAI
30
% Decrease in NAO
25
8.16
20
15.25
PLS3 15
10
IB: Anti-PLSCR3
5
β-actin
0
IB: Anti-Actin - + - + TRAIL
Control RNAi PLS3 RNAi
Fig. 2 PLS3 expression promotes scrambling of mitochondrial was suppressed in Jurkat cells transfected with siRNA against PLS3
membrane lipid. Jurkat cells, either parental or stably transfected compared with Jurkat cells transfected with scrambled siRNA control
with PLS3, were treated with TRAIL for 1 h. Cells were then (Fig. 2c lower panel) from 15.25 to 8.16 which is 46.5% reduction.
recovered and stained with 10-N-Nonyl acridine orange (NAO), a (d) Apoptosis was assessed by DNA content analysis by means of
highly specific stain for cardiolipin in the mitochondria. (a–b) NAO flow cytometry. PLS3 significantly enhanced TRAIL-mediated
assay analysis of cardiolipin. Expression of PLS3 significantly alters apoptosis to 15% as compared to 5% in parental cells and this
TRAIL-induced NAO staining to 30% inhibition as compared with enhancement was blocked by D609. (e) Electron microscopic analysis
15% inhibition in parental cells treated with TRAIL. A pharmaco- of mitochondria structure. Parental cells and PLS3 expressing cells
logical inhibitor or phospholipases, D609, blocked this change of treated with or without 10 ng/ml TRAIL for 2 h. Irregular mitochon-
NAO-staining. (c) Western blotting analysis showing knock-down of drial appearance and loss of cristae was detected in PLS3 expressing
PLS3 siRNA-treated Jurkats cells. Actin is included as a loading cells. The micrographs are shown at the same magnification
control (Fig. 2c upper panel). Cardiolipin changes during apoptosis
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Apoptosis (2008) 13:845–856 851
phosphatidylcholine (PC) caused by altered phospholipase panel). NAO staining revealed that TRAIL-induced cardi-
activity results in TRAIL-induced alterations in cardiolipin olipin changes during apoptosis was suppressed in Jurkat
[5, 36] Therefore, in order to determine whether phos- cells transfected with siRNA against PLS3 compared with
pholipase activity is involved in the F-PLS3 mediated Jurkat cells transfected with scrambled siRNA control
changes in cardiolipin, we utilized a pharmacological (Fig. 2c lower panel) from 15.25 to 8.16 which is 46.5%
inhibitor of phospholipases known as D609 [42]. This reduction. These observations indicate that TRAIL-induced
compound has been generally considered to inhibit PC- changes in cardiolipin (change the mitochondrial potential)
PLC as well as PLD activity and has been shown to protect is mediated at least partially a PLS3-dependent manner.
cells from Fas-induced apoptosis as well as TRAIL- Moreover, our data suggests that cardiolipin externalization
induced apoptosis [36, 42–44]. As shown in Fig. 2a, b, the precedes activation of execution caspases, as 1 h of TRAIL
F-PLS3-induced changes in NAO staining were attenuated treatment did not result in high levels of apoptosis
by D609 indicating that PLS3 can mediate early changes in (Fig. 2d), while there was significant level of cardiolipin
mitochondrial cardiolipin content that is dependent on remodeling (as measured by NAO staining) at this time
phospholipase C and D activity. We further investigated point (Fig. 2b).
cardiolipin changes in response to TRAIL treatment using We next investigated the morphology of mitochondria in
a siRNA pool against PLS3 to silence PLS3 (Fig. 2c upper these cells by electron microscopic analysis. As shown in
F-PLS3+TRAIL
A C10 c
9 d
8
F-PLS3
Activity (Fold)
Control
TRAIL
Vector
7
6
5
15 Cytochrome C 4 b
3
60 2 a
Hsp 60
1
Mitochondrial fraction 0
Control
Vector
F-PLS3
Vector
Vector+LEHD
F-PLS3
F-PLS3+LEHD
F-PLS3+TRAIL
F-PLS3
Control
TRAIL
Vector
TRAIL
15 Cytochrome C Wilcoxon signed-rank test : a. Vector vs. PLS3 (p=0.6625)
GRB 2 b. Vector vs. TRAIL (p=0.0809)
24
c. PLS3 vs. PLS3 +TRAIL (p=0.0809)
Cytosolic fraction
d. TRAIL vs. PLS3+ TRAIL (p=0.0809)
F-PLS3+TRAIL
CONTROL
VECTOR
Vector F-PLS3
F-PLS3
TRAIL
B D
0 1 3 6 0 1 3 6 h TRAIL
kDa
45 Pro-Caspase 9 Pro-Caspase 9 46.5
p37
Cleaved Cas 9 p37
p35
Cleaved Cas 9 p35
30
43 β-Actin 43 β-Actin
Fig. 3 PLS3 expression enhances TRAIL-mediated activation of the performed with antibodies to procaspase-9. (Human Specific. Cell
mitochondrial apoptotic pathway. (a) Expression of PLS3 enhanced Signaling cat # 9502). The molecular mass of the inactive pro-form of
the TRAIL-mediated release of cytochrome c from the mitochondrion the caspase-9 (48 kD) is indicated by arrows. (c) Caspase-9 activity
to the cytoplasm. Mitochondrial and cytoplasmic fractions of TRAIL- was analyzed using a caspase-9 fluorescent assay to confirm the
treated PLS3 stably transfected Jurkat cells were analyzed by Western Western blotting results. TRAIL treatment of parental cells resulted in
blotting with antibodies against cytochrome c (mouse anti cyto- a 3-fold activation of caspase-9, and yielded a 9-fold activation of
chrome c, clone 7H8.2C12. BD Bioscience). Staining for Hsp 60 and caspase-9 when PLS3 was expressed. A caspase-9 inhibitor (LEHD)
GRB 2 were used as loading controls for the mitochondrial and was used to demonstrate that the activity of PLS3 was caspase-9-
cytoplasmic fractions, respectively. Data shown are representative of specific. (d) Time course analysis (0, 1, 3 and 6 h) of activation of
3 independent experiments. (b) PLS3 enhances the TRAIL-induced caspase-9 in TRAIL-treated PLCRS3 stably transfected Jurkat cells.
proteolytic cleavage of caspase-9. PLCRS3 stably transfected Jurkat Expression of PLS3 in Jurkat cells resulted in a significant change in
cells were treated with TRAIL for 6 h. Western blot analysis was the time course of caspase-9 processing
123
852 Apoptosis (2008) 13:845–856
Fig. 2e, F-PLS3 expression causes some changes in the activation and cleavage in F-PLS3-expressing cells was
morphology of mitochondria. Notably, when F-PLS3 observed in as early as 3 h, compared to the 6 h necessary
expressing cells were treated with TRAIL (1 h), there was for cleavage of caspase-9 in parental cells.
a significant morphological change in the mitochondria as These studies demonstrate that F-PLS3 promotes early
compared with parental cells. As shown in Fig. 2e, F-PLS3 mitochondrial changes in response to death receptor-induced
expressing cells that were treated with TRAIL exhibited an apoptosis, hence facilitating both the release of apoptogenic
apparent disruption of the inner membrane and cristae. factors and the full activation and processing of caspase-9.
Micrographs are shown at the same magnification. Toge-
ther, these studies demonstrate that PLS3 expression alters PLS3 expression in Jurkat cells enhances
the integrity of mitochondrial membrane that is dependent TRAIL-mediated activation of caspases-3 and -8
on activity of phospholipases.
It has been previously demonstrated that caspase-9 acti-
PLS3 expression in Jurkat cells enhances vation leads to the processing and activation of caspase-3
TRAIL-mediated activation of the mitochondrial [25, 36, 40, 51]. Additionally, caspase-3 appears to be an
apoptotic pathway
F-PLS3+TRAIL
Localization of PLS3 to the mitochondrion and its
A
involvement in translocation of cardiolipin indicates that
F-PLS3
Control
TRAIL
Vector
PLS3 may be a critical component for the initiation and/or
reinforcement of cell death pathways at mitochondria.
Thus, we sought to determine whether expression of PLS3 48 Pro-Caspase 3
potentiated the activation of apoptotic events downstream
43 β-Actin
of mitochondria, such as the release of apoptogenic factors
(i.e., cyt c) and the activation of caspase-9. As shown in
Fig. 3, we found that while there was no release of cyto- B 35 c
d
chrome c from the mitochondria to the cytosol in parental 30
Activity (Fold)
25
and F-PLS3-expressing cells, TRAIL treatment of parental
20
cells for 6 h resulted in minimal release of cytochrome c,
15
but significant increase in cytochrome c release was b
10
detected by Western blot analysis in F-PLS3-expressing a
5
cells that were treated with TRAIL (Fig. 3). These data 0
Control
F-PLS3
Vector+DEVD
F-PLS3+DEVD
vector
vector
123
Apoptosis (2008) 13:845–856 853
F-PLS3
F-PLS3
b Fig. 5 Expression of PLS3 enhances the activation of caspase-8 in
Control
Vector
Vector
TRAIL-treated cells. (a) Western blot analysis of Jurkat cells
A demonstrated that PLS3 enhanced TRAIL-induced proteolytic cleav-
- - - + + TRAIL age of caspase 8. The molecular mass of the inactive proform
Casp-8 (55 kDa) of caspase 8 is indicated (arrow). (b) A caspase-8
55 fluorescent activity assay confirmed the Western blot results and
Casp-8 p40
demonstrated that expression of PLS3 significantly enhanced the
Casp-8 p20 TRAIL-induced activation of caspase-8. TRAIL treatment of parental
cells resulted in a 3-fold activation of caspase-8, while expression of
PLS3 in these cells yielded a 14-fold activation of caspase-8 upon
43 β-Actin TRAIL treatment. A caspase-8 inhibitor (IETD) was used to show
that the activity of PLS3 was caspase-8-dependent. (c) Ectopic
expression of PLS3 in Jurkat cells enhances TRAIL-induced activa-
B 16 13.5 tion and proteolytic cleavage of the initiator caspase-8. Jurkat cells
14 stably transfected with pcDNA 3.1 FLAG or pcDNA 3.1 FLAG PLS3
12 were treated with TRAIL for 6 h. Western blot analysis with
Apoptosis (%)
Vector
Control
F-PLS3
TRAIL
123
854 Apoptosis (2008) 13:845–856
F-PLS3+TRAIL
amino acid sequence contains a conserved PKCd( phos-
Vector+TRAIL
phorylation site [57], and PLS3 has been shown to be a high
affinity substrate of PKCd( [34, 58]. As, PKCd activation is
F-PLS3
Control
Vector
A
dependent on caspase mediated cleavage resulting in the
kDa release of a constitutively active kinase domain [45], we
66 PKCδ hypothesized that TRAIL-induced regulation of PKCd
might be involved in enhancement of apoptosis by PLS3.
Cleaved PKCδ In order to test this premise, we analyzed the TRAIL-
30
induced cleavage of PKCd in the parental as well as
37
F-PLS3-expressing Jurkat cells. Figure 6 demonstrates an
PLS3
enhancement of PKCd(cleavage in F-PLS3-expressing cells
β.actin (Fig. 6a) and this is accompanied by an increase in the
43
apoptotic response of these cells to TRAIL (40% as com-
pared to 20% cell death in parental Jurkat cells) (Fig. 6b).
B 45 Thus, the TRAIL-induced activation of PKCd regulates the
40 37.5
F-PLS3
Vector
Vector
123
Apoptosis (2008) 13:845–856 855
123
856 Apoptosis (2008) 13:845–856
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