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. Author manuscript; available in PMC: 2009 May 7.
Published in final edited form as: J Neurochem. 2009 Mar 19;109(4):1157–1167. doi: 10.1111/j.1471-4159.2009.06043.x

CXCL12 Increases Human Neural Progenitor Cell Proliferation Through Akt-1/FOXO3a Signaling Pathway

Yumei Wu *, Hui Peng *, Min Cui *, Nicholas P Whitney *, Yunlong Huang *, Jialin C Zheng *,†
PMCID: PMC2679099  NIHMSID: NIHMS104488  PMID: 19302476

Abstract

CXCL12, a ligand for the chemokine receptor CXCR4, is well known in mediating neural progenitor cell (NPC) migration during neural development. However, the effects of CXCL12 on human NPC proliferation and its associated signaling pathways remain unclear. The transcription factor FOXO3a, a downstream target of Akt-1, is critical for cell cycle control and may also play an important role in regulating NPC proliferation. In this study, we found CXCL12 promotes human NPC proliferation as determined by the proliferation marker Ki67 and BrdU incorporation. This CXCL12-mediated NPC proliferation was associated with an increase in Akt-1 and FOXO3a phosphorylation in a time- and dose-dependent manner. The CXCR4 antagonist (T140) or inhibitors for G proteins (PTX) and PI3K (LY294002) abolished CXCL12-mediated NPC proliferation and phosphorylation of Akt-1 and FOXO3a. The roles of Akt-1 and FOXO3a in CXCL12-mediated NPC proliferation were further investigated by using adenoviral over-expression in NPCs. Over-expression of dominant-negative Akt-1 or wild-type FOXO3a in NPC abrogated CXCL12-mediated proliferation. These data suggest CXCL12-mediated NPC proliferation is reliant upon the phosphorylation of Akt-1 and FOXO3a and gives insight to an essential role of CXCL12 in neurogenesis. Understanding this mechanism may facilitate the development of novel therapeutic targets for NPC proliferation during neurogenesis.

Keywords: Neural progenitor cell, CXCL12, CXCR4, Akt-1; FOXO3a; proliferation

INTRODUCTION

CXCL12 (stromal cell-derived factor 1, SDF-1), a small secreted α chemokine protein (8-13 kDa), is the only known physiological ligand for chemokine receptor CXCR4. Although the CXCL12/CXCR4 pathway and its functions were originally identified in the immune system (Arai et al. 2000; Rossi and Zlotnik 2000), increasing evidence suggests that this pathway plays a critical role in the development of the central nervous system (CNS) as well. Targeted disruption of the genes encoding CXCL12 or CXCR4 causes similar abnormalities in the development of brain as well as the cardiovascular system and hematopoietic system, and both result in perinatal lethality in homozygous mutant animals (Jazin et al. 1997; McGrath et al. 1999; Lu et al. 2002; Tran et al. 2004). Mice that lack either the CXCR4 receptor or CXCL12 show abnormal development of both the internal granule layer of the cerebellum (Ma et al. 1998; Zou et al. 1998) and the dentate gyrus of the hippocampus (Bagri et al. 2002; Lu et al. 2002), indicating CXCL12/CXCR4 interactions are critical for the regulation of neural progenitor cell (NPC) function and the neurogenesis process.

It is well known that neurogenesis is dependent upon proper neural progenitor cell (NPC) proliferation, fate specification (differentiation), directed migration, survival, maturation, and functional integration of progeny into neuronal circuits (Fallon et al. 2000; Ming and Song 2005). Throughout brain development, CXCL12 is expressed by cells lining migratory paths and by cells surrounding the end location of migration, while the migrating cells express CXCR4 (Ma et al. 1998; Zou et al. 1998; Peng et al. 2007). This spatial distribution of CXCL12- and CXCR4-expressing cells in the brain is consistent with their roles in regulating the migration and possibly the proliferation/differentiation of NPCs. Self-renewal or proliferation of NPCs is critical to maintain the pool of NPCs during neurogenesis. In vitro studies showed that CXCL12 potentiated the proliferative responses of granule precursor cells to sonic hedgehog (Klein et al. 2001) and increased rat NPC proliferation with basic fibroblast growth factor (bFGF) treatment (Gong et al. 2006). However, the potential individual role of CXCL12 in human NPC proliferation and its associated signaling pathways during neurogenesis remains unclear.

Evidence obtained from neuronal studies showed that stimulation of CXCR4 by CXCL12 leads to the activation of intracellular pathways such as PI3K/Akt-1 and changes in cell cycle proteins affecting neuronal survival (Khan et al. 2003). It is well known that Akt-1 is a serine/threonine kinase and a downstream target of PI3K, which critically regulates cell proliferation, differentiation, and apoptosis and functions as an upstream signaling molecule for many target genes (Fruman et al. 1998; Plas and Thompson 2005). Akt-1 promotes cell proliferation by interacting with 14-3-3 proteins that sequester p21 in the cytoplasm (Muise-Helmericks et al. 1998; Graff et al. 2000; Zhou et al. 2001) or by upregulating cyclin D proteins (Muise-Helmericks et al. 1998), which results in cell cycle progression. More related studies indicate Akt-1 phosphorylates and inhibits the winged-helix family of transcription factors, namely FOXO3a, which is a key negative regulator of cell cycle progression (Nakamura et al. 2000; Brunet et al. 2001).

FOXO3a is one of the FOXO (Forkhead box, class O) subclass of Forkhead transcription factors (Birkenkamp et al. 2007). As a major substrate of Akt-1, FOXO3a plays a critical role in coordinating cell survival and death and regulating stress responses and longevity (Brunet et al. 2001; Birkenkamp et al. 2007). One way in which Akt-1 promotes cell survival and proliferation is by phosphorylating FOXO3a, which results in the sequestration of FOXO3a in the cytoplasm away from cell death-inducing genes (You et al. 2004; Greer and Brunet 2005; Maiese et al. 2007; Cui et al. 2008; Sedding 2008; Yang et al. 2008b). Our previous studies showed CXCL12 phosphorylated Akt-1 in NPCs (Peng et al. 2004), raising the possibility that CXCL12 itself may promote NPC proliferation through activation of Akt-1, and subsequently, inactivation of FOXO3a. Accordingly, the major aim of this study was to investigate whether CXCL12, acting via the PI3K/Akt way, was able to induce the phosphorylation and inactivation of FOXO3a in NPCs and to elucidate the possible role of this event on NPC proliferation.

Using a well-established in vitro culture system, we demonstrated CXCL12 increased human NPC proliferation and phosphorylation of Akt-1 and FOXO3a. To further analyze the role of CXCL12, the CXCR4 antagonist (T140) or inhibitors for G proteins (Pertussis Toxin, PTX) and PI3K (LY294002) were shown to abolish CXCL12-mediated NPC proliferation and phosphorylation of Akt-1 and FOXO3a. Loss-of-function studies showed over-expression of dominant-negative Akt-1 and wild-type FOXO3a in NPC eliminated CXCL12-mediated NPC proliferation. As a whole, our data show that CXCR4/G protein/Akt-1/FOXO3a signaling pathway is responsible for CXCL12-mediated NPC proliferation, further emphasizing that FOXO3a is a major player in the proliferative effects of CXCL12 on NPC.

Methods and materials

Reagents and materials

Human recombinant CXCL12 was obtained from R & D (R&D Systems, Minneapolis, MN), T140, a gift from Dr. Nobutaka Fujii (Kyoto University, Japan), PTX, and LY294002 were from Calbiochem (Calbiochem, San Diego, CA). Anti-phospho-Akt-1, anti-Akt-1, anti-phospho-FOXO3a, and anti-FOXO3a antibodies were purchased from Cell Signaling (Cell Signaling, Danvers, MA). All secondary antibodies conjugated with horseradish peroxidase were purchased from Cell Signaling.

Neural Progenitor Cell Culture

Human cortical NPC were isolated from human brain tissue as previously described (Peng et al. 2004). Briefly, NPCs were cultured in substrate-free tissue culture flasks and grown in suspension as spheres in neurosphere initiation medium (NPIM), which consisted of X-Vivo 15 (BioWhittaker, Walkersville, ME) with N2 supplement (/Invitrogen, Carlsbad, CA), neural cell survival factor-1 (NSF-1, Bio Whittaker), bFGF (20 ng/mL, Sigma-Aldrich), epidermal growth factor (EGF, 20 ng/mL, Sigma-Aldrich), leukemia inhibitory factor (LIF, 10 ng/mL, Chemicon, Temecula, CA), and 60 ng/mL N-acetylcysteine (Sigma-Aldrich). Cells were passaged at two-week intervals as previously described (Peng et al. 2004). All studies utilizing human subjects were performed in full compliance with the University of Nebraska Medical Center and National Institutes of Health's ethical guidelines.

Proliferation Assay

Before each experiment, NPCs were passaged and seeded in 24-well plates (coated with poly-D-lysine, 10 μg/ml) at a density of 4 × 104 cells/well in NPIM for 24 h. Culture medium was replaced with different concentration of CXCL12 in X-Vivo 15 for 72 hours. For the experiment utilizing inhibitors, cells were pretreated with T140 (30 μM), PTX (100 ng/ml), or LY294002 (20 μM) for 2 hours followed by stimulation with 50 ng/ml CXCL12 for 72 h and subjected to immunocytochemistry.

Flow Cytometry Assay

NPC were treated with CXCL12 or NPIM for 3 days. Single cells were harvested by trypsin digestion and mechanical disassociation. Cell proliferation was examined by phycoerythirn (PE)-conjugated Ki67 staining (BD Biosciences, San Diego, CA). Briefly, 1 × 106 cells were washed twice with Ca2+/Mg2+-free phosphate-buffered saline (PBS, Invitrogen), fixed overnight in 75% cold ethanol, digested with RNase A (Sigma-Aldrich), and stained with PE-Ki67 for 30 min; PE-IgG isotype was used for background scanning. For the BrdU incorporation assay, NPC were treated with BrdU (100 μg/ml) for 1-4 h before collected and subjected to FITC-BrdU staining. Data were obtained and analyzed by flow cytometry using the Cell Quest software on a FACScan (BD Biosciences, San Diego, CA).

Immunocytochemistry

Cells were fixed in 4% paraformaldehyde for 10 min and washed in PBS as previously described by Peng et al. (Peng et al. 2004). Subsequently, cells were incubated overnight with mouse anti-Ki67 (1:100, BD Bioscience) and rabbit anti-nestin (chemicon) for the identification of proliferating NPCs, followed by Alexa Fluor secondary antibodies, goat anti-mouse IgG Alexa Fluor 488, and goat anti-rabbit IgG Alexa Fluor 594 (Molecular Probes, Eugene, OR, 1:200) for 1 h at room temperature. All antibodies were diluted in PBS with 0.1% Triton X-100 and 2% BSA. Nuclei were counterstained with Hoechst 33342 (Sigma-Aldrich). Morphological changes were visualized and captured with a Nikon Eclipse E800 microscope equipped with a digital imaging system. Images were imported into Image-ProPlus, version 4.0 (Media Cybernetics, Sliver Spring, MD) for quantification. Thirty random fields (total 800-1000 cells/culture) of immunostained cells were manually counted using a 20× objective.

Western Blotting

To study the effect of CXCL12 on the phosphorylation of various signaling proteins, cells were treated with 50 ng/ml CXCL12 for various time points or with different concentration of CXCL12 (from 10 to 100 ng/ml) for 15 min after overnight starvation of growth factors. For the experiments utilizing inhibitors, cells were pretreated with T140 (30 μM), PTX (100 ng/ml), or LY294002 (20 μM) for 2 hours followed by stimulation with 50 ng/ml CXCL12 for 15 min. After each treatment, cells were rinsed twice with PBS and lysed by M-PER Protein Extraction Buffer (Pierce, Rockford, IL) containing 1× protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Protein concentration was determined using the BCA Protein Assay Kit (Pierce). Proteins (10-20 μg) were separated on a 10% SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred to an Immuno-Blot polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA). After blocking in TBS/Tween (0.1%) with 5% fat-free milk for 2 h, the membrane was incubated with primary antibodies for phosphorylated Akt-1 and FOXO3a, total Akt-1 and FOXO3a (1:1,000; Cell Signaling Technologies) overnight at 4°C followed by horseradish peroxidase-conjugated secondary antibodies (1:10,000; Cell Signaling Technologies) and then developed using Enhanced Chemiluminescent (ECL) solution (Pierce). The molecular sizes of the developed proteins were determined by comparison with pre-stained protein markers (Invitrogen). For data quantification the films were scanned with a CanonScan 9950F scanner and the acquired images were then analyzed using Image J program (developed at the U.S. National Institutes of Health and available on the internet at http://rsb.info.nih.gov/ij).

Over-expression of Akt-1 and FOXO3a by Adenoviral Infection

Replication-defective adenovirus vectors expressing dominant-negative Akt-1 (Ad-DN-Akt-1) and constitutively-active Akt-1 (Ad-myrAkt-1) were generated as described (Shiojima et al. 2002) and provided by K. Walsh (Boston University School of Medicine, Boston, MA). The constitutively-active Akt-1 (Ad-myrAkt) construct has the c-src myristoylation sequence fused in-frame to the N terminus of the HA-Akt-1 (wild-type)-coding sequence. The dominant-negative mutant of Akt-1 construct (Ad-DN-Akt) has a hemagglutinin tag at the N terminus and three amino acid substitutions at lysine 179, threonine 308, and serine 473 to alanine. Replication-defective adenoviral vectors expressing dominant negative FOXO3a (Ad-DN-O3a) and wild-type FOXO3a (Ad-WTO3a) were purchased from Vector Biolabs. Both Ad-DN-O3a and Ad-WT-O3a have a hemagglutinin tag at the N terminus and express GFP. Adenoviral constructs were amplified in HEK 293 cells and purified by ultracentrifugation through a CsCl gradient. NPC were infected with recombinant adenovirus in X-Vivo 15 for 8 h and then recovered for 24 h in NPIM. Infection efficiency was close to 80% as determined by the GFP expression. For proliferation assay, NPCs were treated with CXCL12 for 3 days in basal medium followed by infection with adenovirus.

Statistical Analyses

Data were expressed as means ± SD. The data were evaluated statistically by analysis of variance (ANOVA) followed by the Tukey-test for paired observations. Significance was considered as a p value of < 0.05. To account for any donor-specific differences, all experiments were performed with NPCs from at least three donors. All assays were performed at least two times, with triplicate or quadruplicate samples in each experiment.

RESULTS

1. CXCL12 treatment increased human NPC proliferation

To address whether CXCL12 has a direct effect on NPC proliferation, we employed a well-established in vitro human NPC culture system (Peng et al. 2004; Peng et al. 2005; Peng et al. 2008; Whitney et al. 2008). In order to measure the individual effect of CXCL12 on NPC proliferation, we treated NPC with CXCL12 in the absent of EGF and bFGF to avoid the overwhelming proliferative effect of these strong mitogens. Thus, the capacity of CXCL12 to promote NPC proliferation was investigated by means of Ki67 staining after deprivation of growth factors, EGF and bFGF, with increasing concentration of CXCL12 (10-100 ng/ml) for 1-3 days. NPC proliferation increased after 1-day post-CXCL12 treatment (data not shown) and significantly increased after 3 days of treatment (Figure 1). Quantification data showed that about 37% of NPCs were still proliferating 72 h after growth factor deprivation (Fig. 1 A, B) while the percent of proliferating NPCs increased to 53.4% in 10 ng/ml, 59.4% in 50 ng/ml, and 63.4% in 100 ng/ml CXCL12 stimulation after 3 days. This result showed that CXCL12 caused a dose-dependent increase in the proliferation of NPC with a starting concentration of CXCL12 at 10 ng/ml (Fig. 1 C-F). Similar results were obtained by flow cytometry assay using PE-conjugated Ki-67 Kit (data not shown). In order to further confirm the effect of CXCL12 on NPC proliferation, DNA synthesis was determined by BrdU incorporation and similar results were obtained when NPCs were treated with different doses of CXCL12 in the absence of growth factors (Fig. 2). To exclude the nonspecific NPC proliferative effects of chemokines, we also introduced the other two chemokines, CCL2 (MCP-1) or CXCL8 (IL-8), to cultured human NPC. We determined NPC proliferation in the presence of CCL2 or CXCL8 by BrdU incorporation. Even though receptors for CCL2 (CCR2) and CXCL8 (CXCR2) are expressed at low levels on human NPC (data not shown), both CCL2 and CXCL8 had no significant effect on NPC proliferation (Fig. 2D). These results suggest that CXCL12, but not CCL2 or CXCL8, could increase NPC proliferation after growth factors deprivation.

Figure 1. Proliferation effect of CXCL12 on human NPC by Ki67 staining.

Figure 1

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CXCL12-mediated proliferation of human NPC was determined by Ki67 staining after increasing concentration of CXCL12 stimulation without the presence of growth factors for 3 days. Cell proliferation was assessed by immunostaining for Ki67 positive in NPC in control group (A, B) and with CXCL12 treatment (C, D). NPC cultured with neurosphere initiation medium (NPIM), which consisted of X-Vivo 15 with N2 supplement, neural cell survival factor-1 (NSF-1), basic fibroblast growth factor (bFGF, 20 ng/ml), epidermal growth factor (EGF, 20 ng/ml), leukemia inhibitory factor (LIF, 10 ng/ml), and 60 ng/ml N-acetylcysteine, was used as a positive control. Hoechst staining was used to determine the total cell numbers in the culture and Nestin staining was used to determine total NPC in control and CXCL12 treatment. The percentage of Ki67-positive proliferating NPCs was determined for each treatment with different concentrations of CXCL12 by counting positive cells per microscopic field in 30 pictures per condition (E, F). Results are representative of three independent experiments with NPC from three human donors. ** denotes p < 0.01 in comparison to control. Scale bar=100 μm.

Figure 2. Effect of CXCL12 on human NPC proliferation by BrdU incorporation.

Figure 2

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CXCL12-mediated proliferation of human NPC was determined by BrdU incorporation following different concentrations of CXCL12 stimulation without the presence of growth factors for 3 days. NPIM was used as a positive control (C, D), and both CCL2 and CXCL8 (50 ng/ml) were used to exclude the non-specific NPC proliferation induced by chemokines. The percentage of NPC BrdU incorporation was determined for each treatment with different concentrations of CXCL12 (A, B, D). Results are representative of three independent experiments with NPC from three human donors. **denotes p < 0.01 in comparison to control. Scale bar=100 μm.

2. CXCL12 increased phosphorylation of Akt-1/FOXO3a in human NPC

Akt-1 is identified as converting extracellular stimuli to intracellular signals involved in cell survival and proliferation (Brunet et al. 2001). Evidence showed that PI3K/Akt-1 pathway contributes to the proliferation or self-renewal of embryonic stem cells (Paling et al. 2004; Kim et al. 2005). Akt-1 activation also stimulates breast cancer cell proliferation through multiple downstream targets impinging on cell-cycle regulation (Liang et al. 2002), including inhibition of p27 expression through phosphorylation and inhibition of the FOXO transcription factors (Medema et al. 2000; Greer and Brunet 2005). To explore the intracellular pathways responsible for CXCL12-mediated NPC proliferation, we investigated the effects of CXCL12 on the phosphorylation of Akt-1 and FOXO3a by Western blotting. Cultured NPCs were treated with CXCL12 50 ng/ml for different time points (from 1 min up to 2 days) after starvation overnight. The cell lysates were analyzed for the presence of phosphorylated Akt-1 (activation) and FOXO3a (inactivation) by phospho-specific antibodies to the specific phosphorylation sites of Akt-1 (Ser 473) or FOXO3a (Ser 318). CXCL12 induced an increase in phosphrylation of Akt-1 at Ser 473 site and FOXO3a at Ser 318 site in human NPC as shown in Fig. 3A. CXCL12-induced activation of Akt-1 was clearly evident at 5 min upon CXCL12 stimulation. The peak activation occurred at 10-15 min (1.8 fold, compared with control), lasted up to 1 hour, and then slowly declined.

Figure 3. Effects of CXCL12 on Akt-1, and FOXO3a in neural progenitor cell cultures.

Figure 3

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Human cortical NPCs were deprived of NPIM overnight before treatment with 50 ng/ml CXCL12 for 1, 5, 15, 30 min, 1 h, 2 h, 1 d, and 2 d, and 15 min with 0, 5, 10, 50, and 100 ng/ml CXCL12. Total cell lysates were obtained from each treatment and antibodies specific for the activated and total forms of Akt-1 and FOXO3a were used; β-actin was used as loading control. Human cortical NPCs showed increased activation (phosphorylation) of Akt-1 (A, C), inactivation of FOXO3a (A, C) in a time-dependent manner, and dose-dependency on CXCL12 stimulation. Data is shown as the ratio of phosphorylated and total (B, D). Results are representative of three independent experiments with NPC from three human donors. *denotes p < 0.05 in comparison to control for phosphorylation of Akt-1upon CXCL12 stimulation; †denotes p < 0.05 in comparison to control for phosphorylation of FOXO3a upon CXCL12 stimulation.

FOXO3a is a downstream signaling molecule of Akt-1, which is involved in cell survival and proliferation. Therefore, we examined the changes of FOXO3a phosphorylation under the same treatment. The peak phosphorylation (inactivation) time of FOXO3a in NPC was around 15-30 min (2.2 fold compared with control), which was later than Akt-1 phosphorylation under CXCL12 treatment, and lasted for 2 days (Fig. 3A, B). To determine the dose-dependent effect of CXCL12 on the phosphorylation of Akt-1 and FOXO3a, cultured NPCs were starved overnight before treated with CXCL12 at 0, 5, 10, 50, and 100 ng/ml for 15 min and the cell lysates were analyzed for the phosphorylation of Akt-1 and FOXO3a. Application of CXCL12 in NPC culture for 15 min induced a dose-dependent increase of Akt-1 and FOXO3a phosphorylation. The phosphorylation of Akt-1 and FOXO3a was observed at a minimal concentration of 5 ng/ml CXCL12 and reached maximal levels at about 50 to 100 ng/ml (Fig. 3C, D).

3. CXCL12-mediated NPC proliferation and phosphorylation of Akt-1 and FOXO3a are through CXCR4, G protein, and PI3 kinase pathways

CXCL12 binding to CXCR4 can mediate trimeric GTP-binding protein G inhibitory (Gi) protein activation and induce subsequence intracellular signaling and function changes in NPCs (van Biesen et al. 1996; Bajetto et al. 1999; Vlahakis et al. 2002). To analyze the involvement of CXCR4 in CXCL12-mediated NPC proliferation, we tested NPC proliferation in the presence or absence of a CXCR4 antagonist, T140. In the presence of T140, NPC proliferation upon CXCL12 stimulation was attenuated significantly (Fig. 4A) while T140 treatment alone had no proliferative effect in NPCs (data not shown). To determine whether the NPC proliferation induced by CXCL12 was dependent on the activation of heterotrimetric G proteins, we used a G proteins inhibitor, PTX. NPCs pretreated with PTX (100 ng/ml) showed a dramatic decrease of CXCL12-induced cell proliferation (Fig. 4A), while PTX alone had no significant effect on NPC proliferation. These results indicate CXCR4 regulates second messenger activities through the Gi-Go GTP-binding proteins.

Figure 4. Proliferative effects of CXCL12 on human NPC is through CXCR4, G protein, PI3K, Akt-1 Pathways.

Figure 4

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For blocking, human NPCs were treated with 30 nM T140, 100 ng/ml PTX and 20 μM LY294002 for 2 h before CXCL12 stimulation after deprivation of NPIM overnight. Western blotting analysis showed decreased activation (phosphorylation) of Akt-1 and inactivation (phosphorylation) of FOXO3a upon CXCL12 stimulation (A) in T140, PTX, and LY294002 pretreated NPCs. CXCL12-mediated human NPC proliferation was abolished with pretreatment with T140, PTX, and LY294002 for 2 h followed by CXCL12 treatment for 3 days (B). Results are representative of three independent experiments with NPC from three human donors. ** denotes p < 0.01 in comparison to control and CXCL12 treatment.

G-protein-coupled receptor activation results in PI-3 kinase activation and down stream Akt-1 activation. We next tested whether CXCL12-induced NPC proliferation is through PI-3k/Akt pathway. Human NPC were pretreated with 20 nM of LY294002, a specific PI3K inhibitor, for 2 hours before CXCL12 treatment. NPC proliferation induced by CXCL12 was completely abolished by LY294002 (Fig. 4A). Moreover, the PI3K inhibitor also reduced the basal level of NPC proliferation shown by a decrease in Ki67-positive cells as compared to untreated cells (data not shown). This data suggest that the PI3K pathway is involved in CXCL12-mediated NPC proliferation.

Accordingly, Western blotting showed that phosphorylation of Akt-1 and FOXO3a in NPC upon CXCL12 stimulation was blocked by T140, PTX, or LY294002 pretreatment (Fig. 4B). Thus, in agreement with the results obtained from the proliferation assay, these data suggested that CXCR4/G protein/PI3K pathway is responsible for CXCL12- mediated NPC proliferation through phosphorylation of Akt-1 and FOXO3a.

4. Over-expression of dominant-negative Akt-1 and wild-type FOXO3a abolished CXCL12-mediated NPC proliferation

It has been well documented that Akt-1 and FOXO3a critically regulate cell proliferation, and we also noted CXCL12 induces a significant increase in phosphrylation of Akt-1 and FOXO3a. To further examine the roles of Akt-1 and FOXO3a in CXCL12-mediated NPC proliferation, we first performed an adenovirus delivery system to over-express dominant-negative Akt-1 (Ad-DN-Akt-1) and wild-type FOXO3a (Ad-WT-FOXO3a) in NPC with a serial diluted multiplicity of infection (MOI) 1:500 and MOI 1:1,000 (1K). Adenovirus expressing only GFP (Ad-GFP) was used as a vector control. The infection efficiency of NPC for all vectors was about 80% at the viral titer, with MOI 1:500, no significant loss of cells were observed in Ad-GFP, Ad-DN-Akt, and Ad-WT-FOXO3a groups (Fig. 5A). The over-expression of Ad-DN-Akt and Ad-WT-FOXO3a was confirmed by immunoblotting against total Akt-1 and FOXO3a as shown in figure 5B and 5C. Expression of total Akt-1 and FOXO3a increased dose-dependently corresponding to the titer of virus infection. Based on the florescent images and Western blotting, virus titer MOI 1:500 was chosen for further studies in order to obtain the higher infection efficiency as well as lowest toxicity in NPC after virus infection.

Figure 5. Dominant-negative Akt-1 and wild-type FOXO3a negatively regulates CXCL12-mediated human NPC proliferation.

Figure 5

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Human NPCs were infected with Ad-GFP, Ad-DN-Akt, and Ad-WT-O3 virus at multiplicity of infection (MOI) 1:500 and 1:1K for 8 h and recovered in NPIM overnight. Morphology showed over 80% cells were infected and remained in viable (A). Western blotting showed total Akt-1 expression increased dose-dependently upon Ad-DN-Akt virus infection (B) as well as an increase in total FOXO3a expression upon Ad-WT-O3 virus infection (C). In panel D, human NPCs were infected with Ad-GFP, Ad-DN-Akt, and Ad-WT-O3 virus at MOI 1:500 and subjected to 50 ng/ml CXCL12 for 3 days. Cell proliferation was determined by Ki67 staining. Cell proliferation decreased in Ad-DN-Akt and Ad-WT-O3-infected NPC compared to NPCs carrying GFP vector alone (showed as * in D), and CXCL12-mediated NPC proliferation was abolished in Ad-DN-Akt and Ad-WT-O3-infected NPCs upon CXCL12 stimulation (showed as † in D). Data are shown as mean ± S.E. of three experiments with NPC from three human donors. * denotes p < 0.01 compared to NPCs infected with GFP alone, †indicates p < 0.05 compared to NPCs infected with GFP upon CXCL12 treatment. Scale bar=50 μm.

To specifically target Akt-1, we further delivered Ad-DN-Akt to NPCs using adenovirus delivery system in vitro and Ad-GFP was used as control vector. The Ad-DN-Akt construct has a hemagglutinin tag at the N terminus and three amino acid substitutions at lysine 179, threonine 308, and serine 473 to alanine. Similarly, CXCL12-induced NPC proliferation was observed in NPC over-expressing Ad-GFP as compared with NPC without any over-expression. CXCL12-induced NPC proliferation was decreased in Ad-DN-Akt group as compared to Ad-GFP group under the same treatment (Fig. 5D). Furthermore, similar results were obtained by flow cytometry assay using PE-conjugated Ki-67 Kit (data not shown). This result suggests that endogenous Akt-1 is necessary for CXCL12-induced NPC proliferation.

Similarly, to examine the functional significance of FOXO3a, a downstream target of Akt-1 essential for cell proliferation, in CXCL12-mediated NPC proliferation, we performed over-expression of wild-type Ad-FOXO3a and then examined the CXCL12-mediated proliferative effects in NPCs. NPC over-expressing wild-type FOXO3a (Ad-WT-O3a) were exposed to CXCL12 for 3 days. The result showed CXCL12-mediated NPC proliferation decreased dramatically in Ad-WT-O3a group as compared to Ad-GFP group (Fig. 5D), indicating that FOXO3a, besides Akt-1, is also critical in CXCL12-mediated NPC proliferation.

Discussion

This study reveals that CXCL12 promotes human NPC proliferation in the absence of growth factors, providing evidence that CXCL12 plays another important role in neurogenesis in addition to mediating NPC migration. Furthermore, we demonstrated CXCL12 increased the phosphorylation of Akt-1 and FOXO3a, an important downstream transcription factor that is a key negative regulator of cell cycle progression (Nakamura et al. 2000; Brunet et al. 2001). The CXCR4 antagonist (T140) as well as inhibitors for G proteins (PTX) and PI3K (LY294002) abolished CXCL12-mediated NPC proliferation and phosphorylation of Akt-1 and FOXO3a. Notably, loss-of-function studies showed over-expression of dominant-negative Akt-1 and wild-type FOXO3a in NPCs abolished CXCL12-mediated NPC proliferation, indicating the important role of Akt-1 and FOXO3a in the regulation of human NPC proliferation mediated by CXCL12. As a whole, our data show that CXCR4/G protein/Akt-1/FOXO3a signaling pathway is responsible for CXCL12-mediated NPC proliferation and FOXO3a is a major player in the effects of CXCL12 on NPC proliferation. To our knowledge, this is the first report that the transcription factor FOXO3a is directly associated with human NPC proliferation upon CXCL12 stimulation.

It is now accepted that neurogenesis exists throughout life and is capable of replacing neurons, astrocytes, and oligodendrocytes under conditions of brain injury or disease (Gage 2000; Horner and Gage 2000; Emsley et al. 2005). Notably, recent collective evidence indicates that neurogenesis is affected during brain injury and neurodegenerative disorders by the dysregulation of cytokines, chemokines, neurotransmitters, and reactive oxygen species caused by inflammation and mediated by activated macrophages, microglia and reactive astrocytes (Whitney et al, 2009). Specifically, neurogenesis is dependent upon the proper proliferation, fate specification (differentiation), migration, survival, maturation, and functional integration of progeny into neuronal circuits (Fallon et al. 2000; Ming and Song 2005). How chemokines and their receptors, especially CXCL12 and CXCR4, play a role in these processes has been an intense area of investigation due to the essential roles of CXCL12 and CXCR4 in neuronal development.

CXCR4 is highly expressed during development in the cerebellum, hippocampus and neocortex, and expression persists into adulthood (Jazin et al. 1997; Ma et al. 1998; Zou et al. 1998; Lu et al. 2002; Stumm et al. 2003). On the other hand, CXCL12, the only known physiological ligand for CXCR4 (Rossi and Zlotnik 2000), is predominantly expressed by oligodendrocytes, astrocytes and neurons in the neocortex, hippocampus, and cerebellum (Gleichmann et al. 2000; Stumm et al. 2003). CXCL12/CXCR4 interactions are known to regulate NPC migration in the cerebellum (Ma et al. 1998; Zou et al. 1998), DG (Lu et al. 2002), and cortex (Stumm et al. 2003; Peng et al. 2004; Peng 2007). Notably, it was recently reported that the recruitment of CXCR4-positive progenitor cells to regenerating tissue is mediated by hypoxic gradients via the transcription factor hypoxia-inducible factor-1-induced expression of CXCL12 (Ceradini et al. 2004). In addition, others and we have identified CXCR4 expression on human and mouse NPCs and established CXCL12-mediated NPC migration via CXCR4 (Peng et al. 2004; Tran et al. 2004). Even though CXCL12 is a constitutively-produced chemokine, it is elevated during brain inflammation that is associated with many brain injuries and diseases including HIV-1 associated dementia (Zheng et al. 1999; Peng et al. 2006; Schonemeier et al. 2008). CXCL12 is released by glia in response to activation by inflammatory factors including IL-1β, a product of macrophage activation in HIV-1 infection, indicating the potential role of CXCL12 in the regulation of NPC migration during HIV-1 infection in the CNS (Peng et al. 2006).

Besides acting as a chemoattractant for progenitor and precursor cells, the work by Klein et al. indicated CXCL12 acts synergistically with sonic hedgehog (SHH) to increase proliferation of granule cell precursors (Klein et al. 2001). In addition, CXCL12 has also been shown to increase rat NPC proliferation with bFGF treatment (Gong et al. 2006). However, there has not yet been a report indicating the specific and direct role in CXCL12 in the regulation of human NPC proliferation. Our data demonstrated CXCL12 induced a dose-dependent increase of Ki67-positive NPCs after growth factors deprivation (Fig. 1). NPC proliferation increased 1 day after CXCL12 treatment and significantly increased 3 days after treatment. Further, this effect was blocked by T140 indicating CXCL12 appears to influence NPC proliferation through CXCR4 (Fig. 4). This in vitro observation with human NPCs demonstrated that CXCL12 could also directly affect NPC proliferation in addition to its primary role in the regulation of NPC migration.

To evaluate the comprehensive effect of CXCL12, we have characterized the CXCL12-mediated effects on NPC proliferation and the associated signaling pathways regulating NPC function. Notably, CXCL12-mediated NPC proliferation was associated with increase in Akt-1 and FOXO3a phosphorylation in a time- and dose-dependent manner (Fig. 3). Further, CXCL12-mediated NPC proliferation and phosphorylation of Akt-1 and FOXO3a were abolished by inhibitors for G proteins (PTX) and PI3K (LY294002) (Fig. 4), indicating the importance of Akt-1-associated signaling pathways in the regulation of CXCL12-mediated NPC proliferation.

Akt-1 is a serine/threonine kinase and a downstream target of PI3K, which plays an important role in the survival and proliferation of various cell types, including NPC (Chang et al. 2003; Dugourd et al. 2003; Finnberg and El-Deiry 2004; Dziembowska et al. 2005; Urbich et al. 2005). Activation of the Akt-1 pathway is coupled to transcription factors and cell cycle machinery, such as FOXO3a, via phosphorylation. FOXO3a is a member of the family of Forkhead transcription factors characterized by the presence of a highly conserved Forkhead domain with a winged-helix motif and DNA binding activity (Weigel and Jackle 1990; Kaestner et al. 2000; Tsai et al. 2007) and functions as an important intermediate of several signaling cascades, serving to regulate differentiation, survival, and proliferation (Zou et al. 1998; Urbich et al. 2005; Senf et al. 2008; Yang et al. 2008a; Yang et al. 2008b). As a major substrate of Akt-1, FOXO3a plays a critical role in coordinating cell survival and death and regulating stress responses and longevity (Brunet et al. 2001; You et al. 2004; Greer and Brunet 2005; Birkenkamp et al. 2007; Maiese et al. 2007; Cui et al. 2008; Sedding 2008). One way Akt-1 promotes cell survival is by phosphorylating FOXO3a, which results in its inactivation and sequestration in the cytoplasm away from cell death-inducing genes (You et al. 2004; Greer and Brunet 2005; Maiese et al. 2007; Cui et al. 2008; Sedding 2008). The role of FOXO3a in the apoptosis of neurons and other cell types was first implicated by Brunet and colleagues who found that the expression of unphosphorylated mutant FOXO3a in cerebellar granule neurons, CCL39 fibroblasts, and Jurkat cells induced apoptosis (Brunet et al. 1999; Shin et al. 2001; Zhu et al. 2008). More recently, Yang et al. identified FOXO3a as an important regulating factor for cancer cell proliferation and tumorigeneis (Yang et al. 2008a). However, little is currently known about such downstream signaling events mediated by Akt-1, especially its nuclear targets, in human NPC upon CXCL12 stimulation.

The present results reveal that CXCL12 rapidly induced transient phosphorylation of Akt-1 (peak time at 15-30 min) in human NPC in a concentration- and time-dependent manner (Fig. 3). Subsequently, CXCL12 also phosphorylate FOXO3a in a similar manner (Fig. 3). This process positively enhances NPC proliferation, with a significant increase in NPC proliferation after 3 days of CXCL12 treatment. The phosphorylation of endogenous FOXO3a induced by CXCL12 in human NPC, demonstrates that this transcription factor is indeed a target and a component of CXCL12 receptor signaling in NPCs. Furthermore, a CXCR4 antagonist, G protein inhibitor, and PI3K inhibitor abrogated phosphorylation of both Akt-1 and FOXO3a (Fig. 4), suggesting these two critical proteins are the downstream effectors of CXCL12/CXCR4.

Our result demonstrated that there was transient Akt-1 (minutes to hours) and sustained FOXO3 phosphorylation (hours to days). It is important to note that the phosphorylation of FOXO3 is not only a balance of kinase and phosphatase activity, but polyubiquitination and deubiquitination may also be involved in this process. Following cytoplasmic translocation, FOXO phosphorylation also results in FOXO ubiquitination and proteasomal degradation, further reducing the transcriptional activity of FOXO (Huang et al. 2005; Tothova and Gilliland 2007; Calnan and Brunet 2008; Fu and Tindall 2008). In addition to Akt-1, many other kinases also phosphorylate FOXO on independent sites and inhibit transcription of genes that promote apoptosis (Bim, FasL and TRAIL) and cell-cycle arrest (p27 and p21) (Yang et al. 2008b). These kinases include serum and glucocorticoid-regulated kinase (SGK), c-Jun N-terminal kinase (JNK), extracellular signal-related kinase (ERK), dual specificity tyrosine-phosphorylated and regulated kinase (DYRK1A), mammalian Ste20-like kinase-1 (MST1) and IκB kinase (IKK) (Brunet et al. 1999; Tran et al. 2003; Greer and Brunet 2008; Yang et al. 2008b). Thus, there may be other non-Akt pathways involved in the regulation of FOXO3a and it is associated functions.

In order to further examine the roles of Akt-1 and FOXO3 in CXCL12-mediated NPC proliferation, we over-expressed Akt-1 and FOXO3a in NPCs through an adenovirus delivery system. Interestingly, the over-expression of wild-type FOXO3a and dominant-negative Akt-1 in NPC was shown to attenuate CXCL12-mediated NPC proliferation (Fig. 5C). In contrast, constitutively-active Akt-1 (myrAkt) and dominant-negative FOXO3a in NPCs enhanced NPC proliferation after growth factors deprivation (data not shown). While the other kinases or factors may be also important, our results support the conclusion that Akt-1 pathway is the major pathway to inactivate FOXO3a in NPCs.

How FOXO3a and its associated downstream factors are involved in CXCL12-mediated NPC proliferation is an interesting and important topic that is currently under investigation. FOXO3a has been reported to regulate cell cycle arrest by activation of p27, p130, and p21, repression of cyclin D expression (G1/S arrest) or activation of cyclin G2 (G0/G1 arrest) (Kops et al. 2002; Hauck et al. 2007; Rathbone et al. 2008). In NPC, it may be possible that CXCL12 induces FOXO3a inactivation, leading to the downregulation of p27 or p21 activity and/or upregulation of cyclins that promote the cell cycle progression. On the other hand, FOXO3a is also critical for cell survival and death. In our culture system, deprivation of growth factors EGF and bFGF may induce NPC apoptosis. The proliferative effect of CXCL12 on NPC may be partially based on anti-apoptosis or the promotion of cell survival. Further studies will be carried out to explore the critical roles of FOXO3a in NPC functions including apoptosis and cell survival. Thus, the exact role of FOXO3a and the associated up and down stream factors in the regulation of NPC proliferation certainly warrant further investigation. Nevertheless, the important link between CXCL12-mediated human NPC proliferation and FOXO3a phosphorylation (inactivation) suggests an essential role of FOXO3a in the regulation of NPC proliferation.

In summary, the present study strongly suggests that CXCL12 is capable of stimulating the phosphorylation of the Forkhead transcription factor FOXO3a via the PI3K/Akt kinase pathway in human NPC. The CXCL12/CXCR4 interaction in the regulation of NPC proliferation may act to maintain the neural precursor pool during neurogenesis. Furthermore, the phosphorylation of FOXO3a by CXCL12 may be an additional new mechanism by which CXCL12 promotes cell proliferation in the brain.

Acknowledgements

This work was supported in part by research grants by the National Institutes of Health: R01 NS 41858-01, R01 NS 061642-01, R21 MH 083525-01, P01 NS043985, P20 RR15635-01 (JZ) and F31 NS 062659 (NPW). The authors kindly acknowledge Mr. Matthew Beaver and Ms. Li Wu who provided technical support for this work. Ms. Tess Eidem and Kelly Long provided valuable comments and suggestions about the manuscript. Dr. Charles Kuszynski and Ms. Victoria Smith performed the flow cytometry support. Ms. Julie Ditter, Johna Belling, Robin Taylor, Myhanh Che, Emilie Scoggins, Mary Cavell and Mr. Na Ly provided outstanding administrative support.

Reference

  1. Arai J, Yasukawa M, Yakushijin Y, Miyazaki T, Fujita S. Stromal cells in lymph nodes attract B-lymphoma cells via production of stromal cell-derived factor-1. Eur J Haematol. 2000;64:323–332. doi: 10.1034/j.1600-0609.2000.90147.x. [DOI] [PubMed] [Google Scholar]
  2. Bagri A, Gurney T, He X, Zou YR, Littman DR, Tessier-Lavigne M, Pleasure SJ. The chemokine SDF1 regulates migration of dentate granule cells. Development. 2002;129:4249–4260. doi: 10.1242/dev.129.18.4249. [DOI] [PubMed] [Google Scholar]
  3. Bajetto A, Bonavia R, Barbero S, Piccioli P, Costa A, Florio T, Schettini G. Glial and neuronal cells express functional chemokine receptor CXCR4 and its natural ligand stromal cell-derived factor 1. J Neurochem. 1999;73:2348–2357. doi: 10.1046/j.1471-4159.1999.0732348.x. [DOI] [PubMed] [Google Scholar]
  4. Birkenkamp KU, Essafi A, van der Vos KE, da Costa M, Hui RC, Holstege F, Koenderman L, Lam EW, Coffer PJ. FOXO3a induces differentiation of Bcr-Abl-transformed cells through transcriptional down-regulation of Id1. J Biol Chem. 2007;282:2211–2220. doi: 10.1074/jbc.M606669200. [DOI] [PubMed] [Google Scholar]
  5. Brunet A, Park J, Tran H, Hu LS, Hemmings BA, Greenberg ME. Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a) Mol Cell Biol. 2001;21:952–965. doi: 10.1128/MCB.21.3.952-965.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC, Blenis J, Greenberg ME. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell. 1999;96:857–868. doi: 10.1016/s0092-8674(00)80595-4. [DOI] [PubMed] [Google Scholar]
  7. Calnan DR, Brunet A. The FoxO code. Oncogene. 2008;27:2276–2288. doi: 10.1038/onc.2008.21. [DOI] [PubMed] [Google Scholar]
  8. Ceradini DJ, Kulkarni AR, Callaghan MJ, Tepper OM, Bastidas N, Kleinman ME, Capla JM, Galiano RD, Levine JP, Gurtner GC. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1. Nat Med. 2004;10:858–864. doi: 10.1038/nm1075. [DOI] [PubMed] [Google Scholar]
  9. Chang F, Lee JT, Navolanic PM, Steelman LS, Shelton JG, Blalock WL, Franklin RA, McCubrey JA. Involvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy. Leukemia. 2003;17:590–603. doi: 10.1038/sj.leu.2402824. [DOI] [PubMed] [Google Scholar]
  10. Cui M, Huang Y, Zhao Y, Zheng J. Transcription factor FOXO3a mediates apoptosis in HIV-1-infected macrophages. J Immunol. 2008;180:898–906. doi: 10.4049/jimmunol.180.2.898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dugourd C, Gervais M, Corvol P, Monnot C. Akt is a major downstream target of PI3-kinase involved in angiotensin II-induced proliferation. Hypertension. 2003;41:882–890. doi: 10.1161/01.HYP.0000060821.62417.35. [DOI] [PubMed] [Google Scholar]
  12. Dziembowska M, Tham TN, Lau P, Vitry S, Lazarini F, Dubois-Dalcq M. A role for CXCR4 signaling in survival and migration of neural and oligodendrocyte precursors. Glia. 2005;50:258–269. doi: 10.1002/glia.20170. [DOI] [PubMed] [Google Scholar]
  13. Emsley JG, Mitchell BD, Kempermann G, Macklis JD. Adult neurogenesis and repair of the adult CNS with neural progenitors, precursors, and stem cells. Prog Neurobiol. 2005;75:321–341. doi: 10.1016/j.pneurobio.2005.04.002. [DOI] [PubMed] [Google Scholar]
  14. Fallon J, Reid S, Kinyamu R, Opole I, Opole R, Baratta J, Korc M, Endo TL, Duong A, Nguyen G, Karkehabadhi M, Twardzik D, Patel S, Loughlin S. In vivo induction of massive proliferation, directed migration, and differentiation of neural cells in the adult mammalian brain. Proc Natl Acad Sci U S A. 2000;97:14686–14691. doi: 10.1073/pnas.97.26.14686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Finnberg N, El-Deiry WS. Activating FOXO3a, NF-kappaB and p53 by targeting IKKs: an effective multi-faceted targeting of the tumor-cell phenotype? Cancer Biol Ther. 2004;3:614–616. doi: 10.4161/cbt.3.7.1057. [DOI] [PubMed] [Google Scholar]
  16. Fruman DA, Meyers RE, Cantley LC. Phosphoinositide kinases. Annu Rev Biochem. 1998;67:481–507. doi: 10.1146/annurev.biochem.67.1.481. [DOI] [PubMed] [Google Scholar]
  17. Fu Z, Tindall DJ. FOXOs, cancer and regulation of apoptosis. Oncogene. 2008;27:2312–2319. doi: 10.1038/onc.2008.24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gage FH. Mammalian neural stem cells. Science. 2000;287:1433–1438. doi: 10.1126/science.287.5457.1433. [DOI] [PubMed] [Google Scholar]
  19. Gleichmann M, Gillen C, Czardybon M, Bosse F, Greiner-Petter R, Auer J, Muller HW. Cloning and characterization of SDF-1gamma, a novel SDF-1 chemokine transcript with developmentally regulated expression in the nervous system. Eur J Neurosci. 2000;12:1857–1866. doi: 10.1046/j.1460-9568.2000.00048.x. [DOI] [PubMed] [Google Scholar]
  20. Gong X, He X, Qi L, Zuo H, Xie Z. Stromal cell derived factor-1 acutely promotes neural progenitor cell proliferation in vitro by a mechanism involving the ERK1/2 and PI-3K signal pathways. Cell Biol Int. 2006;30:466–471. doi: 10.1016/j.cellbi.2006.01.007. [DOI] [PubMed] [Google Scholar]
  21. Graff JR, Konicek BW, McNulty AM, Wang Z, Houck K, Allen S, Paul JD, Hbaiu A, Goode RG, Sandusky GE, Vessella RL, Neubauer BL. Increased AKT activity contributes to prostate cancer progression by dramatically accelerating prostate tumor growth and diminishing p27Kip1 expression. J Biol Chem. 2000;275:24500–24505. doi: 10.1074/jbc.M003145200. [DOI] [PubMed] [Google Scholar]
  22. Greer EL, Brunet A. FOXO transcription factors at the interface between longevity and tumor suppression. Oncogene. 2005;24:7410–7425. doi: 10.1038/sj.onc.1209086. [DOI] [PubMed] [Google Scholar]
  23. Greer EL, Brunet A. FOXO transcription factors in ageing and cancer. Acta Physiol (Oxf) 2008;192:19–28. doi: 10.1111/j.1748-1716.2007.01780.x. [DOI] [PubMed] [Google Scholar]
  24. Hauck L, Harms C, Grothe D, An J, Gertz K, Kronenberg G, Dietz R, Endres M, von Harsdorf R. Critical role for FoxO3a-dependent regulation of p21CIP1/WAF1 in response to statin signaling in cardiac myocytes. Circ Res. 2007;100:50–60. doi: 10.1161/01.RES.0000254704.92532.b9. [DOI] [PubMed] [Google Scholar]
  25. Horner PJ, Gage FH. Regenerating the damaged central nervous system. Nature. 2000;407:963–970. doi: 10.1038/35039559. [DOI] [PubMed] [Google Scholar]
  26. Huang H, Regan KM, Wang F, Wang D, Smith DI, van Deursen JM, Tindall DJ. Skp2 inhibits FOXO1 in tumor suppression through ubiquitin-mediated degradation. Proc Natl Acad Sci U S A. 2005;102:1649–1654. doi: 10.1073/pnas.0406789102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jazin E, Soderstrom S, Ebendal T, Larhammar D. Embryonic expression of the mRNA for the rat homologue of the fusin/CXCR4 HIV-1 co-receptor. J. Neuroimmunol. 1997;79:148–154. doi: 10.1016/s0165-5728(97)00117-3. [DOI] [PubMed] [Google Scholar]
  28. Kaestner KH, Knochel W, Martinez DE. Unified nomenclature for the winged helix/forkhead transcription factors. Genes Dev. 2000;14:142–146. [PubMed] [Google Scholar]
  29. Khan MZ, Brandimarti R, Musser BJ, Resue DM, Fatatis A, Meucci O. The chemokine receptor CXCR4 regulates cell-cycle proteins in neurons. J Neurovirol. 2003;9:300–314. doi: 10.1080/13550280390201010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kim SJ, Cheon SH, Yoo SJ, Kwon J, Park JH, Kim CG, Rhee K, You S, Lee JY, Roh SI, Yoon HS. Contribution of the PI3K/Akt/PKB signal pathway to maintenance of self-renewal in human embryonic stem cells. FEBS Lett. 2005;579:534–540. doi: 10.1016/j.febslet.2004.12.024. [DOI] [PubMed] [Google Scholar]
  31. Klein RS, Rubin JB, Gibson HD, DeHaan EN, Alvarez-Hernandez X, Segal RA, Luster AD. SDF-1 alpha induces chemotaxis and enhances Sonic hedgehog-induced proliferation of cerebellar granule cells. Development. 2001;128:1971–1981. doi: 10.1242/dev.128.11.1971. [DOI] [PubMed] [Google Scholar]
  32. Kops GJ, Medema RH, Glassford J, Essers MA, Dijkers PF, Coffer PJ, Lam EW, Burgering BM. Control of cell cycle exit and entry by protein kinase B-regulated forkhead transcription factors. Mol Cell Biol. 2002;22:2025–2036. doi: 10.1128/MCB.22.7.2025-2036.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Liang J, Zubovitz J, Petrocelli T, Kotchetkov R, Connor MK, Han K, Lee JH, Ciarallo S, Catzavelos C, Beniston R, Franssen E, Slingerland JM. PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest. Nat Med. 2002;8:1153–1160. doi: 10.1038/nm761. [DOI] [PubMed] [Google Scholar]
  34. Lu M, Grove EA, Miller RJ. Abnormal development of the hippocampal dentate gyrus in mice lacking the CXCR4 chemokine receptor. Proc Natl Acad Sci U S A. 2002;99:7090–7095. doi: 10.1073/pnas.092013799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ma Q, Jones D, Borghesani PR, Segal RA, Nagasawa T, Kishimoto T, Bronson RT, Springer TA. Impaired B -lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4 and SDF 1 deficient mice. Proc Natl Acad Sci. 1998;95:9448–9453. doi: 10.1073/pnas.95.16.9448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Maiese K, Chong ZZ, Shang YC. “Sly as a FOXO”: new paths with Forkhead signaling in the brain. Curr Neurovasc Res. 2007;4:295–302. doi: 10.2174/156720207782446306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. McGrath KE, Koniski AD, Maltby KM, McGann JK, Palis J. Embryonic expression and function of the chemokine SDF-1 and its receptor, CXCR4. Dev Biol. 1999;213:442–456. doi: 10.1006/dbio.1999.9405. [DOI] [PubMed] [Google Scholar]
  38. Medema R, Kops G, Bos J, Burgering B. AFX-like forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1. Nature. 2000;404:782–787. doi: 10.1038/35008115. [DOI] [PubMed] [Google Scholar]
  39. Ming GL, Song H. Adult neurogenesis in the mammalian central nervous system. Annu Rev Neurosci. 2005;28:223–250. doi: 10.1146/annurev.neuro.28.051804.101459. [DOI] [PubMed] [Google Scholar]
  40. Muise-Helmericks RC, Grimes HL, Bellacosa A, Malstrom SE, Tsichlis PN, Rosen N. Cyclin D expression is controlled post-transcriptionally via a phosphatidylinositol 3-kinase/Akt-dependent pathway. J Biol Chem. 1998;273:29864–29872. doi: 10.1074/jbc.273.45.29864. [DOI] [PubMed] [Google Scholar]
  41. Nakamura N, Ramaswamy S, Vazquez F, Signoretti S, Loda M, Sellers WR. Forkhead transcription factors are critical effectors of cell death and cell cycle arrest downstream of PTEN. Mol Cell Biol. 2000;20:8969–8982. doi: 10.1128/mcb.20.23.8969-8982.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Paling NR, Wheadon H, Bone HK, Welham MJ. Regulation of embryonic stem cell self-renewal by phosphoinositide 3-kinase-dependent signaling. J Biol Chem. 2004;279:48063–48070. doi: 10.1074/jbc.M406467200. [DOI] [PubMed] [Google Scholar]
  43. Peng H, Kolb R, Kennedy JE, Zheng J. Differential expression of CXCL12 and CXCR4 during human fetal neural progenitor cell differentiation. J Neuroimmune Pharmacol. 2007;2:251–258. doi: 10.1007/s11481-007-9081-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Peng H, Huang Y, Duan Z, Erdmann N, Xu D, Herek S, Zheng J. Cellular IAP1 regulates TRAIL-induced apoptosis in human fetal cortical neural progenitor cells. J Neurosci Res. 2005;82:295–305. doi: 10.1002/jnr.20629. [DOI] [PubMed] [Google Scholar]
  45. Peng H, Whitney N, Wu Y, Tian C, Dou H, Zhou Y, Zheng J. HIV-1-infected and/or immune-activated macrophage-secreted TNF-alpha affects human fetal cortical neural progenitor cell proliferation and differentiation. Glia. 2008;56:903–916. doi: 10.1002/glia.20665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Peng H, Huang Y, Rose J, Erichsen D, Herek S, Fujii N, Tamamura H, Zheng J. Stromal cell-derived factor 1 mediated CXCR4 signaling in rat and human cortical neural progenitor cells. Journal of Neuroscience Research. 2004;76:35–50. doi: 10.1002/jnr.20045. [DOI] [PubMed] [Google Scholar]
  47. Peng H, Erdmann N, Whitney N, Dou H, Gorantla S, Gendelman HE, Ghorpade A, Zheng J. HIV-1-infected and/or immune activated macrophages regulate astrocyte SDF-1 production through IL-1beta. Glia. 2006;54:619–629. doi: 10.1002/glia.20409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Peng H, Whitney N, Zheng J. HIV-1-infected and/or immune activated macrophages affect human neural progenitor cell function. The 14th Conference on Retroviruses and Opportunistic Infections; Los Angeles, CA. 2007. [Google Scholar]
  49. Plas DR, Thompson CB. Akt-dependent transformation: there is more to growth than just surviving. Oncogene. 2005;24:7435–7442. doi: 10.1038/sj.onc.1209097. [DOI] [PubMed] [Google Scholar]
  50. Rathbone CR, Booth FW, Lees SJ. FoxO3a preferentially induces p27Kip1 expression while impairing muscle precursor cell-cycle progression. Muscle Nerve. 2008;37:84–89. doi: 10.1002/mus.20897. [DOI] [PubMed] [Google Scholar]
  51. Rossi D, Zlotnik A. The biology of chemokines and their receptors. Annu Rev Immunol. 2000;18:217–242. doi: 10.1146/annurev.immunol.18.1.217. [DOI] [PubMed] [Google Scholar]
  52. Schonemeier B, Schulz S, Hoellt V, Stumm R. Enhanced expression of the CXCl12/SDF-1 chemokine receptor CXCR7 after cerebral ischemia in the rat brain. J Neuroimmunol. 2008;198:39–45. doi: 10.1016/j.jneuroim.2008.04.010. [DOI] [PubMed] [Google Scholar]
  53. Sedding DG. FoxO transcription factors in oxidative stress response and ageing--a new fork on the way to longevity? Biol Chem. 2008;389:279–283. doi: 10.1515/BC.2008.033. [DOI] [PubMed] [Google Scholar]
  54. Senf SM, Dodd SL, McClung JM, Judge AR. Hsp70 overexpression inhibits NF-kappaB and Foxo3a transcriptional activities and prevents skeletal muscle atrophy. Faseb J. 2008;22:3836–3845. doi: 10.1096/fj.08-110163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Shin I, Bakin AV, Rodeck U, Brunet A, Arteaga CL. Transforming growth factor beta enhances epithelial cell survival via Akt-dependent regulation of FKHRL1. Mol Biol Cell. 2001;12:3328–3339. doi: 10.1091/mbc.12.11.3328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Shiojima I, Yefremashvili M, Luo Z, Kureishi Y, Takahashi A, Tao J, Rosenzweig A, Kahn CR, Abel ED, Walsh K. Akt signaling mediates postnatal heart growth in response to insulin and nutritional status. J Biol Chem. 2002;277:37670–37677. doi: 10.1074/jbc.M204572200. [DOI] [PubMed] [Google Scholar]
  57. Stumm RK, Zhou C, Ara T, Lazarini F, Dubois-Dalcq M, Nagasawa T, Hollt V, Schulz S. CXCR4 regulates interneuron migration in the developing neocortex. J Neurosci. 2003;23:5123–5130. doi: 10.1523/JNEUROSCI.23-12-05123.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Tothova Z, Gilliland DG. FoxO transcription factors and stem cell homeostasis: insights from the hematopoietic system. Cell Stem Cell. 2007;1:140–152. doi: 10.1016/j.stem.2007.07.017. [DOI] [PubMed] [Google Scholar]
  59. Tran H, Brunet A, Griffith EC, Greenberg ME. The many forks in FOXO's road. Sci STKE. 2003;2003:RE5. doi: 10.1126/stke.2003.172.re5. [DOI] [PubMed] [Google Scholar]
  60. Tran PB, Ren D, Veldhouse TJ, Miller RJ. Chemokine receptors are expressed widely by embryonic and adult neural progenitor cells. J Neurosci Res. 2004;76:20–34. doi: 10.1002/jnr.20001. [DOI] [PubMed] [Google Scholar]
  61. Tsai KL, Sun YJ, Huang CY, Yang JY, Hung MC, Hsiao CD. Crystal structure of the human FOXO3a-DBD/DNA complex suggests the effects of post-translational modification. Nucleic Acids Res. 2007;35:6984–6994. doi: 10.1093/nar/gkm703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Urbich C, Knau A, Fichtlscherer S, Walter DH, Bruhl T, Potente M, Hofmann WK, de Vos S, Zeiher AM, Dimmeler S. FOXO-dependent expression of the proapoptotic protein Bim: pivotal role for apoptosis signaling in endothelial progenitor cells. Faseb J. 2005;19:974–976. doi: 10.1096/fj.04-2727fje. [DOI] [PubMed] [Google Scholar]
  63. van Biesen T, Luttrell LM, Hawes BE, Lefkowitz RJ. Mitogenic signaling via G protein-coupled receptors. Endocr Rev. 1996;17:698–714. doi: 10.1210/edrv-17-6-698. [DOI] [PubMed] [Google Scholar]
  64. Vlahakis SR, Villasis-Keever A, Gomez T, Vanegas M, Vlahakis N, Paya CV. G protein-coupled chemokine receptors induce both survival and apoptotic signaling pathways. J Immunol. 2002;169:5546–5554. doi: 10.4049/jimmunol.169.10.5546. [DOI] [PubMed] [Google Scholar]
  65. Weigel D, Jackle H. The fork head domain: a novel DNA binding motif of eukaryotic transcription factors? Cell. 1990;63:455–456. doi: 10.1016/0092-8674(90)90439-l. [DOI] [PubMed] [Google Scholar]
  66. Whitney NP, Peng H, Erdmann NB, Tian C, Monaghan DT, Zheng JC. Calcium-permeable AMPA receptors containing Q/R-unedited GluR2 direct human neural progenitor cell differentiation to neurons. Faseb J. 2008 doi: 10.1096/fj.07-104661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Yang JY, Zong CS, Xia W, Yamaguchi H, Ding Q, Xie X, Lang JY, Lai CC, Chang CJ, Huang WC, Huang H, Kuo HP, Lee DF, Li LY, Lien HC, Cheng X, Chang KJ, Hsiao CD, Tsai FJ, Tsai CH, Sahin AA, Muller WJ, Mills GB, Yu D, Hortobagyi GN, Hung MC. ERK promotes tumorigenesis by inhibiting FOXO3a via MDM2-mediated degradation. Nat Cell Biol. 2008a;10:138–148. doi: 10.1038/ncb1676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Yang W, Dolloff NG, El-Deiry WS. ERK and MDM2 prey on FOXO3a. Nat Cell Biol. 2008b;10:125–126. doi: 10.1038/ncb0208-125. [DOI] [PubMed] [Google Scholar]
  69. You H, Jang Y, You-Ten AI, Okada H, Liepa J, Wakeham A, Zaugg K, Mak TW. p53-dependent inhibition of FKHRL1 in response to DNA damage through protein kinase SGK1. Proc Natl Acad Sci U S A. 2004;101:14057–14062. doi: 10.1073/pnas.0406286101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Zheng J, Thylin M, Ghorpade A, Xiong H, Persidsky Y, Cotter R, Niemann D, Che M, Zeng Y, Gelbard H, Shepard R, Swartz J, Gendelman H. Intracellular CXCR4 signaling, neuronal apoptosis and neuropathogenic mechanisms of HIV-1-associated dementia. J Neuroimmunol. 1999;98:185–200. doi: 10.1016/s0165-5728(99)00049-1. [DOI] [PubMed] [Google Scholar]
  71. Zhou BP, Liao Y, Xia W, Spohn B, Lee MH, Hung MC. Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neuoverexpressing cells. Nat Cell Biol. 2001;3:245–252. doi: 10.1038/35060032. [DOI] [PubMed] [Google Scholar]
  72. Zhu S, Evans S, Yan B, Povsic TJ, Tapson V, Goldschmidt-Clermont PJ, Dong C. Transcriptional regulation of Bim by FOXO3a and Akt mediates scleroderma serum-induced apoptosis in endothelial progenitor cells. Circulation. 2008;118:2156–2165. doi: 10.1161/CIRCULATIONAHA.108.787200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Zou YR, Kottmann AH, Kuroda M, Taniuchi I, Littman DR. Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature. 1998;393:595–599. doi: 10.1038/31269. [DOI] [PubMed] [Google Scholar]

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