A novel mechanism of methylglyoxal cytotoxicity ... - Wiley Online Library

JOURNAL OF NEUROCHEMISTRY

| 2009 | 108 | 273–284

doi: 10.1111/j.1471-4159.2008.05764.x

*Department of Physiology, Medical Science Research, Institute, College of Medicine, Dong-A University, Busan, South Korea Department of Pharmacology, Medical Science Research, Institute, College of Medicine, Dong-A University, Busan, South Korea

Abstract Methylglyoxal (MGO) is an endogenous dicarbonyl compound that is highly produced in hyperglycemic conditions. It forms advanced glycation endproducts that are believed to contribute, as etiological factors, to the pathophysiology of diabetic complications. In addition, MGO suppresses cell viability through the induction of apoptosis in vitro. In this study, we have, for the first time, demonstrated the effect of MGO on the gp130 cytokine-induced signal transducer and activator of transcription 3 (STAT3) responses in RT4 schwannoma, PC12 pheochromocytoma and U87MG glioma cells. At dose that very mildly affects cell viability, MGO rapidly induces endocytotic degradation of gp130, which involves the di-leucine internalization motif in the cytoplasmic domain of gp130, without affecting other growth factor receptors. Concomitant

inhibition of basal and interleukin-6-induced STAT3 activation was observed following pre-treatment with MGO. The inhibitory effect of MGO on the gp130/STAT3 signaling was prevented by the pre-treatment with an advanced glycation endproduct scavenger aminoguanidine. Finally, these deleterious effects of MGO on STAT3 signaling led to down-regulation of a STAT3 target gene, Bcl-2, and sensitized cellular toxicity induced by H2O2 and etoposide. Our data indicate that MGO affects cell viability via desensitization of gp130/STAT3 signaling, which is the key signaling pathway for cell survival, and thereby promotes cytotoxicity. Keywords: cell death, cytokines, gp130, methylglyoxal, neuroglial cells, STAT3. J. Neurochem. (2009) 108, 273–284.

Persistent hyperglycemia appears to initiate the formation of advanced glycation end products (AGEs), leading to one possible mechanism of hyperglycemia-induced tissue damage (Brownlee 2005). It is known that the concentrations of potent AGE precursors, glyoxal and methylglyoxal (MGO), are increased in the plasma of diabetic patients (McLellan et al. 1994; Beisswenger et al. 2001; Lapolla et al. 2003). At high concentrations, glyoxal and MGO affect cellular function by reacting with amino and sulfhydryl groups of several proteins, which may result in diabetic complications (Zeng and Davies 2005). For example, covalent modification of signaling intermediates, such as insulin receptor substrate by MGO, is implicated in the impairment of cellular insulin signaling (Riboulet-Chavey et al. 2006). Growth factor receptors can also be glycated by MGO, which may contribute to the detrimental effects of AGEs in cell proliferation and survival (Cantero et al. 2007). Furthermore, AGEs have been suggested to be involved in neurodegenerative diseases such as Alzheimer’s disease since AGEs are found in senile plaques and neurofibrillary tangles (Picklo et al. 2002). Based upon these observations, the blockage of

AGE precursor-induced protein derivatization with AGE scavengers is a promising target for attenuation of the progression of diabetic complications (Brownlee 2005) and neurodegenerative diseases (Grillo and Colombatto 2007). MGO-induced cell death is accompanied by the generation of many apoptotic markers such as caspase-3 activation, DNA ladder formation and cytochrome C release (Du et al. 2001; Chan et al. 2007). There are multiple mechanisms by Received July 24, 2008; revised manuscript received September 16, 2008; accepted October 27, 2008. Address correspondence and reprint requests to Hwan Tae Park, Department of Physiology, Medical Science Research, Institute, College of Medicine, Dong-A University, 3-1, Dongdaesin-Dong, Seo-Gu, Busan 602-714, South Korea. E-mail: [email protected] Abbreviations used: AGD, aminoguanidine; AGE, advanced glycation endproducts; DMEM, Dulbecco’s modified Eagle’s Medium; EEA1, early endosomal antigen 1; EGF, epidermal growth factor; FBS, fetal bovine serum; IL-6, Interleukin-6; JAK, janus kinase; MGO, methylglyoxal; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate buffered saline; PFA, paraformaldehyde; ROS, reactive oxygen species; SDS, sodium dodecyl sulfate; STAT3, signal transducer and activator of transcription 3.

2008 The Authors Journal Compilation 2008 International Society for Neurochemistry, J. Neurochem. (2009) 108, 273–284

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which MGO can induce apoptosis. For example, MGOinduced generation of reactive oxygen species (ROS) by modification of mitochondrial glutathione reductase is known to trigger apoptosis (Du et al. 2001; Amicarelli et al. 2003). In addition, p38 MAPK activation was suggested to be a key signaling intermediate of MGO-induced apoptosis in kidney epithelial cells and Schwann cells (Liu et al. 2003; Fukunaga et al. 2004). Furthermore, Chan et al. (2007) recently suggested that the JNK pathway is important for MGO-induced apoptosis. On the other hand, the MGOinduced alterations in growth factor receptor signaling (Cantero et al. 2007) might be implicated in the development of MGO cytotoxicity because growth factors modulate for cell survival and proliferation. However, the effects of MGO on cytokine signaling have not yet been fully investigated specifically with regard to regulation of cell survival by MGO. Signal transducer and activator of transcription 3 (STAT3) is a critical regulator of cytokine-induced gene expressions (Battle and Frank 2002). Exposure of cells to interleukin(IL)-6 leads to the activation of its cell surface receptors, which consist of IL-6 receptor (IL-6R) and 130-kDa transmembrane signal transducer (gp130), thus resulting in the subsequent activation of the janus kinase (JAK)/STAT3 pathway (Kamimura et al. 2003). STAT3 is activated by phosphorylation at its tyrosine 705 residue, and tyrosine phosphorylated STAT3 translocates into the nucleus, where it functions as a transcription factor (Battle and Frank 2002). In the nervous system, the gp130/STAT3 pathway is important not only for the maintenance of normal functions but also for regenerative capacity of injured nerves. For example, gp130 cytokines have been shown to be implicated in the maintenance of motor neuron survivals and the peripheral myelin sheath (Betz et al. 1998; Schweizer et al. 2002). After nerve injury, the STAT3 signaling cascade mediates protective and regenerative functions in neuroglial cells (Schweizer et al. 2002; Qiu et al. 2005; Miao et al. 2006; Herrmann et al. 2008). It was recently reported that expression of ciliary neurotrophic factor, a gp130 cytokine, was decreased in diabetic neuropathy (Calcutt et al. 1992) and that gp130 cytokines had a protective role in the development of diabetic neuropathy (Mizisin et al. 2004; Cameron and Cotter 2007). Given the importance of the gp130/STAT3 signaling pathway in normal and pathological conditions of the nervous system, the aim of our present work is to investigate whether the gp130/STAT3 signaling pathway is modified by the hyperglycemic metabolite, MGO, in neuroglial cells.

MA, USA). Antibodies to STAT3, Bcl2, gp130, Src, ErbB2 and beta-tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The monoclonal antibody to early endosomal antigen1 (EEA1) and goat antibody to gp130 were obtained from Chemicon (Temecula, CA, USA) and R&D Systems (Minneapolis, MN, USA), respectively. An antibody against the Flag peptide, MGO and all other undesignated reagents were purchased from Sigma (St. Louis, MO, USA).

Materials and methods

Western blot analysis Western blot analysis was performed as described previously (Lee et al. 2006, 2007a). Briefly, cells were harvested and lysed in modified radioimmune precipitation assay lysis buffer [150 mM NaCl, 1% Nonidet P-40 (Sigma), 1 mM EDTA, 0.5% deoxycholic

Chemicals All of the phospho-specific antibodies and recombinant IL-6 used in this work were purchased from Cell Signaling Technology (Beverly,

Molecular cloning of Flag-gp130 mutants The human gp130 (aa 23-918) cDNA lacking its signal peptide sequence was PCR amplified from a human gp130 cDNA template (a gift from Dr. T. Taga and K. Ikenaka). The sense PCR primer (5¢GCCGAAGCTTACGAACTTCTAGATCCA-3¢) contained a Hind III site, and the anti-sense primer contained an in-frame stop codon (5¢-CTCGAGTCACTGAGGCATGAGCC-3¢) and Xho I site. The PCR was performed in a PCR machine (Astek, Fukuoka, Japan) according to the following program: 30 cycles at 95C for 45 s, 56C for 45 s and 72C for 45 s with a final extension at 72C for 5 min. After PCR amplification and restriction enzyme digestion, the PCR fragments were inserted downstream of the signal peptide coding sequence in the AP-5 vector (GeneHunter, Brookline, MA, USA, Lee et al. 2006). The Flag epitope tag (DYKDDD) was inframe inserted between the signal peptide sequence of the AP-5 vector and the 23rd aa of the human gp130, thus leading to generation of a signal peptide-Flag tag-gp130 chimera. A gp130 truncation mutant (aa 23-785) that lacks the C-terminal 133 aa was constructed by inserting a stop codon immediately in front of the dileucine motif of the chimeric full-length Flag-gp130 using the QuickChange Mutagenesis kit (Stratagene, La Jolla, CA, USA) as manufacturer’s instructions. The sequence of the sense primers used for mutagenesis is as follows; 5¢-GAGYCYACCCAGCCCTGATT GTTAGATTCAGAG-3¢. The altered region of the mutant construct was verified by sequencing. Cell culture and transient transfection Rat schwannoma RT4 cells, PC12 cells and U87 glioma cells were obtained from the American Type Culture Collection (Rockville, MD, USA) and maintained as previously described (Lee et al. 2000, 2007b). Cells were starved overnight in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 0.1% fetal bovine serum (FBS) before the addition of cytokines or trophic factors. Approximately 24 h after plating, RT4 cells were rinsed twice with DMEM and transfected with a mixture of 0.5 lg plasmid DNA and 2.5 lL of lipofectamine reagent (Invitrogen, Carlsbad, CA, USA) in 80 lL of DMEM per well of an 8 well Labtek dish (Nunc, Rochester, NY, USA). After 8 h, the reaction mixtures were removed, and fresh DMEM containing 10% FBS was added to each well for additional 12 h. The transfection media were then replaced with the growth medium, and the transfected cells were incubated for 48 h.


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acid, 2 lg/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine, 1 mM sodium orthovanadate, and 1 · protease inhibitor cocktail (Roche, Indianapolis, IN, USA)]. The lysates were centrifuged at 8000 g for 10 min at 4C, and the supernatant was collected and submitted to Western blotting. The concentration of protein in each sample was analyzed by the Bradford method, and 2535 lg of protein was separated by 8% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and then transferred onto a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA). After blocking with 0.1% Tween-20 and 5% non-fat dry milk in Tris-buffered saline (25 mM Tris-HCl pH 7.5, 140 mM NaCl) at 20C for 1 h, the membrane was incubated with primary antibodies (1 : 500–1000) in Tris-buffered saline containing 0.3% Triton-X100 containing 2% non-fat dry milk at 4C overnight. After three 15 min washes with TBST, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1 : 3000) for 1 h at 22C. The signals were detected using the Enhanced Chemiluminescence System (ECL advance kit, Amersham Biosciences). For quantification, X-ray films were then scanned using an HP scanner and analyzed with the LAS image analysis system (Fujifilm, Tokyo, Japan). The intensities of the gp130 bands were normalized to those of beta-tubulin, whereas the intensities of the pSTAT3 bands were normalized to those of STAT3 in three independent experiments.

Fig. 1 MGO treatment decreases of cell surface gp130 expression. (a,b) RT4 cells (a) or U87MG glioma cells (b) were treated MGO for 1 day and cell survival was analyzed using the MTT assay. (c–f) Cells were either untreated or treated with MGO (500 lM), cooled and labeled with an antigp130 antibody on ice. The cells with antibody-labeled gp130 were analyzed by flow cytometry (c) or confocal laser microscopy (d–f). (c) A representative result of flow cytometric analysis showed a dramatic decrease of cell surface gp130 by MGO treatment in 30 min. (d–f) Confocal microscopic analyses in RT4 cells (d,e) and PC12 cells (f) revealed that distinct membrane staining of gp130 (red), but not ErbB2 (green), was dramatically reduced following MGO treatment, and the reduction was blocked by AGD treatment. Scale bar; 30 lm. (e) Quantification of the fluorescent intensity of gp130 labeling in the confocal microscopic image. *p < 0.05, the Bonferroni post hoc test.

Immunofluorescence analysis For cellular immunofluorescent staining, cells were fixed with icecold methanol for 15 min at 4C (for pSTAT3) or 4% paraformaldehyde (PFA) for 15 min at 22C (for other antigens) and then washed three times with phosphate bufferd saline (PBS). The cells were blocked with PBS containing 0.2% Triton X-100 and 10% bovine serum albumin for 1 h. Cells were then incubated with primary antibody (1 : 1000) for 16 h at 4C and washed three times with PBS. Next, cells were incubated with Cy3- or Alexa 488conjugated secondary antibody (1 : 800, Jackson Immunoresearch Laboratories, West Grove, PA, USA) for 3 h at 22C and viewed using a laser confocal microscope (LSM510, Carl Zeiss, Jena, Germany). Since our data have a quantitative element using a confocal microscope (Fig. 1e), we used the same PMT setting (detector gain, amplifier offset and amplifier gain) under a constant laser power for every set of experiments. We morphometrically analyzed the intensity of the fluorescence using software that accompanied the LSM 510 microscope. The intensity of the immunofluorescence was evaluated in five randomly selected 272 · 272 lm2 areas from each sample. A total of 10 areas were counted for each group. Live cell binding assay To label cell surface gp130, a goat anti-gp130 antibody that recognizes the extracellular portion of gp130 (R&D Systems) was

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employed. Live cell binding was performed as previously reported (Kim et al. 2005). Briefly, after a 30-min treatment with MGO, the dishes were cooled at 4C, and then incubated the anti-gp130 antibody was treated for 90 min at 4C. The cells were washed twice with PBS to remove unbound antibody and then fixed with 4% PFA for 15 min. After three washes with PBS, the cells were incubated with Cy3-conjugated secondary antibody (1 : 800) for 2 h at 22C. For double immunofluorescent labeling (Fig. 3), a monoclonal anti-Flag antibody (2 lg/mL) was used for the initial phase of live cell labeling. After fixation, cells were immunostained with a rabbit anti-human gp130 antibody (1 : 1000, Santa Cruz) for 16 h at 4C, followed by incubation with Alexa 488- and Cy3conjugated secondary antibodies. Flow cytometry For the detection of cell surface gp130, cells were detached from culture dishes with 0.025% Trypsin-EDTA. After recovery of cells in DMEM containing 10% FBS for 1 h, cells were treated with MGO (500 lM) for 1 h, and then washed. Cells (6.5 · 105) were incubated with the anti-gp130 antibody (1 : 1000) for 30 min at 22C, fixed with 4% PFA, and then incubated with Cy3-conjugated donkey anti-goat IgG for 30 min at 22C. The cells were analyzed by flow cytometry using a FACScan cytometer (Epics XL, Beckman Coulter, Miami, FL, USA). A total of 5000 cells were counted for each FACS analysis. DNA binding assay Total cellular extracts of RT4 cells were prepared in lysis buffer (15 mM HEPES, pH 7.6, 40 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 5% glycerol) and the extracts (300 lg/500 lL) were incubated with 1.5 lg of STAT3-interacting gel shift oligonucleotide (5¢-GATCCTTCTGGGAATTCCTAGATC-3¢)–agarose bead (Santa Cruz) for 2 h at 22C (Ng et al. 2006). After incubation, the beads were washed extensively with the lysis buffer. The bead-precipitated proteins were eluted with 30 lL of SDS sample buffer, separated by 8% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane for immunoblotting. Preparation of primary Schwann cells Primary Schwann cells from adult sciatic nerves were cultured as previously reported (Lee et al. 2007a). All procedures were performed according to protocols approved by the Dong-A University Committee on animal research, which follows the guide of animal experiment established by the Korean Academy of Medical Sciences. The sciatic nerves of adult Sprague-Dawley rats were axotomized to enhance Schwann cell population Sciatic nerves were sectioned 5 mm proximal to the tibioperoneal bifurcation with a fine iris scissor (FST Inc, Foster City, CA, USA) and the regeneration was inhibited by deflection of the proximal stump. Skin incisions were closed with sutures, and animals were housed in plastic cages for 3–4 days after injury. The sciatic nerves were removed and submitted to chemical digestion in 0.2% collagenase in calcium/magnesium-free Hank’s buffered solution at 37C for 2 h. Nerves were then dissociated by gentle shaking for 2 min followed by two or three triturations using a flame-polished Pasteur pipette. The cell pellets obtained after centrifugation were resuspended in DMEM containing 10% FBS, plated at a density of 20,000 cells/cm2

and grown for 2 days. After two or three subcultures using the coldjet method (Jirsova et al. 1997), the identity and purity of Schwann cells were assessed by immunostaining with an antibody raised against S100 (data not shown, Lee et al. 2007a). The S100 staining data indicated that we consistently obtained Schwann cells of 8590% purity. Survival assay The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to analyze cell survival as previously described (Lee et al. 2007a). Briefly, cells were plated in DMEM containing 10% FBS at a density of 5 · 103 cells/well in a 96 well tissue culture plate overnight. At 1 day after plating, cells were washed twice with PBS and grown in serum-free DMEM for 1 day in the presence or absence of the indicated reagents, followed by incubation for 1 h in the presence of 0.5 mg/mL MTT (Sigma). The medium was then aspirated, and the cells were dissolved in isopropanol (100 lL/well). Absorbance of the converted dye (OD) was measured at 570 nm, with a background subtraction at 630 nm in a microplate leader (Bio-Rad, Hercules, CA, USA). All experiments were repeated at least three times. Statistical analysis Differences in the means between the treatment groups were statistically assessed using an analysis of variance followed by the Bonferroni post hoc test. The differences were considered to be statistically significant at the p < 0.05 level.

Results MGO alters membrane localization of gp130 We first examined the cytotoxic effect of MGO in neuroglial cells. MGO treatment elicited dose-dependent cytotoxicity on RT4 schwannoma cells (Fig. 1a), U87MG glioblastoma (Fig. 1b) and PC12 pheochromocytoma cells (data not shown). MGO very mildly reduced cell survival as measured by the MTT assay at a concentration of 500 lM. In contrast, cell death was readily apparent following incubation with 1 mM MGO. In order to determine whether MGO acutely affects gp130-related cytokine signaling in RT4 cells, we first measured a possible change in the presence of gp130 on the cell surface using flow cytometric analysis. As shown in Fig. 1(c), this experiment showed a significant reduction in gp130 labeling of cells incubated with MGO (500 lM) for 30 min, compared to untreated controls. We next employed a live cell binding assay with confocal microscopic analysis (Fig. 1d and f). In control RT4 cells (Fig. 1d) and PC12 cells (Fig. 1f), the binding assay with the anti-gp130 antibody revealed intense membrane localization of gp130. The membrane labeling of cell surface gp130 was dramatically reduced within 30 min of treatment with MGO. It was previously shown that covalent modification of proteins by MGO is involved in the impairment of several signaling processes by MGO (Chang and Wu 2006; Riboulet-Chavey



et al. 2006). Therefore, we investigated whether pre-treatment of cells with aminoguanidine (AGD), which can prevent protein modifications by MGO, hampers the MGO-induced reduction of cell surface gp130. Indeed, incubation of cells with AGD (2 mM), before and during treatment with MGO (500 lM), protected cells from the MGO-induced reduction of gp130 level on the cell surface (Fig. 1d and e). We obtained similar results with PC12 neuronal cells (Fig. 1f). In order to evaluate whether the reduction in gp130 cell surface expression by MGO was specific to gp130, we examined a possible change in the membrane localization of ErbB2 following MGO treatment. It was found that the distinct membrane staining of ErbB2 was not changed by MGO treatment of RT4 cells (Fig. 1d), indicating the specificity of the MGO action on gp130. These findings indicate that the membrane localization of gp130 was altered by MGO. MGO induces endocytotic degradation of gp130 The rapid disappearance of cell surface gp130 by MGO may indicate endocytotic degradation of gp130. To address this

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Fig. 2 Endocytotic degradation of gp130 by MGO in RT4 cells. (a) In control RT4 cells, diffuse perinuclear and membrane staining of gp130 was observed, whereas MGO treatment (500 lM) induced a punctate gp130 staining pattern in the cytoplasm within 30 min. Scale bar: 20 lm. (b) Enlarged images showed that many MGO-induced gp130 immunoreactive clusters were colocalized with EEA1 immunostained regions. Scale bar: 5 lm. (c) Western blot analysis showed that a significant degradation of gp130, but not ErbB2 or betatubulin (Tub), was observed within 1 h of MGO treatment (500 lM) in RT4 cells. (d) A quantitative result showing the time course of the change in gp130 levels following MGO treatment. Data represent the means ± SE of three independent experiments. (e) The effect of an endocytosis blocker, concanavalin A (conA), on MGO-induced degradation of gp130. Cells were incubated with MGO (500 lM) for the indicated lengths of time in the presence of absence of concanavalin A.

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issue, we first co-immunostained cells with antibodies to gp130 and an endocytotic vesicle marker, EEA1 (Lee et al. 2006), following MGO treatment. Untreated RT4 cells showed gp130 immunoreactivity in the cell membrane and perinuclear region. Interestingly, MGO treatment (500 lM) induced dramatic aggregation of gp130 into numerous clusters in the cytoplasm within 30 min (Fig. 2a and b). In addition, most MGO-induced gp130 immunoreactive clusters were colocalized with the EEA1, as indicted by overlap of gp130 and EEA1 immuno-positive regions (Fig. 2b). This finding suggests that MGO induces endocytosis of gp130. It is possible that MGO-induced gp130 endocytosis leads to gp130 degradation. We thus investigated the total cellular level of gp130 protein following MGO treatment using Western blot analysis (Fig. 2c). Examination of gp130 protein levels following MGO (500 lM) treatment revealed a significant reduction in the amount of gp130 proteins. Furthermore, very faint immunoreactive gp130 bands were observed 3 h after MGO treatment in RT4 cells (Fig. 2c and

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Fig. 3 Intracellular region of gp130 containing di-leucine motif is required for MGO-induced endocytosis of gp130. Wild-type or truncated Flag-gp130 was transiently transfected in RT4 cell. At 2 days posttransfection, cells were stimulated with MGO (500 lM) for 30 min. Next, an anti-Flag antibody was allowed to bind to cell surface on ice, and then anti-Flag immunostaining was co-stained with an anti-human gp130 antibody. (a) Cells transfected with wild-type Flag-gp130

showed numerous intracellular immunoreactive clusters of gp130 following MGO treatment. (b) Cells transfected with the truncated Flaggp130 showed no alteration of the membrane stainings of Flag and gp130 following MGO treatment. Furthermore, MGO was not able to induce the formation of the gp130 immunoreactive clusters. SP, signal peptide. Scale bar: 20 lm. ECD: ectodomain, YSTV: tyrosine-based internalization motif, PLLD: di-leucine-based internalization motif.

d), U87MG cells and PC12 cells (data not shown). By contrast, ErbB2 and beta-tubulin protein levels were stable before and after MGO treatment, indicating that MGO selectively degrades gp130. In order to further confirm the endocytotic degradation of gp130 by MGO, we employed a chemical inhibitor of endocytosis, concanavalin A (Arttamangkul et al. 2006). As shown in Fig. 2(e), concanavalin A treatment (250 lg/mL) significantly blocked MGO-induced degradation of gp130. Furthermore, a slight increase in basal gp130 levels was found following concanavalin A treatment. This increase may be attributed to interpretation of constitutive endocytotic turnover of gp130 as described by Thiel et al. (1998). Taken together, these data suggest that MGO negatively affects gp130/STAT3 signaling by inducing endocytotic degradation of the cytokine receptor gp130.

et al. 1994, 1996). This finding led us to differentiate the roles of the two motifs in MGO-induced gp130 endocytosis. We generated a truncation mutant of human Flag-gp130 that has only a tyrosine-based internalization motif in its cytoplasmic domain (Fig. 3). Wild-type gp130 and mutant gp130 cDNA transfectants showed live cell binding with an antiFlag antibody, whereas no binding was observed for nontransfected cells (Fig. S1a). MGO treatment (500 lM) for 30 min reduced the Flag-specific live cell binding for wildtype gp130 transfected cells but not for cells transfected with truncated Flag-gp130 (Fig. 3a and b). The same cells were immunostained with an anti-human gp130 antibody. This experiment revealed that numerous cytoplasmic punctate foci of gp130 following MGO treatment of cells transfected with wild-type gp130. In contrast, the gp130-immunoreactive clusters were not observed following MGO treatment of cells transfected with the truncated gp130. Furthermore, membrane co-localization of Flag and gp130 stainings persisted even in the presence of MGO, suggesting that the membrane localized truncated gp130 resides in the membrane after MGO treatment. These data were further supported by Western blot analysis of lysates prepared from transfected HEK293 cells (Fig. S1b). Our data suggest that the di-

Cytoplasmic region of gp130 containing the di-leucine internalization motif is required for MGO-induced endocytosis It was reported that the cytoplasmic domain of gp130 contains two internalization motifs, a tyrosine based (YSTV) and a di-leucine based motifs (TQPLLDSEER) (Dittrich



leucine internalization motif, rather than the tyrosine-based internalization motif, might be required for MGO-induced endocytosis of gp130. MGO selectively inhibits basal and IL-6-induced activation of STAT3 To determine whether the MGO-induced reduction of gp130 is functionally related to the impairment of gp130 signaling, we investigated whether MGO treatment affects STAT3 activity, a down-stream signal tranducer of the gp130 receptor. Cellular lysates from the neuroglial cell lines were prepared and analyzed by western blot analysis using an antibody to phospho-STAT3 (residue Tyr705, pSTAT3). In RT4 cells, MGO treatment for 30 min significantly reduced the basal levels of pSTAT3 without affecting the total protein level of STAT3 (Fig. 4a). This inhibition was dose-dependent: 500 lM of MGO was sufficient for the inhibition (data not shown). In accordance with a previous study (Fukunaga et al. 2004), MGO (500 lM) consistently activated p38 MAPK. In U87 glioma cells (Fig. 4f) and PC12 cells (data not shown), a single MGO treatment reduced basal pSTAT3 levels. We next examined whether IL-6-induced tyrosine phosphorylation of STAT3 in RT4 cells would be inhibited by pre-treatment with MGO. As shown in Fig. 4(b) and 4(c), IL-6 (50 ng/mL)-dependent STAT3 tyrosine phosphorylation was severely reduced in cells that were pre-incubated with MGO (500 lM) for 30 min. Furthermore, this reduction was prevented by the pre-treatment of AGD (2 mM). We next investigated whether MGO indeed inhibits STAT3 activity by evaluating the effects of MGO on the nuclear translocation and DNA binding activity of STAT3. As was shown by immunofluorescent staining (Fig. 4d), weak pSTAT3-immunoreactivity was principally observed in the cytoplasm of RT4 cells in the absence of MGO, and MGO treatment (500 lM) further reduced this immunoreactivity. IL-6 dramatically induced nuclear translocation of pSTAT3 within 20 min, which was near completely blocked by pre-treatment with MGO. In controls and MGO-treated cells, the DNA binding activity of STAT3 was barely detectable. The DNA binding activity assay revealed that a considerable amount of pSTAT3 was found to interact with STAT3-interacting oligonucleotide-agarose beads in IL-6stimulated cells. This interaction was abolished by MGO treatment (Fig. 4e), indicating that MGO might inhibit the transcriptional activity of STAT3. We also examined the effect of MGO on epidermal growth factor (EGF) signaling in U87 glioma cells in order to know the specificity of MGO action on the gp130 signaling. The EGF-induced activation of the EGF receptor was mildly reduced by treatment with MGO (500 lM) (Fig. 4f), while MGO completely blocked basal pSTAT3 levels in U87 cells. In addition, the inhibitory effect of MGO on the IL-6/STAT3 pathway did not result from IL-6 modification by MGO because the same inhibitory effect was observed in exper-

iments with a continuous presence of MGO (i.e., when IL-6 was added to the medium containing MGO at the end of the pre-incubation) and in wash-out experiments (i.e., when IL-6 was added after washing out MGO at the end of preincubation) (Fig. 4g, RT4 cells). The activation of p38 MAPK by MGO treatment raised the possibility that the MAPK is involved in MGO-induced down-regulation of gp130 signaling. However, the inhibition of p38 MAPK or Jun kinase with specific inhibitors did not affect the inhibitory action of MGO on gp130 signaling in RT4 cells (Fig. 4h). Taken together, our results confirm that MGOinduced down-regulation of gp130 level indeed results in the inhibition of basal and IL-6-induced STAT3 activation. MGO-induced inhibition of gp130/STAT3 signaling is observed in primary Schwann cells In order to extend our findings to physiological conditions, we tested the negative effects of MGO on gp130/STAT3 signaling in primary Schwann cells obtained from adult rat sciatic nerves. The treatment of MGO (500 lM) dramatically reduced not only the cell surface labeling of gp130 but also IL-6-induced STAT3 phosphorylation in primary Schwann cells. Furthermore, AGD pre-treatment completely blocked MGO inhibition of gp130/STAT3 signaling. This finding indicates that the action of MGO on the gp130/STAT3 signaling is not limited to cancer cells (Fig. 5). MGO enhances the toxic effects of hydrogen peroxide and etoposide Since STAT3 signaling contributes to cell survival by upregulating anti-apoptotic proteins such as Bcl-2 family members, we have tried to know whether STAT3 inhibition by MGO actually affects the basal expression of Bcl-2. Treatment with MGO (500 lM) time-dependently reduced basal expression of Bcl-2 without affecting the expression of other proteins in U87 glioma cells (Fig. 6a and b). This finding indicates that MGO-induced down-regulation of antiapoptosis-related STAT3 target genes may negatively affect on cell survival, thus sensitizing U87 glioma cells to death induced by other cytotoxic reagents in U87 glioma cells. We tested this hypothesis by using H2O2 or the etoposide-induced cellular toxicity model. H2O2 (2 mM) and MGO (500 lM) reduced cell survival rates by about 20% and 10% respectively, whereas co-treatment with H2O2 and MGO reduced the survival rate by about 50% relative to the survival rate of the controls (p < 0.05, Fig. 6c). These findings indicate that MGO enhanced the cytotoxic effect of H2O2. MGO treatment also induced a dramatic reduction in cell viability when combined with etoposide, a cytotoxic reagent. Etoposide (1.3 nM) did not significantly reduce cell survival (10%) on its own, but it induced a significant reduction in cell survival in the presence of MGO (p < 0.05, Fig. 6d). Taken together, these findings indicate that MGO sensitizes cells to the cytotoxic effects of both H2O2 and etoposide.



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Fig. 4 Inhibitory effect of MGO on STAT3 activation. (a) MGO suppressed basal tyrosine phosphorylation of STAT3 in a time-dependent manner while it activated p38 MAPK. pp38, phospho-p38 MAPK; Tub, beta-tubulin. (b) Cells were treated with IL-6 in the presence or absence of MGO (500 lM) for 30 min and cell lysates were immunoblotted with an anti-phospho-STAT3 antibody. Aminoguanidine (AGD, 2 mM) prevented MGO-induced inhibition of STAT3 phosphorylation by IL-6. (c) A quantitative analysis of the data presented in (b). The intensity of the pSTAT3 band was normalized to that of STAT3. (d) After stimulation with IL-6 for 20 min, RT4 cells were fixed and immunostained for pSTAT3 to reveal the nuclear translocation of

Discussion The present data show herein a novel mechanism for dicarbonyl aldehydes-mediated cytotoxic effect in the context of cytokine signaling, and they are consistent with the proposed roles of MGO as a cell death-triggering molecule.

pSTAT3. Scale bar: 50 lm. (e) STAT3 DNA binding activity was analyzed with STAT3-interacting oligonucleotide-agarose beads after IL-6 treatment (20 min). Pulled-down STAT3 was labeled by an antipSTAT3 antibody. (f) In U87 cells, the EGF signaling pathway was not significantly affected by MGO pre-treatment (500 lM), but basal tyrosine phosphorylation of STAT3 was down-regulated by MGO (uppermost panel). (g) Washout of MGO medium (500 lM) before IL-6 addition did not affect the ability of MGO inhibition in IL-6-induced STAT3 phosphorylation. (h) The effects of inhibitors for p38 MAPK (SB: SB203580, 20 lM) and JNK (SP: SP600125, 20 lM) on the MGO-mediated inhibition of IL-6-induced STAT3 phosphorylation.

Because gp130/STAT3 signaling is fundamental to a variety of cellular physiology such as development, proliferation and inflammation (Battle and Frank 2002; Kamimura et al. 2003), impairment of this signaling pathway by the reactive aldehydes may lead to significant tissue damages in hyperglycemic conditions. Furthermore, in the nervous system,



(a)

(b)

Fig. 5 Inhibitory effect of MGO on the gp130/STAT3 pathway in primary Schwann cells. (a) Primary Schwann cells were treated with MGO (500 lM) for 30 min, and then submitted to a live cell binding assay with an anti-gp130 antibody. No-Ig: omission of primary antibody. (b) Primary Schwann cells were pre-treated with MGO for

Fig. 6 MGO sensitizes cells to the cytotoxic effects of H2O2 and etoposide. (a) U87 glioma cells were treated with MGO (500 lM) for the indicated lengths of time, and then cell lysates were immunoblotted with an anti-Bcl-2 or anti-Src or anti-betatubulin antibody. (b) A quantitative analysis of the data presented in (a). The intensity of the Bcl-2 band was normalized to that of beta-tubulin. (c) Cells were treated with MGO (500 lM) or H2O2 (2 mM) or both for 1 day, and cell survival was analyzed by the MTT assay. (d) Cells were treated with MGO (500 lM) or etoposide (Etop, 1.3 nM) or both for 1 day, and cell survival was analyzed by the MTT assay. Data represent the means ± SE of three independent experiments. *p < 0.05, the Bonferroni post hoc test.

30 min, then stimulated with IL-6 for 20 min. The cells were immunostained with an antibody to pSTAT3. The nuclear labeling of pSTAT3 induced by IL-6 was blocked by the pre-treatment with MGO. Scale bar: 20 lm.

(a)

(b)

(c)

(d)

STAT3 plays a central role in neuroprotection, astrogliosis and regeneration after injury (Schweizer et al. 2002; Qiu et al. 2005; Miao et al. 2006; Herrmann et al. 2008). Thus, it seems that the MGO-mediated inhibition of STAT3 activation is related to the development of neuronal complications found in hyperglycemic condition and AGE-related neurodegenerative diseases in vivo. It should be mentioned that the concentration of MGO in diabetic patients is below the level that we used in this experiment (Beisswenger et al. 2001). However, the intracellular concentration of MGO varies widely and is generally higher than that of plasma. For example, 300 lM of MGO was found in Chinese hamster ovary cells (Chaplen et al. 1998). In addition, the MGO

concentrations used in our study is much less than that utilized in a previous study to decipher MGO function in cellular signaling in previous studies (Akhand et al. 2001; Riboulet-Chavey et al. 2006). Moreover, we did not observe significant toxic effects of MGO at 500 lM with regard of cell viability. Taken together, these findings suggest the relevance of our results to demonstrate molecular mechanism of MGO action. Cytotoxic mechanism of MGO MGO is known to induce cellular injury with various apoptotic biochemical changes. It has been consistently reported that oxidative stress-mediated activation of apoptotic



mechanism underlies MGO-induced cytotoxicity (Du et al. 2001; Amicarelli et al. 2003). MGO can increase intracellular ROS via depletion of cellular reducing agents and inactivation of ROS scavenger enzymes (Choudhary et al. 1997; Amicarelli et al. 2003). However in the present study, we could not observe any inhibitory effect of an anti-oxidant (N-acetylcysteine, 2 mM) on the negative effect of MGO on gp130/STAT3 signaling (data not shown). Thus the inhibition of gp130/ STAT3 signaling by MGO does not seem to be mechanistically related to the ROS toxicity induced by MGO. Recently, the activations of JNK and p38 MAPK have been reported to be implicated in the apoptosis induced by MGO in osteoblast and Schwann cells respectively (Fukunaga et al. 2004; Chan et al. 2007). We also found that p38 MAPK activation in RT4 cells by MGO treatment (Fig. 4a). However, since inhibition of JNK or p38 MAPK activation did not completely blocked MGO-induced apoptosis (Fukunaga et al. 2004; Chan et al. 2007), it seems that additional mechanisms might be involved in the cytotoxic effect of MGO. In this study, we have shown that MGO reduced basal and IL-6-induced STAT3 activation in neuroglial cells. STAT3 is implicated in various biological responses, including proliferation, differentiation and apoptosis (Battle and Frank 2002). One of the mechanism by which STAT3 regulates apoptosis is related to transcriptional regulation of Bcl-2 family members. It was shown that decreased expression of Bcl-xl by STAT3 inhibition led to increased susceptibility to Fas-mediated apoptosis (Haga et al. 2003). Inhibition of the JAK/STAT3 pathway using JAK inhibitors down-regulated the expression of Bcl-2 family proteins and thereby decreased cell viability (Epling-Burnette et al. 2001). We here have shown that MGO-induced inhibition of STAT3 activity led to the down-regulation of Bcl-2 and increased the susceptibility of U87MG cells to the cytotoxic effects of H2O2 and etoposide. This finding is consistent with previous reports showing that the inhibition of constitutively active STAT3 by specific inhibitors in glioblastoma cells induced down-regulation of Bcl-2 expression and concomitant apoptosis (Rahaman et al. 2002; Iwamaru et al. 2007). Thus it is possible that a chronic increase in the level of MGO in hyperglycemic conditions may suppress gp130/STAT3 signaling, which would result in lower levels of anti-apoptotic proteins in tissues. This situation may make cells more vulnerable to hostile conditions such as ischemia or injury. Further studies are required to evaluate this hypothesis in hyperglycemic conditions in vivo.

prevented by incubation with an AGE scavenger. We thus investigated whether MGO directly acts on gp130 or on STAT3 to produce Ne-(carboxyethyl)lysine adduct formation. We could not observe MGO-induced glycation of gp130 and STAT3 even though basal glycation of STAT3 was observed (Fig. S1c). Thus it is unlikely that lysine modification of gp130 and/or STAT3 by MGO is the primary mechanism of STAT3 signaling impairment by MGO. Since arginine and cysteine are also important amino acid targets of MGO (Rabbani and Thornalley 2008), it is possible that MGO directly affects gp130/STAT3 signaling through glycation of these residues. On the other hand, MGO may induce modifications of certain molecules which are implicated in the membrane localization of gp130. Clathrin-related proteins, such as adaptor protein-2 and dynamin may be a target because they seem to be implicated in the constitutive endocytosis of gp130 (Thiel et al. 1988). In addition, we found that the cytoplasmic domain containing the di-leucine internalization motif is required for endocytosis of gp130 by MGO, indicating a mechanistic similarity of MGO-induced endocytosis of gp130 and the ligand-mediated endocytosis of gp130 (Dittrich et al. 1996). It is also possible that the receptor for AGE may affect the gp130/STAT3 pathway via unidentified mechanisms (Huang et al. 2001). Finally, JNK and p38 MAPK activation might underlie the alteration of membrane localization of gp130 by MGO. However, we found no evidence of involvement of these molecules using specific inhibitors for JNK and p38 MAPK, SP600125 and SB203580 respectively (Fig. 4h). Further studies are required to identify the molecular targets of MGO involved in downregulation of the gp130/STAT3 pathways are required. Conclusively, we herein provide a novel finding that suggests a molecular mechanism of MGO toxicity in neuroglial cells. Further researches to examine the molecular mechanism of MGO actions in cytotoxicity may shed new light into the development of therapeutic strategies to reduce the pathological changes related to AGE-mediated neurodegeneration.

Molecular mechanism of MGO actions on the gp130 receptor MGO is a highly reactive compound, which is capable of reacting with free amino groups on proteins to form Ne(carboxyethyl)lysine (Lo et al. 1994; Cantero et al. 2007). MGO-mediated suppression of the gp130/STAT3 pathway is related to the formation of AGEs since this suppression was

Additional Supporting Information may be found in the online version of this article: Fig. S1 Glycation-independent degradation of gp130 by MGO. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

Acknowledgements This work was supported by grants from Korea Research Foundation Grant (2006-005-J03502). We thank Dr. T. Taga and K. Ikenaka, for providing gp130 cDNA.

Supporting information



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