Báo cáo khoa học: Mammalian 105 kDa heat shock family proteins suppress hydrogen peroxide-induced apoptosis through a p38 MAPK-dependent mitochondrial pathway in HeLa cells potx

Mammalian 105 kDa heat shock family proteins suppress
hydrogen peroxide-induced apoptosis through a p38
MAPK-dependent mitochondrial pathway in HeLa cells
Nobuyuki Yamagishi, Youhei Saito and Takumi Hatayama
Department of Biochemistry, Division of Biological Sciences, Kyoto Pharmaceutical University, Japan
Heat shock proteins (Hsps) are a set of highly
conserved proteins produced in response to physiologi-
cal and environmental stress that serve to protect cells
from stress-induced damage by preventing protein
denaturation and ⁄ or repairing such damage [1]. Mam-
malian Hsps are classified into several families on the
basis of their apparent molecular weight and function,
such as the HSP105 ⁄ 110, HSP90, HSP70, HSP60,
HSP40, and HSP27 families. The HSP70 family is a
major and well-characterized group of Hsps. Several
species of HSP70 family proteins are present in the
various compartments of eukaryotic cells. These pro-
teins play important roles as molecular chaperones
that prevent the irreversible aggregation of denatured
proteins and assist the folding, assembly and transloca-
tion across membranes of cellular proteins [2,3]. In
addition, Hsp70 protects against apoptosis caused by a
variety of stressors such as heat shock, oxidative stress
and chemotherapeutic agents [4–6], and recent studies
have demonstrated that Hsp70 can modulate the func-
tions of several major components in the apoptotic
process, including the caspase cascade and the c-Jun
N-terminal kinase (JNK) signaling pathway [7–12].
Hsp105a and Hsp105b are mammalian members of
the HSP105 ⁄ 110 family, a subgroup of the HSP70
family. Hsp105a is expressed constitutively and in

response to various forms of stress, while Hsp105b is
an alternatively spliced form of Hsp105a that is
Keywords
apoptosis; H
2
O
2;
Hsp105; JNK; p38 MAPK
Correspondence
T. Hatayama, Department of Biochemistry,
Division of Biological Sciences, 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:
(Received 28 April 2008, revised 23 June
2008, accepted 15 July 2008)
doi:10.1111/j.1742-4658.2008.06598.x
Hsp105a and Hsp105b are major heat shock proteins in mammalian cells
that belong to a subgroup of the HSP70 family, HSP105 ⁄ 110. Previously,
we have shown that Hsp105a has opposite effects on stress-induced apop-
tosis depending on the cell type. However, it is not fully understood how
Hsp105 regulates stress-induced apoptosis. In this study, we examined how
Hsp105a and Hsp105b regulate H
2
O
2
-induced apoptosis by using HeLa

cells in which expression of Hsp105a or Hsp105b was regulated using
doxycycline. Overexpression of Hsp105a and Hsp105b suppressed the acti-
vation of caspase-3 and caspase-9 by preventing the release of cyto-
chrome c from mitochondria in H
2
O
2
-treated cells. Furthermore, both
c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase
(p38 MAPK) were activated by treatment with H
2
O
2
, and the activation of
both kinases was suppressed by overexpression of Hsp105a and Hsp105b.
However, H
2
O
2
-induced apoptosis was suppressed by treatment with a
potent inhibitor of p38 MAPK, SB202190, but not a JNK inhibitor,
SP600125. These findings suggest that Hsp105a and Hsp105b suppress
H
2
O
2
-induced apoptosis by suppression of p38 MAPK signaling, one of
the essential pathways for apoptosis.
Abbreviations
DOX, doxycycline; G3DPH, glyceraldehyde 3-phosphate dehydrogenase; Hsps, Heat shock proteins; JNK, c-Jun N-terminal kinase; p38

MAPK, p38 mitogen-activated protein kinase; PARP, poly(ADP-ribose)polymerase; PBS, phosphate-buffered saline; SDS-PAGE, SDS-
polyacrylamide gel electrophoresis.
4558 FEBS Journal 275 (2008) 4558–4570 ª 2008 The Authors Journal compilation ª 2008 FEBS
expressed specifically during mild heat shock [13–15].
These proteins suppress the aggregation of denatured
proteins caused by heat shock in vitro, as does Hsp70,
but have not been found to have refolding activity
[16]. We have demonstrated previously that Hsp105a
protects neuronal PC12 cells against apoptosis induced
by various stresses such as H
2
O
2
, heat shock and anti-
cancer drugs [17]. In addition, Hsp105a suppresses
protein aggregation and apoptosis caused by the
expression of proteins with expanded polyQ tracts in a
cellular model of spinal and bulbar muscular atrophy
[18]. Recently, we also showed that Hsp105a and
Hsp105b suppress staurosporine-induced apoptosis by
inhibiting the translocation of Bax to mitochondria
[19]. In contrast, Hsp105a has the opposite effects on
apoptosis induced by H
2
O
2
in mouse embryonal F9
cells, suggesting that Hsp105a has opposing effects on
stress-induced apoptosis depending on the cell type
[20]. However, it is not fully understood how Hsp105

regulates apoptosis caused by a variety of stresses.
Here, we examined the mechanisms by which Hsp105
regulates H
2
O
2
-induced apoptosis by using HeLa cells
in which expression of Hsp105a or Hsp105b was regu-
lated using doxycycline. Our results suggest Hsp105a
and Hsp105b suppress H
2
O
2
-induced apoptosis by inhi-
bition of the p38 mitogen-activated protein kinase (p38
MAPK) pathway but not the JNK pathway.
Results
Hsp105a and Hsp105 b suppress apoptosis
induced by H
2
O
2
but not by heat shock in
HeLa cells
Previously, we have established human HeLa cell lines
(HeLa-tet ⁄ Hsp105a and HeLa-tet ⁄ Hsp105b) in which
overexpression of mouse Hsp105a or Hsp105b was
induced by removing DOX from the culture medium.
In HeLa-tet ⁄ Hsp105a cells, the level of Hsp105a was
increased approximately twofold by removal of DOX

from the culture medium, and was comparable to that
of Hsp105 (Hsp105a plus Hsp105b) after heat expo-
sure at 42 °C for 6 h. The inducible level of mouse
Hsp105b in HeLa-tet ⁄ Hsp105b cells after DOX
removal was comparable to that of Hsp105b induced
after heat exposure [19]. Using these cells, we examined
whether overexpression of Hsp105a or Hsp105b sup-
presses cell death induced by treatment with H
2
O
2
and
heat shock. As shown in Fig. 1A, these cells were more
resistant to H
2
O
2
as a result of overexpression of
Hsp105a or Hsp105b. As cell death is classified into
two morphologically and biochemical distinct modes,
apoptosis and necrosis, we next examined whether
apoptotic features such as the fragmentation of chro-
matin and the externalization of phosphatidylserine on
the plasma membrane were induced by treatment with
H
2
O
2
. Treatment with H
2

O
2
did induce the develop-
ment of fragmented and ⁄ or condensed chromatin
(Fig. 1B,C). In addition, externalized phosphatidy-
lserine in the plasma membrane, which is detected by
annexin V binding, was observed on the plasma mem-
brane of H
2
O
2
-treated cells but not on that of
untreated control cells (Fig. 1B). Next, we examined
whether Hsp105a and Hsp105b suppress H
2
O
2
-induced
apoptosis. As shown in Fig. 1C, overexpression of
Hsp105a or Hsp105b reduced the number of apoptotic
cells with fragmented and ⁄ or condensed chromatin. In
contrast, overexpression of Hsp105a or Hsp105b did
not significantly affect cell death induced by heat
shock, although apoptotic cell death was induced by
heat shock treatment (Fig. 2).
Hsp105a and Hsp105 b suppress H
2
O
2
-induced

apoptosis upstream of cytochrome c release
from the mitochondria
Caspases are a family of cysteine aspartic acid protease
that are central regulators of apoptosis and form a pro-
teolytic cascade that results in the cleavage of distinct
and vital proteins [21–23]. In particular, caspase-3 is one
of the key executioners of apoptosis, as it is either par-
tially or totally responsible for the proteolytic cleavage
of many key proteins such as poly(ADP-ribose)polymer-
ase (PARP) [24]. Therefore, we next examined whether
caspase-3 is activated in HeLa cells treated with H
2
O
2
.
As shown in Fig. 3A, active forms of caspase-3 and a
cleaved fragment of PARP were detected in cells treated
with H
2
O
2
. Furthermore, overexpression of Hsp105a or
Hsp105b markedly reduced both the amount of the
active forms of caspase-3 and PARP cleavage by treat-
ment with H
2
O
2
. In contrast, although caspase-3 was
markedly activated after heat shock, activation of cas-

pase-3 was not affected by overexpression of Hsp105a
or Hsp105b (Fig. 3B). These results suggested that
overexpression of Hsp105a and Hsp105b prevents the
activation of caspase-3, resulting in suppression of
H
2
O
2
-induced apoptosis. However, Hsp105a and
Hsp105b seem to suppress H
2
O
2
-induced apoptosis
upstream of rather than by direct inhibition of caspase-3
activation, as they do not suppress the activation of
caspase-3 mediated by heat shock.
In mammalian cells, one of the main pathways that
activates procaspase-3 is via mitochondria. When the
mitochondria receive appropriate signals from a vari-
ety of stresses, pro-apoptotic molecules such as cyto-
chrome c are released from mitochondria into the
N. Yamagishi et al. Suppression of H
2
O
2
-induced apoptosis by Hsp105
FEBS Journal 275 (2008) 4558–4570 ª 2008 The Authors Journal compilation ª 2008 FEBS 4559
A
B

C
Fig. 1. Hsp105 suppresses apoptosis induced by H
2
O
2
in HeLa cells. (A) HeLa-tet ⁄ Hsp105 cells that overexpressed Hsp105a or Hsp105b
()DOX) or not (+DOX) were exposed to 0.2–0.6 m
M H
2
O
2
for 1 h. Then, the cells were further incubated in fresh medium at 37 °C for 5 h.
Cell viability was determined by a neutral red uptake assay. Values are shown as the means ± SD of four independent experiments. The
significance of differences was assessed by an unpaired Student’s t-test: *P < 0.05 for cell viability in +DOX versus )DOX. (B)
HeLa-tet ⁄ Hsp105 cells maintained in the medium with 1 lgÆmL
)1
DOX were exposed to 0.4 mM H
2
O
2
for 1 h, and further incubated in fresh
medium at 37 °C for 5 h. The cells were then stained with annexin V-fluorescein isothiocyanate and Hoechst 33342. Phosphatidylserine
externalization and nuclear morphology were observed using a confocal laser scanning microscope. (C) HeLa-tet ⁄ Hsp105 cells were exposed
to 0.4 m
M H
2
O
2
for 1 h, and further incubated in fresh medium at 37 °C for 5 h. The cells were then stained with Hoechst 33342, and
nuclear morphology of cells was observed using a fluorescence microscope (left panels). Rates of apoptosis were calculated using at least

200 cells in each experiment. Values are the means ± SD of four independent experiments (right panel). The significance of differences was
assessed by an unpaired Student’s t-test: *P < 0.05 for apoptosis in +DOX versus ) DOX.
Suppression of H
2
O
2
-induced apoptosis by Hsp105 N. Yamagishi et al.
4560 FEBS Journal 275 (2008) 4558–4570 ª 2008 The Authors Journal compilation ª 2008 FEBS
cytosol, in which cytochrome c forms a complex with
Apaf-1 and procaspase-9, and activates procaspase-9
and procaspase-3 successively [25–27]. Therefore, we
next examined whether caspase-9 is activated in HeLa
cells treated with H
2
O
2
. When HeLa cells were treated
with H
2
O
2
, procaspase-9 was processed to active forms
(37 and 35 kDa fragments), and the activation of cas-
pase-9 was suppressed by overexpression of Hsp105a
or Hsp105b (Fig. 4A). In contrast, caspase-9 was
not activated by heat shock (Fig. 4B). Furthermore,
when the cellular distribution of cytochrome c in
HeLa-tet ⁄ Hsp105a cells was examined by immuno-
fluorescence microscopy, cytochrome c was found
exclusively in the mitochondria of control cells, but

was observed throughout the cytoplasm after treat-
ment with H
2
O
2
in most cells without overexpression
A
B
C
Fig. 2. Hsp105 does not suppress apoptosis induced by heat shock in HeLa cells. HeLa-tet ⁄ Hsp105 cells were heated at 45 °C for
15–60 min, and then the cells were further incubated at 37 °C for 5 h. Cell viability (A), phosphatidylserine externalization and nuclear morphol-
ogy (B), and rates of apoptosis (C) were determined as described in Fig. 1. Values are the means ± SD of four independent experiments. The
significance of differences was assessed by an unpaired Student’s t-test. There were no significant differences between groups.
N. Yamagishi et al. Suppression of H
2
O
2
-induced apoptosis by Hsp105
FEBS Journal 275 (2008) 4558–4570 ª 2008 The Authors Journal compilation ª 2008 FEBS 4561
of Hsp105a. However, release of cytochrome c was
suppressed in cells overexpressing Hsp105a (Fig. 5A).
In addition, when the cells were fractionated into a
soluble fraction and a particulate fraction containing
mitochondria by treatment with digitonin, a large
amount of cytochrome c was detected in the mitochon-
drial fraction of untreated cells with or without over-
expression of Hsp105a or Hsp105b (Fig. 5B).
However, in the cells treated with H
2
O

2
, the amount
of cytochrome c increased in the soluble fraction
concomitant with its decrease in the particulate frac-
tion. The release of cytochrome c from mitochondria
was suppressed by the overexpression of Hsp105a or
Hsp105b. Several studies have demonstrated that Bax,
A
B
Fig. 3. Hsp105 suppresses the activation of
caspase-3 induced by H
2
O
2
but not by heat
shock in HeLa cells. HeLa-tet ⁄ Hsp105 cells
were treated with 0.5 m
M H
2
O
2
for 0.5 or
1 h (A) or heat shock at 45 °C for 1 h (B).
Aliquots (20 lg of protein) of the cell
extracts were separated by SDS–PAGE and
immunostained using anti-Hsp105, anti-cas-
pase-3 or anti-PARP. The intensity of bands
was quantified using public-domain
NIH IMAGE
software ( />and normalized to the total protein content

detected by Coomassie Brilliant Blue
staining. Values are the means of two
independent experiments.
Suppression of H
2
O
2
-induced apoptosis by Hsp105 N. Yamagishi et al.
4562 FEBS Journal 275 (2008) 4558–4570 ª 2008 The Authors Journal compilation ª 2008 FEBS
a pro-apoptotic Bcl-2 family member, is translocated
from the cytoplasm to the mitochondria, and leads to
the release of cytochrome c and other proteins from the
mitochondrial intermembrane space [28,29]. However,
we did not detect Bax translocation to the mitochondria
in HeLa cells treated with H
2
O
2
(Fig. 5B). Under these
conditions, a mitochondrial matrix protein, Hsp60,
was recovered exclusively in the particulate fraction,
whereas a cytosolic protein, glyceraldehyde-3-phos-
phate dehydrogenase (G3DPH), was recovered exclu-
sively in the soluble fraction. Thus, Hsp105a and
Hsp105b suppress H
2
O
2
-induced apoptosis at or
upstream of cytochrome c release from mitochondria.

Hsp105a and Hsp105 b suppress H
2
O
2
-induced
apoptosis by inhibition of the p38 MAPK
pathway but not the JNK pathway
Both JNK and p38 MAPK have been implicated as
key regulators in some forms of stress-induced apopto-
sis [30,31]. Phosphorylation of JNK at Thr183 and
Typ185 is required for JNK activation [32]. Activation
of p38 MAPK occurs through phosphorylation of
Thr180 and Tyr182. Therefore, we examined whether
JNK and p38 MAPK are phosphorylated at
Thr183 ⁄ Tyr185 and Thr180 ⁄ Tyr182, respectively, in
HeLa cells treated with H
2
O
2
(Fig. 6). Phosphorylated
JNK and p38 MAPK were observed within 15 min
after treatment with H
2
O
2
, and the phosphorylation of
these kinases was maintained for more than 2 h. How-
ever, the phosphorylation of JNK and p38 MAPK
was markedly reduced in the cells overexpressing
Hsp105a or Hsp105b.

Next, we examined whether the activation of JNK or
p38 MAPK is required for H
2
O
2
-induced apoptosis,
using a p38 MAPK inhibitor, SB203580, and a JNK
inhibitor, SP600125. As shown in Fig. 7A, H
2
O
2
-
induced apoptosis was suppressed by treatment with
10 lm SB203580, but not by treatment with 10 lm
SP600125. Under these conditions, SB203580 markedly
suppressed phosphorylation of MAPK-activating pro-
tein kinase-2 (a substrate of p38 MAPK), but did not
affect the phosphorylation of c-Jun (a substrate of JNK).
Conversely, SP600125 markedly suppressed the phos-
phorylation of JNK with no effect on p38 MAPK activ-
ity (Fig. 7B). These results suggest that Hsp105a and
Hsp105b suppress H
2
O
2
-induced apoptosis by inhibiting
the p38 MAPK pathway but not the JNK pathway.
A
B
Fig. 4. Effect of Hsp105 overexpression on

the activation of caspase-9 induced by treat-
ment with H
2
O
2
. HeLa-tet ⁄ Hsp105 cells
were treated with 0.5 m
M H
2
O
2
for 0.5 or
1 h (A) or heat shock at 45 °C for 1 h (B).
Aliquots (20 lg of protein) of the cell
extracts were separated by SDS–PAGE and
immunostained using anti-Hsp105 or anti-
caspase-9. The intensity of bands was quan-
tified by public-domain
NIH IMAGE software,
and normalized to the total protein content
detected by Coomassie Brilliant Blue stain-
ing. Values are the means of two indepen-
dent experiments.
N. Yamagishi et al. Suppression of H
2
O
2
-induced apoptosis by Hsp105
FEBS Journal 275 (2008) 4558–4570 ª 2008 The Authors Journal compilation ª 2008 FEBS 4563
Discussion

We showed previously that Hsp105a has opposing
effects on H
2
O
2
-induced apoptosis depending on the
cell type; an anti-apoptotic effect in neuronal PC12
cells and a pro-apoptotic effect in embryonic F9 cells
[17,20]. However, it is not fully understood how
Hsp105a regulates H
2
O
2
-induced apoptosis. In addi-
tion, the role of Hsp105b in stress-induced apoptosis
remains to be clarified. In the present study, we
showed that, although Hsp105a and Hsp105b suppress
the activation of JNK and p38 MAPK by treatment
with H
2
O
2
in HeLa cells, H
2
O
2
-induced apoptosis was
suppressed by the inhibition of p38 MAPK but not
JNK. Thus, Hsp105a and Hsp105b were suggested to
suppress oxidative stress-induced apoptosis at or

upstream of activation of p38 MAPK.
Several studies have demonstrated that Bax, a pro-
apoptotic Bcl-2 family member, is one of the targets for
JNK ⁄ p38 MAPK signaling [33,34]. In response to stress,
Bax undergoes a conformational change and is redistrib-
uted to the mitochondria [35]. It is likely that Bax (and
related members of this subfamily) mediates the release
of cytochrome c from mitochondria [34]. Here, we
showed that Hsp105a or Hsp105 b suppress H
2
O
2
-
induced apoptosis by inhibiting the release of cyto-
chrome c from mitochondria in HeLa cells. However,
we did not detect Bax translocation to the mitochondria
A
B
Fig. 5. Hsp105 suppresses the release of cytochrome c from mitochondria induced by treatment with H
2
O
2
in HeLa cells. (A) HeLa-tet ⁄
Hsp105a cells were exposed to 0.5 m
M H
2
O
2
for 1 h, and then the cells were further incubated in fresh medium at 37 °C for 5 h. The cellu-
lar distribution of cytochrome c was then detected by indirect immunofluorescence microscopy using anti-cytochrome c antibody (green).

Mitochondria were detected by staining with Mitotracker (red). Arrows indicate cells in which cytochrome c has been released into the cyto-
plasm. (B) HeLa-tet ⁄ Hsp105 cells were exposed to 0.5 m
M H
2
O
2
for 1 h, and then the cells were further incubated in fresh medium at
37 °C for 5 h. The cells were then harvested, and were fractionated into cytosolic and mitochondrial fractions. Both fractions were subjected
to 15% SDS–PAGE, followed by immunostaining using anti-cytochrome c, anti-BAX, anti-Hsp60 or anti-G3PDH. Hsp60 and G3PDH are
marker proteins for the mitochondrial and cytoplasmic fractions, respectively. The intensity of bands was quantified by public-domain
NIH
IMAGE
software, and normalized to the total protein content detected by Coomassie Brilliant Blue staining. The amount of cytochrome c
released from mitochondria was estimated from the amounts of cytochrome c in the immunoblots of mitochondrial fraction. Values are the
means ± SD of three independent experiments. Statistical significance was determined by an unpaired Student’s t-test: *P < 0.05 for
cytochrome c release in +DOX versus )DOX.
Suppression of H
2
O
2
-induced apoptosis by Hsp105 N. Yamagishi et al.
4564 FEBS Journal 275 (2008) 4558–4570 ª 2008 The Authors Journal compilation ª 2008 FEBS
in HeLa cells treated with H
2
O
2
. On the other hand, we
observed previously that Hsp105a and Hsp105b sup-
press staurosporine-induced apoptosis by inhibiting the
translocation of Bax to the mitochondria in HeLa cells

[19]. Therefore, H
2
O
2
may induce apoptosis by the
release of cytochrome c from mitochondria without Bax
translocation to the mitochondria in HeLa cells. It has
been established that JNK phosphorylates the anti-
apoptotic proteins Bcl-2 and Bcl-X
L
and inhibits their
prosurvival function [36–39]. In addition to Bcl-2 and
Bcl-X
L
, previous studies have also implicated the pro-
apoptotic BH3-only proteins Bid and Bim in JNK ⁄ p38
MAPK-stimulated apoptosis [32,38]. Further studies
will be necessary to clarify the targets of p38 MAPK
signaling in HeLa cells treated with H
2
O
2
.
Apoptosis signal-regulating kinases (ASKs) are
serine ⁄ threonine kinases that activate both the p38
MAPK and JNK signaling pathways as MAPK kinase
kinase. ASKs are activated in response to various cyto-
toxic stresses, including oxidative stress and UV irradia-
tion, and play an essential role in stress-induced
apoptosis [40,41]. Therefore, it is likely that Hsp105a

and Hsp105b suppress oxidative stress-induced apopto-
sis through the prevention of ASK activity. ASK1 activ-
ity is stimulated by phosphorylation of ASK1 at
Thr838, and suppressed by phosphorylation at Ser83
[42,43]. However, overexpression of Hsp105a and
Hsp105b did not suppress the phosphorylation of ASK1
at Thr838, and did not affect to the phosphorylation of
ASK1 at Ser83 in HeLa cells treated with H
2
O
2
(data
not shown). Although Hsp105a and Hsp105b may sup-
press oxidative stress-induced apoptosis at steps between
the activation of ASK1 and p38 MAPK, further studies
are necessary to clarify the targets of Hsp105 in the
suppression of oxidative stress-induced apoptosis.
Two pathways are known to be important for trans-
ducing a death signal to the apoptotic machinery. The
‘extrinsic’ pathway involves activation of death recep-
tors such as tumor necrosis factor and Fas by binding
of their respective ligands and the subsequent recruit-
ment of caspase-8 [44]. Caspase-8 can activate effector
caspases, such as caspase-3 and caspase-7, directly [45]
or indirectly by cleaving Bid and inducing the release
of cytochrome c from the mitochondria [46–49]. In
contrast, the ‘intrinsic’ pathway is activated directly by
death stimuli, and induces the release of cytochrome c
from the mitochondria into the cytosol [25,26,50–53].
Cytosolic cytochrome c binds to Apaf-1 and induces

caspase-9-dependent activation of caspase-3 [54–57].
Here, we have shown that Hsp105a and Hsp105b sup-
press cytochrome c release from mitochondria and
activate of caspase-9 and caspase-3 in H
2
O
2
-treated
cells. In contrast, Hsp105a and Hsp105b failed to pre-
vent heat-induced apoptosis. Interestingly, heat shock
treatment activated procaspase-3 but not procaspase-9.
Therefore, Hsp105a and Hsp105b may not be able to
Fig. 6. Effects of Hsp105 overexpression on the activation of JNK and p38 induced by treatment with H
2
O
2
. HeLa-tet ⁄ Hsp105 cells were
exposed to 0.5 m
M H
2
O
2
for 30 min, then further incubated in fresh medium at 37 °C for 15–120 min. Aliquots (20 l g of protein) of the cell
extracts were separated by SDS–PAGE and immunostained using anti-phospho-SAPK ⁄ JNK (Thr183 ⁄ Tyr185), anti-SAPK ⁄ JNK, anti-phospho-
p38 MAPK (Thr180 ⁄ Tyr182) or anti-p38 MAPK. Similar results were obtained from three independent experiments, and typical blots are
shown.
N. Yamagishi et al. Suppression of H
2
O
2

-induced apoptosis by Hsp105
FEBS Journal 275 (2008) 4558–4570 ª 2008 The Authors Journal compilation ª 2008 FEBS 4565
suppress apoptosis induced by the mitochondria-inde-
pendent pathway.
Several Hsps have been shown to modulate the
pathway of apoptosis positively. In particular, Hsp70
has been shown to protect against apoptosis by a vari-
ety of stressors through suppression of JNK activation
and apoptosome formation [7–12]. Interestingly, the
chaperone activity of Hsp70 is required for protection
against heat-induced apoptosis [58]. Recently, mamma-
lian Hsp105 ⁄ 110 and its yeast homologues Sse1p ⁄ 2p
have been shown to act as efficient nucleotide
exchange factors for Hsp70 and for its orthologues in
Saccharomyces cerevisiae, Ssa1p and Ssb1p, respec-
tively, and enhance Hsp70-mediated chaperone activity
[59–61]. However, although HSP105 family proteins
are important components of the Hsp70 chaperone
machinery, excess Hsp110 seems to have a negative
effect on Hsp70-mediated chaperone activity because it
accelerates substrate cycling to such an extent that the
reaction becomes unproductive for folding [60]. There-
fore, Hsp105a and Hsp105b seem not to simply sup-
press oxidative stress-induced apoptosis by regulation
of Hsp70-mediated chaperone activity.
In summary, we have shown here that Hsp105a and
Hsp105b suppress H
2
O
2

-induced apoptosis by suppres-
sion of p38 MAPK. Although further studies are nec-
essary to understand the precise mechanism by which
Hsp105 regulates stress-induced apoptosis, understand-
ing the action of Hsp105a and Hsp105b in apoptosis
may offer novel ways of treating apoptosis-related
diseases, such as cancer, injury after ischemia, and
neurodegenerative disorders.
Experimental procedures
Antibodies
The following antibodies were used for immunoblotting
and immunofluorescence experiments: Hsp105, rabbit
polyclonal anti-mouse Hsp105, which only reacts with
A
B
Fig. 7. H
2
O
2
-induced apoptosis was suppressed by treatment with
a p38 MAPK inhibitor but not a JNK inhibitor in HeLa cells.
(A) HeLa-tet ⁄ Hsp105 cells that did not overexpress Hsp105a or
Hsp105b (+DOX) were exposed to 0.5 m
M H
2
O
2
for 45 min, then
further incubated in fresh medium with or without 10 l
M

SB203580 or 10 lM SP600125 at 37 °C for 8 h. Then the cells
were stained with Hoechst 33342 and the nuclear morphology of
the cells was observed using a fluorescence microscope. Rates of
apoptosis were calculated using at least 200 cells in each experi-
ment. Values are the mean ± SD of three independent experi-
ments. Statistical significance was determined by an unpaired
Student’s t-test. A probability level (P) < 0.05 was considered sta-
tistically significant and is indicated by asterisks. (B) HeLa-
tet ⁄ Hsp105 cells (+DOX) were exposed to 0.5 m
M H
2
O
2
for
45 min, then further incubated in fresh medium with SB203580 or
SP600125 at 37 °C for 1 h. Aliquots (20 lg of protein) of the cell
extracts were separated by SDS–PAGE and immunostained using
anti-phospho-SAPK ⁄ JNK (Thr183 ⁄ Tyr185), anti-SAPK ⁄ JNK, anti-
phospho-p38 MAPK (Thr180 ⁄ Tyr182), anti-p38 MAPK, anti-phos-
pho-MAPKAP-2 (Thr222) or anti-phospho-c-Jun (Ser63).
Suppression of H
2
O
2
-induced apoptosis by Hsp105 N. Yamagishi et al.
4566 FEBS Journal 275 (2008) 4558–4570 ª 2008 The Authors Journal compilation ª 2008 FEBS
mouse Hsp105 [62], or rabbit anti-human Hsp105, which
reacts with human, mouse, rat, and monkey Hsp105 [15];
PARP, rabbit polyclonal anti-PARP (#sc-7150; Santa
Cruz Biotechnology, Santa Cruz, CA, USA); cleaved cas-

pase-3, rabbit polyclonal anti-cleaved caspase-3 (Asp175)
(#9661; Cell Signaling Technology, Danvers, MA, USA);
caspase-9, rabbit polyclonal anti-caspase-9 (#9502; Cell
Signaling Technology); cytochrome c, rabbit polyclonal
anti-cytochrome c (#sc-7159; Santa Cruz); Bax, rabbit
polyclonal anti-Bax (#06-499; Upstate Biotechnology,
Lake Placid, NY, USA); Hsp60, mouse monoclonal anti-
Hsp60 clone LK-1 (#SR-B806; Medical & Biological Lab-
oratories; Nagoya, Japan); G3DPH, rabbit polyclonal
anti-G3PDH (#2275-PC-1; Trevigen, Gaithersburg, MD,
USA); JNK phosphorylated at Thr183 and Tyr185, rabbit
polyclonal anti-phospho-SAPK ⁄ JNK (Thr183 ⁄ Tyr185)
(#9251; Cell Signaling Technology); JNK, rabbit poly-
clonal anti-SAPK ⁄ JNK (#9252; Cell Signaling Technol-
ogy); p38 MAPK phosphorylated at Thr180 and Tyr182,
rabbit polyclonal anti-phospho-p38 MAPK (Thr180 ⁄
Tyr182) (#9211; Cell Signaling Technology); p38 MAPK,
rabbit polyclonal anti-p38 MAPK (#9212; Cell Signaling
Technology); MAPKAP-2 phosphorylated at Thr222, rab-
bit polyclonal anti-phospho-MAPKAP-2 (Thr222) (#9211;
Cell Signaling Technology); phosphorylated c-Jun at
Ser63, rabbit polyclonal anti-phospho-c-Jun (Ser63)
(#9261; Cell Signaling Technology).
Cells
HeLa-tet ⁄ Hsp105a and HeLa-tet ⁄ Hsp105b cells, which
overexpress either mouse Hsp105a or Hsp105b by remov-
ing DOX from the medium, have been described previ-
ously [19]. These cells were maintained in Dulbecco’s
modified Eagle’s medium supplemented with 10% fetal
calf serum and 1 lgÆmL

)1
DOX at 37 ° C with 95% air
and 5% CO
2
.
The induction of mouse Hsp105a and Hsp105b expres-
sion was performed as follows. HeLa-tet ⁄ Hsp105a and
HeLa-tet ⁄ Hsp105b cells were trypsinized, washed twice with
10 mL of fresh medium, and then grown in fresh medium
without DOX for 24 h. The following day, the cells were
washed once with NaCl ⁄ P
i
and incubated in fresh medium
without DOX at 37 °C for an additional 24 h.
For treatment with H
2
O
2
, cells were treated with 0.2–
0.6 mm H
2
O
2
in NaCl ⁄ P
i
containing 0.9 mm CaCl
2
and
0.5 mm MgCl
2

at 37 °C for 1 h, washed with NaCl ⁄ P
i
, and
further incubated in fresh medium at 37 °C. For heat shock
treatment, cells were treated in a water bath set at 45 °C
for 15–120 min.
Cell viability assay
Cells (1 · 10
4
cells per well) in 96-well plates were incu-
bated at 37 °C for 3 h with 50 lgÆmL
)1
neutral red, and
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
absorbance at 540 nm was measured.
Detection of phosphatidylserine externalization
Cells grown on collagenized cover slips (2 · 10
4
cells per
cm
2
in 24-well plates) were washed with buffer A (10 mm
Hepes pH 7.5, 140 mm NaCl and 2.5 mm CaCl
2
), and
stained with 0.5 lgÆmL

)1
annexin V–fluorescein isothiocya-
nate solution (Sigma, St Louis, MO, USA) in buffer A con-
taining 10 lm Hoechst 33342 for 10 min in the dark. Cells
displaying phosphatidylserine externalization were observed
using a confocal laser scanning microscope (LSM410; Zeiss,
Jena, Germany).
Morphological examination of apoptotic cells
Cells grown on collagenized cover slips (2 · 10
4
cells per
cm
2
in 24-well plates) were fixed with 3.7% formaldehyde
for 30 min at room temperature. After washing twice with
NaCl ⁄ P
i
, the cells were stained with 10 lm Hoechst 33342
for 10 min in the dark and observed using a fluorescence
microscope (Nikon, Tokyo, Japan). The cells detached from
cover slips were harvested in a microtube, and stained with
10 lm Hoechst 33342. Cells were scored as apoptotic if they
displayed nuclear fragmentation and ⁄ or chromatin conden-
sation. For assessment of proportion of apoptotic cells, the
number of cells on cover slips and the number of detached
cells were determined.
Immunoblot analysis
Cells (2 · 10
5
cells per 35 mm diameter dish) were lysed

with 0.1% SDS and boiled for 5 min. Aliquots (20 lgof
protein) of cell extracts in SDS sample buffer (62.5 mm
Tris ⁄ HCl pH 6.8, 10% glycerol, 2% SDS, 5% 2-mercapto-
ethanol and 0.00125% bromophenol blue) were subjected
to SDS–PAGE, and then transferred onto nitrocellulose
membranes by electrotransfer. The membranes were
blocked with 5% skim milk in Tris–buffered saline (20 mm
Tris ⁄ HCl pH 7.6, 137 mm NaCl) containing 0.1% Tween-
20 (Wako Pure Chemical, Osaka, Japan), and incubated
with the indicated primary antibodies. Then, the mem-
branes were incubated with horseradish peroxidase-conju-
gated anti-rabbit IgG, and the antibody–antigen complexes
were detected using an ECL ⁄ western blot detection system
(GE Healthcare, Little Chalfont, UK).
Intracellular distribution of cytochrome c
Cells grown on collagenized cover slips (2 · 10
4
cells
per cm
2
in 24-well plates) were stained with 0.2 lgÆ mL
)1
N. Yamagishi et al. Suppression of H
2
O
2
-induced apoptosis by Hsp105
FEBS Journal 275 (2008) 4558–4570 ª 2008 The Authors Journal compilation ª 2008 FEBS 4567
Mitotracker Orange (Invitrogen, Carlsbad, CA, USA) for
15 min. Then, cells were fixed with 3.7% formaldehyde for

30 min at room temperature, and permeabilized with 0.4%
Triton X-100 (Wako Pure Chemical) in NaCl ⁄ P
i
. After
blocking with 5% BSA in NaCl ⁄ P
i
, anti-cytochrome c at a
1 : 100 dilution was added to the cover slips, which were
incubated in a moist chamber for 1 h at 37 °C. After wash-
ing with NaCl ⁄ P
i
, fluorescein isothiocyanate-conjugated
anti-rabbit IgG (1 ⁄ 50, Vector, Burlingame, CA, USA) was
added to the cover slips, which were incubated further at
37 °C for 1 h. After another wash with NaCl ⁄ P
i
, the cells
were observed using a confocal laser scanning microscope.
For fractionation into a soluble fraction and a particulate
fraction containing mitochondria, cells (1 · 10
6
cells per
100 mm diameter dish) were lysed with 100 lgÆmL
)1
digito-
nin in NaCl ⁄ P
i
, and then incubated at 25 °C for 5 min.
After centrifugation at 3000 g for 5 min, the supernatants
were recovered and further centrifuged at 15 000 g for

5 min. The supernatants were recovered as cytosolic frac-
tions. Pellets were dissolved in 0.1% SDS, and centrifuged
at 15 000 g for 15 min. The supernatants were recovered as
mitochondrial fractions. Both fractions were subjected to
15% SDS–PAGE, and analyzed by immunoblotting using
anti-cytochrome c, anti-Hsp60 and anti-G3PDH.
Acknowledgements
This study was supported in part by a grant from the
Ministry of Education, Science, Sports and Culture of
Japan (to T. H. and N.Y.).
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