Tài liệu Báo cáo khoa học: Nucleolin/C23 mediates the antiapoptotic effect of heat shock protein 70 during oxidative stress pptx

Nucleolin/C23 mediates the antiapoptotic effect of heat
shock protein 70 during oxidative stress
Bimei Jiang
1
, Bin Zhang
1
, Pengfei Liang
2
, Juan Song
1
, Hongbing Deng
1
, Zizhi Tu
1
, Gonghua Deng
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
Introduction
Characterized by cellular and nuclear shrinkage, cyto-
plasmic blebbing, chromatin condensation and DNA
fragmentation [1,2], apoptosis can cause irreversible
loss of terminally differentiated cardiac myocytes and,
therefore, contributes significantly to the pathogenesis
of many cardiovascular diseases. Apoptosis has been
identified in cardiac myocytes from patients suffering
from myocardial infarction, diabetic cardiomyopathy,
and end-stage congestive heart failure.
Apoptosis is a highly regulated programme of cell

death and can be mediated by death receptors in the
plasma membrane, as well as in the mitochondria and
the endoplasmic reticulum [3]. Studies on apoptotic
pathways in cardiomyocytes have revealed several
molecules as key regulators [4]. Heat shock protein
70 (Hsp70) is a major stress-inducible heat shock pro-
tein that has been shown to protect cells from
apoptosis induced by heat shock, tumour necrosis
factor, growth factor withdrawal, oxidative stress and
radiation [5,6]. Hsp70 is also a major self-preservation
protein in the heart; its overexpression enhances
myocardial tolerance to ischaemia ⁄ reperfusion injury
in both transgenic animals [7] and cell cultures
[8]. Although substantial progress has been made in
understanding the control and mechanisms of apopto-
sis, how Hsp70 protects cardiomyocytes against apop-
tosis induced by a variety of stresses remains to be
investigated.
Keywords
apoptosis; cardiomyocytes; heat shock
protein 70; hydrogen peroxide;
nucleolin ⁄ C23
Correspondence
Xianzhong Xiao, Department of
Pathophysiology, Xiangya School of
Medicine, Central South University,
Changsha, Hunan 410008, China
Fax: +86 731 82355019
Tel: +86 731 2355019
E-mail:

(Received 22 August 2009, revised 26
October 2009, accepted 23 November
2009)
doi:10.1111/j.1742-4658.2009.07510.x
Although heat shock protein 70 (Hsp70) has been shown to markedly inhi-
bit H
2
O
2
-induced apoptosis in C2C12 cells, and nucleolin ⁄ C23 has also
been implicated in apoptosis, the relationship of these two molecules is still
largely unknown. The aim of the current study was to investigate the
potential involvement of nucleolin ⁄ C23 in the antiapoptotic mechanism of
Hsp70. We found that primary cultures of neonatal rat cardiomyocytes
underwent apoptosis upon H
2
O
2
treatment, and in these cells nucleo-
lin ⁄ C23 protein was highly unstable and had a half-life of less than 4 h.
However, transfection with Hsp70 greatly stabilized nucleolin ⁄ C23 and also
protected the cells from H
2
O
2
-induced apoptosis. When nucleolin ⁄ C23 was
knocked down with an antisense oligomer, H
2
O
2

-induced apoptosis became
more severe, even in Hsp70-overexpressed cells, demonstrating an essential
role of nucleolin ⁄ C23 in the antiapoptotic effects of Hsp70. Similar results
were obtained by both nuclear morphology observation and caspase-3
activity assay. Therefore, these data provide evidence that nucleolin ⁄ C23 is
an essential downstream effecter of Hsp70 in the protection of cardiomyo-
cytes against oxidative stress-induced apoptosis.
Abbreviations
DMEM, Dulbecco’s modified Eagle’s medium; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
Hsp70, heat shock protein 70; PBS, phosphate-buffered saline; PI, pyridine iodination; SEM, standard error of the mean.
642 FEBS Journal 277 (2010) 642–652 ª 2009 The Authors Journal compilation ª 2009 FEBS
Nucleolin ⁄ C23 is a 110 kDa multifunctional phos-
phoprotein. It is abundantly expressed in both normal
and cancerous cells [9], and ubiquitously distributed in
the nucleolus, nucleus and cytoplasm of the cell, as
well as at the cell surface [10]. Nucleolin ⁄ C23 regulates
many aspects of DNA and RNA metabolism, such as
RNA polymerase I-mediated transcription, the folding,
maturation, ribosome assembly and nucleocytoplasmic
transport of newly synthesized pre-rRNAs [11].
Because of its widespread distribution and broad range
of involvement, as well as the cross-talking among
molecules of different cellular processes, dissection of
nucleolin ⁄ C23’s specific functions is particularly chal-
lenging.
Recently, nucleolin ⁄ C23 has also been implicated in
apoptosis. All-trans retinoic acid-induced apoptosis
leads to nucleolin downregulation and bcl-2 mRNA
instability; overexpression of nucleolin ⁄ C23 in chronic
lymphocytic leukaemia cells stabilizes bcl-2 mRNA

[12,13]. Our previous study showed that oxidative
stress induces nucleolin ⁄ C23 cleavage and apoptosis
[14], and that nucleolin downregulation induces apop-
tosis in C2C12 cells [15]. These data suggest the roles
of nucleolin ⁄ C23 in oxidative stress-induced apoptosis,
but whether nucleolin ⁄ C23 participates in Hsp70-medi-
ated protection against H
2
O
2
-induced apoptosis is not
clear. In the present investigation, we further studied
the effects of nucleolin ⁄ C23 ablation on Hsp70-medi-
ated protection from apoptosis and examined the
expression and stability of nucleolin ⁄ C23 with Hsp70
overexpression in response to H
2
O
2
-induced apoptosis
using primary culture cardiomyocytes.
Results
Overexpression of Hsp70 protects
cardiomyocytes from H
2
O
2
-induced apoptosis
First, to overexpress Hsp70 we transiently transfected
cardiomyocytes with a plasmid carrying human Hsp70

cDNA, and examined the protein levels by western
blot analysis with Hsp70 antiserum after 36 h. The effi-
ciency of transfection mediated by the Lipofectamine
Plus reagent ranged between 30 and 50% (data not
shown). As can be seen in Fig. 1A, Hsp70 overexpres-
sion was confirmed in the cells transfected with Hsp70
cDNA, but not in the cells transfected with the vector
alone.
To determine the effect of Hsp70 overexpression on
H
2
O
2
-induced apoptosis, the above transfected cells
were exposed to H
2
O
2
(0.5 mmolÆL
)1
) for the indicated
time periods and the percentages of apoptotic cells
examined by morphological observation (Fig. 1B).
A
B
C
Fig. 1. Overexpression of Hsp70 renders cardiomyocytes resistant
to H
2
O

2
. Cardiomyocytes were transiently transfected with full-
length Hsp70 plasmid pcDNA ⁄ Hsp70 (Hsp70 ⁄ C) or pcDNA vector
(pcDNA ⁄ C). (A) Immunoblot analysis of Hsp70 expression. At 36 h
post-transfection, cells were analysed by immunoblotting with anti-
bodies against Hsp70 or GAPDH (as an internal control of protein
loading). The results shown are representative of three indepen-
dent experiments. (B) Quantification of apoptotic cells by nuclear
staining. Untransfected or transfected cells were exposed to
0.5 mmolÆL
)1
H
2
O
2
for the indicated durations and stained with
Hoechst 33258; apoptotic cells showing condensed chromatin
fragments were scored and expressed as the percentage of the
total cell number counted. The results from three indepen-
dent experiments are presented as mean ± SEM. *P < 0.05 versus
control group; #P < 0.05 versus pcDNA3.1 + H
2
O
2
group (n = 3).
(C) Caspase-3 activity assay. Thirty-six hours after transient
transfection, cells were treated with or without 0.5 mmolÆL
)1
H
2

O
2
.
At different time points, cells were harvested for protease activity
of caspase-3 using the caspase colorimetric assay kit. Data were
obtained from four independent experiments. *P < 0.05 versus
control group; #P < 0.05 versus pcDNA3.1 + H
2
O
2
group.
B. Jiang et al. Nucleolin ⁄ C23 in antiapoptotic effect of Hsp70
FEBS Journal 277 (2010) 642–652 ª 2009 The Authors Journal compilation ª 2009 FEBS 643
After 24 h of H
2
O
2
exposure,  44% of untransfected
control cells and 45% of cells transfected with the
empty vector (pcDNA3.1) exhibited similar features of
apoptotic cells, such as highly condensed nuclei and
decreased cell size. However, only 22% of the cells
transfected with Hsp70 underwent apoptosis (Fig. 1B).
We also investigated the effects of Hsp70 on caspase-3
activation induced by oxidative stress. Using an in vitro
substrate-cleavage reaction, H
2
O
2
treatment signi-

ficantly induced the activation of caspase-3 in the
untransfected control cells as well as cells transfected
with the vector alone in a time-dependent manner, but
Hsp70 overexpression significantly reduced caspase-3
activation in response to H
2
O
2
treatment (Fig. 1C).
Increased nucleolin/C23 protein levels in
Hsp70-transfected cells in response to
H
2
O
2
-induced apoptosis
We next examined the expression of nucleolin ⁄ C23
protein in these cells in response to H
2
O
2
exposure.
Upon treatment with H
2
O
2
for up to 8 h, all cells
showed a time-dependent decrease in the 110 kDa
nucleolin ⁄ C23 fragment accompanied by the appear-
ance and an increase in the 80 kDa fragment (Fig. 2).

Although both the untransfected cells and the cells
transfected with the vector alone started to show the
80 kDa fragment at time points as early as 2–4 h after
H
2
O
2
treatment, this smaller protein fragment
appeared in the Hsp70-overexpressed cells at the 8 h
time point (Fig. 2A,B). Overall, the change in nucleo-
lin ⁄ C23 protein levels was least significant in cells
transfected with Hsp70 cDNA (Fig. 2B), suggesting a
protective effect of Hsp70 on the degradation of
nucleolin ⁄ C23 protein.
Prolonged half-life of nucleolin/C23 in
Hsp70-transfected cells in response to H
2
O
2
exposure
To compare the stability of nucleolin ⁄ C23 protein in
cells transfected with the vector alone and cells trans-
fected with Hsp70 cDNA during H
2
O
2
exposure, the
cells were treated with H
2
O

2
for 2 h. Protein synthesis
was then inhibited by cycloheximide (5 lgÆmL
)1
).
Western blot analysis revealed a time-dependent rapid
decline in nucleolin ⁄ C23 110 kDa bands in H
2
O
2
-trea-
ted cells transfected with the vector alone; the half-life
of the protein was estimated to be less than 2 h
(Fig. 3, left panel). At the same time, the 80 kDa
degraded product was detected in an increasing
amount in H
2
O
2
-treated cells transfected with the
vector alone shortly after cycloheximide treatment
(2–8 h). By contrast, nucleolin ⁄ C23 in cells transfected
with Hsp70 cDNA was more stable in response to
H
2
O
2
exposure, reaching its half-life at 8 h after cyclo-
heximide treatment (Fig. 3, right panel).
Overexpression of nucleolin/C23 protects

cardiomyocytes from H
2
O
2
-induced apoptosis
To determine the effect of nucleolin⁄ C23 overexpres-
sion on H
2
O
2
-induced apoptosis in cardiomyocytes, we
first transiently transfected cells with plasmids carrying
human nucleolin ⁄ C23 cDNA or the empty vector.
Using western blot analysis we confirmed nucleo-
lin ⁄ C23 overexpression only in cells transfected with
nucleolin ⁄ C23 cDNA but not the empty vector
(pcDNA ⁄ C) (Fig. 4A). Then, transfected cells were
exposed to H
2
O
2
(0.5 mmolÆL
)1
) and apoptosis was
assayed by both Hoechst nuclear staining (Fig. 4B)
Ctrl 0 h
0 h 2 h 4 h 8 h 0 h 2 h 4 h 8 h
4 h 8 h 12 h
Nucleolin/C23
Nucleolin/C23

GAPDH
GAPDH
PcDNA/C cells with H
2
O
2
exposure Hsp70/C cells with H
2
O
2
exposure
H
2
O
2
A
B
Fig. 2. Changes in nucleolin ⁄ C23 protein
levels in cells transfected with pcDNA
vector (pcDNA ⁄ C) or Hsp70 cDNA (Hsp70 ⁄ C)
during H
2
O
2
exposure. Cardiomyocytes
were untransfected (A) or transiently
transfected with pcDNA vector (pcDNA ⁄ C)
or pcDNA ⁄ Hsp70 (Hsp70 ⁄ C) for 36 h (B),
and were treated with 0.5 mmolÆL
)1

H
2
O
2
for 0–8 h. Cells were harvested and protein
lysates were prepared for western blot anal-
ysis with monoclonal antibodies against
nucleolin ⁄ C23 or GAPDH. The results
shown are the representatives of three
independent experiments.
Nucleolin ⁄ C23 in antiapoptotic effect of Hsp70 B. Jiang et al.
644 FEBS Journal 277 (2010) 642–652 ª 2009 The Authors Journal compilation ª 2009 FEBS
and caspase-3 activity assays (Fig. 4C). By counting
apoptotic nuclei, at 24 h H
2
O
2
exposure,  45% of
cells transfected with the vector alone were apoptotic.
However, only 22% of cells transfected with nucleo-
lin ⁄ C23 cDNA underwent apoptosis (Fig. 4B). Consis-
tently, H
2
O
2
treatment significantly induced caspase-3
activation in cells transfected with the vector alone in
a time-dependent manner, and nucleolin ⁄ C23 overex-
pression significantly reduced caspase-3 activation
(Fig. 4C). However, simultaneous Hsp70 overexpres-

sion afforded the cells no significant protection by
nucleolin ⁄ C23 against oxidant stress (Fig. 4D).
In addition, H
2
O
2
induced apoptosis in cardiomyo-
cytes with a marked downregulation of Bcl-2 protein,
and nucleolin overexpression could inhibit the down-
regulation of Bcl-2 protein (Fig. 5A). Furthermore, we
compared the stability of Bcl-2 mRNA in cells transfect-
ed with nucleolin cDNA (pcDNA3.1-C23) or vector
control (pcDNA3.1) in the presence or absence of H
2
O
2
by incubating the cells with actinomycin D (5 lgÆmL
)1
)
for up to 3 h. Bcl-2 mRNA in H
2
O
2
-treated vector con-
trol cells was found to be highly unstable. In contrast,
Bcl-2 in H
2
O
2
-treated nucleolin-overexpressed cells was

significantly more stable (Fig. 5B).
Potentiation of apoptosis in Hsp70-transfected
cells by nucleolin/C23 antisense oligomer
Next, we used the gene knockdown approach with an
antisense oligonucleotide to examine whether nucleo-
lin ⁄ C23 is necessary in the antiapoptotic function of
Hsp70. Fluorescein isothiocyanate (FITC)-labelled
phosphorothioate oligodeoxynucleotides were success-
fully transfected into primary cultured cardiomyocytes
with Lipofectamine 2000 and achieved a transfection
efficacy of  80% (Fig. 6A). Transfection of nucleo-
lin ⁄ C23 antisense oligonucleotide selectively reduced
the levels of endogenous nucleolin ⁄ C23 protein by
more than 75% after 36 h, whereas transfection of the
scrambled control oligonucleotide did not alter nucleo-
lin ⁄ C23 protein levels (Fig. 6B).
Then, Hsp70 overexpressing cells were transfected
with nonspecific control or nucleolin ⁄ C23 antisense
oligomers and the percentages of apoptotic cells in
response to H
2
O
2
treatment were examined by three
methods. First, by nuclear staining the apoptotic rates
were 9–25% in cells transfected with the control oligo-
mers and much lower at 13 and 44% in cells transfect-
ed with the nucleolin ⁄ C23 antisense oligomer
(Fig. 6C). Second, the activity of caspase-3 was analy-
sed as a marker of apoptotic cells using an in vitro

substrate-cleaving reaction. As shown in Fig. 6D,
H
2
O
2
-induced apoptosis was increased in cells trans-
fected with nucleolin⁄ C23 antisense oligonucleotide
compared with those transfected with the scrambled
control oligonucleotide, even in the presence of Hsp70
overexpression. Finally, apoptosis was further assayed
by flow cytometry with annexin V–FITC ⁄ pyridine
iodination (PI) double staining, which identified apop-
totic cells as annexin V positive, but PI negative. This
method obtained results consistent with those from the
other two methods, showing that when nucleolin ⁄ C23
was knocked down with an antisense oligomer, H
2
O
2
-
induced apoptosis was enhanced, even with the Hsp70-
overexpressed cells (Fig. 7A,B).
Discussion
Both our previous study [16] and the present study
have provided evidence that oxidative stress induces
apoptosis of cardiomyocytes, which can be character-
ized by nuclear condensation and cell shrinkage. Inter-
estingly, upon transfection with an Hsp70-containing
plasmid, these same cells become much less sensitive to
H

2
O
2
exposure. This observation was confirmed by
both cell morphology and caspase-3 activity analyses.
pcDNA/C cells with
H
2
O
2
exposure 2 h
0 hCHX 2 h 4 h 8 h 0 h 2 h 4 h 8 h
Nucleolin/C23
GAPDH
Hsp70/C cells with
H
2
O
2
exposure 2 h
Fig. 3. Nucleolin ⁄ C23 protein is stabilized in Hsp70-transfected cells during H
2
O
2
exposure. Cardiomyocytes were transiently transfected
with pcDNA vector (pcDNA ⁄ C) or pcDNA ⁄ Hsp70 (Hsp70 ⁄ C). After treatment with 0.5 mmolÆL
)1
H
2
O

2
for 2 h, cycloheximide (CHX;
5 lgÆmL
)1
) was added. At different time points (2–8 h), cells were harvested and protein lysates were prepared for western blot analysis
with monoclonal antibodies against nucleolin ⁄ C23 or GAPDH. The results shown are the representatives of three independent experiments.
B. Jiang et al. Nucleolin ⁄ C23 in antiapoptotic effect of Hsp70
FEBS Journal 277 (2010) 642–652 ª 2009 The Authors Journal compilation ª 2009 FEBS 645
Hsp70 has been shown to regulate apoptotic sig-
nalling, effecter molecule activation, certain down-
stream events of caspase activation, the release of
cathepsins, cytochrome c, apoptosis-inducing factor
and even the death-associated lysosome permeabiliza-
tion [17–21]. Particularly, Hsp70 inhibits the release
of cytochrome c and apoptosis-inducing factor from
mitochondria and prevents apoptosis [22,23]. Our
previous study showed that Hsp70 overexpression
inhibited H
2
O
2
-mediated release of Smac (the second
mitochondria-derived activator of caspases), activa-
tion of caspase-3 and caspase-9, and apoptosis in
C2C12 myogenic cells [24]. Stankiewicz et al. [25]
found that Hsp70 overexpression stabilizes Mcl-1
protein in heat-shocked cells. Hsp70 has also been
suggested to play an important role in precondition-
ing, a phenomenon of protection of a heart from
strong ischaemic insult by prior exposure to mild

ischaemia or other mild stresses. In the present
study, we have shown that Hsp70 renders nucleolin⁄
C23 enhanced stability; however, a decrease in nucle-
olin ⁄ C23 stability restrains such a protective effect of
Hsp70 against the H
2
O
2
-induced apoptosis. These
data suggest that Hsp70 may inhibit apoptosis at
multiple points and through a wide range of
mechanisms.
Nucleolin is a major nucleolar phosphoprotein that
belongs to a large family of RNA-binding proteins
[26]. It plays a role in the pre-rRNA transcription
Ctrl pcDNA3.1 pcDNA3.1-C23
Nucleolin/C23
GAPDH
60
40
Percentage of apoptotic nuclei
Caspase-3 activity (fold increase)
Caspase-3 activity (fold increase)
50
30
20
10
0
3
4

2.5
3.5
3.5
3
2.5
2
1.5
1
0.5
0
0.5 mmol·L
–1
H
2
O
2
2
1.5
1
0.5
0
Ctrl
Ctrl
pcDNA3.1
pcDNA3.1-C23
pcDNA3.1-Hsp70
pcDNA3.1-C23 + pcDNA3.1-Hsp70
pcDNA3.1 + H
2
O

2
Ctrl
pcDNA3.1 + H
2
O
2
*
*
#
#
#
#
#
*
pcDNA3.1-C23 + H
2
O
2
pcDNA3.1-C23 + H
2
O
2
A
B
C
D
Fig. 4. Overexpression of nucleolin ⁄ C23 protects cardiomyocytes
from H
2
O

2
-induced apoptosis. Cardiomyocytes were transiently
transfected with full-length nucleolin plasmid (pcDNA3.1-C23) or
pcDNA3.1 vector. (A) Immunoblot analysis of nucleolin ⁄ C23 expres-
sion. At 36 h post-transfection, cells were analysed by immunoblot-
ting with antibodies against nucleolin ⁄ C23 or GAPDH (as an
internal control of protein loading). The results shown are represen-
tatives of three independent experiments. (B) Quantification of
apoptotic cells by nuclear staining. Untransfected or transfected
cells were exposed to 0.5 mmolÆL
)1
H
2
O
2
for the indicated dura-
tions and stained with Hoechst 33258; apoptotic cells showing con-
densed chromatin fragments were scored and expressed as a
percentage of the total cell number counted. The results from
three independent experiments are presented as means ± SEM.
*P < 0.05 versus control group; #P < 0.05 versus pcDNA3.1 +
H
2
O
2
group (n = 3). (C) Caspase-3 activity assay. Thirty-six hours
after transient transfection, cells were treated with or without
0.5 mmolÆL
)1
H

2
O
2
for 12 h and harvested for the determination of
protease activity of caspase-3 using the caspase colorimetric assay
kit. Data were obtained from four independent experiments.
*P < 0.05 versus control group; #P < 0.05 versus pcDNA3.1 +
H
2
O
2
group. (D) Caspase-3 activity assay. Thirty-six hours after
transfection with pcDNA3.1-C23 or pcDNA3.1-Hsp70 or transient
cotransfection with pcDNA3.1-C23 and pcDNA3.1-Hsp70, cells
were treated with or without 0.5 mmolÆL
)1
H
2
O
2
for 12 h and har-
vested for protease activity of caspase-3. Data were obtained from
four independent experiments. *P < 0.05 versus control group;
#P < 0.05 versus pcDNA3.1 + H
2
O
2
group.
Nucleolin ⁄ C23 in antiapoptotic effect of Hsp70 B. Jiang et al.
646 FEBS Journal 277 (2010) 642–652 ª 2009 The Authors Journal compilation ª 2009 FEBS

and ribosome assembly that is implicated in the early
stage of preribosomal ribonucleoprotein assembly and
processing [27]. Nucleolin has also been suggested to
regulate cell proliferation and growth, cytokinesis, rep-
lication, embryogenesis and nucleogenesis [28] by
forming large molecular complexes with other factors,
such as casein kinase II, c-Myb, midkine, histone H1,
nucleophosmin, p53 and protein phosphatase 1. A
number of studies, including our own, indicate that
nucleolin ⁄ C23 may be one of the key components in
the regulation of apoptosis [12–14,29–31]. In the pres-
ent study, we found that overexpression of nucleo-
lin ⁄ C23 protects cardiomyocytes from H
2
O
2
-induced
apoptosis, and that the mechanism is probably associ-
ated with the increase in Bcl-2 mRNA stability
(Fig. 5). In addition, we have also established a strong
relationship between nucleolin ⁄ C23 and the antiapop-
totic effect of Hsp70. Nucleolin ⁄ C23 in Hsp70-trans-
fected cells is significantly stabilized during H
2
O
2
exposure, and simultaneous Hsp70 overexpression
affords no additional protection against oxidant stress
(Fig. 4D). Therefore, Hsp70-transfected cells with a
higher level of nucleolin ⁄ C23 protein become more

resistant to oxidative stress and less susceptible to
apoptotic death. More importantly, the role of nucleo-
lin ⁄ C23 in this scenario is indispensable, as the sup-
pression of nucleolin ⁄ C23 expression by an antisense
oligomer potentiates apoptosis, even in the Hsp70-
transfected cells. These results also indicate that the
nucleolin ⁄ C23 gene has a functional role in the growth
control of cardiomyocytes, and its regulation may be
closely associated with the susceptibility of the cell to
the induction of apoptosis.
Caspases are important molecular mediators of
apoptosis in the cell [32]. Assays for caspase-3 activ-
ity have also confirmed that H
2
O
2
exposure induces
cardiomyocytes to undergo apoptosis and Hsp70
overexpression prevents H
2
O
2
-induced caspase activa-
tion; when nucleolin ⁄ C23 protein is suppressed by
the antisense oligonucleotide, the protection of
Hsp70 is lost.
Taken together, our results demonstrate an impor-
tant relationship among the regulation of nucleo-
lin ⁄ C23, the activation of caspase-3 and the induction
of apoptosis under the setting of oxidative stress and

Hsp70 overexpression. More importantly, for the first
time, we provide strong evidence that nucleolin ⁄ C23 is
a downstream mediator of Hsp70’s antiapoptosis
effects and it functions at the level of protein stability
in cardiomyocytes. It is our hope that such studies on
the mechanisms of apoptosis in cardiomyocytes will
provide a molecular basis for new therapeutic strate-
gies targeting specific pathways to treat human heart
disease.
Materials and methods
Animals
Neonatal Wistar rats (1-3 days) were purchased from the
Animal Resource Center of Center South University. The
following procedures were approved by the Institutional
Animal Care and Use Committee of the Center South Uni-
versity, and were carried out in accordance with the
National Institutes of Health Guide for the Care and Use
of Laboratory Animals. All efforts were made to minimize
the number of animals used and their suffering.
H
2
O
2
pcDNA3.1
100
10
0 0.5
Time (h)
123
Bcl-2 mRNA/GAPDH mRNA (%)

pcDNA3.1 pcDNA3.1-C23
Bcl-2
GAPDH
A
B
Fig. 5. Effect of nucleolin ⁄ C23 on Bcl-2 protein expression and
mRNA stability in the presence or absence of H
2
O
2
. (A) Western
blot analyses showing the effect of nucleolin ⁄ C23 on Bcl-2 protein
expression. Total cellular proteins were isolated and separated by
12% SDS ⁄ PAGE and analysed by immunoblotting with anti-Bcl-2.
Immunoblot analyses with GAPDH antiserum were used as the
protein loading control. (B) Real-time PCR showing the effect of
nucleolin ⁄ C23 on Bcl-2 mRNA stability. Cells transfected with
pcDNA3.1-C23 or the empty vector were cultured in the absence
or presence of H
2
O
2
(0.5 mM) for 2 h. Actinomycin D (5 lgÆmL
)1
)
was then added to the media and the cells were further incubated
for various times (0–3 h). Total RNAs were prepared and subjected
to real-time PCR analyses. The amount of Bcl-2 mRNA was normal-
ized by that of GAPDH. All experiments were performed in tripli-
cate and shown here by one representative. *P < 0.05 versus

pcDNA3.1-transfected control group.
B. Jiang et al. Nucleolin ⁄ C23 in antiapoptotic effect of Hsp70
FEBS Journal 277 (2010) 642–652 ª 2009 The Authors Journal compilation ª 2009 FEBS 647
Cell culture and treatment
Primary cultures of neonatal rat cardiomyocytes were culti-
vated as previously described [33,34]. Briefly, hearts from
neonatal Wistar rats (1–3 days after birth) were removed,
minced and trypsinized at 37 °C with gentle stirring in
D-Hanks buffer containing 0.1% trypsin (Gibco, Rockville,
MD, USA). The cells were then centrifuged and resus-
pended in Dulbecco’s modified Eagle’s medium (DMEM,
Gibco) containing 15% fetal bovine serum (Hyclone,
Logan, UT, USA). After incubation at 37 °C for 120 min,
the suspended cardiomyocytes were seeded at a density of
5 · 10
5
cellsÆmL
)1
. All cells were cultured in DMEM con-
taining 15% fetal bovine serum for 24 h before the initia-
tion of experiments. 5-Bromo-2¢-deoxyuridine (Sigma, St
Louis, MO, USA) (0.1 mmolÆL
)1
) was added to the culture
for 36 h to inhibit the proliferation of nonmyocytes. H
2
O
2
was first diluted in phosphate-buffered saline (PBS, pH 7.4)
and further diluted in culture medium to a final concentra-

tion of 0.5 mmolÆL
)1
for all treatments. Cycloheximide
(Sigma) was diluted in the medium to a final concentration
of 5 lgÆmL
)1
.
Quantification of apoptotic cells
At the predetermined time points after H
2
O
2
exposure, cells
were detached from tissue culture plates with tryp-
sin ⁄ EDTA or cell scrapers, and collected together with the
nonadherent cells. After centrifugation at 500 g for 5 min,
the cells were fixed with 4% paraformaldehyde for 30 min
at room temperature and then washed once with PBS. The
fixed cells were then incubated with Hoechst 33258
(50 ng ÆmL
)1
) for 30 min at room temperature, and washed
60
Hsp70/C + NCL/C23-AsODNs
Hsp70/C + NCL/C23-ScrODNs
Time (h) of H
2
O
2
exposure

*
*
50
40
30
20
10
0
0 6 12 24 36
Percentage of apoptotic nuclei
Hsp70/C + NCL/C23-AsODNs
Hsp70/C + NCL/C23-ScrODNs
Time (h) of H
2
O
2
exposure
*
*
0
0.5
1
1.5
2
3
2.5
0481224
Caspase-3 activity (fold increase)
Ctrl Lip
Hsp70/C cells

ScrODNs AsODNs
Nucleolin/C23
Hsp70
GAPDH
A
B
C
D
Hoechst 33258 staining GFP
Fig. 6. Potentiation of apoptosis in Hsp70-overexpressed cells by
nucleolin ⁄ C23 antisense oligomer. (A) Transfection efficiency of
phosphorothioate oligodeoxynucleotides in primary cultures of car-
diomyocytes. FITC-labelled phosphorothioate oligodeoxynucleotides
were transfected into cells with Lipofectamine 2000 and cells were
stained with Hoechst 33258. Right, the image under blue fluores-
cence channel showing all cells with nuclear Hoechst staining; left,
image under green fluorescence channel showing only cells posi-
tive for FITC-labelled oligomers. (B) Knocking-down of nucleo-
lin ⁄ C23 in cardiomyocytes. The Hsp70-transfected cardiomyocytes
(Hsp70 ⁄ C) were cotransfected with nucleolin ⁄ C23 scrambled
(NCL ⁄ C23-ScrODNs) or antisense (NCL ⁄ C23-AsODNs) oligomers.
After 36 h, cells were harvested for western blot analysis with
monoclonal antibody against nucleolin ⁄ C23 (NCL ⁄ C23) or GAPDH.
(C) Determination of the percentage of apoptotic cells by Hoechst
staining. Hsp70 ⁄ C cells were cotransfected with NCL ⁄ C23-Scr-
ODNs or NCL ⁄ C23-AsODNs and then treated with H
2
O
2
for

6–36 h. The results from three independent experiments are
shown as mean ± SEM. *P < 0.05 versus NCL ⁄ C23-ScrODNs
group. (D) Determination of percentage of apoptotic cells by cas-
pase-3 activity assay. Hsp70 ⁄ C cells were transfected and treated
as above, and assayed for caspase-3 activity. Data were obtained
from four independent experiments. *P < 0.05 versus NCL ⁄ C23-
ScrODNs group.
Nucleolin ⁄ C23 in antiapoptotic effect of Hsp70 B. Jiang et al.
648 FEBS Journal 277 (2010) 642–652 ª 2009 The Authors Journal compilation ª 2009 FEBS
again with PBS. Cells were mounted on to glass slides and
examined by fluorescence microscopy. Apoptotic cells were
identified by the condensation and fragmentation of their
nuclei. The percentage of apoptotic cells was calculated as
the number of apoptotic cells divided by the total number
of cells counted. A minimum of 500 cells were counted for
each slide.
Flow cytometry
Both adherent and floating cells were collected after treatment,
washed with ice-cold PBS and stained with FITC-conju-
gated annexin V (BD Biosciences, Franklin Lakes, NJ,
USA) and PI for 20 min at room temperature in the dark.
The stained cells were then analysed by a flow cytometer
A
B
Fig. 7. Flow cytometry analysis of apoptosis
with annexin V–FITC ⁄ PI double staining.
Hsp70-transfected cardiomyocytes
(Hsp70 ⁄ C) were cotransfected with the
nucleolin ⁄ C23 scrambled (NCL ⁄ C23-ScrO-
DNs) or antisense (NCL ⁄ C23-AsODNs)

oligomers. Twenty-four hours later, they
were treated with 0.5 mmolÆL
)1
H
2
O
2
for 6
or 12 h. Cells were harvested and pro-
cessed for annexin V–FITC and PI costaining
and analysed by flow cytometry (A). Q3
cells were the control cells, Q4 cells were
cells at the early apoptosis stage, Q2 cells
were cells at the late apoptosis stage, and
Q1 cells were necrotic cells. The percentage
of apoptotic cells was also calculated (B).
The results are representative of three
independent experiments. Data are the
mean ± SEM of triplicate samples.
*Significant difference (P < 0.05) versus the
pcDNA3.1 control group; #significant
difference (P < 0.05) versus the control
group.
B. Jiang et al. Nucleolin ⁄ C23 in antiapoptotic effect of Hsp70
FEBS Journal 277 (2010) 642–652 ª 2009 The Authors Journal compilation ª 2009 FEBS 649
(Beckman Coulter, Fullerton, CA, USA). FITC-conjugated
annexin V binds to phosphatidylserine molecules only pres-
ent at the surface of apoptotic cells where they were trans-
located from the internal side of the plasma membrane
during apoptosis. Cells were simultaneously stained with PI

to discriminate membrane-permeable necrotic cells from
FITC-labelled apoptotic cells. Apoptotic cells were identi-
fied as those positive for annexin V–FITC staining but
negative for PI staining, and the percentage of apoptotic
cells in the total number of cells was calculated.
Western blot analyses
Cells were washed with PBS and collected by centrifuga-
tion. Cell pellets were resuspended with 5 volumes of cold
lysis buffer [50 mmolÆL
)1
Tris ⁄ HCl (pH 7.5), 250 mmo-
lÆL
)1
NaCl, 5 mmolÆL
)1
EDTA, 50 mmolÆL
)1
NaF, 0.5%
Nonidet P-40] containing a protease inhibitor mixture
(Roche Applied Science, Burgess Hill, UK). The cell lysate
was incubated on ice for 30 min and centrifuged at
10 000 g for 10 min at 4 °C. The protein concentration of
the supernatant was determined using the Bradford assay
(Bio-Rad, Hercules, CA, USA). Equal amounts of protein
(5–10 lg) were loaded on to and separated by SDS ⁄ PAGE
and transferred to a nitrocellulose membrane. The blot
was blocked with 2% albumin in 20 mmolÆL
)1
Tris ⁄ HCl,
pH 8.0, 150 mmolÆL

)1
NaCl, 0.1% Tween 20 (TBST)
overnight at 4 °C and then incubated with the rabbit anti-
C23 (Sigma) or anti-(glyceraldehyde-3-phosphate dehydro-
genase) (GAPDH; Santa Cruz Biotechnology, Santa Cruz,
CA, USA) for 2 h. After washing with TBST buffer three
times for 15 min each, the blot was incubated with horse-
radish peroxidase-conjugated goat anti-mouse IgG (diluted
1 : 2000 in TBST buffer) at room temperature for 1 h,
and finally washed three times with TBST for 15 min
each. Immunoreactivity was visualized using the enhanced
chemiluminescence reaction (Amersham, Piscataway, NJ,
USA).
Caspase activity assay
The activity of caspase-3 was measured by in vitro sub-
strate-cleavage reactions using a commercial kit according
to the manufacturer’s protocol (R&D Systems, Minneapo-
lis, MN, USA). Briefly, cells cultured in 60 mm dishes were
treated with 0.5 mmolÆL
)1
H
2
O
2
for the indicated time peri-
ods. The cells (5 · 10
6
) were lysed with 250 lL chilled cell
lysis buffer on ice for 10 min. After centrifugation
(10 000 g, 1 min, 4 °C), the protein concentration in the

supernatant was determined using the BioRad protein
assay. Supernatants containing equal amounts of protein
(corresponding to  5 · 10
5
cells) were used for caspase-3
colorimetric assays. After protein samples were incubated
with substrates at 37 °C for 1.5 h, the absorbance at
405 nm was measured using a microtitre plate reader
(Molecular Devices, Sunnyvale, CA, USA). Fold increases
in caspase-3 activity over that before treatment were deter-
mined.
Lipofectamine-mediated gene transfection
Transfection of cells was carried out following the manu-
facturer’s instructions (Lipofectamine 2000Ô, Invitrogen,
Carlsbad, CA, USA). Briefly,  5 · 10
5
cells in 5 mL
appropriate complete growth medium per flask were grown
at 37 °CinaCO
2
incubator until the cells reached 70–80%
confluence (24 h). After rinsing with serum-free and antibi-
otic-free medium, the cells were transfected with
pcDNA3.1-Hsp70 (HspA1A was a gift from I. Benjamin,
University of Utah Health Sciences Center) (experimental
group) or pcDNA3.1 (vector control), at 8 lg DNA per
20 lL Lipofectamine, followed by incubation at 37 °Cina
CO
2
incubator for 6 h. The medium was then replaced with

DMEM culture medium containing 20% fetal bovine
serum.
Nucleolin/C23 antisense oligodeoxynucleotide
The phosphorothioate oligodeoxynucleotides used in this
study were manufactured by Bioasia Biotech (Shanghai,
China). The sequence of nucleolin ⁄ C23 antisense oli-
godeoxynucleotide (NUL ⁄ C23-AsODNs), corresponding to
the rat nucleolin ⁄ C23 translation initiation site, was 5¢-TG
CGAGTTTCACCATGATGGC-3¢. A scrambled nucleolin ⁄
C23 oligodeoxynucleotide (NUL ⁄ C23-ScrODNs; 5¢-CTGA
TGTCACGTCCATGTGAG-3¢) was used as the control.
The oligodeoxynucleotides were diluted in 10 mmolÆL
)1
Tris (pH 7.4) and 1 mmolÆL
)1
EDTA and kept at )20 °C.
RT-PCR
Total RNAs were prepared with the Rneasy kit (Qiagen,
Cambridge, MA, USA) according to the manufacturer’s
instructions and 2 lg samples were reverse transcribed using
oligo(dT) primers. The levels of gene expression were quanti-
fied by real-time PCR, using a QuantiTect SYBR Green PCR
Kit (Qiagen) and the 7500 Fast Real-Time PCR system
(Applied Biosystems, Foster City, CA, USA) under the
following conditions: 40 sequential cycles, each including
95 °C for 10 s and 60 °C for 10 s. Sequences of primers spe-
cific to each target cDNA were: nucleolin, forward, 5-CA
ATCAGGCTGGAGTTGCAAG-3; and reverse, 5-TGGC
CCAGTCCAAGGTAACTT-3 (amplicon size: 282 bp);
GAPDH forward, 5-ACCACAGTCCATGCCATCAC-3;

and reverse, 5-TCCACCACCCTGTTGCTGTA-3 (size:
440 bp). The specificity of PCR products was verified by melt-
ing curve analysis and electrophoresis on agarose gels. The
comparative threshold cycle method and an internal control
(GAPDH) were used to normalize target gene expression.
Nucleolin ⁄ C23 in antiapoptotic effect of Hsp70 B. Jiang et al.
650 FEBS Journal 277 (2010) 642–652 ª 2009 The Authors Journal compilation ª 2009 FEBS
Statistical analyses
Data are presented as mean ± standard error of the mean
(SEM) of the values obtained from the indicated number of
independent experiments. Differences between two groups
were analysed by unpaired Student’s t-test. Differences
between three or more groups were analysed by one-way
ANOVA followed by Student–Newman–Keuls posthoc test.
P < 0.05 was considered statistically significant.
Acknowledgements
This study was supported by grants from the National
Basic Research Program of China (2007CB512007),
the National Natural Science Foundation of China
(30700290) and Special Funds for PhD Training from
The Ministry of Education of China (20060533009).
References
1 Kerr JFR, Wyllie AH & Currie AR (1972) Apoptosis:
a basic biological phenomenon with wide-ranging impli-
cations in tissue kinetics. Br J Cancer 26, 239–257.
2 Jacobsen MD, Weil M & Raff MC (1997) Programmed
cell death in animal development. Cell 88, 347–354.
3 Lee Y & Gustafsson AB (2009) Role of apoptosis in
cardiovascular disease. Apoptosis 14, 536–548.
4 Mani K (2008) Programmed cell death in cardiac

myocytes: strategies to maximize post-ischemic salvage.
Heart Fail Rev 13, 193–209.
5 Arya R, Mallik M & Lakhotia SC (2007) Heat shock
genes – integrating cell survival and death. J Biosci 32,
595–610.
6 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.
7 Marber MS, Mestril R, Chi RS, Sayen MR, Yellon
DM & Dillmann WH (1995) Overexpression of the rat
inducible 70-kD heat stress protein in a transgenic
mouse increases the resistance of the heart to ischemic
injury. J Clin Invest 95, 1446–1456.
8 Jayakumar J, Suzuki K, Khan M, Smolenski RT,
Farrell A, Latif N, Raisky O, Abunasra H, Sammut
IA, Murtuza B et al. (2000) Gene therapy for
myocardial protection: transfection of donor hearts
with heat shock protein 70 gene protects cardiac func-
tion against ischemia-reperfusion injury. Circulation
102, 302–306.
9 Derenzini M, Sirri V, Trere D & Ochs RL (1995) The
quantity of nucleolar proteins nucleolin and protein
B23 is related to cell doubling time in human cancer
cells. Lab Invest 73, 497–502.
10 Hovanessian AG, Puvion-Dutilleul F, Nisole S, Svab J,
Perret E, Deng JS & Krust B (2000) The cell-surface-
expressed nucleolin is associated with the actin cytoskel-
eton. Exp Cell Res 261, 312–328.
11 Ginisty H, Amalric F & Bouvet P (1998) Nucleolin

functions in the first step of ribosomal RNA processing.
EMBO J 17, 1476–1486.
12 Otake Y, Sengupta TK, Bandyopadhyay S, Spicer EK
& Fernandes DJ (2005) Retinoid-induced apoptosis in
HL-60 cells is associated with nucleolin down-regulation
and destabilization of Bcl-2 mRNA. Mol Pharmacol 67,
319–326.
13 Otake Y, Soundararajan S, Sengupta TK, et al. (2007)
Overexpression of nucleolin in chronic lymphocytic leu-
kemia cells induces stabilization of bcl2 mRNA. Blood,
109, 3069–3075.
14 Wang KK, Jiang L, Yi YX, Liu K, E SM, Tang DL,
Wang JS, Shi YZ, Wang QP & Xiao XZ (2004) Effect
of heat shock response on the cleavage of nucleolin
induced by oxidative stress. Zhong Nan Da Xue Xue
Bao Yi Xue Ban 29, 504–508.
15 Wang KK, Jiang L, E SM, Liu K, Zhang LL, Liu MD
& Xiao XZ (2005) Effect of nucleolin down-regulation
on the proliferation and apoptosis in C2C12 cells.
Zhong Nan Da Xue Xue Bao Yi Xue Ban 30, 125–129.
16 Jiang B, Xiao W, Shi Y, Liu M & Xiao X (2005) Heat
shock pretreatment inhibited the release of Smac from
mitochondria and apoptosis induced by hydrogen per-
oxide in cardiomyocytes and C2C12 myogenic cells. Cell
Stress Chaperones 10, 252–262.
17 Feng X, Bonni S & Riabowol K (2006) HSP70 induc-
tion by ING proteins sensitizes cells to tumor necrosis
factor alpha receptor-mediated apoptosis. Mol Cell Biol
26, 9244–9255.
18 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.
19 Bivik C, Rosdahl I & Ollinger K (2007) HSP70 protects
against UVB induced apoptosis by preventing release of
cathepsins and cytochrome c in human melanocytes.
Carcinogenesis 28 , 537–544.
20 Matsumori Y, Hong SM, Aoyama K, Fan Y, Kayama
T, Sheldon RA, Vexler ZS, Ferriero DM, Weinstein PR
& Liu J (2005) HSP70 overexpression sequesters AIF
and reduces neonatal hypoxic ⁄ ischemic brain injury.
J Cereb Blood Flow Metab 25, 899–910.
21 Nylandsted J, Gyrd-Hansen M, Danielewicz A,
Fehrenbacher N, Lademann U, Høyer-Hansen M,
Weber E, Multhoff G, Rohde M & Ja
¨
a
¨
ttela
¨
M (2004)
Heat shock protein 70 promotes cell survival by inhibit-
ing lysosomal membrane permeabilization. J Exp Med
200, 425–435.
22 Didelot C, Schmitt E, Brunet M, Maingret L, Parcellier
A & Garrido C (2006) Heat shock proteins: endogenous
modulators of apoptotic cell death. Handb Exp Pharma-
col 26, 171–198.
B. Jiang et al. Nucleolin ⁄ C23 in antiapoptotic effect of Hsp70

FEBS Journal 277 (2010) 642–652 ª 2009 The Authors Journal compilation ª 2009 FEBS 651
23 Ravagnan L, Gurbuxani S, Susin SA, Maisse C, Daugas
E, Zamzami N, Mak T, Jaattela M, Penninger JM,
Garrido C et al. (2001) Heat-shock protein 70 antago-
nizes apoptosis-inducing factor. Nat Cell Biol 3, 839–843.
24 Jiang B, Wang K, Xiao W, Wang H & Xiao X (2009)
ATP-binding domain of heat shock protein 70 is essen-
tial for its effects on the inhibition of the release of the
second mitochondria-derived activator of caspase and
apoptosis in C2C12 cells. FEBS J 276, 2615–2624.
25 Stankiewicz AR, Livingstone AM, Mohseni N &
Mosser DD (2009) Regulation of heat-induced
apoptosis by Mcl-1 degradation and its inhibition by
Hsp70. Cell Death Differ 16, 638–647.
26 Shaw PJ & Jordan EG (1995) The nucleolus. Annu Rev
Cell Dev Biol 11, 93–121.
27 Borer RA, Lehner CF, Eppenberger HM & Nigg EA
(1989) Major nucleolar proteins shuttle between nucleus
and cytoplasm. Cell 56, 379–390.
28 Xie M, Kobayashi I, Kiyoshima T, Yamaza H, Honda
JY, Takahashi K, Enoki N, Akamine A & Sakai H
(2007) Functional implication of nucleolin in the mouse
first molar development. J Biol Chem 282, 23275–23283.
29 Wang HY, Liu XL & Jiang BM (2008) Changes of
nucleolin expression and cellular localization during
HUVEC apoptosis induced by hydrogen peroxide.
Zhong Nan Da Xue Xue Bao Yi Xue Ban 33, 488–493.
30 Fogal V, Sugahara KN, Ruoslahti E & Christian S
(2009) Cell surface nucleolin antagonist causes
endothelial cell apoptosis and normalization of tumor

vasculature. Angiogenesis 12, 91–100.
31 Sengupta TK, Bandyopadhyay S, Fernandes DJ &
Spicer EK (2004) Identification of nucleolin as an
AU-rich element binding protein involved in bcl-2
mRNA stabilization. J Biol Chem 279 , 10855–10863.
32 Nicholson DW & Thornberry NA (1997) Caspases:
killer proteases. Trends Biochem Sci 22, 299–306.
33 Long X, Crow MT, Sollott SJ, O’Neill L, Menees DS, de
Lourdes Hipolito M, Boluyt MO, Asai T & Lakatta EG
(1998) Enhanced expression of p53 and apoptosis
induced by blockade of the vacuolar proton ATPase in
cardiomyocytes. J Clin Invest 101, 1453–1461.
34 Long X, Boluyt MO & Hipolito ML (1997) p53 and
hypoxia-induced apoptosis of cultured neonatal cardio-
myocytes. J Clin Invest 99, 2635–2643.
Nucleolin ⁄ C23 in antiapoptotic effect of Hsp70 B. Jiang et al.
652 FEBS Journal 277 (2010) 642–652 ª 2009 The Authors Journal compilation ª 2009 FEBS