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Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway
YTAAP-12846; No. of pages: 7; 4C: Toxicology and Applied Pharmacology xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Toxicology and Applied Pharmacology journal homepage: www.elsevier.com/locate/ytaap
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Qiu-Hua Liang 1, Yuan Liu 1, Shan-Shan Wu, Rong-Rong Cui, Ling-Qing Yuan ⁎, Er-Yuan Liao ⁎
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Institute of Metabolism and Endocrinology, Second Xiang-Ya Hospital, Central South University, Changsha, Hunan, People's Republic of China
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Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway
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Article history: Received 28 April 2013 Revised 24 July 2013 Accepted 26 July 2013 Available online xxxx
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Ghrelin is a 28-amino-acid peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR) and strongly stimulates the release of growth hormone from the hypothalamus–pituitary axis. Previous studies have identified the important physiological effects of ghrelin on bone metabolism, such as regulating proliferation and differentiation of osteoblasts, independent of GH/IGF-1 axis. However, research on effects and mechanisms of ghrelin on osteoblast apoptosis is still rare. In this study, we identified expression of GHSR in MC3T3-E1 cells and determined the effects of ghrelin on the apoptosis of osteoblastic MC3T3-E1 cells and the mechanism involved. Our data demonstrated that ghrelin inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as determined by terminal deoxynucleotidyl transferasemediated deoxyribonucleotide triphosphate nick end-labeling (TUNEL) and ELISA assays. Moreover, ghrelin upregulated Bcl-2 expression and downregulated Bax expression in a dose-dependent manner. Our study also showed decreased activated caspase-3 activity under the treatment of ghrelin. Further study suggested that ghrelin stimulated the phosphorylation of ERK and AKT. Pretreatment of cells with the ERK inhibitor PD98059, PI3K inhibitor LY294002, and GHSR-siRNA blocked the ghrelin-induced activation of ERK and AKT, respectively; however, ghrelin did not stimulate the phosphorylation of p38 or JNK. PD90859, LY294002 and GHSR-siRNA attenuated the anti-apoptosis effect of ghrelin in MC3T3-E1. In conclusion, ghrelin inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, which may be mediated by activating the GHSR/ERK and GHSR/PI3K/AKT signaling pathways. © 2013 Published by Elsevier Inc.
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Keywords: Ghrelin Osteoblast Apoptosis Signaling pathway
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Introduction
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Ghrelin, a 28-amino-acid peptide esterified with octanoic acid on Ser 3, is a growth hormone secretagogue (GHS), and it was originally isolated from both human and rat stomach (Kojima et al., 1999). Subsequent studies have identified that the stomach appears to be the major source of ghrelin (Date et al., 2000). Ghrelin-producing cells are also found in the small and large intestines, central nervous system (hypothalamus, pituitary), immune system, lung, heart, placenta, gonads, kidneys, and pancreas (Nikolopoulos et al., 2010; Sakata et al., 2002). As a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR), ghrelin acts through GHSR and stimulates the release of growth hormone from pituitary both in vivo and in vitro (Kojima et al., 1999). GHSR belongs to the G-protein-coupled receptors family and has two subtypes produced by alternative mRNA processing, which are the fulllength and functional type 1a receptor (GHS-R1a), and the truncated
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⁎ Corresponding authors at: Institute of Metabolism and Endocrinology, the Second Xiang-Ya Hospital, Central South University, Changsha, Hunan 410011, People's Republic of China. Fax: +86 731 85361472. E-mail addresses: [email protected] (L.-Q. Yuan), [email protected] (E.-Y. Liao). 1 These authors contributed equally to this work.
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and biological inactive type 1b receptor (GHS-R1b) (Howard et al., 1996; McKee et al., 1997a, 1997b). Expression of GHS-R1a has been also identified in a wide range of tissues including the hypothalamus, pituitary, stomach, heart, lung, pancreas, intestine, kidney, testis, and ovary. Ghrelin has multiple physiological effects mediated by its receptor (Fukushima et al., 2005; Gaytan et al., 2004; Shuto et al., 2001). The wide distribution of ghrelin and its receptor supports the potential for multiple biological activities of ghrelin both in brain and in peripheral tissues. Evidence indicates that ghrelin performs a variety of biological actions including stimulating food intake, promoting adipogenesis, decreasing energy metabolism, improving cardiovascular function, and stimulating GH, prolactin, and cortisol releases (Fukushima et al., 2005; Liang et al., 2012). GH is well known for promoting bone formation (Olney, 2003) and we hypothesized that ghrelin may play a role in bone metabolism. Evidence suggests that ghrelin may have direct effects on bone metabolism (Nikolopoulos et al., 2010; van der Velde et al., 2008). Recently, studies demonstrated that ghrelin and its receptor GHS-R1a are identified in osteoblast cells, and ghrelin promotes the proliferation and differentiation of rat osteoblasts and increases the BMD in rats (Fukushima et al., 2005; Maccarinelli et al., 2005). Costa et al. reported that ghrelin showed mitogenic activity in osteoblasts and increased the bone-resorbing activity of rat osteoclasts, but it did not alter osteoclast differentiation in a
0041-008X/$ – see front matter © 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.taap.2013.07.018
Please cite this article as: Liang, Q.-H., et al., Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway, Toxicol. Appl. Pharmacol. (2013), http://dx.doi.org/10.1016/j.taap.2013.07.018
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Reagents. Synthetic mouse ghrelin peptide [Gly-Ser-Ser(n-octanoyl)Phe-Leu-Ser-Pro-Glu-His-Gln-Lys-Ala-Gln-Gln-Arg-Lys-Glu-Ser-Lys-LysPro-Pro-Ala-Lys-Leu-Gln-Pro-Arg] was purchased from the Chinese Peptide Company (Hangzhou, China). Anti-extracellular signal-regulated kinase (ERK), p-ERK, p38, p-p38, c-jun N-terminal kinase (JNK), p-JNK, AKT, p-AKT, Bax, Bcl-2, caspase-3, and GHSR antibodies were purchased from Santa Cruz Biotechnology Inc. (Waltham, MA, USA). Anti-mouse monoclonal IgG peroxidase conjugate antibody, anti-Rabbit IgG antibody and anti-β-actin polyclonal antibody were purchased from SigmaAldrich Co. (St. Louis, USA). ERK inhibitor PD98059 and PI3K inhibitor LY294002 was purchased from Calbiochem Corp. (San Diego, CA, USA). GHSR-siRNA and siRNA scramble were purchased from GenePharma Company (Shanghai, China).
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Silence of GHSR by RNA interference. RNA interference was used to silence the expression of GHSR in MC3T3-E1 cells. GHSR-siRNA and scramble siRNA were synthesized by GenePharma Biotechnology (Shanghai, China). Cells were plated in six-well plates and cultured for 24 h in media without antibiotics and then were transfected with siRNAs (100 pmol/well) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells were cultured for 72 h. The efficiency of siRNA was determined by protein analysis (Liang et al., 2012).
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Western blot analysis. Cells were seeded for 24 h followed by culturing for 24 h in a serum-free medium. The cells were then treated with or without 10−11, 10−10, or 10−9 mol/L of ghrelin for 48 h. Immunoblotting was performed as previously described (Yuan et al., 2008; Zhu et al., 2012). In brief, total protein was extracted using RIPA lysis buffer (Beyotime, China). BCA assay was used to determine protein concentration, and equal amounts of protein were loaded onto SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk in PBS for 1 h, and then incubated with anti-Bcl2 antibody, anti-Bax antibody, anti-caspase-3 antibody, anti-GHSR antibody, or anti-β-actin antibody. Resultant protein bands following incubation with an appropriate secondary antibody were visualized by chemiluminescence.
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Measurement of MAPK and PI3K/AKT activation. MC3T3-E1 cells were exposed to 10−9 mol/L of ghrelin for 0–60 min. Cell layers were rinsed twice with cold PBS containing 5 mM EDTA and 0.1 mM Na3VO4 and lysed with a buffer consisting of 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 10 mM NaH2PO4, 10% glycerol, 2 mM Na3VO4, 10 mM NaF, 1 mM ABSF, 10 lg/mL leupeptin, and 10 lg/mL aprotinin. Western blot analysis was performed as before (Yuan et al., 2008; Zhu et al., 2012). An equal amount of protein was transferred onto PVDF membranes. The membranes were incubated with anti-p-ERK1/2, -ERK1/2, p-p38, p-38, p-JNK, JNK, p-AKT, and AKT antibodies and visualized by chemiluminescence.
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Statistical analysis. Data are shown as the mean ± standard deviation (SD). Differences between groups were compared by one-way ANOVA. A p-value less than 0.05 was considered statistically significant. All the experiments were repeated at least three times.
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Results
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Expression of GHSR in MC3T3-E1 cells
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Using RT-PCR, we confirmed that mRNA was expressed in MC3T3E1 cells (Fig. 1A). The results showed a 60-bp fragment specific to GHSR. GHSR mRNA expression in mouse stomach tissue was used as a positive control. No bands were observed in reactions without RT or with H2O as a template. Thus, our results reveal that MC3T3-E1 cells express GHSR mRNA.
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Cell cultures. The mouse osteoblastic cell line MC3T3-E1 was obtained from the American Type Culture Collection (ATCC, Rockville, MD). Cells were cultured in α-MEM (Gibco BRL, Gaithersburg, MD) and supplemented with 10% FBS, 20 mM HEPES, 100 U/mL penicillin, 100 mg/mL streptomycin, and 50 mg/mL ascorbic acid. Cells were maintained in a humidified, 95% air, 5% CO2 atmosphere at 37 °C. The medium was refreshed every two days, and the cells were subcultured using 0.05% trypsin with 0.01% EDTA. We used 10−11 mol/L to 10−9 mol/L of ghrelin to incubate MC3T3-E1 cells in the following experiment. The dose of ghrelin was selected based on previous research (Kim et al., 2005). After incubation, the following measurements were performed.
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Reverse transcription-polymerase chain reaction (RT-PCR) detection of GHSR gene expression. To investigate the expression of GHSR mRNA in mouse MC3T3-E1 cells, RT-PCR was performed. Total RNA from cultured MC3T3E1 cells and mouse stomach was isolated using Trizol reagent (Gibco) according to the manufacturer's recommended protocol, and then cDNA was prepared with a RevertAid First Strand cDNA Synthesis Kit (Fermentas). The PCR primers were as follows: GHSR, sense: 5′ACCGTGATGGTATGGGTGTCG-3′, and anti-sense: 5′-CACAGTGAGGCAG AAGACCG-3′. PCR was performed as follows: 94 °C for 1 min, 58 °C for 45 s, and 72 °C for 1 min for 35 cycles, followed by 10-min incubation at 72 °C. A volume of 10 μL of the reaction mixture was electrophoresed on 1.5% agarose gels and stained with ethidium bromide to verify bands. The identities of PCR products were confirmed by direct sequencing using an automatic DNA sequencer (PE Applied Biosystems).
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Cell death ELISA measurement. Apoptosis was directly detected by measuring cytoplasmic nucleosomes (i.e., DNA complexed with histone in the cytoplasm) with a Cell Death Detection ELISA kit (Roche Diagnostics, Roche Molecular Biochemicals, Mannheim, Germany) as described in the manufacturer's instructions. Cells were seeded at a density of 1.0 × 104 cells/well in 24-well plates for 24 h. After culturing in serum-free media for 24 h, cells were exposed to various concentrations (0, 10−11, 10−10, 10−9 mol/L) of ghrelin for 48 h in the absence of serum. Cells were then washed with PBS, incubated for 30 min with 0.5 mL of lysis buffer at 4 °C, and then centrifuged at 15,000 rpm for 10 min. Nucleosomes detected in the supernatants indicated the extent of apoptosis in the sample. To assay the effect of kinase inhibitor on MC3T3-E1 cell apoptosis, cells were pretreated with 10 μM ERK inhibitor PD98059 and 10 μM PI3K inhibitor LY294002 for 3 h prior to treatment with 10−9 mol/L of ghrelin for 48 h.
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murine bone marrow assay nor bone resorption in ex vivo calvarial cultures (Costa et al., 2011). However, data regarding the potential effects of ghrelin on apoptosis of osteoblasts are rare. Sang Wan Kim et al. found that ghrelin stimulated proliferation and differentiation and inhibited TNF-α-induced apoptosis in osteoblastic MC3T3-E1 cells (Kim et al., 2005). This study was undertaken to investigate the effects of ghrelin on the serum deprivation-induced apoptosis of osteoblastic MC3T3-E1 cells and the involved mechanism.
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TUNEL staining detection of osteoblastic MC3T3-E1 apoptosis. In situ apoptosis of MC3T3-E1 cells was measured by terminal deoxynucleotidyl transferase-mediated deoxyribonucleotide triphosphate nick endlabeling (TUNEL). Cells were cultured in a serum-free medium for 24 h and then treated with absence or presence of 10−9 mol/L of ghrelin for 48 h. The cells were washed three times with PBS and then stained with TUNEL reagent (Roche Diagnostics), according to the manufacturer's protocol. DAPI (4-diamino-2-phenylindole) was used to counter stain the nuclei. Cells were observed using a fluorescence microscope (×400 magnification). Six fields were randomly selected and the percentage of positive cells was calculated as the apoptosis index (AI) using the following equation: AI = (number of positive cells / total number of cells) × 100%, as previously described (Kitamura et al., 2004; Zhu et al., 2012).
Please cite this article as: Liang, Q.-H., et al., Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway, Toxicol. Appl. Pharmacol. (2013), http://dx.doi.org/10.1016/j.taap.2013.07.018
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Fig. 1. GHSR mRNA and protein expression in osteoblastic MC3T3-E1 cells. (A) GHSR mRNA expression in MC3T3-E1 cells. Total RNA from MC3T3-E1 cells was subjected to RT-PCR. The PCR products (60 bp) were visualized in a 1.5% agarose gel stained with ethidium bromide. Lane 1, mouse stomach tissue as the positive control; lane 2, MC3T3-E1 cells; lane 3, MC3T3-E1 cells RNA as negative control; and lane 4, H2O as negative control. (B) GHSR protein expression in MC3T3-E1 cells. Total cellular protein was subjected to an immunoblotting analysis using anti-GHSR antibody and identified a band at 44 kDa. Lane 1, mouse stomach tissue as the positive control; lane 2, lysate of MC3T3-E1 cells; lane 3, lysate from MC3T3-E1 cells treated with siRNA scramble; and lane 4, lysate from MC3T3-E1 cells treated with siRNA-GHSR. β-actin was used as the loading control.
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ghrelin treatment increased Bcl-2 protein expression and decreased Bax protein expression in MC3T3-E1 cells. Fig. 4B shows the decreased activated caspase-3 activity under the treatment of ghrelin. The effects of ghrelin were dose dependent. A concentration of 10−9 mol/L of ghrelin showed the maximal anti-apoptotic effect. When the Bax/Bcl-2 ratio in control cells was set to 1, 10−11–10−9 mol/L ghrelin treatment decreased this ratio gradually (Fig. 4C).
Ghrelin protected osteoblastic MC3T3-E1 cells against serum deprivationinduced apoptosis
Ghrelin activated ERK and PI3K/AKT signaling pathway in osteoblastic 235 MC3T3-E1 cells 236
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Serum-deprived MC3T3-E1 cells were treated with various concentration of ghrelin to determine its effect on apoptosis. Cell Death ELISA indicated that after 72 or 48 h of starvation, apoptotic cells at 10−11 mol/L (2.79 ± 0.19 ELISA absorbance units), 10−10 mol/L (2.37 ±0.18 ELISA absorbance units), and 10−9 mol/L (1.91 ± 0.24 ELISA absorbance units) ghrelin were less than that of vehicle-treated group (3.54 ± 0.21 ELISA absorbance units), showing a maximal anti-apoptotic effect at 10−9 mol/L of ghrelin. The details are shown in Fig. 2. TUNEL analysis was used as a second metric of apoptosis in serumdeprived MC3T3-E1 cells. Our data showed that serum starvation could significantly induce MC3T3-E1 cells apoptosis (Fig. 3A) and that incubation with 10−9 mol/L ghrelin protected cells from apoptosis induced by serum deprivation (Fig. 3B). Fig. 3C shows the details about the apoptosis index of control and ghrelin-treated cells.
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Effects of ghrelin on Bcl-2 and Bax protein expressions and caspase-3 activity in osteoblastic MC3T3-E1 cells
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To further investigate the anti-apoptotic effects of ghrelin on MC3T3E1 cells, Western blot analysis was used to assess the expressions of proteins Bcl-2 and Bax and activated caspase-3 activity. Fig. 4A shows that
Recent studies have demonstrated that MAPK and PI3K/AKT signaling pathways mediate ghrelin actions (Kim et al., 2004, 2005; Liang et al., 2012). In this experiment, Western blot analysis was used to determine the effect of ghrelin on MAPK and PI3K/AKT intracellular signaling pathways. Our data demonstrated that 10−9 mol/L ghrelin induced the phosphorylation of ERK after 5 min of stimulation, and the level of phosphorylation reached a peak at 15 min (Fig. 5A). Meanwhile, treatment with ghrelin also increased phosphorylated AKT (p-AKT) levels after 5 min of incubation, and peak activation of phosphorylated AKT occurred at 15 min (Fig. 5C). Conversely, neither p38 nor JNK responds to ghrelin stimulus but did respond to the positive control hydrogen peroxide (H2O2) (Xu et al., 2011) (Fig. 5B). The phosphorylation of ERK and AKT by ghrelin was abolished by the ERK inhibitor PD98059 and PI3K inhibitor LY294002, respectively (Figs. 5D, E). Taken together, these results indicate that ghrelin activates the MAPK/ERK and PI3K/AKT signaling pathways in MC3T3-E1 cells. Fig. 5F shows that suppression of GHSR with siRNA blocked the activation of ERK and AKT. These data indicate that ghrelin stimulates the ERK and PI3K/AKT signal transduction pathways via GHSR in MC3T3E1 cells.
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Using Western blot analyses, we confirmed that GHSR proteins were expressed in MC3T3-E1 cells (Fig. 1B). GHSR expression in mouse stomach tissue was used as a positive control. Treatment with siRNA-GHSR significantly blocked the expression of GHSR protein in MC3T3-E1 cells since few bands were detected, while no blockade was observed on the treatment with the scrambled siRNA. Our results demonstrate that MC3T3-E1 cells primarily express GHSR.
Fig. 2. Effect of ghrelin on serum deprivation-induced osteoblastic MC3T3-E1 cells apoptosis detected by cell death detection ELISA. Cells were deprived of serum for 24 h, then treated with ghrelin (0, 10−11, 10−10, 10−9 mol/L) for 48 h. ELISA results showed that ghrelin suppressed serum deprivation-induced apoptosis in MC3T3-E1 cells in a dose-dependent manner. Data are shown as the mean ± SD (n = 3).
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GHSR/ERK and GHSR/AKT signaling mediated the anti-apoptotic effect of 257 ghrelin in MC3T3-E1 cells 258 Blockade of ERK, AKT phosphorylation by PD98059 or LY294002, respectively, and knockout of GHSR by siRNA were performed to confirm that GHSR, ERK, and PI3K/AKT were involved in the regulation of MC3T3-E1 cell apoptosis. We used ELISA to determine the apoptosis of MC3T3-E1 cells. Our data confirmed that the anti-apoptotic effect of ghrelin exposure was reversed by pretreatment with both PD98059 and LY294002. Suppression of GHSR with siRNA-GHSR, but not scrambled siRNA, also abolished the anti-apoptotic effect of ghrelin, demonstrating that the anti-apoptotic role of ghrelin in MC3T3-E1 cells is mediated via the GHSR/ERK and GHSR/PI3K/AKT signaling pathways. Details are shown in Fig. 6.
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Discussion
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The present study shows that GHSR expression was identified in MC3T3-E1 cells. Treatment with ghrelin inhibits serum deprivationinduced apoptosis of osteoblastic MC3T3-E1 cells, indicating an antiapoptotic role for ghrelin in MC3T3-E1 cells. We also found that
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Please cite this article as: Liang, Q.-H., et al., Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway, Toxicol. Appl. Pharmacol. (2013), http://dx.doi.org/10.1016/j.taap.2013.07.018
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GHSR/ERK and GHSR/PI3K/AKT signaling pathways mediated the protective effect of ghrelin against serum deprivation-induced apoptosis in osteoblastic MC3T3-E1 cells. Apoptosis is defined as the process of cell death associated with caspase activation or caspase-mediated cell death. It is a necessary component of development and a characteristic of all self-renewing tissues, including bone (Jilka et al., 2007). Osteoblast apoptosis plays a critical role during embryonic limb development, skeletal maturation, and adult bone turnover by modeling and remodeling processes during fracture healing and bone regeneration and is an actively controlled process that is affected once pro-apoptotic signals exceed anti-apoptotic signals (Hock et al., 2001). It is now postulated that all major regulators of bone metabolism including bone morphogenetic proteins (BMPs), Wnts, other growth factors and cytokines, integrins, estrogens, androgens, glucocorticoids, PTH and PTH-related protein (PTHrP), immobilization, and the oxidative stress associated with aging contribute to the regulation of osteoblasts apoptosis (Xie et al., 2007). Our previous studies found that apelin and vaspin inhibit apoptosis of human osteoblasts, and L-Carnitine inhibits apoptosis of murine MC3T3-E1 osteoblastic cells (Baldanzi et al., 2002; Xie et al., 2008; Zhu et al., 2012).
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Fig. 3. Effect of ghrelin on serum deprivation-induced osteoblastic MC3T3-E1 cell apoptosis detected by TUNEL analysis. Cells were deprived of serum for 24 h, then treated with the absence or presence of 10−9 mol/L ghrelin for 48 h. Representative images of TUNEL stained cells are shown for the control group (A) and cells were treated with 10−9 mol/L ghrelin for 48 h (B). Apoptotic cells are indicated by yellow arrows. Representative figures are shown. The apoptosis index for control and ghrelin-treated cells is shown (C). Original magnification of all images, ×400. Data are shown as the mean ± SD (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Ghrelin is a newly discovered bioactive peptide that owns multiple physiological effects (Fukushima et al., 2005; Gaytan et al., 2004; Liang et al., 2012; Shuto et al., 2001). Previous studies have determined the inhibitory effects of ghrelin on cell apoptosis. It has been reported that ghrelin inhibits apoptosis in several cells, such as cardiomyocytes, endothelial cells, adipocytes, adrenal zona glomerulosa cells, pancreatic βcells, intestinal epithelial cells, and hypothalamic neurons (Chung et al., 2007; Granata et al., 2007; Kim et al., 2004, 2006; Kui et al., 2009; Mazzocchi et al., 2004; Rodriguez et al., 2012). In our experiment, we chose Cell Death Detection ELISA kit and TUNEL, which are classic methods to evaluate apoptosis through visualizing nuclei containing fragmented DNA by labeling the exposed termini of DNA and to detect the apoptosis of MC3T3-E1 cells (Zhu et al., 2012). Our data demonstrated that 10−11–10−9 mol/L ghrelin could inhibit serum deprivationinduced apoptosis in osteoblastic MC3T3-E1 cells in a dose-dependent manner. Bcl-2 proteins are a family of cytosolic proteins involved in the apoptotic pathways, which are made up of anti-apoptotic proteins (e.g., Bcl-2, Bcl-XL) and pro-apoptotic proteins (e.g., Bax, Bak) (Gu et al., 2013). When death signals overwhelm survival signals, the actions of anti-apoptotic Bcl-2 proteins are abrogated (Xie et al., 2007).
Please cite this article as: Liang, Q.-H., et al., Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway, Toxicol. Appl. Pharmacol. (2013), http://dx.doi.org/10.1016/j.taap.2013.07.018
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Fig. 4. Effect of ghrelin on Bax and Bcl-2 protein expressions and caspase-3 activity in osteoblastic MC3T3-E1 cells. Cells were deprived of serum for 24 h, then treated with 10−11, 10−10, and 10−9 mol/L ghrelin for 48 h. Representative Western blots are shown for Bax, Bcl-2, activated caspase-3, and β-actin. The expressions of Bax and Bcl-2 proteins and activated caspase-3 activity were determined by Western blot (A, B), and the Western blot data were densitometrically quantified and normalized to inner standard (C) (n = 3).
Fig. 5. Involvement of MAPK and PI3K/AKT phosphorylation in ghrelin-stimulated osteoblastic MC3T3-E1 cells. (A and B) Cells were exposed to 10−9 mol/L ghrelin for 0–60 min, or 50 μM hydrogen peroxide (H2O2) for 15 min as a positive control for p38 and JNK activation. Cell lysates were subjected to Western blotting and incubated with antibodies p-ERK, -ERK, p-p38, p38, p-JNK, JNK, p-AKT, and AKT. (C) Cells were exposed to 10−9 mol/L ghrelin for 0–60 min to assess AKT activation. (D) Cells were pretreated with 10 μM PD98059 for 3 h before exposure to 10−9 mol/L ghrelin for 15 min. (E) Cells were pretreated with 10 μM LY294002 for 3 h before exposure to 10−9 mol/L ghrelin for 15 min. The representative results are shown. (F) Cells were treated with scrambled GHSR siRNA, or GHSR-siRNA in the presence of 10−9 mol/L ghrelin. Representative results are shown.
Please cite this article as: Liang, Q.-H., et al., Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway, Toxicol. Appl. Pharmacol. (2013), http://dx.doi.org/10.1016/j.taap.2013.07.018
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PI3K/AKT signaling pathways. They suggest that ghrelin/GHSR may be 356 involved in bone metabolism through regulation of osteoblast apoptosis. 357 358
Conflict of interest statement The authors declare that there are no conflicts of interest.
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This work was supported by funding from The National Natural Science Foundation of China (Grant nos. 81270962, 81070246, 30801174) and the National Science Foundation for Post-doctoral Scientists of China (Grant no. 2012M521568).
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Acknowledgments
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In 2005, Kim et al. once reported the protective effect of ghrelin on TNFα-induced MC3T3-E1 osteoblastic cell apoptosis (Kim et al., 2005). However, the mechanisms were still unclear. We found that ghrelin up-regulated the expression of Bcl-2 and down-regulated the expression of Bax in MC3T3-E1 cells, indicating that the Bcl-2 family is involved in modulating the apoptosis of MC3T3-E1 cells by ghrelin. Furthermore, our data demonstrated that ghrelin blocked the activity of caspase-3, which is a hallmark of apoptosis that has been previously reported (Chua et al., 2003; Xie et al., 2010). To gain further insight into the mechanism of the capacity of ghrelin to inhibit MC3T3-E1 cell apoptosis, we investigated several intracellular signaling pathways. The MAPKs, including ERK, JNK and p38, are serine/ threonine kinase pathways. PI3K/AKT pathway, which can be activated by growth factors and some extracellular signals, regulates fundamental cellular processes including cell proliferation, differentiation, and survival (Cantrell, 2001). Our previous studies have demonstrated the involvement of many of these signaling pathways in the regulation of osteoblast apoptosis (Xie et al., 2008; Zhu et al., 2012). Kim et al. reported that ghrelin promotes the proliferation and differentiation, and inhibited TNF-α-induced apoptosis of MC3T3-E1 osteoblastic cells through ERK signaling pathway. But the PI3K/AKT signaling pathway was not detected (Kim et al., 2005). Recently, we found that ghrelin attenuated the osteoblastic differentiation of vascular smooth muscle cells through GHSR/ERK pathway (Liang et al., 2012). In this study, we tested the activation of both MAPK and PI3K/AKT. Also, RT-PCR and Western blot were adopted to determine the expression of GHSR in MC3T3-E1 cells. Our data showed that ghrelin activated both ERK and PI3K/AKT in MC3T3-E1 cells; however, neither p38 nor JNK was activated under the stimulus of ghrelin. Pretreatment with the ERK inhibitor PD98059 and PI3K inhibitor LY294002 reversed the activation of ERK and AKT in MC3T3-E1 cells, respectively. Furthermore, the suppression of GHSR with siRNA blocked the effects of ghrelin on ERK and AKT, which suggests that the activation of ERK and PI3K/AKT is mediated through GHSR. Both blockade of ERK and AKT and inhibition of GHSR abolish the protective effects of ghrelin on serum deprivation-induced MC3T3-E1 cell apoptosis, indicating that ghrelin suppresses MC3T3-E1 cell apoptosis by activating the GHSR/ERK and GHSR/PI3K/AKT signaling pathways. In conclusion, our study demonstrates that GHSR is expressed in MC3T3-E1 cells and ghrelin protects MC3T3-E1 cells from serum deprivation-induced apoptosis by activating the GHSR/ERK and GHSR/
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Baldanzi, G., Filigheddu, N., Cutrupi, S., Catapano, F., Bonissoni, S., Fubini, A., Malan, D., Baj, G., Granata, R., Broglio, F., Papotti, M., Surico, N., Bussolino, F., Isgaard, J., Deghenghi, R., Sinigaglia, F., Prat, M., Muccioli, G., Ghigo, E., Graziani, A., 2002. Ghrelin and desacyl ghrelin inhibit cell death in cardiomyocytes and endothelial cells through ERK1/2 and PI 3-kinase/AKT. J. Cell Biol. 159, 1029–1037. Cantrell, D.A., 2001. Phosphoinositide 3-kinase signalling pathways. J. Cell Sci. 114, 1439–1445. Chua, C.C., Chua, B.H., Chen, Z., Landy, C., Hamdy, R.C., 2003. Dexamethasone induces caspase activation in murine osteoblastic MC3T3-E1 cells. Biochim. Biophys. Acta 1642, 79–85. Chung, H., Kim, E., Lee, D.H., Seo, S., Ju, S., Lee, D., Kim, H., Park, S., 2007. Ghrelin inhibits apoptosis in hypothalamic neuronal cells during oxygen–glucose deprivation. Endocrinology 148, 148–159. Costa, J.L., Naot, D., Lin, J.M., Watson, M., Callon, K.E., Reid, I.R., Grey, A.B.., Cornish, J., 2011. Ghrelin is an osteoblast mitogen and increases osteoclastic bone resorption in vitro. Int. J. Pept. 2011, 605193. Date, Y., Kojima, M., Hosoda, H., Sawaguchi, A., Mondal, M.S., Suganuma, T., Matsukura, S., Kangawa, K., Nakazato, M., 2000. Ghrelin, a novel growth hormone-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology 141, 4255–4261. Fukushima, N., Hanada, R., Teranishi, H., Fukue, Y., Tachibana, T., Ishikawa, H., Takeda, S., Takeuchi, Y., Fukumoto, S., Kangawa, K., Nagata, K., Kojima, M., 2005. Ghrelin directly regulates bone formation. J. Bone Miner. Res. 20, 790–798. Gaytan, F., Barreiro, M.L., Caminos, J.E., Chopin, L.K., Herington, A.C., Morales, C., Pinilla, L., Paniagua, R., Nistal, M., Casanueva, F.F., Aguilar, E., Dieguez, C., Tena-Sempere, M., 2004. Expression of ghrelin and its functional receptor, the type 1a growth hormone secretagogue receptor, in normal human testis and testicular tumors. J. Clin. Endocrinol. Metab. 89, 400–409. Granata, R., Settanni, F., Biancone, L., Trovato, L., Nano, R., Bertuzzi, F., Destefanis, S., Annunziata, M., Martinetti, M., Catapano, F., Ghe, C., Isgaard, J., Papotti, M., Ghigo, E., Muccioli, G., 2007. Acylated and unacylated ghrelin promote proliferation and inhibit apoptosis of pancreatic beta-cells and human islets: involvement of 3′,5′-cyclic adenosine monophosphate/protein kinase A, extracellular signal-regulated kinase 1/2, and phosphatidyl inositol 3-Kinase/Akt signaling. Endocrinology 148, 512–529. Gu, Y.X., Du, J., Si, M.S., Mo, J.J., Qiao, S.C., Lai, H.C., 2013. The roles of PI3K/Akt signaling pathway in regulating MC3T3-E1 preosteoblast proliferation and differentiation on SLA and SLActive titanium surfaces. J. Biomed. Mater. Res. A 101, 748–754. Hock, J.M., Krishnan, V., Onyia, J.E., Bidwell, J.P., Milas, J., Stanislaus, D., 2001. Osteoblast apoptosis and bone turnover. J. Bone Miner. Res. 16, 975–984. Howard, A.D., Feighner, S.D., Cully, D.F., Arena, J.P., Liberator, P.A., Rosenblum, C.I., Hamelin, M., Hreniuk, D.L., Palyha, O.C., Anderson, J., Paress, P.S., Diaz, C., Chou, M., Liu, K.K., McKee, K.K., Pong, S.S., Chaung, L.Y., Elbrecht, A., Dashkevicz, M., Heavens, R., Rigby, M., Sirinathsinghji, D.J., Dean, D.C., Melillo, D.G., Patchett, A.A., Nargund, R., Griffin, P.R., DeMartino, J.A., Gupta, S.K., Schaeffer, J.M., Smith, R.G., Van der Ploeg, L.H., 1996. A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 273, 974–977. Jilka, R.L., Weinstein, R.S., Parfitt, A.M., Manolagas, S.C., 2007. Quantifying osteoblast and osteocyte apoptosis: challenges and rewards. J. Bone Miner. Res. 22, 1492–1501. Kim, M.S., Yoon, C.Y., Jang, P.G., Park, Y.J., Shin, C.S., Park, H.S., Ryu, J.W., Pak, Y.K., Park, J.Y., Lee, K.U., Kim, S.Y., Lee, H.K., Kim, Y.B., Park, K.S., 2004. The mitogenic and antiapoptotic actions of ghrelin in 3T3-L1 adipocytes. Mol. Endocrinol. 18, 2291–2301. Kim, S.W., Her, S.J., Park, S.J., Kim, D., Park, K.S., Lee, H.K., Han, B.H., Kim, M.S., Shin, C.S., Kim, S.Y., 2005. Ghrelin stimulates proliferation and differentiation and inhibits apoptosis in osteoblastic MC3T3-E1 cells. Bone 37, 359–369. Kim, H., Rafiuddin-Shah, M., Tu, H.C., Jeffers, J.R., Zambetti, G.P., Hsieh, J.J., Cheng, E.H., 2006. Hierarchical regulation of mitochondrion-dependent apoptosis by BCL-2 subfamilies. Nat. Cell Biol. 8, 1348–1358. Kitamura, T., Itoh, M., Noda, T., Matsuura, M., Wakabayashi, K., 2004. Combined effects of cyclooxygenase-1 and cyclooxygenase-2 selective inhibitors on intestinal tumorigenesis in adenomatous polyposis coli gene knockout mice. Int. J. Cancer 109, 576–580. Kojima, M., Hosoda, H., Date, Y., Nakazato, M., Matsuo, H., Kangawa, K., 1999. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402, 656–660.
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Fig. 6. The GHSR/ERK and GHSR/PI3K/AKT signaling pathways mediate the effect of ghrelin to inhibit serum deprivation-induced apoptosis of osteoblastic MC3T3-E1 cells. Cells were pretreated with 10 μM PD98059 or 10 μM LY294002, respectively, for 3 h before exposure to 10−9 mol/L ghrelin for 48 h. Cells were also treated with the siRNA scramble or GHSR-siRNA in the presence of 10−9 mol/L ghrelin for 48 h. MC3T3-E1 cell apoptosis was analyzed by ELISA. Data are shown as the mean ± SD (n = 3).
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Please cite this article as: Liang, Q.-H., et al., Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway, Toxicol. Appl. Pharmacol. (2013), http://dx.doi.org/10.1016/j.taap.2013.07.018
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Qiu-Hua Liang 1, Yuan Liu 1, Shan-Shan Wu, Rong-Rong Cui, Ling-Qing Yuan ⁎, Er-Yuan Liao ⁎
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Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway
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Article history: Received 28 April 2013 Revised 24 July 2013 Accepted 26 July 2013 Available online xxxx
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Ghrelin is a 28-amino-acid peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR) and strongly stimulates the release of growth hormone from the hypothalamus–pituitary axis. Previous studies have identified the important physiological effects of ghrelin on bone metabolism, such as regulating proliferation and differentiation of osteoblasts, independent of GH/IGF-1 axis. However, research on effects and mechanisms of ghrelin on osteoblast apoptosis is still rare. In this study, we identified expression of GHSR in MC3T3-E1 cells and determined the effects of ghrelin on the apoptosis of osteoblastic MC3T3-E1 cells and the mechanism involved. Our data demonstrated that ghrelin inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as determined by terminal deoxynucleotidyl transferasemediated deoxyribonucleotide triphosphate nick end-labeling (TUNEL) and ELISA assays. Moreover, ghrelin upregulated Bcl-2 expression and downregulated Bax expression in a dose-dependent manner. Our study also showed decreased activated caspase-3 activity under the treatment of ghrelin. Further study suggested that ghrelin stimulated the phosphorylation of ERK and AKT. Pretreatment of cells with the ERK inhibitor PD98059, PI3K inhibitor LY294002, and GHSR-siRNA blocked the ghrelin-induced activation of ERK and AKT, respectively; however, ghrelin did not stimulate the phosphorylation of p38 or JNK. PD90859, LY294002 and GHSR-siRNA attenuated the anti-apoptosis effect of ghrelin in MC3T3-E1. In conclusion, ghrelin inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, which may be mediated by activating the GHSR/ERK and GHSR/PI3K/AKT signaling pathways. © 2013 Published by Elsevier Inc.
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Keywords: Ghrelin Osteoblast Apoptosis Signaling pathway
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Introduction
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Ghrelin, a 28-amino-acid peptide esterified with octanoic acid on Ser 3, is a growth hormone secretagogue (GHS), and it was originally isolated from both human and rat stomach (Kojima et al., 1999). Subsequent studies have identified that the stomach appears to be the major source of ghrelin (Date et al., 2000). Ghrelin-producing cells are also found in the small and large intestines, central nervous system (hypothalamus, pituitary), immune system, lung, heart, placenta, gonads, kidneys, and pancreas (Nikolopoulos et al., 2010; Sakata et al., 2002). As a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR), ghrelin acts through GHSR and stimulates the release of growth hormone from pituitary both in vivo and in vitro (Kojima et al., 1999). GHSR belongs to the G-protein-coupled receptors family and has two subtypes produced by alternative mRNA processing, which are the fulllength and functional type 1a receptor (GHS-R1a), and the truncated
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⁎ Corresponding authors at: Institute of Metabolism and Endocrinology, the Second Xiang-Ya Hospital, Central South University, Changsha, Hunan 410011, People's Republic of China. Fax: +86 731 85361472. E-mail addresses: [email protected] (L.-Q. Yuan), [email protected] (E.-Y. Liao). 1 These authors contributed equally to this work.
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and biological inactive type 1b receptor (GHS-R1b) (Howard et al., 1996; McKee et al., 1997a, 1997b). Expression of GHS-R1a has been also identified in a wide range of tissues including the hypothalamus, pituitary, stomach, heart, lung, pancreas, intestine, kidney, testis, and ovary. Ghrelin has multiple physiological effects mediated by its receptor (Fukushima et al., 2005; Gaytan et al., 2004; Shuto et al., 2001). The wide distribution of ghrelin and its receptor supports the potential for multiple biological activities of ghrelin both in brain and in peripheral tissues. Evidence indicates that ghrelin performs a variety of biological actions including stimulating food intake, promoting adipogenesis, decreasing energy metabolism, improving cardiovascular function, and stimulating GH, prolactin, and cortisol releases (Fukushima et al., 2005; Liang et al., 2012). GH is well known for promoting bone formation (Olney, 2003) and we hypothesized that ghrelin may play a role in bone metabolism. Evidence suggests that ghrelin may have direct effects on bone metabolism (Nikolopoulos et al., 2010; van der Velde et al., 2008). Recently, studies demonstrated that ghrelin and its receptor GHS-R1a are identified in osteoblast cells, and ghrelin promotes the proliferation and differentiation of rat osteoblasts and increases the BMD in rats (Fukushima et al., 2005; Maccarinelli et al., 2005). Costa et al. reported that ghrelin showed mitogenic activity in osteoblasts and increased the bone-resorbing activity of rat osteoclasts, but it did not alter osteoclast differentiation in a
0041-008X/$ – see front matter © 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.taap.2013.07.018
Please cite this article as: Liang, Q.-H., et al., Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway, Toxicol. Appl. Pharmacol. (2013), http://dx.doi.org/10.1016/j.taap.2013.07.018
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Reagents. Synthetic mouse ghrelin peptide [Gly-Ser-Ser(n-octanoyl)Phe-Leu-Ser-Pro-Glu-His-Gln-Lys-Ala-Gln-Gln-Arg-Lys-Glu-Ser-Lys-LysPro-Pro-Ala-Lys-Leu-Gln-Pro-Arg] was purchased from the Chinese Peptide Company (Hangzhou, China). Anti-extracellular signal-regulated kinase (ERK), p-ERK, p38, p-p38, c-jun N-terminal kinase (JNK), p-JNK, AKT, p-AKT, Bax, Bcl-2, caspase-3, and GHSR antibodies were purchased from Santa Cruz Biotechnology Inc. (Waltham, MA, USA). Anti-mouse monoclonal IgG peroxidase conjugate antibody, anti-Rabbit IgG antibody and anti-β-actin polyclonal antibody were purchased from SigmaAldrich Co. (St. Louis, USA). ERK inhibitor PD98059 and PI3K inhibitor LY294002 was purchased from Calbiochem Corp. (San Diego, CA, USA). GHSR-siRNA and siRNA scramble were purchased from GenePharma Company (Shanghai, China).
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Silence of GHSR by RNA interference. RNA interference was used to silence the expression of GHSR in MC3T3-E1 cells. GHSR-siRNA and scramble siRNA were synthesized by GenePharma Biotechnology (Shanghai, China). Cells were plated in six-well plates and cultured for 24 h in media without antibiotics and then were transfected with siRNAs (100 pmol/well) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells were cultured for 72 h. The efficiency of siRNA was determined by protein analysis (Liang et al., 2012).
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Western blot analysis. Cells were seeded for 24 h followed by culturing for 24 h in a serum-free medium. The cells were then treated with or without 10−11, 10−10, or 10−9 mol/L of ghrelin for 48 h. Immunoblotting was performed as previously described (Yuan et al., 2008; Zhu et al., 2012). In brief, total protein was extracted using RIPA lysis buffer (Beyotime, China). BCA assay was used to determine protein concentration, and equal amounts of protein were loaded onto SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk in PBS for 1 h, and then incubated with anti-Bcl2 antibody, anti-Bax antibody, anti-caspase-3 antibody, anti-GHSR antibody, or anti-β-actin antibody. Resultant protein bands following incubation with an appropriate secondary antibody were visualized by chemiluminescence.
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Measurement of MAPK and PI3K/AKT activation. MC3T3-E1 cells were exposed to 10−9 mol/L of ghrelin for 0–60 min. Cell layers were rinsed twice with cold PBS containing 5 mM EDTA and 0.1 mM Na3VO4 and lysed with a buffer consisting of 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 10 mM NaH2PO4, 10% glycerol, 2 mM Na3VO4, 10 mM NaF, 1 mM ABSF, 10 lg/mL leupeptin, and 10 lg/mL aprotinin. Western blot analysis was performed as before (Yuan et al., 2008; Zhu et al., 2012). An equal amount of protein was transferred onto PVDF membranes. The membranes were incubated with anti-p-ERK1/2, -ERK1/2, p-p38, p-38, p-JNK, JNK, p-AKT, and AKT antibodies and visualized by chemiluminescence.
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Statistical analysis. Data are shown as the mean ± standard deviation (SD). Differences between groups were compared by one-way ANOVA. A p-value less than 0.05 was considered statistically significant. All the experiments were repeated at least three times.
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Expression of GHSR in MC3T3-E1 cells
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Using RT-PCR, we confirmed that mRNA was expressed in MC3T3E1 cells (Fig. 1A). The results showed a 60-bp fragment specific to GHSR. GHSR mRNA expression in mouse stomach tissue was used as a positive control. No bands were observed in reactions without RT or with H2O as a template. Thus, our results reveal that MC3T3-E1 cells express GHSR mRNA.
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Cell cultures. The mouse osteoblastic cell line MC3T3-E1 was obtained from the American Type Culture Collection (ATCC, Rockville, MD). Cells were cultured in α-MEM (Gibco BRL, Gaithersburg, MD) and supplemented with 10% FBS, 20 mM HEPES, 100 U/mL penicillin, 100 mg/mL streptomycin, and 50 mg/mL ascorbic acid. Cells were maintained in a humidified, 95% air, 5% CO2 atmosphere at 37 °C. The medium was refreshed every two days, and the cells were subcultured using 0.05% trypsin with 0.01% EDTA. We used 10−11 mol/L to 10−9 mol/L of ghrelin to incubate MC3T3-E1 cells in the following experiment. The dose of ghrelin was selected based on previous research (Kim et al., 2005). After incubation, the following measurements were performed.
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Reverse transcription-polymerase chain reaction (RT-PCR) detection of GHSR gene expression. To investigate the expression of GHSR mRNA in mouse MC3T3-E1 cells, RT-PCR was performed. Total RNA from cultured MC3T3E1 cells and mouse stomach was isolated using Trizol reagent (Gibco) according to the manufacturer's recommended protocol, and then cDNA was prepared with a RevertAid First Strand cDNA Synthesis Kit (Fermentas). The PCR primers were as follows: GHSR, sense: 5′ACCGTGATGGTATGGGTGTCG-3′, and anti-sense: 5′-CACAGTGAGGCAG AAGACCG-3′. PCR was performed as follows: 94 °C for 1 min, 58 °C for 45 s, and 72 °C for 1 min for 35 cycles, followed by 10-min incubation at 72 °C. A volume of 10 μL of the reaction mixture was electrophoresed on 1.5% agarose gels and stained with ethidium bromide to verify bands. The identities of PCR products were confirmed by direct sequencing using an automatic DNA sequencer (PE Applied Biosystems).
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Cell death ELISA measurement. Apoptosis was directly detected by measuring cytoplasmic nucleosomes (i.e., DNA complexed with histone in the cytoplasm) with a Cell Death Detection ELISA kit (Roche Diagnostics, Roche Molecular Biochemicals, Mannheim, Germany) as described in the manufacturer's instructions. Cells were seeded at a density of 1.0 × 104 cells/well in 24-well plates for 24 h. After culturing in serum-free media for 24 h, cells were exposed to various concentrations (0, 10−11, 10−10, 10−9 mol/L) of ghrelin for 48 h in the absence of serum. Cells were then washed with PBS, incubated for 30 min with 0.5 mL of lysis buffer at 4 °C, and then centrifuged at 15,000 rpm for 10 min. Nucleosomes detected in the supernatants indicated the extent of apoptosis in the sample. To assay the effect of kinase inhibitor on MC3T3-E1 cell apoptosis, cells were pretreated with 10 μM ERK inhibitor PD98059 and 10 μM PI3K inhibitor LY294002 for 3 h prior to treatment with 10−9 mol/L of ghrelin for 48 h.
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murine bone marrow assay nor bone resorption in ex vivo calvarial cultures (Costa et al., 2011). However, data regarding the potential effects of ghrelin on apoptosis of osteoblasts are rare. Sang Wan Kim et al. found that ghrelin stimulated proliferation and differentiation and inhibited TNF-α-induced apoptosis in osteoblastic MC3T3-E1 cells (Kim et al., 2005). This study was undertaken to investigate the effects of ghrelin on the serum deprivation-induced apoptosis of osteoblastic MC3T3-E1 cells and the involved mechanism.
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TUNEL staining detection of osteoblastic MC3T3-E1 apoptosis. In situ apoptosis of MC3T3-E1 cells was measured by terminal deoxynucleotidyl transferase-mediated deoxyribonucleotide triphosphate nick endlabeling (TUNEL). Cells were cultured in a serum-free medium for 24 h and then treated with absence or presence of 10−9 mol/L of ghrelin for 48 h. The cells were washed three times with PBS and then stained with TUNEL reagent (Roche Diagnostics), according to the manufacturer's protocol. DAPI (4-diamino-2-phenylindole) was used to counter stain the nuclei. Cells were observed using a fluorescence microscope (×400 magnification). Six fields were randomly selected and the percentage of positive cells was calculated as the apoptosis index (AI) using the following equation: AI = (number of positive cells / total number of cells) × 100%, as previously described (Kitamura et al., 2004; Zhu et al., 2012).
Please cite this article as: Liang, Q.-H., et al., Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway, Toxicol. Appl. Pharmacol. (2013), http://dx.doi.org/10.1016/j.taap.2013.07.018
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Fig. 1. GHSR mRNA and protein expression in osteoblastic MC3T3-E1 cells. (A) GHSR mRNA expression in MC3T3-E1 cells. Total RNA from MC3T3-E1 cells was subjected to RT-PCR. The PCR products (60 bp) were visualized in a 1.5% agarose gel stained with ethidium bromide. Lane 1, mouse stomach tissue as the positive control; lane 2, MC3T3-E1 cells; lane 3, MC3T3-E1 cells RNA as negative control; and lane 4, H2O as negative control. (B) GHSR protein expression in MC3T3-E1 cells. Total cellular protein was subjected to an immunoblotting analysis using anti-GHSR antibody and identified a band at 44 kDa. Lane 1, mouse stomach tissue as the positive control; lane 2, lysate of MC3T3-E1 cells; lane 3, lysate from MC3T3-E1 cells treated with siRNA scramble; and lane 4, lysate from MC3T3-E1 cells treated with siRNA-GHSR. β-actin was used as the loading control.
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ghrelin treatment increased Bcl-2 protein expression and decreased Bax protein expression in MC3T3-E1 cells. Fig. 4B shows the decreased activated caspase-3 activity under the treatment of ghrelin. The effects of ghrelin were dose dependent. A concentration of 10−9 mol/L of ghrelin showed the maximal anti-apoptotic effect. When the Bax/Bcl-2 ratio in control cells was set to 1, 10−11–10−9 mol/L ghrelin treatment decreased this ratio gradually (Fig. 4C).
Ghrelin protected osteoblastic MC3T3-E1 cells against serum deprivationinduced apoptosis
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Serum-deprived MC3T3-E1 cells were treated with various concentration of ghrelin to determine its effect on apoptosis. Cell Death ELISA indicated that after 72 or 48 h of starvation, apoptotic cells at 10−11 mol/L (2.79 ± 0.19 ELISA absorbance units), 10−10 mol/L (2.37 ±0.18 ELISA absorbance units), and 10−9 mol/L (1.91 ± 0.24 ELISA absorbance units) ghrelin were less than that of vehicle-treated group (3.54 ± 0.21 ELISA absorbance units), showing a maximal anti-apoptotic effect at 10−9 mol/L of ghrelin. The details are shown in Fig. 2. TUNEL analysis was used as a second metric of apoptosis in serumdeprived MC3T3-E1 cells. Our data showed that serum starvation could significantly induce MC3T3-E1 cells apoptosis (Fig. 3A) and that incubation with 10−9 mol/L ghrelin protected cells from apoptosis induced by serum deprivation (Fig. 3B). Fig. 3C shows the details about the apoptosis index of control and ghrelin-treated cells.
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Effects of ghrelin on Bcl-2 and Bax protein expressions and caspase-3 activity in osteoblastic MC3T3-E1 cells
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To further investigate the anti-apoptotic effects of ghrelin on MC3T3E1 cells, Western blot analysis was used to assess the expressions of proteins Bcl-2 and Bax and activated caspase-3 activity. Fig. 4A shows that
Recent studies have demonstrated that MAPK and PI3K/AKT signaling pathways mediate ghrelin actions (Kim et al., 2004, 2005; Liang et al., 2012). In this experiment, Western blot analysis was used to determine the effect of ghrelin on MAPK and PI3K/AKT intracellular signaling pathways. Our data demonstrated that 10−9 mol/L ghrelin induced the phosphorylation of ERK after 5 min of stimulation, and the level of phosphorylation reached a peak at 15 min (Fig. 5A). Meanwhile, treatment with ghrelin also increased phosphorylated AKT (p-AKT) levels after 5 min of incubation, and peak activation of phosphorylated AKT occurred at 15 min (Fig. 5C). Conversely, neither p38 nor JNK responds to ghrelin stimulus but did respond to the positive control hydrogen peroxide (H2O2) (Xu et al., 2011) (Fig. 5B). The phosphorylation of ERK and AKT by ghrelin was abolished by the ERK inhibitor PD98059 and PI3K inhibitor LY294002, respectively (Figs. 5D, E). Taken together, these results indicate that ghrelin activates the MAPK/ERK and PI3K/AKT signaling pathways in MC3T3-E1 cells. Fig. 5F shows that suppression of GHSR with siRNA blocked the activation of ERK and AKT. These data indicate that ghrelin stimulates the ERK and PI3K/AKT signal transduction pathways via GHSR in MC3T3E1 cells.
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Using Western blot analyses, we confirmed that GHSR proteins were expressed in MC3T3-E1 cells (Fig. 1B). GHSR expression in mouse stomach tissue was used as a positive control. Treatment with siRNA-GHSR significantly blocked the expression of GHSR protein in MC3T3-E1 cells since few bands were detected, while no blockade was observed on the treatment with the scrambled siRNA. Our results demonstrate that MC3T3-E1 cells primarily express GHSR.
Fig. 2. Effect of ghrelin on serum deprivation-induced osteoblastic MC3T3-E1 cells apoptosis detected by cell death detection ELISA. Cells were deprived of serum for 24 h, then treated with ghrelin (0, 10−11, 10−10, 10−9 mol/L) for 48 h. ELISA results showed that ghrelin suppressed serum deprivation-induced apoptosis in MC3T3-E1 cells in a dose-dependent manner. Data are shown as the mean ± SD (n = 3).
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GHSR/ERK and GHSR/AKT signaling mediated the anti-apoptotic effect of 257 ghrelin in MC3T3-E1 cells 258 Blockade of ERK, AKT phosphorylation by PD98059 or LY294002, respectively, and knockout of GHSR by siRNA were performed to confirm that GHSR, ERK, and PI3K/AKT were involved in the regulation of MC3T3-E1 cell apoptosis. We used ELISA to determine the apoptosis of MC3T3-E1 cells. Our data confirmed that the anti-apoptotic effect of ghrelin exposure was reversed by pretreatment with both PD98059 and LY294002. Suppression of GHSR with siRNA-GHSR, but not scrambled siRNA, also abolished the anti-apoptotic effect of ghrelin, demonstrating that the anti-apoptotic role of ghrelin in MC3T3-E1 cells is mediated via the GHSR/ERK and GHSR/PI3K/AKT signaling pathways. Details are shown in Fig. 6.
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Discussion
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The present study shows that GHSR expression was identified in MC3T3-E1 cells. Treatment with ghrelin inhibits serum deprivationinduced apoptosis of osteoblastic MC3T3-E1 cells, indicating an antiapoptotic role for ghrelin in MC3T3-E1 cells. We also found that
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Please cite this article as: Liang, Q.-H., et al., Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway, Toxicol. Appl. Pharmacol. (2013), http://dx.doi.org/10.1016/j.taap.2013.07.018
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GHSR/ERK and GHSR/PI3K/AKT signaling pathways mediated the protective effect of ghrelin against serum deprivation-induced apoptosis in osteoblastic MC3T3-E1 cells. Apoptosis is defined as the process of cell death associated with caspase activation or caspase-mediated cell death. It is a necessary component of development and a characteristic of all self-renewing tissues, including bone (Jilka et al., 2007). Osteoblast apoptosis plays a critical role during embryonic limb development, skeletal maturation, and adult bone turnover by modeling and remodeling processes during fracture healing and bone regeneration and is an actively controlled process that is affected once pro-apoptotic signals exceed anti-apoptotic signals (Hock et al., 2001). It is now postulated that all major regulators of bone metabolism including bone morphogenetic proteins (BMPs), Wnts, other growth factors and cytokines, integrins, estrogens, androgens, glucocorticoids, PTH and PTH-related protein (PTHrP), immobilization, and the oxidative stress associated with aging contribute to the regulation of osteoblasts apoptosis (Xie et al., 2007). Our previous studies found that apelin and vaspin inhibit apoptosis of human osteoblasts, and L-Carnitine inhibits apoptosis of murine MC3T3-E1 osteoblastic cells (Baldanzi et al., 2002; Xie et al., 2008; Zhu et al., 2012).
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Fig. 3. Effect of ghrelin on serum deprivation-induced osteoblastic MC3T3-E1 cell apoptosis detected by TUNEL analysis. Cells were deprived of serum for 24 h, then treated with the absence or presence of 10−9 mol/L ghrelin for 48 h. Representative images of TUNEL stained cells are shown for the control group (A) and cells were treated with 10−9 mol/L ghrelin for 48 h (B). Apoptotic cells are indicated by yellow arrows. Representative figures are shown. The apoptosis index for control and ghrelin-treated cells is shown (C). Original magnification of all images, ×400. Data are shown as the mean ± SD (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Ghrelin is a newly discovered bioactive peptide that owns multiple physiological effects (Fukushima et al., 2005; Gaytan et al., 2004; Liang et al., 2012; Shuto et al., 2001). Previous studies have determined the inhibitory effects of ghrelin on cell apoptosis. It has been reported that ghrelin inhibits apoptosis in several cells, such as cardiomyocytes, endothelial cells, adipocytes, adrenal zona glomerulosa cells, pancreatic βcells, intestinal epithelial cells, and hypothalamic neurons (Chung et al., 2007; Granata et al., 2007; Kim et al., 2004, 2006; Kui et al., 2009; Mazzocchi et al., 2004; Rodriguez et al., 2012). In our experiment, we chose Cell Death Detection ELISA kit and TUNEL, which are classic methods to evaluate apoptosis through visualizing nuclei containing fragmented DNA by labeling the exposed termini of DNA and to detect the apoptosis of MC3T3-E1 cells (Zhu et al., 2012). Our data demonstrated that 10−11–10−9 mol/L ghrelin could inhibit serum deprivationinduced apoptosis in osteoblastic MC3T3-E1 cells in a dose-dependent manner. Bcl-2 proteins are a family of cytosolic proteins involved in the apoptotic pathways, which are made up of anti-apoptotic proteins (e.g., Bcl-2, Bcl-XL) and pro-apoptotic proteins (e.g., Bax, Bak) (Gu et al., 2013). When death signals overwhelm survival signals, the actions of anti-apoptotic Bcl-2 proteins are abrogated (Xie et al., 2007).
Please cite this article as: Liang, Q.-H., et al., Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway, Toxicol. Appl. Pharmacol. (2013), http://dx.doi.org/10.1016/j.taap.2013.07.018
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Fig. 4. Effect of ghrelin on Bax and Bcl-2 protein expressions and caspase-3 activity in osteoblastic MC3T3-E1 cells. Cells were deprived of serum for 24 h, then treated with 10−11, 10−10, and 10−9 mol/L ghrelin for 48 h. Representative Western blots are shown for Bax, Bcl-2, activated caspase-3, and β-actin. The expressions of Bax and Bcl-2 proteins and activated caspase-3 activity were determined by Western blot (A, B), and the Western blot data were densitometrically quantified and normalized to inner standard (C) (n = 3).
Fig. 5. Involvement of MAPK and PI3K/AKT phosphorylation in ghrelin-stimulated osteoblastic MC3T3-E1 cells. (A and B) Cells were exposed to 10−9 mol/L ghrelin for 0–60 min, or 50 μM hydrogen peroxide (H2O2) for 15 min as a positive control for p38 and JNK activation. Cell lysates were subjected to Western blotting and incubated with antibodies p-ERK, -ERK, p-p38, p38, p-JNK, JNK, p-AKT, and AKT. (C) Cells were exposed to 10−9 mol/L ghrelin for 0–60 min to assess AKT activation. (D) Cells were pretreated with 10 μM PD98059 for 3 h before exposure to 10−9 mol/L ghrelin for 15 min. (E) Cells were pretreated with 10 μM LY294002 for 3 h before exposure to 10−9 mol/L ghrelin for 15 min. The representative results are shown. (F) Cells were treated with scrambled GHSR siRNA, or GHSR-siRNA in the presence of 10−9 mol/L ghrelin. Representative results are shown.
Please cite this article as: Liang, Q.-H., et al., Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway, Toxicol. Appl. Pharmacol. (2013), http://dx.doi.org/10.1016/j.taap.2013.07.018
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PI3K/AKT signaling pathways. They suggest that ghrelin/GHSR may be 356 involved in bone metabolism through regulation of osteoblast apoptosis. 357 358
Conflict of interest statement The authors declare that there are no conflicts of interest.
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This work was supported by funding from The National Natural Science Foundation of China (Grant nos. 81270962, 81070246, 30801174) and the National Science Foundation for Post-doctoral Scientists of China (Grant no. 2012M521568).
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References
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In 2005, Kim et al. once reported the protective effect of ghrelin on TNFα-induced MC3T3-E1 osteoblastic cell apoptosis (Kim et al., 2005). However, the mechanisms were still unclear. We found that ghrelin up-regulated the expression of Bcl-2 and down-regulated the expression of Bax in MC3T3-E1 cells, indicating that the Bcl-2 family is involved in modulating the apoptosis of MC3T3-E1 cells by ghrelin. Furthermore, our data demonstrated that ghrelin blocked the activity of caspase-3, which is a hallmark of apoptosis that has been previously reported (Chua et al., 2003; Xie et al., 2010). To gain further insight into the mechanism of the capacity of ghrelin to inhibit MC3T3-E1 cell apoptosis, we investigated several intracellular signaling pathways. The MAPKs, including ERK, JNK and p38, are serine/ threonine kinase pathways. PI3K/AKT pathway, which can be activated by growth factors and some extracellular signals, regulates fundamental cellular processes including cell proliferation, differentiation, and survival (Cantrell, 2001). Our previous studies have demonstrated the involvement of many of these signaling pathways in the regulation of osteoblast apoptosis (Xie et al., 2008; Zhu et al., 2012). Kim et al. reported that ghrelin promotes the proliferation and differentiation, and inhibited TNF-α-induced apoptosis of MC3T3-E1 osteoblastic cells through ERK signaling pathway. But the PI3K/AKT signaling pathway was not detected (Kim et al., 2005). Recently, we found that ghrelin attenuated the osteoblastic differentiation of vascular smooth muscle cells through GHSR/ERK pathway (Liang et al., 2012). In this study, we tested the activation of both MAPK and PI3K/AKT. Also, RT-PCR and Western blot were adopted to determine the expression of GHSR in MC3T3-E1 cells. Our data showed that ghrelin activated both ERK and PI3K/AKT in MC3T3-E1 cells; however, neither p38 nor JNK was activated under the stimulus of ghrelin. Pretreatment with the ERK inhibitor PD98059 and PI3K inhibitor LY294002 reversed the activation of ERK and AKT in MC3T3-E1 cells, respectively. Furthermore, the suppression of GHSR with siRNA blocked the effects of ghrelin on ERK and AKT, which suggests that the activation of ERK and PI3K/AKT is mediated through GHSR. Both blockade of ERK and AKT and inhibition of GHSR abolish the protective effects of ghrelin on serum deprivation-induced MC3T3-E1 cell apoptosis, indicating that ghrelin suppresses MC3T3-E1 cell apoptosis by activating the GHSR/ERK and GHSR/PI3K/AKT signaling pathways. In conclusion, our study demonstrates that GHSR is expressed in MC3T3-E1 cells and ghrelin protects MC3T3-E1 cells from serum deprivation-induced apoptosis by activating the GHSR/ERK and GHSR/
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Fig. 6. The GHSR/ERK and GHSR/PI3K/AKT signaling pathways mediate the effect of ghrelin to inhibit serum deprivation-induced apoptosis of osteoblastic MC3T3-E1 cells. Cells were pretreated with 10 μM PD98059 or 10 μM LY294002, respectively, for 3 h before exposure to 10−9 mol/L ghrelin for 48 h. Cells were also treated with the siRNA scramble or GHSR-siRNA in the presence of 10−9 mol/L ghrelin for 48 h. MC3T3-E1 cell apoptosis was analyzed by ELISA. Data are shown as the mean ± SD (n = 3).
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Please cite this article as: Liang, Q.-H., et al., Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway, Toxicol. Appl. Pharmacol. (2013), http://dx.doi.org/10.1016/j.taap.2013.07.018
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