ATP-binding domain of heat shock protein 70 is essential
for its effects on the inhibition of the release of the
second mitochondria-derived activator of caspase and
apoptosis in C2C12 cells
Bimei Jiang
1
, Kangkai Wang
1
, Pengfei Liang
2
, Weimin Xiao
1
, Haiyun Wang
1
and Xianzhong Xiao
1
1 Department of Pathophysiology, Xiangya School of Medicine, Central South University, Changsha, Hunan, China
2 Department of Burns and plastic surgery, Xiangya Hospital, Central South University, Changsha, Hunan, China
Apoptosis is characterized by specific morphological
and biochemical hallmarks, including cell shrinkage,
membrane blebbing, nuclear breakdown and DNA
fragmentation. As a form of programmed cell death, it
is indispensable for many normal cellular functions,
such as embryo development, tissue homeostasis and
regulation of the immune system [1]. Malfunctions of
apoptosis have been implicated in human diseases,
including myocardial infarction, neurodegenerative dis-
eases, cancer and ischemic stroke [2–4]. Several factors,
including ATP depletion, calcium fluxes and reactive
oxygen species, have been proposed to cause apoptosis
and ⁄ or cytochrome c release in myocytes [5,6].

apoptosis of many cells. Previously, we have shown that heat shock pre-
treatment blocked the release of the second mitochondria-derived activator
of caspase (Smac) to the cytosol and inhibited apoptosis of C2C12 myo-
blast cells in response to H
2
O
2
. The present study aimed to elucidate the
underlying mechanism by over-expressing a major stress-inducible protein,
heat shock protein (HSP) 70, and characterizing the resulting cellular
changes. We demonstrate that HSP70 over-expression markedly inhibited
the release of Smac and prevented the activation of caspases-9 and -3 and
apoptosis in C2C12 cells under H
2
O
2
treatment. However, no direct inter-
action between HSP70 and Smac was observed by co-immunoprecipitation.
Mutational analysis demonstrated that the ATP-binding domain of HSP70,
rather than the peptide-binding domain, was essential for these observed
HSP functions. Taken together, our results provide evidence supporting the
role of HSP70 in the protection of C2C12 cells from H
2
O
2
-induced and
Smac-promoted apoptosis by preventing the release of Smac from mito-
chondria, thereby inhibiting activation of caspases-9 and -3. This mecha-
nism of HSP70 action is dependent on its ATP-binding domain but
independent of its interaction with Smac protein.

blocks apoptosome formation and activation of
caspase-9 [20], and inhibits the release of apoptosis-
inducing factor (AIF) from mitochondria [21].
In our previous study using mouse myogenic C2C12
cells, heat shock pretreatment also prevented apoptosis
induced by oxidative stress [13]. However, whether the
protective effects of HSP70 are mediated by a mecha-
nism involving the release of Smac from mitochondria
remains to be elucidated. To this end, in the present
study, we over-expressed HSP70 and characterized the
subsequent cellular changes using C2C12 as an in vitro
system.
Results
Over-expression of HSP70 inhibits oxidative
stress-induced release of Smac from
mitochondria in C2C12 myogenic cells
To explore the effect of the change in HSP70 protein
expression on hydrogen peroxide (H
2
O
2
)-induced
apoptosis, C2C12 myogenic cells were transfected with
an expression vector with cDNA encoding the full-
length HSP70 protein or the empty vector. After
selection with G418, stably-transfected C2C12 cell
lines that constitutively expressed human HSP70 were
isolated. Two clones, termed HSP70-1 and HSP70-2,
showing different levels of HSP70 proteins by
immunoblot analysis were selected for further

that mitochondrial integrity was preserved and translo-
cation of Smac from mitochondria to the cytosol was
not due to mitochondrial breakdown.
Over-expression of HSP70 inhibits oxidative
stress-induced apoptosis in C2C12 myogenic cells
We next examined the effects of HSP70 over-expres-
sion on oxidative stress-induced apoptosis in C2C12
myogenic cells. As shown in Fig. 2, after treatment
with H
2
O
2
(0.5 mm) for different times, the vector-
transfected control cells underwent apoptosis, as indi-
cated by an apoptotic cell population in the flow
cytometry analysis. The percentages of apoptotic cells
were decreased in both of the HSP70 over-expressed
lines, indicating that HSP70 over-expression protected
cells from H
2
O
2
-induced cytotoxicity. The protective
effects of HSP70 were correlated with the level of
HSP70 expression because the clone with higher
HSP70 expression demonstrated a more significant
reduction of the apoptotic cell population (Fig. 2B).
Furthermore, over-expression of HSP70 displayed an
inhibitory effect on the activation of caspases-9 and -3
induced by H

2
O
2
-treated (0.5 mm for
2 h) cells, indicating that interaction with Smac is not
required with respect to the role of HSP70 in the inhi-
bition of the release of Smac and apoptosis.
The role of the ATP-binding domain of HSP70
in the prevention of the release of Smac and
apoptosis after exposure to H
2
O
2
To determine which region of HSP70 is responsible for
its anti-apoptotic effects, C2C12 myogenic cells were
transiently transfected with expressing plasmids
pcDNA3.1-HSP70
WT
, and pcDNA3.1-HSP70
DATP-BD
or pcDNA3.1-HSP70
DPBD
. First, correct protein
expression from all cell lysates was confirmed by
western blot analysis with HSP70 antibody, showing
immunoreactive bands of the expected sizes (Fig. 4B).
Next, whether the protective potency of HSP70 would
be annulled by deletion of the ATP-binding domain or
the peptide-binding domain was investigated. As
shown in Fig. 5, over-expression of both mutant

apoptotic cells (P < 0.05) (Fig. 6B) and cell viability
(Fig. 6C). By contrast, in these experiments conducted
under the same treatment conditions, HSP70
DATP-BD
over-expression abolished the function of full-length
HSP70 (P < 0.05). No toxic effects were observed
after transfection with the vectors described above.
Discussion
Our previous study demonstrated that heat shock pre-
treatment led to the up-regulation of HSP70 expression
and the inhibition of H
2
O
2
-mediated Smac release and
pcDNA3.1
A
B
HSP70-1 HSP70-2 HS
HSP70
GAPDH
*
##
HSP70-1
HSP70-2
pcDNA3.1
pcDNA3.1
0
pcDNA3.1 HSP70-1 HSP70-2 HS
Ratio of HSP70 to GAPDH

O
2
50
40
30
20
10
0
C
y
to Mit
Ratio of Smac to loading
control
Mit Cyto Mit Cyto Mit Cyto Mit
Fig. 1. Over-expression of HSP70 inhibited H
2
O
2
-induced Smac
release in C2C12 cells. (A) Cell lysates from C2C12 clones
over-expressing HSP70 or vector control plasmid (pcDNA3.1) were
immunoblotted with monoclonal anti-HSP70 serum. Immunoblot
analysis of b-actin was used as the loading control. A representative
experiment is shown. Hybridization signals were quantified and nor-
malized to GAPDH signals and are presented as the fold increase
over the respective controls. HS, Heat stress. (B) Vector control
(pcDNA3.1) and HSP70-over-expressing (HSP70-1, HSP70-2) C2C12
cells were either kept untreated or treated with 0.5 m
M of H
2

apoptosis. Because these effects were similar to those
of our previous observations for the same cells under-
going heat-shock, HSP70 is most likely to be the key
0
Caspase-3
pcDNA3.1
PI

pcDNA3.1 + H
2
O
2
10
1
10
2
10
3
10
4
10
1
10
2
10
3
10
4
10
1

2
% Apoptotic cells
10
2
10
3
10
4
10
1
10
0
10
2
10
3
10
4
10
1
10
0
10
2
10
3
10
4
Q1
Q2

O
2
Caspase-9
pcDNA3.1
MpcDNA3.1 1 2 pcDNA3.1
HSP70 HSP70
0.5m
M H
2
O
2
1 2
pcDNA3.1 + H
2
O
2
HSP70-1 + H
2
O
2
HSP70-2 + H
2
O
2
500 bp
300 bp
100 bp
HSP70-1
HSP70-2
Caspases activity (folds)

M H
2
O
2
for 24 h. Cells were then processed for annexin V-FITC and pyridine iodination (PI) co-staining and ana-
lyzed by flow cytometry. Q3 cells were regarded as control cells, whereas Q4 cells were considered as a measure of early apoptosis, Q2
cells were considered as cells at late apoptosis and Q1 cells were considered as being under necrosis. Next, quantitation of apoptotic cells
was determined. Results are representative of three independent experiments. Data are the mean ± SEM of triplicate samples. *Significant
difference (P < 0.05) compared to the pcDNA3.1 control group; #Significant difference (P < 0.05) compared to the group (*) that was signifi-
cantly different from the pcDNA3.1 control group. (C) Cytosolic DNA was extracted from control and H
2
O
2
-exposed (24 h) C2C12 cells. DNA
samples (4 lg) were electrophoresed on agarose gels to visualize DNA laddering. M, DNA marker.
ATP-binding domain of HSP70 inhibits Smac release B. Jiang et al.
2618 FEBS Journal 276 (2009) 2615–2624 ª 2009 The Authors Journal compilation ª 2009 FEBS
player mediating the anti-apoptotic effects, which is
consistent with the general functional role of the chap-
erone protein. Our previous studies demonstrated that
H
2
O
2
at 0.5 mmolÆL
)1
induced apoptosis significantly,
but only affected a minimal number of cells (approxi-
mately 10%). In the present study, we demonstrated
that the levels of HSP70 protein expression in C2C12

2
O
2
-induced oxida-
tive stress. This is similar to the protective effects of
another heat-shock protein, HSP27, against apoptosis,
as previously reported [24].
The molecular chaperone HSP70 has been shown
to inhibit stress-induced apoptosis by interacting
with apoptotic-associated factors. For example,
HSP70 directly interacts with JNK, resulting in the
suppression of JNK-mediated apoptosis [25]. HSP70
physically interacts with Apaf-1, blocking Apaf-1 ⁄
cytochrome c-mediated caspase activation [20]. HSP70
also binds to and antagonizes AIF, thereby inhibiting
HSP70
H
S
P7
0
+ H
2
O
2
IB: HSP70
IB: Smac
IgG Serum Lysate HSP70 Smac HSP70 Smac
IP IP IP
Fig. 3. No interaction was found between HSP70 and Smac. Vec-
tor control (C2C12-C) and HSP70-over-expressing (C2C12-HSP70)

ΔATP-BD
HSP70
WT
70 kDa
IB: Hsp70
52 kDa
28 kDa
IB: Actin
HSP70
’ΔPBD
HSP70
’ΔATP-BD
PBD
PBD
Fig. 4. Deletion mutants of HSP70 were constructed and transf-
ected. A schematic drawing is shown of the HSP70 deletion
mutants employed in the present study. (A) Deleted amino acids
are indicated by the dotted lines. ATP-BD, 1-383AA, 42 kDa; PBD,
384-542AA, 18 kDa. (B) Western blot analysis demonstrated the
levels of expression of the HSP70 proteins after deletion mutants
of HSP70 were transfected.
Cyto Mit Cyto Mit Cyto Mit Cyto Mit
pcDNA3.1 HSP70
WT
HSP70
ΔPBD
HSP70
ΔATP-BD
H
2

2
O
2
-mediated and Smac-promoted apoptosis is not
attributable to a direct physical interaction between
HSP70 and Smac.
HSP70 contains three functional regions: the ATP-
binding domain, the peptide-binding domain, and the
EEVD motif. Although the EEVD motif is considered
to be involved in the chaperone function of HSP70,
and was assumed to mediate cytoprotection by restor-
ing damaged or unfolded proteins under stress, the
roles of other domains of HSP70 in anti-apoptosis
remain highly controversial. Some studies have pro-
posed that the ATP-binding domain of human HSP70
is not required in HSP70-mediated JNK suppression,
inhibition of cytochrome c release and caspase activa-
tion, and protection of cells from injury [26]. By con-
trast, other studies have shown that the ATP-binding
domain of HSP70 is essential for its anti-apoptotic role.
For example, deletional analysis demonstrated that
the ATP-binding domain is essential for inhibiting
the release of cytochrome c from mitochondria [27].
3
A
*
*
#
#
#

+ H
2
O
2
HSP70
ΔPBD
+ H
2
O
2
30
20
10
0
12 h 24 h
Time (h)
% Apoptotic cells
B
a
cd e
b
pcDNA3.1
HSP70 + H
2
O
2
HSP70
ΔATP-BD
+ H
2

*
#
#
1
0.8
0.6
0.4
0.2
0
pcDNA3.1
pcDNA3.1 + H
2
O
2
Hsp70 + H
2
O
2
Hsp70
ΔATP-BD
+ H
2
O
2
Hsp70
Δ
PBD
+ H
2
O

after transfer, cells were treated with 0.5 m
M H
2
O
2
for 12 or 24 h,
and then stained with Hoechst 33258. Under a fluorescence micro-
scope, apoptotic cells, which contained condensed chromatin frag-
ments, were scored and expressed as a percentage of the total
cell number counted. Data are the mean ± SEM. *Significant differ-
ence (P < 0.05) compared to the pcDNA3.1 control group; #Signifi-
cant difference (P < 0.05) compared to the group (*) that was
significantly different from the pcDNA3.1 control group (n = 5).
(a–f) Cells incubated with H
2
O
2
for 24 h. (C) Determination of cell
viability. Approximately 2000 cells were plated in each well of
96-well plates. After 24 h of incubation, 0.5 m
M of H
2
O
2
was added
and cell viability was measured by an 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl-tetrazolium bromide assay after exposure to H
2
O
2

[30]. Bcl-2 is the prototype of the bcl-2 family of
proteins and is distributed in the mitochondria,
endoplasmic reticulum and nuclear envelope. With a
well-established role with respect to protecting cells
against a variety of apoptotic stimuli, it mainly acts at
the mitochondrial level [31]. A previous study [32]
demonstrated that HSP70 inhibits heat-induced apop-
tosis by preventing Bax translocation. Furthermore,
over-expression of HSP70 was associated with reduced
apoptotic cell death and an increased expression of the
anti-apoptotic protein, Bcl-2 [33]. On the basis of the
available evidence, HSP70 and HSP70
DPBD
may also
suppress Smac release and apoptosis by regulating the
expression of these pro-apoptotic or anti-apoptotic
bcl-2 family proteins.
In summary, using the H
2
O
2
-induced oxidative stress
model, the present study has revealed an important
anti-apoptotic role of HSP70, which comprises a
mechanism that involves the inhibition of Smac release
from mitochondria, and the suppression of caspase
activation. Such a mechanism is independent of the
interaction of HSP70 with Smac but requires the
ATP-binding domain of the protein. However, it
remains to be determined how these findings are

humidified atmosphere containing 5% CO
2
. As a control,
cells were cultured under normal conditions without hyper-
thermia.
Construction of HSP70 and its truncated mutants
Full-length human HSP70 cDNA was obtained as a gener-
ous gift from I. Benjemin (University of Utah Health
Sciences Center, Salt Lake City, UT, USA) It was direc-
tionally cloned between KpnI and BamHI sites into the
mammalian expression vector pcDNA3.1(-)-His-myc. At
the same time, this cDNA was used as the template for
PCR amplification of two HSP70 truncated mutants with
deletion of the ATP-binding domain (HSP70
DATP-BD
)or
the peptide-binding domain (HSP70
DPBD
) using primer
pairs (Table 1). All DNA digested fragments were purified
using a gel purification kit (Invitrogen, Carlsbad, CA,
USA), and subsequently ligated into pcDNA3.1(-)-His-myc
vector overnight at 4 °C with T4 DNA polymerase (Pro-
mega, Madison, WI, USA). The correct insets were verified
by sequencing and digestion. The final constructs were
named pcDNA3.1-HSP70
WT
, pcDNA3.1-HSP70
DATP-BD
or

C2C12 myogenic cells were cultured to sub-confluence and
transfected with each of the expression plasmids manufac-
tured as described in the above steps, or the empty vector
without the cDNA (control) with a Lipofectamine-mediated
method (Lipofectamine 2000, Invitrogen), as described
previously [13].
Preparation of mitochondrial and cytosolic
fractions
The subcellular fractions of C2C12 myogenic cells treated
with or without H
2
O
2
were isolated as described previously
[13].
Western blot analysis
Western blotting with anti-HSP70 and anti-Smac sera was
performed as described previously [34].
Caspase activity assay
Caspase activation was determined according to the method
described previously [13].
Flow cytometric analysis
Both adherent and floating cells were collected after treat-
ment, washed with ice-cold NaCl ⁄ P
i
, and stained with
fluorescein isothiocyanate (FITC)-conjugated annexin V
(BD Biosciences, Franklin Lakes, NJ, USA) and pyridine
iodination (PI) for 20 min at room temperature in the dark.
The stained cells were then analyzed by a flow cytometer

roform extraction followed by an additional chloroform
extraction. DNA pellet was then washed in 70% ethanol
and resuspended in 1 mm EDTA and 10 mm Tris–HCl
(pH 8.0) at a final concentration of 20 lgÆmL
)1
. Aliquots
were electrophoresed on a 1.5% agarose gel containing ethi-
dium bromide, and photographed under UV illumination. A
GeneRuler 100 bp DNA ladder (MBI Fermentas, Hanover,
MD, USA) was utilized as DNA size marker.
Co-immunoprecipitation assay
For co-immunoprecipitation, transiently transfected C2C12
cells were lyzed with pre-cold RIPA buffer (150 mmolÆL
)1
NaCl, 1% NP40, 0.5% deoxycholic acid sodium salt, 0.1%
SDS, 50 mmolÆL
)1
Tris pH 8.0, 1 mm phenylmethanesulfo-
nyl fluoride and complete protease inhibitor tablet) at 4 °C
for 5 min. To reduce nonspecific combination, lysates con-
taining 500 lg of total protein were pre-immunized with
25 lL of a slurry of protein A ⁄ G coupled to agarose beads
(Invitrogen) overnight at 4 °C on a rotating wheel. Aliquots
of the pre-cleared supernatants were then each incubated
with 2 lg of appropriate mouse monoclonal anti-HSP70
serum, polyclonal rabbit anti-Smac serum (R&D Systems,
Minneapolis, MN, USA), normal mouse immunoglobu-
lin G (control for anti-HSP70) or normal rabbit serum
(control for anti-Smac) added into 25 lL of protein A ⁄ G
slurry coupled to agarose beads (Invitrogen) for 5 h at 4 °C

2622 FEBS Journal 276 (2009) 2615–2624 ª 2009 The Authors Journal compilation ª 2009 FEBS
McClean, VA, USA). The attenuance of formazan
formed in control cells was considered as 100% viability.
Statistical analysis
Data are expressed as the mean ± SEM of the indicated
number of separate experiments. Differences between two
groups were analyzed using an unpaired Student’s t-test.
Differences among three or more groups were analyzed by
one-way analysis of variance followed by the Student–New-
man–Keuls post-hoc test. P < 0.05 was considered statisti-
cally significant.
Acknowledgements
This study was supported by the grants from the
National Basic Research Program of China
(2007CB512007), the National Natural Science Foun-
dation of China (30700290) and Special Funds for
PhD Training from the Ministry of Education of
China (20060533009).
References
1 Steller H (1995) Mechanisms and genes of cellular
suicide. Science 267, 1445–1449.
2 Shishido T, Woo CH, Ding B, McClain C, Molina CA,
Yan C, Yang J & Abe J (2008) Effects of
MEK5 ⁄ ERK5 association on small ubiquitin-related
modification of ERK5: implications for diabetic ventric-
ular dysfunction after myocardial infarction. Circ Res
102, 1416–1425.
3 Burke RE (2008) Programmed cell death and new dis-
coveries in the genetics of parkinsonism. J Neurochem
104, 875–890.

c-dependent caspase activation by eliminating IAP
inhibition. Cell 102, 33–42.
11 Verhagen AM, Ekert PG, Pakusch M, Silke J, Connolly
LM, Reid GE, Moritz RL, Simpson RJ & Vaux DL
(2000) Identification of DIABLO, a mammalian protein
that promotes apoptosis by binding to and antagonizing
IAP proteins. Cell 102, 43–53.
12 Jiang B, Xiao W, Shi Y, Liu M & Xiao X (2005) Role
of Smac ⁄ DIABLO in hydrogen peroxide-induced apop-
tosis in C2C12 myogenic cells. Free Radic Biol Med 39,
658–667.
13 Jiang B, Xiao W, Shi Y, Liu M & Xiao X (2005) Heat
shock pretreatment inhibited the release of Smac ⁄ DIA-
BLO from mitochondria and apoptosis induced by
hydrogen peroxide in cardiomyocytes and C2C12
myogenic cells. Cell Stress Chaperones 10, 252–262.
14 Lui JC & Kong SK (2007) Heat shock protein 70 inhib-
its the nuclear import of apoptosis-inducing factor to
avoid DNA fragmentation in TF-1 cells during erythro-
poiesis. FEBS Lett 581, 109–117.
15 Zhao Y, Wang W & Qian L (2007) Hsp70 may protect
cardiomyocytes from stress-induced injury by inhibiting
Fas-mediated apoptosis. Cell Stress Chaperones 12, 83–
95.
16 Jayakumar J, Suzuki K, Khan M, Smolenski RT, Farrell
A, Latif N, Raisky O, Abunasra H, Sammut IA, Mur-
tuza B et al. (2000) Gene therapy for myocardial protec-
tion: transfection of donor hearts with heat shock protein
70 gene protects cardiac function against ischemia-reper-
fusion injury. Circulation 102, III302–III306.

factor alpha receptor-mediated apoptosis. Mol Cell Biol
26, 9244–9255.
23 Didelot C, Schmitt E, Brunet M, Maingret L, Parcellier
A & Garrido C (2006) Heat shock proteins: endogenous
modulators of apoptotic cell death. Handb. Exp. Phar-
macol. 26, 171–198.
24 Chauhan D, Li G, Hideshima T, Podar K, Mitsiades C,
Mitsiades N, Catley L, Tai YT, Hayashi T, Shringarpure
R et al. (2003) Hsp27 inhibits release of mitochondrial
protein Smac in multiple myeloma cells and confers dexa-
methasone resistance. Blood 102, 3379–3386.
25 Park HS, Lee JS, Huh SH, Seo JS & Choi EJ (2001)
Hsp72 functions as a natural inhibitory protein of c-Jun
N-terminal kinase. EMBO J 20, 446–456.
26 Mayer MP & Bukau B (2005) Hsp70 chaperones: cellu-
lar functions and molecular mechanism. Cell Mol Life
Sci 62, 670–684.
27 Mosser DD, Caron AW, Bourget L, Meriin AB, Sher-
man MY, Morimoto RI & Massie B (2000) The chaper-
one function of hsp70 is required for protection against
stress-induced apoptosis. Mol Cell Biol 20, 7146–7159.
28 Park HS, Cho SG, Kim CK, Hwang HS, Noh KT,
Kim MS, Huh SH, Kim MJ, Ryoo K, Kim EK et al.
(2002) Heat shock protein hsp72 is a negative regulator
of apoptosis signal-regulating kinase 1. Mol Cell Biol
22, 7721–7730.
29 Ruchalski K, Mao H, Li Z, Wang Z, Gillers S, Wang
Y, Mosser DD, Gabai V, Schwartz JH & Borkan SC
(2006) Distinct hsp70 domains mediate apoptosis-induc-
ing factor release and nuclear accumulation. J Biol