- Email: [email protected]
Accelerative effect of leflunomide on recovery from hepatic fibrosis involves TRAIL-mediated hepatic stellate cell apoptosis
Life Sciences 84 (2009) 552–557
Contents lists available at ScienceDirect
Life Sciences 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 / l i f e s c i e
Accelerative effect of leflunomide on recovery from hepatic fibrosis involves TRAIL-mediated hepatic stellate cell apoptosis Xiaoming Tang a,1, Juntao Yang b,1, Jun Li a,⁎ a b
School of Pharmacy, Anhui Medical University, Hefei, Anhui Province 230032, China State Key Laboratory of Proteomics, Beijing Proteomics Research Center, Beijing Institute of Radiation Medicine, Beijing 100850, China
a r t i c l e
i n f o
Article history: Received 29 October 2008 Accepted 28 January 2009 Keywords: Hepatic fibrosis Hepatic stellate cells Kupffer cells TRAIL Leflunomide
a b s t r a c t Aims: Hepatic fibrosis is reversible, associated with apoptosis of activated hepatic stellate cells (HSCs) as injury subsides, thus providing potential targets for therapy. Little is known, however, about the course of this condition. The objective of this study was to elucidate the mechanism by which Kupffer cells regulate HSC biology during regression of hepatic fibrosis and the effect of leflunomide on this process. Main methods: We harvested Kupffer cells from rats during spontaneous recovery from liver fibrosis induced by carbon tetrachloride (CCl4) and prepared recovery Kupffer cell conditioned medium (KCCM). Cultureactivated HSCs were pretreated in the absence or presence of A771726, the active metabolite of leflunomide, and then stimulated with recovery KCCM. Key findings: Following stimulation with recovery KCCM, HSCs showed a decrease in proliferation and an increase in apoptosis by a caspase-dependent mechanism. Furthermore, pretreatment with A771726 markedly enhanced these effects. Real-time quantitative PCR (Q-PCR) analysis showed increased expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in Kupffer cells during the spontaneous recovery phase. The pro-apoptotic function of KCCM prepared from TRAIL siRNA-treated Kupffer cells was obviously decreased, suggesting that TRAIL played an important role in recovery from hepatic fibrosis. Moreover, A771726 enhanced recovery KCCM-induced apoptosis of HSCs by a mechanism involving the inhibition of nuclear factor-kappa B (NF-κB) activation. Significance: Our results showed the role of TRAIL in the apoptosis of activated HSCs that is induced by Kupffer cells prepared from livers recovering from CCI4-induced fibrosis and provided insights into the resolution of fibrosis and the mechanisms by which leflunomide might act upon liver fibrosis. © 2009 Elsevier Inc. All rights reserved.
Introduction Hepatic fibrosis is a common consequence of chronic liver injury of any etiology (Friedman 2008a,b), and hepatic stellate cells (HSCs) are the major source of increased extracellular matrix (ECM) proteins in chronic liver diseases (Iredale 2001). If hepatic injury persists, HSCs proliferate and undergo dramatic transdifferentiation from quiescent vitamin A-storing cells to activated myofibroblast-like cells (Geerts et al. 1991). These activated HSCs secrete large amounts of ECM proteins, which is a seminal event in hepatic fibrogenesis. The factors that modulate HSC activation influence the progression of liver fibrosis (Pinzani and Marra 2001). Kupffer cells, the resident macrophages in the liver, act as a double-edged sword in the progression of and recovery from hepatic fibrosis (Friedman 2005). It has been widely reported that Kupffer cells can promote HSC activation during the progression of fibrosis (Ikejima et al. 1999;
⁎ Corresponding author. Tel./fax: +86 551 5161001. E-mail address: [email protected] (J. Li). 1 These authors contributed equally to this work. 0024-3205/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2009.01.017
Shiratori et al. 1986). On the other hand, Kupffer cells can negatively regulate activated HSCs during recovery from liver fibrosis associated with enhancing matrix degradation (Duffield et al. 2005). The mechanism by which Kupffer cells regulate HSC biology during regression of hepatic fibrosis, however, is still far from being fully understood. Leflunomide, an isoxazole derivative, is a unique immunomodulatory agent, capable of treating rheumatoid arthritis (RA) (Fox et al. 1999), allograft and xenograft rejection (Groth et al. 1999), and systemic lupus erythematosus (Manna et al. 2000). After administration, it is metabolized to its active form, A771726, which leads to the immunosuppressive activity (Davis et al. 1996). It has also been reported to block cell cycle progression in the G0/G1 phase (Xu et al. 1995). Recent evidence has suggested that A771726 can significantly inhibit the proliferation and activation of HSCs by inhibiting the Janus kinase (JAK) and protein kinase B (Akt) pathways (Si et al. 2007). In vivo, A771726 showed an inhibitory effect on carbon tetrachloride (CCl4)-induced hepatic fibrosis in rats (Yao et al. 2004). Based on these results, the aim of this study was to elucidate the mechanism by which Kupffer cells regulate HSC biology during regression of hepatic fibrosis and the effect of leflunomide on this process.
X. Tang et al. / Life Sciences 84 (2009) 552–557
Materials and methods Reagents Antibodies against caspase 3, 9, Poly (ADP-ribose) polymerase (PARP), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), β-actin and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and goat anti-rabbit IgG antibody were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Fluorescein isothiocyanate (FITC)-labeled bromodeoxyuridine (BrdU) antibody and annexin V-FITC were purchased from BD Pharmingen. Special chemical inhibitors Z-VAD-FMK, Z-DEVD-FMK, Z-IETD-FMK, Z-LEHDFMK and BAY 11-7082 were from Calbiochem (La Jolla, CA). Collagenase, proteinase E, DNase I, Nycodenz, Dulbeccos Modified Eagles Medium (DMEM), fetal bovine serum (FBS), and powdered 1640 medium were obtained from Gibco (Grand Island, NY). Leflunomide and its active metabolite, A771726, were kindly donated by Cinkate Co. (Shanghai, China). Animals and treatment Adult male Sprague-Dawley (SD) rats weighing 200–250 g were purchased from the Animal Center of Anhui Medical University. Animals were housed in standard facilities and all animals used in this study received humane care in compliance with the institution's guidelines. Male SD rats were intraperitoneally injected with 1.0 ml/ kg body weight of a 1:1 (v/v) solution of CCl4 and olive oil twice weekly for 8 weeks. The control group received the same volume of olive oil. Animals were sacrificed at 14 days of spontaneous recovery (Issa et al. 2003; Iredale et al. 1998). Isolation and culture of Kupffer cells Kupffer cells were isolated by collagenase digestion, differential centrifugation and selective plating as described previously (Friedman and Roll 1987), with slight modifications. Livers were perfused in situ through the portal vein with collagenase. After digestion, livers were minced in collagenase buffer, and the suspension was filtered through nylon mesh. The filtrate was separated into parenchymal and nonparenchymal fractions by differential centrifugation. The Kupffer cell fraction was collected from the non-parenchymal fraction by density gradient centrifugation. To further purify Kupffer cells, cells were seeded on tissue culture plates and cultured at 37 °C with 5% CO2 for 30 min, followed by a single wash step to discard the non-adherent cells. Cell viability, as determined by trypan blue dye exclusion assay, was 95%. Immunohistochemistry and fluoroscopy revealed a purity of N85% Kupffer cells for the Kupffer cell cultures. Preparation of Kupffer cell conditioned medium (KCCM) Isolated Kupffer cells were cultured in 24-well tissue culture plates. Cells were washed and incubated in serum-free medium for 48 h. Conditioned medium was collected by centrifugation at 6000 g to remove cell debris. KCCM was filtered with a 0.45 μM membrane filter and stored at −70 °C until use. Using the method described above, we harvested Kupffer cells from rats during spontaneous recovery from liver fibrosis induced by CCl4 (the recovery Kupffer cells) and prepared recovery KCCM. We also harvested Kupffer cells from rats in the control group (the normal Kupffer cells) and prepared normal KCCM.
553
centrifugation. HSCs were purified by Nycodenz gradient centrifugation and cultured (at a density of 1 × 105 cells/ml) in DMEM containing 15% fetal calf serum, 2 mmol/l glutamine, and 1% antibiotic solution under an atmosphere containing 5% CO2 at 37 °C. Purity of the HSC preparation (N95%) was assessed by typical light microscopic appearance and vitamin A-specific autofluorescence. HSCs were used for experiments after 7 days of culture to induce activation. Cultures were initiated at a cell density of 1 × 105 cells/ml, and all treatments were given after HSCs were cultured with serum starvation for 24 h, unless otherwise noted. Cell proliferation assay Briefly, HSCs were seeded in triplicate in 6-well plates. They were then pretreated with A771726 (0.1, 1.0, 10 μM) for 2 h and stimulated with KCCM for 48 h. For the cell proliferation assay, cells were incubated with BrdU at a final concentration of 10 μM for 2 h at 37 °C in a controlled atmosphere. Cells were harvested and fixed in cold (4 °C) 70% ethanol and left for at least 30 min at 4 °C. The cells were then resuspended in 2 M hydrochloric acid and left at room temperature for 30 min. The acid was removed by centrifugation, and the cell pellets were washed twice in PBST, followed staining with 20 µl FITC-conjugated anti-BrdU antibody (Becton Dickinson). Incorporation was quantified with a FACScalibur (BD Bioscience). For the apoptosis assay, treated cells were harvested and washed in PBS, followed by staining with 20 µl of FITCconjugated annexin V (Becton Dickinson) and analysis by flow cytometry. All experiments were repeated at least three times. Western blot analysis HSCs (or Kupffer cells) were collected and washed in cold phosphate buffered saline (PBS) and lysed in ice-cold RIPA buffer (10 μM Tris–HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and 10 μl/ml protease inhibitor cocktail) for 30 min on ice. The extracted cell protein from each sample was applied to a 7–12% SDS-polyacrylamide gel and transblotted to nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany). Protein bands were visualized using an ECL assay kit (Amersham Biosciences, Buckinghamshire, UK). Quantitative real-time PCR (Q-PCR) Total RNA was extracted using Trizol reagent (Invitrogen) and digested with RNase-free DNaseI (Qiagen Ltd.). First-strand cDNA synthesis was performed using a Reverse Transcript-PCR kit with oligo(dT) primers (Promega, Madison, WI). Quantitative realtime PCR (Q-PCR) was performed using SYBR® Green I Q-PCR MasterMix (Bio-RED) and a Bio-RED IQ5 (Bio-RED) according to the manufacturer's instructions. The mRNA expression levels of tumor necrosis factor-α (TNF-α), Fas ligand (FasL) and TRAIL were calculated using the threshold cycle (Ct) values. All Ct values were normalized to the housekeeping gene, β-actin. Specific primers used in the Q-PCRs were as follows: TRAIL (5′-GCTTCAGTCAGACATTCACG-3′, 5′GTCCCAAAAATCCCCATCTT-3′), TNF-α (5′- GACCCTCACACTCAGATCATCTTCT-3′, 5 ′ -T G C TAC GAC GT G G G C TAC G - 3 ′ ) , Fa s L ( 5 ′ TGCCTCCACTAAGCCCTCTA-3′, 5′-AGGCTGTGGTTGGTGAACTC-3′), β-actin (5′-GGGAAATCGTGCGTGACATT-3′, 5′-GCGGCAGTGGCCATCTC3′). All experiments were repeated at least three times. RNA interference
Isolation and culture of HSCs HSCs were isolated from normal male SD rats by sequential in situ perfusion with collagenase and protease as described previously (Friedman and Arthur 1989), with slight modifications. Briefly, nonparenchymal liver cells were obtained by enzymatic digestion and
The expression vector for rat TRAIL was prepared by cloning the PCRamplified cDNA into pcDNA 3.1A (Clonetech). The primers used for PCR amplification of rat TRAIL were as follows: Forward, 5′-CGCGGATCCATGGCTTCCACC-3′ and Reverse, 5′- GGAATTCGGCTCCAAAGAAGCTG-3′. The siRNAs designed to knock down rat TRAIL and the control non-specific
554
X. Tang et al. / Life Sciences 84 (2009) 552–557
be statistically significant. Data are presented as mean ± Standard Deviation (S.D.) from three independent experiments. Results Effect of recovery KCCM on activated HSCs To determine the effect of Kupffer cells in the resolution of hepatic fibrosis, we used recovery KCCM to stimulate culture-activated HSCs. BrdU cell proliferation assays showed that recovery KCCM suppressed the proliferation of HSCs. If HSCs were pretreated with A771726, the effect of the recovery KCCM was enhanced (Fig. 1A). The analysis of apoptosis showed that recovery KCCM increased the apoptotic rate of HSCs; this could also be enhanced by pretreatment with A771726 in a dose-dependent manner (Fig. 1B). These results suggest that Kupffer cells from the rats undergoing spontaneous recovery from hepatic fibrosis can negatively regulate culture-activated HSCs and that A771726 can significantly enhance this effect. Recovery KCCM-induced apoptosis of HSCs was caspase-dependent
Fig. 1. Effect of recovery KCCM on activated HSCs. (A) BrdU cell proliferation assays. HSCs labeled in vitro with BrdU were stained with anti-BrdU-FITC and analyzed by flow cytometry. (B) Cell apoptosis assays. HSCs were incubated with annexin V-FITC and examined by flow cytometry. Results are the mean±S.D. from three independent experiments. #Pb 0.01 versus serum control values; ⁎Pb 0.05, ⁎⁎Pb 0.01 versus recovery KCCM values.
We detected caspases activation and PARP cleavage in recovery KCCM-treated HSCs by western blot analysis. The level of the active subunit of caspase 3 was increased; PARP was cleaved to its signature peptide; and procaspase-9 levels were reduced, suggesting that caspase signaling pathways were involved in recovery KCCM-induced apoptosis of HSCs (Fig. 2A). Furthermore, prior treatment with the
siRNA (NS siRNA) were purchased from Genepharm Co (China). Their sequences were as follows: siRNA1, 5′-CGAUGACGGUGAUCUGCAUtt-3′, 5′-AUGCAGAUCACCGUCAUCGTT-3′; siRNA2, 5′-CAACGAGGUGAAACAGCUATT-3′, 5′-UAGCUGUUUCACCUCGUUGTT-3′; siRNA3, 5′-GGAUCACUCGGAGAAGCAATT-3′, 5′-UUGCUUCUCCGAGUGAUCCTT-3′; siRNA4, 5′-CCUUAAUUCCAAUCUCCAATT-3′, 5′-UUGGAGAUUGGAAUUAAGGTT-3′. The siRNAs and TRAIL expression vector were co-transfected into HEK 293 cells using Lipofectamine 2000 Reagent (Invitrogen). Silencing of TRAIL was determined by western blotting of cell lysates with an anti-Myc antibody. The effective siRNA was transfected into the recovery Kupffer cells. Knockdown of TRAIL by siRNA was confirmed by western blotting with an anti-TRAIL antibody. After 24 h, the medium was changed. The recovery Kupffer cells transfected with the effective siRNA were incubated in serum-free medium for 48 h to collect the TRAIL siRNA-treated KCCM. Nuclear factor-kappa B (NF-κB) luciferase reporter gene assay HSCs were transfected with pNFκB-Luc (Clontech) and pRL-CMV (Promega, Madison, WI) plasmids using FuGENE 6 (Boehringer Mannheim) and incubated for 18 h at 37 °C. Cells were pretreated with A771726 (0.1, 1.0, 10 μM) or BAY 11-7082 (5 μM) for 2 h and stimulated with KCCM for 4 h. Cells were lysed in a passive lysis buffer (Promega). Luciferase activity was measured using a Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol. Fold induction of NF-κB-luciferase for each treatment was based on untreated values normalized to the fold induction of pRL-CMV reporter values. All experiments were repeated at least three times. Statistical analysis Student's t-test was used for determination of statistical significance as appropriate. Statistical values of P b 0.05 were considered to
Fig. 2. Recovery KCCM-induced apoptosis of HSCs was caspase-dependent. (A) Western blot analysis of caspase-3, PARP and procaspase-9 in HSCs. Two hours before stimulation, HSCs were treated with A771726 (0.1, 1.0, 10 μM). The cell lysates were collected 24 h later. (B) The effect of caspase inhibitors on recovery KCCM-induced apoptosis of HSCs. HSCs were stimulated with recovery KCCM in the absence or presence of caspase inhibitors (50 μmol/l). Results are the mean ± S.D. from three independent experiments.⁎P b 0.05.
X. Tang et al. / Life Sciences 84 (2009) 552–557
555
Fig. 3. Q-PCR analysis of the expression of cytokines in Kupffer cells. Relative expression levels of TNF-α, FasL and TRAIL mRNA were determined using β-actin as an internal control. Results are the mean ± S.D. from three independent experiments. ⁎P b 0.05.
pancaspase inhibitor Z-VAD-FMK, the caspase-3 inhibitor Z-DEVDFMK, the caspase-8 inhibitor Z-IETD-FMK, or the caspase-9 inhibitor Z-LEHD-FMK could clearly inhibit recovery KCCM-induced apoptosis of HSCs (Fig. 2B). These results indicate that recovery KCCM induced apoptosis of HSCs via caspase-dependent pathways. Role of TRAIL in recovery KCCM-induced apoptosis of HSCs To determine the molecular mechanisms underlying the proapoptotic activity of recovery Kupffer cells, Q-PCR was used to detect the expression of pro-apoptotic cytokines in Kupffer cells. The results showed that the expression of FasL and TNF-α did not clearly change; however, the expression of TRAIL significantly was increased in recovery Kupffer cells (Fig. 3). Furthermore, RNA interference was used to confirm the role of TRAIL in recovery KCCM-induced apoptosis of HSCs. The effective siRNA against TRAIL was obtained from screening. The results showed that 2′, 3′ siRNAs were effective in suppressing the expression of TRAIL (Fig. 4A). Expression of TRAIL in the recovery Kupffer cells was reduced by transfection of 3′ siRNA (Fig. 4B). The siRNA-treated KCCM was collected to stimulate cultureactivated HSCs. The apoptotic rate in HSCs stimulated with TRAIL siRNA-treated KCCM was decreased compared with a negative control (Fig. 4C). Altogether, these data show that TRAIL plays an important role in recovery KCCM-induced apoptosis of HSCs.
Fig. 4. TRAIL was involved in recovery KCCM-induced apoptosis of HSCs. (A) Screening of effective siRNAs targeting TRAIL. The silencing effect of each siRNA was detected by western blot assay. (B) TRAIL expression in siRNA-transfected Kupffer cells. The level of endogenous TRAIL was estimated by western blot assay. (C) Effect of siRNA-treated KCCM on apoptosis of HSCs. Results are the mean ± S.D. from three independent experiments. ⁎P b 0.05.
Effect of A771726 on NF-κB activation in recovery KCCM-induced apoptosis of HSCs A luciferase reporter gene assay showed that recovery KCCM could induce NF-κB activation in HSCs. Pretreatment of HSCs with A771726 could significantly inhibit NF-κB activation in a dose-dependent manner (Fig. 5A). BAY 11-7082, an NF-κB inhibitor, was used to determine whether NF-κB was involved in the enhancing effect of A771726 on recovery KCCM-induced apoptosis of HSCs. Inhibition of NF-κB activity by BAY 11-7082, as well as A771726, was effective in enhancing the cytotoxicity of recovery KCCM in HSCs (Fig. 5B). These results suggest that A771726 can accelerate recovery KCCM-induced apoptosis of HSCs by inhibiting NF-κB activation in HSCs. Discussion Liver fibrosis results from persistent damage to the liver (Guo and Friedman 2007). With stimulation by hepatotoxic agents, resident
Fig. 5. Effect of A771726 on NF-κB activation in recovery KCCM-induced apoptosis of HSCs. (A) NF-κB reporter gene assays. NF-κB-luc-transfected HSCs were pretreated with A771726 or BAY 11-7082 for 2 h followed by treatment with recovery KCCM. Cell lysates were collected to detect NF-κB-luc reporter activity. (B) Cell apoptosis assays. HSCs were pretreated with A771726 or BAY 11-7082 for 2 h followed by treatment with recovery KCCM. Results are the mean ± S.D. from three independent experiments. #P b 0.01 versus serum control values; ⁎P b 0.05, ⁎⁎P b 0.01 versus recovery KCCM values.
556
X. Tang et al. / Life Sciences 84 (2009) 552–557
HSCs are activated into fibrogenic myofibroblasts, which synthesize and release large amounts of ECM proteins and tissue inhibitors of metalloproteinases (TIMPs). The degradation of ECM proteins and collagen is inhibited by cytokines such as TIMPs. Eventually, large amounts of ECM proteins accumulate and lead to liver fibrosis (Benyon and Arthur 2001). Recent research has indicated that hepatic fibrosis is reversible as liver injury subsides (Issa et al. 2004). Spontaneous recovery from hepatic fibrosis includes the degradation of ECM proteins, apoptosis of activated HSCs, and regeneration of hepatocytes. It has been reported that Kupffer cells can negatively regulate activated HSCs during recovery from liver fibrosis (Friedman 2008a,b; Issa et al. 2001; Fischer et al. 2002). The mechanism behind this process, however, is still not clear. In this study, we demonstrated that recovery KCCM could inhibit the proliferation of activated HSCs and induce their apoptosis by a mechanism involving caspase activation. Caspase inhibitors could inhibit recovery KCCM-induced apoptosis of HSCs. Q-PCR showed that the expression of TRAIL in recovery phase Kupffer cells was significantly increased. These results suggest that TRAIL might function during the recovery phase. TNF and TNF-receptor superfamily proteins play important roles in inflammatory responses (Choo-Kang et al. 2005). TRAIL receptors 1 (TRAIL-R1) and 2 (TRAIL-R2) recruit the adapter protein Fasassociated death domain protein (FADD) to the cell membrane, followed by activation of the caspase cascade. TRAIL receptors also activate the NF-κB signaling pathway via an adapter molecule, TNF receptor 1-associated death domain protein (TRADD) (Ravi et al. 2001). TRAIL has recently been demonstrated to be a critical factor in hepatic disorders (Afford and Adams 2005). An increase in TRAIL-R1 and TRAILR2 protein expression in human HSCs was observed during activation by culture. TRAIL-induced apoptosis of activated HSCs could be rescued by leptin, a novel profibrogenic cytokine (Saxena et al. 2004). In this study, we found that the apoptotic rate in HSCs stimulated with TRAIL siRNA-treated KCCM was decreased compared with a negative control, indicating that TRAIL played an essential role in recovery KCCMinduced apoptosis of HSCs. Our results provide new insights into the mechanism of recovery from hepatic fibrosis. Leflunomide is a novel immunosuppressive and anti-inflammatory agent used in the treatment of autoimmune disorders such as RA. In this study, we showed that A771726 significantly inhibited NF-κB activation in a dose-dependent manner in HSCs. Furthermore, A771726, as well as BAY 11-7082, was effective in enhancing recovery KCCM-induced apoptosis of HSCs. It was also reported that leflunomide inhibited NF-κB activation in whole liver in a mouse model of concanavalin A (Con A)-induced hepatitis (Imose et al. 2004). Other studies, however, demonstrated that the hepatoprotective effect of leflunomide against glycochenodeoxycholic acid (GCDCA)-induced primary rat hepatocyte death did not involve ERK1/2, p38, PI3K/Akt or NF-κB activation (Vrenken et al. 2008). Previous studies reported that leflunomide did not affect NF-κB activation in erythroleukemia cells or hepatocytes (Leger et al. 2006; Migita et al. 2005), but could inhibit NFκB activation in Jurkat, cervical epithelial and glioma cells (Manna and Aggarwal 1999; Manna et al. 2000). A potential explanation is that the different toxic factors might activate different signal pathways. Moreover, the different observations seem to be cell type specific. Apoptosis of HSCs is an important therapeutic target for hepatic fibrosis (Bataller and Brenner 2001). Gliotoxin, an inhibitor of NF-κB, induces apoptosis of HSCs and attenuates the extent of liver fibrosis in an animal model (Wright et al. 2001). This finding was important to demonstrate that hepatic fibrosis could be treated by inducing the apoptosis of activated HSCs. Previous work in our laboratory indicated that the active metabolite of leflunomide, A771726, had a preventive and therapeutic effect on animal models of liver fibrosis. A771726 significantly inhibited HSC activation and proliferation in the progression of fibrosis. Our results presented here suggest that A771726 can accelerate recovery KCCM-induced apoptosis of HSCs by inhibiting the activation of NF-κB in HSCs. We identified a new
pathway and mechanism for leflunomide in recovery from hepatic fibrosis and provided new evidence for the use of leflunomide in antifibrotic therapy. The degradation of ECM is associated with many diseases such as RA, fibrosis, and sclerosis. Matrix metalloproteinases (MMPs) play an important role in the degradation of ECM. Previous studies by several groups have looked at the effect of the active form of leflunomide on different MMPs in RA. It was reported that leflunomide could inhibit TNF-α and IL-1α-induced MMP-1 production in human fibroblast-like synoviocytes, which might account for the significant reduction in the rate of disease progression in leflunomide-treated RA patients (Burger et al. 2003). Another group, however, showed that leflunomide facilitated TNF-α and IL-17-induced MMP-1 and MMP-3 expression in synoviocytes, which might be involved in the development of drugrelated adverse events (Alexander et al. 2006). Although the effect of leflunomide on MMPs is currently controversial in synoviocytes, leflunomide may play a role in the regulation of MMPs in synoviocytes. Therefore, it is reasonable to speculate that leflunomide may play a role in the regulation of MMPs in hepatic fibrosis. It is interesting and worthwhile to continue studies of whether the regulation of MMPs by leflunomide might contribute to its antifibrotic effect, shedding new light on the molecular mechanism of this drug. Conclusion In this study, we not only demonstrated the role of TRAIL in recovery Kupffer cell-induced apoptosis of activated HSCs, but also provided novel insight into the resolution of fibrosis and the mechanisms by which leflunomide might act in liver fibrosis. Conflict of interest The authors declare no competing financial interests. Acknowledgments Supported by the Chinese National Natural Science Foundation Project (30873081). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.lfs.2009.01.017. References Afford SC, Adams DH. Following the TRAIL from hepatitis C virus and alcohol to fatty liver. Gut 54 (11), 1518–1520, 2005. Alexander D, Friedrich B, Abruzzese T, Gondolph-Zink B, Wulker N, Aicher WK. The active form of leflunomide, HMR1726, facilitates TNF-alpha and IL-17 induced MMP-1 and MMP-3 expression. Cellular Physiology Biochemistry 17 (1–2), 69–78, 2006. Bataller R, Brenner DA. Hepatic stellate cells as a target for the treatment of liver fibrosis. Seminars in Liver Disease 21 (3), 437–451, 2001. Benyon RC, Arthur MJ. Extracellular matrix degradation and the role of hepatic stellate cells. Seminars in Liver Disease 21 (3), 373–384, 2001. Burger D, Begué-Pastor N, Benavent S, Gruaz L, Kaufmann MT, Chicheportiche R, Dayer JM. The active metabolite of leflunomide, A771726, inhibits the production of prostaglandin E2, matrix metalloproteinase 1 and interleukin 6 in human fibroblast-like synoviocytes. Rheumatology 42 (1), 89–96, 2003. Choo-Kang BS, Hutchison S, Nickdel MB, Bundick RV, Leishman AJ, Brewer JM, McInnes IB, Garside P. TNF-blocking therapies: an alternative mode of action? Trends in Immunology 26 (10), 518–522, 2005. Davis JP, Cain GA, Pitts WJ, Magolda RL, Copeland RA. The immunosuppressive metabolite of leflunomide is a potent inhibitor of human dihydroorotate dehydrogenase. Biochemistry 35 (4), 1270–1273, 1996. Duffield JS, Forbes SJ, Constandinou CM, Clay S, Partolina M, Vuthoori S, Wu S, Lang R, Iredale JP. Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair. The Journal of Clinical Investigation 115 (1), 56–65, 2005. Fischer R, Cariers A, Reinehr R, Häussinger D. Caspase 9-dependent killing of hepatic stellate cells by activated Kupffer cells. Gastroenterology 123 (3), 845–861, 2002.
X. Tang et al. / Life Sciences 84 (2009) 552–557 Fox RI, Herrmann ML, Frangou CG, Wahl GM, Morris RE, Strand V, Kirschbaum BJ. Mechanism of action for leflunomide in rheumatoid arthritis. Clinical Immunology 93 (3), 198–208, 1999. Friedman SL. Mac the knife? Macrophages — the double-edged sword of hepatic fibrosis. The Journal of Clinical Investigation 115 (1), 29–32, 2005. Friedman SL. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiological Reviews 88 (1), 125–172, 2008a. Friedman SL. Mechanisms of hepatic fibrogenesis. Gastroenterology 134 (6), 1655–1669, 2008b. Friedman SL, Arthur MJ. Activation of cultured rat hepatic lipocytes by Kupffer cell conditioned medium. Direct enhancement of matrix synthesis and stimulation of cell proliferation via induction of platelet-derived growth factor receptors. The Journal of Clinical Investigation 84 (6), 1780–1785, 1989. Friedman SL, Roll FJ. Isolation and culture of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells by density gradient centrifugation with Stractan. Analytical Biochemistry 161 (1), 207–218, 1987. Geerts A, Lazou JM, De Bleser P, Wisse E. Tissue distribution, quantitation and proliferation kinetics of fat-storing cells in carbon tetrachloride-injured rat liver. Hepatology 13 (6), 1193–1202, 1991. Groth CG, Tibell A, Wennberg L, Korsgren O. Xenoislet transplantation: experimental and clinical aspects. Journal of Molecular Medicine 77 (1), 153–154, 1999. Guo J, Friedman SL. Hepatic fibrogenesis. Seminars in Liver Disease 27 (4), 413–426, 2007. Ikejima K, Enomoto N, Seabra V, Ikejima A, Brenner DA, Thurman RG. Pronase destroys the lipopolysaccharide receptor CD14 on Kupffer cells. The American Journal of Physiology 276, 591–598, 1999. Imose M, Nagaki M, Kimura K, Takai S, Imao M, Naiki T, Osawa Y, Asano T, Hayashi H, Moriwaki H. Leflunomide protects from T-cell-mediated liver injury in mice through inhibition of nuclear factor kappaB. Hepatology 40 (5), 1160–1169, 2004. Iredale JP. Hepatic stellate cell behavior during resolution of liver injury. Seminars in Liver Disease 21 (3), 427–436, 2001. Iredale JP, Benyon RC, Pickering J, McCullen M, Northrop M, Pawley S, Hovell C, Arthur MJ. Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors. The Journal of Clinical Investigation 102 (3), 538–549, 1998. Issa R, Williams E, Trim N, Kendall T, Arthur MJ, Reichen J, Benyon RC, Iredale JP. Apoptosis of hepatic stellate cells: involvement in resolution of biliary fibrosis and regulation by soluble growth factors. Gut 48 (4), 548–557, 2001. Issa R, Zhou X, Trim N, Millward-Sadler H, Krane S, Benyon C, Iredale J. Mutation in collagen-1 that confers resistance to the action of collagenase results in failure of recovery from CCl4-induced liver fibrosis, persistence of activated hepatic stellate cells, and diminished hepatocyte regeneration. The FASEB Journal 17 (1), 47–49, 2003.
557
Issa R, Zhou X, Constandinou CM, Fallowfield J, Millward-Sadler H, Gaca MD, Sands E, Suliman I, Trim N, Knorr A, Arthur MJ, Benyon RC, Iredale JP. Spontaneous recovery from micronodular cirrhosis: evidence for incomplete resolution associated with matrix cross-linking. Gastroenterology 126 (7), 1795–1808, 2004. Leger DY, Liagre B, Beneytout JL. Low dose leflunomide activates PI3K/Akt signalling in erythroleukemia cells and reduces apoptosis induced by anticancer agents. Apoptosis 11 (10), 1747–1760, 2006. Manna SK, Aggarwal BB. Immunosuppressive leflunomide metabolite (A771726) blocks TNF-dependent nuclear factor-κB activation and gene expression. Journal of Immunology 162 (4), 2095–2102, 1999. Manna SK, Mukhopadhyay A, Aggarwal BB. Leflunomide suppresses TNF-induced cellular responses: effects on NF-kappa B, activator protein-1, c-Jun N-terminal protein kinase, and apoptosis. Journal of Immunology 165 (10), 5962–5969, 2000. Migita K, Miyashita T, Maeda Y, Nakamura M, Yatsuhashi H, Ishibashi H, Eguchi K. An active metabolite of leflunomide, A771726, inhibits the production of serum amyloid A protein in human hepatocytes. Rheumatology 44 (4), 443–448, 2005. Pinzani M, Marra F. Cytokine receptors and signaling in hepatic stellate cells. Seminars in Liver Disease 21 (3), 397–416, 2001. Ravi R, Bedi GC, Engstrom LW, Zeng Q, Mookerjee B, Gélinas C, Fuchs EJ, Bedi A. Regulation of death receptor expression and TRAIL/Apo2L-induced apoptosis by NF-kappaB. Nature Cell Biology 3 (4), 409–416, 2001. Saxena NK, Titus MA, Ding X, Floyd J, Srinivasan S, Sitaraman SV, Anania FA. Leptin as a novel profibrogenic cytokine in hepatic stellate cells: mitogenesis and inhibition of apoptosis mediated by extracellular regulated kinase (Erk) and Akt phosphorylation. The FASEB Journal 18 (13), 1612–1614, 2004. Shiratori Y, Geerts A, Ichida T, Kawase T, Wisse E. Kupffer cells from CCl4-induced fibrotic livers stimulate proliferation of fat-storing cells. Journal of Hepatology 3 (3), 294–303, 1986. Si HF, Li J, Lü XW, Jin Y. Suppressive effects of leflunomide on leptin-induced collagen I production involved in hepatic stellate cell proliferation. Experimental Biology and Medicine (Maywood, N.J.) 232 (3), 427–436, 2007. Vrenken TE, Buist-Homan M, Kalsbeek AJ, Faber KN, Moshage H. The active metabolite of leflunomide, A771726, protects rat hepatocytes against bile acid-induced apoptosis. Journal of Hepatology 49 (5), 799–809, 2008. Wright MC, Issa R, Smart DE, Trim N, Murray GI, Primrose JN, Arthur MJ, Iredale JP, Mann DA. Gliotoxin stimulates the apoptosis of human and rat hepatic stellate cells and enhances the resolution of liver fibrosis in rats. Gastroenterology 121 (3), 685–698, 2001. Xu X, Williams JW, Bremer EG, Finnegan A, Chong AS. Inhibition of protein tyrosine phosphorylation in T cells by a novel immunosuppressive agent, leflunomide. The Journal of Biological Chemistry 270 (21), 12398–12403, 1995. Yao HW, Li J, Chen JQ, Xu SY. Inhibitory effect of leflunomide on hepatic fibrosis induced by CCl4 in rats. Acta Pharmacologica Sinica 25 (7), 915–920, 2004.
Contents lists available at ScienceDirect
Life Sciences 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 / l i f e s c i e
Accelerative effect of leflunomide on recovery from hepatic fibrosis involves TRAIL-mediated hepatic stellate cell apoptosis Xiaoming Tang a,1, Juntao Yang b,1, Jun Li a,⁎ a b
School of Pharmacy, Anhui Medical University, Hefei, Anhui Province 230032, China State Key Laboratory of Proteomics, Beijing Proteomics Research Center, Beijing Institute of Radiation Medicine, Beijing 100850, China
a r t i c l e
i n f o
Article history: Received 29 October 2008 Accepted 28 January 2009 Keywords: Hepatic fibrosis Hepatic stellate cells Kupffer cells TRAIL Leflunomide
a b s t r a c t Aims: Hepatic fibrosis is reversible, associated with apoptosis of activated hepatic stellate cells (HSCs) as injury subsides, thus providing potential targets for therapy. Little is known, however, about the course of this condition. The objective of this study was to elucidate the mechanism by which Kupffer cells regulate HSC biology during regression of hepatic fibrosis and the effect of leflunomide on this process. Main methods: We harvested Kupffer cells from rats during spontaneous recovery from liver fibrosis induced by carbon tetrachloride (CCl4) and prepared recovery Kupffer cell conditioned medium (KCCM). Cultureactivated HSCs were pretreated in the absence or presence of A771726, the active metabolite of leflunomide, and then stimulated with recovery KCCM. Key findings: Following stimulation with recovery KCCM, HSCs showed a decrease in proliferation and an increase in apoptosis by a caspase-dependent mechanism. Furthermore, pretreatment with A771726 markedly enhanced these effects. Real-time quantitative PCR (Q-PCR) analysis showed increased expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in Kupffer cells during the spontaneous recovery phase. The pro-apoptotic function of KCCM prepared from TRAIL siRNA-treated Kupffer cells was obviously decreased, suggesting that TRAIL played an important role in recovery from hepatic fibrosis. Moreover, A771726 enhanced recovery KCCM-induced apoptosis of HSCs by a mechanism involving the inhibition of nuclear factor-kappa B (NF-κB) activation. Significance: Our results showed the role of TRAIL in the apoptosis of activated HSCs that is induced by Kupffer cells prepared from livers recovering from CCI4-induced fibrosis and provided insights into the resolution of fibrosis and the mechanisms by which leflunomide might act upon liver fibrosis. © 2009 Elsevier Inc. All rights reserved.
Introduction Hepatic fibrosis is a common consequence of chronic liver injury of any etiology (Friedman 2008a,b), and hepatic stellate cells (HSCs) are the major source of increased extracellular matrix (ECM) proteins in chronic liver diseases (Iredale 2001). If hepatic injury persists, HSCs proliferate and undergo dramatic transdifferentiation from quiescent vitamin A-storing cells to activated myofibroblast-like cells (Geerts et al. 1991). These activated HSCs secrete large amounts of ECM proteins, which is a seminal event in hepatic fibrogenesis. The factors that modulate HSC activation influence the progression of liver fibrosis (Pinzani and Marra 2001). Kupffer cells, the resident macrophages in the liver, act as a double-edged sword in the progression of and recovery from hepatic fibrosis (Friedman 2005). It has been widely reported that Kupffer cells can promote HSC activation during the progression of fibrosis (Ikejima et al. 1999;
⁎ Corresponding author. Tel./fax: +86 551 5161001. E-mail address: [email protected] (J. Li). 1 These authors contributed equally to this work. 0024-3205/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2009.01.017
Shiratori et al. 1986). On the other hand, Kupffer cells can negatively regulate activated HSCs during recovery from liver fibrosis associated with enhancing matrix degradation (Duffield et al. 2005). The mechanism by which Kupffer cells regulate HSC biology during regression of hepatic fibrosis, however, is still far from being fully understood. Leflunomide, an isoxazole derivative, is a unique immunomodulatory agent, capable of treating rheumatoid arthritis (RA) (Fox et al. 1999), allograft and xenograft rejection (Groth et al. 1999), and systemic lupus erythematosus (Manna et al. 2000). After administration, it is metabolized to its active form, A771726, which leads to the immunosuppressive activity (Davis et al. 1996). It has also been reported to block cell cycle progression in the G0/G1 phase (Xu et al. 1995). Recent evidence has suggested that A771726 can significantly inhibit the proliferation and activation of HSCs by inhibiting the Janus kinase (JAK) and protein kinase B (Akt) pathways (Si et al. 2007). In vivo, A771726 showed an inhibitory effect on carbon tetrachloride (CCl4)-induced hepatic fibrosis in rats (Yao et al. 2004). Based on these results, the aim of this study was to elucidate the mechanism by which Kupffer cells regulate HSC biology during regression of hepatic fibrosis and the effect of leflunomide on this process.
X. Tang et al. / Life Sciences 84 (2009) 552–557
Materials and methods Reagents Antibodies against caspase 3, 9, Poly (ADP-ribose) polymerase (PARP), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), β-actin and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and goat anti-rabbit IgG antibody were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Fluorescein isothiocyanate (FITC)-labeled bromodeoxyuridine (BrdU) antibody and annexin V-FITC were purchased from BD Pharmingen. Special chemical inhibitors Z-VAD-FMK, Z-DEVD-FMK, Z-IETD-FMK, Z-LEHDFMK and BAY 11-7082 were from Calbiochem (La Jolla, CA). Collagenase, proteinase E, DNase I, Nycodenz, Dulbeccos Modified Eagles Medium (DMEM), fetal bovine serum (FBS), and powdered 1640 medium were obtained from Gibco (Grand Island, NY). Leflunomide and its active metabolite, A771726, were kindly donated by Cinkate Co. (Shanghai, China). Animals and treatment Adult male Sprague-Dawley (SD) rats weighing 200–250 g were purchased from the Animal Center of Anhui Medical University. Animals were housed in standard facilities and all animals used in this study received humane care in compliance with the institution's guidelines. Male SD rats were intraperitoneally injected with 1.0 ml/ kg body weight of a 1:1 (v/v) solution of CCl4 and olive oil twice weekly for 8 weeks. The control group received the same volume of olive oil. Animals were sacrificed at 14 days of spontaneous recovery (Issa et al. 2003; Iredale et al. 1998). Isolation and culture of Kupffer cells Kupffer cells were isolated by collagenase digestion, differential centrifugation and selective plating as described previously (Friedman and Roll 1987), with slight modifications. Livers were perfused in situ through the portal vein with collagenase. After digestion, livers were minced in collagenase buffer, and the suspension was filtered through nylon mesh. The filtrate was separated into parenchymal and nonparenchymal fractions by differential centrifugation. The Kupffer cell fraction was collected from the non-parenchymal fraction by density gradient centrifugation. To further purify Kupffer cells, cells were seeded on tissue culture plates and cultured at 37 °C with 5% CO2 for 30 min, followed by a single wash step to discard the non-adherent cells. Cell viability, as determined by trypan blue dye exclusion assay, was 95%. Immunohistochemistry and fluoroscopy revealed a purity of N85% Kupffer cells for the Kupffer cell cultures. Preparation of Kupffer cell conditioned medium (KCCM) Isolated Kupffer cells were cultured in 24-well tissue culture plates. Cells were washed and incubated in serum-free medium for 48 h. Conditioned medium was collected by centrifugation at 6000 g to remove cell debris. KCCM was filtered with a 0.45 μM membrane filter and stored at −70 °C until use. Using the method described above, we harvested Kupffer cells from rats during spontaneous recovery from liver fibrosis induced by CCl4 (the recovery Kupffer cells) and prepared recovery KCCM. We also harvested Kupffer cells from rats in the control group (the normal Kupffer cells) and prepared normal KCCM.
553
centrifugation. HSCs were purified by Nycodenz gradient centrifugation and cultured (at a density of 1 × 105 cells/ml) in DMEM containing 15% fetal calf serum, 2 mmol/l glutamine, and 1% antibiotic solution under an atmosphere containing 5% CO2 at 37 °C. Purity of the HSC preparation (N95%) was assessed by typical light microscopic appearance and vitamin A-specific autofluorescence. HSCs were used for experiments after 7 days of culture to induce activation. Cultures were initiated at a cell density of 1 × 105 cells/ml, and all treatments were given after HSCs were cultured with serum starvation for 24 h, unless otherwise noted. Cell proliferation assay Briefly, HSCs were seeded in triplicate in 6-well plates. They were then pretreated with A771726 (0.1, 1.0, 10 μM) for 2 h and stimulated with KCCM for 48 h. For the cell proliferation assay, cells were incubated with BrdU at a final concentration of 10 μM for 2 h at 37 °C in a controlled atmosphere. Cells were harvested and fixed in cold (4 °C) 70% ethanol and left for at least 30 min at 4 °C. The cells were then resuspended in 2 M hydrochloric acid and left at room temperature for 30 min. The acid was removed by centrifugation, and the cell pellets were washed twice in PBST, followed staining with 20 µl FITC-conjugated anti-BrdU antibody (Becton Dickinson). Incorporation was quantified with a FACScalibur (BD Bioscience). For the apoptosis assay, treated cells were harvested and washed in PBS, followed by staining with 20 µl of FITCconjugated annexin V (Becton Dickinson) and analysis by flow cytometry. All experiments were repeated at least three times. Western blot analysis HSCs (or Kupffer cells) were collected and washed in cold phosphate buffered saline (PBS) and lysed in ice-cold RIPA buffer (10 μM Tris–HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and 10 μl/ml protease inhibitor cocktail) for 30 min on ice. The extracted cell protein from each sample was applied to a 7–12% SDS-polyacrylamide gel and transblotted to nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany). Protein bands were visualized using an ECL assay kit (Amersham Biosciences, Buckinghamshire, UK). Quantitative real-time PCR (Q-PCR) Total RNA was extracted using Trizol reagent (Invitrogen) and digested with RNase-free DNaseI (Qiagen Ltd.). First-strand cDNA synthesis was performed using a Reverse Transcript-PCR kit with oligo(dT) primers (Promega, Madison, WI). Quantitative realtime PCR (Q-PCR) was performed using SYBR® Green I Q-PCR MasterMix (Bio-RED) and a Bio-RED IQ5 (Bio-RED) according to the manufacturer's instructions. The mRNA expression levels of tumor necrosis factor-α (TNF-α), Fas ligand (FasL) and TRAIL were calculated using the threshold cycle (Ct) values. All Ct values were normalized to the housekeeping gene, β-actin. Specific primers used in the Q-PCRs were as follows: TRAIL (5′-GCTTCAGTCAGACATTCACG-3′, 5′GTCCCAAAAATCCCCATCTT-3′), TNF-α (5′- GACCCTCACACTCAGATCATCTTCT-3′, 5 ′ -T G C TAC GAC GT G G G C TAC G - 3 ′ ) , Fa s L ( 5 ′ TGCCTCCACTAAGCCCTCTA-3′, 5′-AGGCTGTGGTTGGTGAACTC-3′), β-actin (5′-GGGAAATCGTGCGTGACATT-3′, 5′-GCGGCAGTGGCCATCTC3′). All experiments were repeated at least three times. RNA interference
Isolation and culture of HSCs HSCs were isolated from normal male SD rats by sequential in situ perfusion with collagenase and protease as described previously (Friedman and Arthur 1989), with slight modifications. Briefly, nonparenchymal liver cells were obtained by enzymatic digestion and
The expression vector for rat TRAIL was prepared by cloning the PCRamplified cDNA into pcDNA 3.1A (Clonetech). The primers used for PCR amplification of rat TRAIL were as follows: Forward, 5′-CGCGGATCCATGGCTTCCACC-3′ and Reverse, 5′- GGAATTCGGCTCCAAAGAAGCTG-3′. The siRNAs designed to knock down rat TRAIL and the control non-specific
554
X. Tang et al. / Life Sciences 84 (2009) 552–557
be statistically significant. Data are presented as mean ± Standard Deviation (S.D.) from three independent experiments. Results Effect of recovery KCCM on activated HSCs To determine the effect of Kupffer cells in the resolution of hepatic fibrosis, we used recovery KCCM to stimulate culture-activated HSCs. BrdU cell proliferation assays showed that recovery KCCM suppressed the proliferation of HSCs. If HSCs were pretreated with A771726, the effect of the recovery KCCM was enhanced (Fig. 1A). The analysis of apoptosis showed that recovery KCCM increased the apoptotic rate of HSCs; this could also be enhanced by pretreatment with A771726 in a dose-dependent manner (Fig. 1B). These results suggest that Kupffer cells from the rats undergoing spontaneous recovery from hepatic fibrosis can negatively regulate culture-activated HSCs and that A771726 can significantly enhance this effect. Recovery KCCM-induced apoptosis of HSCs was caspase-dependent
Fig. 1. Effect of recovery KCCM on activated HSCs. (A) BrdU cell proliferation assays. HSCs labeled in vitro with BrdU were stained with anti-BrdU-FITC and analyzed by flow cytometry. (B) Cell apoptosis assays. HSCs were incubated with annexin V-FITC and examined by flow cytometry. Results are the mean±S.D. from three independent experiments. #Pb 0.01 versus serum control values; ⁎Pb 0.05, ⁎⁎Pb 0.01 versus recovery KCCM values.
We detected caspases activation and PARP cleavage in recovery KCCM-treated HSCs by western blot analysis. The level of the active subunit of caspase 3 was increased; PARP was cleaved to its signature peptide; and procaspase-9 levels were reduced, suggesting that caspase signaling pathways were involved in recovery KCCM-induced apoptosis of HSCs (Fig. 2A). Furthermore, prior treatment with the
siRNA (NS siRNA) were purchased from Genepharm Co (China). Their sequences were as follows: siRNA1, 5′-CGAUGACGGUGAUCUGCAUtt-3′, 5′-AUGCAGAUCACCGUCAUCGTT-3′; siRNA2, 5′-CAACGAGGUGAAACAGCUATT-3′, 5′-UAGCUGUUUCACCUCGUUGTT-3′; siRNA3, 5′-GGAUCACUCGGAGAAGCAATT-3′, 5′-UUGCUUCUCCGAGUGAUCCTT-3′; siRNA4, 5′-CCUUAAUUCCAAUCUCCAATT-3′, 5′-UUGGAGAUUGGAAUUAAGGTT-3′. The siRNAs and TRAIL expression vector were co-transfected into HEK 293 cells using Lipofectamine 2000 Reagent (Invitrogen). Silencing of TRAIL was determined by western blotting of cell lysates with an anti-Myc antibody. The effective siRNA was transfected into the recovery Kupffer cells. Knockdown of TRAIL by siRNA was confirmed by western blotting with an anti-TRAIL antibody. After 24 h, the medium was changed. The recovery Kupffer cells transfected with the effective siRNA were incubated in serum-free medium for 48 h to collect the TRAIL siRNA-treated KCCM. Nuclear factor-kappa B (NF-κB) luciferase reporter gene assay HSCs were transfected with pNFκB-Luc (Clontech) and pRL-CMV (Promega, Madison, WI) plasmids using FuGENE 6 (Boehringer Mannheim) and incubated for 18 h at 37 °C. Cells were pretreated with A771726 (0.1, 1.0, 10 μM) or BAY 11-7082 (5 μM) for 2 h and stimulated with KCCM for 4 h. Cells were lysed in a passive lysis buffer (Promega). Luciferase activity was measured using a Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol. Fold induction of NF-κB-luciferase for each treatment was based on untreated values normalized to the fold induction of pRL-CMV reporter values. All experiments were repeated at least three times. Statistical analysis Student's t-test was used for determination of statistical significance as appropriate. Statistical values of P b 0.05 were considered to
Fig. 2. Recovery KCCM-induced apoptosis of HSCs was caspase-dependent. (A) Western blot analysis of caspase-3, PARP and procaspase-9 in HSCs. Two hours before stimulation, HSCs were treated with A771726 (0.1, 1.0, 10 μM). The cell lysates were collected 24 h later. (B) The effect of caspase inhibitors on recovery KCCM-induced apoptosis of HSCs. HSCs were stimulated with recovery KCCM in the absence or presence of caspase inhibitors (50 μmol/l). Results are the mean ± S.D. from three independent experiments.⁎P b 0.05.
X. Tang et al. / Life Sciences 84 (2009) 552–557
555
Fig. 3. Q-PCR analysis of the expression of cytokines in Kupffer cells. Relative expression levels of TNF-α, FasL and TRAIL mRNA were determined using β-actin as an internal control. Results are the mean ± S.D. from three independent experiments. ⁎P b 0.05.
pancaspase inhibitor Z-VAD-FMK, the caspase-3 inhibitor Z-DEVDFMK, the caspase-8 inhibitor Z-IETD-FMK, or the caspase-9 inhibitor Z-LEHD-FMK could clearly inhibit recovery KCCM-induced apoptosis of HSCs (Fig. 2B). These results indicate that recovery KCCM induced apoptosis of HSCs via caspase-dependent pathways. Role of TRAIL in recovery KCCM-induced apoptosis of HSCs To determine the molecular mechanisms underlying the proapoptotic activity of recovery Kupffer cells, Q-PCR was used to detect the expression of pro-apoptotic cytokines in Kupffer cells. The results showed that the expression of FasL and TNF-α did not clearly change; however, the expression of TRAIL significantly was increased in recovery Kupffer cells (Fig. 3). Furthermore, RNA interference was used to confirm the role of TRAIL in recovery KCCM-induced apoptosis of HSCs. The effective siRNA against TRAIL was obtained from screening. The results showed that 2′, 3′ siRNAs were effective in suppressing the expression of TRAIL (Fig. 4A). Expression of TRAIL in the recovery Kupffer cells was reduced by transfection of 3′ siRNA (Fig. 4B). The siRNA-treated KCCM was collected to stimulate cultureactivated HSCs. The apoptotic rate in HSCs stimulated with TRAIL siRNA-treated KCCM was decreased compared with a negative control (Fig. 4C). Altogether, these data show that TRAIL plays an important role in recovery KCCM-induced apoptosis of HSCs.
Fig. 4. TRAIL was involved in recovery KCCM-induced apoptosis of HSCs. (A) Screening of effective siRNAs targeting TRAIL. The silencing effect of each siRNA was detected by western blot assay. (B) TRAIL expression in siRNA-transfected Kupffer cells. The level of endogenous TRAIL was estimated by western blot assay. (C) Effect of siRNA-treated KCCM on apoptosis of HSCs. Results are the mean ± S.D. from three independent experiments. ⁎P b 0.05.
Effect of A771726 on NF-κB activation in recovery KCCM-induced apoptosis of HSCs A luciferase reporter gene assay showed that recovery KCCM could induce NF-κB activation in HSCs. Pretreatment of HSCs with A771726 could significantly inhibit NF-κB activation in a dose-dependent manner (Fig. 5A). BAY 11-7082, an NF-κB inhibitor, was used to determine whether NF-κB was involved in the enhancing effect of A771726 on recovery KCCM-induced apoptosis of HSCs. Inhibition of NF-κB activity by BAY 11-7082, as well as A771726, was effective in enhancing the cytotoxicity of recovery KCCM in HSCs (Fig. 5B). These results suggest that A771726 can accelerate recovery KCCM-induced apoptosis of HSCs by inhibiting NF-κB activation in HSCs. Discussion Liver fibrosis results from persistent damage to the liver (Guo and Friedman 2007). With stimulation by hepatotoxic agents, resident
Fig. 5. Effect of A771726 on NF-κB activation in recovery KCCM-induced apoptosis of HSCs. (A) NF-κB reporter gene assays. NF-κB-luc-transfected HSCs were pretreated with A771726 or BAY 11-7082 for 2 h followed by treatment with recovery KCCM. Cell lysates were collected to detect NF-κB-luc reporter activity. (B) Cell apoptosis assays. HSCs were pretreated with A771726 or BAY 11-7082 for 2 h followed by treatment with recovery KCCM. Results are the mean ± S.D. from three independent experiments. #P b 0.01 versus serum control values; ⁎P b 0.05, ⁎⁎P b 0.01 versus recovery KCCM values.
556
X. Tang et al. / Life Sciences 84 (2009) 552–557
HSCs are activated into fibrogenic myofibroblasts, which synthesize and release large amounts of ECM proteins and tissue inhibitors of metalloproteinases (TIMPs). The degradation of ECM proteins and collagen is inhibited by cytokines such as TIMPs. Eventually, large amounts of ECM proteins accumulate and lead to liver fibrosis (Benyon and Arthur 2001). Recent research has indicated that hepatic fibrosis is reversible as liver injury subsides (Issa et al. 2004). Spontaneous recovery from hepatic fibrosis includes the degradation of ECM proteins, apoptosis of activated HSCs, and regeneration of hepatocytes. It has been reported that Kupffer cells can negatively regulate activated HSCs during recovery from liver fibrosis (Friedman 2008a,b; Issa et al. 2001; Fischer et al. 2002). The mechanism behind this process, however, is still not clear. In this study, we demonstrated that recovery KCCM could inhibit the proliferation of activated HSCs and induce their apoptosis by a mechanism involving caspase activation. Caspase inhibitors could inhibit recovery KCCM-induced apoptosis of HSCs. Q-PCR showed that the expression of TRAIL in recovery phase Kupffer cells was significantly increased. These results suggest that TRAIL might function during the recovery phase. TNF and TNF-receptor superfamily proteins play important roles in inflammatory responses (Choo-Kang et al. 2005). TRAIL receptors 1 (TRAIL-R1) and 2 (TRAIL-R2) recruit the adapter protein Fasassociated death domain protein (FADD) to the cell membrane, followed by activation of the caspase cascade. TRAIL receptors also activate the NF-κB signaling pathway via an adapter molecule, TNF receptor 1-associated death domain protein (TRADD) (Ravi et al. 2001). TRAIL has recently been demonstrated to be a critical factor in hepatic disorders (Afford and Adams 2005). An increase in TRAIL-R1 and TRAILR2 protein expression in human HSCs was observed during activation by culture. TRAIL-induced apoptosis of activated HSCs could be rescued by leptin, a novel profibrogenic cytokine (Saxena et al. 2004). In this study, we found that the apoptotic rate in HSCs stimulated with TRAIL siRNA-treated KCCM was decreased compared with a negative control, indicating that TRAIL played an essential role in recovery KCCMinduced apoptosis of HSCs. Our results provide new insights into the mechanism of recovery from hepatic fibrosis. Leflunomide is a novel immunosuppressive and anti-inflammatory agent used in the treatment of autoimmune disorders such as RA. In this study, we showed that A771726 significantly inhibited NF-κB activation in a dose-dependent manner in HSCs. Furthermore, A771726, as well as BAY 11-7082, was effective in enhancing recovery KCCM-induced apoptosis of HSCs. It was also reported that leflunomide inhibited NF-κB activation in whole liver in a mouse model of concanavalin A (Con A)-induced hepatitis (Imose et al. 2004). Other studies, however, demonstrated that the hepatoprotective effect of leflunomide against glycochenodeoxycholic acid (GCDCA)-induced primary rat hepatocyte death did not involve ERK1/2, p38, PI3K/Akt or NF-κB activation (Vrenken et al. 2008). Previous studies reported that leflunomide did not affect NF-κB activation in erythroleukemia cells or hepatocytes (Leger et al. 2006; Migita et al. 2005), but could inhibit NFκB activation in Jurkat, cervical epithelial and glioma cells (Manna and Aggarwal 1999; Manna et al. 2000). A potential explanation is that the different toxic factors might activate different signal pathways. Moreover, the different observations seem to be cell type specific. Apoptosis of HSCs is an important therapeutic target for hepatic fibrosis (Bataller and Brenner 2001). Gliotoxin, an inhibitor of NF-κB, induces apoptosis of HSCs and attenuates the extent of liver fibrosis in an animal model (Wright et al. 2001). This finding was important to demonstrate that hepatic fibrosis could be treated by inducing the apoptosis of activated HSCs. Previous work in our laboratory indicated that the active metabolite of leflunomide, A771726, had a preventive and therapeutic effect on animal models of liver fibrosis. A771726 significantly inhibited HSC activation and proliferation in the progression of fibrosis. Our results presented here suggest that A771726 can accelerate recovery KCCM-induced apoptosis of HSCs by inhibiting the activation of NF-κB in HSCs. We identified a new
pathway and mechanism for leflunomide in recovery from hepatic fibrosis and provided new evidence for the use of leflunomide in antifibrotic therapy. The degradation of ECM is associated with many diseases such as RA, fibrosis, and sclerosis. Matrix metalloproteinases (MMPs) play an important role in the degradation of ECM. Previous studies by several groups have looked at the effect of the active form of leflunomide on different MMPs in RA. It was reported that leflunomide could inhibit TNF-α and IL-1α-induced MMP-1 production in human fibroblast-like synoviocytes, which might account for the significant reduction in the rate of disease progression in leflunomide-treated RA patients (Burger et al. 2003). Another group, however, showed that leflunomide facilitated TNF-α and IL-17-induced MMP-1 and MMP-3 expression in synoviocytes, which might be involved in the development of drugrelated adverse events (Alexander et al. 2006). Although the effect of leflunomide on MMPs is currently controversial in synoviocytes, leflunomide may play a role in the regulation of MMPs in synoviocytes. Therefore, it is reasonable to speculate that leflunomide may play a role in the regulation of MMPs in hepatic fibrosis. It is interesting and worthwhile to continue studies of whether the regulation of MMPs by leflunomide might contribute to its antifibrotic effect, shedding new light on the molecular mechanism of this drug. Conclusion In this study, we not only demonstrated the role of TRAIL in recovery Kupffer cell-induced apoptosis of activated HSCs, but also provided novel insight into the resolution of fibrosis and the mechanisms by which leflunomide might act in liver fibrosis. Conflict of interest The authors declare no competing financial interests. Acknowledgments Supported by the Chinese National Natural Science Foundation Project (30873081). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.lfs.2009.01.017. References Afford SC, Adams DH. Following the TRAIL from hepatitis C virus and alcohol to fatty liver. Gut 54 (11), 1518–1520, 2005. Alexander D, Friedrich B, Abruzzese T, Gondolph-Zink B, Wulker N, Aicher WK. The active form of leflunomide, HMR1726, facilitates TNF-alpha and IL-17 induced MMP-1 and MMP-3 expression. Cellular Physiology Biochemistry 17 (1–2), 69–78, 2006. Bataller R, Brenner DA. Hepatic stellate cells as a target for the treatment of liver fibrosis. Seminars in Liver Disease 21 (3), 437–451, 2001. Benyon RC, Arthur MJ. Extracellular matrix degradation and the role of hepatic stellate cells. Seminars in Liver Disease 21 (3), 373–384, 2001. Burger D, Begué-Pastor N, Benavent S, Gruaz L, Kaufmann MT, Chicheportiche R, Dayer JM. The active metabolite of leflunomide, A771726, inhibits the production of prostaglandin E2, matrix metalloproteinase 1 and interleukin 6 in human fibroblast-like synoviocytes. Rheumatology 42 (1), 89–96, 2003. Choo-Kang BS, Hutchison S, Nickdel MB, Bundick RV, Leishman AJ, Brewer JM, McInnes IB, Garside P. TNF-blocking therapies: an alternative mode of action? Trends in Immunology 26 (10), 518–522, 2005. Davis JP, Cain GA, Pitts WJ, Magolda RL, Copeland RA. The immunosuppressive metabolite of leflunomide is a potent inhibitor of human dihydroorotate dehydrogenase. Biochemistry 35 (4), 1270–1273, 1996. Duffield JS, Forbes SJ, Constandinou CM, Clay S, Partolina M, Vuthoori S, Wu S, Lang R, Iredale JP. Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair. The Journal of Clinical Investigation 115 (1), 56–65, 2005. Fischer R, Cariers A, Reinehr R, Häussinger D. Caspase 9-dependent killing of hepatic stellate cells by activated Kupffer cells. Gastroenterology 123 (3), 845–861, 2002.
X. Tang et al. / Life Sciences 84 (2009) 552–557 Fox RI, Herrmann ML, Frangou CG, Wahl GM, Morris RE, Strand V, Kirschbaum BJ. Mechanism of action for leflunomide in rheumatoid arthritis. Clinical Immunology 93 (3), 198–208, 1999. Friedman SL. Mac the knife? Macrophages — the double-edged sword of hepatic fibrosis. The Journal of Clinical Investigation 115 (1), 29–32, 2005. Friedman SL. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiological Reviews 88 (1), 125–172, 2008a. Friedman SL. Mechanisms of hepatic fibrogenesis. Gastroenterology 134 (6), 1655–1669, 2008b. Friedman SL, Arthur MJ. Activation of cultured rat hepatic lipocytes by Kupffer cell conditioned medium. Direct enhancement of matrix synthesis and stimulation of cell proliferation via induction of platelet-derived growth factor receptors. The Journal of Clinical Investigation 84 (6), 1780–1785, 1989. Friedman SL, Roll FJ. Isolation and culture of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells by density gradient centrifugation with Stractan. Analytical Biochemistry 161 (1), 207–218, 1987. Geerts A, Lazou JM, De Bleser P, Wisse E. Tissue distribution, quantitation and proliferation kinetics of fat-storing cells in carbon tetrachloride-injured rat liver. Hepatology 13 (6), 1193–1202, 1991. Groth CG, Tibell A, Wennberg L, Korsgren O. Xenoislet transplantation: experimental and clinical aspects. Journal of Molecular Medicine 77 (1), 153–154, 1999. Guo J, Friedman SL. Hepatic fibrogenesis. Seminars in Liver Disease 27 (4), 413–426, 2007. Ikejima K, Enomoto N, Seabra V, Ikejima A, Brenner DA, Thurman RG. Pronase destroys the lipopolysaccharide receptor CD14 on Kupffer cells. The American Journal of Physiology 276, 591–598, 1999. Imose M, Nagaki M, Kimura K, Takai S, Imao M, Naiki T, Osawa Y, Asano T, Hayashi H, Moriwaki H. Leflunomide protects from T-cell-mediated liver injury in mice through inhibition of nuclear factor kappaB. Hepatology 40 (5), 1160–1169, 2004. Iredale JP. Hepatic stellate cell behavior during resolution of liver injury. Seminars in Liver Disease 21 (3), 427–436, 2001. Iredale JP, Benyon RC, Pickering J, McCullen M, Northrop M, Pawley S, Hovell C, Arthur MJ. Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors. The Journal of Clinical Investigation 102 (3), 538–549, 1998. Issa R, Williams E, Trim N, Kendall T, Arthur MJ, Reichen J, Benyon RC, Iredale JP. Apoptosis of hepatic stellate cells: involvement in resolution of biliary fibrosis and regulation by soluble growth factors. Gut 48 (4), 548–557, 2001. Issa R, Zhou X, Trim N, Millward-Sadler H, Krane S, Benyon C, Iredale J. Mutation in collagen-1 that confers resistance to the action of collagenase results in failure of recovery from CCl4-induced liver fibrosis, persistence of activated hepatic stellate cells, and diminished hepatocyte regeneration. The FASEB Journal 17 (1), 47–49, 2003.
557
Issa R, Zhou X, Constandinou CM, Fallowfield J, Millward-Sadler H, Gaca MD, Sands E, Suliman I, Trim N, Knorr A, Arthur MJ, Benyon RC, Iredale JP. Spontaneous recovery from micronodular cirrhosis: evidence for incomplete resolution associated with matrix cross-linking. Gastroenterology 126 (7), 1795–1808, 2004. Leger DY, Liagre B, Beneytout JL. Low dose leflunomide activates PI3K/Akt signalling in erythroleukemia cells and reduces apoptosis induced by anticancer agents. Apoptosis 11 (10), 1747–1760, 2006. Manna SK, Aggarwal BB. Immunosuppressive leflunomide metabolite (A771726) blocks TNF-dependent nuclear factor-κB activation and gene expression. Journal of Immunology 162 (4), 2095–2102, 1999. Manna SK, Mukhopadhyay A, Aggarwal BB. Leflunomide suppresses TNF-induced cellular responses: effects on NF-kappa B, activator protein-1, c-Jun N-terminal protein kinase, and apoptosis. Journal of Immunology 165 (10), 5962–5969, 2000. Migita K, Miyashita T, Maeda Y, Nakamura M, Yatsuhashi H, Ishibashi H, Eguchi K. An active metabolite of leflunomide, A771726, inhibits the production of serum amyloid A protein in human hepatocytes. Rheumatology 44 (4), 443–448, 2005. Pinzani M, Marra F. Cytokine receptors and signaling in hepatic stellate cells. Seminars in Liver Disease 21 (3), 397–416, 2001. Ravi R, Bedi GC, Engstrom LW, Zeng Q, Mookerjee B, Gélinas C, Fuchs EJ, Bedi A. Regulation of death receptor expression and TRAIL/Apo2L-induced apoptosis by NF-kappaB. Nature Cell Biology 3 (4), 409–416, 2001. Saxena NK, Titus MA, Ding X, Floyd J, Srinivasan S, Sitaraman SV, Anania FA. Leptin as a novel profibrogenic cytokine in hepatic stellate cells: mitogenesis and inhibition of apoptosis mediated by extracellular regulated kinase (Erk) and Akt phosphorylation. The FASEB Journal 18 (13), 1612–1614, 2004. Shiratori Y, Geerts A, Ichida T, Kawase T, Wisse E. Kupffer cells from CCl4-induced fibrotic livers stimulate proliferation of fat-storing cells. Journal of Hepatology 3 (3), 294–303, 1986. Si HF, Li J, Lü XW, Jin Y. Suppressive effects of leflunomide on leptin-induced collagen I production involved in hepatic stellate cell proliferation. Experimental Biology and Medicine (Maywood, N.J.) 232 (3), 427–436, 2007. Vrenken TE, Buist-Homan M, Kalsbeek AJ, Faber KN, Moshage H. The active metabolite of leflunomide, A771726, protects rat hepatocytes against bile acid-induced apoptosis. Journal of Hepatology 49 (5), 799–809, 2008. Wright MC, Issa R, Smart DE, Trim N, Murray GI, Primrose JN, Arthur MJ, Iredale JP, Mann DA. Gliotoxin stimulates the apoptosis of human and rat hepatic stellate cells and enhances the resolution of liver fibrosis in rats. Gastroenterology 121 (3), 685–698, 2001. Xu X, Williams JW, Bremer EG, Finnegan A, Chong AS. Inhibition of protein tyrosine phosphorylation in T cells by a novel immunosuppressive agent, leflunomide. The Journal of Biological Chemistry 270 (21), 12398–12403, 1995. Yao HW, Li J, Chen JQ, Xu SY. Inhibitory effect of leflunomide on hepatic fibrosis induced by CCl4 in rats. Acta Pharmacologica Sinica 25 (7), 915–920, 2004.