Báo cáo khoa học: 7-Ketocholesterol-induced apoptosis Involvement of several pro-apoptotic but also anti-apoptotic calcium-dependent transduction pathways ppt

7-Ketocholesterol-induced apoptosis
Involvement of several pro-apoptotic but also anti-apoptotic
calcium-dependent transduction pathways
Arnaud Berthier, Ste
´
phanie Lemaire-Ewing, Ce
´
line Prunet, Thomas Montange, Anne Vejux,
Jean Paul Pais de Barros, Serge Monier, Philippe Gambert, Ge
´
rard Lizard and Dominique Ne
´
el
INSERM U498 – Me
´
tabolisme des lipoprote
´
ines et interactions vasculaires, Dijon Cedex, France
Oxysterols are probably the components of oxidized
low-density lipoproteins which have the strongest
involvement in the genesis and development of athero-
sclerosis [1–3]. Oxysterols mediate the early events of
atherosclerosis observed during the development of the
disease, such as the production of proinflammatory
cytokines, expression of adhesion molecules, and cyto-
toxicity to the cells of the vascular wall and mono-
cytes ⁄ macrophages [4–6]. This cytotoxicity appears to
be mainly related to the induction of apoptosis [6].
Among cytotoxic oxysterols found in atheromatous
lesions, 7-ketocholesterol is one of the most abundant
and one of the most studied [7]. In a previous

report, we demonstrated the involvement of the
calcium-dependent activation of calcineurin (PP2B)
leading to dephosphorylation of the pro-apoptotic
protein BAD in 7-ketocholesterol-induced apoptosis of
THP-1 cells. The rise in free-Ca
2+
activating calcineu-
rin is induced by the translocation of Trpc-1, a com-
ponent of the store-operated Ca
2+
entry channel, into
lipid raft domains, which are microdomains of the
plasma membrane formed by the lateral packing of
glycosphingolipids and cholesterol [8]. However, in this
study, we show that BAD was dephosphorylated at
serine 99 prior to dephosphorylation at serine 75, and
the use of calcineurin inhibitors does not completely
inhibit BAD dephosphorylation, suggesting the activa-
tion of other pro- or anti-apoptotic pathways.
Keywords
apoptosis; calcium; 7-ketocholesterol; signal
transduction; THP-1 cells
Correspondence
D. Ne
´
el, INSERM U498 – Laboratoire de
Biochimie Me
´
dicale, CHU ⁄ Ho
ˆ

pital du
Bocage, 2 Bd Mare
´
chal de Lattre de
Tassigny, BP 77908, 21079 Dijon Cedex,
France
Fax: +33 3 80 29 36 61
Tel: +33 3 80 29 50 03
E-mail:
(Received 28 January 2005, revised 11 April
2005, accepted 18 April 2005)
doi:10.1111/j.1742-4658.2005.04723.x
Oxysterols, and particularly 7-ketocholesterol, appear to be strongly
involved in the physiopathology of atherosclerosis. These molecules are
suspected to be cytotoxic to the cells of the vascular wall and mono-
cytes ⁄ macrophages, particularly by inducing apoptosis. Previous studies
have demonstrated that 7-ketocholesterol-induced apoptosis is triggered by
a sustained increase of cytosolic-free Ca
2+
, which elicits the mitochondrial
pathway of apoptosis by activation of the calcium-dependent phosphatase
calcineurin, leading to dephosphorylation of the ‘BH3 only’ protein BAD.
However, thorough study of the results suggests that other pathways are
implicated in 7-ketocholesterol-induced cytotoxicity. In this study, we dem-
onstrate the involvement of two other calcium-dependent pathways during
7-ketocholesterol-induced apoptosis. The activation of the MEK fi ERK
pathway by the calcium-dependent tyrosine kinase PYK 2, a survival path-
way which delays apoptosis as shown by the use of the MEK inhibitor
U0126, and a pathway involving another pro-apoptotic BH3 only protein,
Bim. Indeed, 7-ketocholesterol treatment of human monocytic THP-1 cells

induces the release of Bim-LC8 from the microtubule-associated dynein
motor complex, and its association with Bcl-2. Therefore, it appears that
7-ketocholesterol-induced apoptosis is a complex phenomenon resulting
from calcium-dependent activation of several pro-apoptotic pathways and
also one survival pathway.
Abbreviations
MEK 1 ⁄ 2, MAPK-Erk kinase-1 and )2; MSB, microtubule-stabilizing buffer; PTK, protein tyrosine kinases.
FEBS Journal 272 (2005) 3093–3104 ª 2005 FEBS 3093
Of the various pathways that are involved in cell
survival, the Ras ⁄ Raf ⁄ MEK ⁄ Erk pathway plays a crit-
ical role. Indeed, Ras-activated Raf operates by phos-
phorylating and activating MAPK-Erk kinase-1 and
-2 (MEK 1 ⁄ 2) [9,10]. MEKs have very narrow sub-
strate specificity, restricted to p44
Erk1
and p42
Erk2
.
Phosphorylation of these kinases by MEKs results in
phosphorylation of further downstream targets inclu-
ding p90
Rsk
, which could phosphorylate and inactivate
BAD at serine 75 [11]. On the other hand, it has been
shown that protein tyrosine kinases, such as proline-
rich tyrosine kinase-2 (PYK 2), transduce key extracel-
lular signals through the activation of the MEK ⁄ Erk
pathway [12]. Moreover, studies have shown that
PYK 2 is activated by an increase in intracellular
Ca

2+
concentration, confirming the well-known pro-
cess of Ca
2+
-induced ERK activation [13].
Bim, like BAD, is a ‘BH3-only’ protein of the Bcl-2
family and an important mediator of apoptosis in
response to loss of survival signal [14,15]. There are
three major splice variants of Bim: short (Bim
S
), long
(Bim
L
) and extra-long (Bim
EL
) [16]. In most cells,
including THP-1 cells, Bim
EL
is the major species
expressed [17]. This isoform of Bim induces apoptosis
by antagonizing the activity of the anti-apoptotic Bcl-2
family members [14]. In lymphocytes, Bim has been
shown to be a major transducer of several apoptotic
signals including microtubule destabilization [18].
In this study, we show that 7-ketocholesterol induced,
via the calcium-sensitive tyrosine kinase PYK 2 ⁄
CAKb ⁄ RAFTK ⁄ CADTK, a calcium-dependent activa-
tion of the MEK 1 ⁄ 2 fi ERK 1 ⁄ 2 survival pathway
delaying several apoptotic mechanisms initiated by the
oxysterol. We also demonstrate the involvement of Bim

in 7-ketocholesterol-induced cytotoxicity. Indeed, we
show that Ca
2+
influx leads to the translocation of the
protein from the microtubule dynein motor complex to
mitochondria, and thus its interaction with Bcl-2 [19].
Therefore, 7-ketocholesterol-induced apoptosis appears
to be a complex phenomenon involving several calcium-
dependent transduction pathways.
Results
ERK 1 ⁄ 2 is activated during the first steps of
7-ketocholesterol-induced apoptosis
The effects of 7-ketocholesterol were examined in rela-
tion to the expression of various signalling proteins
in THP-1 cells. Exposure of cells to 7-ketocholesterol
resulted in a rapid phosphorylation of ERK 1 ⁄ 2 within
1 h, as monitored through the use of phospho-specific
antibodies (Fig. 1A). ERK phosphorylation peaked at
2–3 h, where phosphoERK reached 30% of total ERK
vs. 13% in control, and then declined back towards
basal levels at 12 h after exposure to 7-ketocholesterol
(Fig. 1A,B), whereas apoptosis increased significantly
(Fig. 1D). No activation of p38 MAPK or JNK was
observed (data not shown) and dephosphorylation of
PKB at threonine 308 was noted (Fig. 1C). This
PKB dephosphorylation, leading to its inactivation,
appeared as soon as 3 h after 7-ketocholesterol treat-
ment. However, no phosphorylation of PKB was
observed at serine 473 in either control cells or treated
7-keto

A
B
C
D
P Erk1/2
Erk1/2
7-keto
Ctrl 1h 2h 3h 6h 12h 18h
Ctrl 1h 2h 3h 6h 12h 18h
300
200
100
150
250
50
0
Ctrl
Arbitrary Unit
P Erk1/2 protein
1h 2h 3h 6h 12h 18h
*
*
*
*
*
Time of 7-keto
treatment
P PKB (Thr 308)
% of Apoptotic Cells
Ctrl

7k20
80
70
60
50
40
30
20
10
0
0 6 12 18 24 30 36 42 48
Time (h)
% condensed/fragmented nuclei
PKB
Fig. 1. 7-Ketocholesterol induces ERK activation and Akt ⁄ PKB inac-
tivation. THP-1 cells were treated with 7-ketocholesterol (7-keto,
40 lgÆmL
)1
) for various incubation times. Cell extracts were collec-
ted, subjected to SDS ⁄ PAGE and immunoblotted with ERK 1 ⁄ 2,
phospho-ERK 1 ⁄ 2, Akt and phospho-Akt thr308 antibodies. Repre-
sentative western blots of ERK 1 ⁄ 2–phospho-ERK 1 ⁄ 2 and Akt–
phospho-Akt are shown (A and C, respectively); (B) phospho-ERK
blot densitometry analysis. Values are means ± SD (n ¼ 3).
*P<0.05 vs. control group. (D) Microscopic quantification of cells
with fragmented and ⁄ or condensed nuclei was performed using
Hoechst 33342 and the percentage of apoptotic cells was deter-
mined. Data are means ± SD (n > 5).
Pathways in 7-ketocholesterol-induced apoptosis A. Berthier et al.
3094 FEBS Journal 272 (2005) 3093–3104 ª 2005 FEBS

cells (data not shown). As ERK 1 ⁄ 2 activation is
known to be involved in survival pathways [20], we
then focused our attention on the role of the ERK
pathway, and looked for the possibility of a relation-
ship between ERK 1 ⁄ 2 activation and apoptosis
induced by 7-ketocholesterol.
Inhibition of the MEK fi ERK pathway accelerates
7-ketocholesterol-induced THP-1 apoptosis
To investigate the exact role of ERK in 7-ketocholes-
terol-induced apoptosis, we used the MEK inhibitor
U0126. Treatment of THP-1 cells with U0126 resulted
in the inactivation of ERK 1 ⁄ 2, almost disappearance
of the phospho-ERK spot, in agreement with the inhi-
bition of MEK, on control cells and at both 3 and 6 h
after addition of 7-ketocholesterol (Fig. 2A,B). In light
of these results, we investigated the effect of the inhibi-
tion of the MEK ERK 1 ⁄ 2 pathway on THP-1 cell
viability after 7-ketocholesterol treatment. Whereas
U0126 alone has no effect on cell viability, we found
that the addition of U0126 accelerates 7-ketocholesterol-
induced apoptosis, as shown by the number of apop-
totic cells (Fig. 2C). Indeed, apoptosis of THP-1 cells
is observed 6 h earlier when cotreated with U0126
and 7-ketocholesterol than when treated with 7-keto-
cholesterol alone. This difference of 6 h correlates with
the transient activation of ERK 1 ⁄ 2. Thus 7-ketocho-
lesterol, which induces THP-1 apoptosis, also activates
an anti-apoptotic pathway.
Calcium-dependent activation of ERK 1 ⁄ 2
As calcium signals could be involved in MAPK activa-

tion and as we and others have previously described a
calcium influx in oxysterol-induced apoptosis [8,21,22],
we tested whether or not calcium could mediate
ERK 1 ⁄ 2 phosphorylation. The role of calcium in
MAPK activation was investigated by the cotreatment
of THP-1 with 7-ketocholesterol and verapamil, a cal-
cium channel blocker which has been described as
a potential inhibitor of oxysterol-induced apoptosis.
Under these conditions, verapamil inhibited ERK acti-
vation early as 3 h after treatment with 7-ketocholes-
terol (Fig. 3A,B). These results were strengthened by
the use of the intracellular calcium chelator BAPTA
which also completly inhibited ERK activation (data
not shown).
Next, we wondered how calcium could activate the
ERK 1 ⁄ 2 pathway. As the cytosolic calcium-dependent
PYK 2, a Src kinase activator, could mediate the acti-
vation of the Ras-Raf-Mek-Erk pathway [12,23,24],
activation by phosphorylation of tyrosines 579⁄ 580 in
PYK 2 was investigated using a phosphorylation site-
specific antibody. Figure 3C,D shows that PYK 2 was
phosphorylated at tyrosines 579 ⁄ 580 as early as 1 h
after the THP-1 cells were treated with 7-ketochole-
sterol. This phosphorylation peaked at 2 h and then
declined back toward basal level at 12 h after the
addition of 7-ketocholesterol. Phosphorylated PYK 2
peaked around 56% of total PYK 2 at 2 h vs. 17% in
control. However, only weak phosphorylation (15% of
PYK phosphorylated as in controls) was detected on
these tyrosine residues when cells were cotreated with

7-ketocholesterol and verapamil (Fig. 3E,F) suggesting
that calcium is responsible for the activation of
PYK 2 as previously demonstrated by Lev et al. [13].
Taken together, these results suggest that calcium
uptake activates ERK 1 ⁄ 2 via the activation of
PYK 2.
7-keto
A
B
C
P Erk1/2
% of Apoptotic Cells
Erk1/2
Ctrl U 3h 3h+U 6h 6h+U
Arbitrary Unit
P Erk1/2 protein
250
150
200
100
50
0
Ctrl
Ctrl
U
U
3h 6h3h+U
7k20
7k20 + U
*

*
*
*
*
*
45
35
25
15
5
0
0
%condensed/fragmented nuclei
6121824
Time (h)
40
30
20
10
6h+U
Time of 7-keto
treatment
Fig. 2. The MEK blocker U0126 inhibits 7-ketocholesterol-induced
ERK activation and accelerates apoptosis. THP-1 cells were either
untreated (Ctrl) or incubated with U0126 (U, 10 lmolÆL
)1
) alone or
in association with 7-ketocholesterol (7-keto) for the indicated
times. (A) Cell lysates were subjected to SDS ⁄ PAGE, and immuno-
blot analysis with antibodies against ERK 1 ⁄ 2 or phospho-ERK 1 ⁄ 2

was performed. (B) Phospho-ERK blot densitometry analysis. Val-
ues are means ± SD (n ¼ 3). (C) Microscopic quantification of cells
with fragmented and ⁄ or condensed nuclei was performed using
Hoechst 33342 and the percentage of apoptotic cells was deter-
mined. Data are means ± SD (n ¼ 4) (*P < 0.05).
A. Berthier et al. Pathways in 7-ketocholesterol-induced apoptosis
FEBS Journal 272 (2005) 3093–3104 ª 2005 FEBS 3095
ERK 1/2 inhibits 7-ketocholesterol-induced
apoptosis by phosphorylation of BAD
Having established the role of ERK 1 ⁄ 2 in the 7-keto-
cholesterol survival pathway, we wondered how ERK
inhibited cell death. During apoptosis involving mito-
chondria, Bcl-2 family members play a critical role.
We have previously described that 7-ketocholesterol
induced BAD dephosphorylation at serines 75 and 99,
but BAD dephosphorylation at serine 75 was incom-
plete [8]. Moreover, Scheid et al. [11] showed that ERK
could phosphorylate BAD at serine 75, via p90
RSK
.We
therefore tested the hypothesis that ERK could reduce
apoptosis by phosphorylating BAD. To characterize
the importance of the ERK pathway in the activa-
tion ⁄ inhibition of BAD during the early steps of
7-ketocholesterol-induced apoptosis, the status of BAD
phosphorylation at serine 75 was investigated following
treatment with the MEK inhibitor U0126 (Fig. 4A,B).
Western blot analysis revealed that BAD was only
slightly dephosphorylated at serine 75 at 12 h of treat-
ment with 7-ketocholesterol alone (a 15–20% decrease

of phosphorylated BAD), whereas cotreatment of
THP-1 with the oxysterol and U0126 induced the same
rate of dephosphorylation of BAD as early as 3 h.
We next asked whether the acceleration of BAD
dephosphorylation induced by U0126 could have an
impact on mitochondria integrity. Cells were pretreated
with U0126, followed by various 7-ketocholesterol
treatments, and the transmembrane mitochondrial
potential was measured (Fig. 4C). In the absence of a
MEK inhibitor, mitochondria depolarization first
appeared no later than 12 h after the beginning of
treatment, whereas in the presence of U0126, the trans-
membrane mitochondrial potential declined as early as
6 h. We next wondered whether U0126 could also
accelerate cytochrome c and Smac ⁄ Diablo leakage
from the mitochondria. Western blot analysis was per-
formed and we showed that U0126 increased the
release of cytochome c and Smac ⁄ Diablo induced by
7-ketocholesterol treatment (Fig. 4D). Taken together,
these results indicate that BAD is phosphorylated at
ser75 after ERK activation by 7-ketocholesterol and
this phosphorylation delays apoptosis by preventing
mitochondrial damage.
Secondary decreases of PYK 2 and ERK 1 ⁄ 2 acti-
vities do not appear to be related to a decrease in
cytoplasmic calcium.
As we previously described that 7-ketocholesterol
treatment of THP-1 cells induced an increase of cyto-
plasmic free calcium [8], a time course of intracellular
calcium fluctuations was performed between addition

of 7-ketocholesterol and appearance of a significant
number of apoptotic cells to investigate the relation-
ship between changes in intracellular calcium, activa-
tion of PYK 2-ERK 1 ⁄ 2 pathway and apoptosis. As
shown in Fig. 5 calcium concentration increased until
12 h when, whereas PYK 2 and ERK 1 ⁄ 2 activities
peaked at 2–3 h as shown above. So the secondary
decrease of PYK 2 and ERK 1 ⁄ 2 activities did not
appear to be related to a decrease in cytoplasmic free
calcium.
7-keto
A
B
C
D
E
F
7-keto
P Erk1/2
Erk1/2
P PYK2
PYK2
P PYK2
PYK2
Arbitrary Unit
200
Ctrl
Ctrl 1h 2h 3h 6h 12h 18h
7-keto
Ctrl 1h 2h 3h 6h 12h 18h

Vera 3h 6h 6h+Vera3h+Vera
Ctrl Vera 3h 6h 6h+Vera3h+Vera
Ctrl Vera 3h 6h 6h+Vera3h+Vera
Ctrl Vera 3h 6h 6h+Vera
Time of 7-keto
treatment
Time of 7-keto
treatment
Time of 7-keto
treatment
*
*
*
*
3h+Vera
100
150
50
0
P Erk1/2 protein
Arbitrary Unit
200
120
160
80
40
0
P PYK2 protein
Arbitrary Unit
200

100
150
50
0
P PYK2 protein
Fig. 3. 7-Ketocholesterol-induced calcium influx activates ERK phos-
phorylation through PYK 2. THP-1 cells were either untreated (Ctrl)
or incubated with
L-type calcium channel blocker, verapamil (Vera,
100 lmolÆL
)1
) alone or in association with 7-ketocholesterol for
the indicated times. Cell extracts were collected, subjected to
SDS ⁄ PAGE and immunoblotted with ERK 1 ⁄ 2 and phospho-
ERK 1 ⁄ 2 (A), or PYK 2 and phospho-PYK 2 (C, E) antibodies. (B, D, F)
Respective group densitometry results. Values are means ± SD
(n ¼ 3). *P<0.05 vs. control group.
Pathways in 7-ketocholesterol-induced apoptosis A. Berthier et al.
3096 FEBS Journal 272 (2005) 3093–3104 ª 2005 FEBS
Bim activation depends on calcium uptake
Besides BAD, Bim functions also as a sensor toward
apoptotic stimuli by heterodimerization and inactiva-
tion of anti-apoptotic multi-BH domain proteins.
Moreover, it has been shown that Bim could be under
control of the MEK fi ERK pathway [25]. Indeed,
ERK 1 ⁄ 2 is known to exert its anti-apoptotic activity
in promoting phosphorylation and consequently the
proteasome-dependent degradation of Bim [26]. Inter-
estingly, in THP-1 cells, Bim
EL

is expressed in control
cells and the level of Bim
EL
, as well as Bcl-2, is not
apparently changed by 7-ketocholesterol treatment
(Fig. 6A). Moreover, the use of the ERK inhibitor,
U0126, does not affect Bim
EL
or Bcl-2 expression, sug-
gesting that the transient activation of ERK 1 ⁄ 2by
Calcium Influx
Ctrl
Vera
7-keto
7-keto + Vera
0 2h 4h 6h 8h 10h 12h
Time of 7-keto treatment
(Hours)
70
60
50
40
30
20
10
0
Fluo-3 Fluorescence Arbitrary Unit
Fig. 5. Time course of intracellular calcium after 7-ketocholesterol
treatment of THP-1 cells. THP-1 cells were incubated in the pres-
ence of 7-ketocholesterol (40 lgÆmL

)1
, 7-keto), with or without
verapamil (100 lmolÆL
)1
, Vera) or in the absence of oxysterol (Ctrl).
After different incubation times, cells were loaded with fluo-3 ⁄ AM
and the dye fluorescence was measured by flow cytometry. Each
fluorescent data point is normalized to the maximal fluo-3 fluores-
cence induced in cells treated with ionomycin (2 lmolÆL
)1
). Data
are the means ± SD.
P Bad (S75)
Bad
Hsc 70
Ctrl
800
600
400
200
0
U
3h 6h 12h 12h+U3h+U
7-keto
A
B
C
D
% of Cells with Depolarized Mitochondria
Smac / Diablo

S100
Fraction
Cytochrome c
Hsc 70
Time (h)
% DiOC
6
(3) negative cells
6h+U
Ctrl U
18h 24h 30h18h+U 24h+U 30h+U
7-keto
Ctrl U
7K20
7K20 + U
70
60
50
40
30
20
10
0
0 6 12 18 24
3h 6h 12h 12h+U
Time of 7-keto
treatment
*
*
†

*
*
*
*
*
†
†
3h+U 6h+U
Arbitrary Unit
P Bad protein
Fig. 4. 7-Ketocholesterol-induced ERK phosphorylation inhibits BAD
dephosphorylation, mitochondria depolarization and cytochrome
c–Smac ⁄ Diablo release. (A) Western blot analysis of BAD and
phospho-BAD was performed during 7-ketocholesterol (7-keto) and
U0126 (U) treatment of THP-1 cells. (B) Phospho-BAD blot densi-
tometry analysis. Values are means ± SD (n ¼ 3). *P<0.05 vs.
Ctrl or U group; P<0.05 vs. Ctrl or oxysterol-treated cells. (C)
Transmembrane mitochondrial potential was measured by flow
cytometry using DiOC
6
(3) dye. After the incubation period, fluores-
cence associated with DiOC
6
(3) was measured by flow cytometry
and 10 000 cells were analysed for each assay. Results represent
the means ± SD (n ¼ 4) (*P<0.05). (D) THP-1 cells were either
untreated (Ctrl), treated with U0126 (U) or with 7-ketocholesterol
(7-keto) for 18, 24 or 30 h alone or in association with U0126, and
cytosol fractions (S100 fraction) were collected. Subcellular frac-
tions were subject to SDS ⁄ PAGE and immunoblot analyses were

performed with antibodies against cytochrome c or Smac ⁄ Diablo.
Hsc70 was used as internal control loading. The blots are represen-
tative of two independent experiments.
7-ketoA
B
7-keto
Ctrl
Ctrl
Bim
EL
Bcl-2
Bim
EL
Bcl-2
U3h 6h3h+U 6h+U
1h 2h 3h 6h 12h 18h
Fig. 6. 7-Ketocholesterol and ERK activation do not regulate Bim
expression. THP-1 cells were either untreated (Ctrl), treated with
U0126 (U) or with 7-ketocholesterol (7-keto) alone or in association
with U0126 for the indicated period, and cell extracts were collec-
ted. Lysates were analysed by western blotting using Bim and
Bcl-2 antibodies (A, B).
A. Berthier et al. Pathways in 7-ketocholesterol-induced apoptosis
FEBS Journal 272 (2005) 3093–3104 ª 2005 FEBS 3097
7-ketocholesterol does not induce the degradation of
Bim
EL
in THP-1 cells (Fig. 6B).
In light of these results, we wondered if Bim
EL

was
involved in 7-ketocholesterol-induced apoptosis, because
BAD does not seem to be the only pro-apoptotic
molecule involved [27]. Indeed, Puthalakath et al.
[19] described that the pro-apoptotic activity of Bim
can also be regulated by interaction with the dynein
motor complex and the microtubules. As we previously
described that 7-ketocholesterol induced a calcium
uptake and as it is known that calcium uptake could
destabilize microtubules [28] dissociating Bim from
microtubule dynein motor complex, we wondered if
7-ketocholesterol-induced calcium influx could modify
microtubule structure and Bim
EL
localization. Thus,
we examined the effects of 7-ketocholesterol, alone or
in association with verapamil, on microtubule organ-
ization using classical fluorescence microscopy. Both
control cells and verapamil treated cells displayed a
typical randomly oriented, intact microtubular network
(Fig. 7A,B). In 7-ketocholesterol-treated cells, a pro-
gressive disorganization ⁄ reorganization of microtubules
occurred. These modifications were time-dependent and
affected more than 90% of the cells (Fig. 7C,E and
data not shown). This reorganization ⁄ disorganization
was partially prevented in the presence of verapamil
tubulin α
α
Hoechst 33342 Overlay
Ctrl

A
B
C
D
E
F
Vera
7-keto
6h
7-keto
6h +
7-keto
12h
7-keto
12h +
Vera
Vera
Fig. 7. Analysis of calcium involvement in
7-ketocholesterol-induced THP-1-microtubule-
network disruption. Cells were analysed by
indirect immunofluorescence microscopy
using anti-(a-tubulin) Igs and Hoechst 33342
as described in Experimental procedures
showing microtubules and nuclear structure
of (A) untreated cells (Ctrl) (B) verapamil-
treated cells (Vera), 7-ketocholesterol-trea-
ted cells alone (C, E) or in association with
verapamil (D, F).
Pathways in 7-ketocholesterol-induced apoptosis A. Berthier et al.
3098 FEBS Journal 272 (2005) 3093–3104 ª 2005 FEBS

(Fig. 7D,F), suggesting that calcium uptake could be
the event that initiates microtubule destabilization.
Thus, we wondered if inactivated Bim is targeted
to microtubules and if 7-ketocholesterol treatment of
THP-1 cells could change its localization. Co-immuno-
precipitation experiments revealed that in untreated
cells Bim
EL
is complexed with microtubules and not
with Bcl-2, whereas as 7-ketocholesterol treatment
time progresses, Bim
EL
colocalized with Bcl-2 and
not with microtubules, the part of Bcl2 in anti-Bim
immunoprecipitate (IP Bim) growing from 18% in
control to 50% after 24 h of treatment, a 2.7-fold
increase. (Fig. 8A,B). Moreover, cotreatment of the
cells with 7-ketocholesterol and verapamil appears to
abolish the dissociation of Bim
EL
from the microtu-
bules and consequently its translocation to Bcl-2, the
part of Bcl2 in IP Bim is only of 24% after 24 h of
cotreatment with verapamil. We next investigated the
localization of Bim
EL
at the mitochondrial level under
7-ketocholesterol treatment alone or in association
with verapamil. The results from this investigation
confirmed the increase of Bim

EL
levels in the mito-
chondria and that this increase is inhibited by the
presence of verapamil (Fig. 8C). So, Bim
EL
relocaliza-
tion at the mitochondrial level is dependent on cal-
cium influx induced by 7-ketocholesterol.
Discussion
In a prior report [8], we demonstrated that a major step
in the apoptotic response to 7-ketocholesterol of
human monocytic cell line THP-1 is the sustained
influx of extracellular Ca
2+
leading to the dephospho-
rylation of the pro-apoptotic ‘BH3-only’ protein BAD.
Moreover we demonstrated that this dephosphoryla-
tion was mediated by calcineurin (PP2B). However, this
dephosphorylation was incomplete and occurred more
quickly at serine 99 than at serine 75, suggesting the
existence of alternative mechanisms leading to apopto-
sis. Moreover, this idea was confirmed by the work of
Panini et al. describing that calcium-dependent activa-
tion of cPLA2 led to the stimulation of arachidonate
release and to apoptosis [29]. However, the lack of a
complete inhibition of apoptosis in cPLA2 (– ⁄ –) macro-
phages also led this group to point out the existence of
other pathways regulating oxysterol-induced cell death.
Therefore, oxysterol-induced apoptosis appears to be a
complex phenomenon with multiple initiation pathways

and several apoptotic mechanisms.
In this study, we examined two paradoxical effects
induced by 7-ketocholesterol in THP-1 cells. Indeed,
our results demonstrate a calcium-dependent activation
of one survival pathway, the PYK 2 fi MEK 1 ⁄ 2 fi
IP Bim
A
B
C
7-keto 7-keto
Ctrl Vera 6h 6h
+Vera
Tubulin α
tubulin
500
400
300
200
100
0
Arbitrary Unit
Ctrl
Vera
6h
6h+Vera
12h+Vera
18h+Vera
24h+Ve
ra
12h

18h
24h
Bcl-2
Time of 7-keto
treatment
7-keto
Ctrl 6h 12h 18h
WB : Bim
- Vera
+ Vera
Mitochondria
*
*
*
*
†
†
†
†
Bcl-2
Bim
EL
+Vera +Vera +Vera
12h 12h 18h 18h 24h
Ctrl 6h 12h 18h 24h
24h
Total Extract
Fig. 8. 7-Ketocholesterol-induced calcium
influx activates the dissociation of Bim from
microtubules and consequently its transloca-

tion to Bcl-2 at the mitochondrial level. (A)
THP-1 cells were untreated (Ctrl), treated
with verapamil (Vera) or with 7-ketocholes-
terol alone (7-keto) or in association with
verapamil for 6, 12, 18 or 24 h. After
treatment, anti-Bim immunoprecipitates
were collected, subjected to SDS ⁄ PAGE,
and immunoblotted with antibodies specific
for a-tubulin, Bcl-2 or Bim. Western blots
were also performed on total extract to
check the levels of a-tubulin, Bcl-2 or Bim.
(B) Tubulin and Bcl-2 blot densitometry
analysis. Values are means ± SD, (n ¼ 3).
*P<0.05 vs. Ctrl or Vera group; P<0.05
vs. Ctrl or oxysterol-treated cells. (C) THP-1
cells were untreated (Ctrl), treated with
verapamil (Vera) or with 7-ketocholesterol
alone (7-keto) or in association with verapa-
mil for 6, 12 or 18 h. Mitochondrial fractions
were collected and subjected to western
blot analysis with antibodies against Bim.
The blots are representative of three
independent experiments.
A. Berthier et al. Pathways in 7-ketocholesterol-induced apoptosis
FEBS Journal 272 (2005) 3093–3104 ª 2005 FEBS 3099
ERK 1 ⁄ 2 pathway allowing BAD phosphorylation on
serine 75, as well as one additional apoptotic pathway
inducing the translocation of Bim from microtubules
to Bcl-2 at the mitochondrial level, which leads to mit-
ochondrial damage and apoptosis. In fact, the use of

U0126, a MEK 1 ⁄ 2 inhibitor which accelerates 7-keto-
cholesterol-induced THP-1 apoptosis, clearly suggests
that this signalling pathway acts as a survival pathway.
In most, but not all systems studied, activation of
MEK 1 ⁄ 2 and ERK 1⁄ 2 is associated with the inhibi-
tion of cell death [30,31]. The mechanism by which this
occurs is not completely understood, but could be rela-
ted to the phosphorylation of BAD, and thus, to its
inactivation [11]. Moreover, direct anti-apoptotic
actions of ERK 1 ⁄ 2 related to the phosphoryla-
tion ⁄ inactivation of caspase-9 [32] and to the phos-
phorylation ⁄ degradation of Bim [26] have been
recently described. It is therefore surprising that expo-
sure of leukaemic cells to 7-ketocholesterol at concen-
trations that trigger apoptosis was associated with the
clear activation of ERK 1 ⁄ 2, even if it is a transient
phenomenon. Moreover, it seems that other MAPKs
(JNK or p38 MAPK) were not activated under 7-keto-
cholesterol treatment of THP-1 cells, suggesting that
ERK activation is not under the activation of other
MAPKs as described by Numazawa et al. [33]. Con-
trary to Rusin
˜
ol et al. [27], who described Akt ⁄ PKB
degradation under oxysterol treatment of macrophage
cells, we never noticed a degradation of Akt ⁄ PKB in
THP-1 cells during the course of 7-ketocholesterol
treatment but we did show that Akt ⁄ PKB was inacti-
vated by dephosphorylation at threonine 308.
As we previously described a calcium influx in

7-ketocholesterol-induced THP-1 apoptosis, we exam-
ined the role of calcium in ERK 1 ⁄ 2 activation.
Hence, the L-Type calcium channel blocker verapamil
and intracellular calcium chelator BAPTA completely
inhibited 7-ketocholesterol-induced ERK activation.
Interestingly, calcium-dependent activation of the
MEK 1 ⁄ 2 fi ERK 1 ⁄ 2 pathway has been previously
described in 7b-hydroxycholesterol-treated aortic
smooth muscle cells [21]. Nevertheless, the authors
did not describe the effects or the mechanism of
ERK activation. Protein tyrosine kinases (PTKs)
transduce key extracellular signals that trigger various
biological events, such as cytoskeletal rearrangement
and mitogenesis. Among the PTKs, PYK 2 (also
known as CAKb, RAFTK or CADTK), which exists
mainly in the cytoplasm [34], is abundantly expressed
in haemopoeitic cells and in the brain. Moreover,
PYK 2 is activated by stimuli that increase the
concentrations of intracellular Ca
2+
. Indeed elevation
of intracellular calcium triggers activation of PYK 2
as described by Lev et al. [13] and a maximal cata-
lytic activity was observed after phosphorylation of
PYK 2 at tyrosines 579 and 580 in the kinase domain
activation loop [35]. Thus, in untreated THP-1 cells,
as described by Yamasaki et al. [36], PYK 2 was
poorly phosphorylated at tyrosines 579 ⁄ 580, but after
7-ketocholesterol treatment we saw an increase of its
phosphorylation. Moreover, our data demonstrate

that verapamil inhibits 7-ketocholesterol-mediated-
PYK 2 phosphorylation, suggesting the role of this
protein tyrosine kinase in ERK activation. However
the secondary decreases of PYK 2 and ERK1 ⁄ 2 acti-
vites are not related to a decrease of cytoplasmic free
calcium suggesting that 7-ketocholesterol treatment of
THP-1 induces others transduction pathways leading
to a secondary inactivation of these kinases.
We next examined the ability of the MAPK signal-
ling pathway to inhibit, via p90
RSK
, the apoptotic
effect of 7-ketocholesterol by phosphorylating BAD.
Treatment of cells with the combination of the MEK
inhibitor U0126 and 7-ketocholesterol caused an ear-
lier dephosphophorylation of BAD than with the oxy-
sterol alone. The induction of cell death was also
faster following cotreatment than treatment with
7-ketocholesterol alone, indicating that inhibition of
BAD phosphorylation at serine 75 increases apoptosis.
This process could accelerate the disruption of the mito-
chondrial transmembrane potential and Smac ⁄ diablo
and cytochrome c release into the cytosol. Hence, our
findings suggest that the MAPK signalling pathway pro-
motes cell survival by a mechanism that modulates the
cell death machinery directly by phosphorylating and
thereby inactivating the pro-apoptotic protein BAD.
Our previous results also suggest the possibility of
the involvement of other Ca
2+

-initiated cell death
pathways. Therefore our results showing the release of
the pro-apoptotic ‘BH3 only’ protein Bim and its
association with Bcl-2 in a calcium-dependent man-
ner complement our knowledge on the mechanism
of 7-ketocholesterol-induced apoptosis. A calcium-
dependent destabilization of microtubules, as previ-
ously described by Keith et al. [33], probably induces
the dissociation of Bim from the microtubule dynein
motor complex, allowing inactivation of anti-apopto-
tic, multi-BH domain proteins such as Bcl-2 or Bcl-X
L
in the mitochondria. Previously Palladini et al. des-
cribed a 7-ketocholesterol induced destabilization of
vimentin filament architecture without significant alter-
ations of the microtubule network on a bovine aortic
endothelial cell line. Nevertheless they showed that
7-ketocholesterol induced tubulin aggregates and so
they did not exclude a possible role of microtubules in
oxysterol-induced endothelial cell apoptosis [37]. It has
Pathways in 7-ketocholesterol-induced apoptosis A. Berthier et al.
3100 FEBS Journal 272 (2005) 3093–3104 ª 2005 FEBS
been reported that activation of the ERK 1 ⁄ 2 signaling
pathway promoted phosphorylation and proteasome-
dependent degradation of Bim, but this result was not
obtained in our study. The possible explanation could
be that the length and intensity of activation of
ERK 1 ⁄ 2 is not sufficient to induce a detectable
change in Bim concentration. The short length of
ERK activation and the dephosphorylation of Akt ⁄

PKB could be related to the activation of phosphatases
such as protein phosphatase 1a [38]. Hence, 7-keto-
cholesterol-induced apoptosis appears to be a com-
plex phenomenom implicating several transduction
pathways, two pro-apoptotics and surprisingly one
anti-apoptotic, that to our knowledge have not been
previously described. However further studies will be
necessary to try to quantify the part of these different
pathways in 7-ketocholesterol induced cytotoxicity. All
of these mechanisms seem to be related to the sus-
tained rise of free cytosolic calcium triggered by the
transfer of Trpc-1 into lipid raft domains that we have
previously described [8] and involve the mitochondrial
pathway of apoptosis with the implication of the pro-
apoptotic BH3 proteins BAD and Bim.
As all oxysterols are not able to induce apoptosis and
as cytotoxicity can vary according cell type, it will be
interesting to use other oxysterols and other cell types
and especially other cell lines of the monocytes ⁄ macro-
phages lineage to investigate the implication of these
different pathways and particularly sustained calcium
ion influx, which appears to be the key event in oxy-
sterol-mediated cytotoxicity. In this way the first experi-
ments performed by our group on U937, another
monocytic cell line, with 7-ketocholesterol (A. Berthier,
unpublished results) produced results quite similar to
those obtained with THP-1 cells.
Experimental procedures
Reagents and antibodies
The THP-1 human monocytic cell line was from the Ameri-

can Tissue and Culture Collection (Manassas, VA, USA).
DiOC
6
(3) and the anti-mouse IgG-AlexaFluor 565 were
from Molecular Probes, Inc. (Eugene, OR, USA). 7-Keto-
cholesterol, IgePal, EGTA, Pipes, MgSO
4
, mannitol,
dimethylsulfoxide, verapamil, pepstatin A, aprotinin, tryp-
sine inhibitor, leupeptin, phenylmethylsulfonylfluoride,
paraformaldehyde, Hoechst 33342, NaF, b-glycerophos-
phate and Triton X-100 were from Sigma (Sigma-Aldrich,
L’Isles d’Abeau-Chesnes, France). The anti-Smac ⁄ Diablo
polyclonal antibody was from Imgenex (San Diego,
CA, USA). The anti-BAD monoclonal antibody and the
anti-BAD phospho Ser112 (human Ser75) polyclonal anti-
body were from Upstate Biotechnology (Lake Placid, NY,
USA), and the anti-cytochrome c monoclonal antibody was
from Pharmingen (San Diego, CA, USA). The anti-(Hsc
70), anti-PYK 2, anti-PYK 2 phospho tyr279 ⁄ tyr580 and
Bim polyclonal antibodies, the anti-(a-tubulin) monoclonal
antibody, protein G-Agarose, and total mouse IgG were
from Santa Cruz Biotechnologies (Santa Cruz, CA, USA),
the anti-Erk 1 ⁄ 2, anti-Erk 1 ⁄ 2 phospho Thr202 ⁄ Tyr204,
anti-PKB and anti-PKB phospho Thr308 and U0126 were
from Cell Signaling Technology (Cell Signaling Technology,
Hitchin, UK) and the anti-Bcl-2 Ig was from Dako (Dako,
Trappes, France).
Cell culture
Human monocytic THP-1 cells were grown in RPMI 1640

with glutamax-I (Gibco, Eragny, France) and antibiotics
(100 UÆmL
)1
penicillin, 100 lgÆmL
)1
streptomycin) (Gibco),
supplemented with 10% (v ⁄ v) heat-inactivated fetal bovine
serum (Gibco). The cells were incubated at 37 °C under a
5% CO
2
⁄ 95% air atmosphere (v ⁄ v).
Cell treatment
For all experiments, a 7-ketocholesterol stock solution was
prepared at a concentration of 800 lgÆmL
)1
as previously
described [39]. 7-Ketocholesterol was added to the culture
medium for a final concentration of 40 lgÆmL
)1
. This con-
centration is in the range of levels measured in human plasma
after a meal rich in fat [40]. Verapamil, an L-type calcium
channel inhibitor, was added to the culture medium at a final
concentration of 100 lmolÆL
)1
. The MEK fi Erk inhibitor
U0126 was used at a final concentration of 10 lmolÆL
)1
.In
all experiments, verapamil and U0126 were introduced in the

culture medium 30 min before 7-ketocholesterol.
Characterization of nuclear morphology
by staining with Hoechst 33342
Nuclear morphology of control and treated cells was studied
by fluorescence microscopy after staining with Hoechst 33342
(kEx
max
, 346 nm; kEm
max
, 420 nm) used at 10 lgÆmL
)1
.
The morphological aspect of cell deposits, applied to glass
slides by cytocentrifugation with a cytospin 4 centrifuge
(Shandon, UK), was observed with an Axioskop light micro-
scope (Zeiss, Jena, Germany) by using UV light excitation.
Three hundred cells were examined for each sample.
Flow cytometric measurement of mitochondrial
transmembrane potential ( DW
m
) with the dye
DiOC
6
(3)
Variations of the mitochondrial transmembrane potential
(DY
m
) were measured with 3,3¢-dihexyloxacarbocyanine
A. Berthier et al. Pathways in 7-ketocholesterol-induced apoptosis
FEBS Journal 272 (2005) 3093–3104 ª 2005 FEBS 3101

iodide (DiOC
6
(3); kEx
max
: 484 nm, kEm
max
: 501 nm) used
at a final concentration of 40 nmolÆL
)1
[41]. The flow cyto-
metric analyses were performed on a Galaxy flow cytometer
(Partec, Munster, Germany) and the green fluorescence was
collected through a 524 ⁄ 44-nm band pass filter. Fluorescent
signals were measured on a logarithmic scale of four dec-
ades of log. For each sample, 10 000 cells were acquired
and the data were analysed with flomax software (Partec).
Flow cytometric measurement of cytosolic
calcium with the dye Fluo-3
THP-1 cells were washed with NaCl ⁄ P
i
(pH 7.4) and then
incubated with Fluo-3 ⁄ AM (6 lmolÆL
)1
; kEx
max
: 506 nm,
kEm
max
: 526 nm) for 30 min at 37 °C in Hank’s balanced
salt buffer (pH 7.2) with Pluronic F-127. After loading,

cells were suspended in Hepes buffer (pH 7.4) supplemented
with probenecid (5 mm) to prevent leakage of the dye.
Fluorescence was measured by flow cytometry with a Gal-
axy flow cytometer (Partec) using a 524 ⁄ 44-nm band pass
filter. For each sample, events were acquired for 60 s and
the data were analysed with flomax software (Partec).
Staining for microtubules
Treated or control cells were rinsed twice in microtubule-
stabilizing buffer (MSB; 50 mm Pipes pH 6.9, 10 mm
EGTA, and 10 mm MgSO4). Cells were immediately fixed
for 1 h in MSB containing 4% (w ⁄ v) freshly prepared para-
formaldehyde and 0.2 mm mannitol. Cells were then
washed twice with MSB, fixed on polylysine glass and per-
meabilized for 20 min with 0.5% (w ⁄ v) Triton X-100 in
MSB containing 0.2 molÆL
)1
mannitol. After two washes in
MSB, cells were treated for 2 h at room temperature with
5% (w ⁄ v) BSA in MSB to block nonspecific antibody bind-
ing sites. Cells were then incubated overnight at 4 °C in the
presence of the primary mouse monoclonal antibody
against a-tubulin [1 : 100 dilution in MSB containing 0.1%
(v ⁄ v) MSB] and rinsed twice in MSB containing 0.1%
(w ⁄ v) BSA. Cells were then incubated for 1 h in the pres-
ence of the secondary AlexaFluor 565-conjugated rabbit
anti-mouse antibody (1 : 250 dilution in MSB containing
0.1% MSB). After two washes in MSB, cells were mounted
in Fluoprep. At least 50 cells were examined for each
experiment and three independent experiments were per-
formed for each treatment. Observations were performed

on an Axioskop light microscope (Zeiss, Jena, Germany)
by using UV light excitation.
Immunoprecipitation and western blotting
For the immunoprecipitation of the Bim protein, cells were
suspended in immunoprecipitation buffer [10 mm Tris ⁄ HCl,
140 mm NaCl, 0.1% (w ⁄ v) IgePal] containing a mixture of
protease inhibitors (0.1 mm phenylmethanesulfonyl fluoride,
2.5 lgÆL
)1
aprotinin, 10 l g ÆL
)1
pepstatin A, 2.5 lgÆL
)1
trypsin inhibitor and 2.5 lgÆL
)1
leupeptin). After a 20-min
incubation at 4 °C in the lysis buffer, the cell debris were
eliminated by centrifugation for 10 min at 10 000 g. The
resulting supernatant was precleared by adding 1 lg total
mouse IgG and 50 lL protein G-agarose for 30 min. After
a 10 000 g centrifugation (4 °C, 10 min), the supernatant
was collected, adjusted to 500 lL in lysis buffer and incu-
bated overnight at 4 °C with 10 lg of the anti-Bim Ig on a
rotating device. After the incubation period, 50 lL protein
G-agarose were added and the sample was incubated for
2 h before collecting the immunoprecipitates by centrifuga-
tion at 10 000 g (4 °C, 5 min). After washing the pellet four
times, the immunoprecipitation extract was suspended in
Laemmli’s buffer [1% (w ⁄ v) SDS, 1 mm sodium orthovana-
date, 10 mm Tris ⁄ HCl].

Alternatively, cells were resuspended in Ripa lysis buffer
[0.1% (w ⁄ v) SDS, 1% (w ⁄ v) IgePal, 0.5% (w ⁄ v) Na-deso-
xycholate, 50 mm Tris ⁄ Hcl pH 8.0, 150 mm NaCl] contain-
ing a mixture of protease and phosphatase inhibitors
(0.1 mm phenylmethanesulfonyl fluoride, 2.5 lgÆL
)1
aproti-
nin, 10 lgÆL
)1
pepstatin A, 2.5 lgÆL
)1
trypsin inhibitor,
2.5 lgÆL
)1
leupeptin, 0.1 mm Na-orthovanadate, 40 mm
b-glycerophosphate, 100 mm NaF). After a 30-min incuba-
tion at 4 °C in the lysis buffer, the cell debris were eliminated
by centrifugation for 20 min at 10 000 g and the supernatant
was collected.
The release of cytochrome c and Smac ⁄ Diablo from mito-
chondria to the cytosol was investigated by western blot ana-
lysis of THP-1 cells incubated for 18, 24 or 30 h with
7-ketocholesterol alone or in association with U0126 as pre-
viously described [42]. Mitochondria were obtained before
the last 1000 g centrifugation generating the cytosol fraction.
The protein concentrations were measured by using
bicinchoninic acid reagent (Pierce, Rockford, IL, USA)
according to the method of Smith et al. [43]. Seventy micro-
grams of protein were incubated in loading buffer
[125 mmolÆL

)1
Tris ⁄ HCl, pH ¼ 6.8, 10% (w ⁄ v) 2-merca-
ptoethanol, 4.6% (w ⁄ v) SDS, 20% (v ⁄ v) glycerol, 0.003%
(w ⁄ v) Bromophenol blue], boiled for 3 min, separated by
SDS ⁄ PAGE and electroblotted onto a polyvinylidine diflu-
oride membrane (Bio-Rad, Ivry sur Seine, France). After
blocking nonspecific binding sites for 2 h at room tempera-
ture in TPBS [NaCl ⁄ P
i
, 0.1% (v ⁄ v) Tween-20], the mem-
branes were incubated overnight at 4 °C with the primary
antibody diluted in TPBS. After three 10-min washes with
TPBS, the membranes were incubated with horseradish
peroxidase-conjugated secondary antibody at a dilution of
1 : 2500 for 1 h at room temperature and washed three
times in TPBS for 10 min. Autoradiography of the immu-
noblots was performed using an enhanced chemolumines-
cence detection kit (Amersham, Les Ulis, France). Western
blots were quantified using a JS800 densitometer using
Pathways in 7-ketocholesterol-induced apoptosis A. Berthier et al.
3102 FEBS Journal 272 (2005) 3093–3104 ª 2005 FEBS
quantity one software (Bio-Rad). Each experiment was
repeated three times with identical results.
Statistical methods
Statistical analysis were performed with statview software
(Cary, NC, USA) using a two-way analysis of variance fol-
lowed by Student’s t-test.
Acknowledgements
This work was supported by the University of Bourgo-
gne, the Institut National de la Sante

´
et de la Recher-
che Me
´
dicale (INSERM) and the Conseil Re
´
gional de
Bourgogne. We are grateful to Dr David Wendehenne
and Dr Olivier Lamotte for their gift of anti-ERK 1 ⁄ 2
and anti-phospho-ERK 1 ⁄ 2, and to Jonathan Ewing
for reviewing the English version of this manuscript.
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