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Ghrelin inhibits apoptosis signal-regulating kinase 1 activity via upregulating heat-shock protein 70
Biochemical and Biophysical Research Communications 359 (2007) 373–378 www.elsevier.com/locate/ybbrc
Ghrelin inhibits apoptosis signal-regulating kinase 1 activity via upregulating heat-shock protein 70 Min Yang, Shengdi Hu, Bin Wu, Yanying Miao, Hui Pan, Shigong Zhu
*
Department of Physiology and Pathophysiology, Peking University Health Science Center, 38 Xueyuan Road, Beijing 100083, People’s Republic of China Received 13 May 2007 Available online 25 May 2007
Abstract Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), which has been originally isolated from rat stomach. It has been reported that ghrelin inhibited apoptosis in several cells, such as cardiomyocytes, endothelial cells, adipocyte, adrenal zona glomerulosa cells, pancreatic b-cells, osteoblastic MC3T3-E1 cells, intestinal epithelial cells and hypothalamic neurons. However, it is unknown whether heat-shock protein 70 (HSP70) or apoptosis signal-regulating kinase 1 (ASK1) is the important target molecule which mediates the anti-apoptotic effects of ghrelin. We show that ghrelin inhibited ASK1 activity induced by sodium nitroprusside (SNP), inhibited ASK1-mediated caspase 3 activation and apoptosis in PC12 cells. Ghrelin promoted expression of HSP70. Quercetin, an inhibitor of HSP70, blocked the effects of ghrelin on ASK1 activity. Thus, ghrelin inhibits ASK1-mediated apoptosis and ASK1 activation by a mechanism involving induction of HSP70 expression. The results of the present study suggest the therapeutic potential of ghrelin for some pathological processes or disorders. 2007 Elsevier Inc. All rights reserved. Keywords: Ghrelin; Apoptosis; ASK1; HSP70
Ghrelin is a newly discovered gut–brain peptide, predominantly produced by the stomach, and has been identified as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R) [1]. It has been reported that ghrelin inhibited apoptosis in several cells, such as cardiomyocytes, endothelial cells, adipocyte, adrenal zona glomerulosa cells, pancreatic b-cells, osteoblastic MC3T3E1 cells, intestinal epithelial cells and hypothalamic neurons [2–8]. However, the mechanism and the target molecule on which ghrelin exerts its anti-apoptotic effects remain to be elucidated. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) family member that acts upstream of JNK and p38 kinases by directly phosphorylating their respective MAPKKs (MKK4/7 for JNK and MKK3/6 for p38) [9,10]. ASK1
*
Corresponding author. Fax: +86 10 82801443. E-mail address: [email protected] (S. Zhu).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.05.118
has been shown to be an important signaling kinase in apoptotic cell death in response to various stimuli, including serum or trophic factor withdrawal, TNF-a, reactive nitrogen species (RNS), reactive oxygen species (ROS), ischemia insult, genotoxic stress, and possibly FasL [11– 18]. The active ASK1 is sufficient to induce cell apoptosis through mitochondrial-dependent caspase activation in several cell types [9,11,17,19]. Because of the important role of ASK1 in apoptosis, considerable attention has been focused on the key molecules which regulate ASK1 activity in the cells. One widely pursued strategy is to identify endogenous inhibitors of ASK1 that attenuate ASK1 pathway signaling. It has been reported that several molecules can modulate ASK1 activity, including thioredoxin, protein serine/threonine phosphatase 5 (PP5), glutathione-Stransferase, 14-3-3 protein, Akt, and heat-shock protein 70 (HSP70) [20–25]. It is well known the HSP70 is a molecular chaperone among these molecules and posses important function in cytoprotection. Study has shown that HSP70 inhibited the activation of ASK1 and prevented
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ASK1-dependent apoptotic cell death in NIH3T3 cell line [25]. However, it is unknown whether HSP70 or ASK1 is the important target molecule which mediates the antiapoptotic effects of ghrelin. In the present study, we explored the effects of ghrelin on ASK1 activity, and expression of HSP70, as well as relation among them. Materials and methods Materials. Human ghrelin was purchased from Phoenix Pharmaceuticals (Belmont, CA). RPMI-1640 medium, penicillin, and streptomycin were purchased from Gibco-BRL (Life Technologies, Inc., Gaithersburg, MD, USA). Fetal bovine serum (FBS) and horse serum were purchased from Hyclone (UT, USA). All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Sodium nitroprusside (SNP) and quercetin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The reagents of the BCA method for protein quantification and restoration of Western blot stripping buffer were purchased from Pierce (Pierce Co., Rockford, IL, USA). Cell culture and treatments. Rat pheochromocytoma (PC12) cells were cultured in RPMI-1640 medium supplemented with 10% horse serum, 5% fetal bovine serum (FBS), 50 U/ml penicillin, 50 lg/ml streptomycin, at 37 C in the presence of 95% air and 5% CO2. They were cultured for passage once 5 days. Experiments were performed using dishes coated with poly-L-lysine (10 lg/ml for 12 h) to assist cell adhesion. When cells were 80–90% confluent, the medium was exchanged for RPMI-1640 medium containing 1% FBS, the cells were used for experiments after 24 h. The pretreatment of ghrelin was performed for 30 min. The activation of ASK1 and apoptosis of PC12 cells were induced by the indicated doses of SNP as a NO donor for 18 h. When indicated, the cells were pretreated with quercetin for 60 min. Plasmids and transfection. Mammalian expression plasmids for ASK1 of the wild type (ASK1-WT) in PC-DNA3 (Invitrogen, Groningen, The Netherlands) were kindly provided by Dr. Hidenori Ichijo (Tokyo Medical and Dental University, Tokyo, Japan). Transfections with either ASK1 expressing PC-DNA3 plasmids or the corresponding empty vectors were performed using Lipofectamine 2000 (Life Technologies Inc., Rockville, MD, USA). For transient transfection, PC12 cells were plated in a washed glass coverslip or 60-mm dish for 24 h before transfection and transfected with plasmids (1 lg for coverslip or 2 lg for 60-mm dish) using Lipofectamine 2000 according to the manufacturer’s recommendation. Thirty-six hours following transfection, cells were exposed to SNP in the presence or absence of ghrelin. Protein extractions and Western blot analysis. Following experimental treatments, the cells were washed with PBS (pH 7.4) two times and sonicated on ice in a lysis buffer containing 150 mM NaCl, 100 mM Tris–HCl pH 7.4, 1% Triton X-100 and 1 mM Phenylmethyl sulphonyl fluoride (PMSF). Then, cellular debris were pelleted by centrifugation at 12,000g for 10 min at 4 C, the supernatant was collected by Western blotting. Protein concentrations were measured by the Bio-Rad Assay (Bio-Rad Laboratories, Hercules, CA). Proteins (30 lg per lane) were separated in 10% SDS–PAGE gels and electrophoretically transferred onto nitrocellulose for 2 h at 0.2 A. Membranes were blocked with blocking buffer (5% non-fat dried milk and 0.1% Tween 20 in TBS buffer) at room temperature for 1 h. Primary antibody incubations were overnight at 4 C using the appropriate dilutions (polyclonal ASK1 1:1000, polyclonal phosphoASK1 1:1000, monoclonal HSP70 1:1000, polyclonal caspase 3 1:1000, polyclonal b-actin 1:1000). Membranes were washed for 30 min with three changes, incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibody. Specific antibody–antigen complexes were detected using the ECL Western Blot Detection Kit. After immunoblotting, digitized images of the immunoreactive bands were quantitated by image analysis with use of the NIH Image software (version 1.62). Additional background measurements were taken from each film and subtracted from these values.
Flow-cytometry analysis. Apoptosis was measured by FCM analysis of the percentage of subdiploid apoptotic nuclei. Thirty-six hours after transfection with ASK1 expression plasmid and the corresponding empty vector, cells were treated with SNP in the presence or absence of ghrelin for 18 h. About 1 · 106/ml cells were harvested, washed with PBS, and fixed in 70% ethanol at 4 C for 24 h. The cells were washed again and incubated with 50 ll RNase (50 lg/ml) at 37 C for 30 min. Resuspending in PBS containing propidium iodide (50 lg/ml) in the dark for 10 min, the percentage of apoptotic nuclei, recognized by their subdiploid DNA content, was determined using the Becton–Dickinson FACS scan Flow Cytometer (Becton–Dickinson, Heidelberg, Germany). Statistical analysis. Each experiment was repeated at least three times. The data were presented as means ± SEM and analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s test. Statistical significance was set at p < 0.05.
Results Ghrelin prevents ASK1-mediated apoptosis of PC12 cells To determine whether ghrelin prevents ASK1-mediated apoptosis, we subjected PC12 cells to transient transfection with PC-DNA3-ASK1 plasmids, and then examined the effects of ghrelin on the cleavage status of caspase 3 and apoptosis induced by SNP. As shown in Fig. 1A, the activity of ASK1 was quite low in the corresponding empty vector-transfected PC12 cells. However, the activity of ASK1 was significantly increased by SNP with a concentration-dependent manner in ASK1-transfected PC12 cells (Fig. 1A). In the corresponding empty vector-transfected PC12 cells, the expression of caspase 3 cleavage was quite low, caspase 3 activity was increased by SNP, and was reduced by ghrelin in SNP-treated PC12 cells (Fig. 1B). In ASK1-transfected PC12 cells, there are the expression of caspase 3 cleavage but the level of caspase 3 cleavage was low, caspase 3 activity was increased significantly by SNP, whereas ghrelin markedly blocked caspase 3 activation (p < 0.05) (Fig. 1B). Apoptosis of PC12 cells were measured by flow-cytometric analysis. As shown in Fig. 2, the number of apoptosis was rare in PC12 cells transfection with ASK1 alone and no significant variance compared with the control vectors, apoptosis was markedly increased by SNP in ASK1 transfected cells compared with the control vectors transfected cells (p < 0.05). However, ghrelin treatment effectively reduced SNP-induced apoptosis in the corresponding empty vector-transfected, and significantly decreased SNP-induced apoptosis in ASK1PC12 cells (p < 0.01) (Fig. 2). These data show that ghrelin inhibited ASK1-mediated apoptosis of PC12 cells. Ghrelin inhibits ASK1 activation To test whether ghrelin is able to suppress the activity of ASK1, we examined the effects of ghrelin on the SNPinduced activation of ASK1. Western blot analysis showed that there was the expression of phosphorylated ASK1 in PC12 cells transfection with ASK1 alone, but the activity of ASK1 was dropped upon ghrelin treatment (Fig. 3).
M. Yang et al. / Biochemical and Biophysical Research Communications 359 (2007) 373–378
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32KDa caspase-3
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Fig. 1. Effects of ghrelin on SNP-induced caspase 3 activation in ASK1-transfected PC12 cells. (A) SNP induces the activity of ASK1. PC-DNA3-ASK1 or PC-DNA3 plasmids were transfected into PC12 cells and incubated for 36 h. PC12-ASK1 cells were treated with 0.1, 0.3 mM SNP for 18 h. The cell proteins were subjected to Western blot analysis with antibodies against phospho-ASK1 or ASK1, and relative amounts of expression of phospho-ASK were compared with the intensity of ASK1. *p < 0.05, **p < 0.01 vs the empty vector-transfected PC12 cells. (B) Effects of ghrelin on SNP-induced caspase 3 activation in ASK1-transfected PC12 cells. PC12-ASK1 and PC12-PC-DNA3 cells were cultured in the absence or presence of 100 nM ghrelin for 30 min, and then treated with 0.3 mM SNP for 18 h. The cell proteins were subjected to Western blot analysis with antibody against caspase 3, and relative amounts of expression of caspase 3 cleavage were compared with the intensity of b-actin. *p < 0.05.
p-ASK1
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G SN hre P lin+
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Fig. 2. Effects of ghrelin on ASK1-mediated apoptosis of PC12 cells. PC12-ASK1 and PC12-PC-DNA3 cells were cultured in the absence or presence of 100 nM ghrelin for 30 min, and then treated with 0.3 mM SNP for 18 h. Apoptosis was measured by FCM analysis of the percentage of subdiploid apoptotic nuclei. *p < 0.05, **p < 0.01.
The activity of ASK1 was markedly increased by SNP in ASK1-transfected PC12 cells, whereas ghrelin significantly decreased the amount of phosphorylated ASK1 (p < 0.01) (Fig. 3). These results suggest that ghrelin inhibited SNPinduced ASK1 activation.
SNP Ghrelin ASK1
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Fig. 3. Effects of ghrelin on SNP-induced activation of ASK1. PC-DNA3ASK1 plasmids were transfected into PC12 cells and incubated for 36 h. PC12-ASK1 cells were in the absence or presence of 100 nM ghrelin for 30 min, and then treated with 0.3 mM SNP for 18 h. The cell proteins were subjected to Western blot analysis with antibodies against phospho-ASK1 or ASK1, and relative amounts of expression of phospho-ASK were compared with the intensity of ASK1. *p < 0.05, **p < 0.01.
Ghrelin inhibition of ASK1 activity is mediated by induction of HSP70 The effects of ghrelin on expression of HSP70 were shown in Fig. 4A. The expression of HSP70 was quite
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HSP70
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Fig. 4. Ghrelin inhibition of ASK1 activity is mediated by induction of HSP70. (A) Effects of ghrelin on the protein expression of HSP70 in PC12 cells transfected with ASK1 induced by SNP. PC-DNA3-ASK1 plasmids were transfected into cells and incubated for 36 h. PC12-ASK1 cells were in the absence or presence of 100 nM ghrelin for 30 min, and then treated with 0.3 mM SNP for 18 h. The cell proteins were subjected to Western blot analysis with antibody against HSP70, and relative amounts of expression of HSP70 were compared with the intensity of b-actin. **p < 0.01 vs the SNP-treated PCDNA3-ASK1 cells. (B) Effects of quercetin on SNP-induced phosphorylation of ASK1 in response to ghrelin. PC12-ASK1 cells were cultured in the absence or presence of 10-lM quercetin for 60 min followed by the presence of 100 nM ghrelin. After 30 min, cells were exposed to 0.3 mM SNP for 18 h. The cell proteins were subjected to Western blot analysis with antibodies against HSP70 or phospho-ASK1, and relative amounts of expression of HSP70 or phospho-ASK1 were compared with the intensity of b-actin. *p < 0.05 vs the SNP-treated PC-DNA3-ASK1 cells, #p < 0.05 vs Ghrelin + SNP + ASK1.
low in PC12 cells transfection with ASK1 alone. Exposure to SNP upregulated HSP70 in ASK1-transfected PC12 cells, whereas the levels of HSP70 seem to be mild. Compared with ASK1-transfected PC12 cells exposed to SNP, ghrelin treatment significantly enhanced the expression of HSP70 in the absence or presence SNP (p < 0.01). These results suggest that ghrelin promoted the expression of HSP70 in PC12 with or without SNP injury. To examine the role of HSP70 in inhibition of ASK1 activity by ghrelin, quercetin, an inhibitor of HSP70 protein synthesis was used in experiments. The results show that SNP increased ASK1 phosphorylation, and produced a low level of HSP70 in ASK1-transfected PC12 cells (Fig. 4B). Compared with SNP treatment, the expression of HSP70 was attenuated, and associated with the activity of ASK1 was increased in ASK1-transfected PC12 cells exposed to quercetin and SNP (p < 0.05). On the other hand, ghrelin pretreatment markedly upregulated HSP70 and produced a low level of ASK1 phosphorylation. Compared with ghrelin pretreatment exposed to SNP, quercetin blocked the accumulation of HSP70, and connected with increased ASK1 activity (p < 0.05) (Fig. 4B). The results suggest that ghrelin inhibited ASK1 activity via upregulating HSP70. Discussion In the present study, we demonstrate that ghrelin inhibits ASK1 activity and ASK1-mediated apoptosis in PC12 cells. The results also suggest that ghrelin is a potent endog-
enous inducer of HSP70, by which ghrelin inhibited ASK1 activity. Ghrelin is a newly discovered gut–brain peptide. Apart from a potent GH-releasing action, it has been reported that ghrelin inhibited apoptosis in several cells, such as cardiomyocytes, endothelial cells, adipocyte, adrenal zona glomerulosa cells, pancreatic b-cells, osteoblastic MC3T3E1 cells, intestinal epithelial cells, and hypothalamic neurons [2–8]. Ghrelin has also been shown to activate the MAPK, phosphatidylinositol 3 kinase(PI3K)-Akt, PKA and PKC pathways, all of which mediate the anti-apoptotic effects of ghrelin [2–8]. In addition, ghrelin inhibited apoptosis by increasing Bcl-2/Bax ratio, prevention of cytochrome c release, inhibiting reactive oxygen species generation, stabilizing mitochondrial transmembrane potential and inhibition of caspase 3 activation [8]. However, the mechanism and the target molecule which ghrelin exerts its anti-apoptotic effects remain to be elucidated. Numerous studies have shown that activation of ASK1 is positively linked to apoptosis. Activation of ASK1 induces apoptotic cell death by triggering mitochondrial events that include the release of cytochrome c from mitochondria and the subsequent activation of caspase 9 and caspase 3 [19]. In primary neurons derived from ASK1 / mice, ER stress- and polyglutamine-induced JNK activation and apoptosis were reduced [15]. It was recently shown that ASK1 was also required for amyloid b (Ab)-induced neuronal cell apoptosis, and ASK1-JNK pathway was activated in a ROS-dependent manner. ASK1 / neurons were
M. Yang et al. / Biochemical and Biophysical Research Communications 359 (2007) 373–378
resistant to Ab-induced cell apoptosis [16]. Moreover, ASK1/MKK4/JNK play a central role in signaling pathways related to oxidative stress-induced apoptosis in neuronal cells [17,18]. These reports on the roles of ASK1 in apoptosis suggest the importance of ASK1 in the pathogenesis of neurodegenerative diseases. ASK1 may be a potential therapeutic target for prevention and treatment of various neurodegenerative diseases. To elucidate whether ASK1 is the important target molecule which mediates the anti-apoptotic effects of ghrelin, we explore the effects of ghrelin on ASK1 activity and its mechanism. Our data show that ghrelin inhibited ASK1mediated apoptosis of PC12 cells and suppressed the ASK1 activation. Other studies have proposed that selenite directly inhibiting ASK1 activity may be related to a cysteine-rich domain in its NH2 terminus of ASK1, and to the change redox potential of sulfhydryl groups of ASK1 [26]. Overexpression of CDC25A diminishes homo-oligomerization of ASK1, the binding of CDC25A to ASK1 at the region adjacent to the kinase domain could inhibit oligomerization of ASK1 [27]. Structurally, ghrelin does not resemble known kinase inhibitors. It was found that ghrelin inhibited the activity of ASK1 even 18 h after ghrelin treatment, but not short-term. Its time course of action suggested that ghrelin may function by facilitating production of an endogenous ASK1 antagonist. The biological function of ASK1 is negatively regulated by ASK1-interacting proteins, which include thioredoxin and other proteins. Thioredoxin in a reduced form binds to the NH2-terminal part of ASK1 and blocks activation of ASK1 by TNF [20]. The 14-3-3 protein, a phosphoserine-binding molecule, binds to ASK1 specifically via Ser967 of ASK1 and suppresses ASK1-induced apoptosis [23]. Protein serine/threonine phosphatase 5 (PP5) directly dephosphorylates Thr845 and inactivates ASK1 activity [21]. ASK1 may be a physiological target of Akt, as activation of the phosphoinositide 3-kinase (PI3-K)/Akt pathway is associated with a decrease in ASK1 kinase activity [24]. HSP70 may bind to ASK1 and prevent ASK1 homo-oligomerization, which negatively regulates ASK1 activity [25]. It is little known whether suppression of ASK1 activation by ghrelin is related to induction of HSP70. Our data demonstrate that ghrelin treatment results in the upregulation of HSP70 with or without SNP injury in PC12 cells. It seems that ASK1 inhibition by ghrelin is due to induction of HSP70. As shown in Fig. 4B, the prevention of HSP70 increased ASK1 activity induced by SNP. Blocking the ability of ghrelin to inhibit ASK1 phosphorylation was observed in cells treated with quercetin. These results suggested that ghrelin suppresses apoptotic signaling through ASK1 inhibition due to the induction of HSP70. In summary, we demonstrate for the first time that ghrelin may prevent ASK1-mediated apoptosis of PC12 cells and inhibit ASK1 activation. The suppression of ASK1 activity by ghrelin was mediated through inducing HSP70 expression. Our results show a novel mechanism of anti-
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apoptosis of ghrelin through the pathway of HSP70 and ASK1. Ghrelin may serve as a useful tool for basic research, and may have therapeutic potential for some pathological processes or disorders. Acknowledgments This work was supported by the National Natural Science Foundation of China (30370557), the Ministry of Education for Research Program of China (20020001083), and in part by the National Major Basic Research Program of China (G2000056908). PC-DNA3ASK1 plasmids were kindly provided by Dr. Hidenori Ichijo (Tokyo Medical and Dental University, Tokyo, Japan). References [1] M. Kojima, H. Hosoda, Y. Date, M. Nakazato, H. Matsuo, K. Kangawa, Ghrelin is a growth-hormone-releasing acylated peptide from stomach, Nature. 402 (1999) 656–660. [2] G. Baldanzi, N. Filigheddu, S. Cutrupi, F. Catapano, S. Bonissoni, A. Fubini, D. Malan, G. Baj, R. Granata, F. Broglio, M. Papotti, N. Surico, F. Bussolino, J. Isgaard, R. Deghenghi, F. Sinigaglia, M. Prat, G. Muccioli, E. Ghigo, A. Graziani, Ghrelin and des-acyl ghrelin inhibit cell death in cardiomyocytes and endothelial cells through ERK1/2 and PI3-kinase/AKT, J. Cell Biol. 159 (2002) 1029–1037. [3] M.S. Kim, C.Y. Yoon, P.G. Jang, Y.J. Park, C.S. Shin, H.S. Park, J.W. Ryu, Y.K. Pak, J.Y. Park, K.U. Lee, S.Y. Kim, H.K. Lee, Y.B. Kim, K.S. Park, The mitogenic and antiapoptotic actions of ghrelin in 3T3-L1 adipocytes, Mol. Endocrinol. 18 (2004) 2291–2301. [4] G. Mazzocchi, G. Neri, M. Rucinski, P. Rebuffat, R. Spinazzi, L.K. Malendowicz, G.G. Nussdorfer, Ghrelin enhances the growth of cultured human adrenal zona glomerulosa cells by exerting MAPK-mediated proliferogenic and antiapoptotic effects, Peptides 25 (2004) 1269–1277. [5] S.W. Kim, S.J. Her, S.J. Park, D. Kim, K.S. Park, H.K. Lee, B.H. Han, M.S. Kim, C.S. Shin, S.Y. Kim, Ghrelin stimulates proliferation and differentiation and inhibits apoptosis in osteoblastic MC3T3-E1 cells, Bone 37 (2005) 359–369. [6] T. Waseem, M. Duxbury, H. Ito, F. Rocha, D. Lautz, E. Whang, S. Ashley, M. Robinson, Ghrelin ameliorates TNF-a induced antiproliferative and pro-apoptotic effects and promotes intestinal epithelial restitution, J. Am. Coll. Surg. 199 (2004) 16. [7] R. Granata, F. Settanni, L. Biancone, L. Trovato, R. Nano, F. Bertuzzi, S. Destefanis, M. Annunziata, M. Martinetti, F. Catapano, C. Ghe`, J. Isgaard, M. Papotti, E. Ghigo, G. Muccioli, Acylated and unacylated ghrelin promote proliferation and inhibit apoptosis of pancreatic b-cells and human islets: Involvement of 3’,5’-cyclic adenosine monophosphate/protein kinase A, extracellular signalregulated kinase 1/2, and phosphatidyl inositol 3-kinase/Akt signaling, Endocrinology 148 (2007) 512–529. [8] H. Chung, E. Kim, D.H. Lee, S. Seo, S. Ju, D. Lee, H. Kim, S. Park, Ghrelin inhibits apoptosis in hypothalamic neuronal cells during oxygen–glucose deprivation, Endocrinology 148 (2007) 148–159. [9] H. Ichijo, E. Nishida, K. Irie, P. Dijke, M. Saitoh, T. Moriguchi, M. Takagi, K. Matsumoto, K. Miyazono, Y. Gotoh, Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/ JNK and p38 signaling pathways, Science 275 (1997) 90–94. [10] H. Ichijo, From receptors to stress-activated MAP kinases, Oncogene 18 (1999) 6087–6093. [11] H.Y. Chang, H. Nishitoh, X. Yang, H. Ichijo, D. Baltimore, Activation of apoptosis signal-regulating kinase 1 (ASK1) by the adapter protein Daxx, Science 281 (1998) 1860–1863. [12] V.V. Sumbayev, I.M. Yasinska, Regulation of MAP kinase-dependent apoptotic pathway: implication of reactive oxygen and nitrogen species, Arch. Biochem. Biophys. 436 (2005) 406–412.
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Ghrelin inhibits apoptosis signal-regulating kinase 1 activity via upregulating heat-shock protein 70 Min Yang, Shengdi Hu, Bin Wu, Yanying Miao, Hui Pan, Shigong Zhu
*
Department of Physiology and Pathophysiology, Peking University Health Science Center, 38 Xueyuan Road, Beijing 100083, People’s Republic of China Received 13 May 2007 Available online 25 May 2007
Abstract Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), which has been originally isolated from rat stomach. It has been reported that ghrelin inhibited apoptosis in several cells, such as cardiomyocytes, endothelial cells, adipocyte, adrenal zona glomerulosa cells, pancreatic b-cells, osteoblastic MC3T3-E1 cells, intestinal epithelial cells and hypothalamic neurons. However, it is unknown whether heat-shock protein 70 (HSP70) or apoptosis signal-regulating kinase 1 (ASK1) is the important target molecule which mediates the anti-apoptotic effects of ghrelin. We show that ghrelin inhibited ASK1 activity induced by sodium nitroprusside (SNP), inhibited ASK1-mediated caspase 3 activation and apoptosis in PC12 cells. Ghrelin promoted expression of HSP70. Quercetin, an inhibitor of HSP70, blocked the effects of ghrelin on ASK1 activity. Thus, ghrelin inhibits ASK1-mediated apoptosis and ASK1 activation by a mechanism involving induction of HSP70 expression. The results of the present study suggest the therapeutic potential of ghrelin for some pathological processes or disorders. 2007 Elsevier Inc. All rights reserved. Keywords: Ghrelin; Apoptosis; ASK1; HSP70
Ghrelin is a newly discovered gut–brain peptide, predominantly produced by the stomach, and has been identified as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R) [1]. It has been reported that ghrelin inhibited apoptosis in several cells, such as cardiomyocytes, endothelial cells, adipocyte, adrenal zona glomerulosa cells, pancreatic b-cells, osteoblastic MC3T3E1 cells, intestinal epithelial cells and hypothalamic neurons [2–8]. However, the mechanism and the target molecule on which ghrelin exerts its anti-apoptotic effects remain to be elucidated. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) family member that acts upstream of JNK and p38 kinases by directly phosphorylating their respective MAPKKs (MKK4/7 for JNK and MKK3/6 for p38) [9,10]. ASK1
*
Corresponding author. Fax: +86 10 82801443. E-mail address: [email protected] (S. Zhu).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.05.118
has been shown to be an important signaling kinase in apoptotic cell death in response to various stimuli, including serum or trophic factor withdrawal, TNF-a, reactive nitrogen species (RNS), reactive oxygen species (ROS), ischemia insult, genotoxic stress, and possibly FasL [11– 18]. The active ASK1 is sufficient to induce cell apoptosis through mitochondrial-dependent caspase activation in several cell types [9,11,17,19]. Because of the important role of ASK1 in apoptosis, considerable attention has been focused on the key molecules which regulate ASK1 activity in the cells. One widely pursued strategy is to identify endogenous inhibitors of ASK1 that attenuate ASK1 pathway signaling. It has been reported that several molecules can modulate ASK1 activity, including thioredoxin, protein serine/threonine phosphatase 5 (PP5), glutathione-Stransferase, 14-3-3 protein, Akt, and heat-shock protein 70 (HSP70) [20–25]. It is well known the HSP70 is a molecular chaperone among these molecules and posses important function in cytoprotection. Study has shown that HSP70 inhibited the activation of ASK1 and prevented
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ASK1-dependent apoptotic cell death in NIH3T3 cell line [25]. However, it is unknown whether HSP70 or ASK1 is the important target molecule which mediates the antiapoptotic effects of ghrelin. In the present study, we explored the effects of ghrelin on ASK1 activity, and expression of HSP70, as well as relation among them. Materials and methods Materials. Human ghrelin was purchased from Phoenix Pharmaceuticals (Belmont, CA). RPMI-1640 medium, penicillin, and streptomycin were purchased from Gibco-BRL (Life Technologies, Inc., Gaithersburg, MD, USA). Fetal bovine serum (FBS) and horse serum were purchased from Hyclone (UT, USA). All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Sodium nitroprusside (SNP) and quercetin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The reagents of the BCA method for protein quantification and restoration of Western blot stripping buffer were purchased from Pierce (Pierce Co., Rockford, IL, USA). Cell culture and treatments. Rat pheochromocytoma (PC12) cells were cultured in RPMI-1640 medium supplemented with 10% horse serum, 5% fetal bovine serum (FBS), 50 U/ml penicillin, 50 lg/ml streptomycin, at 37 C in the presence of 95% air and 5% CO2. They were cultured for passage once 5 days. Experiments were performed using dishes coated with poly-L-lysine (10 lg/ml for 12 h) to assist cell adhesion. When cells were 80–90% confluent, the medium was exchanged for RPMI-1640 medium containing 1% FBS, the cells were used for experiments after 24 h. The pretreatment of ghrelin was performed for 30 min. The activation of ASK1 and apoptosis of PC12 cells were induced by the indicated doses of SNP as a NO donor for 18 h. When indicated, the cells were pretreated with quercetin for 60 min. Plasmids and transfection. Mammalian expression plasmids for ASK1 of the wild type (ASK1-WT) in PC-DNA3 (Invitrogen, Groningen, The Netherlands) were kindly provided by Dr. Hidenori Ichijo (Tokyo Medical and Dental University, Tokyo, Japan). Transfections with either ASK1 expressing PC-DNA3 plasmids or the corresponding empty vectors were performed using Lipofectamine 2000 (Life Technologies Inc., Rockville, MD, USA). For transient transfection, PC12 cells were plated in a washed glass coverslip or 60-mm dish for 24 h before transfection and transfected with plasmids (1 lg for coverslip or 2 lg for 60-mm dish) using Lipofectamine 2000 according to the manufacturer’s recommendation. Thirty-six hours following transfection, cells were exposed to SNP in the presence or absence of ghrelin. Protein extractions and Western blot analysis. Following experimental treatments, the cells were washed with PBS (pH 7.4) two times and sonicated on ice in a lysis buffer containing 150 mM NaCl, 100 mM Tris–HCl pH 7.4, 1% Triton X-100 and 1 mM Phenylmethyl sulphonyl fluoride (PMSF). Then, cellular debris were pelleted by centrifugation at 12,000g for 10 min at 4 C, the supernatant was collected by Western blotting. Protein concentrations were measured by the Bio-Rad Assay (Bio-Rad Laboratories, Hercules, CA). Proteins (30 lg per lane) were separated in 10% SDS–PAGE gels and electrophoretically transferred onto nitrocellulose for 2 h at 0.2 A. Membranes were blocked with blocking buffer (5% non-fat dried milk and 0.1% Tween 20 in TBS buffer) at room temperature for 1 h. Primary antibody incubations were overnight at 4 C using the appropriate dilutions (polyclonal ASK1 1:1000, polyclonal phosphoASK1 1:1000, monoclonal HSP70 1:1000, polyclonal caspase 3 1:1000, polyclonal b-actin 1:1000). Membranes were washed for 30 min with three changes, incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibody. Specific antibody–antigen complexes were detected using the ECL Western Blot Detection Kit. After immunoblotting, digitized images of the immunoreactive bands were quantitated by image analysis with use of the NIH Image software (version 1.62). Additional background measurements were taken from each film and subtracted from these values.
Flow-cytometry analysis. Apoptosis was measured by FCM analysis of the percentage of subdiploid apoptotic nuclei. Thirty-six hours after transfection with ASK1 expression plasmid and the corresponding empty vector, cells were treated with SNP in the presence or absence of ghrelin for 18 h. About 1 · 106/ml cells were harvested, washed with PBS, and fixed in 70% ethanol at 4 C for 24 h. The cells were washed again and incubated with 50 ll RNase (50 lg/ml) at 37 C for 30 min. Resuspending in PBS containing propidium iodide (50 lg/ml) in the dark for 10 min, the percentage of apoptotic nuclei, recognized by their subdiploid DNA content, was determined using the Becton–Dickinson FACS scan Flow Cytometer (Becton–Dickinson, Heidelberg, Germany). Statistical analysis. Each experiment was repeated at least three times. The data were presented as means ± SEM and analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s test. Statistical significance was set at p < 0.05.
Results Ghrelin prevents ASK1-mediated apoptosis of PC12 cells To determine whether ghrelin prevents ASK1-mediated apoptosis, we subjected PC12 cells to transient transfection with PC-DNA3-ASK1 plasmids, and then examined the effects of ghrelin on the cleavage status of caspase 3 and apoptosis induced by SNP. As shown in Fig. 1A, the activity of ASK1 was quite low in the corresponding empty vector-transfected PC12 cells. However, the activity of ASK1 was significantly increased by SNP with a concentration-dependent manner in ASK1-transfected PC12 cells (Fig. 1A). In the corresponding empty vector-transfected PC12 cells, the expression of caspase 3 cleavage was quite low, caspase 3 activity was increased by SNP, and was reduced by ghrelin in SNP-treated PC12 cells (Fig. 1B). In ASK1-transfected PC12 cells, there are the expression of caspase 3 cleavage but the level of caspase 3 cleavage was low, caspase 3 activity was increased significantly by SNP, whereas ghrelin markedly blocked caspase 3 activation (p < 0.05) (Fig. 1B). Apoptosis of PC12 cells were measured by flow-cytometric analysis. As shown in Fig. 2, the number of apoptosis was rare in PC12 cells transfection with ASK1 alone and no significant variance compared with the control vectors, apoptosis was markedly increased by SNP in ASK1 transfected cells compared with the control vectors transfected cells (p < 0.05). However, ghrelin treatment effectively reduced SNP-induced apoptosis in the corresponding empty vector-transfected, and significantly decreased SNP-induced apoptosis in ASK1PC12 cells (p < 0.01) (Fig. 2). These data show that ghrelin inhibited ASK1-mediated apoptosis of PC12 cells. Ghrelin inhibits ASK1 activation To test whether ghrelin is able to suppress the activity of ASK1, we examined the effects of ghrelin on the SNPinduced activation of ASK1. Western blot analysis showed that there was the expression of phosphorylated ASK1 in PC12 cells transfection with ASK1 alone, but the activity of ASK1 was dropped upon ghrelin treatment (Fig. 3).
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32KDa caspase-3
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Fig. 1. Effects of ghrelin on SNP-induced caspase 3 activation in ASK1-transfected PC12 cells. (A) SNP induces the activity of ASK1. PC-DNA3-ASK1 or PC-DNA3 plasmids were transfected into PC12 cells and incubated for 36 h. PC12-ASK1 cells were treated with 0.1, 0.3 mM SNP for 18 h. The cell proteins were subjected to Western blot analysis with antibodies against phospho-ASK1 or ASK1, and relative amounts of expression of phospho-ASK were compared with the intensity of ASK1. *p < 0.05, **p < 0.01 vs the empty vector-transfected PC12 cells. (B) Effects of ghrelin on SNP-induced caspase 3 activation in ASK1-transfected PC12 cells. PC12-ASK1 and PC12-PC-DNA3 cells were cultured in the absence or presence of 100 nM ghrelin for 30 min, and then treated with 0.3 mM SNP for 18 h. The cell proteins were subjected to Western blot analysis with antibody against caspase 3, and relative amounts of expression of caspase 3 cleavage were compared with the intensity of b-actin. *p < 0.05.
p-ASK1
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Fig. 2. Effects of ghrelin on ASK1-mediated apoptosis of PC12 cells. PC12-ASK1 and PC12-PC-DNA3 cells were cultured in the absence or presence of 100 nM ghrelin for 30 min, and then treated with 0.3 mM SNP for 18 h. Apoptosis was measured by FCM analysis of the percentage of subdiploid apoptotic nuclei. *p < 0.05, **p < 0.01.
The activity of ASK1 was markedly increased by SNP in ASK1-transfected PC12 cells, whereas ghrelin significantly decreased the amount of phosphorylated ASK1 (p < 0.01) (Fig. 3). These results suggest that ghrelin inhibited SNPinduced ASK1 activation.
SNP Ghrelin ASK1
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Fig. 3. Effects of ghrelin on SNP-induced activation of ASK1. PC-DNA3ASK1 plasmids were transfected into PC12 cells and incubated for 36 h. PC12-ASK1 cells were in the absence or presence of 100 nM ghrelin for 30 min, and then treated with 0.3 mM SNP for 18 h. The cell proteins were subjected to Western blot analysis with antibodies against phospho-ASK1 or ASK1, and relative amounts of expression of phospho-ASK were compared with the intensity of ASK1. *p < 0.05, **p < 0.01.
Ghrelin inhibition of ASK1 activity is mediated by induction of HSP70 The effects of ghrelin on expression of HSP70 were shown in Fig. 4A. The expression of HSP70 was quite
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HSP70
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Fig. 4. Ghrelin inhibition of ASK1 activity is mediated by induction of HSP70. (A) Effects of ghrelin on the protein expression of HSP70 in PC12 cells transfected with ASK1 induced by SNP. PC-DNA3-ASK1 plasmids were transfected into cells and incubated for 36 h. PC12-ASK1 cells were in the absence or presence of 100 nM ghrelin for 30 min, and then treated with 0.3 mM SNP for 18 h. The cell proteins were subjected to Western blot analysis with antibody against HSP70, and relative amounts of expression of HSP70 were compared with the intensity of b-actin. **p < 0.01 vs the SNP-treated PCDNA3-ASK1 cells. (B) Effects of quercetin on SNP-induced phosphorylation of ASK1 in response to ghrelin. PC12-ASK1 cells were cultured in the absence or presence of 10-lM quercetin for 60 min followed by the presence of 100 nM ghrelin. After 30 min, cells were exposed to 0.3 mM SNP for 18 h. The cell proteins were subjected to Western blot analysis with antibodies against HSP70 or phospho-ASK1, and relative amounts of expression of HSP70 or phospho-ASK1 were compared with the intensity of b-actin. *p < 0.05 vs the SNP-treated PC-DNA3-ASK1 cells, #p < 0.05 vs Ghrelin + SNP + ASK1.
low in PC12 cells transfection with ASK1 alone. Exposure to SNP upregulated HSP70 in ASK1-transfected PC12 cells, whereas the levels of HSP70 seem to be mild. Compared with ASK1-transfected PC12 cells exposed to SNP, ghrelin treatment significantly enhanced the expression of HSP70 in the absence or presence SNP (p < 0.01). These results suggest that ghrelin promoted the expression of HSP70 in PC12 with or without SNP injury. To examine the role of HSP70 in inhibition of ASK1 activity by ghrelin, quercetin, an inhibitor of HSP70 protein synthesis was used in experiments. The results show that SNP increased ASK1 phosphorylation, and produced a low level of HSP70 in ASK1-transfected PC12 cells (Fig. 4B). Compared with SNP treatment, the expression of HSP70 was attenuated, and associated with the activity of ASK1 was increased in ASK1-transfected PC12 cells exposed to quercetin and SNP (p < 0.05). On the other hand, ghrelin pretreatment markedly upregulated HSP70 and produced a low level of ASK1 phosphorylation. Compared with ghrelin pretreatment exposed to SNP, quercetin blocked the accumulation of HSP70, and connected with increased ASK1 activity (p < 0.05) (Fig. 4B). The results suggest that ghrelin inhibited ASK1 activity via upregulating HSP70. Discussion In the present study, we demonstrate that ghrelin inhibits ASK1 activity and ASK1-mediated apoptosis in PC12 cells. The results also suggest that ghrelin is a potent endog-
enous inducer of HSP70, by which ghrelin inhibited ASK1 activity. Ghrelin is a newly discovered gut–brain peptide. Apart from a potent GH-releasing action, it has been reported that ghrelin inhibited apoptosis in several cells, such as cardiomyocytes, endothelial cells, adipocyte, adrenal zona glomerulosa cells, pancreatic b-cells, osteoblastic MC3T3E1 cells, intestinal epithelial cells, and hypothalamic neurons [2–8]. Ghrelin has also been shown to activate the MAPK, phosphatidylinositol 3 kinase(PI3K)-Akt, PKA and PKC pathways, all of which mediate the anti-apoptotic effects of ghrelin [2–8]. In addition, ghrelin inhibited apoptosis by increasing Bcl-2/Bax ratio, prevention of cytochrome c release, inhibiting reactive oxygen species generation, stabilizing mitochondrial transmembrane potential and inhibition of caspase 3 activation [8]. However, the mechanism and the target molecule which ghrelin exerts its anti-apoptotic effects remain to be elucidated. Numerous studies have shown that activation of ASK1 is positively linked to apoptosis. Activation of ASK1 induces apoptotic cell death by triggering mitochondrial events that include the release of cytochrome c from mitochondria and the subsequent activation of caspase 9 and caspase 3 [19]. In primary neurons derived from ASK1 / mice, ER stress- and polyglutamine-induced JNK activation and apoptosis were reduced [15]. It was recently shown that ASK1 was also required for amyloid b (Ab)-induced neuronal cell apoptosis, and ASK1-JNK pathway was activated in a ROS-dependent manner. ASK1 / neurons were
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resistant to Ab-induced cell apoptosis [16]. Moreover, ASK1/MKK4/JNK play a central role in signaling pathways related to oxidative stress-induced apoptosis in neuronal cells [17,18]. These reports on the roles of ASK1 in apoptosis suggest the importance of ASK1 in the pathogenesis of neurodegenerative diseases. ASK1 may be a potential therapeutic target for prevention and treatment of various neurodegenerative diseases. To elucidate whether ASK1 is the important target molecule which mediates the anti-apoptotic effects of ghrelin, we explore the effects of ghrelin on ASK1 activity and its mechanism. Our data show that ghrelin inhibited ASK1mediated apoptosis of PC12 cells and suppressed the ASK1 activation. Other studies have proposed that selenite directly inhibiting ASK1 activity may be related to a cysteine-rich domain in its NH2 terminus of ASK1, and to the change redox potential of sulfhydryl groups of ASK1 [26]. Overexpression of CDC25A diminishes homo-oligomerization of ASK1, the binding of CDC25A to ASK1 at the region adjacent to the kinase domain could inhibit oligomerization of ASK1 [27]. Structurally, ghrelin does not resemble known kinase inhibitors. It was found that ghrelin inhibited the activity of ASK1 even 18 h after ghrelin treatment, but not short-term. Its time course of action suggested that ghrelin may function by facilitating production of an endogenous ASK1 antagonist. The biological function of ASK1 is negatively regulated by ASK1-interacting proteins, which include thioredoxin and other proteins. Thioredoxin in a reduced form binds to the NH2-terminal part of ASK1 and blocks activation of ASK1 by TNF [20]. The 14-3-3 protein, a phosphoserine-binding molecule, binds to ASK1 specifically via Ser967 of ASK1 and suppresses ASK1-induced apoptosis [23]. Protein serine/threonine phosphatase 5 (PP5) directly dephosphorylates Thr845 and inactivates ASK1 activity [21]. ASK1 may be a physiological target of Akt, as activation of the phosphoinositide 3-kinase (PI3-K)/Akt pathway is associated with a decrease in ASK1 kinase activity [24]. HSP70 may bind to ASK1 and prevent ASK1 homo-oligomerization, which negatively regulates ASK1 activity [25]. It is little known whether suppression of ASK1 activation by ghrelin is related to induction of HSP70. Our data demonstrate that ghrelin treatment results in the upregulation of HSP70 with or without SNP injury in PC12 cells. It seems that ASK1 inhibition by ghrelin is due to induction of HSP70. As shown in Fig. 4B, the prevention of HSP70 increased ASK1 activity induced by SNP. Blocking the ability of ghrelin to inhibit ASK1 phosphorylation was observed in cells treated with quercetin. These results suggested that ghrelin suppresses apoptotic signaling through ASK1 inhibition due to the induction of HSP70. In summary, we demonstrate for the first time that ghrelin may prevent ASK1-mediated apoptosis of PC12 cells and inhibit ASK1 activation. The suppression of ASK1 activity by ghrelin was mediated through inducing HSP70 expression. Our results show a novel mechanism of anti-
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apoptosis of ghrelin through the pathway of HSP70 and ASK1. Ghrelin may serve as a useful tool for basic research, and may have therapeutic potential for some pathological processes or disorders. Acknowledgments This work was supported by the National Natural Science Foundation of China (30370557), the Ministry of Education for Research Program of China (20020001083), and in part by the National Major Basic Research Program of China (G2000056908). PC-DNA3ASK1 plasmids were kindly provided by Dr. Hidenori Ichijo (Tokyo Medical and Dental University, Tokyo, Japan). References [1] M. Kojima, H. Hosoda, Y. Date, M. Nakazato, H. Matsuo, K. Kangawa, Ghrelin is a growth-hormone-releasing acylated peptide from stomach, Nature. 402 (1999) 656–660. [2] G. Baldanzi, N. Filigheddu, S. Cutrupi, F. Catapano, S. Bonissoni, A. Fubini, D. Malan, G. Baj, R. Granata, F. Broglio, M. Papotti, N. Surico, F. Bussolino, J. Isgaard, R. Deghenghi, F. Sinigaglia, M. Prat, G. Muccioli, E. Ghigo, A. Graziani, Ghrelin and des-acyl ghrelin inhibit cell death in cardiomyocytes and endothelial cells through ERK1/2 and PI3-kinase/AKT, J. Cell Biol. 159 (2002) 1029–1037. [3] M.S. Kim, C.Y. Yoon, P.G. Jang, Y.J. Park, C.S. Shin, H.S. Park, J.W. Ryu, Y.K. Pak, J.Y. Park, K.U. Lee, S.Y. Kim, H.K. Lee, Y.B. Kim, K.S. Park, The mitogenic and antiapoptotic actions of ghrelin in 3T3-L1 adipocytes, Mol. Endocrinol. 18 (2004) 2291–2301. [4] G. Mazzocchi, G. Neri, M. Rucinski, P. Rebuffat, R. Spinazzi, L.K. Malendowicz, G.G. Nussdorfer, Ghrelin enhances the growth of cultured human adrenal zona glomerulosa cells by exerting MAPK-mediated proliferogenic and antiapoptotic effects, Peptides 25 (2004) 1269–1277. [5] S.W. Kim, S.J. Her, S.J. Park, D. Kim, K.S. Park, H.K. Lee, B.H. Han, M.S. Kim, C.S. Shin, S.Y. Kim, Ghrelin stimulates proliferation and differentiation and inhibits apoptosis in osteoblastic MC3T3-E1 cells, Bone 37 (2005) 359–369. [6] T. Waseem, M. Duxbury, H. Ito, F. Rocha, D. Lautz, E. Whang, S. Ashley, M. Robinson, Ghrelin ameliorates TNF-a induced antiproliferative and pro-apoptotic effects and promotes intestinal epithelial restitution, J. Am. Coll. Surg. 199 (2004) 16. [7] R. Granata, F. Settanni, L. Biancone, L. Trovato, R. Nano, F. Bertuzzi, S. Destefanis, M. Annunziata, M. Martinetti, F. Catapano, C. Ghe`, J. Isgaard, M. Papotti, E. Ghigo, G. Muccioli, Acylated and unacylated ghrelin promote proliferation and inhibit apoptosis of pancreatic b-cells and human islets: Involvement of 3’,5’-cyclic adenosine monophosphate/protein kinase A, extracellular signalregulated kinase 1/2, and phosphatidyl inositol 3-kinase/Akt signaling, Endocrinology 148 (2007) 512–529. [8] H. Chung, E. Kim, D.H. Lee, S. Seo, S. Ju, D. Lee, H. Kim, S. Park, Ghrelin inhibits apoptosis in hypothalamic neuronal cells during oxygen–glucose deprivation, Endocrinology 148 (2007) 148–159. [9] H. Ichijo, E. Nishida, K. Irie, P. Dijke, M. Saitoh, T. Moriguchi, M. Takagi, K. Matsumoto, K. Miyazono, Y. Gotoh, Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/ JNK and p38 signaling pathways, Science 275 (1997) 90–94. [10] H. Ichijo, From receptors to stress-activated MAP kinases, Oncogene 18 (1999) 6087–6093. [11] H.Y. Chang, H. Nishitoh, X. Yang, H. Ichijo, D. Baltimore, Activation of apoptosis signal-regulating kinase 1 (ASK1) by the adapter protein Daxx, Science 281 (1998) 1860–1863. [12] V.V. Sumbayev, I.M. Yasinska, Regulation of MAP kinase-dependent apoptotic pathway: implication of reactive oxygen and nitrogen species, Arch. Biochem. Biophys. 436 (2005) 406–412.
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