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

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],

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

, 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

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

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).

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

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,

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

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-

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-

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

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

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

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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