Hepatic stimulator substance mitigates hepatic cell injury through suppression of the mitochondrial permeability transition Yuan Wu1,*, Jing Zhang1,*, Lingyue Dong1, Wen Li1, Jidong Jia2 and Wei An1 Department of Cell Biology and Municipal Key Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, Beijing, China Liver Unit, Beijing Friendship Hospital, Capital Medical University, Beijing, China 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: anwei@ccmu.edu.cn *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 possible 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 inhibitor of MPT Therefore, we propose that the protective effect of HSS may be associated with blockade of MPT 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 promote hepatocyte proliferation and liver regeneration after partial hepatectomy [1–3] The HSS-mediated 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; wm, inner transmembrane potential FEBS Journal 277 (2010) 1297–1309 ª 2010 The Authors Journal compilation ª 2010 FEBS 1297 HSS and mitochondrial permeability transition Y Wu et al 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 carcinoma and cholangiocellular carcinoma [5] This increased expression of HSS in liver tumors may due to its ability to stimulate DNA synthesis [6–8] In addition to its ability to promote liver regeneration, HSS has been shown to protect the liver from acute injury caused by several compounds, including CCl4 [9], d-galactosamine [10], ethanol [11], H2O2 [12], and cadmium [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 mitogenic effects of other growth factors, such as epidermal 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 transforming growth factor-a, ALR alone showed little effect on hepatocyte proliferation in vitro, suggesting that hepatocytes might not contain surface receptors specific 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 molecules with regard to their cDNA and protein sequences, there are a few disagreements For example, 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] 1298 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 critical role in the regulation of cell death [33–35], and mitochondrial permeability transition (MPT) is considered to be the pivotal event of mitochondria-mediated cell death [36] MPT leads to loss of the inner transmembrane potential (wm) [37], reduction of the intracellular 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 identified the mitochondrial location of HSS After a mitochondrial lesion specifically caused by carbonyl cyanide m-chlorophenylhydrazone (CCCP), a classic protonophore-type uncoupling agent [38], we analyzed Dwm, 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, cyclosporin 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 protection is strongly associated with the mitochondrial membrane pore Knockdown of HSS expression by RNA interference destroyed MP, leading to a great FEBS Journal 277 (2010) 1297–1309 ª 2010 The Authors Journal compilation ª 2010 FEBS Y Wu et al 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 significantly 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 HSS and mitochondrial permeability transition 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 condensation and fragmentation being observable using fluorescence microscopy As detected using Hoechst 33342 staining, vector-transfected cells and cells expressing mutant HSS underwent typical apoptotic changes [39], including shrinkage, membrane blebbing, chromatin condensation, and the formation of apoptotic bodies, after being treated with CCCP However, 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%, respectively) However, the proportion of apoptotic cells following 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 wm Fig (A) The level of HSS RNA in the three types of cells was quantified by real-time RT-PCR, and is expressed as genomic equivalents 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 differential 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 Alteration of wm 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 CCCPinduced 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- FEBS Journal 277 (2010) 1297–1309 ª 2010 The Authors Journal compilation ª 2010 FEBS 1299 HSS and mitochondrial permeability transition Y Wu et al Fig 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 experiments dent increase in wm At the maximal dose of HSS (10 lgỈmL)1), however, addition of CsA to the mitochondria did not increase wm 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 protection 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 mitochondria 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 1300 cells CsA could rescue MP effectively in HSSexpressing 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 cytochrome c was inhibited Exposure of cells to CCCP resulted in substantial loss of wm, and, as a consequence, the mitochondrial membranes were easily damaged and there was massive leakage of cytochrome 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 FEBS Journal 277 (2010) 1297–1309 ª 2010 The Authors Journal compilation ª 2010 FEBS Y Wu et al HSS and mitochondrial permeability transition HSS-expressing cells (Fig 4A, lanes and 7) A densitometric 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-transfected 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 HSSexpressing cells was about 45% higher than that in vector-transfected cells, suggesting that HSS-expressing cells were less affected by the impaired energy production After addition of CsA, the ATP level in HSSexpressing 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 HSSexpressing cells, with a dose-dependent pattern The maximal effects of CsA and NIM811 could be obtained Effect of HSS on CCCP-induced apoptosis Fig Effect of CCCP on MPT (A, B) Equal cell numbers from different cultures were treated with CCCP (30 lM) for h The mitochondria 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) Dwm following treatment with CCCP and CsA Dwm in HSS-expressing cells was less severe than in vector-transfected cells; **P < 0.05 as compared with vector-transfected cells ctrl, control (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 Caspase activation is a key step in DNA damageinduced apoptosis To further understand the protective effect of HSS against CCCP-induced apoptosis, the activation of caspase-3 was examined using the enzymatic Caspase-Glo ⁄ assay After CCCP treatment, caspase-3 activity increased markedly in vectortransfected 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 transfected cells, we investigated the intracellular ATP level, caspase-3 activity, and cytochrome c level As shown FEBS Journal 277 (2010) 1297–1309 ª 2010 The Authors Journal compilation ª 2010 FEBS 1301 HSS and mitochondrial permeability transition Y Wu et al Fig Western blot of mitochondrial cytochrome c (Cyt c) Mitochondria were isolated from wild-type cells, vector-transfected cells, HSS-expressing cells, and mutant HSS-expressing cells, and analyzed using western blot with an antibody against cytochrome c All blots were blotted with an antibody against COX IV to control for equal loading *P < 0.001 and **P < 0.05, respectively, as compared with vector-transfected cells (A, B); **P < 0.05 as compared with mutant HSS-expressing cells (C, D) ctrl, control Fig Caspase-3 activity Following treatment with CCCP and CsA, caspase-3 activities were analyzed in wild-type cells, vectortransfected cells, and HSS-expressing cells **P < 0.05 as compared with vector-transfected cells and mutant HSS-expressing cells ctrl, control Fig Intracellular ATP level (A) HSS-transfected or vector-transfected 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 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% reduction) In addition, caspase-3 activity was greatly increased in HSS siRNA-transfected cells (Fig 7B), 1302 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 CCCPinduced cytochrome c release Having demonstrated that HSS expression can inhibit apoptosis, we next aimed to determine whether the FEBS Journal 277 (2010) 1297–1309 ª 2010 The Authors Journal compilation ª 2010 FEBS Y Wu et al HSS and mitochondrial permeability transition Fig 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 mitochondria **P < 0.05 as compared with the other panels Fig Silencing HSS expression accelerates CCCP-induced apoptosis 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 lM 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-transfected 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 measured using western blot The blots were reprobed for the mitochondrial marker COX IV to confirm equal protein loading ctrl, control addition of rHSS to isolated mitochondria would prevent 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 °C) for h resulted in substantial cytochrome c release as compared with the control (lane 2) The administration of rHSS reduced cytochrome 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 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- FEBS Journal 277 (2010) 1297–1309 ª 2010 The Authors Journal compilation ª 2010 FEBS 1303 HSS and mitochondrial permeability transition Y Wu et al able to all ions, including protons The charge imbalance that results from the generation of an electrochemical gradient across the IM forms the basis of the IM wm During cell death, MP often increases, allowing for the release of soluble proteins The only mechanism underlying mitochondrial membrane permeabilization 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 mitochondrial IM [39] The exact molecular composition of the PTP remains unclear When MPT occurs, MP collapses, leading to the failure of oxidative phosphorylation and necrotic cell death [41–43] In addition, MPT causes large-amplitude swelling, outer membrane rupture, and release of cytochrome c from the intermembrane 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 pathways are the extrinsic and intrinsic pathways In the intrinsic pathway (also known as the ‘mitochondrial pathway’), apoptosis results from an intracellular cascade 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-dependent apoptosis [45] Of the released mitochondrial proteins, 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 caspase cascade, which ultimately culminates in apoptotic cell death CCCP is a protonophore that renders the mitochondrial IM permeable to protons and causes dissipation of the proton gradient across the IM CCCP also uncouples the transfer of electrons through the electron transfer chain from ATP production CCCPinduced 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 protects cells from apoptosis, the present in vitro study used CCCP to explore the influence of mitochondrial uncoupling on hepatocytes The mitochondria of 1304 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] Therefore, as seen in Fig 5, ATP levels may drop substantially after CCCP treatment The results of the current experiments provide evidence 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 important intracellular survival factor for hepatocytes [24]; however, the mechanism by which HSS protects hepatocytes remains unclear It has recently been demonstrated that HSS is a novel component of the mitochondrial 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 H2O2-mediated injury [12] Therefore, in this study, we investigated whether HSS could function as an MPT inhibitor, thereby alleviating 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 follow-up to our initial report [12], in this article we demonstrate that HSS represses the onset of MPT, substantially decreasing mitochondrial depolarization (Fig 3), alleviating cellular ATP level (Fig 5), and therefore enhancing cell survival (Fig 2) Furthermore, HSS-deficient hepatocytes were sensitive to CCCP-mediated damage (Figs 2–6) A similar phenomenon was observed with H2O2-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 inhibiting 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 mechanism underlying HSS-mediated liver protection and to identify candidate HSS-binding molecules Nevertheless, in this study, we provide the first evidence FEBS Journal 277 (2010) 1297–1309 ª 2010 The Authors Journal compilation ª 2010 FEBS Y Wu et al HSS and mitochondrial permeability transition that HSS is equivalent to CsA in inhibiting the onset of MPT using G418 (400 lgỈmL)1) for 14 days The cells resistant to G418 were used for further study Experimental procedures RNA extraction and real-time PCR Reagents DMEM and TRIzol were purchased from Gibco BRL (Paisley, UK), and fetal bovine serum was purchased from Hyclone (Victoria, Australia) Both Lipofectamine 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 ⁄ Assay were purchased from Promega (Madison, WI, USA) Fluorescein isothiocyanate-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 Technologies (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 GraviTrap 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 purchased from Cell Signaling Technology (Beverly, MA, USA) Bisbenzimide Hoechst 33342, CCCP, CsA, 5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-tetraethylbenzimidazolocarbocyanine iodide (JC-1), dimethylsulfoxide and other chemical 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 mL)1 penicillin and 100 lgỈmL)1 streptomycin in a 5% CO2 humidified atmosphere incubator A total of · 106 BEL-7402 cells were seeded and allowed to grow to 50– 70% confluence The cells were transfected with lg of either HSS–pcDNA 3.0 or pcDNA 3.0 vector with Lipofectamine 2000, following the manufacturer’s recommendations Eight hours post-transfection, the cells were selected 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 reversetranscribed into cDNA, using the SuperScript III FirstStrand Synthesis System cDNA was synthesized from lg 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 internal 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 105 cellsỈmL)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 lL of 10 mgỈmL)1 Hoechst 33342, a DNA-specific fluorescent dye, was added to each well, and the plates were incubated for 10 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 attachment, 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 ⁄ Pi, the cells were resuspended in binding buffer [10 mm Hepes ⁄ NaOH (pH 7.4), 140 mm NaCl, 2.5 mm CaCl2] Fluorescein isothiocyanate-conjugated annexin V was added to a final concentration of mgỈmL)1 The mixture was incubated for 10 in the dark at room temperature The cells were then resuspended in propidium iodide solution, and incubated again in the dark for another 30 at room temperature The stained cells were analyzed using a FACScan flow cytometer (Becton Dickinson, 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 homogenized in 1.5 mL of ice-cold Mito-Cyto Buffer with a Dounce homogenizer After centrifugation twice at 800 g for at °C, the supernatant was collected, trans- FEBS Journal 277 (2010) 1297–1309 ª 2010 The Authors Journal compilation ª 2010 FEBS 1305 HSS and mitochondrial permeability transition Y Wu et al ferred to a fresh microcentrifuge tube, and centrifuged at 12 000 g for 10 at °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 wm in isolated mitochondria JC-1 is a mitochondrion-specific dye that can be used to determine wm Mitochondria with high wm 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 lm) for 15 min, the cells were incubated with CCCP (30 lm) for h, and the mitochondria were then isolated as mentioned above.The wm was measured after JC-1 staining, mainly as described by van der Toorn M et al [60] The wm 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 CellTiter-Glo Luminescent Cell Viability Assay Kit The HSSexpressing cells were plated in 96-well plates at 2.5 · 104 cells per well The cells were treated with CCCP (30 lm) for 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 ⁄ activity Caspase activity was detected by using the Caspase-Glo ⁄ 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 ⁄ reagent (100 lL) was then added to each well, and the plate was incubated on a rotary shaker for 30 at room temperature Luminescence was recorded for each well The caspase-3 ⁄ 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 · 105 cells per well) and grown 1306 in 10% fetal bovine serum ⁄ DMEM without antibiotics for 24 h to 70–80% confluence To prepare the transfection complex, 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 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 overnight in antibiotic-free 0.1% fetal bovine serum ⁄ DMEM, and the cells were then treated with 15 lm CCCP or dimethylsulfoxide 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 constructed 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 differential centrifugation as described above To determine the effect of rHSS protein on the release of cytochrome c, mitochondria (50 lg) were incubated with protein (rHSS, mutant rHSS, or mock protein; 100 lgỈmL)1 each) in 25 lL of buffer for h at °C CCCP (300 lm) was then added to the mitochondria, and the CCCP-treated mitochondria were incubated at °C for h The mitochondrial suspension was then centrifuged at 12 000 g for 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 FEBS Journal 277 (2010) 1297–1309 ª 2010 The Authors Journal compilation ª 2010 FEBS Y Wu et al 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 Technology China (2006AA02A410) References LaBrecque DR & Pesch LA (1975) Preparation and partial characterization of hepatic regenerative stimulator substance (SS) from rat liver J Physiol 248, 273– 284 Gandhi CR, Kuddus R, Subbotin VM, Prelich J, Murase N, Rao AS, Nalesnik MA, Watkins SC, DeLeo A, Trucco M et al (1999) A fresh look at augmenter of liver regeneration in rats Hepatology 29, 1435–1445 LaBrecque DR (1982) In vitro stimulation of cell growth by hepatic stimulator substance Am J Physiol 242, G289–G295 Tanigawa K, Sakaida I, Masuhara M, Hagiya M & Okita K (2000) Augmenter of liver regeneration (ALR) may promote liver regeneration by reducing natural killer (NK) cell activity in human liver diseases J Gastroenterol 35, 112–119 Thasler WE, Schlott T, Thelen P, Hellerbrand C, Bataille F, Lichtenauer M, Schlitt HJ, Jauch KW & Weiss TS (2005) Expression of augmenter of liver regeneration (ALR) in human liver cirrhosis and carcinoma Histopathology 47, 57–66 Labrecque DR, Wilson M & Fogerty S (1984) Stimulation of HTC hepatoma cell growth in vitro by hepatic stimulator substance (HSS) Interactions with serum, insulin, glucagon, epidermal growth factor and platelet derived growth factor Exp Cell Res 150, 419–429 Cui CP, Zhang DJ, Shi BX, Du SJ, Wu DL, Wei P, Zhong GS, Guo ZK, Liu Y, Wang LS et al (2008) Isolation and functional identification of a novel human hepatic growth factor: hepatopoietin Cn Hepatology 47, 986–995 Zhang YD, Zhou L, Zhao JF, Peng J, Liu XD, Liu XS & Jia ZM (2006) Expression, purification and bioactivity of human augmenter of liver regeneration World J Gastroenterol 12, 4401–4405 Mei MH, An W, Zhang BH, Shao Q & Gong DZ (1993) Hepatic stimulator substance protects against acute liver-failure induced by carbon-tetrachloride poisoning in mice Hepatology 17, 638–644 10 Ho DW, Fan ST, To J, Woo YH, Zhang Z, Lau C & Wong J (2002) Selective plasma filtration for treatment of fulminant hepatic failure induced by D-galactosamine in a pig model Gut 50, 869–876 HSS and mitochondrial permeability transition 11 Liatsos GD, Mykoniatis MG, Margeli A, Liakos AA & Theocharis SE (2003) Effect of acute ethanol exposure on hepatic stimulator substance (HSS) levels during liver regeneration: protective function of HSS Dig Dis Sci 48, 1929–1938 12 Wu Y, Chen L, Yu H, Liu HJ & An W (2007) Transfection of hepatic stimulator substance gene desensitizes hepatoma cells to H2O2-induced cell apoptosis via preservation of mitochondria Arch Biochem Biophys 464, 48–56 13 Tzirogiannis KN, Panoutsopoulos GI, Demonakou MD, Hereti RI, Alexandropoulou KN & Mykoniatis MG (2004) Effect of hepatic stimulator substance (HSS) on cadmium-induced acute hepatotoxicity in the rat liver Dig Dis Sci 49, 1019–1028 14 Gribilas G, Zarros A, Zira A, Giaginis C, Tsourouflis G, Liapi C, Spiliopoulou C & Theocharis SE (2009) Involvement of hepatic stimulator substance in experimentally induced fibrosis and cirrhosis in the rat Dig Dis Sci 54, 2367–2376 15 Kiso S, Kawata S, Tamura S, Higashiyama S, Ito N, Tsushima H, Taniguchi N & Matsuzawa Y (1995) Role of heparin-binding epidermal growth factor-like growth factor as a hepatotrophic factor in rat liver regeneration after partial hepatectomy Hepatology 22, 1584–1590 16 Francavilla A, Ove P, Polimeno L, Coetzee M, Makowka L, Rose J, Van Thiel DH & Starzl TE (1987) Extraction and partial purification of a hepatic stimulatory substance in rats, mice, and dogs Cancer Res 47, 5600–5605 17 Giorda R, Hagiya M, Seki T, Shimonishi M, Sakai H, Michaelson J, Francavilla A, Starzl TE & Trucco M (1996) Analysis of the structure and expression of the augmenter of liver regeneration (ALR) gene Mol Med 2, 97–108 18 Wang G, Yang XM, Zhang Y, Wang QM, Chen HP, Wei HD, Xing GC, Hu ZY, Zhang CG, Fang DC et al (1999) Identification and characterization of receptor for mammalian hepatopoietin that is homologous to yeast ERV1 J Biol Chem 274, 11469–11472 19 Tury A, Mairet-Coello G, Lisowsky T, Griffond B & Fellmann D (2005) Expression of the sulfhydryl oxidase ALR (Augmenter of Liver Regeneration) in adult rat brain Brain Res 1048, 87–97 20 Polimeno L, Pesetti B, Giorgio F, Moretti B, Resta L, Rossi R, Annoscia E, Patella V, Notarnicola A, Mallamaci R et al (2009) Expression and localization of augmenter of liver regeneration in human muscle tissue Int J Exp Pathol 90, 423–430 21 Hofhaus G, Stein G, Polimeno L, Francavilla A & Lisowsky T (1999) Highly divergent amino termini of the homologous human ALR and yeast scERV1 gene products define species specific differences in cellular localization Eur J Cell Biol 78, 349–356 FEBS Journal 277 (2010) 1297–1309 ª 2010 The Authors Journal compilation ª 2010 FEBS 1307 HSS and mitochondrial permeability transition Y Wu et al 22 Gatzidou E, Kouraklis G & Theocharis S (2006) Insights on augmenter of liver regeneration cloning and function World J Gastroenterol 12, 4951–4958 23 Lange H, Lisowsky T, Gerber J, Muhlenhoff U, Kispal G & Lill R (2001) An essential function of the mitochondrial sulfhydryl oxidase Erv1p ⁄ ALR in the maturation of cytosolic Fe ⁄ S proteins EMBO Rep 2, 715–720 24 Thirunavukkarasu C, Wang LF, Harvey SA, Watkins SC, Chaillet JR, Prelich J, Starzl TE & Gandhi CR (2008) Augmenter of liver regeneration: an important intracellular survival factor for hepatocytes J Hepatol 48, 578–588 25 Lisowsky T, Lee JE, Polimeno L, Francavilla A & Hofhaus G (2001) Mammalian augmenter of liver regeneration protein is a sulfhydryl oxidase Dig Liver Dis 33, 173–180 26 Lisowsky T, Weinstat-Saslow DL, Barton N, Reeders ST & Schneider MC (1995) A new human gene located in the PKD1 region of chromosome-16 is a functional homolog to ERV1 of yeast Genomics 29, 690–697 27 Hagiya M, Francavilla A, Polimeno L, Ihara I, Sakai H, Seki T, Shimonishi M, Porter KA & Starzl TE (1994) Cloning and sequence-analysis of the rat augmentor of liver-regeneration (ALR) gene: expression of biologically-active recombinant ALR and demonstration of tissue distribution Proc Natl Acad Sci USA 91, 8142–8146 28 Yanez RJ, Rodriguez JM, Nogal ML, Yuste L, Enriquez C, Rodriguez JF & Vinuela E (1995) Analysis of the complete nucleotide-sequence of African swine fever virus Virology 208, 249–278 29 Polimeno L, Lisowsky T & Francavilla A (1999) From yeast to man – from mitochondria to liver regeneration: a new essential gene family Ital J Gastroenterol 31, 494–500 30 Polimeno L, Capuano F, Marangi LC, Margiotta M, Lisowsky T, Lerardi E, Francavilla R & Francavilla A (2000) The augmenter of liver regeneration induces mitochondrial gene expression in rat liver and enhances oxidative phosphorylation capacity of liver mitochondria Dig Liver Dis 32, 510–517 31 Thasler WE, Dayoub R, Muhlbauer M, Hellerbrand C, Singer T, Grabe A, Jauch KW, Schlitt HJ & Weiss TS (2006) Repression of cytochrome P450 activity in human hepatocytes in vitro by a novel hepatotrophic factor, augmenter of liver regeneration J Pharmacol Exp Ther 316, 822–829 32 Farrell SR & Thorpe C (2005) Augmenter of liver regeneration: a flavin-dependent sulfhydryl oxidase with cytochrome c reductase activity Biochemistry 44, 1532– 1541 33 Green DR & Reed JC (1998) Mitochondria and apoptosis Science 281, 1309–1312 34 Kroemer G & Reed JC (2000) Mitochondrial control of cell death Nat Med 6, 513–519 1308 35 Joza N, Susin SA, Daugas E, Stanford WL, Cho SK, Li CY, Sasaki T, Elia AJ, Cheng HY, Ravagnan L et al (2001) Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death Nature 410, 549–554 36 Zamzami N & Kroemer G (2001) The mitochondrion in apoptosis: how Pandora’s box opens Nat Rev Mol Cell Biol 2, 67–71 37 Zamzami N, Marchetti P, Castedo M, Zanin C, Vayssiere JL, Petit PX & Kroemer G (1995) Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo J Exp Med 181, 1661–1672 38 Yang JH, Gross RL, Basinger SF & Wu SM (2001) Apoptotic cell death of cultured salamander photoreceptors induced by cccp: CsA-insensitive mitochondrial permeability transition J Cell Sci 114, 1655–1664 39 Martinou JC & Green DR (2001) Breaking the mitochondrial barrier Nat Rev Mol Cell Biol 2, 63–67 40 Kroemer G (2001) Mitochondrial control of apoptosis Bull Acad Natl Med 185, 1135–1143 41 Zamzami N, Susin SA, Marchetti P, Hirsch T, GomezMonterrey I, Castedo M & Kroemer G (1996) Mitochondrial control of nuclear apoptosis J Exp Med 183, 1533–1544 42 Kim JS, He L, Qian T & Lemasters JJ (2003) Role of the mitochondrial permeability transition in apoptotic and necrotic death after ischemia ⁄ reperfusion injury to hepatocytes Curr Mol Med 3, 527–535 43 Kim JS, He L & Lemasters JJ (2003) Mitochondrial permeability transition: a common pathway to necrosis and apoptosis Biochem Biophys Res Commun 304, 463– 470 44 Kantrow SP, Tatro LG & Piantadosi CA (2000) Oxidative stress and adenine nucleotide control of mitochondrial permeability transition Free Radic Biol Med 28, 251–260 45 Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES & Wang X (1997) Cytochrome c and dATP-dependent formation of Apaf1 ⁄ caspase-9 complex initiates an apoptotic protease cascade Cell 91, 479–489 46 O’Brien KA, Muscarella DE & Bloom SE (2001) Differential induction of apoptosis and MAP kinase signaling by mitochondrial toxicants in drug-sensitive compared to drug-resistant B-lineage lymphoid cell lines Toxicol Appl Pharmacol 174, 245–256 47 Mlejnek P (2001) Caspase-3 activity and carbonyl cyanide m-chlorophenylhydrazone-induced apoptosis in HL-60 Altern Lab Anim 29, 243–249 48 Muscarella DE, O’Brien KA, Lemley AT & Bloom SE (2003) Reversal of Bcl-2-mediated resistance of the EW36 human B-cell lymphoma cell line to arsenite- and pesticide-induced apoptosis by PK11195, a ligand of the FEBS Journal 277 (2010) 1297–1309 ª 2010 The Authors Journal compilation ª 2010 FEBS Y Wu et al 49 50 51 52 53 54 mitochondrial benzodiazepine receptor Toxicol Sci 74, 66–73 de Graaf AO, van den Heuvel LP, Dijkman HB, de Abreu RA, Birkenkamp KU, de Witte T, van der Reijden BA, Smeitink JA & Jansen JH (2004) Bcl-2 prevents loss of mitochondria in CCCP-induced apoptosis Exp Cell Res 299, 533–540 Chavin KD, Yang SQ, Lin HZ, Chatham J, Chacko VP, Hoek JB, Walajtys-Rode E, Rashid A, Chen CH, Huang CC et al (1999) Obesity induces expression of uncoupling protein-2 in hepatocytes and promotes liver ATP depletion J Biol Chem 274, 5692–5700 Halestrap AP & Davidson AM (1990) Inhibition of Ca2(+)-induced large amplitude swelling of liver and heart mitochondria by cyclosporine is probably caused by the inhibitor binding to mitochondrial-matrix peptidyl-prolyl cis–trans isomerase and preventing it interacting with the adenine nucleotide translocase Biochem J 268, 153–160 Zoratti M & Szabo I (1995) The mitochondrial permeability transition Biochim Biophys Acta 1241, 139–176 Halloran PF (1996) Molecular mechanisms of new immunosuppressants Clin Transplant 10, 118–123 Fournier N, Ducet G & Crevat A (1987) Action of cyclosporine on mitochondrial calcium fluxes J Bioenerg Biomembr 19, 297–303 HSS and mitochondrial permeability transition 55 Hernandez-Munoz R, Sanchez-Sevilla L, MartinezGomez A & Dent MA (2003) Changes in mitochondrial adenine nucleotides and in permeability transition in two models of rat liver regeneration Hepatology 37, 842–851 56 An W, Liu XJ, Lei TG, Dai J & Du GG (1999) Growth induction of hepatic stimulator substance in hepatocytes through its regulation on EGF receptors Cell Res 9, 37–49 57 Tian ZJ & An W (2004) ERK1 ⁄ contributes negative regulation to STAT3 activity in HSS-transfected HepG2 cells Cell Res 14, 141–147 58 Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ & Klenk DC (1985) Measurement of protein using bicinchoninic acid Anal Biochem 150, 76–85 59 Reers M, Smith TW & Chen LB (1991) J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential Biochemistry 30, 4480–4486 60 van der Toorn M, Kauffman HF, van der Deen M, Slebos DJ, Koeter GH, Gans RO & Bakker SJ (2007) Cyclosporin A-induced oxidative stress is not the consequence of an increase in mitochondrial membrane potential FEBS J 274, 3003–3012 FEBS Journal 277 (2010) 1297–1309 ª 2010 The Authors Journal compilation ª 2010 FEBS 1309 ... generation of an electrochemical gradient across the IM forms the basis of the IM wm During cell death, MP often increases, allowing for the release of soluble proteins The only mechanism underlying mitochondrial. .. 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. .. HSS and mitochondrial permeability transition HSS-expressing cells (Fig 4A, lanes and 7) A densitometric analysis of the cytochrome c content is shown in Fig 4B,D Effect of HSS on the intracellular