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NO signaling regulates apoptosis induced by glycochenodeoxycholate in hepatocytes
Cellular Signalling 23 (2011) 1677–1685
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Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c e l l s i g
iNOS/NO signaling regulates apoptosis induced by glycochenodeoxycholate in hepatocytes Kewei Wang a,⁎, John J. Brems b, Richard L. Gamelli b, Ai-Xuan Holterman a a b
Departments of Pediatrics and Surgery/Section of Pediatric Surgery, Rush University Medical Center, Chicago, IL 60612, United States Department of Surgery, Loyola University Medical Center, Maywood, IL 60153, United States
a r t i c l e
i n f o
Article history: Received 22 April 2011 Accepted 6 June 2011 Available online 13 June 2011 Keywords: iNOS Hepatocyte Apoptosis Cholestatic liver disease
a b s t r a c t Inducible nitric oxide synthase (iNOS) and nitric oxide (NO) can ameliorate apoptosis induced by toxic glycochenodeoxycholate (GCDC) in hepatocytes. However, the underlying molecular mechanisms are not yet understood in detail. This study is to clarify the function of iNOS/NO and its mechanisms during the apoptotic process. The apoptosis was brought about by GCDC in rat primary hepatocytes. iNOS/NO signaling was then investigated. iNOS inhibitor 1400 W enhanced the GCDC-induced apoptosis as reflected by caspase-3 activity and TUNEL assay. Exogenous NO regulated the apoptosis subsequent to NO donor S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP). The GCDC-induced apoptosis was decreased with 0.1 mM SNAP or 0.15 mM SNP, while it was increased with 0.8 mM SNAP or 1.2 mM SNP. The endogenous iNOS inhibited apoptosis, but the exogenous NO played a dual role during the GCDC-induced apoptosis. There was a potential iNOS/Akt/survivin axis that inhibited the hepatocyte apoptosis in low doses of NO donors. In contrast, high doses of NO donors activated CHOP through p38MAP-kinase (p38MAPK), upregulated TRAIL receptor DR5, and suppressed survivin. Consequently the high doses of NO donors promoted the apoptosis in hepatocytes. Our data suggest that the iNOS/NO signaling can modulate Akt/survivin and p38MAPK/CHOP pathways to mediate the GCDC-induced the apoptosis in hepatocytes. These signaling pathways may serve as targets for therapeutic intervention in cholestatic liver disease. © 2011 Elsevier Inc. All rights reserved.
1. Introduction iNOS can synthesize NO from the terminal nitrogen atom of in the presence of NADPH and molecular oxygen [1]. NO as a short-lived free radical plays an important role in the regulation of many pathophysiologic processes [2,3]. Previous studies have demonstrated that the induced NO is liver-protective. iNOS inhibitors significantly increase hepatic damage [4–6]. The upregulation of iNOS/NO can suppress apoptosis via interrupting caspase activation and mitochondrial dysfunction [7–9]. NO is pro-apoptotic as well, despite having an anti-apoptotic function [10]. NO increases TRAIL-induced cytotoxicity by facilitating the mitochondria-mediated caspase signal transduction pathway [11]. The iNOS expression is regulated at multiple stages along the signaling pathway. iNOS/NO signaling affects liver function in different degrees [12,13]. The numerous mechanisms have been
L-arginine
Abbreviations: iNOS, inducible nitric oxide synthase; NO, nitric oxide; GCDC, glycochenodeoxycholate; SNAP, S-nitroso-N-acetyl-penicillamine; SNP, sodium nitroprusside; PI3K, Phosphoinositide 3-kinase; NF-κB, nuclear factor kappaB; p38MAPK, P38 mitogen-activated protein kinases; CHOP, CCAAT/enhancer-binding protein homologous protein; TRAIL, TNF-related apoptosis-inducing ligand. ⁎ Corresponding author at: Rush University Medical Center, 1725 W. Harrison St., Suite 718, Chicago, IL 60612, United States. Tel.: +1 312 942 3358; fax: +1 312 942 5202. E-mail address: [email protected] (K. Wang). 0898-6568/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2011.06.003
associated with the regulation of iNOS expression. The study on iNOS/ NO signaling may find new targets for the treatment of liver injury. GCDC is a predominant toxic bile acid. In cholestatic patients or experimental animal models, the accumulated GCDC is correlated with apoptotic or necrotic cell death in vivo [14,15]. GCDC also causes apoptotic injury in primary hepatocytes in vitro. GCDC induces the apoptosis in hepatocytes via Fas ligand-independent mechanisms by cellular trafficking of the death receptor plasma membrane [16]. Once activated, Fas signals can bring about mitochondrial dysfunction and ultimately activation of caspase-3, which results in cell death [17]. GCDC decreases NF-κB DNA-binding capacity through inhibiting IκBα degradation and thereby iNOS expression is reduced [18]. GCDC can inhibit iNOS expression and NO synthesis in hepatocytes through the inactivation of iNOS promoter as well [19]. In vivo adenoviral iNOS pre-treatment ameliorates rat liver transplant preservation injury and improves survival rate [20]. Functionally, the exogenous or endogenous NO production inhibits the GCDC-induced apoptosis [21,22]. The GCDC-induced hepatocyte apoptosis has already become a classical model through which to investigate apoptosis, bile toxicity, and liver injury [23]. iNOS/NO signaling regulates the GCDC-induced hepatocyte apoptosis, but some detailed links are still unclear. To examine the relationship between iNOS expression and apoptosis, GCDC was chosen as an apoptosis-inducer to trigger apoptotic injury in
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hepatocytes. Through the GCDC-hepatocyte model, iNOS expression was analyzed at multiple levels. The endogenous iNOS might be antiapoptotic and cytoprotective, whereas the exogenous NO was a bifunctional regulator of apoptosis. At low concentrations, the role of NO was similar to the upregulation of the endogenous iNOS. NO activated the expression of survivin to exert an antiapoptotic role in hepatocytes. The iNOS/survivin pathway was affected by the modification of Akt/NF-κB signaling. However, high NO concentrations initiated the activation of p38MAPK/CHOP/DR5 as well as suppression of survivin. High doses of NO donors took a wide range of actions and boosted the hepatocyte apoptosis. The current study aims to evaluate the pathophysiologic role of iNOS/NO during the GCDCinduced apoptosis in hepatocytes. These results refine the molecular mechanism that iNOS/NO regulates apoptosis. 2. Materials and methods
2.7. TUNEL assay The TdT-FragELTM DNA fragmentation detection kit was obtained from Calbiochem (#QIA33). We followed the manufacturer instruction with only minor modifications [8].
2.8. Western blotting assay Protein samples were resolved by 8–10% SDS-polyacrylamide gel, transferred to nitrocellulose membrane, and blotted with appropriate primary antibodies at dilution of 1: 500–1000. Peroxidase-conjugated secondary antibodies were incubated at a dilution of 1: 2000–3000. Bound antibody was visualized through chemiluminescent substrate (ECL; Amersham Biosciences). β–actin was used as equal loading control.
2.1. Hepatocyte isolation and culture 2.9. Quantitative real-time PCR (qRT-PCR) Hepatocytes were isolated from adult Sprague–Dawley rats along standard liver perfusion procedure. Dead cells were removed by Percoll (Sigma) gradient centrifugation. Following the last wash, highly purified hepatocytes (N98% viability) were suspended in cold Williams E medium, diluted to a density of 5.5 × 10 5, plated into collagen-coated dishes (Falcon), and cultured as described previously [8,23,24].
Total RNA was isolated from hepatocytes with ready-to-use TRIZOL Reagent. The first-strand cDNA was synthesized using standard protocol. For qRT-PCR, reactions were amplified with the appropriate primer sets and analyzed in triplicate. The relative level of gene expression was normalized to the expression level of 18S rRNA.
2.2. Reagents and treatment
2.10. Electrophoretic mobility shift assay
The iNOS-specific inhibitor N-(3-(aminomethyl)benzyl)acetamidine (1400 W) (50 μM), the PI3K/Akt inhibitor LY294002 (15 μM), and the p38 MAPK inhibitor SB203580 (15 μM) were purchased from Calbiochem. GCDC, SNAP, and SNP were obtained from Sigma. A concentration of 50 μM GCDC was used unless otherwise indicated. Control groups were treated with PBS buffer.
Nuclear extract was prepared with the modified Dignam protocol [8]. Protein-DNA complexes were separated from the unbound DNA probe by electrophoresis through 6% native polyacrylamide gels containing 0.5 × Tris borate/EDTA.
2.3. iNOS Plasmids The eukaryotic expression vector pcDNA3-iNOS was constructed as previously published [25,26]. The rat iNOS coding sequence was cloned into the expression vector pcDNA3.1. Sequence analysis confirmed that the coding sequence was identical to the database entry. 2.4. Caspase assay 100 μg of cell lysate was utilized to assay the activity of caspase-3. Caspase assay kit was bought from Calbiochem. The reaction system employed the colorimetric substrate IETD-pNA. The activity of caspase-3 was calculated as pmol/min [23]. 2.5. Cell-cycle analysis As previously described [27], hepatocytes were removed using the trypsin/EGTA solution. Nuclei were fixed in ice-cold 70% EtOH, resuspended in PI staining solution, and analyzed by flow cytometry. 2.6. Transfection of siRNA The double stranded siRNA against Survivin was composed of the following oligonucleotides: UGAGCCUGAUUUGGCCCAG and CUGGGCCAAAUCAGGCUCA [28]. The scramble siRNA having no known homology with mammalian genes was used for nonspecific silencing effects. The cells were transfected with 200 nM double-stranded siRNA in lipophilic transfection reagent.
2.11. Statistical analysis All data represent at least three experiments using cells or extracts from a minimum of three separate isolations. Results are expressed as mean ± SD unless otherwise indicated. Comparisons were performed using either Student's t test for independent samples or repeated measures for the ANOVA followed by Bonferroni correction where appropriate. P values b 0·05 were considered significant.
3. Results 3.1. iNOS involves GCDC-induced hepatocyte apoptosis When the hepatocytes were stimulated with different concentrations of GCDC for 4 h, iNOS expression was decreased by the high GCDC dosages (Fig. 1A). At GCDC concentration of 50 μM, iNOS was upregulated at 2 h and then reduced to a low level at 16 h (Fig. 1B). Interestingly, the levels of iNOS were negatively correlated to the caspase-3 activity after GCDC treatment for 4 h (P = 0.021). As the rat hepatocytes were treated with iNOS-specific inhibitor 1400 W, the GCDC-induced apoptosis was reflected with caspase-3 activity and TUNEL assay (Fig. 1C, D). The iNOS inhibitor 1400 W could enhance the GCDC-induced apoptosis. Evidently, endogenous iNOS was related to the apoptotic injury in hepatocytes. After the transfection with iNOS plasmids in the cultured rat hepatocytes for 16 h, GCDC (60 μM) treatment was then started for another 4 h. iNOS plasmids inhibited the GCDC-induced apoptosis as indicated by caspase-3 and TUNEL assay (Fig. 1E, F). Following the application of iNOS inhibitor or the transfection of iNOS plasmids, the iNOS expression significantly affected the profile of GCDC-induced apoptosis in hepatocytes.
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Fig. 1. iNOS/NO involves liver injury. 1A and 1B, iNOS expression was affected in GCDC dose- and time-dependent manners. Values are mean ± SD (n = 6 in each group). Control groups were treated with PBS buffer. **P b 0.01; *P b 0.05 vs. control. Rat hepatocytes were pre-incubated with iNOS inhibitor 1400 W for 1 hour before GCDC treatment. Apoptosis was detected with caspase-3 activity (1C) and TUNEL assay (1D). 1E, After iNOS plasmids (0.25 μg) were transfected into rat hepatocytes for 16 h, 60 μM of GCDC was added for another 4 h. The iNOS plasmids decreased the caspase-3 activity. 1F, The iNOS plasmids inhibited the GCDC-induced apoptosis as shown by TUNEL assay.
3.2. The exogenous NO plays a dual role during the GCDC-induced hepatocyte apoptosis SNAP and SNP were selected as NO donors to test the role of exogenous NO during the GCDC-induced apoptosis in hepatocytes. After SNAP (0–1 mM) or SNP (0–1.2 mM) was co-administered with GCDC (50 μM), the apoptotic response was observed with the caspase-3 activity (Fig. 2A, B) and TUNEL assay (Fig. 2C). The GCDCinduced hepatocyte apoptosis was diminished at low concentrations of SNAP (0.1 mM) or SNP (0.15 mM). However, the apoptosis was increased at high concentrations of SNAP (0.8 mM) or SNP (1.0 mM). Of note, toxic effects of the high NO concentrations appeared not only in hepatocytes, but in CHO cells as well (data not shown). The high NO concentrations significantly increased the release of cytochrome c (Fig. 2D). The exogenous NO exposure could inhibit apoptosis that only occurred in a specific range of NO concentrations. Besides apoptosis, cell proliferation was another aspect used to estimate the role of NO in hepatocytes. The high NO concentrations (SNAP 0.8 mM and SNP 1.0 mM) hindered cell growth in hepatocytes, whereas the low NO concentrations (SNAP 0.1 mM and SNP 0.15 mM) stimulated
cell proliferation (Fig. 2E). Cell-cycle arrest was detected using flow cytometry (Fig. 2F). In the untreated hepatocytes, 42.6% of the cells appeared in G0/G1, 35.8% in S phase, and 21.6% in G2/M phases of the cell cycle. After a 4 h treatment with 0.8 mM SNAP or 1.2 mM SNP, the G2/M population decreased to 8.6% and 7.1% respectively. When the treatment was prolonged for 8 h, the G2/M population was similar to that observed at 4 h, which suggested that these agents could prevent cells from progressing into the G2/M phases. The combination of both the antiapoptosis and cell proliferation might actually reflect the pathophysiologic role of iNOS/NO. The exogenous NO can promote or inhibit apoptosis, which plays a dual role during the GCDC-induced hepatocyte injury. 3.3. iNOS/NO counteracts hepatocyte apoptosis through the upregulation of survivin GCDC at a concentration of 50 μM could activate the endogenous expression of iNOS (Fig. 3A). The amplification of iNOS gene was accompanied by the upregulation of survivin expression (Fig. 3B). The level of survivin was positively correlated to the expression of iNOS
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Fig. 2. The exogenous NO plays a dual role. 2A and 2B, The GCDC-induced caspase-3 activity was altered following the different concentrations of SNAP or SNP. Control: GCDC treatment for 4 h without SNAP or SNP (0 μM). *P b 0.05; **P b 0.01 vs. control. 2C, Apoptosis was observed with the TUNEL staining. 2D, Release of cytochrome c from hepatocytes was assessed by immunoblotting. 2E, Hepatocytes were incubated with the indicated concentrations of SNAP or SNP. The cell proliferation was analyzed by the MTT assay. 2F, Cellcycle arrest, induced by 0.8 mM SNAP or 1.2 mM SNP, was determined using flow cytometry.
(Pb 0.01) (Fig. 3C). There was a certain relationship between iNOS and survivin expression, but we did not know if the levels of iNOS/NO could alter the survivin expression. To test this hypothesis, we treated hepatocytes with iNOS plasmids or NO donors. Thereafter, the survivin expression was analyzed. The survivin expression was increased by the transfection of 0.25 μg iNOS plasmids and NO donors at the concentration of 0.1 mM SNAP or 0.15 mM SNP (Fig. 3D). In contrast, the survivin expression was downregulated by the transfection of 2.5 μg iNOS plasmids and the high NO concentrations of 0.8 mM SNAP or 1.2 mM SNP (Fig. 3E). At the low NO concentrations, the upregulated survivin was abrogated by the iNOS inhibitor 1400 W (data not shown). Through enhancement of survivin expression, the low NO concentrations might lead to the augmentation of antiapoptotic factors contributing to cell survival. Importantly, a siRNA-mediated silence of survivin significantly blocked the antiapoptotic effect of the low NO concentrations (Fig. 3F). As the iNOS-specific inhibitor 1400 W cooperated with survivin siRNA (Fig. 3G, H), this combination could synergistically elevate the GCDCinduced apoptosis as shown by caspase-3 activity and TUNEL assay (Fig. 3I, J).
3.4. Survivin expression induced by the low NO concentrations may be regulated via PI3K/Akt/NF-κB signaling crosstalk The low NO concentrations activate the survivin expression, but the molecular basis for this observation remains undetermined. The upregulation of survivin requires an activation of PI3K/Akt in tumor cells [29], but we are unsure if the same applies in hepatocytes. Next, hepatocytes were pre-treated with the PI3K/Akt inhibitor LY294002 (15 μM, 1 h) and then added with NO donors (0.1 mM SNAP or 0.15 mM SNP). PI3K/Akt inhibitor reduced the expression of phosphorylated Akt (Fig. 4A, B), resulted in the downregulation of survivin (Fig. 4C), and enhanced the GCDC-induced apoptosis as reflected through caspase-3 activity (Fig. 4D). These data indicated that the NO-induced survivin expression in hepatocytes also required the activation of the PI3K/Akt signaling. Moreover, PI3K/Akt-inhibitor LY294002 suppressed the activation of NF-κB as shown by EMSA (Fig. 4E). Because the NF-κB inhibitor could downregulate the survivin expression (Fig. 4F), perhaps there was a potential PI3K/Akt/NF-κB axis to modulate the level of survivin. In addition, p38MAP-kinase (p38MAPK) was reported to
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Fig. 3. iNOS/NO counteracts hepatocyte apoptosis through the upregulation of survivin. 3A, 50 μM GCDC stimulated iNOS expression. Controls were treated with PBS buffer. *P b 0.05; **P b 0.01 vs. control. 3B, 50 μM GCDC can also upregulate the survivin expression. 3 C, The upregulation of survivin was positively correlated to the expression of iNOS (P b 0.01). 3D, The survivin expression was elevated by the transfection of 0.25 μg iNOS plasmids, 0.1 mM SNAP, and 0.15 mM SNP. 3E, The survivin expression was downregulated by 2.5 μg iNOS plasmids, 0.8 mM SNAP, and 1.2 mM SNP. 3F, Survivin siRNA eliminated the antiapoptotic effects of low NO concentrations. The GCDC-induced apoptosis was detected by TUNEL assay. 3G and 3H, The iNOS-specific inhibitor 1400 W could cooperate with survivin siRNA, which synergistically downregulated the expression of survivin. 3I and 3J, The combination of 1400W with survivin siRNA induced the serious apoptosis as shown by caspase-3 activity and TUNEL assay.
involve the iNOS/NO signaling pathway [30]. The p38MAPK expression was then evaluated under the treatment of exogenous NO. Anti-p38 and anti-phospho-p38 antibodies were utilized in Western blotting. The
p38MAPK expression showed no difference after the treatment with 0.1 mM SNAP or 0.15 mM SNP (Fig. 4G). Subsequently, the p38MAPKspecific inhibitor SB203580 was used to assess its effect on survivin
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expression and apoptosis. Results showed that the survivin expression, significantly upregulated by low concentrations of SNAP/SNP, was not altered by p38MAPK inhibitor SB203580 (Fig. 4H). Likewise, the GCDC-
induced apoptosis was not significantly affected by SB203580 (Fig. 4I). The activation of p38MAPK may be not important under low NO concentrations.
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3.5. High NO concentrations activate CHOP/DR5 and inhibit survivin through p38MAPK pathway The low concentrations of the NO donors (0.1 mM SNAP or 0.15 mM SNP) drove Akt/survivin signaling pathway, whereas the high concentrations of NO donors (0.8 mM SNAP and 1.2 mM SNP) stimulated pp38/p38 expression (Fig. 5A, B). Obviously, the activation of NO downstream signals such as Akt or p38MAPK is NO concentration-dependent. The high NO concentration could induce apoptosis through the endoplasmic reticulum (ER) stress pathway involving CHOP/GADD153 (Fig. 5C, D). The current data imply that two mechanisms of apoptosis induced by the high NO concentrations should be considered. One is that the high NO concentration mediates an early activation of CHOP, causes an upregulation of TRAIL receptor DR5, thus sensitizes hepatocytes to TRAIL (Fig. 5E, F); the other is that the high NO concentration leads to the downregulation of antiapoptotic factors such as survivin. The downregulation of the survivin expression was counteracted by the p38MAPK-specific inhibitor SB203580 (Fig. 5G, H). The mechanisms that the high NO concentration suppresses survivin remain unknown. Apoptosis induced by high NO donors or overexpression of iNOS was associated with the activation of p38MAPK/CHOP, which was blocked by the p38MAPKspecific inhibitor SB203580 (Fig. 5I, J). Taken together, the high NO concentration regulates the hepatocyte apoptosis through the modulation of the p38MAPK/CHOP and survivin expression. Here, we provide novel evidences for the molecular mechanism as to how NO signaling contributes to the apoptosis in hepatocytes. These results suggest that the pathway p38MAPK/CHOP and p38MAPK/survivin may be regulatory targets for the interference of the hepatocyte apoptosis. 4. Discussion Our results confirm that iNOS regulates the hepatocyte apoptosis through pathway Akt, p38MAPK, and their downstream targeting genes. The endogenous iNOS is antiapoptotic in the GCDC-hepatocyte model. However the antiapoptotic ability of the endogenous iNOS is limited. A high concentration of GCDC or a low concentration of GCDC in long time exposure can completely neutralize the resistance of the endogenous iNOS. The exogenous NO can protect hepatocytes against apoptosis, but the benefits of the exogenous NO are concentrationdependent. At low NO levels, iNOS activates PI3K/Akt/ NF-κB signals to stimulate survivin expression. The upregulated survivin inhibits caspase activity, thereby leading to negative regulation of apoptosis. The survival rate of hepatocytes is increased following NO stimulation at the low concentrations, whereas the high NO dosage exacerbates the apoptosis as shown by caspase-3 activity and TUNEL assay. The high NO level activates the CHOP/DR5 pathway to sensitize hepatocytes to TRAIL, which combines with the downregulated survivin to result in severe apoptosis subsequent to GCDC treatment. The present data demonstrate that the low NO concentrations protect hepatocytes from apoptotic damage. In fact, hepatic iNOS expression is increased by a variety of stimuli, such as acute and chronic inflammation, hemorrhagic shock, and pro-inflammatory cytokines [31]. The endogenous iNOS itself is not enough to overcome toxic bile-induced cholestatic liver injury, although the iNOS can be amplified by cytokine-inducers or iNOS gene transfection [19]. The
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exogenous iNOS/NO has shown to inhibit the GCDC-induced apoptosis. For example, adenoviral iNOS pre-treatment could decrease acute cholestatic liver injury in bile duct ligated rats [19,20]. However, iNOS expression and NO production can elicit either protective or detrimental effects, depending on the local conditions and redox state [32]. iNOS/NO plays the protective role only in a limited range. The high level of NO donors induces the hepatocyte apoptosis and thus is noxious, which restricts the iNOS/NO application in the treatment of cholestatic disease. We have to look for other possible targets. The current study provides new signaling pathways or Akt/ survivin and p38MAPK/CHOP axis. These may be potential targets to design therapeutic intervention for the treatment of cholestatic liver disease. Further studies are still required to elucidate the pathophysiologic function of iNOS/NO during GCDC-induced liver injury. These data were derived from rat primary hepatocytes, but the results were similar to previous studies in other tissues or cells. Several examples demonstrated the role of iNOS/survivin in cancer cells. iNOS/ survivin was actually a common denominator that plays a comparable role in a number of different kinds of cancer. iNOS/survivin signaling contributed chemoresistance in head and neck squamous cell carcinoma (HNSCC) [30,33]. Cell death induced by high amounts of SNAP/SNP or by strong overexpression of iNOS involved the activation of p38MAPK, which was counteracted by the p38MAPK inhibitor SB202190. NO signaling contributes to therapy resistance in HNSCC by modulating survivin expression. Application of the iNOS-specific inhibitor 1400 W combined with RNAi-mediated downregulation of survivin could mutually enhance drug-induced cell death [34]. In ovarian carcinoma, NO donors SNAP/SNP or strong overexpression of iNOS suppressed survivin levels via the p38MAPK pathway and triggered apoptosis [35]. Low NO concentrations maintained resistance against anticancer agentsinduced apoptosis. Cytoprotection was mediated by survivin through its upregulation subsequent to low SNAP/SNP doses. RNAi-mediated depletion of survivin blocked the antiapoptotic effects of NO signaling [35,36]. In human lung carcinoma cells, survivin was downregulated by high SNAP/SNP to cause cell growth arrest and apoptosis. The specific p38MAPK inhibitor significantly decreased the cytotoxicity and increased the survivin levels in NO donor-treated and iNOS-transfected cells [37]. Cancer cells produce increased amounts of iNOS/NO. The increased formation of iNOS/NO induces the synthesis of survivin. The survivin, as an inhibitor of programmed cell death, are in turn exploited by the cancer cells to protect themselves against attack by chemo- or radiotherapy so that cancer cells employ the iNOS/Akt/survivin axis as a survival aid [35]. The antiapoptotic mechanisms of NO involve a series of NO target interactions that range from indirect and nonspecific to direct interaction with apoptotic machinery. NO directly inhibits caspase activity through S-nitrosylation of cysteine thiol in hepatocytes, endothelial cells, and several tumor cell lines [21,38,39]. The antiapoptotic action of NO is to inhibit the most apical caspase-8 by S-nitrosylation, subsequently preventing Bid cleavage, mitochondrial cytochrome c release, caspases-9 and caspases-3 activation [40]. Moreover, S-nitrosylation of caspases occurs extremely efficiently in hepatocytes [41]. The protective effect of NO does not require expression of the cytoprotective genes. An important feature of NO inhibition of caspase activity is that NO can rescue a cell from apoptosis even after the caspase cascade has been activated. Because
Fig. 4. The low NO concentrations induce survivin expression via PI3K/Akt/NF-κB signaling crosstalk. 4A, PI3K/Akt inhibitor LY294002 blocked the expression of phosphorylated Akt. Rat hepatocytes were pre-treated with 15 μM of LY294002 for 1 h, incubated with 0.1 mM SNAP or 0.15 mM SNP for 10 h, and then challenged cells with GCDC for 4 h. 4B, Quantitation of α-p-Akt immunostaining. α-p-Akt expression was normalized with α-Akt. Ratios of α-p-Akt to α-Akt were expressed as arbitrary units. **P b 0.01 vs. control. 4 C, LY294002 caused a downregulation of survivin, **P b 0.01. 4D, LY294002 increased caspase-3 activity, **P b 0.01. 4E, LY294002 suppressed the activation of NF-κB as demonstrated through EMSA with oct1 as control. 4F, NF-κB inhibitor BAY 11–7082 could downregulate the survivin expression. *P b 0.05; **P b 0.01 vs. control. 4G, p38MAPK (p38) and phosphorylated p38MAPK (pp38) expression were not affected by the low NO concentrations (0.1 mM SNAP; 0.15 mM SNP). 4H, The survivin expression through real-time PCR analysis. Hepatocytes were pre-incubated with 15 μM of p38MAPK-specific inhibitor SB203580 for 1 h, treated with 0.1 mM SNAP or 0.15 mM SNP for 12 h, and then insulted with 50 μM of GCDC for 4 h. Controls were treated with PBS buffer. 4I, Caspase-3 activity was not significantly altered by SB203580. *P b 0.05.
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Fig. 5. The high NO concentrations activate CHOP/DR5 and inhibit survivin through p38MAPK pathway. 5A, The high concentration of NO donors (0.8 mM SNAP and 1.2 mM SNP) activated the phosphorylated p38MAPK (pp38) expression by Western blotting. 5B, Quantitation of pp38 immunostaining. pp38 was standardized with p38MAPK (p38) and ratios were expressed as arbitrary units. *P b 0.05; **P b 0.01 vs. control. 5C, The high NO concentrations induced CHOP/GADD153 expression as demonstrated by Western blotting. 5D, CHOP/GADD153 expression was normalized against β-actin, **P b 0.01. 5E and 5F, The expression of DR5 was increased by the high NO concentrations in time-dependent manner. 5G and 5H, The survivin expression, downregulated by high NO concentrations or by strong overexpression of iNOS plasmids, could be reversed by the p38MAPK-specific inhibitor SB203580. Blank was treated with PBS buffer. **P b 0.01. 6I and 6J, Apoptosis, induced by the high NO concentrations or by strong overexpression of iNOS plasmids, was significantly reduced by inhibitor SB 203580, **P b 0.01.
NO easily diffuses within a cell, as well as from cell to cell, NO can efficiently guard against aberrant activation of caspases. In addition to S-nitrosylation of caspases, several mechanisms for the antiapoptotic effect of NO have been proposed. They include (i) induction of cytoprotective stress proteins, e.g. HSP32 and HSP70; (ii) cGMP-
dependent inhibition of apoptotic signal transduction [41,42]. NO/ cGMP inhibition of apoptosis can involve the activation of cGMPdependent protein kinase and the inhibition of caspase activation. NO increases cGMP level through the activation of soluble guanylyl cyclase. The cGMP interrupts apoptotic signaling in some cell types,
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including hepatocytes, splenocytes, and neuronal PC12 cells [32]. NO activation of soluble guanylyl cyclase contributes to NO-mediated protection from TNFα/ActD-induced apoptosis, which is the dominant mechanism for protection in some cell types [40]. Through these mechanisms, NO can prevent the activation of downstream caspases and the terminal events in apoptosis such as the cleavage of the inhibitor of caspase-dependent activated DNase and the activation of caspase-dependent activated DNase [43,32]. In summary, iNOS/NO signaling regulated the GCDC-induced apoptosis in rat hepatocytes. Two mechanisms were involved. The high NO concentrations mediated survivin inhibition and CHOP activation that caused upregulation of TRAIL receptor DR5 and thus sensitized hepatocytes to TRAIL; The low NO concentrations led to the upregulation of survivin expression, contributing to cell survival. The antiapoptotic survivin, modulated by Akt/NF-κB, might be an essential factor in decreasing the GCDC-induced apoptosis in hepatocytes. Akt/ survivin and p38MAPK/CHOP will be new targets for designing hepatoprotective strategies and developing therapeutic application in the treatment of liver diseases. Acknowledgments This work was supported in part by the Falk Medical Research Foundation grant and National Institute of Health grant R21 DK070784-02. We appreciated Xiao Wang's help during preparation of the manuscript. References [1] A.K. Udit, W. Belliston-Bittner, E.C. Glazer, et al., J. Am. Chem. Soc. 127 (32) (2005) 11212–11213. [2] W.W. Chang, I.J. Su, W.T. Chang, W. Huang, H.Y. Lei, J. Viral Hepat. 15 (7) (2008) 490–497. [3] J. Abrams, Am. J. Cardiol. 77 (13) (1996) 31–37. [4] E.T. Crockett, J.J. Galligan, B.D. Uhal, J. Harkema, R. Roth, K. Pandya, BMC Clin. Pathol. 6 (2006) 3. [5] B.S. Taylor, L.H. Alarcon, T.R. Billiar, Biochemistry (Mosc) 63 (7) (1998) 766–781. [6] V. Baron, J. Hernandez, M. Noyola, B. Escalante, P. Muriel, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 127 (3) (2000) 243–249. [7] J. Li, C.A. Bombeck, S. Yang, Y.M. Kim, T.R. Billiar, J. Biol. Chem. 274 (24) (1999) 17325–17333. [8] K. Wang, J.J. Brems, R.L. Gamelli, J. Ding, J. Biol. Chem. 280 (25) (2005) 23490–23495. [9] B. Yerushalmi, R. Dahl, M.W. Devereaux, E. Gumpricht, R.J. Sokol, Hepatology 33 (3) (2001) 616–626.
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Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c e l l s i g
iNOS/NO signaling regulates apoptosis induced by glycochenodeoxycholate in hepatocytes Kewei Wang a,⁎, John J. Brems b, Richard L. Gamelli b, Ai-Xuan Holterman a a b
Departments of Pediatrics and Surgery/Section of Pediatric Surgery, Rush University Medical Center, Chicago, IL 60612, United States Department of Surgery, Loyola University Medical Center, Maywood, IL 60153, United States
a r t i c l e
i n f o
Article history: Received 22 April 2011 Accepted 6 June 2011 Available online 13 June 2011 Keywords: iNOS Hepatocyte Apoptosis Cholestatic liver disease
a b s t r a c t Inducible nitric oxide synthase (iNOS) and nitric oxide (NO) can ameliorate apoptosis induced by toxic glycochenodeoxycholate (GCDC) in hepatocytes. However, the underlying molecular mechanisms are not yet understood in detail. This study is to clarify the function of iNOS/NO and its mechanisms during the apoptotic process. The apoptosis was brought about by GCDC in rat primary hepatocytes. iNOS/NO signaling was then investigated. iNOS inhibitor 1400 W enhanced the GCDC-induced apoptosis as reflected by caspase-3 activity and TUNEL assay. Exogenous NO regulated the apoptosis subsequent to NO donor S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP). The GCDC-induced apoptosis was decreased with 0.1 mM SNAP or 0.15 mM SNP, while it was increased with 0.8 mM SNAP or 1.2 mM SNP. The endogenous iNOS inhibited apoptosis, but the exogenous NO played a dual role during the GCDC-induced apoptosis. There was a potential iNOS/Akt/survivin axis that inhibited the hepatocyte apoptosis in low doses of NO donors. In contrast, high doses of NO donors activated CHOP through p38MAP-kinase (p38MAPK), upregulated TRAIL receptor DR5, and suppressed survivin. Consequently the high doses of NO donors promoted the apoptosis in hepatocytes. Our data suggest that the iNOS/NO signaling can modulate Akt/survivin and p38MAPK/CHOP pathways to mediate the GCDC-induced the apoptosis in hepatocytes. These signaling pathways may serve as targets for therapeutic intervention in cholestatic liver disease. © 2011 Elsevier Inc. All rights reserved.
1. Introduction iNOS can synthesize NO from the terminal nitrogen atom of in the presence of NADPH and molecular oxygen [1]. NO as a short-lived free radical plays an important role in the regulation of many pathophysiologic processes [2,3]. Previous studies have demonstrated that the induced NO is liver-protective. iNOS inhibitors significantly increase hepatic damage [4–6]. The upregulation of iNOS/NO can suppress apoptosis via interrupting caspase activation and mitochondrial dysfunction [7–9]. NO is pro-apoptotic as well, despite having an anti-apoptotic function [10]. NO increases TRAIL-induced cytotoxicity by facilitating the mitochondria-mediated caspase signal transduction pathway [11]. The iNOS expression is regulated at multiple stages along the signaling pathway. iNOS/NO signaling affects liver function in different degrees [12,13]. The numerous mechanisms have been
L-arginine
Abbreviations: iNOS, inducible nitric oxide synthase; NO, nitric oxide; GCDC, glycochenodeoxycholate; SNAP, S-nitroso-N-acetyl-penicillamine; SNP, sodium nitroprusside; PI3K, Phosphoinositide 3-kinase; NF-κB, nuclear factor kappaB; p38MAPK, P38 mitogen-activated protein kinases; CHOP, CCAAT/enhancer-binding protein homologous protein; TRAIL, TNF-related apoptosis-inducing ligand. ⁎ Corresponding author at: Rush University Medical Center, 1725 W. Harrison St., Suite 718, Chicago, IL 60612, United States. Tel.: +1 312 942 3358; fax: +1 312 942 5202. E-mail address: [email protected] (K. Wang). 0898-6568/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2011.06.003
associated with the regulation of iNOS expression. The study on iNOS/ NO signaling may find new targets for the treatment of liver injury. GCDC is a predominant toxic bile acid. In cholestatic patients or experimental animal models, the accumulated GCDC is correlated with apoptotic or necrotic cell death in vivo [14,15]. GCDC also causes apoptotic injury in primary hepatocytes in vitro. GCDC induces the apoptosis in hepatocytes via Fas ligand-independent mechanisms by cellular trafficking of the death receptor plasma membrane [16]. Once activated, Fas signals can bring about mitochondrial dysfunction and ultimately activation of caspase-3, which results in cell death [17]. GCDC decreases NF-κB DNA-binding capacity through inhibiting IκBα degradation and thereby iNOS expression is reduced [18]. GCDC can inhibit iNOS expression and NO synthesis in hepatocytes through the inactivation of iNOS promoter as well [19]. In vivo adenoviral iNOS pre-treatment ameliorates rat liver transplant preservation injury and improves survival rate [20]. Functionally, the exogenous or endogenous NO production inhibits the GCDC-induced apoptosis [21,22]. The GCDC-induced hepatocyte apoptosis has already become a classical model through which to investigate apoptosis, bile toxicity, and liver injury [23]. iNOS/NO signaling regulates the GCDC-induced hepatocyte apoptosis, but some detailed links are still unclear. To examine the relationship between iNOS expression and apoptosis, GCDC was chosen as an apoptosis-inducer to trigger apoptotic injury in
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hepatocytes. Through the GCDC-hepatocyte model, iNOS expression was analyzed at multiple levels. The endogenous iNOS might be antiapoptotic and cytoprotective, whereas the exogenous NO was a bifunctional regulator of apoptosis. At low concentrations, the role of NO was similar to the upregulation of the endogenous iNOS. NO activated the expression of survivin to exert an antiapoptotic role in hepatocytes. The iNOS/survivin pathway was affected by the modification of Akt/NF-κB signaling. However, high NO concentrations initiated the activation of p38MAPK/CHOP/DR5 as well as suppression of survivin. High doses of NO donors took a wide range of actions and boosted the hepatocyte apoptosis. The current study aims to evaluate the pathophysiologic role of iNOS/NO during the GCDCinduced apoptosis in hepatocytes. These results refine the molecular mechanism that iNOS/NO regulates apoptosis. 2. Materials and methods
2.7. TUNEL assay The TdT-FragELTM DNA fragmentation detection kit was obtained from Calbiochem (#QIA33). We followed the manufacturer instruction with only minor modifications [8].
2.8. Western blotting assay Protein samples were resolved by 8–10% SDS-polyacrylamide gel, transferred to nitrocellulose membrane, and blotted with appropriate primary antibodies at dilution of 1: 500–1000. Peroxidase-conjugated secondary antibodies were incubated at a dilution of 1: 2000–3000. Bound antibody was visualized through chemiluminescent substrate (ECL; Amersham Biosciences). β–actin was used as equal loading control.
2.1. Hepatocyte isolation and culture 2.9. Quantitative real-time PCR (qRT-PCR) Hepatocytes were isolated from adult Sprague–Dawley rats along standard liver perfusion procedure. Dead cells were removed by Percoll (Sigma) gradient centrifugation. Following the last wash, highly purified hepatocytes (N98% viability) were suspended in cold Williams E medium, diluted to a density of 5.5 × 10 5, plated into collagen-coated dishes (Falcon), and cultured as described previously [8,23,24].
Total RNA was isolated from hepatocytes with ready-to-use TRIZOL Reagent. The first-strand cDNA was synthesized using standard protocol. For qRT-PCR, reactions were amplified with the appropriate primer sets and analyzed in triplicate. The relative level of gene expression was normalized to the expression level of 18S rRNA.
2.2. Reagents and treatment
2.10. Electrophoretic mobility shift assay
The iNOS-specific inhibitor N-(3-(aminomethyl)benzyl)acetamidine (1400 W) (50 μM), the PI3K/Akt inhibitor LY294002 (15 μM), and the p38 MAPK inhibitor SB203580 (15 μM) were purchased from Calbiochem. GCDC, SNAP, and SNP were obtained from Sigma. A concentration of 50 μM GCDC was used unless otherwise indicated. Control groups were treated with PBS buffer.
Nuclear extract was prepared with the modified Dignam protocol [8]. Protein-DNA complexes were separated from the unbound DNA probe by electrophoresis through 6% native polyacrylamide gels containing 0.5 × Tris borate/EDTA.
2.3. iNOS Plasmids The eukaryotic expression vector pcDNA3-iNOS was constructed as previously published [25,26]. The rat iNOS coding sequence was cloned into the expression vector pcDNA3.1. Sequence analysis confirmed that the coding sequence was identical to the database entry. 2.4. Caspase assay 100 μg of cell lysate was utilized to assay the activity of caspase-3. Caspase assay kit was bought from Calbiochem. The reaction system employed the colorimetric substrate IETD-pNA. The activity of caspase-3 was calculated as pmol/min [23]. 2.5. Cell-cycle analysis As previously described [27], hepatocytes were removed using the trypsin/EGTA solution. Nuclei were fixed in ice-cold 70% EtOH, resuspended in PI staining solution, and analyzed by flow cytometry. 2.6. Transfection of siRNA The double stranded siRNA against Survivin was composed of the following oligonucleotides: UGAGCCUGAUUUGGCCCAG and CUGGGCCAAAUCAGGCUCA [28]. The scramble siRNA having no known homology with mammalian genes was used for nonspecific silencing effects. The cells were transfected with 200 nM double-stranded siRNA in lipophilic transfection reagent.
2.11. Statistical analysis All data represent at least three experiments using cells or extracts from a minimum of three separate isolations. Results are expressed as mean ± SD unless otherwise indicated. Comparisons were performed using either Student's t test for independent samples or repeated measures for the ANOVA followed by Bonferroni correction where appropriate. P values b 0·05 were considered significant.
3. Results 3.1. iNOS involves GCDC-induced hepatocyte apoptosis When the hepatocytes were stimulated with different concentrations of GCDC for 4 h, iNOS expression was decreased by the high GCDC dosages (Fig. 1A). At GCDC concentration of 50 μM, iNOS was upregulated at 2 h and then reduced to a low level at 16 h (Fig. 1B). Interestingly, the levels of iNOS were negatively correlated to the caspase-3 activity after GCDC treatment for 4 h (P = 0.021). As the rat hepatocytes were treated with iNOS-specific inhibitor 1400 W, the GCDC-induced apoptosis was reflected with caspase-3 activity and TUNEL assay (Fig. 1C, D). The iNOS inhibitor 1400 W could enhance the GCDC-induced apoptosis. Evidently, endogenous iNOS was related to the apoptotic injury in hepatocytes. After the transfection with iNOS plasmids in the cultured rat hepatocytes for 16 h, GCDC (60 μM) treatment was then started for another 4 h. iNOS plasmids inhibited the GCDC-induced apoptosis as indicated by caspase-3 and TUNEL assay (Fig. 1E, F). Following the application of iNOS inhibitor or the transfection of iNOS plasmids, the iNOS expression significantly affected the profile of GCDC-induced apoptosis in hepatocytes.
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Fig. 1. iNOS/NO involves liver injury. 1A and 1B, iNOS expression was affected in GCDC dose- and time-dependent manners. Values are mean ± SD (n = 6 in each group). Control groups were treated with PBS buffer. **P b 0.01; *P b 0.05 vs. control. Rat hepatocytes were pre-incubated with iNOS inhibitor 1400 W for 1 hour before GCDC treatment. Apoptosis was detected with caspase-3 activity (1C) and TUNEL assay (1D). 1E, After iNOS plasmids (0.25 μg) were transfected into rat hepatocytes for 16 h, 60 μM of GCDC was added for another 4 h. The iNOS plasmids decreased the caspase-3 activity. 1F, The iNOS plasmids inhibited the GCDC-induced apoptosis as shown by TUNEL assay.
3.2. The exogenous NO plays a dual role during the GCDC-induced hepatocyte apoptosis SNAP and SNP were selected as NO donors to test the role of exogenous NO during the GCDC-induced apoptosis in hepatocytes. After SNAP (0–1 mM) or SNP (0–1.2 mM) was co-administered with GCDC (50 μM), the apoptotic response was observed with the caspase-3 activity (Fig. 2A, B) and TUNEL assay (Fig. 2C). The GCDCinduced hepatocyte apoptosis was diminished at low concentrations of SNAP (0.1 mM) or SNP (0.15 mM). However, the apoptosis was increased at high concentrations of SNAP (0.8 mM) or SNP (1.0 mM). Of note, toxic effects of the high NO concentrations appeared not only in hepatocytes, but in CHO cells as well (data not shown). The high NO concentrations significantly increased the release of cytochrome c (Fig. 2D). The exogenous NO exposure could inhibit apoptosis that only occurred in a specific range of NO concentrations. Besides apoptosis, cell proliferation was another aspect used to estimate the role of NO in hepatocytes. The high NO concentrations (SNAP 0.8 mM and SNP 1.0 mM) hindered cell growth in hepatocytes, whereas the low NO concentrations (SNAP 0.1 mM and SNP 0.15 mM) stimulated
cell proliferation (Fig. 2E). Cell-cycle arrest was detected using flow cytometry (Fig. 2F). In the untreated hepatocytes, 42.6% of the cells appeared in G0/G1, 35.8% in S phase, and 21.6% in G2/M phases of the cell cycle. After a 4 h treatment with 0.8 mM SNAP or 1.2 mM SNP, the G2/M population decreased to 8.6% and 7.1% respectively. When the treatment was prolonged for 8 h, the G2/M population was similar to that observed at 4 h, which suggested that these agents could prevent cells from progressing into the G2/M phases. The combination of both the antiapoptosis and cell proliferation might actually reflect the pathophysiologic role of iNOS/NO. The exogenous NO can promote or inhibit apoptosis, which plays a dual role during the GCDC-induced hepatocyte injury. 3.3. iNOS/NO counteracts hepatocyte apoptosis through the upregulation of survivin GCDC at a concentration of 50 μM could activate the endogenous expression of iNOS (Fig. 3A). The amplification of iNOS gene was accompanied by the upregulation of survivin expression (Fig. 3B). The level of survivin was positively correlated to the expression of iNOS
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Fig. 2. The exogenous NO plays a dual role. 2A and 2B, The GCDC-induced caspase-3 activity was altered following the different concentrations of SNAP or SNP. Control: GCDC treatment for 4 h without SNAP or SNP (0 μM). *P b 0.05; **P b 0.01 vs. control. 2C, Apoptosis was observed with the TUNEL staining. 2D, Release of cytochrome c from hepatocytes was assessed by immunoblotting. 2E, Hepatocytes were incubated with the indicated concentrations of SNAP or SNP. The cell proliferation was analyzed by the MTT assay. 2F, Cellcycle arrest, induced by 0.8 mM SNAP or 1.2 mM SNP, was determined using flow cytometry.
(Pb 0.01) (Fig. 3C). There was a certain relationship between iNOS and survivin expression, but we did not know if the levels of iNOS/NO could alter the survivin expression. To test this hypothesis, we treated hepatocytes with iNOS plasmids or NO donors. Thereafter, the survivin expression was analyzed. The survivin expression was increased by the transfection of 0.25 μg iNOS plasmids and NO donors at the concentration of 0.1 mM SNAP or 0.15 mM SNP (Fig. 3D). In contrast, the survivin expression was downregulated by the transfection of 2.5 μg iNOS plasmids and the high NO concentrations of 0.8 mM SNAP or 1.2 mM SNP (Fig. 3E). At the low NO concentrations, the upregulated survivin was abrogated by the iNOS inhibitor 1400 W (data not shown). Through enhancement of survivin expression, the low NO concentrations might lead to the augmentation of antiapoptotic factors contributing to cell survival. Importantly, a siRNA-mediated silence of survivin significantly blocked the antiapoptotic effect of the low NO concentrations (Fig. 3F). As the iNOS-specific inhibitor 1400 W cooperated with survivin siRNA (Fig. 3G, H), this combination could synergistically elevate the GCDCinduced apoptosis as shown by caspase-3 activity and TUNEL assay (Fig. 3I, J).
3.4. Survivin expression induced by the low NO concentrations may be regulated via PI3K/Akt/NF-κB signaling crosstalk The low NO concentrations activate the survivin expression, but the molecular basis for this observation remains undetermined. The upregulation of survivin requires an activation of PI3K/Akt in tumor cells [29], but we are unsure if the same applies in hepatocytes. Next, hepatocytes were pre-treated with the PI3K/Akt inhibitor LY294002 (15 μM, 1 h) and then added with NO donors (0.1 mM SNAP or 0.15 mM SNP). PI3K/Akt inhibitor reduced the expression of phosphorylated Akt (Fig. 4A, B), resulted in the downregulation of survivin (Fig. 4C), and enhanced the GCDC-induced apoptosis as reflected through caspase-3 activity (Fig. 4D). These data indicated that the NO-induced survivin expression in hepatocytes also required the activation of the PI3K/Akt signaling. Moreover, PI3K/Akt-inhibitor LY294002 suppressed the activation of NF-κB as shown by EMSA (Fig. 4E). Because the NF-κB inhibitor could downregulate the survivin expression (Fig. 4F), perhaps there was a potential PI3K/Akt/NF-κB axis to modulate the level of survivin. In addition, p38MAP-kinase (p38MAPK) was reported to
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Fig. 3. iNOS/NO counteracts hepatocyte apoptosis through the upregulation of survivin. 3A, 50 μM GCDC stimulated iNOS expression. Controls were treated with PBS buffer. *P b 0.05; **P b 0.01 vs. control. 3B, 50 μM GCDC can also upregulate the survivin expression. 3 C, The upregulation of survivin was positively correlated to the expression of iNOS (P b 0.01). 3D, The survivin expression was elevated by the transfection of 0.25 μg iNOS plasmids, 0.1 mM SNAP, and 0.15 mM SNP. 3E, The survivin expression was downregulated by 2.5 μg iNOS plasmids, 0.8 mM SNAP, and 1.2 mM SNP. 3F, Survivin siRNA eliminated the antiapoptotic effects of low NO concentrations. The GCDC-induced apoptosis was detected by TUNEL assay. 3G and 3H, The iNOS-specific inhibitor 1400 W could cooperate with survivin siRNA, which synergistically downregulated the expression of survivin. 3I and 3J, The combination of 1400W with survivin siRNA induced the serious apoptosis as shown by caspase-3 activity and TUNEL assay.
involve the iNOS/NO signaling pathway [30]. The p38MAPK expression was then evaluated under the treatment of exogenous NO. Anti-p38 and anti-phospho-p38 antibodies were utilized in Western blotting. The
p38MAPK expression showed no difference after the treatment with 0.1 mM SNAP or 0.15 mM SNP (Fig. 4G). Subsequently, the p38MAPKspecific inhibitor SB203580 was used to assess its effect on survivin
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expression and apoptosis. Results showed that the survivin expression, significantly upregulated by low concentrations of SNAP/SNP, was not altered by p38MAPK inhibitor SB203580 (Fig. 4H). Likewise, the GCDC-
induced apoptosis was not significantly affected by SB203580 (Fig. 4I). The activation of p38MAPK may be not important under low NO concentrations.
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3.5. High NO concentrations activate CHOP/DR5 and inhibit survivin through p38MAPK pathway The low concentrations of the NO donors (0.1 mM SNAP or 0.15 mM SNP) drove Akt/survivin signaling pathway, whereas the high concentrations of NO donors (0.8 mM SNAP and 1.2 mM SNP) stimulated pp38/p38 expression (Fig. 5A, B). Obviously, the activation of NO downstream signals such as Akt or p38MAPK is NO concentration-dependent. The high NO concentration could induce apoptosis through the endoplasmic reticulum (ER) stress pathway involving CHOP/GADD153 (Fig. 5C, D). The current data imply that two mechanisms of apoptosis induced by the high NO concentrations should be considered. One is that the high NO concentration mediates an early activation of CHOP, causes an upregulation of TRAIL receptor DR5, thus sensitizes hepatocytes to TRAIL (Fig. 5E, F); the other is that the high NO concentration leads to the downregulation of antiapoptotic factors such as survivin. The downregulation of the survivin expression was counteracted by the p38MAPK-specific inhibitor SB203580 (Fig. 5G, H). The mechanisms that the high NO concentration suppresses survivin remain unknown. Apoptosis induced by high NO donors or overexpression of iNOS was associated with the activation of p38MAPK/CHOP, which was blocked by the p38MAPKspecific inhibitor SB203580 (Fig. 5I, J). Taken together, the high NO concentration regulates the hepatocyte apoptosis through the modulation of the p38MAPK/CHOP and survivin expression. Here, we provide novel evidences for the molecular mechanism as to how NO signaling contributes to the apoptosis in hepatocytes. These results suggest that the pathway p38MAPK/CHOP and p38MAPK/survivin may be regulatory targets for the interference of the hepatocyte apoptosis. 4. Discussion Our results confirm that iNOS regulates the hepatocyte apoptosis through pathway Akt, p38MAPK, and their downstream targeting genes. The endogenous iNOS is antiapoptotic in the GCDC-hepatocyte model. However the antiapoptotic ability of the endogenous iNOS is limited. A high concentration of GCDC or a low concentration of GCDC in long time exposure can completely neutralize the resistance of the endogenous iNOS. The exogenous NO can protect hepatocytes against apoptosis, but the benefits of the exogenous NO are concentrationdependent. At low NO levels, iNOS activates PI3K/Akt/ NF-κB signals to stimulate survivin expression. The upregulated survivin inhibits caspase activity, thereby leading to negative regulation of apoptosis. The survival rate of hepatocytes is increased following NO stimulation at the low concentrations, whereas the high NO dosage exacerbates the apoptosis as shown by caspase-3 activity and TUNEL assay. The high NO level activates the CHOP/DR5 pathway to sensitize hepatocytes to TRAIL, which combines with the downregulated survivin to result in severe apoptosis subsequent to GCDC treatment. The present data demonstrate that the low NO concentrations protect hepatocytes from apoptotic damage. In fact, hepatic iNOS expression is increased by a variety of stimuli, such as acute and chronic inflammation, hemorrhagic shock, and pro-inflammatory cytokines [31]. The endogenous iNOS itself is not enough to overcome toxic bile-induced cholestatic liver injury, although the iNOS can be amplified by cytokine-inducers or iNOS gene transfection [19]. The
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exogenous iNOS/NO has shown to inhibit the GCDC-induced apoptosis. For example, adenoviral iNOS pre-treatment could decrease acute cholestatic liver injury in bile duct ligated rats [19,20]. However, iNOS expression and NO production can elicit either protective or detrimental effects, depending on the local conditions and redox state [32]. iNOS/NO plays the protective role only in a limited range. The high level of NO donors induces the hepatocyte apoptosis and thus is noxious, which restricts the iNOS/NO application in the treatment of cholestatic disease. We have to look for other possible targets. The current study provides new signaling pathways or Akt/ survivin and p38MAPK/CHOP axis. These may be potential targets to design therapeutic intervention for the treatment of cholestatic liver disease. Further studies are still required to elucidate the pathophysiologic function of iNOS/NO during GCDC-induced liver injury. These data were derived from rat primary hepatocytes, but the results were similar to previous studies in other tissues or cells. Several examples demonstrated the role of iNOS/survivin in cancer cells. iNOS/ survivin was actually a common denominator that plays a comparable role in a number of different kinds of cancer. iNOS/survivin signaling contributed chemoresistance in head and neck squamous cell carcinoma (HNSCC) [30,33]. Cell death induced by high amounts of SNAP/SNP or by strong overexpression of iNOS involved the activation of p38MAPK, which was counteracted by the p38MAPK inhibitor SB202190. NO signaling contributes to therapy resistance in HNSCC by modulating survivin expression. Application of the iNOS-specific inhibitor 1400 W combined with RNAi-mediated downregulation of survivin could mutually enhance drug-induced cell death [34]. In ovarian carcinoma, NO donors SNAP/SNP or strong overexpression of iNOS suppressed survivin levels via the p38MAPK pathway and triggered apoptosis [35]. Low NO concentrations maintained resistance against anticancer agentsinduced apoptosis. Cytoprotection was mediated by survivin through its upregulation subsequent to low SNAP/SNP doses. RNAi-mediated depletion of survivin blocked the antiapoptotic effects of NO signaling [35,36]. In human lung carcinoma cells, survivin was downregulated by high SNAP/SNP to cause cell growth arrest and apoptosis. The specific p38MAPK inhibitor significantly decreased the cytotoxicity and increased the survivin levels in NO donor-treated and iNOS-transfected cells [37]. Cancer cells produce increased amounts of iNOS/NO. The increased formation of iNOS/NO induces the synthesis of survivin. The survivin, as an inhibitor of programmed cell death, are in turn exploited by the cancer cells to protect themselves against attack by chemo- or radiotherapy so that cancer cells employ the iNOS/Akt/survivin axis as a survival aid [35]. The antiapoptotic mechanisms of NO involve a series of NO target interactions that range from indirect and nonspecific to direct interaction with apoptotic machinery. NO directly inhibits caspase activity through S-nitrosylation of cysteine thiol in hepatocytes, endothelial cells, and several tumor cell lines [21,38,39]. The antiapoptotic action of NO is to inhibit the most apical caspase-8 by S-nitrosylation, subsequently preventing Bid cleavage, mitochondrial cytochrome c release, caspases-9 and caspases-3 activation [40]. Moreover, S-nitrosylation of caspases occurs extremely efficiently in hepatocytes [41]. The protective effect of NO does not require expression of the cytoprotective genes. An important feature of NO inhibition of caspase activity is that NO can rescue a cell from apoptosis even after the caspase cascade has been activated. Because
Fig. 4. The low NO concentrations induce survivin expression via PI3K/Akt/NF-κB signaling crosstalk. 4A, PI3K/Akt inhibitor LY294002 blocked the expression of phosphorylated Akt. Rat hepatocytes were pre-treated with 15 μM of LY294002 for 1 h, incubated with 0.1 mM SNAP or 0.15 mM SNP for 10 h, and then challenged cells with GCDC for 4 h. 4B, Quantitation of α-p-Akt immunostaining. α-p-Akt expression was normalized with α-Akt. Ratios of α-p-Akt to α-Akt were expressed as arbitrary units. **P b 0.01 vs. control. 4 C, LY294002 caused a downregulation of survivin, **P b 0.01. 4D, LY294002 increased caspase-3 activity, **P b 0.01. 4E, LY294002 suppressed the activation of NF-κB as demonstrated through EMSA with oct1 as control. 4F, NF-κB inhibitor BAY 11–7082 could downregulate the survivin expression. *P b 0.05; **P b 0.01 vs. control. 4G, p38MAPK (p38) and phosphorylated p38MAPK (pp38) expression were not affected by the low NO concentrations (0.1 mM SNAP; 0.15 mM SNP). 4H, The survivin expression through real-time PCR analysis. Hepatocytes were pre-incubated with 15 μM of p38MAPK-specific inhibitor SB203580 for 1 h, treated with 0.1 mM SNAP or 0.15 mM SNP for 12 h, and then insulted with 50 μM of GCDC for 4 h. Controls were treated with PBS buffer. 4I, Caspase-3 activity was not significantly altered by SB203580. *P b 0.05.
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Fig. 5. The high NO concentrations activate CHOP/DR5 and inhibit survivin through p38MAPK pathway. 5A, The high concentration of NO donors (0.8 mM SNAP and 1.2 mM SNP) activated the phosphorylated p38MAPK (pp38) expression by Western blotting. 5B, Quantitation of pp38 immunostaining. pp38 was standardized with p38MAPK (p38) and ratios were expressed as arbitrary units. *P b 0.05; **P b 0.01 vs. control. 5C, The high NO concentrations induced CHOP/GADD153 expression as demonstrated by Western blotting. 5D, CHOP/GADD153 expression was normalized against β-actin, **P b 0.01. 5E and 5F, The expression of DR5 was increased by the high NO concentrations in time-dependent manner. 5G and 5H, The survivin expression, downregulated by high NO concentrations or by strong overexpression of iNOS plasmids, could be reversed by the p38MAPK-specific inhibitor SB203580. Blank was treated with PBS buffer. **P b 0.01. 6I and 6J, Apoptosis, induced by the high NO concentrations or by strong overexpression of iNOS plasmids, was significantly reduced by inhibitor SB 203580, **P b 0.01.
NO easily diffuses within a cell, as well as from cell to cell, NO can efficiently guard against aberrant activation of caspases. In addition to S-nitrosylation of caspases, several mechanisms for the antiapoptotic effect of NO have been proposed. They include (i) induction of cytoprotective stress proteins, e.g. HSP32 and HSP70; (ii) cGMP-
dependent inhibition of apoptotic signal transduction [41,42]. NO/ cGMP inhibition of apoptosis can involve the activation of cGMPdependent protein kinase and the inhibition of caspase activation. NO increases cGMP level through the activation of soluble guanylyl cyclase. The cGMP interrupts apoptotic signaling in some cell types,
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including hepatocytes, splenocytes, and neuronal PC12 cells [32]. NO activation of soluble guanylyl cyclase contributes to NO-mediated protection from TNFα/ActD-induced apoptosis, which is the dominant mechanism for protection in some cell types [40]. Through these mechanisms, NO can prevent the activation of downstream caspases and the terminal events in apoptosis such as the cleavage of the inhibitor of caspase-dependent activated DNase and the activation of caspase-dependent activated DNase [43,32]. In summary, iNOS/NO signaling regulated the GCDC-induced apoptosis in rat hepatocytes. Two mechanisms were involved. The high NO concentrations mediated survivin inhibition and CHOP activation that caused upregulation of TRAIL receptor DR5 and thus sensitized hepatocytes to TRAIL; The low NO concentrations led to the upregulation of survivin expression, contributing to cell survival. The antiapoptotic survivin, modulated by Akt/NF-κB, might be an essential factor in decreasing the GCDC-induced apoptosis in hepatocytes. Akt/ survivin and p38MAPK/CHOP will be new targets for designing hepatoprotective strategies and developing therapeutic application in the treatment of liver diseases. Acknowledgments This work was supported in part by the Falk Medical Research Foundation grant and National Institute of Health grant R21 DK070784-02. We appreciated Xiao Wang's help during preparation of the manuscript. References [1] A.K. Udit, W. Belliston-Bittner, E.C. Glazer, et al., J. Am. Chem. Soc. 127 (32) (2005) 11212–11213. [2] W.W. Chang, I.J. Su, W.T. Chang, W. Huang, H.Y. Lei, J. Viral Hepat. 15 (7) (2008) 490–497. [3] J. Abrams, Am. J. Cardiol. 77 (13) (1996) 31–37. [4] E.T. Crockett, J.J. Galligan, B.D. Uhal, J. Harkema, R. Roth, K. Pandya, BMC Clin. Pathol. 6 (2006) 3. [5] B.S. Taylor, L.H. Alarcon, T.R. Billiar, Biochemistry (Mosc) 63 (7) (1998) 766–781. [6] V. Baron, J. Hernandez, M. Noyola, B. Escalante, P. Muriel, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 127 (3) (2000) 243–249. [7] J. Li, C.A. Bombeck, S. Yang, Y.M. Kim, T.R. Billiar, J. Biol. Chem. 274 (24) (1999) 17325–17333. [8] K. Wang, J.J. Brems, R.L. Gamelli, J. Ding, J. Biol. Chem. 280 (25) (2005) 23490–23495. [9] B. Yerushalmi, R. Dahl, M.W. Devereaux, E. Gumpricht, R.J. Sokol, Hepatology 33 (3) (2001) 616–626.
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