Ghrelin regulates cell cycle-related gene expression in cultured hippocampal neural stem cells

in Journal of Endocrinology

We have previously demonstrated that ghrelin stimulates the cellular proliferation of cultured adult rat hippocampal neural stem cells (NSCs). However, little is known about the molecular mechanisms by which ghrelin regulates cell cycle progression. The purpose of this study was to investigate the potential effects of ghrelin on cell cycle regulatory molecules in cultured hippocampal NSCs. Ghrelin treatment increased proliferation assessed by CCK-8 proliferation assay. The expression levels of proliferating cell nuclear antigen and cell division control 2, well-known cell-proliferating markers, were also increased by ghrelin. Fluorescence-activated cell sorting analysis revealed that ghrelin promoted progression of cell cycle from G0/G1 to S phase, whereas this progression was attenuated by the pretreatment with specific inhibitors of MEK/extracellular signal-regulated kinase 1/2, phosphoinositide 3-kinase/Akt, mammalian target of rapamycin, and janus kinase 2/signal transducer and activator of transcription 3. Ghrelin-induced proliferative effect was associated with increased expression of E2F1 transcription factor in the nucleus, as determined by Western blotting and immunofluorescence. We also found that ghrelin caused an increase in protein levels of positive regulators of cell cycle, such as cyclin A and cyclin-dependent kinase (CDK) 2. Moreover, p27KIP1 and p57KIP2 protein levels were reduced when cell were exposed to ghrelin, suggesting downregulation of CDK inhibitors may contribute to proliferative effect of ghrelin. Our data suggest that ghrelin targets both cell cycle positive and negative regulators to stimulate proliferation of cultured hippocampal NSCs.

Abstract

We have previously demonstrated that ghrelin stimulates the cellular proliferation of cultured adult rat hippocampal neural stem cells (NSCs). However, little is known about the molecular mechanisms by which ghrelin regulates cell cycle progression. The purpose of this study was to investigate the potential effects of ghrelin on cell cycle regulatory molecules in cultured hippocampal NSCs. Ghrelin treatment increased proliferation assessed by CCK-8 proliferation assay. The expression levels of proliferating cell nuclear antigen and cell division control 2, well-known cell-proliferating markers, were also increased by ghrelin. Fluorescence-activated cell sorting analysis revealed that ghrelin promoted progression of cell cycle from G0/G1 to S phase, whereas this progression was attenuated by the pretreatment with specific inhibitors of MEK/extracellular signal-regulated kinase 1/2, phosphoinositide 3-kinase/Akt, mammalian target of rapamycin, and janus kinase 2/signal transducer and activator of transcription 3. Ghrelin-induced proliferative effect was associated with increased expression of E2F1 transcription factor in the nucleus, as determined by Western blotting and immunofluorescence. We also found that ghrelin caused an increase in protein levels of positive regulators of cell cycle, such as cyclin A and cyclin-dependent kinase (CDK) 2. Moreover, p27KIP1 and p57KIP2 protein levels were reduced when cell were exposed to ghrelin, suggesting downregulation of CDK inhibitors may contribute to proliferative effect of ghrelin. Our data suggest that ghrelin targets both cell cycle positive and negative regulators to stimulate proliferation of cultured hippocampal NSCs.

Introduction

Ghrelin is a unique 28-amino acid peptide hormone mainly produced in the stomach. The peptide is known to act through the activation of the growth hormone (GH) secretagogue receptor 1a (Kojima et al. 1999). Earlier studies revealed that ghrelin exerts a stimulatory influence on GH release primarily at the levels of anterior pituitary and hypothalamus and enhances food intake to regulate body weight and energy homeostasis (Date et al. 2000). Ghrelin also has wide variety of physiological actions throughout the body, including effects on exocrine and endocrine pancreatic functions, carbohydrate metabolism, the cardiovascular system, gastric secretion, stomach motility and sleep (Van der Lely et al. 2004, Ghigo et al. 2005, Kojima & Kangawa 2005). In the central nervous system (CNS), ghrelin shows various nonendocrine effects to control different brain functions, such as enhancement of learning and memory (Diano et al. 2006), attenuation of anxiety and depression (Carlini et al. 2004, Lutter et al. 2008), modulation of reward and motivation (Naleid et al. 2005, Abizaid et al. 2006, Jiang et al. 2006), and protection of neuronal cells (Chung et al. 2007, 2008, Miao et al. 2007, Jiang et al. 2008, Hwang et al. 2009, Moon et al. 2009a, Lee et al. 2010a,b).

Neurogenesis is known to persist throughout adulthood in several mammalian species in the hippocampus, especially the subgranular zone of the dentate gyrus (DG) and in the subventricular zone of the lateral ventricle (Ming & Song 2005, Zhao et al. 2008). Hippocampus plays an important role in the process of learning, memory and emotional responses (Zhao et al. 2008); hence, a lot of attention has been paid on hippocampal neurogenesis. Exact understanding of the mechanisms involved in the adult hippocampal neurogenesis may augment the development of new therapy using stem-cell-based strategies against neurodegenerative diseases. We have previously reported that ghrelin regulates adult hippocampal neurogenesis in vivo and in vitro. Specifically, ghrelin administration enhances hippocampal neurogenesis in adult mice (Moon et al. 2009b). Our studies using ghrelin-knockout mice demonstrated that deletion of ghrelin resulted in lower numbers of progenitor cells in the DG, whereas ghrelin treatment restored progenitor cell numbers to those of wild-type controls (Li et al. 2013). Furthermore, we also reported that ghrelin induces cellular proliferation of cultured adult rat hippocampal neural stem cells (NSCs) via the activation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), phosphoinositide 3-kinase (PI3K)/Akt/glycogen synthase kinase (GSK)-3β, PI3K/Akt/mammalian target of rapamycin (mTOR)/p70S6K and janus kinase (JAK) 2/signal transducer, and activator of transcription (STAT) 3 signaling pathways (Chung et al. 2013). Collectively, these data suggest that ghrelin acts as a neurogenic agent. However, the precise mechanism of this stimulatory effect of ghrelin on hippocampal NSCs proliferation has not been fully understood.

Several cell cycle molecules and signaling pathways have been implicated in the regulation of adult hippocampal neurogenesis (Patricio et al. 2013). The progression of cell cycle is regulated by the activities of two important cell cycle regulators, cyclins and cyclin-dependent kinases (CDKs) (Beukelaers et al. 2012). During the G1 phase, cyclin D-CDK4/6 complexes are activated. Cyclin E and A, together with their catalytic enzyme, CDK2, are necessary for the advancement of the cell cycle from the G1 into S phase and progression through S phase, respectively. Cyclin B-CDK1 complexes contribute to M phase entry. The transcription factor E2F1 is involved in the regulation of cell cycle regulation (Yoshikawa 2000). Additionally, the activity of cyclin–CDK complex can be regulated by the inhibitory interaction with CDK inhibitors (CKIs), such as p21CIP1, p27KIP1 and p57KIP2 (Vidal & Koff 2000). Therefore, the balance between the levels of promitogenic cyclins and antimitogenic CKIs is thought to be important for the progression into S phase. Mitogenic signals, such as insulin-like growth factor-1 (IGF-1) and basic fibroblast growth factor (bFGF), are known to stimulate proliferation of cortical precursors through upregulation of cyclins and downregulation of CKIs (Li & Cicco-Bloom 2004, Mairet-Coello et al. 2009). In contrast, pituitary adenylate cyclase-activating polypeptide exerts antimitogenic effect via augmenting expression of p57KIP2 (Carey et al. 2002). Moreover, it has been reported that ghrelin promotes cell cycle progression in intestinal epithelial cells (Waseem et al. 2014) and augments the expression of positive regulators of cell cycle progression in murine T-cells (Lee et al. 2014). Based on these findings, we hypothesized that the proliferative actions of ghrelin on hippocampal NSCs may be related to control of cell cycle molecules. At present, no data are available on the effects of ghrelin on cell cycle regulators in hippocampal NSCs. Therefore, in this study, we performed a series of experiments in order to pursue further the biological effects of ghrelin in the CNS and determine the neurogenic mechanism of action of ghrelin in adult rat hippocampal NSCs. We found that ghrelin-mediated proliferation was associated with the induction of nuclear E2F1 and regulation of other cell cycle-related genes.

Materials and methods

Materials

Rat ghrelin was obtained from Peptides International (Louisville, KT, USA). NSC expansion media, Dulbecco’s modified Eagle’s medium (DMEM)/F12 and B27 supplement were obtained from Gibco/Invitrogen. B-27 is an optimized serum substitute developed for low-density plating and long-term viability and growth of CNS neurons (Brewer et al. 1993). PD98059, U0126, LY294002, rapamycin and cucurbitacin I were obtained from Tocris (Ellisville, MO, USA) and Akt inhibitor VIII was procured from Santa Cruz Biotechnology. All tissue culture reagents were obtained from Gibco/Invitrogen, and all other reagents were purchased from Sigma, unless otherwise indicated.

Adult rat hippocampal NSC cultures and treatments

Adult rat hippocampal NSCs were obtained from Chemicon (Catalog No. SCR022). These cells are ready-to-use primary NSCs isolated from the hippocampus of adult Fisher 344 rats. They were grown in an NSC expansion medium containing DMEM/F12 medium with l-glutamine, B27 supplement, 1× solution of penicillin, streptomycin and fungizone, and bFGF (20ng/mL). Tissue culture plastic- or glass wares that were used to culture hippocampal NSCs were coated with poly-l-ornithine (10µg/mL) and laminin (5µg/mL). The hippocampal NSCs were maintained at 37°C in a 5% CO2 humidified incubator and passaged once every 5–6days. To determine whether ghrelin stimulates the proliferation of hippocampal NSCs, cells were treated with ghrelin (100nM) for 24, 48, and 72h. This dose of ghrelin (100nM) was selected on the basis of our previous report showing that it increased the proliferation of hippocampal NSCs and it was the lowest dose with the maximum response (Chung et al. 2013). Experiments were also performed by incubating the cells with the following pharmacological inhibitors: 20µM PD98059 for 0.5h, 10µM U0126 for 0.5h, 20µM LY294002 for 1h, 100nM Akt inhibitor VIII for 1h, 200nM rapamycin for 1h or 1nM cucurbitacin I for 0.5h. All experiments were performed three times in duplicate.

Evaluation of cell proliferation using CCK-8 proliferation assay

Experiments for cell proliferation were performed in 24-well plates. Cells were seeded at a density of 1×105cells/mL of neuronal expansion media containing bFGF. After 24h, the media were replaced with fresh media with ghrelin (100nM) or a vehicle and incubated for 24, 48 and 72h. The cell proliferation was determined using the CCK-8 proliferation assay (Enzo Life Sciences, Lausen, Switzerland) according to the manufacturer’s instructions. In brief, 20µL CCK-8 solution were added into each well (containing 200µL medium), and further cultured for 2h at 37°C. The absorbance of each group at 450nm was detected (n=3) using an absorbance microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA) and it was directly proportional to the number of living cells.

Fluorescence-activated cell sorting (FACS) analysis

Cell cycle distribution was examined by FACS analysis. 1×106 cells were collected and fixed with 3.7% paraformaldehyde. After fixation, the propidium iodide (50µg/mL)-staining solution with RNase A (BD Biosciences, San Jose, CA, USA) was added. After 30-min incubation, the cells were filtered on a nylon mesh filter. Stained samples were analyzed by flow cytometric analysis using FACSCalibur (BD Biosciences, San Jose, CA, USA).

Real-time RT-PCR

Total RNA from hippocampal NSCs was extracted using the Qiagen RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions and was reverse transcribed using the Superscript II reverse transcriptase (Life Technologies) at 42°C with random hexamer priming. An RNA control tube containing all reverse transcription reagents except reverse transcriptase was included as a negative control to monitor genomic DNA contamination. Real-time RT-PCR was used to determine mRNA levels of proliferating cell nuclear antigen (PCNA) and cell division control (CDC) 2 in hippocampal NSCs using primers specific for PCNA and CDC2 (The Rat Cell Cycle RT2 Profiler PCR Array, Qiagen), and β-actin (sense: 5′-ATG GGT CAG AAG GAC TCC TAC G-3′ and antisense: 5′-AGT GGT ACG ACC AGA GGC ATA C-3′). Reactions were performed using the StepOnePlus Real-Time PCR System (Applied Biosystems).

Western blotting

Cells were lysed in a buffer containing 20mM Tris–HCl (pH 7.4), 1mM EDTA, 140mM NaCl, 1% (w/v) Nonidet P-40, 1mM Na3VO4, 1mM phenylmethylsulfonyl fluoride, 50mM NaF and 10µg/mL aprotinin. Cell lysates were separated by 12% SDS–PAGE and electrotransferred onto polyvinylidene difluoride membranes (Bio-Rad). For the detection of E2F1, cells were fractionated into nuclear and cytoplasmic contents using the Nuclear/Cytoplasmic Extraction Kit (Pierce Biotechnology) according to the manufacturer’s instructions. The membranes were soaked in a blocking buffer (1× Tris-buffered saline (TBS), 1% bovine serum albumin (BSA) and 1% nonfat dry milk) for 1h and incubated overnight at 4°C with the primary antibodies against PCNA, CDC2, Laminin A, GAPDH (Cell Signaling; 1:1000), and CDK2, cyclin A, p27KIP1 and p57KIP2, E2F1 (Santa Cruz Biotechnology; 1:1000). Blots were developed using a peroxidase-conjugated antirabbit IgG and a chemiluminescent detection system (Santa Cruz Biotechnology). The bands were visualized using a ChemiDoc XRS system (Bio-Rad) and quantified using Quantity One imaging software (Bio-Rad).

Immunocytochemistry

Adult rat hippocampal NSCs were fixed in 4% paraformaldehyde for 20min at room temperature. Cells were washed twice with phosphate-buffered saline (PBS) and permeabilized with PBS containing 0.4% Triton X-100 for 20min, followed by blocking with TBS containing 0.02% Tween 20 and 10% normal goat serum for 1h at room temperature. Primary antibody incubations were performed at 4°C overnight in TBS containing 0.02% Tween 20 and 3% BSA. Cells were incubated in a blocking solution (0.3% Triton X-100, 1% BSA and 3% normal goat serum in PBS) for 2h. After overnight incubation with primary antibody against E2F1 (1:400; Santa Cruz Biotechnology) at 4°C, cells were rinsed with PBS, followed by incubation with a secondary antibody (Alexa Fluor 488 goat antirabbit IgG, 1:400, Invitrogen) for 4h at room temperature. Images were acquired by the Carl Zeiss LSM 700 Meta confocal microscope and analyzed using Carl Zeiss ZEN image software.

Statistical analysis

Data are presented as mean±s.e.m. of three different experiments (each experiment was performed in duplicate). Statistical analysis between groups was performed using Student’s t-test for the comparison of two means or one-way ANOVA and the Holm–Sidak method for multiple comparisons using SigmaStat for Windows Version 3.10 (Systat Software, Inc, Point Richmond, CA, USA). P<0.05 was considered statistically significant.

Results

Ghrelin stimulates proliferation of adult rat hippocampal NSCs

Based on our previous results demonstrating that ghrelin increases BrdU incorporation of cultured adult rat hippocampal NSCs (Chung et al. 2013), CCK-8 proliferation assay was used to confirm the effect of ghrelin on cell proliferative ability. As shown in Fig. 1A, ghrelin (100nM) significantly increased cell proliferation in a time-dependent manner. Next, we further analyzed well-defined cell-proliferating markers, PCNA and CDC2, by Western blotting. After 24-h treatment with ghrelin (100nM), protein levels of PCNA and CDC2 were significantly increased by 126.3±7.9 and 139.0±7.8%, respectively (P<0.05 vs vehicle-treated control), as shown in Fig. 1B. These results indicate that ghrelin increased the expression of well-known cell cycle-related genes, PCNA and CDC2, which are essential for progression through mitosis. Ghrelin also promoted the levels of mRNA for PCNA and CDC2 assessed by real-time RT-PCR (Fig. 1C).

Figure 1
Figure 1

Ghrelin stimulates proliferation of adult rat hippocampal NSCs. (A) Cells were treated with ghrelin (100nM) for 24, 48 and 72h, and the cell proliferation was assayed using the CCK-8 proliferation assay. (B) Cells were treated with ghrelin (100nM) for 24h. Protein lysates were prepared and assayed by Western blotting using specific anti-PCNA and anti-CDC2 antibodies. The band intensities of PCNA and CDC2 were normalized to the band intensity of β-actin. (C) Time course of ghrelin-induced effects on PCNA and CDC2 mRNA levels. Cells were treated with ghrelin (100nM) for 4, 8, 12 and 24h. Real-time RT-PCR was performed to measure mRNA levels of PCNA and CDC2 and adjusted by β-actin. The data are expressed as percentage of vehicle and are shown as the mean±s.e.m. of three different experiments (each experiment was performed in duplicate). *P<0.05 vs vehicle-treated control.

Citation: Journal of Endocrinology 230, 2; 10.1530/JOE-16-0126

Multiple pathways are involved in ghrelin-induced cell cycle progression of adult rat hippocampal NSCs

Our previous data indicated that pretreatment of cells with specific inhibitors of MEK/ERK1/2, PI3K/Akt, mTOR, and JAK2/STAT3 attenuated ghrelin-induced hippocampal NSCs proliferation (Chung et al. 2013). To further investigate ghrelin-induced cell proliferation, we analyzed the effect of ghrelin on cell cycle entry using FACS analysis of distribution of cells in each phase of the cell cycle. Following growth factor deprivation for 16h, cells were treated with vehicle or ghrelin (100nM) for 8h. In vehicle-treated cells, 84.4±0.2% of cells were in G0/G1, 6.0±0.6% were in S and 9.7±0.6% were in G2/M phase (Fig. 2A). When cells were incubated with ghrelin for 8h, the G0/G1 population was decreased to 80.7±0.5%, the S phase population was increased to 10.9±0.8% and the G2/M population was decreased to 8.4±0.7% (Fig. 2A). These results indicated that hippocampal NSCs entered the S phase after 8-h treatment with ghrelin. However, when cells were exposed to MEK1 inhibitor PD98059, MEK1/2 inhibitor U0126, PI3K inhibitor LY294002, Akt inhibitor VIII, mTOR inhibitor rapamycin or JAK2/STAT3 inhibitor cucurbitacin I, ghrelin-induced increase in S phase ratio was significantly attenuated (Fig. 2B).

Figure 2
Figure 2

Fluorescence-activated cell sorting (FACS) analysis of cell cycle distribution. (A) Following 16-h starvation and pretreatment with 20µM PD98059 for 0.5h, 10µM U0126 for 0.5h, 20µM LY294002 for 1h, 100nM Akt inhibitor VIII for 1h, 200nM rapamycin for 1h or 1nM cucurbitacin I for 0.5h, cells were treated with ghrelin (100nM) for 8h. The cells were harvested by propidium iodide (50µg/mL)-staining solution and subjected to FACS analysis. Results were expressed as percentage of total cells in G0/G1, S or G2/M phase of the cell cycle. Values represent the mean±s.e.m. of three different experiments (each experiment was performed in duplicate). (B) The bar graphs represent the S phase ratio expressed as the percentage of vehicle. *P<0.05 vs vehicle-treated cells, P<0.05 vs ghrelin-treated cells.

Citation: Journal of Endocrinology 230, 2; 10.1530/JOE-16-0126

Ghrelin increases nuclear expression of transcription factor E2F1 in adult rat hippocampal NSCs

It has been demonstrated that the transcription factor E2F1 is involved in controlling proliferation and neuronal cell numbers in the postnatal and adult brain (Cooper-Kuhn et al. 2002). To identify changes in subcellular localization of E2F1, Western blotting was conducted to detect E2F1 in nuclear and cytoplasmic fractions. Figure 3 shows ghrelin-induced increase in E2F1 protein both in the nuclear (Fig. 3A) and cytoplasmic (Fig. 3B) fractions of cells. We also calculated the nuclear/cytoplasmic E2F1 ratio and found that ghrelin significantly increased the ratio after 8-h treatment (Fig. 3C), suggesting increased expression of E2F1 in the nucleus. These results were confirmed by immunofluorescence confocal microscopy, which showed an increased staining pattern when cells were treated with ghrelin (Fig. 4).

Figure 3
Figure 3

Ghrelin increases the nuclear expression of transcription factor E2F1. Cells were treated with ghrelin (100nM) for 4, 8, 12 and 24h. Nuclear and cytoplasmic lysates were prepared and assayed by Western blotting using specific anti-E2F1, anti-laminin A and anti-GAPDH antibodies. (A) The nuclear E2F1 band intensity was normalized to the laminin A band intensity. (B) The cytoplasmic E2F1 band intensity was normalized to the GAPDH band intensity. Representative Western blotting images are shown in the upper insets. The data are expressed as the mean±s.e.m. of three different experiments (each experiment was performed in duplicate). (C) The bar graphs represent the nuclear/cytoplasmic E2F1 ratio expressed as the percentage of control. *P<0.05 vs control.

Citation: Journal of Endocrinology 230, 2; 10.1530/JOE-16-0126

Figure 4
Figure 4

Confocal microscopic analysis of E2F1 expression. Cells were treated with ghrelin (100nM) for 4, 8, 12 and 24h. Cells were incubated with primary antibody to E2F1. Cells were counterstained with DAPI, and images were captured using confocal microscopy; scale bar, 5µm.

Citation: Journal of Endocrinology 230, 2; 10.1530/JOE-16-0126

Ghrelin increases the expression of cyclin A and CDK2 in adult rat hippocampal NSCs

To determine the mechanisms involved in ghrelin-induced proliferation of hippocampal NSCs, we examined the expression of cyclin A, which is accumulated during S phase (Yam et al. 2002). Regulation of expression of cyclin A is mediated by the transcription factor E2F1 (Schulze et al. 1995). As shown in Fig. 5A, ghrelin increased cyclin A protein levels in a time-dependent manner, with a significant rise after 8-h treatment. Similar increase in protein levels of CDK2 was also observed after 4-h ghrelin treatment (Fig. 5B). We further determined which signaling pathways are involved in ghrelin-induced increase in cyclin A and CDK2 expression. We found that pretreatment of cells with PD98059, LY294002, rapamycin or cucurbitacin I significantly attenuated ghrelin-mediated enhancement of cyclin A and CDK2 protein levels (Fig. 5C and D).

Figure 5
Figure 5

Ghrelin increases the expression of cyclin A and CDK2. (A and B) Cells were treated with ghrelin (100nM) for 4, 8, 12 and 24h. (C and D) Cells were preincubated with 20µM PD98059 for 0.5h, 20µM LY294002 for 1h, 200nM rapamycin for 1h or 1nM cucurbitacin I for 0.5h and then treated with ghrelin (100nM) for 8h. Lysates were prepared and assayed by Western blotting using specific anti-cyclin A, anti-CDK2 and anti-β-actin antibodies. Cyclin A (A and C) and CDK2 (B and D) band intensities were normalized to the β-actin band intensity and expressed as relative band intensity. Representative Western blotting images are shown in the upper insets. The data are expressed as the mean±s.e.m. of three different experiments (each experiment was performed in duplicate). *P<0.05 vs control; P<0.05 vs ghrelin-treated cells.

Citation: Journal of Endocrinology 230, 2; 10.1530/JOE-16-0126

Ghrelin decreases the expression of p27KIP1 and p57KIP2 in adult rat hippocampal NSCs

We also examined the expression of two negative regulators of cell cycle, p27KIP1 and p57KIP2. We first analyzed the expression of p27KIP1, because it is likely an important CKI that induces cell cycle exit in proliferative cells (Cobrinik 2005). As shown in Fig. 6A, ghrelin treatment significantly decreased p27KIP1 protein levels by 65% at 4h, 70% at 8h and 58% at 12h. The protein levels of p27KIP1 remained reduced even 24h after ghrelin treatment, exhibiting a 47% decrease. We also examined the effect of ghrelin on the expression of p57KIP2. Ghrelin elicited a significant decrease in p57KIP2 protein levels (from 75 to 62%) at 4, 8, 12 and 24h (Fig. 6B). Next, the pathways mediating the ghrelin-induced decrease in p27KIP1 and p57KIP2 expression were examined by exposure of cells to PD98059, LY294002, rapamycin or cucurbitacin I. As shown in Fig. 6C and D, these inhibitors attenuated the decrease in p27KIP1 and p57KIP2 protein levels of cells subjected to ghrelin treatment.

Figure 6
Figure 6

Ghrelin decreases the expression of p27KIP1 and p57KIP2. (A and B) Cells were treated with ghrelin (100nM) for 4, 8, 12 and 24h. (C and D) Cells were preincubated with 20µM PD98059 for 0.5h, 20µM LY294002 for 1h, 200nM rapamycin for 1h or 1nM cucurbitacin I for 0.5h and then treated with ghrelin (100nM) for 8h. Lysates were prepared and assayed by Western blotting using specific anti-p27KIP1, anti-p57KIP2 and anti-β-actin antibodies. p27KIP1 (A and C) and p57KIP2 (B and D) band intensities were normalized to the β-actin band intensity and expressed as relative band intensity. Representative Western blotting images are shown in the upper insets. The data are expressed as the mean±s.e.m. of three different experiments (each experiment was performed in duplicate). *P<0.05 vs control; P<0.05 vs ghrelin-treated cells.

Citation: Journal of Endocrinology 230, 2; 10.1530/JOE-16-0126

Discussion

In the current study, our data indicate that ghrelin-induced increase in nuclear expression of E2F1 is associated with ghrelin-mediated promotion of cell cycle progression in cultured adult rat hippocampal NSCs. The proliferative effect of ghrelin appears to be mediated by increased expression of genes that promote progression through the cell cycle, such as cyclin A and CDK2, while inhibiting expression of genes that trigger cell cycle exit, such as p27KIP1 and p57KIP2. Ghrelin-induced cell cycle progression from G0/G1 to S phase was dependent on the activities of MEK/ERK1/2, PI3K/Akt/mTOR and JAK2/STAT3 signaling pathways.

In previous paper, we found that ghrelin increased the proliferation of cultured hippocampal NSCs assessed by BrdU incorporation, suggesting mitogenic potential of ghrelin (Chung et al. 2013). In this study, the proliferative effect of ghrelin has been confirmed by CCK-8 proliferation assay and enhancement of expression of PCNA and CDC2, well-defined and commonly used cell proliferation markers (Castedo et al. 2002, Moldovan et al. 2007). PCNA, a marker for G1 and S phase, acts as a homotrimer and helps increase the processivity of leading strand synthesis during DNA replication (Moldovan et al. 2007). CDC2, also called cyclin-dependent kinase 1, has to be active to allow cells to progress from the G2 to the M phase (Castedo et al. 2002). In the current study, 24-h incubation of cells with ghrelin significantly increased the expression of PCNA and CDC2, suggesting that ghrelin augmented entry into and progression of hippocampal NSCs through the cell cycle.

To investigate the ability of ghrelin to cause hippocampal NSCs entry into the cell cycle, we performed FACS analysis of cell distribution in each phase of the cell cycle. We found that ghrelin increased the number of cells in S phase with a decrease in number of cells in G0/G1 phase, indicating ghrelin-mediated promotion of entry into S phase. Multiple signaling pathways and molecules are involved in regulation of the proliferation of neural progenitor cells. For example, a dominant negative Akt inhibits cell proliferation (Peltier et al. 2007) and the activation of ERK is important for the proliferation of neural progenitor cells (Learish et al. 2000, Zhou et al. 2004). Inhibitor of GSK-3β, a downstream effector of Akt, promotes the proliferation of progenitor cells, while β-catenin overexpression increases the proliferation (Adachi et al. 2007). The activation of mTOR/p70S6K signaling has been reported to increase the proliferation of NSCs in vitro (Ryu et al. 2003). In addition, STAT3 inhibition blocked leptin-induced proliferation of adult hippocampal neural progenitor cells (Garza et al. 2008). These findings were supported by our previous report, where we showed that ghrelin increased the phosphorylation of ERK1/2, Akt, STAT3, GSK-3β, mTOR and p70S6K, and chemical inhibition of MEK/ERK1/2, PI3K/Akt, mTOR and STAT3 reversed ghrelin-induced increase in the proliferation of hippocampal NSCs (Chung et al. 2013). In agreement with these observations, we found that ghrelin-induced progression into S phase in hippocampal NSCs was attenuated when cells were pretreated with specific inhibitors of MEK/ERK1/2, PI3K/Akt, mTOR and JAK2/STAT3. Taken together, our previous and current results suggest that multiple signaling pathways, such as MEK/ERK1/2, PI3K/Akt/GSK-3β, PI3K/Akt/mTOR/p70S6K and JAK2/STAT3, are involved in ghrelin-induced cell cycle progression and proliferation of hippocampal NSCs.

In this study, we have shown that ghrelin increases the nuclear expression of E2F1, which is requisite for enabling E2F1 to reach target DNA sequences (Muller & Helin 2000). Considering that E2F1 expression is essential in adult neurogenesis (Cooper-Kuhn et al. 2002), the increase in expression of nuclear E2F1 may contribute to ghrelin-mediated proliferation of hippocampal NSCs. The E2F1 transcription factor is known to bind exclusively to retinoblastoma (Rb) protein in a cell cycle-dependent manner (Yoshikawa 2000). During the G1 phase, the cyclin D/CDK4-6 complex phosphorylates the Rb protein, resulting in sequential phosphorylation by cyclin E/CDK2 and the release of free E2F1 (Abrous et al. 2005). CDK2 then combines with cyclin A to maintain the cells in S phase (Beukelaers et al. 2012). Indeed, cyclin A, a marker of S phase, was elevated by ghrelin and this increase was paralleled by enhanced protein levels of CDK2. Moreover, exposure of cells to PD98059, LY294002, rapamycin or cucurbitacin I resulted in significant attenuation of the stimulatory effect of ghrelin on cyclin A and CDK2 protein levels, indicating that this peptide increased the expression of cyclin A and CDK2 through the activation of MEK/ERK1/2, PI3K/Akt/mTOR and JAK2/STAT3 pathways. Therefore, we propose that mitotic ghrelin signaling increases cyclin A levels by stimulating nuclear expression of E2F1, leading to formation of active cyclin A/CDK2 complexes and further hyperphosphorylation of Rb. Increased activity of cyclin A/CDK2 triggers the initiation of DNA replication and thus entry into the S phase of the cell cycle.

Our results suggest that, besides stimulating promitogenic cyclin A and CDK2, ghrelin also induces downregulation of cell cycle inhibitors, p27KIP1 and p57KIP2, to promote S phase entry of hippocampal NSCs. Our data also suggest that the activation of MEK/ERK1/2, PI3K/Akt/mTOR and JAK2/STAT3 is involved in the regulation by ghrelin in p27KIP1 and p57KIP2 expression. The antimitogenic molecules p27KIP1 and p57KIP2 negatively regulate the progression of cell cycle by binding and inhibiting complexes of cyclin E/CDK2 and cyclin A/CDK2 and thus promote cell cycle exit in proliferative cells (Vidal & Koff 2000, Abrous et al. 2005). Reduced levels of p27KIP1 and p57KIP2 have been implicated in the mechanism of IGF-1- or bFGF-mediated proliferative action in cortical precursor cells (Li & Cicco-Bloom 2004, Mairet-Coello et al. 2009). Similar findings were also observed in IGF-1- and FGF-2-stimulated oligodendrocyte progenitor cells (Frederick & Wood 2004). In the current study, we have shown that ghrelin downregulates p27KIP1 and p57KIP2, helping to trigger entry into S phase of hippocampal NSCs. It is likely that ghrelin mitigates inhibitory influence on cyclin/CDK activity by the reduced expression of p27KIP1 and p57KIP2.

Combining the current results and previous studies, ghrelin seems to be a plausible candidate that improves hippocampus-dependent cognitive function. The supporting evidences are as follows. (1) Adult neurogenesis in the DG has been proposed to mediate hippocampus-dependent learning and memory (Zhao et al. 2008). (2) We and others have previously reported that ghrelin-deleted mice showed impairments in memory performance, which was rescued by exogenous peripheral administration of ghrelin (Diano et al. 2006, Li et al. 2013). (3) In calorie-restricted animals, where plasma ghrelin levels are elevated (Lutter et al. 2008), survival of newborn cells in the DG has been increased in wild-type mice (Lee et al. 2002), but not in ghrelin-knockout mice (Kim et al. 2015) and enhanced memory performance in hippocampus-dependent behavioral test was observed (Fontan-Lozano et al. 2007, Adams et al. 2008).

In conclusion, our observations indicate that ghrelin has a mitogenic effect in cultured hippocampal NSCs. Ghrelin-induced mitogenic regulation may be mediated via enhanced progression from G1 to S phase due to enhanced nuclear expression of E2F1 and opposite regulation of positive and negative cell cycle regulators. Therefore, ghrelin may be a possible beneficial approach for the impairments in learning and memory performance observed in various neurodegenerative diseases.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2011-0030072 to S P and No. 2014R1A1A2058316 to H C)

Author contribution statement

Hyunju Chung: carried out the laboratory experiments, collected the data and wrote the manuscript. Seungjoon Park: designed the study, analyzed and interpreted the data and wrote the manuscript.

References

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    Ghrelin stimulates proliferation of adult rat hippocampal NSCs. (A) Cells were treated with ghrelin (100nM) for 24, 48 and 72h, and the cell proliferation was assayed using the CCK-8 proliferation assay. (B) Cells were treated with ghrelin (100nM) for 24h. Protein lysates were prepared and assayed by Western blotting using specific anti-PCNA and anti-CDC2 antibodies. The band intensities of PCNA and CDC2 were normalized to the band intensity of β-actin. (C) Time course of ghrelin-induced effects on PCNA and CDC2 mRNA levels. Cells were treated with ghrelin (100nM) for 4, 8, 12 and 24h. Real-time RT-PCR was performed to measure mRNA levels of PCNA and CDC2 and adjusted by β-actin. The data are expressed as percentage of vehicle and are shown as the mean±s.e.m. of three different experiments (each experiment was performed in duplicate). *P<0.05 vs vehicle-treated control.

  • View in gallery

    Fluorescence-activated cell sorting (FACS) analysis of cell cycle distribution. (A) Following 16-h starvation and pretreatment with 20µM PD98059 for 0.5h, 10µM U0126 for 0.5h, 20µM LY294002 for 1h, 100nM Akt inhibitor VIII for 1h, 200nM rapamycin for 1h or 1nM cucurbitacin I for 0.5h, cells were treated with ghrelin (100nM) for 8h. The cells were harvested by propidium iodide (50µg/mL)-staining solution and subjected to FACS analysis. Results were expressed as percentage of total cells in G0/G1, S or G2/M phase of the cell cycle. Values represent the mean±s.e.m. of three different experiments (each experiment was performed in duplicate). (B) The bar graphs represent the S phase ratio expressed as the percentage of vehicle. *P<0.05 vs vehicle-treated cells, P<0.05 vs ghrelin-treated cells.

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    Ghrelin increases the nuclear expression of transcription factor E2F1. Cells were treated with ghrelin (100nM) for 4, 8, 12 and 24h. Nuclear and cytoplasmic lysates were prepared and assayed by Western blotting using specific anti-E2F1, anti-laminin A and anti-GAPDH antibodies. (A) The nuclear E2F1 band intensity was normalized to the laminin A band intensity. (B) The cytoplasmic E2F1 band intensity was normalized to the GAPDH band intensity. Representative Western blotting images are shown in the upper insets. The data are expressed as the mean±s.e.m. of three different experiments (each experiment was performed in duplicate). (C) The bar graphs represent the nuclear/cytoplasmic E2F1 ratio expressed as the percentage of control. *P<0.05 vs control.

  • View in gallery

    Confocal microscopic analysis of E2F1 expression. Cells were treated with ghrelin (100nM) for 4, 8, 12 and 24h. Cells were incubated with primary antibody to E2F1. Cells were counterstained with DAPI, and images were captured using confocal microscopy; scale bar, 5µm.

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    Ghrelin increases the expression of cyclin A and CDK2. (A and B) Cells were treated with ghrelin (100nM) for 4, 8, 12 and 24h. (C and D) Cells were preincubated with 20µM PD98059 for 0.5h, 20µM LY294002 for 1h, 200nM rapamycin for 1h or 1nM cucurbitacin I for 0.5h and then treated with ghrelin (100nM) for 8h. Lysates were prepared and assayed by Western blotting using specific anti-cyclin A, anti-CDK2 and anti-β-actin antibodies. Cyclin A (A and C) and CDK2 (B and D) band intensities were normalized to the β-actin band intensity and expressed as relative band intensity. Representative Western blotting images are shown in the upper insets. The data are expressed as the mean±s.e.m. of three different experiments (each experiment was performed in duplicate). *P<0.05 vs control; P<0.05 vs ghrelin-treated cells.

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    Ghrelin decreases the expression of p27KIP1 and p57KIP2. (A and B) Cells were treated with ghrelin (100nM) for 4, 8, 12 and 24h. (C and D) Cells were preincubated with 20µM PD98059 for 0.5h, 20µM LY294002 for 1h, 200nM rapamycin for 1h or 1nM cucurbitacin I for 0.5h and then treated with ghrelin (100nM) for 8h. Lysates were prepared and assayed by Western blotting using specific anti-p27KIP1, anti-p57KIP2 and anti-β-actin antibodies. p27KIP1 (A and C) and p57KIP2 (B and D) band intensities were normalized to the β-actin band intensity and expressed as relative band intensity. Representative Western blotting images are shown in the upper insets. The data are expressed as the mean±s.e.m. of three different experiments (each experiment was performed in duplicate). *P<0.05 vs control; P<0.05 vs ghrelin-treated cells.

  • AbizaidALiuZWAndrewsZBShanabroughMBorokEElsworthJDRothRHSleemanMWPicciottoMRTschopMH2006Ghrelin modulates the activity and synaptic input organization of midbrain dopamine neurons while promoting appetite. Journal of Clinical Investigation11632293239. (doi:10.1172/JCI29867)

    • Search Google Scholar
    • Export Citation
  • AbrousDNKoehlMLeMM2005Adult neurogenesis: from precursors to network and physiology. Physiological Reviews85523569. (doi:10.1152/physrev.00055.2003)

    • Search Google Scholar
    • Export Citation
  • AdachiKMirzadehZSakaguchiMYamashitaTNikolchevaTGotohYPeltzGGongLKawaseTAlvarez-BuyllaA2007Beta-catenin signaling promotes proliferation of progenitor cells in the adult mouse subventricular zone. Stem Cells2528272836. (doi:10.1634/stemcells.2007-0177)

    • Search Google Scholar
    • Export Citation
  • AdamsMMShiLLinvilleMCForbesMELongABBennettCNewtonIGCarterCSSonntagWERiddleDR2008Caloric restriction and age affect synaptic proteins in hippocampal CA3 and spatial learning ability. Experimental Neurology211141149. (doi:10.1016/j.expneurol.2008.01.016)

    • Search Google Scholar
    • Export Citation
  • BeukelaersPVandenboschRCaronNNguyenLMoonenGMalgrangeB2012Cycling or not cycling: cell cycle regulatory molecules and adult neurogenesis. Cellular and Molecular Life Sciences6914931503. (doi:10.1007/s00018-011-0880-6)

    • Search Google Scholar
    • Export Citation
  • BrewerGJTorricelliJREvegeEKPricePJ1993Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. Journal of Neuroscience Research35567576. (doi:10.1002/jnr.490350513)

    • Search Google Scholar
    • Export Citation
  • CareyRGLiBCicco-BloomE2002Pituitary adenylate cyclase activating polypeptide anti-mitogenic signaling in cerebral cortical progenitors is regulated by p57Kip2-dependent CDK2 activity. Journal of Neuroscience2215831591.

    • Search Google Scholar
    • Export Citation
  • CarliniVPVarasMMCragnoliniABSchiothHBScimonelliTNde BarioglioSR2004Differential role of the hippocampus, amygdala, and dorsal raphe nucleus in regulating feeding, memory, and anxiety-like behavioral responses to ghrelin. Biochemical and Biophysical Research Communications313635641. (doi:10.1016/j.bbrc.2003.11.150)

    • Search Google Scholar
    • Export Citation
  • CastedoMPerfettiniJLRoumierTKroemerG2002Cyclin-dependent kinase-1: linking apoptosis to cell cycle and mitotic catastrophe. Cell Death & Differentiation912871293. (doi:10.1038/sj.cdd.4401130)

    • Search Google Scholar
    • Export Citation
  • ChungHKimELeeDHSeoSJuSLeeDKimHParkS2007Ghrelin inhibits apoptosis in hypothalamic neuronal cells during oxygen-glucose deprivation. Endocrinology148148159. (doi:10.1210/en.2006-0991)

    • Search Google Scholar
    • Export Citation
  • ChungHSeoSMoonMParkS2008Phosphatidylinositol-3-kinase/Akt/glycogen synthase kinase-3 beta and ERK1/2 pathways mediate protective effects of acylated and unacylated ghrelin against oxygen-glucose deprivation-induced apoptosis in primary rat cortical neuronal cells. Journal of Endocrinology198511521. (doi:10.1677/JOE-08-0160)

    • Search Google Scholar
    • Export Citation
  • ChungHLiEKimYKimSParkS2013Multiple signaling pathways mediate ghrelin-induced proliferation of hippocampal neural stem cells. Journal of Endocrinology2184959. (doi:10.1530/JOE-13-0045)

    • Search Google Scholar
    • Export Citation
  • CobrinikD2005Pocket proteins and cell cycle control. Oncogene2427962809. (doi:10.1038/sj.onc.1208619)

  • Cooper-KuhnCMVroemenMBrownJYeHThompsonMAWinklerJKuhnHG2002Impaired adult neurogenesis in mice lacking the transcription factor E2F1. Molecular and Cellular Neuroscience21312323. (doi:10.1006/mcne.2002.1176)

    • Search Google Scholar
    • Export Citation
  • DateYKojimaMHosodaHSawaguchiAMondalMSSuganumaTMatsukuraSKangawaKNakazatoM2000Ghrelin, a novel growth hormone-relasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology14142554261. (doi:10.1210/en.141.11.4255)

    • Search Google Scholar
    • Export Citation
  • DianoSFarrSABenoitSCMcNayECda SilvaIHorvathBGaskinFSNonakaNJaegerLBBanksWA2006Ghrelin controls hippocampal spine synapse density and memory performance. Nature Neuroscience9381388. (doi:10.1038/nn1656)

    • Search Google Scholar
    • Export Citation
  • Fontan-LozanoASaez-CassanelliJLIndaMCde los Santos-ArteagaMSierra-DominguezSALopez-LluchG gado-GarciaCarrionAM2007Caloric restriction increases learning consolidation and facilitates synaptic plasticity through mechanisms dependent on NR2B subunits of the NMDA receptor. Journal of Neuroscience271018510195. (doi:10.1523/JNEUROSCI.2757-07.2007)

    • Search Google Scholar
    • Export Citation
  • FrederickTJWoodTL2004IGF-I and FGF-2 coordinately enhance cyclin D1 and cyclin E-cdk2 association and activity to promote G1 progression in oligodendrocyte progenitor cells. Molecular and Cellular Neuroscience25480492. (doi:10.1016/j.mcn.2003.11.015)

    • Search Google Scholar
    • Export Citation
  • GarzaJCGuoMZhangWLuXY2008Leptin increases adult hippocampal neurogenesis in vivo and in vitro. Journal of Biological Chemistry2831823818247. (doi:10.1074/jbc.M800053200)

    • Search Google Scholar
    • Export Citation
  • GhigoEBroglioFArvatEMaccarioMPapottiMMuccioliG2005Ghrelin: more than a natural GH secretagogue and/or an orexigenic factor. Clinical Endocrinology62117. (doi:10.1111/j.1365-2265.2004.02160.x)

    • Search Google Scholar
    • Export Citation
  • HwangSMoonMKimSHwangLAhnKJParkS2009Neuroprotective effect of ghrelin is associated with decreased expression of prostate apoptosis response-4. Endocrine Journal56609617. (doi:10.1507/endocrj.K09E-072)

    • Search Google Scholar
    • Export Citation
  • JiangHBetancourtLSmithRG2006Ghrelin amplifies dopamine signaling by cross talk involving formation of growth hormone secretagogue receptor/dopamine receptor subtype 1 heterodimers. Molecular Endocrinology2017721785. (doi:10.1210/me.2005-0084)

    • Search Google Scholar
    • Export Citation
  • JiangHLiLJWangJXieJX2008Ghrelin antagonizes MPTP-induced neurotoxicity to the dopaminergic neurons in mouse substantia nigra. Experimental Neurology212532537. (doi:10.1016/j.expneurol.2008.05.006)

    • Search Google Scholar
    • Export Citation
  • KimYKimSKimCSatoTKojimaMParkS2015Ghrelin is required for dietary restriction-induced enhancement of hippocampal neurogenesis: lessons from ghrelin knockout mice. Endocrine Journal62269275. (doi:10.1507/endocrj.EJ14-0436)

    • Search Google Scholar
    • Export Citation
  • KojimaMKangawaK2005Ghrelin: structure and function. Physiological Reviews85495522. (doi:10.1152/physrev.00012.2004)

  • KojimaMHosodaHDateYNakazatoMMatsuoHKangawaK1999Ghrelin is a growth hormone-relesasing acylated peptide from stomach. Nature402656660. (doi:10.1038/45230)

    • Search Google Scholar
    • Export Citation
  • LearishRDBrussMDHaak-FrendschoM2000Inhibition of mitogen-activated protein kinase kinase blocks proliferation of neural progenitor cells. Developmental Brain Research12297109. (doi:10.1016/S0165-3806(00)00064-X)

    • Search Google Scholar
    • Export Citation
  • LeeJSeroogyKBMattsonMP2002Dietary restriction enhances neurotrophin expression and neurogenesis in the hippocampus of adult mice. Journal of Neurochemistry80539547. (doi:10.1046/j.0022-3042.2001.00747.x)

    • Search Google Scholar
    • Export Citation
  • LeeJLimEKimYLiEParkS2010aGhrelin attenuates kainic acid-induced neuronal cell death in the mouse hippocampusJournal of Endocrinology205263270. (doi:10.1677/joe-10-0040)

    • Search Google Scholar
    • Export Citation
  • LeeJYChungHYooYSOhYJOhTHParkSYuneTY2010bInhibition of apoptotic cell death by ghrelin improves functional recovery after spinal cord injury. Endocrinology15138153826. (doi:10.1210/en.2009-1416)

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