A Proteomic Approach in Investigating the Hepatoprotective Mechanism of Schisandrin B: Role of Raf Kinase Inhibitor Protein Yan Chen,† Siu-Po Ip,*,† Kam-Ming Ko,‡ Terence C. W. Poon,§ Eddy W. Y. Ng,§ Paul B. S. Lai,| Qing-Qiu Mao,† Yan-Fang Xian,† and Chun-Tao Che† School of Chinese Medicine, The Chinese University of Hong Kong, Department of Biochemistry, The Hong Kong University of Science of Technology, and Li Ka Shing Institute of Health Sciences, Department of Medicine and Therapeutics, and Department of Surgery, The Chinese University of Hong Kong, Hong Kong SAR, China Received August 26, 2010
To identify key proteins involved in the hepatoprotection afforded by schisandrin B (Sch B), we used a proteomic approach to screen proteins that were specifically regulated by Sch B in mouse livers and to investigate the role of the proteins in hepatoprotection. Thirteen proteins were specifically activated or suppressed by Sch B treatment. Among the 13 proteins, Raf kinase inhibitor protein (RKIP) was postulated to be the key regulator involved in the development of hepatotoxin-induced cellular damage. The results indicated that the downregulation of RKIP by antisense RKIP vector transfection led to the activation of the Raf-1/MEK/ERK signaling pathway, as evidenced by increases in the level of MEK/ERK phosphorylation and the level of nuclear factor erythroid 2-related factor 2 in the nucleus. The signaling effect produced by RKIP downregulation resembled that triggered by Sch B, wherein both treatments resulted in a decrease in the extent of carbon tetrachloride-induced apoptotic cell death in AML12 hepatocytes. Overexpression of RKIP by the sense RKIP transfection vector or the inhibition of MEK kinase by PD98059 was able to abrogate the cytoprotective effect of Sch B in the hepatocytes. The results indicate that Sch B triggers the Raf/MEK/ERK signaling pathway, presumably by downregulating RKIP, thereby protecting against carbon tetrachloride-induced cytotoxicity. Keywords: Proteome • hepatoprotective agent • schisandrin B • carbon tetrachloride • hepatotoxicity • RKIP
Introduction Schisandrin B (Sch B), a dibenzocyclooctadiene derivative, is a potent hepatoprotective agent. Previous studies in our laboratory have shown that Sch B pretreatment protected against hepatotoxicity induced by various toxins in mice.1-3 The hepatoprotection was mediated by the enhancement of the mitochondrial antioxidant system.3-5 However, the molecular mechanism underlying the hepatoprotective action of Sch B has not been clearly defined. Our proteomic screening study showed that Raf kinase inhibitor protein (RKIP) might be involved in the hepatoprotective mechanism of Sch B. Therefore, the effect of Sch B on the RKIP-mediated signaling pathway was investigated. RKIP has been identified as an endogenous inhibitor of the Raf-1/MEK/ERK signaling pathway.6 RKIP directly disrupts the interaction between Raf-1 and MEK, thereby preventing the activation of MEK and its downstream signal transduction.6 * To whom correspondence should be addressed: School of Chinese Medicine, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China. Telephone: (852)31634457. Fax: (852)31634459. E-mail: paulip@ cuhk.edu.hk. † School of Chinese Medicine, The Chinese University of Hong Kong. ‡ The Hong Kong University of Science of Technology. § Li Ka Shing Institute of Health Sciences and Department of Medicine and Therapeutics, The Chinese University of Hong Kong. | Department of Surgery, The Chinese University of Hong Kong. 10.1021/pr100871h
2011 American Chemical Society
The Raf-1/MEK/ERK signaling pathway activates nuclear factor erythroid 2-related factor 2 (Nrf2) and the subsequent binding to the antioxidant response element (ARE), with resultant induction of phase II enzymes that catalyze the detoxification of xenobiotics and enhance cell survival under intoxicated conditions.7,8 Impairment of the detoxification cascade may increase the sensitivity of cells to xenobiotic-induced apoptosis.9-11 Studies using cell culture and animal models have demonstrated that overexpression of RKIP could sensitize cancer cells to radiation and druginduced apoptosis, whereas the loss of RKIP function confers protection.12,13 Tsai et al. also reported that RKIP is a survivalrelated protein, and the reduction in the RKIP level in rats suffering from acidic fibroblast growth factor-induced spinal cord injury may aid the partial recovery.14 However, the inter-relationship between RKIP expression and hepatoprotection of Sch B against toxins is still unknown. RKIP is the most upstream protein, while other proteins such as glutathione S-transferase A3, S-adenosylmethionine synthetase, and dehydrogenase enzymes are downstream proteins that may be regulated by the level of RKIP. We therefore select RKIP as the first target of investigation and hypothesized that RKIP plays an important role in the hepatoprotective action of Sch B. To test this hypothesis, we examined the effect of downregulation and overexpression of RKIP on carbon tetrachloride (CCl4)-induced apoptosis in AML12 hepatocytes and Journal of Proteome Research 2011, 10, 299–304 299 Published on Web 11/13/2010
research articles
Chen et al.
investigated the effect of RKIP overexpression on the hepatoprotection afforded by Sch B. The effect of Sch B on the Raf1/MEK/ERK signaling pathway, which is downstream of RKIP, was also investigated.
Materials and Methods Materials. Immobiline pH-gradient (IPG) DryStrips (pH 3-10, length of 13 cm), IPG buffer (pH 3-10), DryStrip cover fluids, thiourea, urea, CHAPS, dithiothreitol (DTT), Pharmalyte (pH 3-10), bromophenol blue, TEMED, Coomassie brilliant blue G-250, molecular weight markers, Tris base, SDS, glycine, and the enhanced chemiluminescence (ECL) system were purchased from Amersham Biosciences (Stockholm, Sweden). PVDF membrane was obtained from Millipore (Boston, MA). Polyclonal goat anti-RKIP, polyclonal rabbit anti-Nrf2, and polyclonal goat anti-lamin B antibodies, horseradish peroxidase-conjugated goat anti-rabbit IgG, goat anti-mouse IgG, and rabbit anti-goat IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal rabbit anti-phospho-MEK, anti-MEK, anti-phospho-ERK, and anti-ERK antibodies were obtained from Cell Signaling Technology (Beverly, MA). Mercaptoethanol, iodoacetamide, R-cyano-4-hydroxycinnamic acid (CCA), and HCl were supplied by Sigma-Aldrich (St. Louis, MO). All buffers were prepared with Milli-Q water. Sch B was purified from the petroleum extract of Schisandra chinensis as described in our previous report.15 Alternatively, it can be purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Animal and Cell Treatment. Male Balb/c mice (25-30 g) were maintained on a 12 h light-dark cycle at 22 °C and allowed food and water ad libitum. Animals were randomly assigned to groups of five individuals. In the treatment group, animals were treated intragastrically with Sch B at a daily dose of 3 mmol/kg for 3 days. Control animals received the vehicle (i.e., olive oil, 10 mL/kg). Animals were sacrificed 24 h after receiving the last dose, and hepatic tissue samples were taken for proteomic and Western blot analysis. All animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (Bethesda, MD). Ethical approval for all experimental procedures was obtained from the Animal Ethical Committee of the Chinese University of Hong Kong. The AML12 cell line, a well-differentiated, nontransformed murine hepatocyte cell line derived from transforming growth factor-R transgenic mice,16 was used for all experiments and purchased from ATTC (Rockville, MD). AML12 hepatocytes were cultured in a Dulbecco’s modified Eagle’s medium (DMEM)/F12 (GIBCO BRL) mixture supplemented with 10% (v/v) fetal bovine serum. Sch B was dissolved in dimethyl sulfoxide (DMSO) at 10 mg/mL. For cell treatments, the Sch B solution was further diluted in serum free culture medium and the final concentration of DMSO was less than 0.2% (v/v). Control cells were given the medium containing 0.2% DMSO only. Previous studies in our laboratory indicated that pretreatment of AML12 hepatocytes with 6.25 µmol/L Sch B for 16 h yielded an optimal protection against oxidative injury.17 Immediately after Sch B treatment, the cells were challenged with CCl4 (dissolved in 0.2% ethanol) at 0.2 mmol/L for 3 h.18 To investigate the effect of MEK inhibitor (PD98059) on the hepatoprotective effect of Sch B, 20 µmol/L PD98059 (Calbiochem, San Diego, CA) was added after serum starvation 300
Journal of Proteome Research • Vol. 10, No. 1, 2011
Figure 1. (A) Representative 2-DE maps of hepatic tissue of a mouse treated with Sch B (left) and the control (right). Thirteen differential protein spots are marked with arrows. (B) Close-up image of the region of 2-DE gels showing the downregulation of protein spot 7 (RKIP) in Sch B-treated mouse liver when compared with the control. (C) Western blot analysis of RKIP in Sch B-treated mouse liver (S1-S5) and the control (C1-C5). β-Actin was used as an internal control. 2-DE was conducted three times for each sample.
overnight and Sch B treatment, and then the cells were challenged with CCl4 as described above. Two-Dimensional Gel Electrophoresis (2-DE), Protein Identification by Mass Spectrometry (MS), Stable Transfection, DNA Fragmentation Analysis, Nuclear Extract Preparation, and Western Blot Analysis. Details of the methods are provided in the Supporting Information and this section. In brief, 2-DE was performed to separate proteins from control and Sch B-treated liver tissue samples,19 and the images of the gels were analyzed with PDQuest. All the differential protein spots were excised from stained gels using punch, and in-gel trypsin digestion was performed.19 Plasmids were kindly provided by E. T. Keller (University of Michigan, Ann Arbor, MI).20 DNA transfection is described in the Supporting Information and this section. Quantification of DNA fragmentation was assessed with a Cell Death Detection ELISAPlus kit (Roche Applied Sciences, Mannheim, Germany) according to the manufacturer’s protocol. Nuclear protein was extracted by using a kit from Panomics (Fremont, CA) following the manufacturer’s instructions. Western blot analysis was conducted as previously described.19 Statistical Analysis. The statistical software package SPSS 13.0 was used in this study. Single comparisons were performed using Mann-Whitney’s U test, and multiple comparisons were performed using the Kruskal-Wallis test (one-way analysis of variance). All statistical tests were two-sided. Differences were considered statistically significant for P values of
