Sivelestat Suppresses iNOS Gene Expression in Proinflammatory Cytokine-Stimulated Hepatocytes
Abstract
Background Recent evidence has indicated that sivele- stat, a neutrophil elastase inhibitor, has liver-protective effects in a variety of liver injuries. Proinflammatory cytokines including interleukin (IL)-1b stimulate the induction of inducible nitric oxide synthase (iNOS) gene expression, leading to excess production of NO and resulting in liver damage. We hypothesized that inhibition of iNOS induction underlies the protective effects of sivelestat on the liver. The objective of this study was to investigate whether sivelestat directly influences iNOS induction in cultured hepatocytes, which is used as a simple in vitro injury model, and to determine the mech- anism involved.
Methods Primary cultured rat hepatocytes were treated with IL-1b in the presence or absence of sivelestat. The induction of iNOS and its signaling pathway were analyzed.
Results Sivelestat inhibited the induction of iNOS mRNA and its protein, followed by decreased production of NO. Transfection and iNOS gene antisense-transcript experi- ments revealed that sivelestat reduced the levels of iNOS mRNA at both the promoter activation and mRNA stabil- ization steps. However, sivelestat had no effects on the degradation of IjB and nuclear translocation of NF-jB subunit p65, although it moderately blocked the activation of NF-jB. In contrast, sivelestat blocked the upregulation of IL-1 receptor I through the inactivation of phosphati- dylinositol 3-kinase/Akt.
Conclusions Delayed sivelestat addition experiments demonstrated that the destabilization of the iNOS mRNA contributed more significantly to the inhibitory effects of sivelestat than the reduction in iNOS mRNA synthesis. Sivelestat may provide useful therapeutic effects through the suppression of iNOS induction involved in liver injury.
Keywords Sivelestat · Inducible nitric oxide synthase · Liver injury · Primary cultured hepatocytes · Interleukin-1b · Antisense-transcript · mRNA stabilization · Nuclear factor-jB · Type I interleukin-1 receptor
Introduction
Post-extended hepatectomy frequently causes not only acute liver injury but also acute lung injury with systemic inflammatory response syndrome (SIRS) owing to either surgical stress or surgical site infection, which are related to postoperative death [1]. Sivelestat (ONO-5046) is a novel specific neutrophil elastase inhibitor [2], and is clinically used for acute lung injury with SIRS [3–5] mainly in Japan, although its efficacy is controversial worldwide [6]. Recent accumulated evidence has indicated that sivelestat also has liver-protective effects. Sivelestat reduced neutrophil chemoattractant production and mono- cyte chemoattractant protein-1 expression after ischemia– reperfusion (IR) in the rat liver [7, 8]. Sivelestat ameliorated IR damage in the canine and rat liver [9, 10] and after rat liver transplantation [11]. Sivelestat attenuated lipopolysaccharide (LPS)-induced hepatic microvascular dysfunction in mice [12] and prevented LPS-induced liver injury following experimental partial hepatectomy in rats [13]. Recently, Shimoda et al. [14] found that sivelestat reduced hepatic injury and stabilized the hemodynamics after IR in a pig hepatectomy model. However, the detailed mechanisms involved in the liver-protective effects of sivelestat against various injuries remain unclear.
In the inflamed liver, LPS and proinflammatory cyto- kines including interleukin (IL)-1b, tumor necrosis factor (TNF)-a, and interferon-c stimulate the induction of inducible nitric oxide synthase (iNOS) gene expression, followed by excess levels of nitric oxide (NO) production. NO production by iNOS has been implicated as one of the factors in liver injury, although NO has been reported to exert either detrimental or beneficial effects depending the insults and tissues involved. In animal models of liver injury caused by various insults, such as IR, partial hepa- tectomy and endotoxin shock, the induction of iNOS and NO production is upregulated concomitantly with the production of cytokines including TNF-a, IL-6, interferon-c, and cytokine-induced neutrophil chemoattractant-1 in the liver, as we reported previously [15–19]. In these studies, drugs showing liver-protective effects inhibited the induc- tion of iNOS and NO production as well as the decreased production of various inflammatory mediators. Further- more, in vitro experiments with primary cultured rat hepatocytes revealed that these drugs inhibited the induc- tion of iNOS and NO production [17, 20, 21]. Thus, the prevention of NO production is considered to be one of the indicators of liver protection. In the present study, we examined IL-1b-stimulated cultured hepatocytes as a sim- ple in vitro injury model for in vivo animal models. We hypothesized that sivelestat would directly inhibit the induction of iNOS and NO production in this in vitro system, and investigated the mechanism involved in its inhibitory effects.
Materials and Methods
Materials
Sivelestat sodium hydrate (ONO-5046 Na, sodium N-{2-[4-(2,2-dimethylpropionyloxy) phenylsulfonylamino] benzoyl} aminoacetate tetrahydrate) was generously pro- vided by Ono Pharmaceutical (Osaka, Japan). It was dis- solved in culture medium by sonication and adjusted to pH 7.4 with NaHCO3. Recombinant human IL-1b (2 9 107 U/mg protein) was provided by Otsuka Pharmaceutical Co. Ltd. (Tokushima, Japan). [c-32P]Adenosine-50-triphosphate
(ATP; -222 TBq/mmol) was obtained from DuPont-New England Nuclear Japan (Tokyo, Japan). Rats were kept at 22°C under a 12-h/12-h light/dark cycle, and received food and water ad libitum. All animal experiments were per- formed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health, and approved by the Animal Care Committee of Kansai Medical University.
Primary Cultures of Hepatocytes
Hepatocytes were isolated from male Wistar strain rats (200–220 g; Charles River, Tokyo, Japan) by collagenase (Wako Pure Chemicals, Osaka, Japan) perfusion [22, 23]. Isolated hepatocytes were suspended in culture medium at 6 9 105 cells/ml, seeded into 35-mm plastic dishes (2 ml/ dish; Falcon Plastic, Oxnard, CA, USA) and cultured at 37°C in a CO2 incubator under a humidified atmosphere of 5% CO2 in air. The culture medium was Williams’ medium E (WE) supplemented with 10% newborn calf serum, Hepes (5 mM), penicillin (100 U/ml), streptomycin (0.1 mg/ml), dexamethasone (10 nM), and insulin (10 nM). After 5 h, the medium was replaced with fresh serum- and hormone- free WE, and the cells were cultured overnight before use in experiments. The number of cells attached to the dishes were calculated by counting the nuclei [24] and using a ratio of 1.37 ± 0.04 nuclei/cell (mean ± SE, n = 7 experiments).
Treatment of Cells with Sivelestat
On day 1, the cells were washed with fresh serum- and hormone-free WE, and incubated with IL-1b (1 nM) in the same medium in the presence or absence of sivelestat. The doses of sivelestat used are indicated in the appropriate figures and their legends.
Determinations of NO Production and Lactate Dehydrogenase
Culture medium was used for measurements of nitrite (a stable metabolite of NO) to reflect NO production by the Griess method [25]. Culture medium was also used for measurements of LDH activity to reflect cell viability using a commercial kit (Wako Pure Chemicals).
Western-Blot Analysis
Total cell lysates were obtained from cultured cells as described previously [20] with minor modifications as fol- lows. Cells (1 9 106 cells/35-mm dish) were lysed in 100–200 ll of solubilizing buffer (10 mM Tris–HCl, pH 7.4, containing 1% Triton X-100, 0.5% Nonidet P-40, 1 mM EDTA, 1 mM EGTA, phosphatase inhibitor cocktail (Nacalai Tesque, Kyoto, Japan), 1 mM phenylmethylsulfo- nylfluoride (PMSF), and protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany)), passed through a 26-gauge needle, allowed to stand on ice for 30 min, and then centrifuged (16,000 9 g for 15 min). The supernatant (total cell lysate) was mixed with sodium dodecyl sulfate-poly- acrylamide gel electrophoresis (SDS-PAGE) sample buffer (final: 125 mM Tris–HCl, pH 6.8, containing 5% glycerol, 2% SDS and 1% 2-mercaptoethanol), subjected to SDS- PAGE and electroblotted onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA). Immunostaining was performed using primary antibodies against mouse iNOS (Affinity BioReagents, Golden, CO, USA), human IjBa, human IjBb, mouse type I IL-1 receptor (IL-1RI) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rat b-tubulin (internal control; Clone TUB2.1; Sigma Chemical Co., St. Louis, MO, USA), followed by visualization with an ECL blotting detection reagent (GE Healthcare Biosciences Corp., Piscataway, NJ, USA).
In the case of Akt, total cell lysates prepared from 100-mm dishes (5 9 106 cells/dish) were precleared with Protein A (Sigma Chemical Co.), and then mixed with a mouse monoclonal antibody against human Akt1 (Akt5G3; Cell Signaling, Beverly, MA, USA) and Protein G-Sepharose (Pharmacia LKB Biotech, Uppsala, Sweden). After incubation overnight at 4°C, the immunocomplexes were centrifuged (16,000 9 g for 5 min). The beads were washed with solubilizing buffer, dissolved in SDS-PAGE sample buffer, and analyzed by Western blotting using rabbit polyclonal antibodies against human Akt and phos- pho-(Ser473) Akt (Cell Signaling) as primary antibodies. In the case of p65, nuclear extracts were immunoprecipi- tated with an anti-p65 antibody (H286; Santa Cruz Bio- technology). The bands were analyzed by Western blotting using an antibody against human NF-jB p65 (BD Transduction Laboratories, Lexington, KY, USA).
Reverse Transcriptase-Polymerase Chain Reaction
Total RNA was extracted from cultured hepatocytes using a guanidinium-phenol–chloroform method [26] with Trizol reagent (Invitrogen, Carlsbad, CA, USA) or a phenol-free, filter-based total RNA isolation kit (RNAqueous Kit; Ambion, Austin, TX, USA) according to the manufacturer’s instructions, and then treated with a TURBO DNA-free Kit (Ambion) if necessary. For strand-specific RT-PCR analy- sis, cDNAs were synthesized from total RNA with strand- specific primers, and step-down PCR was performed using PC708 (Astec, Fukuoka, Japan), as previously described [27, 28] with minor modifications. For iNOS, IL-1RI and elongation factor-1a (EF; internal control) mRNAs, an oligo(dT) primer was used for RT and the primer sets 50-CCAACCTGCAGGTCTTCGATG-30 and 50-GTCGA TGCACAACTGGGTGAAC-30 (257-bp product), 50-CGA AGACTATCAGTTTTTGGAAC-30 and 50-GTCTTTCCA TCTGAAGCTTTTGG-30 (327-bp product), and 50-TCTG GTTGGAATGGTGACAACATGC-30 and 50-CCAGGAAGAGCTTCACTCAAAGCTT-30 (307-bp product) were used for PCR, respectively. For the antisense-transcript of iNOS, the sense primer 50-CCTTTGCCTCATACTTCCTC AGA-30 was used for RT and the primer set 50-ACCAGGAG GCGCCATCCCGCTGC-30 and 50-ATCTTCATCAAG-
GAATTATACACGG-30 (211-bp product) was used for PCR. The PCR protocols for iNOS and EF, or IL-1RI were: 10 cycles of (94°C, 1 min; 72°C, 2 min); 15 cycles of (94°C, 1 min; 65°C, 1 min 30 s; 72°C, 20 s); and 5, or 15 cycles of (94°C, 1 min; 60°C, 1 min 30 s; 72°C, 20 s), respectively. The PCR protocol for the antisense-transcript was: 10 cycles of (94°C, 1 min; 65°C, 1 min 30 s; 72°C, 20 s); 15 cycles of (94°C, 1 min; 60°C, 1 min 30 s; 72°C, 20 s); and 5 cycles of (94°C, 1 min; 55°C, 1 min 30 s; 72°C, 20 s). The amplified products were analyzed by 3% agarose gel electrophoresis with ethidium bromide, and the levels of iNOS, IL-1RI, EF and antisense-transcript were semi-quantified using a UV transilluminator. The cDNAs for the rat iNOS mRNA and antisense-transcript were deposited in DDBJ/EMBL/Gen- Bank under Accession Nos. AB250951 and AB250952, respectively.
Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared according to Schreiber et al. [29] with minor modifications [30]. Briefly, the dishes were placed on ice, washed with Tris–HCl-buffered saline, harvested with the same buffer using a rubber policeman and centrifuged (1,840 9 g for 1 min). The precipitate (2 9 106 cells from two 35-mm dishes) was suspended in 400 ll of lysis buffer (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 500 U/ml trasylol,
0.5 mM PMSF, and 1 mM dithiothreitol) and incubated on ice for 15 min. After addition of Nonidet P-40 (final: 0.625%), the cells were lysed by vortexing (2–3 times for 1 min each) and centrifuged (15,000 9 g for 1 min). The nuclear pellet was resuspended with extraction buffer (10 mM Hepes, pH 7.9, 0.4 M NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 500 U/ml trasylol, 0.5 mM PMSF and 1 mM dithiothreitol), followed by continuous mixing for 20 min and centrifugation (15,000 9 g for 5 min). Ali- quots of the supernatant (nuclear extract) were frozen in liquid nitrogen and stored at -80°C until use.
Binding reactions (total: 15 ll) were performed by incubating nuclear extract aliquots (4 lg of protein) in reaction buffer (20 mM Hepes, pH 7.9, 1 mM EDTA, 60 mM KCl, 10% glycerol and 1 mg of poly(dI-dC)) with the probe (approximately 40,000 cpm) for 20 min at room temperature. In the case of supershift assays, the nuclear extracts were incubated in the presence of anti-p50 and anti-p65 antibodies (nuclear factor (NF)-jBp50 (NLS) and NF-jBp65 (H286); Santa Cruz Biotech) or cold probes as a competitor (250-fold excess) for 30 min at 4°C, followed by incubation with the labeled probe. The products were electrophoresed at 100 V in a 4.8% polyacrylamide gel in high ionic strength buffer (50 mM Tris–HCl, 380 mM glycine, 2 mM EDTA, pH 8.5) and the dried gels were analyzed by autoradiography. An NF-jB consensus oli- gonucleotide (50-AGTTGAGGGGA-CTTTCCCAGGC-30) from the mouse immunoglobulin j light chain was pur- chased (Promega, Madison, WI, USA) and labeled with [c-32P]ATP and T4 polynucleotide kinase. The protein concentration was measured by the method of Bradford [31] with a binding assay kit (Bio-Rad) using bovine serum albumin as a standard.
Transfection and Luciferase Assay
Transfection of cultured hepatocytes was performed as described previously [32, 33]. Briefly, hepatocytes were digested with BamH I and Xba I. This cDNA for the iNOS 30-UTR (submitted to DDBJ/EMBL/GenBank under Accession No. AB250951) was used to replace the SV40 polyadenylation signal (SVpA) of pRiNOS-Luc to create pRiNOS-Luc-30UTR.
Construction of Luciferase Reporter Plasmids and Expression Plasmids
The 1.2-kb 50-flanking region including the TATA box of the rat iNOS gene was inserted into the pGL3-Basic vector (Promega) to create pRiNOS-Luc-SVpA [30]. A rat cDNA for the 30-untranslated region (UTR) of the iNOS mRNA was amplified with the primers 50-tgctctaGACAGTGAGG GGTTTGGAGAGA-30 and 50-gcggatcctttaTTCTTGATCA AACACTCATTTT-30, and the resultant cDNA was cultured at 4 9 105 cells/dish (35 9 10 mm) in WE sup- plemented with serum, dexamethasone and insulin for 7 h, before being subjected to magnet-assisted transfection (MATra). Reporter plasmids pRiNOS-Luc-SVpA or pRiNOS-Luc-30UTR (1 lg) and the CMV promoter-driven b-galactosidase plasmid pCMV-LacZ (1 ng) as an internal control were mixed with MATra-A reagent (1 ll; IBA GmbH, Go¨ttingen, Germany). After incubation for 15 min on a magnetic plate at room temperature, the medium was replaced with fresh WE containing serum. The cells were cultured overnight, and then treated with IL-1b in the presence or absence of sivelestat. The luciferase and (1 nM) in the presence or absence of sivelestat (5 mM) for 8 h for pRiNOS-Luc-SVpA (b) and 5 h for pRiNOS-Luc-30UTR (c). The luciferase activities were normalized by the b-galactosidase activity. The fold activation was calculated by dividing the luciferase activity by the control activity (without IL-1b and sivelestat). Data are means ± SD for n = 4 dishes. *p \ 0.05 vs. IL-1b alone. d Cells were treated with IL-1b (1 nM) in the presence or absence of sivelestat (5 mM) for the indicated times. Total RNA was analyzed by strand-specific RT-PCR to detect the iNOS gene antisense-transcript (AST). RT(-): a negative control PCR using total RNA without RT b-galactosidase activities of cell extracts were measured using PicaGene (Wako Pure Chemicals) and Beta-Glo (Promega) kits, respectively.
Fig. 2 Effects of sivelestat on cellular cytotoxicity. Cells were treated with IL-1b (1 nM) in the presence or absence of sivelestat (1–5 mM) for 8 h. The LDH activities were measured in the culture medium (data are means ± SD for n = 3 dishes/point).
Fig. 1 Effects of sivelestat on the induction of NO production and iNOS in proinflammatory cytokine-stimulated hepatocytes. Cultured hepatocytes were treated with IL-1b (1 nM) in the presence or absence of sivelestat (1–5 mM). a Effects of sivelestat (5 mM) treatment for the indicated times on NO production (IL-1b, open circles; IL-1b + sivelestat, filled circles; sivelestat, filled triangles; controls (without IL-1b and sivelestat), open triangles). b Effects of treatment with various doses of sivelestat (1-5 mM) for 10 h on NO production (top) and iNOS protein (middle). The levels of nitrite were measured in the culture medium (data are means ±SD for n = 3 dishes/point; *p \ 0.05 vs. IL-1b alone). In the Western-blotting panels, cell lysates (20 lg of protein) were subjected to SDS-PAGE in a 7.5% gel, and immunoblotted with an anti-iNOS or anti-b-tubulin antibody. c Effects of sivelestat (5 mM) treatment for the indicated times on the expression of iNOS mRNA. Total RNA was analyzed by strand-specific RT-PCR to detect iNOS mRNA, using EF mRNA as an internal control.
Fig. 3 Effects of sivelestat on the transactivation of the iNOS promoter and the expression of the iNOS gene antisense-transcript. a Schematic representation of the promoter region of the iNOS gene. Two reporter constructs are shown beneath the iNOS gene and mRNA. The constructs consist of the rat iNOS promoter (1.2 kb), a luciferase gene and the SV40 poly(A) region (pRiNOS-Luc-SVpA) or iNOS 30-UTR (pRiNOS-Luc-30UTR). ‘An’ indicates the presence of a poly(A) tail. The iNOS 3’-UTR contains AREs (AUUU(U)A 9 6), which contribute to mRNA stabilization. b, c Each construct was introduced into hepatocytes, and the cells were treated with IL-1b.
Statistical Analysis
The results shown in the figures are representative of 3–4 independent experiments yielding similar findings. Differ- ences were analyzed by the Bonferroni-Dunn test, and values of p \ 0.05 were considered to indicate statistical significance.
Results
Sivelestat Inhibits the Induction of iNOS in IL-1b-Stimulated Hepatocytes
The proinflammatory cytokine IL-1b stimulates iNOS induction, which is followed by the production of NO in primary cultured rat hepatocytes [34]. Simultaneous addi- tion of sivelestat with IL-1b inhibited the levels of nitrite (an NO metabolite) in the culture medium in time- and dose-dependent manners (Fig. 1a, 1b). Sivelestat exerted its maximal effects at concentrations of 4–5 mM, and decreased NO production to almost basal levels. Western-blotting analysis revealed that sivelestat also inhibited the expression of iNOS protein in a dose-dependent manner, with maximal effects at 4–5 mM (Fig. 1b, middle). Furthermore, RT-PCR analysis revealed that sivelestat reduced the levels of iNOS mRNA in a time-dependent manner (Fig. 1c). These results suggested that sivelestat inhibited the induction of iNOS gene expression at a transcriptional and/or post-transcrip- tional step. Although the concentrations of sivelestat used were higher than those used in clinical treatment, it showed no cellular cytotoxicity, as evaluated by the release of LDH into the culture medium (Fig. 2) and Trypan blue exclusion by hepatocytes (data not shown).
Effects of Sivelestat on iNOS Promoter Activation and iNOS mRNA Stabilization
We examined the mechanisms involved in the inhibition of iNOS expression. It is known that the levels of iNOS mRNA are regulated by iNOS promoter transactivation with tran- scription factors such as NF-jB and by posttranscriptional modifications such as mRNA stabilization [35]. Therefore, we carried out transfection experiments with iNOS promoter- firefly luciferase constructs, namely pRiNOS-Luc-SVpA and pRiNOS-Luc-30UTR (untranslated region) (Fig. 3a), which detect iNOS mRNA synthesis and its stabilization, respec- tively [36, 37]. IL-1b increased the luciferase activities of c Nuclear translocation of NF-jB subunit p65. Nuclear extracts were immunoprecipitated, and the immunoprecipitates were analyzed by Western blotting with an anti-p65 antibody. d Supershift assay. Nuclear extracts were incubated with a labeled NF-jB consensus oligonucleotide in the presence of an anti-p50 antibody, anti-p65 antibody or cold probe as a competitor (Comp, 250-fold excess), and were analyzed by EMSA. Open and filled arrows show supershifted bands these constructs, and sivelestat significantly reduced both of these luciferase activities (Fig. 3b, 3c). We recently reported the expression of an iNOS gene antisense-transcript that interacts with the 30-UTR containing AU-rich elements (AREs) of iNOS mRNA and its ARE-binding proteins, thereby leading to iNOS mRNA stabilization in cytokine- stimulated hepatocytes [38]. In support of the latter obser- vation (Fig. 3c), iNOS antisense-transcript analysis by RT-PCR revealed that IL-1b increased the expression of the iNOS gene antisense-transcript in a time-dependent manner, and that sivelestat markedly reduced the levels of the anti- sense-transcript (Fig. 3d).
Fig. 4 Effects of sivelestat on the degradation of IjB proteins and activation of NF-jB. Cells were treated with IL-1b (1 nM) in the presence or absence of sivelestat (5 mM) for the indicated times. a Cell lysates (20 lg of protein) were subjected to SDS-PAGE in a 12.5% gel, followed by immunoblotting with an anti-IjBa, anti-IjBb, or anti-b-tubulin antibody. b Activation of NF-jB. Nuclear extracts (4 lg of protein) were analyzed by EMSA (upper). The bands corresponding to NF-jB were quantified by densitometry (lower, means ± SD for n = 5 experiments; *p \ 0.05 vs. IL-1b alone).
Effects of Sivelestat on the Activation of NF-jB and Upregulation of IL-1RI
Two signaling pathways, the activation of NF-jB (its translocation from the cytoplasm to the nucleus, and DNA binding) through the degradation of IjB and the upregula- tion of IL-1RI through the activation of phosphatidylino- sitol 3-kinase (PI3K)/Akt, are essential for iNOS induction [20, 21, 39, 40]. IL-1b stimulates the degradation of IjB proteins, which is followed by the activation of NF-jB. Sivelestat did not inhibit the degradation of IjBa and IjBb at 0.5 h and rather decreased their recovery at 1 h and thereafter (Fig. 4a). EMSAs with nuclear extracts revealed that sivelestat had no effects at 1–3 h, but suppressed NF-jB activation moderately at 4–5 h (Fig. 4b). However, Western blotting of nuclear extracts revealed that sivelestat did not reduce the levels of NF-jB subunit p65 in the nucleus at 4 h (Fig. 4c). Furthermore, supershift assay demonstrated that sivelestat did not influence the compo- nents of NF-jB, p50, and p65 (Fig. 4d).
IL-1b also stimulates the upregulation of IL-1RI through the activation of PI3 K/Akt [39]. We found that sivelestat inhibited the phosphorylation of Akt (Fig. 5a). RT-PCR and Western-blot analyses revealed that sivelestat reduced the expression of IL-1RI mRNA and protein, respectively (Figs. 5b, 5c).
Fig. 5 Effects of sivelestat on the upregulation of IL-1RI. Cells were treated with IL-1b (1 nM) in the presence or absence of sivelestat (5 mM) for the indicated times. a Phosphorylation of Akt. Total cell lysates were immunoprecipitated with an anti-Akt antibody, followed by immunoblotting with an anti-phospho-Akt or anti-Akt antibody. b Total RNA was analyzed by strand-specific RT-PCR to detect IL-1RI mRNA, using EF mRNA as an internal control. c Cell lysates (50 lg of protein) were subjected to SDS-PAGE in a 7.5% gel, and immunoblotted with an anti-IL-1RI or anti-b-tubulin antibody the steps of both its mRNA synthesis and stabilization in proinflammatory cytokine-stimulated hepatocytes (Fig. 3). In the former, sivelestat probably reduced the transactivation of the iNOS promoter (Fig. 3b) at least partly through the inhibition of NF-jB activation (Fig. 4b). However, sivele- stat did not inhibit IjBa and IjBb degradations (Fig. 4a) and p65 nuclear translocation (Fig. 4c), indicating that sivelestat cannot influence the nuclear translocation of NF-jB through IjB degradation. In concert with NF-jB translocation, the upregulation of IL-1RI, which stimulates the phosphoryla- tion of NF-jB subunit p65, is required for transcriptional activation of the iNOS gene, as we reported previously [39]. In the present study, we found that sivelestat decreased the expression of IL-1RI mRNA and protein (Fig. 5b and 5c) through the inhibition of Akt phosphorylation (Fig. 5a), presumably leading to the inhibition of p65 phosphorylation and decreased DNA binding of NF-jB and resulting in decreased activities of iNOS promoter transactivation. However, it cannot negate the possibility that sivelestat may affect iNOS mRNA synthesis through other transcription factors rather than NF-jB.
Regarding the iNOS mRNA stabilization, the 30-UTR of the iNOS mRNA in rats has six AREs (AUUU(U)A), which are associated with ARE-binding proteins such as HuR and heterogeneous nuclear ribonucleoproteins L/I (PTB), thus contributing to the stabilization of the mRNA [41]. Recently, we found that the antisense strand corre- sponding to the 30-UTR of iNOS mRNA is transcribed from the iNOS gene, and that the iNOS mRNA antisense- transcript plays a key role in stabilizing the iNOS mRNA by interacting with the 30-UTR and ARE-binding proteins [38]. In our in vitro model, sivelestat destabilized the iNOS mRNA through the inhibition of iNOS gene antisense- transcript expression (Fig. 3c and 3d). Drugs such as eda- ravone (free-radical scavenger) [21], FR183998 (Na+/H+ exchanger inhibitor) [17, 19] and insulin-like growth factor I [18] were found to inhibit iNOS induction partly by suppressing iNOS antisense-transcript production in dam- aged liver and primary cultured hepatocytes.
Delayed treatment with sivelestat was found to cause a significant reduction in NO production and iNOS induction (Fig. 6a, 6b and 6d). These observations may be of clinical importance, since the initiation of therapeutic sivelestat treatment is usually delayed from the onset of diseases. In the case of the 3-h-delayed treatment, sivelestat was found to have no effect on the activation of NF-jB (Fig. 6c). We supposed that delayed treatment with sivelestat could not influence the increased expression of IL-1RI mRNA by Akt activation, which are involved in iNOS promoter transac- tivation through the phosphorylation of NF-jB subunit p65 as mentioned before, since these events stimulated by IL-1b were almost finished and returned to the basal levels at 3 h (Fig. 5a and 5b). In contrast, delayed sivelestat treatment maintained the inhibitory effect on the iNOS antisense-transcript expression (Fig. 6e), suggesting that the destabilization of iNOS mRNA largely contributed to the decreased levels of iNOS mRNA and protein. Delayed sivelestat treatment was previously found to attenuate acute lung injury in hamsters [42].
These results suggest that sivelestat can inhibit the induction of iNOS expression in liver injury, which is involved in the liver-protective effects of sivelestat. Shimoda et al. [14] reported that sivelestat decreased the anti-b-tubulin antibody. c–e Cells were treated with sivelestat (5 mM) at 3 h after IL-1b addition. The effects of sivelestat on the activation of NF-jB (c), and the expressions of iNOS mRNA (d) and iNOS gene antisense-transcript (AST) (e) were analyzed at the indicated times after IL-1b addition. Nuclear extracts (4 lg of protein) were analyzed by EMSA. Total RNA was analyzed by strand-specific RT-PCR to detect iNOS mRNA, using EF mRNA as an internal control, and the iNOS gene antisense-transcript (AST) serum levels of NO in a pig hepatectomy model with hepatic IR injury. Sivelestat inhibition of iNOS induction seems to be common in other inflammatory cells. Hagiwara et al. [43] reported that LPS stimulated the induction of iNOS and NO production in addition to inflammatory mediators such as TNF-a and IL-6 in the mouse cell line RAW264.7. Sivelestat decreased the production of these mediators, and consequently inhibited iNOS induction through the inhibition of NF-jB activation by blocking IjB phosphorylation and its degradation. These findings are partly similar to our present findings in hepatocytes, and suggest that the inhibitory effects of sivelestat are not specific for hepatocytes.
Fig. 6 Effects of delayed sivelestat administration on the induction of iNOS in hepatocytes. Cultured hepatocytes were treated with sivelestat (5 mM) at 0–4 h after the addition of IL-1b (1 nM). a, b The effects of sivelestat on NO production (a) and iNOS protein (b) were analyzed at 8 h after IL-1b addition. The levels of nitrite were measured in the culture medium (data are means ± SD for n = 3 dishes/point; *p \ 0.05 vs. IL-1b alone). In the Western- blotting panels, cell lysates (20 lg of protein) were subjected to SDS-PAGE in a 7.5% gel, and immunoblotted with an anti-iNOS.
Recently, we found that another neutrophil elastase inhibitor, FR136706, attenuated the lethal acute liver fail- ure induced by D-galactosamine/LPS in rats, and sup- pressed the induction of iNOS [44]. Thus, our simple in vitro experiment with cultured hepatocytes may be ade- quate for screening of liver-protective drugs, because it is rapid and inexpensive compared with in vivo animal models of liver injury. However, the liver-protective effects of drugs deduced from this model need to be examined and supported in in vivo animal models.
In conclusion, the neutrophil elastase inhibitor sivelestat inhibited iNOS gene expression at transcriptional and posttranscriptional steps in cultured rat hepatocytes in an in vitro liver injury model. Sivelestat may have therapeutic potential not only for acute lung injury but also for various liver injuries.
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