Tài liệu Báo cáo khoa học: Enhancement of oxidative stress-induced apoptosis by Hsp105a in mouse embryonal F9 cells pptx

Enhancement of oxidative stress-induced apoptosis by Hsp105a
in mouse embryonal F9 cells
Nobuyuki Yamagishi, Youhei Saito, Keiichi Ishihara and Takumi Hatayama
Department of Biochemistry, Kyoto Pharmaceutical University, Japan
Hsp105a is one of the major mammalian heat shock proteins
that belongs to the HSP105/110 family, and is expressed at
especially high levels in the brain as compared with other
tissues in mammals. Previously, we showed that Hsp105a
prevents stress-induced apoptosis in neuronal PC12 cells,
and is a novel anti-apoptotic neuroprotective factor in
the mammalian brain. On the other hand, we have also
demonstrated that Hsp105a is expressed transiently at
high levels during mouse embryogenesis and is found not
only in various tissues but also in apoptotic cells. In the
present study, to elucidate the role of Hsp105a during
mouse embryogenesis, we established mouse embryonal F9
cell lines that constitutively over-express Hsp105a.Over-
expression of Hsp105a enhanced hydrogen peroxide-
induced apoptosis by enhancing the activation of caspase-3,
poly(ADP-ribose)polymerasecleavage,cytochrome crelease
and activation of p38 mitogen-activated protein kinase
(p38). Furthermore, oxidative stress-induced apoptosis was
suppressed by SB202190, a potent inhibitor of p38, in F9
cells. These findings indicated that the activation of p38 is an
essential step for apoptosis in F9 cells and that Hsp105a
enhances activation of p38, release of cytochrome c and
caspase activation. Hsp105a may play important roles in
organogenesis, during which marked apoptosis occurs, by
enhancing apoptosis during mouse embryogenesis.
Keywords: apoptosis; F9 cells; Hsp105; p38 MAPK; oxida-
tive stress.

Cell death is classified into two major morphologically and
biochemically distinct modes, necrosis and apoptosis [1].
Necrosis is characterized by swelling of organelles and cells,
followed by lysis of the plasma membrane and random
DNA degradation. In contrast, apoptosis is a process that is
characterized by cell shrinkage, plasma membrane blebbing,
nuclear condensation and endonucleolytic cleavage of DNA
into fragments of oligonucleosomal length, and is a funda-
mental and indispensable process during normal embryonic
development, tissue homeostasis and regulation of the
immune system [2–4]. In addition, environmental stresses
such as heat shock, radiation, chemical agents and oxidative
stress can also induce apoptosis.
Heat shock proteins (Hsps) are a set of highly conserved
proteins that are induced in response to physiological and
environmental stress, and are classified into several families
on the basis of their apparent molecular weights, such as
HSP105/110, HSP90, HSP70, HSP60, HSP40 and HSP27
[5,6]. Several studies have shown that Hsp70, Hsp90 and
Hsp27 protect against cell death through apoptosis by a
variety of stressors, such as heat shock, oxidative stress and
chemotherapeutic agents [7–9]. In addition, recent studies
have demonstrated that Hsp70, Hsp90, Hsp60 and Hsp27
can modulate the functions of several major components of
apoptotic processes, including the caspase cascade and the
c-Jun N-terminal kinase (JNK) signalling pathway [10–19].
We have previously characterized two heat shock pro-
teins, Hsp105a and Hsp105b, which belong to the HSP105/
110 family and are expressed in various mammals including
human, mouse and rat [20–22]. Hsp105a is a constitutively

expressed 105-kDa stress protein and is induced by a variety
of stressors, whereas Hsp105b is an alternatively spliced
form of Hsp105a that is specifically induced by heat shock at
42 °C. These proteins exist as complexes associated with
Hsp70/Hsc70 [23,24], and negatively regulate Hsp70/Hsc70
chaperone activity [25]. In addition, our recent study demon-
strated that Hsp105a protects neuronal cells against the
apoptosis induced by various stresses [26]. On the other
hand, we have shown previously that the level of Hsp105a
increases transiently in most tissues of mouse embryos from
gestational day 9–11, and that Hsp105a is localized not only
in various tissues, but also in apoptotic cells and apoptotic
bodies at the interdigital regions of limbs, suggesting that
Hsp105a may play important roles in apoptosis dur-
ing mouse embryogenesis [27].
In the present study, to examine the role of Hsp105a
during mouse embryogenesis, we established mouse embry-
onal F9 cells that constitutively express Hsp105a,and
showed that Hsp105a enhanced the oxidative stress-induced
apoptosis at or upstream of p38 mitogen-activated protein
kinase (p38) activation.
EXPERIMENTAL PROCEDURES
Cell culture
Mouse teratocarcinoma F9 cells were obtained from the
Japanese Cancer Research Resources Bank and maintained
Correspondence to T. Hatayama, Department of Biochemistry,
Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi,
Yamashina-ku, Kyoto 607-8414, Japan.
Fax: + 81 75 595 4758, Tel.: + 81 75 595 4653,
E-mail:

Abbreviations: HSP, heat shock protein; JNK, c-Jun N-terminal
kinase; PARP, poly (ADP-ribose) polymerase; Ac-DEVD-pNA,
N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide.
(Received 19 March 2002, revised 19 June 2002,
accepted 11 July 2002)
Eur. J. Biochem. 269, 4143–4151 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03109.x
in Dulbecco’s modified Eagle’s minimal essential medium
(Nissui Pharmaceutical) supplemented with 10% foetal
bovine serum (Life Technologies) in a humidified atmo-
sphere of 5% CO
2
in air at 37 °C. To induce cell differen-
tiation, cells grown on collagen-coated culture dishes were
incubated in the presence of 100 n
M
retinoic acid or 1 m
M
dibutyryl-cAMP/100 n
M
retinoic acid at 37 °Cfor6days.
Cell morphology was examined using a difference interfer-
ence contrast microscope.
Construction of mouse Hsp105a expression plasmid
and isolation of Hsp105a-over-expressing cells
Plasmid pcDNA105a wasusedtoexpressmouseHsp105a
in F9 cells. To construct this plasmid, the mouse Hsp105a
cDNA derived from pB105-1 plasmid [21] was subcloned
into EcoRV–XbaI sites of the mammalian expression vector
pcDNA3 (Invitrogen).
F9 cells were transfected with pcDNA105a or pcDNA3

empty vector by lipofection using Superfect reagent (Qiagen)
according to the manufacturer’s instructions. Forty-eight
hours after transfection, the cells were maintained in
complete medium containing 400 lgÆmL
)1
geneticin (Life
Technologies) for 3 weeks to select geneticin-resistant cells.
The surviving cell clones were isolated, grown in complete
medium containing 200 lgÆmL
)1
geneticin, and the expres-
sion levels of Hsp105a were analysed by Western blotting
using anti-mouse Hsp105 Ig [28].
Oxidative stress treatment
Cells were grown exponentially on culture dishes at 37 °C
for 24 h and then treated with 0.25–2 m
M
hydrogen
peroxide in NaCl/P
i
containing 0.9 m
M
CaCl
2
and 0.5 m
M
MgCl
2
at 37 °C for 30–60 min, washed with NaCl/P
i

and
further incubated in fresh medium at 37 °Cfor24h.
Cell viability assay
After stress treatment, cells were incubated in medium
containing 50 lgÆmL
)1
neutral red at 37 °C for 3 h, then
fixed with 1% formaldehyde containing 1% CaCl
2
for
1 min. The dye incorporated into viable cells was extracted
with 50% ethanol containing 1% acetic acid, and absorb-
ance at 540 nm was measured.
DNA fragmentation analysis
DNA fragmentation was analysed essentially as described
by Ishizawa et al. [29]. Cells were lysed at 37 °Cfor30min
in 200 lLlysisbuffer(10m
M
Tris/HCl pH 8.0, 150 m
M
NaCl, 10 m
M
EDTA, 0.1% SDS, 0.5 mgÆmL
)1
ribonuc-
lease A, 0.5 mgÆmL
)1
proteinase K), and the cell lysates
were mixed with 300 lLNaIsolution(6
M

NaI, 10 m
M
Tris/HCl pH 8.0, 13 m
M
EDTA, 0.5% sodium N-lauroyl-
sarcosine, 30 lgÆmL
)1
glycogen) and incubated at 60 °Cfor
15 min. An equal volume of isopropanol was added to the
mixtures, which were shaken vigorously and kept for
15 min at room temperature. After centrifugation at
15 000 g for 15 min, the precipitate was washed successively
with 50% and 100% isopropanol, dried in air, and resolved
in Tris/HCl/EDTA buffer (10 m
M
Tris/HCl pH 8.0, 1 m
M
EDTA). Aliquots of 5 lg of DNA were electrophoresed on
2% agarose gels and stained with 1 lgÆmL
)1
ethidium
bromide.
Morphological examination of apoptotic cells
Cells were plated onto coverslips at a density of 1 · 10
5
cellsÆcm
)2
and grown at 37 °C for 24 h. After treatment,
cells were washed with NaCl/P
i

, fixed with 3.7% formalde-
hyde for 30 min at room temperature, and stained with
10 l
M
Hoechst 33342 for 10 min in the dark. After washing
with NaCl/P
i
, the stained cells were observed using a
fluorescence microscope (Zeiss). Cells were scored as
apoptotic if they displayed nuclear fragmentation and/or
chromatin condensation.
Assay for poly (ADP-ribose) polymerase (PARP) cleavage
Cells (1 · 10
6
cells) were lysed with 200 lLlysisbuffer
(50 m
M
Tris/HCl pH 8.0, 150 m
M
NaCl, 1% NP-40, 0.1%
SDS, 5 lgÆmL
)1
aprotinin, 5 lgÆmL
)1
leupeptin, 2 lgÆmL
)1
pepstatin A, 1 m
M
phenylmethanesulfonyl fluoride) on ice
for 1 h. The lysate was then sonicated for 10 s and

centrifuged at 20 000 g for 15 min at 4 °C. The supernatant
was recoverd as the cell extract. Aliquots (20 lgprotein)of
cell extracts in SDS sample buffer containing urea (62.5 m
M
Tris/HCl pH 6.8, 6
M
urea, 10% glycerol, 2% SDS, 5% 2-
mercaptoethanol, 0.00125% Bromophenol blue) were sub-
jected to 7.5% SDS/PAGE, then transferred onto nitrocel-
lulose membranes by electrotransfer. The membranes were
blocked with 10% skim milk in NaCl/Tris (20 m
M
Tris/HCl
pH 7.6, 137 m
M
NaCl) containing 0.1% Tween 20 (NaCl/
Tris/Tween), and incubated with anti-PARP Ig (Santa
Cruz). Then, the membranes were incubated with horse-
radish peroxidase-conjugated anti-rabbit IgG, and the
antibody–antigen complexes were detected using the ECL-
Western blot detection system (Amersham Pharmacia
Biotech).
Measurement of caspase-3 activity
Caspase-3 activity was measured using the colorimetric
CaspACE assay system according to the manufacturer’s
instructions (Promega). Briefly, cells (1 · 10
6
cells) were
suspended in 50 lL cell lysis buffer on ice for 10 min, and
lysed by freezing and thawing. After centrifugation at

20 000 g for 20 min at 4 °C, the supernatants were recov-
ered as cell extracts. Cell extracts (50 lgprotein)were
incubated in caspase assay buffer (100 m
M
Hepes pH 7.5,
10 m
M
sucrose, 0.1% Chaps, 10 m
M
dithiothreitol) con-
taining 200 l
M
N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide
(Ac-DEVD-pNA) at 25 °C for 2 h, and then absorbance
at 405 nm was measured using a microplate reader.
Release of cytochrome
c
from mitochondria
Release of cytochrome c was analysed essentially as des-
cribed by Yano et al. [30]. Briefly, cell suspensions were
mixed with an equal volume of 100 lgÆmL
)1
digitonin in
NaCl/P
i
, and incubated at 25 °C for 5 min. After centrif-
ugation at 15 000 g for 2 min, supernatants were recovered
as cytosolic fractions. Pellets were dissolved in NaCl/P
i
containing 0.5% Triton X-100, and centrifuged, then

supernatants were recovered as mitochondrial fractions.
4144 N. Yamagishi et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Both fractions were subjected to 15% SDS/PAGE, and
analysed by Western blotting using anti-cytochrome c Ig
(Santa Cruz).
Activation of JNK and p38
Phosphoryled JNK and p38 were detected by Western
blotting using PhosphoPlus JNK (Thr183/Tyr185) and
PhosphoPlus p38 (Thr180/Tyr182) antibody kits (Cell
Signaling Technology, Inc.), respectively. Cell extracts
(20 lg protein) were separated by SDS/PAGE (10%
polyacrylamide), and analysed by Western blotting using
phosphorylation state-specific anti-JNK Ig or anti-p38 Ig.
Then, the membranes were incubated at 50 °Cfor30minin
stripping buffer (62.5 m
M
Tris/HCl pH 6.7, 2% SDS,
100 m
M
2-mercaptoethanol), and total JNK or p38 on the
same membranes were detected using anti-JNK Ig or anti-
p38 Ig, respectively.
Kinase activity of p38 was assayed by its ability to
phosphorylate MAPKAPK-2. Cell extracts (20 lgprotein)
were separated by SDS/PAGE (10% polyacrylamide), and
analysed by Western blotting using phosphorylation state-
specific anti-(MAPKAPK-2) Ig (Cell Signaling Technology,
Inc.).
RESULTS
Characterization of stable transfectants over-expressing

Hsp105a
To determine the role of Hsp105a in embryonal cells, we
established mouse embryonal F9 cell clones that express
Hsp105a at high levels. In this study, we used two
Hsp105a over-expressing F9 cell lines, S3 and S23, in
which the expression levels of Hsp105a were about two-
and threefold higher than that in parental F9 cells or
controls transfected with empty pcDNA3 vector (V1),
respectively (Fig. 1A). The growth rates of S3 and S23
cells were not significantly different from those of F9
and V1 cells (Fig. 1B). F9 cells can be differentiated
toward primitive endoderm-like and parietal endoderm-
like cells by exposure to retinoic acid and dibutyryl-
cAMP/retinoic acid, respectively [31,32]. Upon exposure
to retinoic acid, S3 and S23 cells showed an enlarged and
flattened morphology as seen in F9 or V1 cells, which is
characteristic of primitive endoderm-like cells (Fig. 1C,
e–h). In addition, the Hsp105a over-expressing cells
were also induced to differentiate toward parietal endo-
derm-like cells by treatment with retinoic acid and
dibutyryl-cAMP; they also showed the typical changes
in morphology (Fig. 1C, i–l). Thus, over-expression of
Hsp105a did not affect cell growth and differentiation
toward primitive endoderm-like and parietal endoderm-
like phenotypes in F9 cells.
Enhancement of oxidative stress-induced apoptosis
by over-expression of Hsp105a in F9 cells
As Hsp105a is expressed transiently at high levels
during mouse embryogenesis and is localized in apop-
totic cells [27], we next examined the effects of over-

expression of Hsp105a on stress-induced cell death in
embryonal F9 cells. When F9, V1, S3 and S23 cells
weretreatedwith0.25–1m
M
hydrogen peroxide for 1 h,
Fig. 1. Over-expression of Hsp105a in F9 cells and its effects on growth and differentiation. (A) Cell extracts (20 lg protein) from parental F9 cells
(F9) and cell clones stably transfected with either pcDNA3 vector (V1) or pcDNA105a (S3 and S23) were separated by SDS/PAGE, and the levels
of Hsp105a were determined by Western blotting using anti-mouse Hsp105 Ig. (B) F9. V1, S3 and S23 cells were plated into 35 mm culture dishes at
adensityof1· 10
4
cells per dish, and cultured for 6 days at 37 °C. At the indicated times, cell numbers were counted. (C) Cells (1 · 10
5
cells per
dish) were cultured in the presence of 100 n
M
retinoic acid (+ RA) or 1 m
M
dibutyryl-cAMP/100 n
M
retinoic acid (+ RA/cAMP) at 37 °Cfor
6 days. Cell morphology was observed using a difference interference contrast microscope. Scale bars ¼ 10 lm.
Ó FEBS 2002 Hsp105 enhances oxidative stress-induced apoptosis (Eur. J. Biochem. 269) 4145
S3 and S23 cells were more sensitive to the oxidative
stress than F9 or V1 cells (Fig. 2A). Therefore, we
further analysed the hydrogen peroxide-induced cell
death of F9 cells.
Cell death is classified into two morphologically and
biochemical distinct modes, apoptosis and necrosis [1].
To characterize the hydrogen peroxide-induced cell
death in F9 cells, we examined whether DNA fragmen-

tation, characteristic of apoptosis, occurred in these cells.
As shown in Fig. 2B, nucleosomal-length DNA frag-
mentation was observed in the hydrogen peroxide-
treated F9 cells, and the amounts of fragmented DNA
caused by low doses of hydrogen peroxide were
increased in the Hsp105a over-expressing cells as
compared with those in S3 and S23 cells. In addition,
apoptotic morphology such as nuclear condensation and
chromatin fragmentation was also prominently observed
by Hoechst 33342 staining in these cells (Fig. 3A), and
the rate of apoptotic cells was approximately threefold
higher in the Hsp105a over-expressing cells than in F9
or V1 cells (Fig. 3B). However, these morphological
changes were suppressed by treatment with a cell-
permeable caspase inhibitor zVAD-fmk. Thus, Hsp105a
was demonstrated to enhance oxidative stress-induced
apoptosis in F9 cells.
Over-expression of Hsp105a enhances caspase-3 activity
and PARP cleavage after treatment with hydrogen
peroxide
A common event in the apoptotic pathway is the
activation of caspases. These enzymes participate in a
cascade that is triggered in response to pro-apoptotic
signals and results in cleavage of a set of proteins,
resulting in disassembly of the cells. Caspase-3 is a major
effector caspase, and induces cleavage of several substrate
proteins and is responsible for several apoptotic processes.
We next assayed caspase-3 activity in extracts from cells
treated with oxidative stress. As shown in Fig. 4A,
although caspase-3 activity increased slightly in F9 and

V1 cells treated with hydrogen peroxide, its activity was
increased markedly in S3 and S23 cells by this treatment.
In addition, the activation of caspase-3 was suppressed by
the treatment with a cell-permeable caspase inhibitor
zVAD-fmk.
PARP, a DNA repair-related enzyme, is an important
substrate of caspase-3, and is cleaved from a 116-kDa
protein to an 85-kDa fragment and inactivated during
apoptosis [33,34]. Western blotting analysis clearly revealed
cleavage of PARP in F9 cells treated with hydrogen
peroxide (Fig. 4B). Furthermore, in accordance with the
Fig. 2. Effects of Hsp105a over-expression on
the sensitivity of F9 cells to hydrogen peroxide.
F9, V1, S3 and S23 cells were exposed to
0.1–1 m
M
hydrogen peroxide for 1 h, and
further incubated for 24 h at 37 °C. (A) Cell
viability was then determined by neutral red
assay. Experiments were repeated at least
three times and essentially the same results
were obtained each time. (B) DNA was
extracted from the cells treated with hydrogen
peroxide, and aliquots (5 lg each) of DNA
were electrophoresed on a 2% agarose gel and
visualized by staining with ethidium bromide.
4146 N. Yamagishi et al. (Eur. J. Biochem. 269) Ó FEBS 2002
enhancement of caspase-3 activity, the cleavage of PARP
by hydrogen peroxide was more intense in S3 and S23
cells than in F9 or V1 cells. Thus, over-expression of

Hsp105a was shown to markedly enhance the activation
of procaspase-3 during apoptosis induced by hydrogen
peroxide.
Over-expression of Hsp105a enhances release
of cytochrome
c
from mitochondria after treatment
with hydrogen peroxide
In mammalian cells, one of the main pathways that activates
caspase-3 is via mitochondria. When mitochondria receive
appropriate signals from a variety of stresses or are
damaged irreversibly, pro-apoptotic molecules such as
cytochrome c are released from mitochondria into the
cytosol [35–37]. In the cytosol, cytochrome c forms a
complex with Apaf-1 and procaspase-9, and activates
caspase-9, which in turn converts procaspase-3 into its
active form, resulting in apoptosis [38–40]. We next analysed
whether cytochrome c is released from mitochondria in F9
cells by oxidative stress. When F9 cells were fractionated
into mitochondrial and cytosolic fractions and cytochrome c
was examined by Western blotting, a large amount of
cytochrome c was found in mitochondria with only a small
amount in the cytosolic fraction of control cells under these
experimental conditions (Fig. 5A). However, although the
amounts of cytochrome c released by hydrogen peroxide
increased slightly in F9 or V1 cells, the release was increased
to a greater extent in S3 and S23 cells than in control and V1
cells (Fig. 5B). Thus, over-expression of Hsp105a seemed to
enhance the release of cytochrome c from mitochondria in
F9 cells by hydrogen peroxide.

Fig. 3. Enhancement of oxidative stress-induced apoptosis by over-expression of Hsp105a. (A) F9, V1, S3 and S23 cells were grown on coverslips,
exposedto1m
M
hydrogen peroxide for 1 h, and further incubated at 37 °C for 24 h. Ten l
M
z-VAD-fmk was added to the medium 1 h before
treatment. Cells were washed with NaCl/P
i
, fixed with 3.7% formaldehyde, and stained with 10 l
M
Hoechst 33342. Nuclear morphology of cells
was observed using a fluorescence microscope. (B) Rates of apoptotic cells were obtained from at least 200 cells in each experiment. Data are shown
as the means ± SD of three independent experiments. The significance of differences was assessed by unpaired Student’t t-test.
Fig. 4. Effects of over-expression of Hsp105a on caspase-3 activity. F9,
V1, S3 and S23 cells were exposed to 1 m
M
hydrogen peroxide for 1 h,
and further incubated for 24 h at 37 °C. (A) Caspase-3 activity in cell
extracts was measured with caspase-3 substrate, Ac-DEVD-pNA.
Data represent the means ± SD of three independent experiments. (B)
Aliquots (20 lg protein) of cell extracts were separated by SDS/
PAGE, and PARP (116 kDa) and the cleaved fragment (85 kDa) were
detected by Western blotting using anti-PARP Ig.
Ó FEBS 2002 Hsp105 enhances oxidative stress-induced apoptosis (Eur. J. Biochem. 269) 4147
Over-expression of Hsp105a enhances activation
of p38 after treatment with hydrogen peroxide
JNK and p38 pathways are activated by cellular stresses and
inflammatory cytokines, resulting in growth arrest and
apoptosis, and have been implicated as key regulators of
stress-induced apoptosis in many cell types [41,42]. Fur-

thermore, mitochondria are influenced by proapoptotic
signals through the JNK pathway [43]. To determine the
effects of Hsp105a on JNK and p38 signalling pathways, we
examined the activation of JNK and p38 in F9 cells by
oxidative stress. As shown in Fig. 6A, JNK was not
activated in control F9 or V1 cells by treatment with
hydrogen peroxide. In contrast, hydrogen peroxide-treat-
ment induced marked activation of JNK within 30 min in
S23 cells, but not in S3 cells. Therefore, the enhancement of
the oxidative stress-induced JNK-activation seemed not to
be solely due to the over-expression of Hsp105a in F9 cells.
On the other hand, p38 was activated at low levels within
1–2 h in F9 and V1 cells after treatment with hydrogen
peroxide, and the activation of p38 by hydrogen peroxide
was enhanced in both S3 and S23 cells compared with F9
and V1 cells (Fig. 6B). Thus, over-expression of Hsp105a
seemed to enhance the oxidative stress-induced activation of
p38 but not JNK in F9 cells.
As Hsp105a enhances the activation of p38 induced by
oxidative stress, we further examined whether its activation
is responsible for the induction of apoptosis in F9 cells using
SB202190, a potent inhibitor of p38. As shown in Fig. 7A,
although hydrogen peroxide-treatment induced apoptosis in
F9 cells as described above, the apoptosis was significantly
suppressed by SB202190. Under these conditions, although
phosporylation of MAPKAPK-2, a substrate of p38, was
enhanced by the hydrogen peroxide treatment, it was
suppressed to basal level by treatment with 10 l
M
SB202190

(Fig. 7B). These findings indicate that the activation of p38
is an essential step for induction of apoptosis by hydrogen
peroxide, and Hsp105a is suggested to enhance the oxida-
Fig. 6. Effects of Hsp105a on activation of
JNK and p38 by oxidative stress. F9, V1, S3
andS23cellswereexposedto1m
M
hydrogen
peroxide for 30 min and further incubated for
0.5, 1 or 2 h at 37 °C. Aliquots (20 lgprotein)
of cell extracts were separated by SDS/PAGE.
Activated and total JNK or p38 were detected
by Western blotting, as described in Experi-
mental procedures. (A) JNK, (B) p38. Upper
and lower panels represent activated and total
JNK or p38, respectively.
Fig. 5. Effects of Hsp105a over-expression on release of cytochrome c
from mitochondria. F9, V1, S3 and S23 cells were exposed to 1 m
M
hydrogen peroxide for 45 min, and further incubated for 24 h at
37 °C. Cells were then fractionated into cytosolic and mitochondrial
fractions, as described in Experimental procedures. (A) Both fractions
were subjected to 15% SDS/PAGE, and analysed by Western blotting
using anti-cytochrome c Ig.C,cytosolicfraction;M,mitochondrial
fraction. (B) The densities the cytochrome c bands were quantified by
densitometry, and the rates release into the cytosol are shown. Data
represent the means ± SD of three independent experiments. Filled
bars, untreated cells; open bars, hydrogen peroxide-treated cells.
4148 N. Yamagishi et al. (Eur. J. Biochem. 269) Ó FEBS 2002
tive stress-induced apoptosis at or upstream of p38 activa-

tion in F9 cells.
DISCUSSION
Hsp105a is expressed in most tissues, but its levels are
especially high in the brain of adult mammals such as rats,
mice and humans [21–23]. We have shown that Hsp105a
plays an important role in protection of neuronal cells
against stress-induced apoptosis [26]. In accordance with
these findings, ischemia/reperfusion in the rat forebrain
induces the expression of HSP105/110 family proteins,
Hsp105a, APG-1 (testis-specific homologue of Hsp105) and
APG-2 [44,45], and Hsp110 (hamster homologue of
Hsp105a) confers heat resistance on rat fibroblasts and
human epithelial carcinoma cells [46]. On the other hand, we
have also shown that the levels of Hsp105a increase
transiently in embryonic tissues during mouse embryogen-
esis, and this protein is localized not only in various tissues,
but also in apoptotic cells and apoptotic bodies at the
interdigital regions of limbs [27]. In the present study, to
explore the function of Hsp105a in embryogenesis, we
established mouse embryonal F9 cell lines that constitutively
express Hsp105a. Although growth rate and differentiation
of F9 cells were not affected by the over-expression of
Hsp105a, the sensitivity of cells to oxidative stress was
enhanced by the over-expression of Hsp105a,andthese
findings were in clear contrast with those in neuronal PC12
cells. However, as sensitivity of F9 cells to stresses such as
heat shock, etoposide, actinomycin D and serum depriva-
tion was also enhanced by over-expression of Hsp105a
(unpublished data), the enhancement of cell death by
Hsp105a seemed to be a general phenomenon in embryonal

F9 cells. The present findings together with previous
observations in neuronal PC12 cells suggested that Hsp105a
has a pro-apoptotic effect in embryonal cells and an anti-
apoptotic effect in neuronal cells. Thus, these observations
provide the first evidence that Hsp105a can function as an
enhancer or suppressor of apoptosis depending on the cell
type in mammals.
Apoptosis is an active process resulting in characteristic
morphological changes such as cell shrinkage, condensation
of chromatin and membrane blebbing [2–4]. The common
pathway of apoptosis involves a family of proteases known
as the caspases, which are activated in a proteolytic cascade
to cleave specific substrates. The release of cytochrome c
from mitochondria triggers the formation of apoptosome
complex with Apaf-1 and pro-caspase-9, and activates
caspase-9, which then in turn activates downstream effector
caspases such as caspase-3 [38–40]. Active caspase-3 cleaves
several substrates such as PARP [33,34], and activates death
effector molecules or triggers the structural changes char-
acteristic of apoptotic cells. Here, we showed that over-
expression of Hsp105a enhances PARP cleavage, caspase-3
activation and release of cytochrome c from mitochondria
in F9 cells exposed to hydrogen peroxide, and our results
Fig. 7. Suppression of oxidative stress-induced
apoptosis in F9 cells by a potent p38 inhibitor.
F9, V1, S3 and S23 cells were grown on
coverslips, treated with or without 10 l
M
SB202190 for 1 h before and during the
1 h-treatment with 1 m

M
hydrogen peroxide,
and further incubated at 37 °C for 24 h (A) or
2 h (B). (A) Cells were then washed with
NaCl/P
i
, fixed with 3.7% formaldehyde, and
stainedwith10l
M
Hoechst 33342. Nuclear
morphology of cells was observed using a
fluorescence microscope. Rates of apoptotic
cells were obtained from at least 300 cells in
each experiment. Data are shown as the means
± SD of at least three independent experi-
ments. The significance of differences was
assessed by unpaired Student’t t-test. (B) Cell
extracts (20 lg proteins) of S23 cells were
separated by SDS/PAGE, and phosphory-
lated MAPKAPK-2 was detected by Western
blotting using anti-phospho-MAPKAPK-2
Ig.
Ó FEBS 2002 Hsp105 enhances oxidative stress-induced apoptosis (Eur. J. Biochem. 269) 4149
suggested that Hsp105a enhances the apoptosis at or
upstream of cytochrome c release from mitochondria.
Furthermore, the transmission of signals from external
stresses is accompanied by the activation of a family of
stress-activated protein kinases, JNK and p38. Activation of
these signalling pathways leads to apoptosis [41,42], and
mitochondria is influenced by proapoptotic signal trans-

duction through the JNK pathway [43]. As the activation of
p38 is an essential step for apoptosis induced by hydrogen
peroxide in F9 cells as shown in Fig. 7, Hsp105a was
suggested to enhance the oxidative stress-induced apoptosis
directly or indirectly at or upstream of activation of p38. In
contrast, although Hsp105a prevents the apoptosis induced
by several stresses including hydrogen peroxide in neuronal
PC12 cells, p38 is not activated by these stresses in neuronal
cells [26]. The p38 signalling pathway may be a possible
target at which Hsp105a enhances the apoptosis induced by
hydrogen peroxide in embryonal cells.
Several Hsps have been shown to modulate the pathway
of apoptosis positively or negatively. Hsp60 with or without
Hsp10 directly stimulates apoptosis by promoting the
proteolytic maturation of caspase-3 [17,18]. In contrast,
Hsp70, Hsp90 and Hsp27 exert negative influences on
apoptotic signalling. In particular, Hsp70 has been shown to
protect against apoptosis by a variety of stressors through
suppression of JNK activation [10–13] and apoptosome
formation [14,15]. Interestingly, a recent study demonstra-
ted that the chaperone activity of Hsp70 is required for
protection against heat-induced apoptosis [47]. In contrast,
we cannot detect the chaperone activity of Hsp105a, but the
protein exists as complexes associated with Hsp70/Hsc70
[23,24] and suppresses the Hsc70 chaperone activity [25].
Therefore, it is possible that Hsp105a may stimulate stress-
induced apoptosis by negative regulation of Hsp70/Hsc70
chaperone that is required for suppression of apoptosis.
In conclusion, we showed here that Hsp105a enhances
apoptosis at or upstream of p38 activation. The apoptosis-

enhancing activity of Hsp105a may play important roles in
organogenesis, in which marked apoptosis occurs during
mouse embryogenesis, although further studies are neces-
sary to understand the precise mechanism by which Hsp105a
enhances stress-induced apoptosis.
ACKNOWLEDGMENTS
This study was supported in part by grant from the Ministry of
Education, Science, Sports and Culture of Japan (T. H.).
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