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Recombinant insulin-like growth factor binding protein-4 inhibits proliferation and promotes differentiation of neural progenitor cells
Neuroscience Letters 642 (2017) 71–76
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Recombinant insulin-like growth factor binding protein-4 inhibits proliferation and promotes differentiation of neural progenitor cells Hanxu Niu, Rongbin Gou, Qunyuan Xu, Deyi Duan ∗ Department of Neurobiology, Beijing Center of Neural Regeneration and Repair, Beijing Institute for Brain Disorders, Laboratory of Neurodegenerative Disorders of the Ministry of Education, Capital Medical University, Beijing, 100069, China
h i g h l i g h t s • • • •
Blockade of IGF-IR in NPCs inhibits cell growth and enhances neuronal differentiation. Exogenous IGFBP4 inhibits proliferation and enhances neuronal differentiation of NPCs. IGFBP4 decreases Akt activation without affecting Erk and p38 phosphorylation in NPCs. IGFBP4 affects proliferation and differentiation of NPCs via IGF-IR signaling pathway.
a r t i c l e
i n f o
Article history: Received 21 November 2016 Received in revised form 16 January 2017 Accepted 28 January 2017 Available online 5 February 2017 Keywords: IGFBP4 Neural progenitor cells Cell proliferation Neural differentiation IGF-IR
a b s t r a c t Insulin-like growth factor (IGF) is involved in regulating many processes during neural development, and IGF binding protein-4 (IGFBP4) functions as a modulator of IGF actions or in an IGF-independent manner (e.g., via inhibiting Wnt/-catenin signaling). In the present study, neural progenitor cells (NPCs) were isolated from the forebrain of newborn mice to investigate effects of IGFBP4 on the proliferation and differentiation of NPCs. The proliferation of NPCs was evaluated using Cell Counting Kit-8 (CCK-8) after treatment with or without IGFBP4 as well as blockers of IGF-IR and -catenin. Phosphorylation levels of Akt, Erk1, 2 and p38 were analyzed by Western blotting. The differentiation of NPCs was evaluated using immunofluorescence and Western blotting. It was shown that exogenous IGFBP4 significantly inhibited the proliferation of NPCs and it did not induce a more pronounced inhibition of cell proliferation after blockade of IGF-IR but it did after antagonism of -catenin. Akt phosphorylation was significantly decreased and phosphorylation levels of Erk1, 2 and p38 were not significantly changed in IGFBP4treated NPCs. Excessive IGFBP4 significantly promoted NPCs to differentiate into astrocytes and neurons. These data suggested that exogenous IGFBP4 inhibits proliferation and promotes differentiation of neural progenitor cells mainly through IGF-IR signaling pathway. © 2017 Elsevier B.V. All rights reserved.
1. Introduction It has well been established that insulin-like growth factor (IGF) system plays an essential role in the normal growth and development of the brain [11]. IGF-I exerts pleiotropic effects on neural stem cells and mature neurons in the developing brain and neurogenesis, axon remodeling and de novo synaptogenesis in the embryonic and adult brain [11,14]. High affinity IGF binding proteins (IGFBPs), designated IGFBP1 through IGFBP6, have been proposed to inhibit the biological actions of IGFs by hindering their binding to IGF receptors in most circumstances [1,14], or to
∗ Corresponding author. E-mail address: [email protected] (D. Duan). http://dx.doi.org/10.1016/j.neulet.2017.01.066 0304-3940/© 2017 Elsevier B.V. All rights reserved.
enhance IGF actions in certain conditions [1]. Some IGFBPs were also reported to have IGF-independent actions which are mediated by interaction with cell surface ‘receptors’ (such as integrins, pertussis toxin-sensitive and –insensitive G-protein coupled receptors) or nuclear hormone receptors [1]. One IGF-independent action of IGFBP4 was identified to promote cardiogenesis of induced pluripotent stem cells through inhibiting -catenin signaling [18]. It was demonstrated that IGFBPs are relevant in development and maturation of the brain. The inhibitory effects of IGFBPs on the brain growth and the number of oligodendrocytes, astrocytes and neurons have been extensively investigated in transgenic mice that over-express IGFBP1-3,5,6 [14]. However, the roles of IGFBP4 in the brain remain to be elucidated. In the present study, the effects of IGFBP4 on the proliferation and differentiation of primary neural progenitor cells (NPCs) were
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observed and its IGF-dependent or -independent actions were evaluated after treatment of NPCs with blockers of IGF-IR and -catenin. The phosphorylation levels of Akt, p38 and Erk molecules were analyzed by Western blotting. 2. Materials and methods 2.1. Suspension culture of NPCs [6] Newborn mice were dissected in a laminar flow hood and the brain was transferred to a 35 mm dish containing cold PBS with penicillin-streptomycin (500 UI/ml–500 g/ml) (Merck Millipore, Darmstadt, Germany). The cortex and hippocampus were dissected under stereomicroscope, then minced mechanically in DMEM/F12 (Gibco-Invitrogen, Carlsbad, CA). Tissue fragments were further dissociated by gentle trituration to release single cells. The cell suspensions were centrifuged at 1000 rpm for 5 min, filtered through a 100-m filter. Cells were then transferred to 6-well plates and maintained at 37 ◦ C in DMEM/F12 growth medium supplied with 2% B27 serum-free supplements (Gibco-Invitrogen), 2 mM lglutamine (Gibco-Invitrogen), 20 ng/ml epidermal growth factor (EGF; R&D Systems, Minneapolis, MN), 20 ng/ml basic fibroblast growth factor (bFGF; R&D Systems), and penicillin-streptomycin (100 UI/ml–100 g/ml) [19]. Culture medium was changed and cells passaged every 3-4 days. NPCs at passage 4 were used in all experiments. The care and handling of the newborn mice was approved by the Beijing Municipal Administration Office for Laboratory Animals (approval number SCXK(Jing)2009-0007). 2.2. Adherent monolayer culture of NPCs An adherent monolayer of NPCs was cultured on substrates of poly-d-lysine (PDL) and laminin (R&D System, Gaithersburg, USA). PDL-laminin coated culturewares were prepared according to the Technical Manual Version 2.1.0 (2016) for In Vitro Proliferation and Differentiation of Mouse Neural Stem and Progenitor Cells Using NeuroCult (STEMCELL Technologies Inc, Vancouver, BC, Canada. Data available from: https://www.stemcell.com). The suspension cultures (free-floating neurospheres) of NPCs were harvested into a 15 ml polypropylene tube and centrifuged at 1000 rpm for 5 min. The cell pellets were suspended in 1 ml fresh medium, triturated several times until a single cell suspension was achieved, and then filtered through a 40 m filter. The cell suspensions were then seeded onto the pre-prepared PDL-laminin coated culturewares for cell proliferation test or immunocytochemistry. 2.3. Cell proliferation Dissociated single cell suspensions (105 /ml) were inoculated into a PDL-laminin coated 96-well plate (100 l/well) in triplicate or quintuplicate and cultured in a humidified incubator (37 ◦ C, 5% CO2). The culture medium was supplemented with the following reagents: a neutralized antibody against IGFBP4 (4 g/ml, R&D Systems, Minneapolis, MN), IGFBP4 (0.5 g/ml, R&D Systems) [17], the half maximal inhibitory concentration of AG1024 (AG1024 IC50, dissolved in DMSO at a final concentration of 7 M, Calbiochem, Merck Millipore, Billerica, MA, USA) [7], AG1024 (20 M), IGFBP4 + AG1024/AG1024 IC50, FH535 (dissolved in DMSO at 20 M, Tocris Bioscience, Avonmouth, Bristol, UK) [5,9], and IGFBP4 + FH535. Treatment with DMSO only was used as control. The cultures were fed with new medium every three days. Each day, 10 l of CCK-8 solution was added to each well and incubated for 4 h. The absorbance was measured at 450 nm using a microplate reader (ELx808, Biotek Instruments, Winooski, VT, USA). The experiments were repeated three times.
2.4. Cell differentiation Dissociated single cells were seeded onto PDL-laminin coated culturewares and cultured in DMEM/F12 differentiation medium with withdrawal of bFGF and EGF, and supplemented with 2% B27, 2 mM l-glutamine, 10% fetal bovine serum, and penicillinstreptomycin (100 UI/ml–100 g/ml) [19]. The cells grown on the coated glass coverslips placed in 24-well plates were used to evaluate the number of GFAP- and MAP2-positive cells by fluorescent immunocytochemistry, and the cells on the coated 90-mm Petri dishes were collected to analyze the expression of GFAP and MAP2 by Western blotting. 2.5. Immunocytochemistry The adherent monolayer cultures of NPCs on the coated glass coverslips were fixed by immersing in 4% paraformaldehyde for 30 min. The endogenous peroxidase was quenched by 1% hydrogen peroxide in 50% ethanol for 30 min at room temperature. After washes in PBS, the coverslips were blocked for 1 h at room temperature in a blocking solution (5% goat serum in PBS), followed by incubation at 4 ◦ C overnight with the following antibodies: a mouse monoclonal antibody to Nestin (1:200; Abcam, Cambridge, UK), rabbit anti-IGFBP4 (1:200; Santa Cruz, CA, USA), rabbit antiGFAP (1:1000; Abcam) and anti-MAP2 (1:500; Abcam). The cells were then incubated at room temperature for 2 h with goat antirabbit secondary antibodies conjugated with Fluor-488 (1:250; EarthOx, San Fransico, CA, USA) or a goat anti-mouse secondary antibody conjugated with Alexa Fluor-594 (1:250; EarthOx) in the dark. The coverslips were coverslipped with fluorescent mounting medium (Dako) and the fluorochrome labeled antibody was visualized and photographed under a fluorescent microscope (Olympus IX71) equipped with an Olympus camera (DP73). The number of GFAP- and MAP2-immunoreactive cells was determined related to the number of DAPI-stained nuclei. Approximately 200 cells were counted within randomly selected visual fields through fluorescence microscopy at 40× magnification. 2.6. Western blotting An adherent monolayer of NPCs was cultured on PDL-laminin coated 90 mm dishes with or without IGFBP4 for 5 d. The cells were collected and lysed for 30 min at 4 ◦ C with a 360-1 lysis buffer containing 50 mM Tris (pH7.2), 150 mM NaCl, 0.5% Nonidet P-40 (NP40), 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, and 0.1% leupeptin. The cell lysates were harvested and centrifuged at 4 ◦ C for 10 min at 12,000g to collect supernatant protein extracts. The concentration of total protein was tested using a BCA protein assay kit (Pierce, Rockford, IL, USA). Equal amounts of protein from each lysate (10 g) were loaded and size-fractionated by SDS polyacrylamide gel electrophoresis at a constant voltage of 120 V at 4 ◦ C. The proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (0.2 m; Millipore, Bedford, USA) at a constant current of 100 mA for 3 h at 4 ◦ C. The membrane was rinsed for 10 min with Tris-buffered saline supplemented with 0.05% Tween 20 (TBST, pH 7.4) followed by nonspecific binding with a blocking solution (10% non-fat dry milk in TBST). The blocked membrane was probed with antibodies raised against GFAP (1:10000; Abcam); MAP2 (1:1000; Abcam); Akt, p38 and Erk1,2, phospho-Akt (p-Akt), p-p38, and p-Erk1,2 (1:1000 each, CST, Danvers, MA, USA) overnight at 4 ◦ C. After wash three times (10 min each) in TBST, the membrane was incubated with a secondary goat anti-rabbit IgG or a goat anti-mouse IgG conjugated to horseradish peroxidase (1:5000 each; EarthOx) for 2 h at room temperature. Finally, the blots were developed by use of a SuperEnhanced chemiluminescence detection kit (Applygen Technologies
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Fig. 1. Culture and identification of the NPCs. NPCs formed suspension neurosphere-like structures in serum-free medium 10 d later (A) and adherent monolayer-structures on PDL-laminin substrate 24 h after re-seeding (B). Immunofluorescent staining revealed that NPCs expressed Nestin (C) and IGFBP4 (D).
Inc., Beijing, China), and the images of protein bands were captured on Kodak X-ray film. As an internal control, the expression of -actin was detected by a mouse monoclonal antibody against actin (1:2000; Beijing GuanXingYun Sci & Tech Co, Beijing, China) in the same membrane with the same procedures. Intensities of the blotted bands were acquired by scanning of the X-ray film with a FluorChem Q imaging system (Proteinsimple, Sata Clara, CA, USA) and quantified using a densitometric software ImageJ 1.49 v (Wayne Rasband, NIH, USA). The densitometric units of the bands were expressed relative to the values for -actin or unphosphorylated proteins. 2.7. Statistical analysis Data were expressed as means ± SEM. For comparisons between two groups, two-tailed Student’s t-test was performed. For multiple comparisons, one-way Analysis of Variance (ANOVA) followed by Bonferroni post hoc test was conducted using SPSS version 13.0. Evidence of a statistically significant difference between mean values was considered to be at P < 0.05. 3. Results
filament protein found in NPCs [8], was expressed mainly in the cell soma and the cellular processes of the adherent NPCs (Fig. 1C). IGFBP4 was expressed mainly in the cell soma (Fig. 1D).
3.2. Proliferation When physiologically secreted IGFBP4 protein was neutralized with a specific antibody and recombinant IGFBP4 protein was added into culture medium, the proliferation of NPCs was significantly suppressed (Fig. 2A). Most IGF actions, if not all, are mediated by IGF-IR, and IGFIR signaling is essential for the normal neural development [14] (Fig. 2B). To explore whether IGFBP4 action on NPC proliferation is IGF-dependent, AG1024 (a specific inhibitor of IGF-IR) and FH535 (a -catenin inhibitor) were separately added into adherent monolayer cultures of NPCs. Both AG1024 and FH535 significantly inhibited NPC proliferation compared with DMSO control. The addition of IGFBP4 and AG1024 did not lead to a more marked inhibition than AG1024 or IGFBP4 alone (Fig. 2C), neither did IGFBP4 and AG1024 IC50 in combination (data not shown). IGFBP4 and FH535 in combination inhibited NPC proliferation more strongly than FH535 or IGFBP4 alone (Fig. 2D).
3.1. Identification 3.3. IGF-IR signaling Cells isolated from newborn mice formed non-adherent (suspension) neurosphere-like structures in serum-free medium after 10 days of culture (Fig. 1A). Single cell suspensions seeded onto previously PDL-laminin coated plates formed an adherent monolayer of cells after 24 h (Fig. 1B). Nestin, a cytoskeletal intermediate
IGF-IR signaling events were measured by probing Western blots with antibodies against p-Akt, Akt, p-p38, p38, p-Erk1, 2, and Erk1, 2. The phosphorylation level of Akt was significantly decreased in cells treated with IGFBP4, and the activation of p38
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Fig. 2. Effects of IGFBP4 on proliferation of NPCs. When secreted IGFBP4 protein was neutralized or exogenous IGFBP4 protein was added, NPC proliferation was significantly suppressed (A). When IGF-IR and -catenin were inhibited with AG1024 and FH535 (B), cell proliferation was significantly inhibited (C, D). AG1024 and IGFBP4 in combination did not produce a more marked inhibition compared with AG1024 or IGFBP4 alone (C) but FH535 and IGFBP4 together did (D). Absorbance data were expressed as means ± SEM in bar graphs A, C and D.
Fig. 3. Phosphorylation levels of signaling molecules downstream IGF-IR. Western blotting analysis showed that IGFBP4 treatment resulted in significantly decreased Akt phosphorylation (A) and unchangeable phosphorylation of p38 (B) and Erk1, 2 (C) in the NPCs.
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Fig. 4. Differentiation of NPCs in FBS-medium without bFGF and EGF. Fluorescent immunocytochemistry (A) and bar graph (B), and Western blotting (C, D) revealed that IGFBP4 significantly increased glial and neuronal differentiation. Blockade of IGF-IR significantly enhanced neuronal differentiation (E).
and Erk1, 2 was not significantly changed after IGFBP4 treatment (Fig. 3). 3.4. Differentiation After culture in FBS-medium without bFGF and EGF, NPCs were induced to differentiate into astrocytes and neurons on PDL-laminin coated 24-well plates. Immunostaining with antibodies specific for GFAP and MAP2 (Fig. 4A) showed that IGFBP4 significantly promoted differentiation of NPCs (Fig. 4B). The differentiation promotion was further confirmed by Western blotting analysis of GFAP and MAP2 expression (Fig. 4C, D). Blockade of IGFIR significantly enhanced neuronal differentiation and IGFBP4 did not lead to a more marked neuronal differentiation in the presence of AG1024 (Fig. 4A, E). Glial differentiation remained unchangeable after treatment with AG1024 or AG1024 + IGFBP4 (Fig. 4E). 4. Discussion The present study demonstrated that blocking IGF-I signaling inhibits NPC proliferation and enhances neuronal differentiation, and exogenous IGFBP4 inhibits proliferation and enhances neuronal differentiation of NPCs as a regulator of IGF-I signaling mainly via down-regulating Akt activation. The influence of IGF-I on regulating proliferation of neural stem cells remains elusive. More proliferative cells are found in the
dentate gyrus of IGF-I knockout mice and bigger clonal neurospheres from IGF-I knockout mice are formed in culture [11,12]. In contrast, proliferation of neural stem cells is up-regulated in the hippocampus of IGF-I transgenic mice [11,20] and exogenous IGF-I promotes the proliferation of progenitor cells in the adult hippocampus [11]. Wnt signaling is implicated in many aspects of neural development through canonical (-catenin-dependent) or non-canonical (-catenin-independent) pathway, and canonical Wnt/-catenin signaling has been linked to promoting proliferation and specification of neural stem cells [10]. In the present study, blockade of IGF-IR and -catenin leads to significant decreases in the proliferation of NPCs in vitro, supporting the stimulatory effects of IGF-I [20] and Wnt/-catenin [10,15] on the proliferation of neural stem cells. We found that immunodepletion of endogenous IGFBP4 and administration of exogenous IGFBP4 suppress NPC proliferation. The role of IGFBP4 is very complex, and it is cell type and context dependent. IGFBP4 functions via predominantly inhibiting IGF actions [1]. However, genetic ablation of IGFBP4 was reported to reduce body mass of mice by 10–15% from embryonic day 14.5 to postnatal age of 14 weeks [13]. The beneficial influence of endogenous IGFBP4 on NPC proliferation was consistent with the requirement of IGFBP4 for normal prenatal growth in IGFBP4 knockout mice [13]. IGFBP4 was reported to trigger cellular senescence in mesenchymal stem cells [17], and inhibitive effects of exogenous IGFBP4 on NPC proliferation perhaps result from its pro-senescent effects. Nonetheless, further work will be needed
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to clarify the relationship between reduced cell proliferation and induced apoptosis in IGFBP4-treated NPCs. If the inhibitory effect of IGFBP4 is IGF-dependent, a more remarkable inhibition cannot be induced in the presence of AG1024, otherwise a cumulative inhibition of IGFBP4 (via IGFindependent) and AG1024 can be produced. Indeed, we did not find a more marked inhibitory effect on the proliferation of the NPCs treated with AG1024 and IGFBP4 in combination than with AG1024 or IGFBP4 alone. However, a more pronounced inhibitory effect was observed in the NPCs treated with IGFBP4 and FH535 than with IGFBP4 or FH535 alone. The data demonstrated that IGF-IR signaling pathway is mainly involved in IGFBP4 action on inhibiting NPC proliferation. Accumulating data show that IGF signals through Ras-Raf-MAPK and PI3K-Akt pathways in IGF neural actions and PI3K-Akt pathway is required for IGF-stimulated neural proliferation [14]. In the present study, a significantly decreased level of Akt phosphorylation was found in IGFBP4-treated NPCs, consistent with decreased Akt phosphorylation in IGF-IR-ablated olfactory neural stem cells [2]. Akt kinase can phosphorylate and inactivate glycogen synthase kinase 3 (GSK3), which in turn stabilizes cyclin D1 and promotes neural cell proliferation and survival [14]. It can be supposed that IGFBP4-induced decrease of NPC proliferation may mainly result from a significant decrease in Akt activation. The majority of accumulating findings show that IGF-I is crucial for differentiation of NPCs into neuronal lineage. Over-expression of IGF-I specifically in neural stem cells up-regulates glial and neuronal differentiation in adult transgenic mice [20]. However, conditional knockout of IGF-IR increases cumulative production of neuroblasts and neurons in the olfactory bulb of the mouse [2]. We found that blockade of IGF-IR promotes neuronal differentiation, consistent with the beneficial effect of suppressed IGF signaling on fostering olfactory bulb neurogenesis [2]. Administration of IGFBP4 is found to promote neuronal differentiation, but it did not promote more pronounced differentiation in the presence of AG1024, suggesting an involvement of the repression of IGF-IR signaling in IGFBP4-induced differentiation. Phosphorylated Akt is known to phosphorylate and inactivate GSK3, which in turn stabilizes -catenin by reducing its phosphorylation degradation [14]. Inhibition of PI3K/Akt signal pathway is reported to promote motor neuron differentiation of human endometrial stem cells [3], and loss of -catenin promotes neuronal differentiation [15]. Therefore, IGFBP4-induced decrease of Akt phosphorylation and thus degradation of -catenin perhaps underlie its stimulatory effects on neuronal differentiation. Erk [4,16] but not Akt [4] signaling is involved in astrocyte differentiation of neural stem cells, and inhibition of Erk signaling is reported to block astrocyte differentiation [4]. IGFBP4-promoted astrocyte differentiation is perhaps mediated by Erk signaling, which, unlike p-Akt, is not significantly decreased in IGFBP4-treated NPCs. AG1024 promoted neuronal differentiation more obviously than IGFBP4, and it perhaps decreased the phosphorylation levels of Erk1, 2, thereby preventing astrocyte differentiation. A further study will be needed to clarify the relationship between phosphorylation levels of Erk1, 2 and differentiation potential of NPCs into astrocytes after AG1024 treatment. The effects of IGFBP4 on NPC differentiation in the present study are in contrast to those of other members of IGFBP family. The reduced number of oligodendrocytes, astrocytes or neurons is observed in transgenic mice that over-express IGFBPs driven by promoters phosphoglycerate kinase (IGFBP-1,3), metallothioneinI (IGFBP-1,5), cytomegalovirus (IGFBP2), actin (IGFBP5), and GFAP (IGFBP6) [14]. The roles of IGFBP4 in regulating neural stem cell growth and development in the brain are being explored in recently-established transgenic mice over-expressing IGFBP4 specifically in neurons.
Conflict of interest The authors report no conflict of interest related to this study. References [1] L.A. Bach, Insulin-like growth factor binding proteins?an update, Pediatr. Endocrinol. Rev. 13 (2015) 521–530. [2] Z. Chaker, S. Aid, H. Berry, M. Holzenberger, Suppression of IGF-I signals in neural stem cells enhances neurogenesis and olfactory function during aging, Aging Cell 14 (2015) 847–856. [3] S. Ebrahimi-Barough, E. Hoveizi, M. Yazdankhah, J. Ai, M. Khakbiz, F. Faghihi, R. Tajerian, N. Bayat, Inhibitor of PI3 K/Akt signaling pathway small molecule promotes motor neuron differentiation of human endometrial stem cells cultured on electrospun biocomposite polycaprolactone/collagen scaffolds, Mol. Neurobiol. (2016). [4] I. Fujimoto, K. Hasegawa, K. Fujiwara, M. Yamada, K. Yoshikawa, Necdin controls EGFR signaling linked to astrocyte differentiation in primary cortical progenitor cells, Cell Signal. 28 (2016) 94–107. [5] S. Handeli, J.A. Simon, A small-molecule inhibitor of Tcf/beta-catenin signaling down-regulates PPARgamma and PPARdelta activities, Mol. Cancer Ther. 7 (2008) 521–529. [6] Z. Li, K. Li, L. Zhu, Q. Kan, Y. Yan, P. Kumar, H. Xu, A. Rostami, G.X. Zhang, Inhibitory effect of IL-17 on neural stem cell proliferation and neural cell differentiation, BMC Immunol. 14 (2013) 20. [7] J. Ligeza, A. Klein, Growth factor/growth factor receptor loops in autocrine growth regulation of human prostate cancer DU145 cells, Acta Biochim. Pol. 58 (2011) 391–396. [8] C. Liu, Y. Zhong, A. Apostolou, S. Fang, Neural differentiation of human embryonic stem cells as an in vitro tool for the study of the expression patterns of the neuronal cytoskeleton during neurogenesis, Biochem. Biophys. Res. Commun. 439 (2013) 154–159. [9] L. Montorsi, S. Parenti, L. Losi, F. Ferrarini, C. Gemelli, A. Rossi, G. Manco, S. Ferrari, B. Calabretta, E. Tagliafico, T. Zanocco-Marani, A. Grande, Expression of mu-protocadherin is negatively regulated by the activation of the beta-catenin signaling pathway in normal and cancer colorectal enterocytes, Cell Death Dis. 7 (2016) e2263. [10] K.A. Mulligan, B.N. Cheyette, Wnt signaling in vertebrate neural development and function, J. Neuroimmune Pharmacol. 7 (2012) 774–787. [11] V. Nieto-Estevez, C. Defterali, C. Vicario-Abejon, IGF-I: a key growth factor that regulates neurogenesis and synaptogenesis from embryonic to adult stages of the brain, Front. Neurosci. 10 (2016) 52. [12] V. Nieto-Estevez, C.O. Oueslati-Morales, L. Li, J. Pickel, A.V. Morales, C. Vicario-Abejon, Brain insulin-like growth factor-i directs the transition from stem cells to mature neurons during postnatal/adult hippocampal neurogenesis, Stem Cells 34 (2016) 2194–2209. [13] Y. Ning, A.G. Schuller, C.A. Conover, J.E. Pintar, Insulin-like growth factor (IGF) binding protein-4 is both a positive and negative regulator of IGF activity in vivo, Mol. Endocrinol. 22 (2008) 1213–1225. [14] J. O’Kusky, P. Ye, Neurodevelopmental effects of insulin-like growth factor signaling, Front. Neuroendocrinol. 33 (2012) 230–251. [15] Y. Pei, S.N. Brun, S.L. Markant, W. Lento, P. Gibson, M.M. Taketo, M. Giovannini, R.J. Gilbertson, R.J. Wechsler-Reya, WNT signaling increases proliferation and impairs differentiation of stem cells in the developing cerebellum, Development 139 (2012) 1724–1733. [16] P. Selvaraj, L. Xiao, C. Lee, S.R. Murthy, N.X. Cawley, M. Lane, I. Merchenthaler, S. Ahn, Y.P. Loh, Neurotrophic factor-alpha1: a key Wnt-beta-catenin dependent anti-proliferation factor and ERK-Sox9 activated inducer of embryonic neural stem cell differentiation to astrocytes in neurodevelopment, Stem Cells (2016). [17] V. Severino, N. Alessio, A. Farina, A. Sandomenico, M. Cipollaro, G. Peluso, U. Galderisi, A. Chambery, Insulin-like growth factor binding proteins 4 and 7 released by senescent cells promote premature senescence in mesenchymal stem cells, Cell Death Dis. 4 (2013) e911. [18] Y. Xue, Y. Yan, H. Gong, B. Fang, Y. Zhou, Z. Ding, P. Yin, G. Zhang, Y. Ye, C. Yang, J. Ge, Y. Zou, Insulin-like growth factor binding protein 4 enhances cardiomyocytes induction in murine-induced pluripotent stem cells, J. Cell Biochem. 115 (2014) 1495–1504. [19] J. Yang, P. Gu, S. Menges, H. Klassen, Quantitative changes in gene transcription during induction of differentiation in porcine neural progenitor cells, Mol. Vis. 18 (2012) 1484–1504. [20] H. Yuan, R. Chen, L. Wu, Q. Chen, A. Hu, T. Zhang, Z. Wang, X. Zhu, The regulatory mechanism of neurogenesis by IGF-1 in adult mice, Mol. Neurobiol. 51 (2015) 512–522.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Recombinant insulin-like growth factor binding protein-4 inhibits proliferation and promotes differentiation of neural progenitor cells Hanxu Niu, Rongbin Gou, Qunyuan Xu, Deyi Duan ∗ Department of Neurobiology, Beijing Center of Neural Regeneration and Repair, Beijing Institute for Brain Disorders, Laboratory of Neurodegenerative Disorders of the Ministry of Education, Capital Medical University, Beijing, 100069, China
h i g h l i g h t s • • • •
Blockade of IGF-IR in NPCs inhibits cell growth and enhances neuronal differentiation. Exogenous IGFBP4 inhibits proliferation and enhances neuronal differentiation of NPCs. IGFBP4 decreases Akt activation without affecting Erk and p38 phosphorylation in NPCs. IGFBP4 affects proliferation and differentiation of NPCs via IGF-IR signaling pathway.
a r t i c l e
i n f o
Article history: Received 21 November 2016 Received in revised form 16 January 2017 Accepted 28 January 2017 Available online 5 February 2017 Keywords: IGFBP4 Neural progenitor cells Cell proliferation Neural differentiation IGF-IR
a b s t r a c t Insulin-like growth factor (IGF) is involved in regulating many processes during neural development, and IGF binding protein-4 (IGFBP4) functions as a modulator of IGF actions or in an IGF-independent manner (e.g., via inhibiting Wnt/-catenin signaling). In the present study, neural progenitor cells (NPCs) were isolated from the forebrain of newborn mice to investigate effects of IGFBP4 on the proliferation and differentiation of NPCs. The proliferation of NPCs was evaluated using Cell Counting Kit-8 (CCK-8) after treatment with or without IGFBP4 as well as blockers of IGF-IR and -catenin. Phosphorylation levels of Akt, Erk1, 2 and p38 were analyzed by Western blotting. The differentiation of NPCs was evaluated using immunofluorescence and Western blotting. It was shown that exogenous IGFBP4 significantly inhibited the proliferation of NPCs and it did not induce a more pronounced inhibition of cell proliferation after blockade of IGF-IR but it did after antagonism of -catenin. Akt phosphorylation was significantly decreased and phosphorylation levels of Erk1, 2 and p38 were not significantly changed in IGFBP4treated NPCs. Excessive IGFBP4 significantly promoted NPCs to differentiate into astrocytes and neurons. These data suggested that exogenous IGFBP4 inhibits proliferation and promotes differentiation of neural progenitor cells mainly through IGF-IR signaling pathway. © 2017 Elsevier B.V. All rights reserved.
1. Introduction It has well been established that insulin-like growth factor (IGF) system plays an essential role in the normal growth and development of the brain [11]. IGF-I exerts pleiotropic effects on neural stem cells and mature neurons in the developing brain and neurogenesis, axon remodeling and de novo synaptogenesis in the embryonic and adult brain [11,14]. High affinity IGF binding proteins (IGFBPs), designated IGFBP1 through IGFBP6, have been proposed to inhibit the biological actions of IGFs by hindering their binding to IGF receptors in most circumstances [1,14], or to
∗ Corresponding author. E-mail address: [email protected] (D. Duan). http://dx.doi.org/10.1016/j.neulet.2017.01.066 0304-3940/© 2017 Elsevier B.V. All rights reserved.
enhance IGF actions in certain conditions [1]. Some IGFBPs were also reported to have IGF-independent actions which are mediated by interaction with cell surface ‘receptors’ (such as integrins, pertussis toxin-sensitive and –insensitive G-protein coupled receptors) or nuclear hormone receptors [1]. One IGF-independent action of IGFBP4 was identified to promote cardiogenesis of induced pluripotent stem cells through inhibiting -catenin signaling [18]. It was demonstrated that IGFBPs are relevant in development and maturation of the brain. The inhibitory effects of IGFBPs on the brain growth and the number of oligodendrocytes, astrocytes and neurons have been extensively investigated in transgenic mice that over-express IGFBP1-3,5,6 [14]. However, the roles of IGFBP4 in the brain remain to be elucidated. In the present study, the effects of IGFBP4 on the proliferation and differentiation of primary neural progenitor cells (NPCs) were
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observed and its IGF-dependent or -independent actions were evaluated after treatment of NPCs with blockers of IGF-IR and -catenin. The phosphorylation levels of Akt, p38 and Erk molecules were analyzed by Western blotting. 2. Materials and methods 2.1. Suspension culture of NPCs [6] Newborn mice were dissected in a laminar flow hood and the brain was transferred to a 35 mm dish containing cold PBS with penicillin-streptomycin (500 UI/ml–500 g/ml) (Merck Millipore, Darmstadt, Germany). The cortex and hippocampus were dissected under stereomicroscope, then minced mechanically in DMEM/F12 (Gibco-Invitrogen, Carlsbad, CA). Tissue fragments were further dissociated by gentle trituration to release single cells. The cell suspensions were centrifuged at 1000 rpm for 5 min, filtered through a 100-m filter. Cells were then transferred to 6-well plates and maintained at 37 ◦ C in DMEM/F12 growth medium supplied with 2% B27 serum-free supplements (Gibco-Invitrogen), 2 mM lglutamine (Gibco-Invitrogen), 20 ng/ml epidermal growth factor (EGF; R&D Systems, Minneapolis, MN), 20 ng/ml basic fibroblast growth factor (bFGF; R&D Systems), and penicillin-streptomycin (100 UI/ml–100 g/ml) [19]. Culture medium was changed and cells passaged every 3-4 days. NPCs at passage 4 were used in all experiments. The care and handling of the newborn mice was approved by the Beijing Municipal Administration Office for Laboratory Animals (approval number SCXK(Jing)2009-0007). 2.2. Adherent monolayer culture of NPCs An adherent monolayer of NPCs was cultured on substrates of poly-d-lysine (PDL) and laminin (R&D System, Gaithersburg, USA). PDL-laminin coated culturewares were prepared according to the Technical Manual Version 2.1.0 (2016) for In Vitro Proliferation and Differentiation of Mouse Neural Stem and Progenitor Cells Using NeuroCult (STEMCELL Technologies Inc, Vancouver, BC, Canada. Data available from: https://www.stemcell.com). The suspension cultures (free-floating neurospheres) of NPCs were harvested into a 15 ml polypropylene tube and centrifuged at 1000 rpm for 5 min. The cell pellets were suspended in 1 ml fresh medium, triturated several times until a single cell suspension was achieved, and then filtered through a 40 m filter. The cell suspensions were then seeded onto the pre-prepared PDL-laminin coated culturewares for cell proliferation test or immunocytochemistry. 2.3. Cell proliferation Dissociated single cell suspensions (105 /ml) were inoculated into a PDL-laminin coated 96-well plate (100 l/well) in triplicate or quintuplicate and cultured in a humidified incubator (37 ◦ C, 5% CO2). The culture medium was supplemented with the following reagents: a neutralized antibody against IGFBP4 (4 g/ml, R&D Systems, Minneapolis, MN), IGFBP4 (0.5 g/ml, R&D Systems) [17], the half maximal inhibitory concentration of AG1024 (AG1024 IC50, dissolved in DMSO at a final concentration of 7 M, Calbiochem, Merck Millipore, Billerica, MA, USA) [7], AG1024 (20 M), IGFBP4 + AG1024/AG1024 IC50, FH535 (dissolved in DMSO at 20 M, Tocris Bioscience, Avonmouth, Bristol, UK) [5,9], and IGFBP4 + FH535. Treatment with DMSO only was used as control. The cultures were fed with new medium every three days. Each day, 10 l of CCK-8 solution was added to each well and incubated for 4 h. The absorbance was measured at 450 nm using a microplate reader (ELx808, Biotek Instruments, Winooski, VT, USA). The experiments were repeated three times.
2.4. Cell differentiation Dissociated single cells were seeded onto PDL-laminin coated culturewares and cultured in DMEM/F12 differentiation medium with withdrawal of bFGF and EGF, and supplemented with 2% B27, 2 mM l-glutamine, 10% fetal bovine serum, and penicillinstreptomycin (100 UI/ml–100 g/ml) [19]. The cells grown on the coated glass coverslips placed in 24-well plates were used to evaluate the number of GFAP- and MAP2-positive cells by fluorescent immunocytochemistry, and the cells on the coated 90-mm Petri dishes were collected to analyze the expression of GFAP and MAP2 by Western blotting. 2.5. Immunocytochemistry The adherent monolayer cultures of NPCs on the coated glass coverslips were fixed by immersing in 4% paraformaldehyde for 30 min. The endogenous peroxidase was quenched by 1% hydrogen peroxide in 50% ethanol for 30 min at room temperature. After washes in PBS, the coverslips were blocked for 1 h at room temperature in a blocking solution (5% goat serum in PBS), followed by incubation at 4 ◦ C overnight with the following antibodies: a mouse monoclonal antibody to Nestin (1:200; Abcam, Cambridge, UK), rabbit anti-IGFBP4 (1:200; Santa Cruz, CA, USA), rabbit antiGFAP (1:1000; Abcam) and anti-MAP2 (1:500; Abcam). The cells were then incubated at room temperature for 2 h with goat antirabbit secondary antibodies conjugated with Fluor-488 (1:250; EarthOx, San Fransico, CA, USA) or a goat anti-mouse secondary antibody conjugated with Alexa Fluor-594 (1:250; EarthOx) in the dark. The coverslips were coverslipped with fluorescent mounting medium (Dako) and the fluorochrome labeled antibody was visualized and photographed under a fluorescent microscope (Olympus IX71) equipped with an Olympus camera (DP73). The number of GFAP- and MAP2-immunoreactive cells was determined related to the number of DAPI-stained nuclei. Approximately 200 cells were counted within randomly selected visual fields through fluorescence microscopy at 40× magnification. 2.6. Western blotting An adherent monolayer of NPCs was cultured on PDL-laminin coated 90 mm dishes with or without IGFBP4 for 5 d. The cells were collected and lysed for 30 min at 4 ◦ C with a 360-1 lysis buffer containing 50 mM Tris (pH7.2), 150 mM NaCl, 0.5% Nonidet P-40 (NP40), 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, and 0.1% leupeptin. The cell lysates were harvested and centrifuged at 4 ◦ C for 10 min at 12,000g to collect supernatant protein extracts. The concentration of total protein was tested using a BCA protein assay kit (Pierce, Rockford, IL, USA). Equal amounts of protein from each lysate (10 g) were loaded and size-fractionated by SDS polyacrylamide gel electrophoresis at a constant voltage of 120 V at 4 ◦ C. The proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (0.2 m; Millipore, Bedford, USA) at a constant current of 100 mA for 3 h at 4 ◦ C. The membrane was rinsed for 10 min with Tris-buffered saline supplemented with 0.05% Tween 20 (TBST, pH 7.4) followed by nonspecific binding with a blocking solution (10% non-fat dry milk in TBST). The blocked membrane was probed with antibodies raised against GFAP (1:10000; Abcam); MAP2 (1:1000; Abcam); Akt, p38 and Erk1,2, phospho-Akt (p-Akt), p-p38, and p-Erk1,2 (1:1000 each, CST, Danvers, MA, USA) overnight at 4 ◦ C. After wash three times (10 min each) in TBST, the membrane was incubated with a secondary goat anti-rabbit IgG or a goat anti-mouse IgG conjugated to horseradish peroxidase (1:5000 each; EarthOx) for 2 h at room temperature. Finally, the blots were developed by use of a SuperEnhanced chemiluminescence detection kit (Applygen Technologies
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Fig. 1. Culture and identification of the NPCs. NPCs formed suspension neurosphere-like structures in serum-free medium 10 d later (A) and adherent monolayer-structures on PDL-laminin substrate 24 h after re-seeding (B). Immunofluorescent staining revealed that NPCs expressed Nestin (C) and IGFBP4 (D).
Inc., Beijing, China), and the images of protein bands were captured on Kodak X-ray film. As an internal control, the expression of -actin was detected by a mouse monoclonal antibody against actin (1:2000; Beijing GuanXingYun Sci & Tech Co, Beijing, China) in the same membrane with the same procedures. Intensities of the blotted bands were acquired by scanning of the X-ray film with a FluorChem Q imaging system (Proteinsimple, Sata Clara, CA, USA) and quantified using a densitometric software ImageJ 1.49 v (Wayne Rasband, NIH, USA). The densitometric units of the bands were expressed relative to the values for -actin or unphosphorylated proteins. 2.7. Statistical analysis Data were expressed as means ± SEM. For comparisons between two groups, two-tailed Student’s t-test was performed. For multiple comparisons, one-way Analysis of Variance (ANOVA) followed by Bonferroni post hoc test was conducted using SPSS version 13.0. Evidence of a statistically significant difference between mean values was considered to be at P < 0.05. 3. Results
filament protein found in NPCs [8], was expressed mainly in the cell soma and the cellular processes of the adherent NPCs (Fig. 1C). IGFBP4 was expressed mainly in the cell soma (Fig. 1D).
3.2. Proliferation When physiologically secreted IGFBP4 protein was neutralized with a specific antibody and recombinant IGFBP4 protein was added into culture medium, the proliferation of NPCs was significantly suppressed (Fig. 2A). Most IGF actions, if not all, are mediated by IGF-IR, and IGFIR signaling is essential for the normal neural development [14] (Fig. 2B). To explore whether IGFBP4 action on NPC proliferation is IGF-dependent, AG1024 (a specific inhibitor of IGF-IR) and FH535 (a -catenin inhibitor) were separately added into adherent monolayer cultures of NPCs. Both AG1024 and FH535 significantly inhibited NPC proliferation compared with DMSO control. The addition of IGFBP4 and AG1024 did not lead to a more marked inhibition than AG1024 or IGFBP4 alone (Fig. 2C), neither did IGFBP4 and AG1024 IC50 in combination (data not shown). IGFBP4 and FH535 in combination inhibited NPC proliferation more strongly than FH535 or IGFBP4 alone (Fig. 2D).
3.1. Identification 3.3. IGF-IR signaling Cells isolated from newborn mice formed non-adherent (suspension) neurosphere-like structures in serum-free medium after 10 days of culture (Fig. 1A). Single cell suspensions seeded onto previously PDL-laminin coated plates formed an adherent monolayer of cells after 24 h (Fig. 1B). Nestin, a cytoskeletal intermediate
IGF-IR signaling events were measured by probing Western blots with antibodies against p-Akt, Akt, p-p38, p38, p-Erk1, 2, and Erk1, 2. The phosphorylation level of Akt was significantly decreased in cells treated with IGFBP4, and the activation of p38
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Fig. 2. Effects of IGFBP4 on proliferation of NPCs. When secreted IGFBP4 protein was neutralized or exogenous IGFBP4 protein was added, NPC proliferation was significantly suppressed (A). When IGF-IR and -catenin were inhibited with AG1024 and FH535 (B), cell proliferation was significantly inhibited (C, D). AG1024 and IGFBP4 in combination did not produce a more marked inhibition compared with AG1024 or IGFBP4 alone (C) but FH535 and IGFBP4 together did (D). Absorbance data were expressed as means ± SEM in bar graphs A, C and D.
Fig. 3. Phosphorylation levels of signaling molecules downstream IGF-IR. Western blotting analysis showed that IGFBP4 treatment resulted in significantly decreased Akt phosphorylation (A) and unchangeable phosphorylation of p38 (B) and Erk1, 2 (C) in the NPCs.
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Fig. 4. Differentiation of NPCs in FBS-medium without bFGF and EGF. Fluorescent immunocytochemistry (A) and bar graph (B), and Western blotting (C, D) revealed that IGFBP4 significantly increased glial and neuronal differentiation. Blockade of IGF-IR significantly enhanced neuronal differentiation (E).
and Erk1, 2 was not significantly changed after IGFBP4 treatment (Fig. 3). 3.4. Differentiation After culture in FBS-medium without bFGF and EGF, NPCs were induced to differentiate into astrocytes and neurons on PDL-laminin coated 24-well plates. Immunostaining with antibodies specific for GFAP and MAP2 (Fig. 4A) showed that IGFBP4 significantly promoted differentiation of NPCs (Fig. 4B). The differentiation promotion was further confirmed by Western blotting analysis of GFAP and MAP2 expression (Fig. 4C, D). Blockade of IGFIR significantly enhanced neuronal differentiation and IGFBP4 did not lead to a more marked neuronal differentiation in the presence of AG1024 (Fig. 4A, E). Glial differentiation remained unchangeable after treatment with AG1024 or AG1024 + IGFBP4 (Fig. 4E). 4. Discussion The present study demonstrated that blocking IGF-I signaling inhibits NPC proliferation and enhances neuronal differentiation, and exogenous IGFBP4 inhibits proliferation and enhances neuronal differentiation of NPCs as a regulator of IGF-I signaling mainly via down-regulating Akt activation. The influence of IGF-I on regulating proliferation of neural stem cells remains elusive. More proliferative cells are found in the
dentate gyrus of IGF-I knockout mice and bigger clonal neurospheres from IGF-I knockout mice are formed in culture [11,12]. In contrast, proliferation of neural stem cells is up-regulated in the hippocampus of IGF-I transgenic mice [11,20] and exogenous IGF-I promotes the proliferation of progenitor cells in the adult hippocampus [11]. Wnt signaling is implicated in many aspects of neural development through canonical (-catenin-dependent) or non-canonical (-catenin-independent) pathway, and canonical Wnt/-catenin signaling has been linked to promoting proliferation and specification of neural stem cells [10]. In the present study, blockade of IGF-IR and -catenin leads to significant decreases in the proliferation of NPCs in vitro, supporting the stimulatory effects of IGF-I [20] and Wnt/-catenin [10,15] on the proliferation of neural stem cells. We found that immunodepletion of endogenous IGFBP4 and administration of exogenous IGFBP4 suppress NPC proliferation. The role of IGFBP4 is very complex, and it is cell type and context dependent. IGFBP4 functions via predominantly inhibiting IGF actions [1]. However, genetic ablation of IGFBP4 was reported to reduce body mass of mice by 10–15% from embryonic day 14.5 to postnatal age of 14 weeks [13]. The beneficial influence of endogenous IGFBP4 on NPC proliferation was consistent with the requirement of IGFBP4 for normal prenatal growth in IGFBP4 knockout mice [13]. IGFBP4 was reported to trigger cellular senescence in mesenchymal stem cells [17], and inhibitive effects of exogenous IGFBP4 on NPC proliferation perhaps result from its pro-senescent effects. Nonetheless, further work will be needed
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to clarify the relationship between reduced cell proliferation and induced apoptosis in IGFBP4-treated NPCs. If the inhibitory effect of IGFBP4 is IGF-dependent, a more remarkable inhibition cannot be induced in the presence of AG1024, otherwise a cumulative inhibition of IGFBP4 (via IGFindependent) and AG1024 can be produced. Indeed, we did not find a more marked inhibitory effect on the proliferation of the NPCs treated with AG1024 and IGFBP4 in combination than with AG1024 or IGFBP4 alone. However, a more pronounced inhibitory effect was observed in the NPCs treated with IGFBP4 and FH535 than with IGFBP4 or FH535 alone. The data demonstrated that IGF-IR signaling pathway is mainly involved in IGFBP4 action on inhibiting NPC proliferation. Accumulating data show that IGF signals through Ras-Raf-MAPK and PI3K-Akt pathways in IGF neural actions and PI3K-Akt pathway is required for IGF-stimulated neural proliferation [14]. In the present study, a significantly decreased level of Akt phosphorylation was found in IGFBP4-treated NPCs, consistent with decreased Akt phosphorylation in IGF-IR-ablated olfactory neural stem cells [2]. Akt kinase can phosphorylate and inactivate glycogen synthase kinase 3 (GSK3), which in turn stabilizes cyclin D1 and promotes neural cell proliferation and survival [14]. It can be supposed that IGFBP4-induced decrease of NPC proliferation may mainly result from a significant decrease in Akt activation. The majority of accumulating findings show that IGF-I is crucial for differentiation of NPCs into neuronal lineage. Over-expression of IGF-I specifically in neural stem cells up-regulates glial and neuronal differentiation in adult transgenic mice [20]. However, conditional knockout of IGF-IR increases cumulative production of neuroblasts and neurons in the olfactory bulb of the mouse [2]. We found that blockade of IGF-IR promotes neuronal differentiation, consistent with the beneficial effect of suppressed IGF signaling on fostering olfactory bulb neurogenesis [2]. Administration of IGFBP4 is found to promote neuronal differentiation, but it did not promote more pronounced differentiation in the presence of AG1024, suggesting an involvement of the repression of IGF-IR signaling in IGFBP4-induced differentiation. Phosphorylated Akt is known to phosphorylate and inactivate GSK3, which in turn stabilizes -catenin by reducing its phosphorylation degradation [14]. Inhibition of PI3K/Akt signal pathway is reported to promote motor neuron differentiation of human endometrial stem cells [3], and loss of -catenin promotes neuronal differentiation [15]. Therefore, IGFBP4-induced decrease of Akt phosphorylation and thus degradation of -catenin perhaps underlie its stimulatory effects on neuronal differentiation. Erk [4,16] but not Akt [4] signaling is involved in astrocyte differentiation of neural stem cells, and inhibition of Erk signaling is reported to block astrocyte differentiation [4]. IGFBP4-promoted astrocyte differentiation is perhaps mediated by Erk signaling, which, unlike p-Akt, is not significantly decreased in IGFBP4-treated NPCs. AG1024 promoted neuronal differentiation more obviously than IGFBP4, and it perhaps decreased the phosphorylation levels of Erk1, 2, thereby preventing astrocyte differentiation. A further study will be needed to clarify the relationship between phosphorylation levels of Erk1, 2 and differentiation potential of NPCs into astrocytes after AG1024 treatment. The effects of IGFBP4 on NPC differentiation in the present study are in contrast to those of other members of IGFBP family. The reduced number of oligodendrocytes, astrocytes or neurons is observed in transgenic mice that over-express IGFBPs driven by promoters phosphoglycerate kinase (IGFBP-1,3), metallothioneinI (IGFBP-1,5), cytomegalovirus (IGFBP2), actin (IGFBP5), and GFAP (IGFBP6) [14]. The roles of IGFBP4 in regulating neural stem cell growth and development in the brain are being explored in recently-established transgenic mice over-expressing IGFBP4 specifically in neurons.
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