Mixed lineage kinase 3 connects reactive oxygen species to c-Jun NH2-terminal kinase-induced mitochondrial apoptosis in genipin-treated PC3 human prostate cancer cells

Biochemical and Biophysical Research Communications 362 (2007) 307–312 www.elsevier.com/locate/ybbrc

Mixed lineage kinase 3 connects reactive oxygen species to c-Jun NH2-terminal kinase-induced mitochondrial apoptosis in genipin-treated PC3 human prostate cancer cells Hye-Young Hong a, Byung-Chul Kim b

a,b,*

a Division of Life Sciences, Kangwon National University, Chuncheon 200-701, Republic of Korea Research Institute of Life Sciences, Kangwon National University, Chuncheon 200-701, Republic of Korea

Received 22 July 2007 Available online 9 August 2007

Abstract It has been reported that genipin, the aglycone of geniposide, induces apoptotic cell death in human hepatoma cells via a NADPH oxidase-reactive oxygen species (ROS)-c-Jun NH2-terminal kinase (JNK)-dependent activation of mitochondrial pathway. This continuing work aimed to define that mixed lineage kinase 3 (MLK3) is a key mediator, which connect between ROS and JNK in genipin-induced cell death signaling. In PC3 human prostate cancer cells, genipin stimulated MLK3 activity in concentration- and time-dependent manner. The PC3 cells stably transfected with dominant-negative form of MLK3 was less susceptible to population of the sub-G1 apoptotic cells, activation of caspase, collapse of mitochondrial membrane potential, and release of cytochrome c triggered by genipin, suggesting a crucial role of MLK3 in genipin signaling to apoptotic cell death. Diphenyleneiodonium (DPI), a specific inhibitor of NADPH oxidase, markedly inhibited ROS generation and MLK3 phosphorylation in the genipin-treated cells. Pretreatment with SP0600125, a specific inhibitor of JNK but neither U0126, a specific inhibitor of MEK1/2 nor PD169316, a specific inhibitor of p38 suppressed genipininduced apoptotic cell death. Notably, both the phosphorylation of JNK and induction of c-Jun induced by genipin were markedly inhibited in PC3-EGFP-MLK3 (K144R) cells expressing a dominant-negative MLK3 mutant. Taken together, our observations suggest genipin signaling to apoptosis of PC3 cells is mediated via activation of ROS-dependent MLK3, which leads to downstream activation of JNK.  2007 Elsevier Inc. All rights reserved. Keywords: Genipin; Apoptosis; Mixed lineage kinase 3; c-Jun NH2-terminal kinase; Reactive oxygen species

Apoptosis, programmed cell death, is known to be essential to develop and maintain homeostasis during cell growth and elimination of damaged cells in multicellular organisms. Failure of apoptosis, a genetically and evolutionarily conserved process, is regarded as a major determinant in development of carcinoma [1]. Reactive oxygen species (ROS) such as singlet oxygen, superoxide anion radical ðO2  Þ, hydrogen peroxide *

Corresponding author. Address: Division of Life Sciences and Research Institute of Life Sciences, Kangwon National University, 192-1 Hyoja-2-dong, Chuncheon 200-701, Republic of Korea. Fax: +82 33 242 0459. E-mail address: [email protected] (B.-C. Kim). 0006-291X/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.07.165

(H2O2), and hydroxyl radical ðOH Þ is produced in a variety of cells stimulated with cytokines, peptide growth factors, irradiations or inflammatory system and contributes to the regulation of the various biological processes [2]. ROS at low concentration function as physiological mediators in many cellular events, including cell proliferation, glucose transport and lipid synthesis, while the production of excess amounts of ROS contributes to apoptosis [3]. This implies that a redox state of a cell is a critical factor in deciding its susceptibility to apoptotic stimuli [4]. Mitogen-activated protein (MAP) kinases are a group of protein serine/threonine kinases that are activated in response to a wide variety of external signals. The p42/44 MAPK usually regulates mitogenic cellular processes

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whereas the stress-activated protein kinase (SAPK), also termed c-Jun N-terminal kinase (JNK), is a target molecule of ROS, which is produced in response to a wide variety of stress signals such as UV irradiation, heat shock, glutathione depletion, and many chemotherapeutic drugs, and function as a key mediator in signal transduction cascade that leads to apoptosis [5]. The mixed lineage kinase 3 (MLK3) is a recently described member of MLK subfamily of mammalian serine/threonine protein kinases that function as mitogen-activated protein kinases (MAPK) kinase kinases (MAPKKKs) [6]. Studies on neuronal cell types suggest that MLK3 predominantly activates the JNK pathway and plays a pivotal role in neuronal cell death evoked by NGF-deprivation or ischemia injury [7,8]. Cdc42, a member of Rho family GTPase, induces activation loop phosphorylation and membrane targeting of MLK3 [9] and promotes neuronal apoptosis through activating MLK3JNK3 cascade during ischemic reperfusion in rat hippocampus [10]. However, an implication of ROS in MLK3 signaling cascade that mediates apoptosis has never been reported. Geniposide is one of the main irioid-glyco compounds in Gardenia jasminoides Ellis fruits used as an oriental folk medicine, and is converted to the aglycone genipin by bacterial enzymes, b-D-glycosidases in the digestive organs and then observed into the blood [11,12]. Although genipin was not studied in detail, its pharmacological activities have been documented in recent years. Genipin shows the antifibrogenic effect by decreasing TGF-b1 expression in human subconjunctional fibroblasts [13]. In PC12h cells, genipin exerts neuritogenic effect by activating mitogenactivated protein kinase (MAPK) through the soluble guanylate cyclase-cGMP-dependent protein kinase-signaling pathway [14,15]. In addition to these actions, genipin has also been proved to possess antioxidant and antiinflammatory activities [16,17]. We previously reported that genipin induces apoptotic cell death in human hepatoma cells via a NADPH oxidase-reactive oxygen species (ROS)-c-Jun NH2-terminal kinase (JNK)-dependent activation of mitochondrial pathway [18]. In the present study, we carried out further studies to define that MLK3 is a key mediator, which connect between ROS and JNK in genipin-induced cell death signaling. Our present data suggest that genipin signaling to apoptosis of PC3 cells is mediated via activation of ROSdependent MLK3, which leads to downstream activation of JNK. Materials and methods Reagents. The MEK1/2 inhibitor U0126, p38 kinase inhibitor PD169316, JNK inhibitor SP600125, and NADPH oxidase inhibitor diphenyleneiodonium were from Calbiochem (La Jolla, CA). The Nacetyl-Asp-Glu-Val-Asp-q-nitroanlide (Ac-DEVD-pNA) was purchased from Enzyme Systems Products (Dublin, CA). DNA constructs. The expression plasmid DNAs encoding wild type of the human MLK3 (pCMS-EGFP-MLK3) and dominant-negative mutant

form of the human MLK3 (pCMS-EGFP-MLK3 (K144R)) were from Zhiheng Xu (Columbia University, New York, NY). Cell culture and generation of stable cell lines. PC3 human prostate cancer cell line obtained from American Type Culture Collection (Manassas, VA) was grown in RPMI1640 medium supplemented with 10% heat-inactivated bovine serum, 100 U/ml penicillin, and 100 lg/ml streptomycin at 37 C under a humidified 95/5% (v/v) mixture of air and CO2. For stable expression of dominant-negative MLK3, PC3 cells were co-transfected with pCMS-EGFP or pCMS-EGFP-MLK3 (K144R) expression plasmid and pCDNA3 expression plasmid using FuGENE 6 (Roche, Mannheim, Germany). Stably transfected clones were selected with 0.5 mg/ml neomycin (Invitrogen, Carlsbad, CA). After 2 weeks of selection, neomycin-resistant and fluorescence-positive colonies were populated, and analyzed for GFP-MLK3 expression by immunoblotting the cell lysates with anti-GFP antibody. DNA transfection and luciferase assay. The PC3 cells were transfected using FuGENE 6 (Roche, Mannheim, Germany). To control for variation in transfection efficiency, all clones were co-transfected with 0.2 lg of CMV-b-GAL, a eukaryotic expression vector in which Escherichia coli bgalactosidase (Lac Z) structural gene is under the transcriptional control of the CMV promoter. Luciferase reporter activity was assessed on a luminometer with a luciferase assay system (Promega, Madison, WI) according to the manufacturer’s protocol. Transfection experiments were performed in triplicate with two independently isolated sets, and the results were averaged. Caspase-3 assay. Caspase-3 activity in cytosolic extract was determined with a spectrophotometric assay, as described previously [19]. Briefly, the peptide substrate N-acetyl-Asp-Glu-Val-Asp-q-nitroanlide (Ac-DEVDpNA) was added to the cell lysates in assay buffer (50 mM Hepes, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 10 mM dithiothreitol, 1 mM EDTA, 10% glycerol) and incubated at 37 C. The cleavage of the substrate was monitored at 405 nm. Immunoblotting analysis. Cytosolic extracts were obtained in 1% Triton X-100 lysis buffer (50 mM Tris–Cl, pH 8.0, 150 mM sodium chloride, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM b-glycerophosphate, 1 lg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Western blotting was performed using anti-phospho-JNK (G9; Cell Signaling Technology, Beverly, MA), anti-phospho-MEK1/2 (47E6; Cell Signaling Technology, Beverly, MA), anti-phospho-p38 (3D7; Cell Signaling Technology, Beverly, MA), antiphospho-MLK3 (Cell Signaling Technology, Beverly, MA), anti-phosphoc-Jun (54B3; Cell Signaling Technology, Beverly, MA), anti-cleaved caspase-3 (5A1; Cell Signaling Technology, Beverly, MA), anti-cytochrome c (7H8.2C12; PharMingen, San Diego, CA), anti-GFP (FL; Santa Cruz Biotechnology, Santa Cruz, CA), anti-MKK7 (H160; Santa Cruz Biotechnology, Santa Cruz, CA), anti-JNK (FL; Santa Cruz Biotechnology, Santa Cruz, CA), anti-c-Jun (H-79; Santa Cruz Biotechnology, Santa Cruz, CA), anti-MLK3 (C-20; Santa Cruz Biotechnology, Santa Cruz, CA), and anti-b-actin (AC-15; Sigma, St. Louis, MO) antibodies. Protein samples were heated at 95 C for 5 min and analyzed by SDS–PAGE. Immunoblot signals were developed using Super Signal Ultra chemiluminescence detection reagents (Pierce Biotechnology, Rockford, IL). Immuno complex protein kinase assay. Kinases MKK7 and JNK were immunoprecipitated from control cell lysates using the appropriate antibody, respectively. MLK3 was obtained from cell lysates that were harvested after incubation with or without genipin for the indicated times. Immunoprecipitates were recovered with the aid of GammaBind beads and were washed twice with lysis buffer containing 500 mM NaCl, twice with lysis buffer, and twice again with kinase buffer (20 mM MOPS, pH 7.2, 2 mM EGTA, 20 mM a-glycerol phosphate). The MLK3 precipitates were mixed with MKK7 and JNK. The kinase reaction was initiated by addition of 200 lM ATP and 2 lg of c-Jun. Following 15-min incubation at 30 C, the reaction were terminated by the addition of SDS loading buffer. Immunoblots were performed using anti-phospho-c-Jun antibody. JNK protein kinase assay. JNK activity was determined using a JNK assay kit according to the manufacturer’s protocol (Cell Signaling Technology, Beverly, MA). Briefly, an amino-terminal c-Jun (amino acid residues 1–89) fusion protein bound to glutathione-Sepharose beads was used

H.-Y. Hong, B.-C. Kim / Biochemical and Biophysical Research Communications 362 (2007) 307–312 to pull down JNK from cell lysates. The kinase reaction (50 ll) was then carried out using the c-Jun fusion protein as a substrate in the presence of cold ATP. Phosphorylation of the c-Jun fusion protein at Ser-63 was measured by Western blot using an anti-phospho-c-Jun rabbit polyclonal antibody that detects only catalytically activated c-Jun phosphorylated at Ser-63. Protein samples were heated to 95 C for 5 min and subjected to SDS–PAGE on 10% acrylamide gels. Immunoblots were performed using anti-phospho-c-Jun antibody. Analysis of cytochrome c release. For mitochondrial cytochrome c release assay [20], PC3 cells were scraped off in isotonic isolation buffer (10 mM Hepes, pH 7.6, 1 mM EDTA, 250 mM sucrose), collected by centrifugation at 2500g for 5 min at 4 C, and resuspended in hypotonic isolation buffer (10 mM Hepes, pH 7.6, 1 mM EDTA, 50 mM sucrose). Cells were disrupted by passing through a 27-gauge needle 5–10 times and checked for cracked cells by trypan blue staining. Hypertonic isolation buffer (10 mM Hepes, pH 7.6, 1 mM EDTA, 450 mM sucrose) was added to balance the buffer’s tonicity. Samples were centrifuged at 100,000g at 4 C for 1 h, and supernatants containing the cytosolic proteins were recovered, and analyzed by Western blotting. Measurement of intracellular ROS. For analysis of intracellular ROS, the redox-sensitive fluorescent probe DCFH-DA was used, as previously described [21]. Cells were incubated with 5 lM DCFH-DA for 30 min at 37 C. The harvested cells were immediately analyzed by a flow cytometry. Assessment of mitochondrial transmembrane potential. Changes in mitochondria membrane potential were determined using staining cells with the fluorescence probe dihydrorhodamine 123 (Molecular Probes, Eugene, OR). Cells were incubated in phosphate-buffered saline (PBS) containing 10 lM dihydrorhodamine 123 (Rh-123) for 30 min at 37 C in the dark and analyzed in a FACScalibur flow cytometry (Becton Dickinson, San Jose, CA). For control purposes, the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) (50 lM; Sigma, St. Louis, MO) was used. The fluorescence was excited with an Argon laser (excitation wavelength, 488 nm) and analyzed in FL-1 (wavelength, 520 nm; photomultiplier tube [PMT] voltage, 437 V). At least 2 · 104 events were acquired in list mode and analyzed with CELLQuest software (Becton Dicknson, San Jose, CA). Statistical analysis. All data presented are expressed as means ± SD, and a representative of three or more independent experiments. Statistical analyses were assessed by Student’s t test for paired data. Results were considered significant at p < 0.05.

Results and discussion Activation of MLK3 by genipin To identify the involvement of mixed-lineage kinase 3 (MLK3), the levels of phosphorylated and total MLK3 were determined in PC3 cells treated with 100 lM genipin for the various times. Genipin treatment of PC3 cells induced phosphorylation of MLK3 that reached a maximum within 30 min (Fig. 1A). Treatment with genipin at 10, 50, 100, 200 lM for 30 min enhanced MLK3 phosphorylation in PC3 cells in a concentration-dependent manner (Fig. 1B). Because MLK3 has been known to act upstream of both MKK4 and MKK7, which, in turn, lie upstream of JNKs and c-Jun in the apoptotic pathway, the activity of MLK3 at different times after treatment of genipin was determined by in vitro kinase assay toward c-Jun as described in Materials and methods. The time-course pattern for the maximum activation of c-Jun phosphorylation correlated well with that of maximum MLK3 activation (Fig. 1A and C).

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α-β-actin Fig. 1. Genipin stimulates phosphorylation and activation of MLK3 in PC3 cells. Cells were treated with 100 lM genipin for the indicated times (A) or at the indicated concentrations for 30 min (B). Cell lysates were subjected to SDS–PAGE followed by immunoblot analysis using antibody specific to phosphorylated form of MLK3. (C) MLK3 was obtained from cell lysates that were harvested after incubation with or without genipin for the indicated times. The MLK3 immunoprecipitates were mixed with MKK7 and JNK, and in vitro kinase assay was performed using c-Jun as substrate.

Requirement for MLK3 in genipin-induced apoptosis To further investigate the role of MLK3 in genipin signaling to apoptosis, we generated cell lines stably expressing a GFP-fused dominant-negative MLK3 mutant (EGFP-MLK3 (K144R)) in PC3 cells. Because MLK3 was cloned into the pCMS-EGFP vector, stable cells expressing the dominant-negative form of MLK3 could be visualized by green fluorescence. Exposure of PC3EGFP cells to 100-lM genipin for 24 h resulted in the apparent apoptotic cell death with cell shrinkage and appearance of condensed nuclei whereas exposure to the same concentration of genipin on PC3-EGFP-MLK3 (K144R) cells was unable to give apoptotic cell death (Fig. 2A). Fluorescence-activated cell sorter (FACS) analysis also showed that the cell death percentage of genipintreated PC3-EGFP-MLK3 (K144R) cells in sub-G1 phase was low to the genipin-treated control PC3-EGFP cells (Fig. 2B). Recent findings reveal that activation of the JNK pathway can cause cytochrome c release and that apoptotic stimuli fail to release cytochrome c in JNK null cells [22]. Accordingly, to test whether apoptotic cell death evoked by MLK3 is propagated in this manner, the efflux of cytochrome c from mitochondria to cytosol was examined in genipin-treated PC3-EGFP and PC3-EGFPMLK3 (K144R) cells by Western blotting. As shown in Fig. 2C, cytochrome c content was clearly diminished in

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Fig. 2. MLK3 activity is required for genipin-induced apoptosis. (A) PC3-EGFP or PC3-EGFP-MLK3 (K144R) cells were treated with DMSO or 100 lM genipin for 24 h. Changes in cellular morphologies were observed by phase-contrast microscopy (magnification, 200·). A fluorescence image of PC3 cells stably transfected with control vector (pEGFP) or dominant-negative mutant of MLK3 (pEGFP-MLK3 (K144R) was shown from the picture of above. (B) A representative illustration is shown of propodium iodide (PI) incorporation measured in control and genipin-treated conditions by flow cytometric analysis. Cell death is expressed as the percentage of cells counted in sub-G1 phase. (C) Release of cytochrome c from mitochondria in PC3EGFP or PC3-EGFP-MLK3 (K144R) cells treated with 100 lM genipin or vehicle, which was detected by Western blot analysis. (D) Changes of mitochondrial transmembrane potential (Dwm) in PC3-EGFP or PC3-EGFP-MLK3 (K144R) cells pretreated with DMSO (gray) or 100 lM genipin (black) before adding Rh-123, which were measured in FACScan flow cytometer. For a positive control, carbonyl cyanide m-chlorophenylhydrazone (CCCP), an uncoupling agent, was used. (E) Caspase-3 activity in PC3-EGFP or PC3-EGFP-MLK3 (K144R) cells treated with 100 lM genipin or vehicle. The enzymatic activity is represented as DA405/min/mg protein. (F) Immunoblot for cleaved caspase-3. For the detection of cleaved caspase-3, PC3-EGFP or PC3-EGFP-MLK3 (K144R) cells were treated with 100 lM genipin or vehicle for 24 h.

the cytosol of genipin-treated PC3-EGFP-MLK3 (K144R) cells than in control PC3-EGFP cells. Because high amplitude mitochondrial swelling causes release of mitochondrial proteins (e.g., cytochrome c) through collapse of mitochondrial outer membrane [23], the effect of genipin on mitochondrial membrane potential (Dwm) also was examined in PC3-EGFP and PC3-EGFP-MLK3 (K144R) cells. The fluorescent mitochondrial-specific dye dihydrorhodamine 123 (Rh-123) was used as a probe of Dwm. Treatment with genipin induced a decrease of Rh-123 fluorescence (depolarization) in PC3-EGFP cells whereas these changes were not seen in PC3-EGFP-MLK3 (K144R) cells (Fig. 2D). Decrease in Rh-123 fluorescence by genipin was comparable with the one induced by treatment with the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler that collapse Dwm (Fig. 2D). The sequential activation of caspase in the transduction of apoptotic signal is also characteristic of the mitochondrial apoptotic pathway [24]. As shown in Fig. 2E, less stimulation of caspase-3 activity in the presence of genipin was time-dependently observed in MLK3 (K144R)-expressing PC3 cells compared to control cells. The decreased proteolytic activation of caspase-3 in genipin-treated PC3-EGFP-MLK3 (K144R) cells also was defined by Western blotting (Fig. 2F). These results suggest

that activation of MLK3 is ultimately responsible for genipin-induced apoptotic process in PC3 human prostate cancer cells. ROS acts upstream of MLK3 in genipin signaling Reactive oxygen species (ROS) has been recognized as an important mediator of the genipin signaling to apoptosis in hepatoma cells [18]. We next determined any role of ROS for the activation of MLK3 in response to genipin. In PC3 cells, genipin enhanced ROS levels, and this effect was dramatically inhibited by DPI, a NADPH-like flavoenzyme inhibitor (Fig. 3A). Moreover, DPI blocked genipin-induced MLK3 phosphorylation in a concentrationdependent manner (Fig. 3B). However, levels of genipininduced ROS production were similar in PC3-EGFP and PC3-EGFP-MLK3 (K144R) cells (Fig. 3C). It thus appears that genipin stimulates generation of ROS, and this leads to MLK3 activation and subsequently to apoptosis. JNK acts downstream of MLK3 in genipin signaling The use of SP600125, a specific inhibitor of JNK, U0126, a specific inhibitor of MEK1/2 and PD169316, a specific inhibitor of p38 MAPK, confirmed requirement

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Fig. 3. Genipin stimulates MLK3 via a ROS-dependent pathway. (A) PC3 cells were pretreated with the redox-sensitive fluorescence dye DCFH-DA for 30 min, and then, treated with 100 lM genipin in the presence or absence of diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase. (B) PC3 cells were exposed to 100 lM genipin in the presence or absence of DPI. Phosphorylation of MLK3 was analyzed by immunoblot analysis. (C) PC3-EGFP or PC3-EGFP-MLK3 (K144R) cells were incubated as described in (A), after which DCF fluorescence was analyzed.

for JNK activation during genipin-induced apoptosis in PC3 cells. SP600125 markedly suppressed the activation of caspase-3 in genipin-treated PC3 cells (Fig. 4A). In con-

trast, other inhibitors, UO126 and PD169316, were unable to suppress the activation of caspase-3 in the same conditions (Fig. 4A). To determine whether MLK3 acts

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Fig. 4. Genipin-induced apoptosis is mediated via MLK3-JNK-linked cascade. (A) PC3 cells were treated with 100 lM genipin or vehicle for 24 h in the absence (control) or presence of JNK inhibitor SP600125 (20 lM), MEK1/2 inhibitor U0126 (10 lM) or p38 MAPK inhibitor PD169316 (5 lM). Caspase3 activity was determined as described under ‘‘Materials and methods’’. The enzymatic activity is represented as DA405/min/mg protein. *p < 0.05 compared with control cells treated with genipin. (B) PC3-EGFP or PC3-EGFP-MLK3 (K144R) cells were incubated with 100 lM genipin or vehicle for 30 min. Equal amount of protein samples was then assayed for JNK activity using c-Jun fusion protein (1–89) as a substrate. (C) PC3 cells, which were cotransfected with the pc-Jun-Luc reporter plasmid with pCMS-EGFP, pCMS-EGFP-MLK3 (WT), or pCMS-EGFP-MLK3 (K144R), were incubated without or with 100 lM genipin. The relative luciferase activity was calculated as described under ‘‘Materials and methods’’. *p < 0.05 compared with control cells treated with genipin.

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upstream of JNK during genipin-induced apoptosis, levels of JNK activation was compared between PC3-EGFP and PC3-EGFP-MLK3 (K144R) cells. As shown in Fig. 4B, genipin-induced JNK activation was dramatically reduced in PC3-EGFP-MLK3 (K144R) cells, indicating the importance of MLK3 activity in that case. Additional evidence on the participation of MLK3 as an upstream activator of JNK in genipin signaling was obtained using a reporter plasmid pc-Jun-Luc containing c-Jun promoter fused to luciferase coding sequences. Co-transfection with pCMSMLK3 (K144R) encoding a dominant-negative MLK3 mutant significantly and dose-dependently diminished genipin-induced stimulation of c-Jun-luciferase activity, whereas co-transfection with pCMS-MLK3 (WT) further enhanced genipin-induced c-Jun promoter activity (Fig. 4C). These findings indicate that MLK3 acts upstream of JNK in apoptotic signaling cascade triggered by genipin. Collectively, our data strongly suggest that genipin signaling to apoptosis of PC3 cells is mediated via activation of ROSdependent MLK3, which leads to downstream activation of JNK. Acknowledgments This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF2005-003-C00137). References [1] C. Rust, G.J. Gores, Apoptosis and liver disease, Am. J. Med. 108 (2000) 567–574. [2] M. Valko, D. Leibfritz, J. Moncol, M.T. Cronin, M. Mazur, J. Telser, Free radicals and antioxidants in normal physiological functions and human disease, Int. J. Biochem. Cell Biol. 39 (2007) 44–84. [3] J.M. McCord, Human diseases, free radicals, and the oxidant/ antioxidant balance, Clin. Biochem. 26 (1993) 351–357. [4] E. Cadenas, K.J.A. Davies, Mitochondrial free radical generation, oxidative stress, and aging, Free Radic. Biol. Med. 29 (2000) 222–230. [5] H.M. Shen, Z.G. Liu, JNK signaling pathway is a key modulator in cell death mediated by reactive oxygen and nitrogen species, Free Radic. Biol. Med. 40 (2006) 928–939. [6] K.A. Gallo, G.L. Johnson, Mixed-lineage kinase control of JNK and p38 MAPK pathways, Nat. Rev. Mol. Cell Biol. 3 (2002) 663–672. [7] Z. Xu, A.C. Maroney, P. Dobrzanski, N.V. Kukekov, L.A. Greene, The MLK family mediates c-Jun N-terminal kinase activation in neuronal apoptosis, Mol. Cell Biol. 21 (2001) 4713–4724. [8] Q.G. Zhang, R.M. Wang, X.H. Yin, J. Pan, T.L. Xu, G.Y. Zhang, Knock-down of POSH expression is neuroprotective through downregulating activation of the MLK3-MKK4-JNK pathway following cerebral ischemia in the rat hippocampal CA1 subfield, J. Neurochem. 95 (2005) 784–795. [9] Y. Du, B.C. Bock, K.A. Schachter, M. Chao, K.A. Gallo, Cdc42 induces activation loop phosphorylation and membrane targeting of mixed lineage kinase 3, J. Biol. Chem. 280 (2005) 42984–42993.

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