Yonago Acta medica 2012;55:11–20

Mechanisms Underlying Production and Externalization of
Oxidized Phosphatidylserine in Apoptosis: Involvement ofMitochondria
Atsushi Yamashita, Hitoshi Morikawa, Naoko Tajima, Mari Teraoka, Chiaki Kusumoto,
Kazuhiro Nakaso and Tatsuya Matsura
Division of Medical Biochemistry, Department of Pathophysiological and Therapeutic Science,
School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8503, Japan

The present study was performed by using selective inhibitors of caspase-8 and caspase-3functioning upstream and downstream from mitochondria, respectively to determine
whether mitochondria are involved in the mechanisms underlying production and externalization of oxidized phosphatidylserine (PSox) during Fas-mediated apoptosis. Treatment with anti-Fas antibody induced caspase-3 activation, chromatin condensation, releaseof cytochrome c (cyt c) from mitochondria into the cytosol as well as production of PSox
and its exposure to the cell surface in Jurkat cells. Inhibition of caspase-8 by pretreatment with Z-IETD-FMK, a membrane permeable selective caspase-8 inhibitor reduced
mitochondrial cyt c release, the amount of PSox not only within but also on the surface ofJurkat cells, caspase-3 activation, and apoptotic cell number after treatment with anti-Fas
antibody. In contrast, Z-DEVD-FMK, a membrane permeable selective caspase-3 inhibitor was unable to inhibit cyt c release, and the amount of PSox both within and on thesurface of the cells after anti-Fas antibody, although it suppressed caspase-3 activation andapoptosis. Thus, these results strongly suggest that mitochondria play an important role inproduction of PSox and subsequent its externalization during apoptosis.
Key words: apoptosis; caspase inhibitor; cytochrome c; mitochondria; oxidized phosphatidylserine
Phospholipids in plasma membrane are distributed
asymmetrically between two leaflets (Bretscher,1972), and the choline-containing phospholipids,
phosphatidylcholine and sphingomyelin, residemainly in the outer (extracellular) leaflet, whereas
the aminophospholipids, phosphatidylserine (PS)and, to a lesser extent, phosphatidylethanolamine(PE), are confined to the inner (cytosolic) leaf-

let (Verkleij and Post, 2000). This asymmetry is
considered to be established and maintained by atleast three lipid translocators: i) ATP-dependent
flippases, members of type 4 P-type ATPases, also
referred to as aminophospholipid translocases,that are specific for aminophospholipids, with apreference for PS over PE, and catalyze the inward
translocation of aminophospholipids (Folmer etAbbreviations: AMC, 7-amino-4-methyl-coumarin; BHT, butylated hydroxytoluene; CHAPS, 3-[(3-cholamidopropyl)
dimethylammonio]-1-propane-sulfonate; CL, cardiolipin; CLox, oxidized CL; cyt c, cytochrome c; DTT, dithiothreitol; DISC,
death-inducing signaling complex; FCS, fetal calf serum; HPTLC, high-performance thin-layer chromatography; LPE,
1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine; MOMP, mitochondrial outer membrane permeabilization; PBS,
phosphate-buffered saline; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PMSF, phenylmethyl-sulfonyl fluoride;PS, phosphatidylserine; PSox, oxidized PS; ROS, reactive oxygen species; SAGOOH, 1-stearoyl-2-arachidonylglycerol hydroperoxide; TMPD, N,N,N’,N’-tetramethyl-p-phenylenediamine dihydrochloride

11

A. Yamashita et al.

externalization (Kagan et al., 2004). Therefore, we
determined whether departure of cyt c from mitochondria is required for production of PSox as wellas its externalization during apoptosis using two
selective caspase inhibitors functioning upstream
or downstream from mitochondria in the present
study.

al., 2009); ii) another ATP-dependent translocator
floppases, members of ATP-binding cassette transporters, which catalyze the efflux of phospholipids
from the inner to the outer leaflet with little selectivity for polar head group of the phospholipids(Contreras et al., 2010) and iii) energy-independent
scramblases that are non-selective and catalyze
bidirectional transbilayer movement of phospholipids Ca 2+-dependently but ATP-independently(Bevers and Williamson, 2010). It is generally accepted that PS predominantly localized in the innerleaflet migrates to the outer leaflet of the plasma
membrane during apoptosis, and is recognized bymacrophages as an “eat-me” signal, resulting insuccessful clearance of apoptotic cells (Verhoven etal., 1995). This PS egress during apoptosis seems
to be associated with the inhibition of flippases aswell as the activation of scramblases in the plasma
membrane (Fadeel et al, 2010).
The generation of reactive oxygen species(ROS) and subsequent oxidative stress are commonly involved in the initiation and/or the execution phases of apoptosis (Banki et al., 1999; Li etal., 1999). Mitochondrial permeability transition
and the release of cytochrome c (cyt c) from mitochodria into the cytosol during apoptosis are related
to excessive ROS generation (Cai and Jones, 1998;
Matsura et al., 2005). Recent studies have reported
almost simultaneous oxidation and externalizationof an anionic phospholipid PS during apoptosis(Matsura et al., 2003; Kagan et al., 2004). Selective
oxidation of another anionic phospholipids such as
cardiolipin (CL) and phosphatidylinositol (PI) has
also been induced during apoptosis (Matsura et al.,
2003; Kagan et al., 2004; Matsura et al., 2005).
We revealed that H2O2 is important not only
for PS oxidation but also for its externalization during apoptosis using catalase overexpressing HP100
cells (Matsura et al., 2004). We also found that
there is oxidized PS (PSox) both within and on thesurface of Jurkat cells undergoing apoptosis after
treatment with anti-Fas antibody (Matsura et al.,2005).
It has been suggested that peroxidase activity of cyt c is closely related to PS oxidation andMaterials and Methods
Materials
Fluorescamine was purchased from Sigma Chemical (St. Louis, MO). Anti-Fas antibody (Clone CH11) was purchased from Medical and Biological
Laboratories (Nagoya, Japan). Fetal calf serum(FCS) was purchased from PAA Laboratories
GmbH (Linz, Austria). Membrane-permeable caspase-8 inhibitor (Z-IETD-FMK) and caspase-3 inhibitor (Z-DEVD-FMK) were purchased from R &D Systems (Minneapolis, MN). Methanol (HPLCgrade), acetone (HPLC-grade), acetic acid, iodine,ninhydrin spray, butylated hydroxytoluene (BHT)
and N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD) were obtained from Wako
Pure Chemical Industries (Osaka, Japan). Chloroform (HPLC-grade) was purchased from Kanto
Kagaku (Tokyo, Japan). 7-Amino-4-methyl-coumarin (AMC) and acetyl-Asp-Glu-Val-Asp-AMC(Ac-DEVD-AMC) were purchased from Peptide
Institute (Osaka, Japan). RPMI 1640 medium
with phenol red was obtained from Nissui Pharmaceutical (Tokyo). RPMI 1640 medium without
phenol red was obtained from Cosmo Bio (Tokyo).
Hoechst 33342 was purchased from Molecular
Probes (Eugene, OR). High-performance thin-layer
chromatography (HPTLC) plates (10 × 10 cm,
Silica Gel 60) were obtained from Merck (Darmstadt, Germany). 1-Oleoyl-2-hydroxy-sn-glycero-3phosphoethanolamine (LPE) was purchased from
Avanti Polar Lipids (Alabaster, AL). 1-Stearoyl2-arachidonylglycerol hydroperoxide (SAGOOH)
was synthesized as described previously (Takekoshi
et al., 1995). All other chemicals used were of analytical grade.
12Mitochondria involved in PSox productionof caspase activity, 10 µg of lysate diluted to 20 µL
with lysis buffer was mixed with 20 µL of 2 × ICE
buffer containing 40 mM HEPES-KOH (pH 7.4),
20% (v/v) glycerol, 1 mM PMSF and 4 mM DTT
supplemented with 40 µM Ac-DEVD-AMC (afluorogenic peptide substrate) and incubated for 60
min at 37 ˚C. After 60 min, 160 µL of distilled water was added and the fluorescence was measured
in a CytoFluor II (Applied Biosystems, Foster City,CA) fluorescence microplate reader using excitation
at 360 ± 40 nm and emission at 460 ± 40 nm. One
unit of caspase activity was defined as the amountof enzyme required to release 1 pmol AMC per
minute at 37 ˚C.
The protein concentration of cell lysates wasmeasured by the method of Bradford (Bradford,
1976).

Cell cultures
Jurkat T cell line was obtained from Riken Cell
Bank (Tsukuba, Japan), and grown in RPMI 1640
medium supplemented with 10% heat-inactivated
FCS, 2 mM glutamine and antibiotics (100 units/mL penicillin, 100 µg/mL streptomycin) at 37 ˚Cin a humidified incubator with 5% CO2. All the experiments were performed using RPMI 1640 medium containing 10% FCS. Cells were seeded at aconcentration of 2 × 105 cells/mL and logarithmic
growth was maintained by passaging every 2 or 3
days.
Determination of apoptotic cells
Apoptotic cells were assessed by nuclear morphology as described previously (Matsura et al., 2002,
2004). Jurkat cells (1.0 × 106 cells/mL) were incubated with anti-Fas antibody at 37 ˚C, washedwith phosphate-buffered saline (PBS), and thenfixed with 1% glutaraldehyde overnight. Samples
were centrifuged, resuspended in PBS, stainedwith Hoechst 33342 (1 mM), and then mounted ona glass slide, and observed under fluorescence microscope. Results were expressed as the percentageof the cells showing characteristic nuclear morphological features of apoptosis (nuclear condensationand fragmentation) relative to the total number ofcounted cells (≥ 200 cells per each point).

Flow cytometry of PS externalization
Annexin V binding to cells was determined using
a commercially available Annexin V-FITC Apoptosis Detection Kit (Medical & Biological Laboratories, Nagoya, Japan) and flow cytometry as previously described (Matsura et al., 2005). Briefly, after
treatment of cells (1 × 106 cells/mL) with anti-Fas
antibody, cells were recovered and washed oncewith pre-warmed RPMI 1640 medium without
phenol red. Cells were incubated with fluorescein
isothiocyanate-conjugated annexin V (0.5 µg/mL)
for 5 min and then were collected by centrifugation
and washed with binding buffer. Propidium iodide(0.6 µg/mL) was added, and cells were immediately
analyzed with an Epics cytofluorometer (Beckman
Coulter, Fullerton, CA).

Measurement of caspase-3 activity
The activity of caspase-3 was determined as described previously (Matsura et al., 2002, 2004).
Briefly, after treatment of Jurkat cells (1 × 106 cells/mL) with anti-Fas antibody (200 ng/mL), cells werecollected, washed in PBS, and lysed for 20 min
on ice in lysis buffer containing 10 mM HEPESKOH (pH 7.4), 2 mM EDTA, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate)(CHAPS), 1 mM phenylmethyl-sulfonyl fluoride(PMSF) and 5 mM dithiothreitol (DTT). The suspensions were centrifuged at 4 ˚C and the supernatants were collected as lysates. For measurement

Determination of cyt c release
Jurkat cells were treated with 200 ng/mL anti-Fas
antibody, harvested and washed three times with
ice-cold PBS. Cells were resuspended in isotonic
buffer containing 10 mM HEPES, 0.3 M mannitol,
0.1% bovine serum albumin, 5 mM EDTA and 1
mM PMSF supplemented with 0.1 mM digitonin(5 × 107 cells/mL), left on ice for 5 min and imme13

A. Yamashita et al.

diately centrifuged at 8,500 × g for 5 min at 4 ˚C.
The supernatant collected was used as the cytosolic
fraction. The cyt c amount in the cytosolic fraction
was measured by Western blotting using cyt c (6H2)
monoclonal antibody (Santa Cruz Biotechnology,
Santa Cruz, CA) (1:1000) as described previously(Matsura et al., 2005). The densitometric quantitative analysis was performed by using NIH Image.

N2 stream. The phospholipid classes in the extracts
were analyzed by two-dimensional HPTLC using a solvent system of chloroform:methanol:water(55:35:4, v/v/v) in the first direction and chloroform:acetone:methanol:acetic acid:water (50:20:10:10:5,
v/v/v/v/v) in the second. The phospholipids werevisualized by exposure to iodine vapor. The identity of each phospholipid was established by comparison with the Rf values of authentic standards(Phospholipid Kit; DOOSAN Serdary ResearchLaboratories, Yongin City, Korea). Fluorescaminemodified PS and PE were localized by exposure of
HPTLC plates to UV light with a Gel Doc 2000
(Bio-Rad Laboratories, Hercules, CA). LPE (2nmol) was used as an internal standard for fluorescamine-modified phospholipids. We photographed
the plate, scanned PS, PE and LPE modified by fluorescamine (referred to as mPS, mPE and mLPE,
respectively) in control and anti-Fas antibodytreated cells, and quantified the ratios mPS/mLPE
in both cells by using NIH Image. In addition, we
measured the phosphorus content in the total lipids
extracted from both control and Fas-ligated cells(8 × 107 cells) and expressed the content of mPS as
the following relative density: 104 × mPS/mLPE/
phosphorus of 8 × 107 cells.
To determine PSox on the cell surface, colorimetric spray detection with TMPD was carriedout (Matsura et al., 2005). After chromatography,
the plate was dried under an N2 stream and sprayed
immediately with freshly prepared 1% (w/v) TMPD
in methanol:water:acetic acid (50:50:1, v/v/v). Synthetic SAGOOH was used as an external standard
as described previously (Matsura et al., 2005). We
quantified the ratio of TMPD-visualized phospholipid/TMPD-visualized SAGOOH in control andanti-Fas antibody-treated cells by using NIH Imageand expressed the content as the following relative
density: 10 4 × TMPD-visualized phospholipid/
TMPD-visualized SAGOOH/phosphorus of 8 ×
107 cells. Then, the same plate was subjected to
UV light exposure to determine whether the PSox
was externalized. Thereafter, the content of PSox
exposed on the cell surface was expressed as thefollowing relative ratio: (TMPD-visualized mPS/

Fluorescamine labeling of externalized aminophospholipids and measurement of phosphorus
Labeling of externalized aminophospholipids,
PS and PE with membrane-impermeable fluorescamine, a probe for visualizing lipids that contain
primary amino groups, was carried out by methods
previously described (Matsura et al., 2005). Briefly,
Jurkat cells (1 × 106 cells/mL) treated with antiFas antibody (200 ng/mL) for 8 h at 37 ˚C weresuspended in labeling buffer (150 mM NaCl, 5 mM
KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM NaHCO3,
5 mM glucose, and 20 mM HEPES/KOH, pH 8.0).
Cells were gently agitated in the presence of fluorescamine (200 mM) for 30 s. The reaction was
stopped by the addition of 40 mM Tris-HCl, pH 7.5.
Cells were counted again and collected by centrifugation, and lipids in 8 × 107 cells were extracted
by the Folch procedure (Folch et al., 1957) andanalyzed by HPTLC. To protect lipids from autoxidation during the extraction procedure, the antioxidant BHT (1.3 mM) was added to each sample.
Aliquots of the cells (1 × 107) were stored at –80
˚C until phosphorus measurement. The phosphorus
content of lipids was determined by the methodof Chalvardjian and Rudnicki (Chalvardjian and
Rudnicki, 1970).
Detection of externalized and oxidized PS
by HPTLC
Externalized and oxidized PS was determined as
described previously (Matsura et al., 2005). Briefly,
lipid extracts redissolved in chloroform:methanol(2:1, v/v) were applied to the HPTLC plate under an
14

Mitochondria involved in PSox production

TMPD-visualized SAGOOH)/(mPS/mLPE). Additionally, to determine unmodified PS, i.e., PS
within the cells, the same plate was sprayed withninhydrin, another probe that reacts with primaryamino groups.

2B) by 87% and 54%, respectively, compared with
anti-Fas antibody alone.
Given these results, we used 5 µM as the concentration of caspase inhibitors in the following
experiments.

Statistical evaluations

Effect of caspase inhibitors on PS externalization in Jurkat cells following Fas ligation

Data were expressed as mean ± SE. Changes invariables for different assays were analyzed either
by Student’s t test (single comparisons) or by oneway ANOVA for multiple comparisons. Differences were considered to be significant at P < 0.05.

We next examined whether there is difference inthe effect of caspase-8 inhibitor and caspase-3 inhibitor functioning upstream and downstream frommitochondria, respectively, on PS externalization
during Fas-mediated apoptosis. As shown in Fig. 3,

Results
Inhibition by caspase inhibitors of Fasmediated apoptosis and caspase-3 activation
According to the results of our previous experiments (Matsura et al., 2005), we induced apoptosis
by treatment of Jurkat cells with 200 ng/mL antiFas antibody for 8 h at 37 ˚C. To determine theconcentration of caspase inhibitors required toinhibit Fas-mediated apoptosis in Jurkat cells, we
treated the cells with Z-IETD-FMK, a membranepermeable caspase-8 inhibitor, or Z-DEVD-FMK,a membrane-permeable caspase-3 inhibitor atconcentrations ranging from 0.5 to 5 µM 30 min
before addition of anti-Fas antibody. Z-IETD-FMK
inhibited Fas-mediated apoptosis in a dose dependent manner, and the numbers of apoptotic cells
were significantly reduced to 14% of cells exposed
to anti-Fas antibody alone by treatment with 5 µM
Z-IETD-FMK (Fig. 1A). Pretreatment of the cellswith Z-IETD-FMK also inhibited Fas-mediatedcaspase-3 activation dose-dependently (Fig. 1B).
Treatment with 5 µM Z-IETD-FMK significantly
inhibited caspase-3 activity by 46% compared with
anti-Fas antibody alone (Fig. 1B). Z-DEVD-FMK
pretreatment similarly inhibited Fas-mediatedapoptosis in a dose-dependent manner. Treatment
with 5 µM of Z-DEVD-FMK suppressed apoptotic
cell number (Fig. 2A) and caspase-3 activity (Fig.

Fig. 1. Effect of caspase-8 inhibitor on Fas-mediatedapoptosis and caspase-3 activation in Jurkat cells. Cells
were incubated with anti-Fas antibody (200 ng/mL) in
10% fetal calf serum containing RPMI 1640 for 8 h at 37
˚C under a 5% CO2/95% air atmosphere. Z-IETD-FMK,a caspase-8 inhibitor at the indicated concentrations wasadded 30 min before anti-Fas antibody treatment. The
ratio of apoptotic cells (A) was determined by examining nuclear morphology after Hoechst 33342 staining.
Caspase-3 activities (B) were measured with a fluorescent
substrate as described in Materials and Methods. Data
points represent the means ± SE (n = 3). *P < 0.01 versus
control, †P < 0.01 versus anti-Fas antibody alone.

15

A. Yamashita et al.

ever, Z-DEVD-FMK was unable to suppress Fasmediated mitochondrial cyt c release (Fig. 4).

Z-IETD-FMK inhibited PS externalization in Jurkat cells after Fas ligation by 40%. In contrast, ZDEVD-FMK failed to suppress PS externalization.

Effect of caspase inhibitors on the amountof PSox transmigrated to the cell surface
during Fas-mediated apoptosis

Effect of caspase inhibitors on release of
mitochondrial cytochrome c in Jurkat cells
following Fas ligation

As reported previously (Matsura et al., 2005), we
developed the fluorescamine-TMPD method todetect PSox externalized after Fas ligation using
two probes on HPTLC chromatogram: TMPD for
phospholipid hydroperoxides and fluorescamine
for external aminophospholipids. The amount of
externalized PS, i.e., mPS increased after treatment of the cells with anti-Fas antibody (200 ng/mL) for 8 h (Fig. 5A). TMPD-visualized mPS, i.e.,oxidized mPS also increased after Fas ligation (Fig.
5B). Z-IETD-FMK reduced both levels of mPS
and oxidized mPS to control level (Figs. 5A and B).
In contrast, Z-DEVD-FMK failed to suppress bothlevels (Figs. 5A and B). Semi-quantitatively, Fas
ligation increased the amount of PSox on the cellsurface 1.7 times compared with control (Fig. 5C).
Z-IETD-FMK significantly decreased the amountby 38% (Fig. 5C). However, Z-DEVD-FMK did

To examine effect of caspase inhibitors on the release of cyt c from mitochondria into the cytosol,
we measured cyt c in the cytosol of Jurkat cells at 8h after treatment with anti-Fas antibody by Western
blotting. Z-IETD-FMK prevented the efflux of cyt
c from mitochondria into the cytosol during Fasmediated apoptosis in Jurkat cells (Fig. 4). How-

Fig. 2. Effect of caspase-3 inhibitor on Fas-mediatedapoptosis and caspase-3 activation in Jurkat cells. Cells
were incubated with anti-Fas antibody (200 ng/mL) as
described in the legend to Fig. 1. Z-DEVD-FMK, a caspase-3 inhibitor at the indicated concentrations was added
30 min before anti-Fas antibody treatment. The ratio of
apoptotic cells (A) was determined by examining nuclear
morphology after Hoechst 33342 staining. Caspase-3 activities (B) were measured with a fluorescent substrate as
described in Materials and Methods. Data points represent
the means ± SE (n = 3). *P < 0.01 versus control, †P < 0.01
versus anti-Fas antibody alone.

Fig. 3. Effect of caspase inhibitors on phosphatidylserine(PS) exposure to the surface of Jurkat cells after treatment
with anti-Fas antibody. Cells were incubated with anti-Fas
antibody (200 ng/mL) as described in the legend to Fig.
1. Z-IETD-FMK (5 µM) or Z-DEVD-FMK (5 µM) was
added 30 min before anti-Fas antibody treatment. At theend of incubation, cells were collected and PS externalization was determined by flow cytometry with an Annexin
V-FITC Apoptosis Detection Kit as described in Materials and Methods. Data points represent the means ± SE
(n = 3). *P < 0.01 versus control, †P <0.01 versus anti-Fas
antibody alone (black column).

16

Mitochondria involved in PSox production

not inhibit the increase in the amount (Fig. 5C).
Furthermore, PS unmodified by fluorescamine, i.e.,
PS within the cells (referred to as PSin) was determined using ninhydrin staining. Although anti-Fas
antibody treatment decreased PSin, Z-IETD-FMK
but not Z-DEVD-FMK prevented the decrease
in PSin after Fas ligation (data not shown). As
mentioned above, the amount of PSin in the cells
treated with anti-Fas antibody or anti-Fas antibody
plus Z-DEVD-FMK was lesser than that in control
cells or the cells treated with anti-Fas antibody plus
Z-IETD-FMK. However, the degree of PSin oxidation was stronger in the former cells (Fig. 5B).

Fig. 4. Effect of caspase inhibitors on cytochrome c (cyt c)release into the cytosol in Jurkat cells after treatment with
anti-Fas antibody. Cells were incubated with anti-Fas antibody (200 ng/mL) as described in the legend
to Fig. 1. Z-IETD-FMK (5 µM) or Z-DEVDFMK (5 µM) was added 30 min before anti-Fas
antibody treatment. At the end of incubation,
cytosolic fractions were prepared and analyzed
for cyt c content by Western blotting as described in Materials and Methods. The resultsof densitometric analysis are shown as fold increase relative to control. Data points represent
the means ± SE (n = 3). *P < 0.01 versus control, †P < 0.01 versus anti-Fas antibody alone(black column).

LPE lysoPE
mLPE LPE modified by fluorescaminemPE PE modified by fluorescaminemPS PS modified by fluorescamine
NL neutral lipid
PE phosphatidylethanolamine
PI phosphatidylinositol
PS phosphatidylserine
SAGOOH 1-stearoyl-2-arachidonylglycerolhydroperoxideFig. 5. Effect of caspase inhibitors on oxidation of PS exposed to the surface of Jurkat cells after treatment with anti-Fas
antibody. Cells were incubated with anti-Fas antibody (200 ng/mL) as described in the legend to Fig. 1. Z-IETD-FMK(5 µM) or Z-DEVD-FMK (5 µM) was added 30 min before anti-Fas antibody treatment. At the end of incubation, total
lipids of cells were extracted after a short treatment with fluorescamine and subjected to HPTLC.
A: Fluorescamine-modified PS and PE were localized by exposure of HPTLC plates to UV light. LPE was used as aninternal standard.
B: To determine PSox on the cell surface, colorimetric spray detection with TMPD was carried out as described in Materials and Methods. SAGOOH was used as an external standard as described in Materials and Methods. Representative data from three separate experiments are shown.
C: The content of PSox exposed on the cell surface was shown as the relative ratio as described in Materials and Methods. Data points represent the means ± SE of 3 separate experiments. *P < 0.01 versus control, †P < 0.01 versus antiFas antibody alone (black column).
HPTLC, high-performance thin-layer chromatography; TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride.

17

A. Yamashita et al.

of intermembrane space proteins such as cyt c into
the cytosol and apoptosome formation responsible
for the activation of executioner caspases including
caspase-3. Jurkat cells we used in this study belong
to type II cells (Shawgo et al., 2009).
Recent studies have suggested that peroxidase
activity of cyt c is closely related to PS oxidationand externalization (Kagan et al., 2004; Bayir et al.,2006). Furthermore, it has been reported that PS
oxidation in lung epithelial A549 cells undergoing
apoptosis after Fas ligation was sensitive to a pancaspase inhibitor, Z-VAD, which inhibited cyt cefflux from mitochondria but was insensitive toa caspase-3 inhibitor, Z-DQMD, which failed toprevent mitochondrial disruption and cyt c release(Jiang et al., 2004). However, they did not examine
the effect of caspase inhibitors on PSox externalization in their study.
Previously we developed fluorescamineTMPD method to directly determine the presenceof PSox on the surface of apoptotic cells (Matsura
et al., 2005). This method is semiquantitative but
can easily detect PS externalization and oxidationon the same HPTLC plate (i.e., PSox on the cellsurface). Additionally, we can detect PSox within
the cells on the same HPTLC plate by a combination of staining with ninhydrin, another probe thatreacts with primary amino groups. We revealedby these methods that treatment of Jurkat cellswith anti-Fas antibody increased PSox within thecells and caused PSox to appear on the cell surface(Matsura et al., 2005).
To illustrate mitochondrial involvement inproduction and exposure of PSox, we examined byfluorescamine-TMPD method the effect of selective
inhibitors of caspase-8 and caspase-3 on production of PSox and its externalization during extrinsic type II cell apoptosis in the present study. As
mentioned above, caspase-8 inhibitor can block the
apoptotic signal from DISC to mitochondria. By
contrast, caspase-3 inhibitor functions downstream
from mitochondria (more precisely downstream of
apoptosome formation) to suppress characteristic
morphological and biochemical changes in apoptosis. Inhibition of caspase-8 by Z-IETD-FMK, a

Discussion
In the present study, we demonstrated using selective inhibitors of caspase-8 and caspase-3 that mitochondria play an important role in both production and externalization of PSox during apoptosis.
Caspases are a special family of proteases
characterized by a cysteine in the catalytic site andan unusual cleavage specificity for aspartic acidresidues (Grütter, 2000). To date the human caspase family has 11 members, which are classified
into two major subfamilies, the apoptotic and theinflammatory caspases (Siegel, 2006). The apoptotic caspases are further divided into two classes,
initiator and executioner caspases (Kantari and
Walczak, 2011). Caspase-8 is an initiator caspaseand caspase-3 is an executioner one.
There are two signaling pathways for apoptosis, intrinsic (mitochondrial) and extrinsic (receptor-mediated) pathways (Olsson and Zhivotovsky,2011). The intrinsic apoptosis induced by stresses
such as DNA damages, ROS, ultraviolet and anticancer drugs, involves disruption of mitochondrial
integrity accompanied by the release of cyt c into
the cytosol. In the cytosol, released cyt c binds toapoptotic protease-activating factor 1, ATP, andprocaspase-9 to form the apoptosome and lead toactivation of executioner caspases, which finally induce cell death. In the extrinsic apoptosis, extracellular cognate ligands (e.g., Fas ligand) or agonistic
antibodies (e.g., anti-Fas antibody CH-11) stimulateoligomerization of receptor (e.g., Fas), the recruitment of adaptor proteins (e.g., FADD) and initiator
procaspase-8 molecules to the cytosolic side of
the receptor to form the death-inducing signaling
complex (DISC) (Shawgo et al., 2009). Within the
DISC, procaspase-8 molecules are activated bydimerization and subsequently undergo auto-processing. Active caspase-8 can directly activate theexecutioner caspase-3 (type I cells) or cleaves thecytosolic BH3-only protein Bid to truncated Bid(tBid) (type II cells). In turn, tBid translocates to themitochondria and stimulates mitochondrial outermembrane permeabilization (MOMP), the release18

Mitochondria involved in PSox production

released into the cytosol forms cyt c/PS complexes
in the cytosolic leaflet of plasma membrane asmentioned above and probably oxidizes PS by its
peroxidase activity (Matsura et al., 2003). We previously observed using HP100 cells overexpressing
catalase derived from HL-60 cells that endogenous
H2O2 is a key oxidant for cyt c release from mitochondria as well as PS oxidation during apoptosis(Matsura et al., 2004). Taken together, these findings support strongly the notion that interaction ofcyt c/anionic phospholipid (CL in mitochondria or
PS in plasma membranes) complexes with H2O2 isimportant for PS oxidation via peroxidase activity
of cyt c.
In respect to externalization of PSox, recent
report demonstrated that flippase can recognize
PSox and PS as its substrates, and that PSox may
act as a “non-enzymatic scramblase” and stimulate scrambling of PS and/or PSox (Tyurina et al.,2004). However, its precise mechanisms remain tobe elucidated. At least, it must be clarified how theaffinity of membrane phospholipid translocators
including flippases and scramblases for PSox is.
In conclusion, the present study has shownthat mitochondria play an essential role in production of PSox and subsequent its transmigration
to the cell surface during extrinsic apoptosis byblocking signal transduction from DISC upstream
and downstream from mitochondria using selective
caspase-8 inhibitor and caspase-3 inhibitor, respectively.

membrane permeable selective caspase-8 inhibitor
reduced mitochondrial cyt c release into the cytosol, the amount of PSox not only within but also on
the surface of type II Jurkat cells, caspase-3 activation, and apoptotic cell number after treatment with
anti-Fas antibody (CH-11). In contrast, Z-DEVDFMK, a membrane permeable selective caspase-3
inhibitor was unable to inhibit cyt c release, andthe amount of PSox both within and on the surfaceof the cells after anti-Fas antibody as expected,
although it suppressed activation of the executioner
caspase-3 and apoptosis. These results strongly
suggested that mitochondrial event, especially cyt cdeparture from mitochondria plays a critical role inproduction of PSox within the cells and subsequent
its appearance on the cell surface during extrinsic
apoptosis.
It is considered that a potential mechanism
underlying PSox production during apoptosis relies on the specific interactions between positively
charged cyt c and negatively charged phospholipids
such as PS in the cytosolic leaflet of the plasma
membrane and CL in the mitochondria (Kaganet al., 2004, 2009). As for CL, high-affinity binding of CL with cyt c—accompanied by unfolding of the protein—is realized largely through anelectrostatic interface between negatively charged
phosphates on CL and positively charged lysines oncyt c, as well as through hydrophobic interactionsof CL’s acyl groups with a hydrophobic domain of
the protein (Kagan et al., 2009). PS may also bindand unfold cyt c via analogous mechanisms. It has
been reported that the interaction of cyt c with CL
causes a marked negative shift of cyt c’s redox potential, thus precluding its operation as an electron
acceptor from mitochondrial complex III (Kaganet al., 2009). It is proposed that cyt c in cyt c/CL
complexes functions as peroxidase to oxidize CL in
the presence of ROS (O2– and H2O2) produced bydisrupted mitochondrial electron transport (Kaganet al., 2009; Hüttemann et al., 2011). Since cyt c
has lower affinity for oxidized CL (CLox) than for
CL, it gets detached from membranes. Thereafter,
cyt c is released into the cytosol following stimulation of MOMP by CLox (Kagan et al., 2009). cyt cAcknowledgments: This work was supported in part by
Grants-in-Aid for Scientific Research from the Ministryof Education, Culture, Sports, Science and Technology ofJapan.
We thank Dr. Susumu Takekoshi (Department of Pathology, Tokai University School of Medicine) for supplyof 1-stearoyl-2-arachidonylglycerol hydroperoxide.
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Received December 28, 2011; accepted January 17, 2012
Corresponding author: Tatsuya Matsura, MD, PhD

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