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

(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

[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

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

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).

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

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

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

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
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

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
-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

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

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-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-

*
*
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

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)

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

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

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).
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