Tài liệu Báo cáo khoa học: Hepatic stimulator substance mitigates hepatic cell injury through suppression of the mitochondrial permeability transition pdf

Hepatic stimulator substance mitigates hepatic cell injury
through suppression of the mitochondrial permeability
transition
Yuan Wu
1,
*, Jing Zhang
1,
*, Lingyue Dong
1
, Wen Li
1
, Jidong Jia
2
and Wei An
1
1 Department of Cell Biology and Municipal Key Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University,
Beijing, China
2 Liver Unit, Beijing Friendship Hospital, Capital Medical University, Beijing, China
Introduction
Hepatic stimulator substance (HSS) is expressed in the
liver cytosol of weanling or partially hepatectomized
adult rats, and was first described by LaBrecque and
Pesch [1]. A major function of this protein is to pro-
mote hepatocyte proliferation and liver regeneration
after partial hepatectomy [1–3]. The HSS-mediated
Keywords
apoptosis; hepatic stimulator substance;
mitochondria; mitochondrial membrane
potential; mitochondrial permeability
transition
Correspondence

W. An, Department of Cell Biology,
Municipal Key Laboratory for Liver
Protection and Regulation of Regeneration,
Capital Medical University, Beijing 100069,
China
Fax: +86 10 83911480
Tel: +86 10 83911480
E-mail:
*These authors contributed equally to this
work
(Received 12 October 2009, revised 18
December 2009, accepted 23 December
2009)
doi:10.1111/j.1742-4658.2010.07560.x
Hepatic stimulator substance (HSS) has been shown to protect liver cells
from various toxins. However, the mechanism by which HSS protects
hepatocytes remains unclear. In this study, we established BEL-7402
cells that stably express HSS and analyzed the protective ability of HSS on
cells through mitochondrial permeability (MP). After administration of
carbonyl cyanide m-chlorophenylhydrazone (CCCP), a specific agent that
leads to depolarization of the mitochondrial transmembrane potential, the
apoptosis rate of HSS-expressing cells was significantly reduced, as
measured using Hoechst staining and flow cytometry. The mitochondrial
membrane transition and cytochrome c leakage were significantly inhibited
in the HSS-expressing cells as compared with the untransfected cells, and,
as a consequence, the cellular ATP content in the HSS-expressing cells was
relatively preserved. Additionally, decreased caspase-3 activity was
observed in the HSS-expressing cells treated with CCCP as compared with
the vector-transfected cells and cells expressing mutant HSS. Furthermore,
silencing of HSS expression using small interfering RNA accelerated

CCCP-induced apoptosis. In isolated mitochondria, recombinant HSS
reduced the release of cytochrome c induced by CCCP, indicating a possi-
ble role for HSS in regulation of mitochondrial permeability transition
(MPT). HSS-expressing BEL-7402 cells are resistant to CCCP injury, and
HSS protection is identical to that observed with cyclosporin A, an inhibi-
tor of MPT. Therefore, we propose that the protective effect of HSS may
be associated with blockade of MPT.
Abbreviations
ALR, augmenter of liver regeneration; CCCP, carbonyl cyanide m-chlorophenylhydrazone; COX IV, cytochrome c oxidase subunit IV;
CsA, cyclosporin A; HSS, hepatic stimulator substance; IM, inner membrane; JC-1, 5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-
tetraethylbenzimidazolocarbocyanine iodide; MP, mitochondrial permeability; MPT, mitochondrial permeability transition;
PTP, permeabilization transition pore; rHSS, recombinant hepatic stimulator substance; siRNA, small interfering RNA; w
m
, inner
transmembrane potential.
FEBS Journal 277 (2010) 1297–1309 ª 2010 The Authors Journal compilation ª 2010 FEBS 1297
promotion of liver regeneration has been demonstrated
to be related to its inhibition of hepatic natural killer
cell activity in an acute liver injury model [4]. HSS
expression has also been reported to be increased in
cirrhotic human livers, and the mRNA level of HSS
was elevated in tissue samples of hepatocellular carci-
noma and cholangiocellular carcinoma [5]. This
increased expression of HSS in liver tumors may due
to its ability to stimulate DNA synthesis [6–8]. In addi-
tion to its ability to promote liver regeneration, HSS
has been shown to protect the liver from acute injury
caused by several compounds, including CCl
4
[9],

d-galactosamine [10], ethanol [11], H
2
O
2
[12], and cad-
mium [13]. HSS has also been shown to have clinical
potential, as exogenous HSS administration to rats
with thioacetamide-induced liver fibrosis ⁄ cirrhosis is
able to significantly decrease fibrosis and to suppress
the onset of cirrhosis [14].
Several in vitro studies have demonstrated that
although HSS promotes cell growth in dividing
hepatocytes, it is unable to stimulate cell division of
primary cultured or mature hepatocytes. When added
to cultures of primary hepatocytes, HSS had minimal
effects on cell growth; instead, it augmented the mito-
genic effects of other growth factors, such as epider-
mal growth factor [15]. Thereafter, HSS crude extract
was further purified, and a fraction (· 830 000) that
was responsible for the growth-augmenting activity
was referred to as augmenter of liver regeneration
(ALR) [16]. In 1996, the cDNA sequence of ALR
was reported by Giorda et al. [17]. Unlike complete
mitogens such as hepatic growth factor or transform-
ing growth factor-a, ALR alone showed little effect
on hepatocyte proliferation in vitro, suggesting that
hepatocytes might not contain surface receptors spe-
cific for ALR. This hypothesis has now been refuted,
as high-affinity receptors for ALR have been found
on the surface of hepatic cells [18]. Although current

data suggest that HSS and ALR are very similar mol-
ecules with regard to their cDNA and protein
sequences, there are a few disagreements. For exam-
ple, HSS was present only in the liver, but ALR was
found to be expressed in many tissues [19,20], with
different subcellular localizations [21], and seems to
have remarkably diverse functions related not only to
liver regeneration [22]. More recent publications have
demonstrated that HSS has diverse functions. For
example, it regulates FAD-linked disulfide bridges in
proteins, the biogenesis of cytosolic Fe–S proteins,
and electron transfer via FAD to cytochrome c [23].
Most recently, Thirunavukkarasu et al. have reported
that HSS is an important intracellular survival factor
for hepatocytes [24].
In 2001, Lisowsky et al. [25] first reported that
mammalian HSS is an FAD-linked sulfhydryl oxidase
with a CXXC active motif in the C-terminal domain.
Yeast Erv1p (essential for respiration and vegetative
growth) has about 42% amino acid homology with
mammalian HSS in the C-terminal domain [26]. Yeast
ERV1, the mouse [17], rat [27] and human [26] HSS
genes, and some orthologous genes that have been
identified in dsDNA viruses [28] have together been
defined as the ERV ⁄ HSS gene family [29]. Members of
this family have a highly conserved C-terminal
domain, and this conserved C-terminus is functionally
interchangeable between yeast and human. Like Erv1p,
HSS is located in the mitochondrial intermembrane
space, and they both help with the maturation of Fe–S

proteins outside of the mitochondria [23]. Additionally,
HSS has been shown to induce mitochondrial gene
expression and enhance the oxidative phosphorylation
capacity of liver mitochondria [30]. Studies that have
focused on HSS and the mitochondria have indicated
that HSS not only affects the activity of P450 via gene
repression [31], but also interacts with the respiratory
chain via the modification of cytochrome c [32].
It is widely accepted that mitochondria play a criti-
cal role in the regulation of cell death [33–35], and
mitochondrial permeability transition (MPT) is consi-
dered to be the pivotal event of mitochondria-mediated
cell death [36]. MPT leads to loss of the inner trans-
membrane potential (w
m
) [37], reduction of the intra-
cellular ATP level, matrix swelling, and the release of
proapoptotic proteins such as cytochrome c.
As mitochondria play an essential role during cell
death, we aimed to determine the relationship between
HSS expression and mitochondrial protection, and
whether mitochondrial permeability (MP) would be
targeted by HSS. In this study, we established a
BEL-7402 cell line that stably expresses HSS and iden-
tified the mitochondrial location of HSS. After a mito-
chondrial lesion specifically caused by carbonyl
cyanide m-chlorophenylhydrazone (CCCP), a classic
protonophore-type uncoupling agent [38], we analyzed
Dw
m

, the intracellular ATP level, and the leakage of
cytochrome c. HSS demonstrated a protective effect
against CCCP-induced apoptosis, inhibiting MPT. The
protective effect of HSS was compared, in parallel,
with that of another known MPT inhibitor, cyclospo-
rin A (CsA), and its analog NIM811. CsA and
NIM811 displayed inhibitory effects on MPT, in a
dose–response manner; similarly, HSS also inhibited
MPT, further supporting our hypothesis that HSS pro-
tection is strongly associated with the mitochondrial
membrane pore. Knockdown of HSS expression by
RNA interference destroyed MP, leading to a great
HSS and mitochondrial permeability transition Y. Wu et al.
1298 FEBS Journal 277 (2010) 1297–1309 ª 2010 The Authors Journal compilation ª 2010 FEBS
increase in the ability of CCCP to damage the
mitochondria. In conclusion, HSS protects liver cells
from CCCP-induced apoptosis. From this result, we
propose that the potential mechanism by which HSS
mediates its antiapoptotic effect is related to regulation
of MPT.
Results
HSS expression in the cells
Real-time RT-PCR demonstrated that HSS was signif-
icantly expressed in transfected cells as compared with
vector-transfected cells (pcDNA3.0 alone) or wild-type
cells (Fig. 1A). Protein expression of HSS in the three
cell lines (untransfected cells, vector-transfected cells,
and HSS-expressing cells) was detected using western
blot. As shown in Fig. 1B, a 15 kDa band was
detected after hybridization with the antibody against

HSS in the cells transfected with the HSS expression
construct. By contrast, HSS expression in wild-type
and vector-transfected cells was minimal.
Morphological evidence of the antiapoptotic
effect of HSS
As shown in Fig. 2A,B, the treatment of cells with
50 lm CCCP induced profound changes in the nuclear
morphology of hepatoma cells, with chromatin con-
densation and fragmentation being observable using
fluorescence microscopy. As detected using
Hoechst 33342 staining, vector-transfected cells and
cells expressing mutant HSS underwent typical apopto-
tic changes [39], including shrinkage, membrane bleb-
bing, chromatin condensation, and the formation of
apoptotic bodies, after being treated with CCCP. How-
ever, in cells expressing HSS, the apoptotic rate was
significantly decreased following treatment with CCCP.
To verify this putative protective effect by HSS, we set
up a parallel control using 10 lm CsA, which is a
potent inhibitor of MPT. Both CsA treatment and the
expression of HSS decreased the number of apoptotic
cells, and CsA alone was able to greatly alleviate
CCCP-induced apoptosis.
Apoptosis evaluated by flow cytometry
As shown in Fig. 2C,D, the proportions of apoptotic
vector-transfected cells and cells expressing mutant
HSS treated with 50 lm CCCP were comparatively
high (74.24% ± 3.32% and 75.11% ± 4.40%, respec-
tively). However, the proportion of apoptotic cells fol-
lowing treatment with CCCP was significantly reduced

in HSS-expressing cells (52.4% ± 3.90%). Thus, the
apoptotic rate in HSS-expressing cells was decreased
by about 30% as compared with vector-transfected
cells and mutant HSS-expressing cells. Similarly,
CCCP-induced apoptosis was also inhibited by CsA.
Effect of HSS on alteration of w
m
Alteration of w
m
is known to be an early event in the
apoptotic signaling cascade [40]. As it has been shown
that HSS is localized to the mitochondria of hepatoma
cells [12], we explored whether HSS plays an important
role in protecting the mitochondria from CCCP-
induced damage and apoptosis. As shown in
Fig. 3A,B, the addition of 30 lm CCCP to the isolated
mitochondria induced MPT-dependent swelling, as
shown by a large decrease in the fluorescence intensity
in cells. CsA, NIM811 and HSS induced a dose-depen-
Fig. 1. (A) The level of HSS RNA in the three types of cells was
quantified by real-time RT-PCR, and is expressed as genomic equiv-
alents per culture. The expression of HSS in cells stably transfected
(Tr) with the HSS expression construct is significantly greater than
that in wild-type cells and vector-transfected cells. (B) The differen-
tial expression of HSS in the three cell lines. Mitochondrial protein
extracts (25 lg) from each of the cell cultures were analyzed using
western blot and an antibody against HSS.
Y. Wu et al. HSS and mitochondrial permeability transition
FEBS Journal 277 (2010) 1297–1309 ª 2010 The Authors Journal compilation ª 2010 FEBS 1299
dent increase in w

m
. At the maximal dose of HSS
(10 lgÆmL
)1
), however, addition of CsA to the mito-
chondria did not increase w
m
further, indicating that
HSS protection against MPT could not be enhanced
by CsA (Fig. 3A). In order to determine whether HSS
can augment the protective effect of CsA against
MPT, we added various doses of CsA to the isolated
mitochondria subjected to CCCP injury. Both CsA
and its analog NIM811 showed potent protection
against MPT; however, similar to the finding shown in
Fig. 3A, the protection provided by CsA and NIM811,
if obtained at the maximal doses, could not be further
enhanced by HSS. These results imply that HSS pro-
tection of mitochondria might be due to inhibition of
the MP disruption, and the inhibition of MPT,
although not fully clarified yet, could be due to a
mechanism similar to that responsible for the effect of
CsA or NIM811. In addition, we investigated whether
HSS protection could be obtained in the isolated mito-
chondria of HSS-expressing cells. As seen in Fig. 3C,
MP in the mitochondria of HSS-transfected cells was
affected less by CCCP than that of vector-transfected
cells. CsA could rescue MP effectively in HSS-
expressing cells, indicating that HSS transfection could
alleviate mitochondrial damage through inhibition of

MP collapse. Moreover, damage to the mitochondrial
membrane pores following treatment with CCCP led
to a remarkable amount of leakage of cytochrome c,
whereas in HSS-expressing cells, the leakage of cyto-
chrome c was inhibited. Exposure of cells to CCCP
resulted in substantial loss of w
m
, and, as a
consequence, the mitochondrial membranes were easily
damaged and there was massive leakage of cyto-
chrome c (Fig. 4).
Effect of HSS on alteration of cytochrome c
leakage
As shown in Fig. 4, treatment of cells with CCCP
(50 lm) led to serious damage to the mitochondrial
membrane, resulting in the leakage of cytochrome c
from the mitochondria in vector-transfected cells
(Fig. 4A, lane 4). However, the cytochrome c content
was significantly preserved in cells expressing HSS
Fig. 2. Assessment of apoptosis by Hoechst 33342. (A) Wild-type cells, vector-transfected cells and HSS-expressing cells were analyzed
after Hoechst 33342 staining. The cells treated with CCCP have undergone chromatin condensation and margination. (B) The number of
apoptotic cells decreased as compared with the control (ctrl) group and cells treated with CsA. (C) Apoptosis was analyzed using flow
cytometry. The CCCP treatment significantly increased the apoptotic rate in vector-transfected and mutant HSS-expressing cells. However,
apoptosis was markedly inhibited in HSS-expressing cells. (D) Statistical evaluation of the apoptotic rate from three independent experi-
ments.
HSS and mitochondrial permeability transition Y. Wu et al.
1300 FEBS Journal 277 (2010) 1297–1309 ª 2010 The Authors Journal compilation ª 2010 FEBS
(Fig. 4A, lane 5); the cytochrome c leakage was
reduced by approximately 75% in HSS-expressing cells
as compared with vector-transfected cells (P < 0.05).

The preservation of mitochondrial cytochrome c was
not observed in cells expressing mutant HSS (Fig. 4A,
lane 4). Treatment with CsA decreased cytochrome c
leakage in both vector-transfected and HSS-expressing
cells, but CsA appeared to have more of an effect in
HSS-expressing cells (Fig. 4A, lanes 6 and 7). A densi-
tometric analysis of the cytochrome c content is shown
in Fig. 4B,D.
Effect of HSS on the intracellular ATP level
To examine the energy production of mitochondria in
CCCP-treated cells, the intracellular ATP level was
measured. The intracellular ATP level in vector-trans-
fected cells and HSS-expressing cells was greatly
reduced after treatment with CCCP (Fig. 5A).
Although the ATP level of HSS-expressing cells fell to
a low level, the relative amount of ATP in HSS-
expressing cells was about 45% higher than that in
vector-transfected cells, suggesting that HSS-expressing
cells were less affected by the impaired energy produc-
tion. After addition of CsA, the ATP level in HSS-
expressing cells was more readily restored than that in
vector-transfected cells. Moreover, we examined the
effects of CsA and NIM811 on the restoration of
intracellular ATP level. Figure 5B shows that both
agents were able to increase the ATP levels in HSS-
expressing cells, with a dose-dependent pattern. The
maximal effects of CsA and NIM811 could be
obtained.
Effect of HSS on CCCP-induced apoptosis
Caspase activation is a key step in DNA damage-

induced apoptosis. To further understand the protec-
tive effect of HSS against CCCP-induced apoptosis,
the activation of caspase-3 was examined using the
enzymatic Caspase-Glo 3 ⁄ 7 assay. After CCCP treat-
ment, caspase-3 activity increased markedly in vector-
transfected cells, and this increase was inhibited in
HSS-expressing cells (P < 0.05; Fig. 6), but not in
cells expressing mutant HSS. Similarly, CsA showed a
potent inhibitory effect on cell apoptosis by decreasing
the activity of caspase-3 in the three cell lines.
Effect of HSS knockdown on CCCP-induced
apoptosis
To further elucidate the functional role of HSS in
CCCP-induced apoptosis, the expression of HSS was
inhibited at the post-transcriptional level by using a
gene silencing strategy [small interfering RNAs
(siRNAs)]. As demonstrated by western blot, the HSS
level in cells transfected with the HSS-specific siRNA
was much lower than that in cells transfected with a
scrambled siRNA (data not shown). Using these trans-
fected cells, we investigated the intracellular ATP level,
caspase-3 activity, and cytochrome c level. As shown
Fig. 3. Effect of CCCP on MPT. (A, B) Equal cell numbers from dif-
ferent cultures were treated with CCCP (30 l
M) for 1 h. The mito-
chondria were then isolated. HSS, CsA and its analog NIM811
were added to the mitochondria, and their effects on MPT were
analyzed. In (A) and (B), MPT was increased in a dose-dependent
pattern; **P < 0.05 versus treatment with CCCP. (C) Dw
m

follow-
ing treatment with CCCP and CsA. Dw
m
in HSS-expressing cells
was less severe than in vector-transfected cells; **P < 0.05 as
compared with vector-transfected cells. ctrl, control.
Y. Wu et al. HSS and mitochondrial permeability transition
FEBS Journal 277 (2010) 1297–1309 ª 2010 The Authors Journal compilation ª 2010 FEBS 1301
in Fig. 7A, following treatment with 15 lm CCCP, the
intracellular ATP level in HSS siRNA-transfected cells
was significantly reduced as compared with that in
scrambled siRNA-transfected cells (about 40% reduc-
tion). In addition, caspase-3 activity was greatly
increased in HSS siRNA-transfected cells (Fig. 7B),
suggesting that knockdown of HSS expression
increases cellular susceptibility to CCCP damage. As a
result, the cytochrome c content markedly declined in
the mitochondrial compartment in CCCP-treated cells
transfected with the HSS siRNA as compared with
controls (Fig. 7C). This indicates that there was an
impairment of the permeabilization transition pore
(PTP) of the mitochondrial membrane and that HSS
expression is a critical factor that protects cells from
CCCP-induced apoptosis.
Effect of recombinant HSS (rHSS) on CCCP-
induced cytochrome c release
Having demonstrated that HSS expression can inhibit
apoptosis, we next aimed to determine whether the
Fig. 4. Western blot of mitochondrial cyto-
chrome c (Cyt c). Mitochondria were iso-

lated from wild-type cells, vector-transfected
cells, HSS-expressing cells, and mutant
HSS-expressing cells, and analyzed using
western blot with an antibody against cyto-
chrome c. All blots were blotted with an
antibody against COX IV to control for equal
loading. *P < 0.001 and **P < 0.05, respec-
tively, as compared with vector-transfected
cells (A, B); **P < 0.05 as compared with
mutant HSS-expressing cells (C, D). ctrl,
control.
Fig. 5. Intracellular ATP level. (A) HSS-transfected or vector-trans-
fected cells were treated with CCCP and CsA, and the intracellular
ATP level was measured. **P < 0.05 as compared with the ATP
level in vector-transfected cells. (B) Effect of CsA and its analog
NIM811 on ATP in HSS-transfected cells. **P < 0.05 versus CCCP
treatment. ctrl, control.
Fig. 6. Caspase-3 activity. Following treatment with CCCP and
CsA, caspase-3 activities were analyzed in wild-type cells, vector-
transfected cells, and HSS-expressing cells. **P < 0.05 as com-
pared with vector-transfected cells and mutant HSS-expressing
cells. ctrl, control.
HSS and mitochondrial permeability transition Y. Wu et al.
1302 FEBS Journal 277 (2010) 1297–1309 ª 2010 The Authors Journal compilation ª 2010 FEBS
addition of rHSS to isolated mitochondria would pre-
vent impairment of the PTP. rHSS and mutant rHSS
(Cys62 fi Ser, Cys65 fi Ser) were expressed in and
purified from prokaryotic cells. The resulting protein
had a molecular mass of 15 kDa, as determined using
SDS ⁄ PAGE (data not shown). As shown in Fig. 8,

incubation of isolated mitochondria with CCCP
(300 lm at 4 °C) for 4 h resulted in substantial cyto-
chrome c release as compared with the control
(lane 2). The administration of rHSS reduced cyto-
chrome c release (Fig. 8, lane 4), suggesting that HSS
protects the mitochondria from CCCP-induced injury.
However, the mutant protein and a mock protein were
both incapable of protecting the mitochondria (Fig. 8,
lanes 3 and 5, respectively). These results suggest a
possible role for HSS in the regulation of MPT. This
protective role in mitochondria may be dependent
upon the intact form of HSS, and if the CXXC motif
at the C-terminus, which is essential for its enzymatic
activity [34], is mutated, then HSS loses its protective
effect (Fig. 8, lane 5).
Discussion
Mitochondria are the energy producers of the cell, and
are essential for the maintenance of cell life. However,
in the last 10 years, it has also become apparent that
mitochondria are the control centers for cell death. In
healthy cells, the mitochondrial inner membrane (IM),
which is the boundary between the intermembrane ⁄
intercristae space and the matrix, is nearly imperme-
Fig. 7. Silencing HSS expression accelerates CCCP-induced apop-
tosis. Cells were transfected with an HSS-specific siRNA or a
scrambled siRNA as a control. Forty-eight hours post-transfection,
the cells were treated with 15 l
M CCCP for 24 h and harvested for
determination of the intracellular ATP level, the caspase-3 activity,
and the cytochrome c (Cyt c) level. (A) Intracellular ATP level. The

ATP level was measured as described in Experimental procedures.
The data are presented as the mean value from three independent
experiments; **P < 0.05 as compared with control siRNA-trans-
fected cells. (B) Caspase-3 activity was determined as described in
Experimental procedures. The data are presented as the mean of
triplicate determinations from three independent experiments;
**P < 0.05 as compared with scrambled siRNA-transfected cells.
(C) The cytochrome c level in the mitochondrial pellet was mea-
sured using western blot. The blots were reprobed for the mito-
chondrial marker COX IV to confirm equal protein loading. ctrl,
control.
Fig. 8. rHSS inhibits CCCP-induced cytochrome c (Cyt c) release
from isolated mitochondria. The cytochrome c and COX IV levels
were analyzed using western blot. The control is untreated mito-
chondria. **P < 0.05 as compared with the other panels.
Y. Wu et al. HSS and mitochondrial permeability transition
FEBS Journal 277 (2010) 1297–1309 ª 2010 The Authors Journal compilation ª 2010 FEBS 1303
able to all ions, including protons. The charge imbal-
ance that results from the generation of an electro-
chemical gradient across the IM forms the basis of the
IM w
m
. During cell death, MP often increases, allow-
ing for the release of soluble proteins. The only
mechanism underlying mitochondrial membrane per-
meabilization that has been described to date is MPT,
which is generally studied in isolated mitochondria and
compromises the normal integrity of the mitochondrial
IM. This results in the IM becoming freely permeable
to protons, leading to the uncoupling of oxidative

phosphorylation. MPT is caused by the opening of the
nonselective, highly conductive PTP in the mitochon-
drial IM [39]. The exact molecular composition of the
PTP remains unclear. When MPT occurs, MP
collapses, leading to the failure of oxidative phosphor-
ylation and necrotic cell death [41–43]. In addition,
MPT causes large-amplitude swelling, outer membrane
rupture, and release of cytochrome c from the inter-
membrane space, triggering activation of caspases and
apoptosis [41,44].
Apoptosis is a genetically predetermined mechanism
that may be activated by several molecular pathways.
The best characterized and the most prominent path-
ways are the extrinsic and intrinsic pathways. In the
intrinsic pathway (also known as the ‘mitochondrial
pathway’), apoptosis results from an intracellular cas-
cade of events in which mitochondrial permeabilization
plays a crucial role. During apoptosis, MPT generally
precedes apoptotic cell death, both in vitro and in vivo
[41]. The release of cytochrome c as a consequence of
MPT is one of the key events in mitochondria-depen-
dent apoptosis [45]. Of the released mitochondrial pro-
teins, cytochrome c is considered to be the most
important, because it can trigger a critical step in the
activation of mitochondria-dependent apoptosis [12],
the assembly of the apoptosome. Upon formation of
this complex, caspase-9 acquires the ability to trigger
the processing and activation of the downstream cas-
pase cascade, which ultimately culminates in apoptotic
cell death.

CCCP is a protonophore that renders the mito-
chondrial IM permeable to protons and causes dissi-
pation of the proton gradient across the IM. CCCP
also uncouples the transfer of electrons through the
electron transfer chain from ATP production. CCCP-
induced apoptosis has been reported in many cell
lines, such as Jurkatneo, FL5.12, HL-60, and ST486
[46–49].
To test the hypothesis that HSS overexpression pro-
tects cells from apoptosis, the present in vitro study
used CCCP to explore the influence of mitochondrial
uncoupling on hepatocytes. The mitochondria of
hepatocytes became depolarized 24 h after exposure to
CCCP. Uncoupling may further lead to an impairment
in mitochondrial ATP formation and the hydrolysis of
ATP by the uncoupler-stimulated ATPase [50]. There-
fore, as seen in Fig. 5, ATP levels may drop substan-
tially after CCCP treatment.
The results of the current experiments provide evi-
dence that mitochondrial uncoupling in hepatocytes
leads to PTP opening and cell swelling, an event that
is probably reduced in extent by CsA. CsA specifically
inhibits PTP opening by binding to cyclophilin D in
the matrix and on the inner surface of the IM [51–54].
Growing evidence has implicated MPT in the necrotic
and apoptotic death of hepatocytes [42,43,55]. In a
previous report, HSS was considered to be an impor-
tant intracellular survival factor for hepatocytes [24];
however, the mechanism by which HSS protects
hepatocytes remains unclear. It has recently been dem-

onstrated that HSS is a novel component of the mito-
chondrial intermembrane space that is specifically
required for maturation of Fe–S-binding proteins [23].
Subsequently, we found that the overexpression of
HSS protects hepatic cells from H
2
O
2
-mediated injury
[12]. Therefore, in this study, we investigated whether
HSS could function as an MPT inhibitor, thereby alle-
viating hepatic injury and promoting the survival of
hepatocytes, after transfection of HSS into the cells or
the administration of HSS to isolated mitochondria
in vitro.
HSS exerts a potent hepatocyte protective effect
by a hitherto unknown mechanism [56,57]. As a fol-
low-up to our initial report [12], in this article we
demonstrate that HSS represses the onset of MPT,
substantially decreasing mitochondrial depolari-
zation (Fig. 3), alleviating cellular ATP level (Fig. 5),
and therefore enhancing cell survival (Fig. 2). Fur-
thermore, HSS-deficient hepatocytes were sensitive to
CCCP-mediated damage (Figs 2–6). A similar phe-
nomenon was observed with H
2
O
2
-mediated
injury (data not shown), indicating that endogenous

HSS has an important role in the protection of
hepatocytes from apoptotic death resulting from
MPT.
Our results suggest that MPT probably plays a
critical role in the damage induced by CCCP. HSS
is able to protect the hepatocytes, probably by inhib-
iting MPT resulting from the mitochondrial PTP.
However, more precise investigations of the protein–
protein interactions of HSS within the mitochondria
will be required to elucidate the molecular mecha-
nism underlying HSS-mediated liver protection and
to identify candidate HSS-binding molecules. Never-
theless, in this study, we provide the first evidence
HSS and mitochondrial permeability transition Y. Wu et al.
1304 FEBS Journal 277 (2010) 1297–1309 ª 2010 The Authors Journal compilation ª 2010 FEBS
that HSS is equivalent to CsA in inhibiting the onset
of MPT.
Experimental procedures
Reagents
DMEM and TRIzol were purchased from Gibco BRL
(Paisley, UK), and fetal bovine serum was purchased
from Hyclone (Victoria, Australia). Both Lipofecta-
mine 2000 and the SuperScript III First-Strand Synthesis
System were purchased from Invitrogen (Carlsbad, CA,
USA). The gentamicin analog G418 was purchased from
Gibco BRL. The power SYBR Green PCR Master Mix
was purchased from Applied Biosystems (Warrington,
UK). The CellTiter-Glo Luminescent Cell Viability Kit
and the Caspase-Glo 3 ⁄ 7 Assay were purchased from
Promega (Madison, WI, USA). Fluorescein isothiocya-

nate-conjugated annexin V was purchased from Biosea
(Beijing, China). The Mitochondria ⁄ Cytosol Isolation Kit
was purchased from Applygen Technologies (Beijing,
China). The siRNA and nontargeting control (scrambled)
siRNA were purchased from Dharmacon RNA Technolo-
gies (Shanghai, China). The QuikChange Site-Directed
Mutagenesis Kit was purchased from Stratagene (La
Jolla, CA, USA). The His-tag vector pET-15b and the
Escherichia coli strain Origami (DE3) were purchased
from Novagen (Darmstadt, Germany). The His Gravi-
Trap Kit was purchased from Phamarcia (Little Chalfont,
UK). The bicinchoninic acid kit was purchased from
Pierce (Rockford, IL, USA). The antibody against HSS,
the antibody against cytochrome c and the enhanced
chemiluminescence kit were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). The horseradish
peroxidase-conjugated goat anti-(mouse IgG) was pur-
chased from Cell Signaling Technology (Beverly, MA,
USA). Bisbenzimide Hoechst 33342, CCCP, CsA,
5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-tetraethylbenzimidazolocarbo-
cyanine iodide (JC-1), dimethylsulfoxide and other chemi-
cal reagents were all purchased from Sigma Aldrich (St
Louis, MO, USA). The CsA analog NIM811 was kindly
provided by Novartis (Basel, Switzerland).
Cell culture and plasmid DNA transfection
BEL-7402 hepatoma cells were cultured at 37 °C in DMEM
supplemented with 10% fetal bovine serum, 100 UÆmL
)1
penicillin and 100 lgÆmL
)1

streptomycin in a 5% CO
2
humidified atmosphere incubator. A total of 2 · 10
6
BEL-7402 cells were seeded and allowed to grow to 50–
70% confluence. The cells were transfected with 5 lgof
either HSS–pcDNA 3.0 or pcDNA 3.0 vector with Lipofec-
tamine 2000, following the manufacturer’s recommenda-
tions. Eight hours post-transfection, the cells were selected
using G418 (400 lgÆmL
)1
) for 14 days. The cells resistant
to G418 were used for further study.
RNA extraction and real-time PCR
Total RNA from HSS-expressing cells, vector-transfected
cells and wild-type cells was extracted using the QIAamp
RNA Purification Kit. The extracted RNA was reverse-
transcribed into cDNA, using the SuperScript III First-
Strand Synthesis System. cDNA was synthesized from 3 l g
of total RNA in 20 lL of reaction mixture. Real-time PCR
was performed using the Power SYBR Green Master Mix,
as recommended by the manufacturer. The HSS gene was
amplified using the ABI Prism 7300 Sequence Detection
System (Applied Biosystems, Foster City, CA, USA) with
specific primers. The 18S rRNA was amplified as an inter-
nal standard. Primers were designed using the primer design
software primer express (Applied Biosystems).
Microscopic observation of cellular morphology
The cells were plated in 24-well plates at 10
5

cellsÆmL
)1
.
Sixteen hours after plating, the cells were treated with either
50 lm CCCP or 50 lm CCCP and 10 lm CsA for 24 h. After
24 h, 1.5 lLof10mgÆmL
)1
Hoechst 33342, a DNA-specific
fluorescent dye, was added to each well, and the plates were
incubated for 10 min at 37 °C. The stained cells were then
observed using a Leica DMILH fluorescence microscope.
Flow cytometric analysis
Cells were seeded in 100 mm culture dishes. After attach-
ment, the cells were incubated with either 50 lm CCCP or
50 lm CCCP and 10 lm CsA for 24 h. After being washed
twice with NaCl ⁄ P
i
, the cells were resuspended in binding
buffer [10 mm Hepes ⁄ NaOH (pH 7.4), 140 mm NaCl,
2.5 mm CaCl
2
]. Fluorescein isothiocyanate-conjugated ann-
exin V was added to a final concentration of 1 mgÆmL
)1
.
The mixture was incubated for 10 min in the dark at room
temperature. The cells were then resuspended in propidium
iodide solution, and incubated again in the dark for
another 30 min at room temperature. The stained cells were
analyzed using a FACScan flow cytometer (Becton Dickin-

son, Franklin Lakes, NJ, USA). The data were analyzed
using cellquest software (Becton Dickinson).
Isolation of mitochondria
The isolation of mitochondria was performed according to
the instructions for the Mitochondria ⁄ Cytosol Isolation Kit
for Cultured Cells. The cells were harvested and homoge-
nized in 1.5 mL of ice-cold Mito-Cyto Buffer with a
Dounce homogenizer. After centrifugation twice at 800 g
for 5 min at 4 °C, the supernatant was collected, trans-
Y. Wu et al. HSS and mitochondrial permeability transition
FEBS Journal 277 (2010) 1297–1309 ª 2010 The Authors Journal compilation ª 2010 FEBS 1305
ferred to a fresh microcentrifuge tube, and centrifuged at
12 000 g for 10 min at 4 °C. The pellet, which contained
the mitochondria, was resuspended in 30 lL of Mito-Cyto
Buffer. The protein concentration was determined using the
bicinchoninic acid method [58], with BSA as a standard.
The isolated mitochondria were stored on ice prior to the
experiments, and all experiments were performed up to
1–5 h after preparation.
Measurement of w
m
in isolated mitochondria
JC-1 is a mitochondrion-specific dye that can be used to
determine w
m
. Mitochondria with high w
m
will form JC-1
aggregates and fluoresce red ( 590 nm); consequently,
mitochondrial depolarization is indicated by a decrease in

the red fluorescence intensity [59]. The cells were grown to
80–90% confluence. After pretreatment with a gradient of
either CsA (0.1, 1.0, 10 or 15 lm ) or its analog NIM811
(0.1, 1.0, 10 or 15 l m) for 15 min, the cells were incubated
with CCCP (30 lm) for 1 h, and the mitochondria were
then isolated as mentioned above.The w
m
was measured
after JC-1 staining, mainly as described by van der Toorn
M et al. [60]. The w
m
was obtained with 485 nm excitation,
using a 590 nm bandpass filter in SPECTRA max M2
(Molecular Devices, Sunnyvale, CA, USA).
Determination of the intracellular ATP level
The intracellular ATP level was measured using the Cell-
Titer-Glo Luminescent Cell Viability Assay Kit. The HSS-
expressing cells were plated in 96-well plates at
2.5 · 10
4
cells per well. The cells were treated with CCCP
(30 lm) for 1 h, and subsequently lysed with 100 lL of lysis
buffer. The ATP concentration was immediately measured
using a Glomax 96 Microplate Luminometer (Promega).
Caspase-3
⁄
7 activity
Caspase activity was detected by using the Caspase-Glo 3 ⁄ 7
Assay Kit. Briefly, the cells were seeded in a 96-well plate
and incubated for 24 h at 37 °C. The cells were treated with

either 50 lm CCCP or 50 lm CCCP and 100 lm CsA for
24 h. The caspase-3 ⁄ 7 reagent (100 lL) was then added to
each well, and the plate was incubated on a rotary shaker
for 30 min at room temperature. Luminescence was
recorded for each well. The caspase-3 ⁄ 7 activity is presented
as the mean of results from three experiments.
Small interfering RNA-mediated gene silencing
BEL-7402 cells were transfected with an HSS-specific siRNA
or nontargeting control (scrambled) siRNA, according to
standard protocols. Briefly, confluent BEL-7402 cells were
replated in six-well plates (3 · 10
5
cells per well) and grown
in 10% fetal bovine serum ⁄ DMEM without antibiotics for
24 h to 70–80% confluence. To prepare the transfection com-
plex, DharmaFECT-4 transfection reagent (4 lL per well)
was incubated with the HSS-specific siRNA or the scrambled
siRNA in antibiotic-free and serum-free medium for 30 min
at room temperature. The cells were then incubated with the
siRNA–DharmaFECT-4 complexes for 24 h at 37 °C. For
recovery, the cells were cultured in 10% fetal bovine
serum ⁄ DMEM (antibiotic-free) for another 24 h. Before the
CCCP treatment, BEL-7402 cells were serum-deprived over-
night in antibiotic-free 0.1% fetal bovine serum ⁄ DMEM,
and the cells were then treated with 15 lm CCCP or dimeth-
ylsulfoxide for 24 h and harvested to determine the ATP
content, caspase-3 activity, and the cytochrome c level as
described above.
Preparation of recombinant protein
The HSS cDNA (375 bp) was amplified by PCR. The

Cys62 fi Ser and Cys65 fi Ser mutants of HSS were con-
structed using the QuikChange Site-Directed Mutagenesis
Kit. All constructs were verified by DNA sequencing. The
NdeI and BamHI restriction sites were used for cloning
the PCR fragments into the His-tag vector, pET-15b. The
recombinant proteins were generated in the E. coli strain
Origami (DE3), and the N-terminal His-tagged proteins
were purified using a His GraviTrap kit according to the
manufacturer’s protocols. Purification to homogeneity was
verified using SDS ⁄ PAGE gels and by antibody tests. The
pure proteins were desalted, concentrated, and stored at –
80 °C until further use. The concentration of the protein
was estimated using the bicinchoninic acid assay.
Cytochrome c release in CCCP-treated
mitochondria
Mitochondria were isolated from BEL-7402 cells by differ-
ential centrifugation as described above. To determine the
effect of rHSS protein on the release of cytochrome c, mito-
chondria (50 lg) were incubated with protein (rHSS,
mutant rHSS, or mock protein; 100 lgÆmL
)1
each) in
25 lL of buffer for 1 h at 4 °C. CCCP (300 lm) was then
added to the mitochondria, and the CCCP-treated mito-
chondria were incubated at 4 °C for 4 h. The mitochondrial
suspension was then centrifuged at 12 000 g for 5 min, and
the resulting supernatants were analyzed for the release of
cytochrome c with western blot. The mitochondrial pellets
were probed with the cytochrome c oxidase subunit IV
(COX IV) antibody to normalize for loading.

Statistical analysis
All values are expressed as mean ± standard deviation.
Statistical significance was determined using a one-way
HSS and mitochondrial permeability transition Y. Wu et al.
1306 FEBS Journal 277 (2010) 1297–1309 ª 2010 The Authors Journal compilation ª 2010 FEBS
ANOVA. A P-value < 0.05 was considered to be
significant.
Acknowledgement
This work was supported by grant from the National
‘863’ Project of the Ministry of Science and Technol-
ogy China (2006AA02A410).
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