The mood stabilizer valproate activates human ...

JOURNAL OF NEUROCHEMISTRY

| 2013

doi: 10.1111/jnc.12292

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*Division of Regenerative Medicine, Institute of Cellular and System Medicine, National Health Research Institutes, Miaoli, Taiwan †Graduate Program of Biotechnology in Medicine, Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan ‡Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan

Abstract Valproic acid (VPA) is the primary mood-stabilizing drug to exert neuroprotective effects and to treat bipolar disorder in clinic. Fibroblast growth factor 1 (FGF1) has been shown to regulate cell proliferation, cell division, and neurogenesis. Human FGF1 gene 1B promoter ( 540 to +31)-driven green fluorescence (F1BGFP) has been shown to recapitulate endogenous FGF1 gene expression and facilitates the isolation of neural stem/progenitor cells (NSPCs) from developing and adult mouse brains. In this study, we provide several lines of evidence to demonstrate the underlying mechanisms of VPA in activating FGF-1B promoter activity: (i) VPA significantly increased the FGF-1B mRNA expression and the percentage of F1BGFP(+) cells; (ii) the increase of F1BGFP expression by VPA involves changes of regulatory factor X

(RFX) 1-3 transcriptional complexes and the increase of histone H3 acetylation on the 18-bp cis-element of FGF-1B promoter; (iii) treatments of other histone deacetylases (HDAC) inhibitors, sodium butyrate and trichostatin A, significantly increased the expression levels of FGF-1B, RFX2, and RFX3 transcripts; (iv) treatments of glycogen synthase kinase 3 (GSK-3) inhibitor, lithium, or GSK-3 siRNAs also significantly activated FGF-1B promoter; (v) VPA specifically enhanced neuronal differentiation in F1BGFP(+) embryonic stem cells and NSPCs rather than GFP( ) cells. This study suggested, for the first time, that VPA activates human FGF1 gene promoter through inhibiting HDAC and GSK-3 activities. Keywords: FGF1, GSK-3, HDACs, lithium, RFX, VPA. J. Neurochem. (2013) 10.1111/jnc.12292

Valproic acid (VPA) and lithium chloride (LiCl) are two mood-stabilizing drugs used to treat patients with bipolar disorder (Kazantsev and Thompson 2008; Li et al. 2012). It has been reported that the major pharmacological actions of VPA are to inhibit histone deacetylase (HDAC) and glycogen synthase kinase-3 (GSK-3) activities (Phiel et al. 2001; Werstuck et al. 2004), while LiCl is the inhibitor of GSK-3 (Stambolic et al. 1996; Zhang et al. 2003). Recent studies also demonstrate that VPA or LiCl treatment increases the expression of brain-derived neurotrophic factor (BDNF) (Yasuda et al. 2007). It has been shown that both the VPA and LiCl can expand the number of neural stem cells in adult mouse brains (Higashi et al. 2008). VPA can further induce neuronal differentiation (Hsieh et al. 2004; Yu et al. 2009). However, the mechanisms of their effects on neurogenesis remain to be investigated.

Fibroblast growth factors (FGF) play important roles in many cellular processes, such as cell growth, cell differentiation, and neurogenesis (Beenken and Mohammadi 2009). Disruption of FGF signaling results in loss of neural Received April 22, 2013; accepted April 22, 2013. Address correspondence and reprint requests to Ing-Ming Chiu, Institute of Cellular and System Medicine, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 35053, Taiwan. E-mail: [email protected] 1 Present address: Institute of Biomedical Sciences, Mackay Medical College, New Taipei City, Taiwan. 2 These two authors contribute equally for this study. Abbreviations used: ERK, extracellular signal-regulated kinases; FGF, fibroblast growth factor; GFAP, glial fibrillary acidic protein; GSK-3, glycogen synthase kinase 3; HDACs, histone deacetylases; MAP2, microtubule-associated protein 2; Tuj1, neuron-specific class III betatubulin; VPA, valproic acid.

© 2013 International Society for Neurochemistry, J. Neurochem. (2013) 10.1111/jnc.12292

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stem cells, and leads to the decreased size in mouse hippocampus and subventricular zone (Ohkubo et al. 2004). FGF1 is the prototype member of FGF family that functions as a mitogen for mesoderm- and neuroectodermderived cells (Chiu et al. 2001). FGF1 has been demonstrated to be expressed at embryonic to adult tissues and predominantly in the neural tissues, suggesting its involvement in neural developmental processes (Thisse and Thisse 2005; Mason 2007; Zakrzewska et al. 2008). Activation of FGFRs by FGF1 enhances cell growth and neurite outgrowth in neurons (Lin et al. 2009). Furthermore, FGF1 stimulates self-renewal and proliferation of neural stem/progenitor cells (NSPCs) (Lee et al. 2009; Hsu et al. 2010). The dysregulation of FGF1 has been linked to major depressive disorders (Evans et al. 2004). It has been shown that single nucleotide polymorphism in FGF1 gene is highly associated with the risk of schizophrenia development (Jungerius et al. 2007). Remarkably, the electroconvulsive therapy (ECT)-induced neurogenesis in hippocampus is accompanied by up-regulation of FGF-1B promoter in dentate gyrus (Ma et al. 2009). The human FGF1 gene is over 120 kb long and contains three protein-coding exons as well as a long 3′-untranslated region. It also contains at least four upstream untranslated exons, designated 1A, 1B, 1C, and 1D, which are alternatively spliced to the first protein-coding exon (Chiu et al. 2001). This tissue-specific expression of the four mRNA generated by alternative splicing relies on the use of different promoters. Thus, FGF-1A transcript predominates in the human kidney (Myers et al. 1993), and FGF-1C and -1D transcripts predominate in vascular smooth muscle cells and fibroblasts (Chotani et al. 2000). FGF-1A transcript is enhanced when mice were feed high-fat diet and this enhancement is regulated through a PPAR response element in the FGF-1A promoter (Jonker et al. 2012). FGF-1B is the major transcript within the human brain and retina (Myers et al. 1993, 1995). We have previously utilized FGF-1B promoter-driven green fluorescence (F1BGFP) reporter to monitor endogenous FGF1 expression in brain cells and to isolate NSPCs (Hsu et al. 2009; Lee et al. 2009). The 540-bp ( 540 to +31) sequence upstream of the FGF-1B transcription initiation site is critical for the activation of FGF-1B promoter (Ray et al. 1997). The 18-bp sequence ( 484 to 467) within the regulatory region RR2 contains RFX binding sites. RFX1, RFX2 and RFX3 transcription factors could directly bind the 18-bp cis-element ( 484 to 467), activate FGF-1B promoter and contribute to the maintenance of F1BGFP(+) NSPCs (Hsu et al. 2010, 2012). We previously showed that RFX1 is the transcriptional repressor of FGF-1B promoter through competing the 18-bpbinding site with RFX2 and RFX3 (Hsu et al. 2010, 2012). HDAC and GSK-3 have been identified as targets for the treatments of mood disorders and neurodegenerative diseases. It has been suggested that VPA and LiCl exert therapeutic effects through HDACs and GSK-3 inhibition.

This study was undertaken to investigate the underlying mechanism of VPA in activating FGF-1B promoter and neuronal differentiation. Here, we show that VPA treatment affects the RFX transcription factor binding to FGF-1B promoter. Moreover, RFX transcription factors mimic the effects of VPA on FGF-1B promoter. This study suggested, for the first time, that VPA activates human FGF1 gene promoter through inhibiting HDAC and GSK-3 activities.

Materials and methods Cell culture U-1240 MG and U-1242 MG glioblastoma cells were cultured in minimal essential media (MEM) supplemented with 10% calf serum (Hyclone, Logan, UT, USA), 100 units/mL penicillin, and 100 lg/ mL streptomycin (Gibco, NY, USA) at 37°C as previous described (Hsu et al. 2012). U-1240 MG/F1BGFP cells were cultured in standard culture medium containing 100 lg/mL G418 (EMD Millipore, Billerica, MA, USA) (Hsu et al. 2012). CD133(+) glioblastoma cells were derived from human GBM tissues and cultured in MEM containing 10% fetal bovine serum (FBS, Hyclone), 1X non-essential amino acid (Gibco), 1X sodium pyruvate (Gibco), and 19 penicillin/streptomycin (Gibco) in 5% CO2 at 37°C incubator. In this study, CD133(+) glioblastoma cells were transfected with pF1BGFP reporter by electroporation with microporator MP-100 (Digital Bio Tech Co., Seoul, Korea) and cultured in medium in standard culture medium containing 500 lg/mL G418 (EMD Millipore). Neurosphere culture and differentiation assay U-1240 MG/F1BGFP(+) and U-1240 MG/F1BGFP( ) cells were washed with basal medium and seeded at a maximal density of 1 9 104 cells in 60-mm Petri dish (Falcon Industries, Oxnard, CA, USA) with 5-mL neurosphere medium (NS medium): DMEMHG/ F12 supplemented with B27 (Invitrogen, Carlsbad, CA, USA), N2 (Invitrogen), 20 ng/mL epidermal growth factor, 20 ng/mL FGF2, and 2 lg/mL heparin (Hsu et al. 2010, 2012). Subsequently, cells were cultured in 5% CO2 at 37°C incubator. For differentiation assay, U-1240 MG/F1BGFP(+) and U-1240 MG/F1BGFP( ) cells were cultured in neural stem cell medium to a cell density of 10 cells/lL. After 7 days, neurospheres were dissociated to single cells by HyQTase and cultured the single cells in tissue culture dish (Corning, NY, USA) with Dulbecco’s modified Eagle’s medium/ F12 medium (DMEM/F12; Invitrogen) supplemented with 2% FBS for 7 days. Immunostaining For immunostaining, cells dissociated from neurospheres were plated in tissue culture chamber slides (Lab-Tek, Naper- ville, IL, USA) with DMEM/F12 (Invitrogen) supplemented with 2% FCS. After 7 days, differentiated cells were fixed in 2% paraformaldehyde for 20 min at 25°C, and then washed three times with phosphate buffered saline (PBS). For blocking, cells were incubated with 1% FBS plus 0.1% Triton-X 100 in PBS for 1 h at 25°C. Cells were then incubated with Anti-Tubulin Antibody (1 : 400, EMD Millipore, Bedford, MA, USA; MAB1637), Anti-MAP2A Antibody (1 : 500, EMD Millipore, MAB378), and Anti-GFP Antibody


VPA activates FGF-1B promoter

(1 : 1000, Invitrogen, A11122) for overnight at 4°C. Subsequently, the cells were incubated with appropriate FITC or rhodamineconjugated secondary antibody (1 : 500) for 1 h at 25°C. All sections were observed under a fluorescent microscope (Olympus, Tokyo, Japan). Electrophoretic mobility shift assay (EMSA) Nuclear protein extraction and EMSA experiments were performed as described previously (Hsu et al. 2012). The sequences of probes and cold competitors used in the EMSA experiments were listed as follows: 26-bp, (5′-ACGACCTGCTGTTTCCCTGGCAACTC-3′); 18-bp sequence, (5′-CTGTTTCCCTGGCAACTC-3′); U-1240 MG, U-1242 MG, U-1240 MG/F1BGFP( ) cells were treated with VPA (2 mM) for 72 h. For the supershift assays, 2 lg of anti-RFX1 (I19X, Santa Cruz Biotechnology, CA, USA), anti-RFX2 (C-15X, Santa Cruz Biotechnology), or anti-RFX3 (T-17X, Santa Cruz Biotechnology) polyclonal antibody was added. Treatment of VPA or LiCl, and siRNA knockdown VPA and LiCl were purchased form Sigma-Aldrich (St Louis, MO, USA). Double-stranded small interfering RNA oligos were designed using BLOCK-iTTM RNAi Designer software (Invitrogen). Representative Stealth RNAi oligos selected from three different RNAi against targeted gene are listed as follows: GSK-3a (validated stealth siRNA), GSK-3b (validated stealth siRNA), RFX2-RNAi (HSS109207), and RFX3-RNAi (HSS184279). Cells were transfected with siRNA against GSK-3a, GSK-3b, RFX2, or RFX3 using Lipofectamine RNAiMAX transfection reagent (Invitrogen) according to the manufacturer’s instructions. Three different RNAi against RFX2 and RFX3 were tested, and representative results for RNAi knockdown using RFX2-RNAi (HSS109207), RFX3-RNAi (HSS184279) were shown. Reverse transcription and quantitative polymerase chain reaction (Q-PCR) To assay mRNA expression, total RNA was prepared using RNeasy mini kit (Qiagen, Valencia, CA, USA) and reverse transcribed (Superscript II reverse transcriptase) according to the protocols supplied by the manufacturer. Quantitative-PCR analysis was performed using an ABI prism 7500 machine (Applied Biosystems, Foster City, CA, USA) using SYBR Green method (Kapa Biosystems, Woburn, MA, USA). The primers used in the Q-PCR were listed in Table 1. A standard thermal protocol was applied for all QPCR: 95°C for 3 min, followed by 40 cycles of 95°C for 15 s, 60°C for 60 s. Western blot analyses Forty micrograms of total protein fraction was separated on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto Immobilon polyvinylidene fluoride membrane (EMD Millipore). Blots were incubated initially with blocking buffer (5% milk) for 1 h at 25°C, and then with specific primary antibodies against b-actin (EMD Millipore, MAB 1510), tubulin (EMD Millipore, MAB1637), tyrosine hydroxylase (EMD Millipore, AB152), galactocerebroside (EMD Millipore, MAB342), GFAP (Invitrogen, A-21282), GSK-3a (Ser21) (EMD Millipore, 07-393), phospho-GSK-3b (Ser9) (EMD Millipore, 05-643), GSK3a (#4337, Cell Signaling Technology, Danvers, MA, USA), GSK-

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Table 1 Primer sequences used in Q-PCR analysis of mRNA Gene

Sequence

FGF-1B

5′- TGAGCGAGTGTGGAGAGAGGTA-3′ 5′- GCTGTGAAGGTGGTGATTTCC-3′ 5′-GCGATTGAAAACCTCCAAAA-3′ 5′-GGCTTCAGACGAATCCCATA-3′ 5′-AAACTGGACCCAGTCAATGC-3′ 5′-TGTTGCATGGGTTGTTGTCT-3′ 5′-CACTGACTTTTGCTGCTGCTTCT-3′ 5′-TGGCGCTCGCGTGTG-3′ 5′-TTGTTAAACCTCGGCAAAATCG-3′ 5′-AGACTATTGGAGGTATTGCTGTTCATT-3′ 5′-GCAAAAGGAAAGCAACTAAGAC-3′ 5′-CCATCTCTCTGTCTCTCTCTC-3′ 5′-GACTGAACGCGGCGCTAGAC-3′ 5′-CGGCGGAGGCTTAACGTGGA-3′

RFX2 RFX3 ASCl MYT1L BRN2 NEUROD1

3b (#9315, Cell Signaling Technology), and acetyl-Histone H3 (EMD Millipore, 06-599). Primary antibodies had been diluted (1 : 2000) with Tris-buffered saline-Tween 20 (TBS-T) containing 5% milk and 0.01% sodium azide. After antibody incubation, the blots were washed with TBS-T for 1 h and incubated with anti-goat IgG conjugated with horseradish peroxidase (Santa Cruz Biotechnology) for 1 h at 25°C. After the washing of the secondary antibodies (1 : 2000) three times with TBS-T, bands were detected by chemiluminescent horseradish peroxidase substrate (EMD Millipore) and exposed to X-ray film. Chromatin immunoprecipitation (ChIP) ChIP assays were performed using EZ-Magna ChIP kit (EMD Millipore) according to the manufacturer’s instructions. Briefly, U-1240 MG, U1242 MG, and 2 mM VPA-treated U-1242 MG cells were cross-linked with 1% formaldehyde and chromatin was sheared by sonication. ChIP were performed with Anti-acetylHistone H3 (AcH3) (EMD Millipore, 06-599) and Normal Rabbit IgG (EMD Millipore, 12-370). ChIPed DNA was subjected to PCR using specific primers for FGF-1B promoter region (Table 1). PCR reaction was conducted using Maxima Hot Start Green PCR Master Mix (K1061, Thermo Scientific, Rockford, IL, USA) with following thermal protocol: one cycle of 4 min at 95°C, 32 cycles of 30 s at 95°C, 30 s at 59°C, 30 s at 72°C, followed by 5 min at 72°C. PCR products were analyzed by agarose gel electrophoresis. Specific primers for FGF-1B regulatory region were used in PCR reaction: 5′-GCAGGGATGCCAGATGACA-3′ and 5′- TGTGTGAGCCGAATGGACTTC-3′ with the amplicon size of 166 bp. To quantify ChIP results, PCR gel bands intensity from the triplicate ChIP experiment were measured using ImageJ (NIH, Bethesda, MD, USA). The fold enrichment values for the ChIP experiment were determined by dividing the AcH3 ChIP signal by corresponding IgG control. Maintenance and neuronal differentiation of mouse embryonic stem cells The mouse embryonic stem cells (ESCs) E14tg2a was cultured in DMEM containing 1% FBS, 10% knock-out serum, 0.1 mM


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2-mercaptoethanol, 1% non-essential amino acid, 1% sodium pyruvate, and 1000 U/mL leukemic inhibitory factor (ESGRO– LIF, EMD Millipore) on 0.1% gelatin-coated dishes. To establish E14tg2a/F1BGFP cells, E14tg2a cells were transfected with F1BGFP reporter (Hsu et al. 2009) and cultured in normal growth medium. Three days post-transfection, cells were subjected to 2 weeks of G418 selection (250 lg/mL). For neuronal differentiation, ESCs were seeded on gelatin-coated 100 mm tissue culture plate (Corning) at density 1 9 104 cells/cm and cultured in neuronal differentiation medium (N2B27) [1 : 1 mixture of DMEM/F12 supplemented with 1% N2 (Invitrogen) and Neurobasal supplemented with 2% B27] for 3 days. Medium was replaced every 2 days. Flow cytometry analysis For monitoring F1BGFP reporter, cells were trypsinized and resuspended in PBS, and then analyzed on flow cytometer (FACSCalibur, BD Biosciences, Franklin Lakes, NJ, USA). For cell surface marker analysis, the cells were stained with Anti-CD24-PE antibody (553262, BD Biosciences) or Anti-CD133-PE antibody (12-1331-82, eBioscience, San Diego, CA, USA) and analyzed by FACSCalibur.

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Results VPA treatment up-regulated the expression of F1BGFP in both FGF-1B positive and negative glioblastoma cell lines FGF-1B is the major transcript of FGF1 in the brain. F1BGFP reporter has been suggested as a useful tool for the isolation of NSPCs with higher neuronal lineage differentiation potential (Hsu et al. 2009; Lee et al. 2009). Here, we first investigated the effects of VPA on the activation of FGF1B promoter. Human glioblastoma U-1240 MG/F1BGFP(+), U-1240 MG/F1BGFP( ), and U-1242 MG/F1BGFP( ) cells were treated with 0.5 mM and 2 mM VPA for 72 h. The cells were harvested and analyzed for the expression of F1BGFP by flow cytometry. After treated with VPA for 72 h, the F1BGFP(+) cell population in the U-1240 MG/F1BGFP (+) cells showed a significant increase (p < 0.05, Fig. 1a). Furthermore, VPA increased the percentage of F1BGFP(+) cells in U-1240 MG/F1BGFP( ) cells in a dose-dependent manner (Fig. 1a). In addition, VPA also increased the percentage of F1BGFP(+) cells in FGF-1B( ) U-1242 MG cells, which lack RFX2/3 transcriptional activators for FGF1B promoter. CD133 is a general marker for neural stem cells. Here, we also use another glioblastoma cell line, CD133(+)/F1BGFP( ). The percentage of F1BGFP(+) cells in CD133(+)/F1BGFP( ) cells can be significantly increased by 4 mM VPA treatment. To further understand whether VPA increased the FGF-1B transcript, we performed a quantitative-PCR (Q-PCR) analysis to measure the expression of FGF-1B mRNA. Treatment of VPA increased the FGF-1B transcript in U-1240 MG cells (Fig. 1b). As expected, FGF-1B transcript was also significantly increased in VPA-treated U-1242 MG cells (Fig. 1c). Based on our

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Fig. 1 Valproic acid (VPA) up-regulated the F1BGFP expression. (a) U-1240 MG/F1BGFP(+), U-1240 MG/F1BGFP( ), U-1242 MG/ F1BGFP( ) and CD133(+)/F1BGFP( ) glioblastoma cells were treated with the indicated concentration of VPA for 72 h. When assayed by flow cytometry, VPA treatment increased F1BGFP(+) cell population. (b, c) U-1240 MG and U-1242 MG glioblastoma cell lines were treated with indicated concentration VPA for 72 h. The expression levels of FGF-1B, RFX2 and RFX3 transcripts were determined by quantitative-PCR, using b-actin as an internal control. Data are represented as mean SEM of three independent experiments and (*) indicates p ≤ 0.05, (**) indicates p < 0.01 versus vehicle control.



earlier findings, RFX2/3 complex is crucial for the activation of FGF-1B promoter. We next examined the RFX2 and RFX3 mRNA levels in VPA-treated cells. Our results showed that the expression levels of RFX2 and RFX3 were significantly increased in VPA-treated U-1240 MG and U-1242 MG cells (Fig. 1b and c). VPA treatment differentially regulated RFX transcription factors binding to 18-bp cis-element of FGF-1B promoter Previously, we have demonstrated that RFX1, RFX2 and RFX3 transcription factors were implicated in the regulation of FGF-1B promoter (Hsu et al. 2010, 2012). We next investigated whether VPA affects RFX1-3 proteins binding to the 18-bp cis-element ( 484 to 467) of FGF-1B promoter. As shown in the results of EMSA, three RFX complexes were shown to bind to the 26-bp probe ( 492 to 467). Using antibody supershift assay in the EMSA, we have previously identified that the slowest, intermediate and the fastest complexes are RFX1/1, RFX1/2, 1/3 and RFX2/3, respectively (Fig. 2a, lane 1) (Hsu et al. 2012). The changes of binding properties of RFX complexes were observed in both FGF-1B(+) U-1240 MG and FGF-1B( ) U-1242 MG cells after VPA treatment (Fig. 2a, lanes 5 and 10). Treatment of VPA dose-dependently reduced the 26-bp-

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Fig. 2 Effects of valproic acid (VPA) on the binding of RFX transcription factors to 18-bp cis-element of F1B promoter. (a) U-1240 MG and U-1242 MG cells were treated with VPA. After 72 h, nuclear extracts were prepared for EMSA. For competition assays, 18-bp was used as specific cold competitor (lanes 2). Supershift analysis was conducted using RFX1, 2, and 3 antibodies (lanes 11, 12, and 13). VPA could down-regulate RFX1/1, RFX1/2 and RFX1/3 complex formation (lanes 5 and 10, arrows) in U-1240 MG and U-1242 MG cells. RFX2/3

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binding activity of RFX1/1, RFX1/2, and RFX1/3 complexes in both U-1240 MG and U-1242 MG cells (Fig. 2a, lanes 4, 5, 9, and 10, arrowhead). Interestingly, in U-1242 MG cells, treatment of 2 mM VPA resulted in the formation of an additional EMSA complex with retarded mobility than RFX2/3 complex (Fig. 2a, lane 10, arrowhead). To determine the component of this additional complex, we next performed EMSA supershift assay with antibodies specifically against RFX1, 2 and 3, respectively. As illustrated in Fig. 2a lanes 12 and 13, the additional complex could be supershifted with anti-RFX2 and anti-RFX3 antibodies, indicating that this complex was RFX2/3 heterodimer with an unknown modification. To further study the mechanism of VPA-mediated FGF-1B promoter activation, the U-1240 MG/F1BGFP( ) cells were treated with VPA and nuclear extracts were analyzed by EMSA. As shown in Fig. 2b, RFX2/3 complex was much more abundant in U-1240 MG/ F1BGFP(+) cells than in U-1240 MG/F1BGFP( ) cells. After treated with 2 mM VPA, the 26-bp-binding activity of RFX1/1, RFX1/2, and RFX1/3 in U-1240 MG/F1BGFP( ) cells were markedly decreased (Fig. 2b, lane 5, arrow). Notably, an additional complex was also observed after treated with VPA. These observations were consistent in VPA-treated U-1242 MG cells. Together, these findings

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complex in U-1240 MG was retarded by VPA treatment (lanes 4 and 5, arrowheads). The binding of RFX2/3 complex was also increased in U1242 MG cells (lane 10, arrowhead). (b) U-1240 MG/F1BGFP(+) and U-1240 MG/F1BGFP( ) cells were treated with VPA for 72 h. After VPA treatment, a retarded RFX2/3 complex was also observed (lane 5, arrowhead). Treatment of VPA also diminished the RFX1/1, RFX1/2 and 1/3 complexes formation in U-1240 MG/F1BGFP( ) cells (lane 5, arrows).


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Fig. 3 FGF-1B promoter activity is regulated by histone deacetylases (HDAC) activities. (a) Effects of valproic acid (VPA) on acetylation of histion H3 and inhibitory site phosphorylation of glycogen synthase kinase 3 (GSK-3) in U-1242 MG cells. U-1242 MG cells were treated with 2 mM VPA for 72 h and the VPA effects were analyzed by western blot. (b) The upper penal shows a schematic diagram of the genomic region subjected to chromatin immunoprecipitation (ChIP) analysis. ChIP experiment indicates that VPA treatment increases acetylated histone H3 occupancy on the FGF-1B promoter in U-1242

MG cells. *p < 0.01. (c) U-1240 MG/F1BGFP( ) cells treated with indicated concentration of sodium butyrate (SB) or TSA (d) for 72 h, and the promoter activity was measured by monitoring green fluorescent protein (GFP) expression using flow cytometry. (e) VPA or TSAtreated U-1240 MG/F1BGFP( ) cells were simultaneously treated with either RFX2-siRNA or RFX3-siRNA. Data are represented as mean SEM of three independent experiments and (*) indicates p < 0.05, (**) indicates p < 0.01 versus vehicle control. (##) indicates p < 0.01 versus VPA treatment only.

suggested that RFX transcription factors were implicated in VPA-mediated FGF-1B promoter activation.

after VPA treatment, we performed ChIP assay with antiacetylated histone H3 antibodies (AcH3) in control and VPAtreated U-1242 MG cells. ChIP assays revealed that VPA treatment enhanced the acetylation of histone H3 around the regulatory region RR2 of FGF-1B promoter (Fig. 3b). These results indicated that VPA activated FGF-1B promoter through inducing histone acetylation around the RR2 region. We next test whether blocking the HDAC activity gives the similar effect of VPA in activating FGF-1B promoter, we treated the U-1240 MG/F1BGFP( ) cells with two different HDAC inhibitor, sodium butyrate (SB) and trichostatin A (TSA). Treatment of SB dose-dependently increased the percentage of F1BGFP(+) cells in U-1240/F1BGFP(+) cells

Activation of FGF-1B promoter is regulated by HDAC activities Previous studies have shown that VPA inhibits HDAC activity (Gottlicher et al. 2001; Phiel et al. 2001; De Sarno et al. 2002; Abel and Zukin 2008). To determine the effect of VPA on histone acetylation, western blot was performed with antiacetylated histones H3 antibodies (Fig. 3a). We found that the acetylation level of histone H3 was increased in VPA-treated U1242 MG cells. To examine the change in acetylation status of histone around the regulatory region RR2 of FGF-1B promoter



(Fig. 3c). The percentage of F1BGFP(+) cells in U-1240/ F1BGFP(+) cells could be significantly induced about four fold at 2 mM SB. In addition, we also used a structurally unrelated HDAC inhibitor, TSA, to confirm the effect of HDAC inhibition on FGF-1B promoter. As shown in Fig. 3d, treatment with TSA could increase FGF-1B promoter activity in U-1240 MG/F1BGFP( ), U-1242 MG/ F1BGFP( ), and CD133(+)/F1BGFP( ) cells in a dosedependent manner. These results suggested that HDAC inhibition played an important role in activating FGF-1B promoter. To clarify the mechanism of FGF-1B activation by HDAC inhibitors, U-1240 MG/F1BGFP( ) cells were further treated with RFX2-siRNA and RFX3-siRNA simultaneously in VPA and TSA-treated cells (Fig. 3e). Intriguingly, we observed a significant decrease in VPA-induced FGF-1B promoter activation after siRNA knockdown of RFX2. siRNA. Knockdown of RFX3 did not affect the level of VPA-induced FGF-1B activation. These results suggested the essential role of RFX2 transcription factor and FGF1 signaling in VPA-mediated activation of FGF-1B promoter. Interestingly, siRNA knockdown of RFX2 or RFX3 did not affect TSA-induced FGF-1B activation (Fig. 3e). Using EMSA, TSA treatment did not affect RFX2/3 complex (data not shown). Therefore, RFX2 and RFX3 transcription factors may not contribute to the TSA-induced FGF-1B promoter activation.

FGF-1B promoter is regulated by GSK-3 activities Recent studies have suggested that GSK-3 is a potential target for mood-disorder therapy (Wada 2009; Polter et al. 2010). It has been reported that the mood stabilizer VPA directly blocked GSK-3 activity (Chen et al. 1999). Interestingly, we observed that the phosphorylation of GSK-3 at ser21, which has been reported as an inhibitory site of GSK-3, was increased in VPA-treated U-1242 MG cells. To further investigate whether GSK-3 is implicated in the regulation of FGF-1B promoter, we next investigated the effect of LiCl, a well-known GSK-3 inhibitor, on the activation of FGF-1B promoter. We showed that LiCl treatment dose-dependently increased the percentage of F1BGFP(+) cells in U-1240 MG/F1BGFP( ) cells (Fig. 4a). Furthermore, we performed siRNA knockdown experiment. U-1240 MG/F1BGFP( ) cells were treated either with GSK-3a or GSK-3b siRNA. After 48 h, cells were harvested and analyzed for F1BGFP expression by flow cytometry. As shown in Fig. 4b, our results revealed that GSK-3a or GSK-3b siRNA treatment efficiently reduced the expression of target gene. The percentage of F1BGFP(+) cells was significantly increased by knockdown of GSK-3a or GSK-3b (Fig. 4c). Our result suggested that FGF-1B promoter activity could also be regulated by GSK-3 activities. In addition, we also investigated the effects of LiCl on the expression of

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FGF-1B, RFX2 and RFX3 and found that LiCl could significantly enhance the expression levels of FGF-1B and RFX2 transcripts in U-1240 MG and U-1242 MG cells (Fig. 4d and e). Notably, RFX3 mRNA expression was not affected after treatment of LiCl. These results suggested that the FGF-1B promoter can be regulated by HDAC and GSK-3 activities. VPA treatment enhanced neuronal differentiation of F1BGFP(+) NSPCs in vitro It has been shown that HDAC inhibition induces neuronal differentiation of NSPCs (Hsieh et al. 2004; Yu et al. 2009). Furthermore, VPA has been used to generate neuron cells form embryonic stem cells (ESCs) (Gaspard et al. 2009; Juliandi et al. 2012). To elucidate the correlation between VPA-mediated FGF-1B promoter activation and neuronal differentiation, we next investigated the effects of HDAC inhibition on neuronal differentiation efficiency in ESCs and NSPCs. To induce neuronal differentiation in ESCs, E14TG2a/F1BGFP cells were cultured in N2B27 medium for 3 days. As shown in Fig. 5a, the neuronal marker microtubule-associated protein 2 (MAP2) remains unchanged in E14TG2a/F1BGFP cells when cultured in N2B27 medium. Notably, administration of VPA or SB increased the expression of MAP2. We also evaluated neuronal differentiation efficiency of ESCs by cell surface marker CD24 and CD133. CD24 has been defined as a neural lineage differentiation of stem cells (Pruszak et al. 2009), and CD133 has been used in isolation of neural stem cells in embryonic brain (Pfenninger et al. 2007). By flow cytometry, we found that treatment of VPA and SB in N2B27-cultured ESCs significantly increased the expression of CD24 and CD133 (Fig. 5b and c). These findings are consistent with the effect that VPA and SB increase neuronal differentiation efficiency (Hsieh et al. 2004; Juliandi et al. 2012). Furthermore, the percentage of F1BGFP(+) cells in E14TG2a/F1BGFP cells in ES medium was low (17.2 0.3%), and was increased by 1.3-fold (p < 0.05) in N2B27 medium. Notably, In the presence of HDAC inhibitors, VPA and SB, the percentage of F1BGFP(+) cells were significantly increased (p < 0.01), suggesting a positive correlation between FGF-1B promoter activation and neuronal differentiation of ESCs (Fig. 5d). We have previously shown that F1BGFP(+) NSPCs exhibit enhanced neuronal differentiation ability than F1BGFP( ) cells (Lee et al. 2009), we explore the effect of VPA in these two different cell populations. To enrich the NSPCs, U-1240 MG/F1BGFP(+) and U-1240 MG/F1BGFP ( ) cells were cultured as neurosphere in neural stem cell media. After 7 days, neurospheres was dissociated into single cells and then placed in differentiation medium with or without VPA for another 7 days (Fig. 6a). After 7 days differentiation, F1BGFP(+) NSPCs were observed to be


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(a)

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Fig. 4 FGF-1B promoter activity is regulated by glycogen synthase kinase 3 (GSK-3) activities. (a) U-1240 MG/F1BGFP( ) cells were treated with 10, 20, or 40 mM lithium chloride (LiCl) for 72 h. LiCl treatment decreased the percentage of F1BGFP(+) cells. (b) Confirmation of the effectiveness of siRNA against GSK-3a or GSK-3b. U1240 MG cells were transfected with GSK-3a or GSK-3b siRNA at indicated concentrations for 48 h. (c) U-1240 MG cells were transiently transfected with GSK-3a or GSK-3b siRNA at indicated concentrations

for 48 h. F1BGFP expression was measured by flow cytometry. (d, e) U-1240 MG and U-1242 MG glioblastoma cell lines were treated with indicated concentration of LiCl for 72 h. The expression levels of FGF1B, RFX2, and RFX3 transcripts were analyzed by quantitative-PCR. Data are represented as mean SEM of three independent experiments and (*) indicates p < 0.05, (**) indicates p < 0.01 versus vehicle control.

differentiated into MAP2(+) and neuron-specific class III beta-tubulin (TuJ1)(+) cells. Notably, VPA could significantly enhance the neuronal differentiation of F1BGFP(+) NSPCs into MAP2(+) and TuJ1(+) cells (Fig. 6b). The expression levels of tyrosine hydroxylase and MAP2 can be significantly enhanced during the differentiation of VPAtreated F1BGFP(+) NSPCs (Fig. 6c). Furthermore, treatment of VPA reduced the expression of astrocyte marker GFAP expression in either differentiated F1BGFP(+) and F1BGFP ( ) cells. It has been shown that FGF receptors (FGFRs), ERK and GSK-3 signaling pathways play important roles during neuronal differentiation (Hao et al. 2004; Chen et al. 2010a; Hur and Zhou 2010). To investigate whether these signaling pathways are involved in VPA-induced neuronal differentiation, we performed western blot to investigate these signaling molecules during VPA-induced neuronal differentiation (Fig. 6c). It has been reported that GSK-3a (Ser21) and GSK-3b (Ser9) phosphorylation inhibit GSK-3 activity (Zhang et al. 2003). Treatment of VPA in F1BGFP (+) NSPCs increased the phosphorylation of GSK-3a. Although treatment of VPA decreased GSK-3b (Ser9) phosphorylation in both F1BGFP(+) and ( ) cells, VPAtreated F1BGFP(+) NSPCs still exhibit higher phosphorylation levels at GSK-3b (Ser9) than VPA-treated F1BGFP( ) cells. These results suggested that F1BGFP(+) NSPCs was a

primary target of VPA in VPA-induced neuronal differentiation. Effects of VPA on the expression levels of ASCL1, BRN2, MYT1L, and NEUROD1 transcription factors between F1BGFP(+) and ( ) cells The pro-neurogenic transcription factors ASCL1, BRN2, MYT1L, and NEUROD1 have been shown to induced the reprogramming of human skin fibroblasts to neurons (Pang et al. 2011). To further compare the effects of VPA on the mRNA expression levels of pro-neurogenic transcription factors between F1BGFP(+) and ( ) cells, we performed quantitative-PCR to analyze mRNA expression levels of ASCL1, BRN2, MYT1L, and NEUROD1. The mRNA expression level of ASCL1 was sustained in VPA-treated F1BGFP(+) NSPCs, but decreased in VPA-treated F1BGFP ( ) cells (Fig. 6d). mRNA expression levels of NEUROD1 and MYT1L were significantly up-regulated in VPA-treated F1BGFP(+) NSPCs (Fig. 6d). VPA treatment did not affect the expression of NEUROD1, but decreased the MYT1L expression in VPA-treated F1BGFP( ) cells. Interestingly, treatment of VPA up-regulated the expression of BRN2 in F1BGFP( ) cells. Our results indicated that VPA-induced neuronal differentiation in F1BGFP(+) NSPCs might be also through up-regulating the expression of MYT1L and NEUROD1 in F1BGFP(+) NSPCs.



(a)

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Fig. 5 FGF-1B promoter is activated upon neuronal differentiation induced by valproic acid (VPA). E14TG2a/F1BGFP cells cultured in N2B27 and treated with indicated concentration of VPA or sodium butyrate (SB) for 72 h. (a) E14TG2a/F1BGFP cells were cultured in the indicated conditions. mRNA expression of microtubule-associated protein 2 (MAP2) was analyzed by Q-PCR, and b-actin was as an

Discussion In this study, we provide several lines of evidence to demonstrate the underlying mechanisms of VPA in activating FGF-1B promoter activity: (i) VPA significantly upregulated the FGF-1B mRNA expression and the percentage of F1BGFP(+) cells (Fig. 1); (ii) the increases of FGF-1B mRNA expression and the percentage of F1BGFP(+) cells involve down-regulation of RFX1-related complex, upregulation of RFX2/3 complex and the increase in histone acetylation on FGF-1B promoter (Figs 2 and 3); (iii) treatments of other HDAC inhibitors, SB and TSA, activated FGF-1B promoter (Fig. 3); (iv) treatments of other GSK inhibitor, lithium, or transfection with GSK-3 siRNAs also

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internal control. (b, c) Flow cytometry of CD133 and CD24 expression in E14TG2a/F1BGFP cells cultured in different conditions. (d) Flow cytometry analysis of F1BGFP in E14TG2a/F1BGFP cells cultured in indicated conditions. Data are represented as mean SEM of three independent experiments and (#) indicates p < 0.05, (*) indicates p < 0.01 versus N2B27 control.

activated FGF-1B promoter (Fig. 4); (v) VPA specifically enhanced neuronal differentiation in F1BGFP(+) ESCs and NSPCs rather than GFP( ) cells (Fig. 6). This study suggested, for the first time, that FGF1 is an important target for VPA. Many studies have revealed that glioblastoma contains NSPC-like cells which have multipotent and self-renewal capacities (Galli et al. 2004). As a result of the difficulties in obtaining normal human brain NSPCs, we therefore used human glioblastoma cells and mouse ES-derived NSPCs in this study to evaluate the effects of VPA on FGF1 and neuronal differentiation. To exclude the possibility that the endogenous FGF-1B expression may influence the Q-PCR results, we have used FGF-1B(+) U-1240 MG glioblastoma


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(a)

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Fig. 6 Treatment with valproic acid (VPA) enhanced neuronal differentiation of F1BGFP(+) NSPCs in vitro. (a) Schematic illustration of the process for the isolation of neural stem/progenitor cells (NSPCs) from U-1240 MG/F1BGFP cells using neural stem cell medium. U-1240 MG/ F1BGFP cells generated neurospheres in neural stem cell medium. These neurospheres were dissociated into single cells and subsequently placed in 2% fetal bovine serum (FBS) differentiation medium with or without 2 mM VPA. Scale bar = 200 lm. (b) Representative result of microtubule-associated protein 2 (MAP2) and neuron-specific class III beta-tubulin (TuJ1) immunostaining. Treatment of U-1240 MG/ F1BGFP(+) NPCs with 2 mM VPA under the differentiation condition (in the presence of 2% FBS, Diff.) for 7 days resulted in an increase expression of MAP2 and TuJ1. (c) VPA enhanced the protein levels of

TuJ1 (neuron marker) and tyrosine hydroxylase in U-1240 MG/ F1BGFP(+) NPCs under the differentiation condition. Western blot of GFAP, MAP2, Tyrosine hydroxylase, Galactocerebroside (GalC), phospho-glycogen synthase kinase 3a (GSK-3a) (Ser21), phosphorGSK-3b (Ser9) in differentiated cells treated NPCs treated with 2 mM VPA or vehicle. b-actin was used as a internal control. (d) Effects of VPA on the mRNA expression levels of neuronal lineage specific transcription factors between U-1240 MG/F1BGFP(+) and ( ) cells. mRNA expression levels of neuronal lineage specific transcription factors, ASCL1, NEUROD1, MYT1L, and BRN2 were analyzed by QPCR, and using b-actin as an internal control. Data are represented as mean SEM of three independent experiments and (*) indicates p < 0.01 versus VPA-treated F1BGFP(+) cells.

cell line and FGF-1B( ) U-1242 MG glioblastoma cell line to evaluate the VPA effects on FGF1B mRNA expression. From our results, VPA could significantly increase the mRNA expression of FGF-1B in both U-1240 MG and U-1242 MG cells (Fig. 1b and c). In neurogenesis, HDACs are known to be critical regulators of neuronal cell fate determination (Montgomery et al. 2009; Sun et al. 2011). The role of HDACs in regulating the function and development of nervous system has been studied in several animal models (Fischer et al. 2007; Ma et al. 2010). HDAC1 controls the differentiation of neural progenitor to mature neurons in mouse and zebra fish (Harrison et al. 2011; Sun et al. 2011). Furthermore, HDAC inhibition by VPA or SB shows benefits in the treatment of neurodegenerative diseases (Chuang et al. 2009). It has been shown that stroke-induced brain damage in mouse can be reduced by VPA treatment (Kim et al. 2007). HDAC inhibitors also promote neurogenesis and exert neuroprotective effect in models of brain ischemia (Kim et al. 2009). In clinical applications, HDAC inhibitors have been used in the treatment of central nerve system disorders and mood disorders. VPA is one of the most widely used HDAC inhibitors in clinic, and has often been used as an anticonvulsant and mood stabilizer (Phiel et al. 2001; Chen et al. 2010b). VPA has been shown to induce neuronal differentiation of NSPCs in vitro and in vivo (Hsieh et al. 2004). Previous studies indicated that VPA treatment increased the expression of BDNF and neurogenic basic helix-loop-helix (bHLH) transcription factor NeuroD in neuron cells (Hsieh et al. 2004; Yasuda et al. 2007). In our study, we found that VPA treatment increased acetylation of histone H3 on FGF1B promoter, suggesting that VPA activates FGF-1B promoter through inhibition of HDAC (Figs 1 and 3). In addition to HDAC inhibition, several studies have proposed that GSK-3 might also the therapeutic target of VPA (Chen et al. 1999; De Sarno et al. 2002; Jin et al. 2005; Leng et al. 2008). The impaired regulation of GSK-3 signaling is involved in mood disorders (Zhang et al. 2003). Inhibition of GSK-3 by mood stabilizers was previously described as important actions in treatment of mood disorders (De Sarno et al. 2002; Zhang et al. 2003; Kozlovsky

et al. 2006; Aubry et al. 2009). However, the downstream effects upon GSK-3 inhibition remain to be investigated. Therefore, we were interested to see if GSK-3 is implicated in the regulation of FGF-1B promoter. Our data showed that VPA could inhibit GSK-3a (Fig. 3b). In addition, FGF-1B promoter activity is elevated through inhibition of GSK-3 by LiCl or siRNA treatment (Fig. 4). Although the therapeutic plasma lithium levels are in the range from 0.4 to 1.2 mM, we observed that 20 or 40 mM of LiCl treatment did not cause significant cell death in our experiment, this may be because of the possibility that glioblastoma cells are more resistant to LiCl than normal brain cells. Therefore, we should use this concentration to see the effect of GSK-3 inhibition on FGF-1B promoter activity. In addition, the combined treatment with both GSK-3a and GSK-3b siRNAs did not show stronger effects on F1BGFP expression (data not shown). In LiCl treatment experiment, the up-regulation of FGF-1B promoter could only be observed in long-term treatment of lithium (up to 72 h). However, using the more specific and efficient siRNA against GSK-3a or GSK-3b, as shown in Fig. 4b and c, we demonstrated that knockdown of GSK-3a or GSK-3b could up-regulate FGF-1B promoter in 48 h of transfection. This may be because of the different inhibitory efficiency between lithium and GSK-3 siRNA. The important role of FGFs in nervous system development has been reported. It has also been demonstrated that the dysregulation of FGF2 is implicated in the development of mood disorders. FGF2 has been shown to improve learning and memory in mouse brain injury models (McDermott et al. 1997; Valouskova and Gschanes 1999; Turner et al. 2012). In a rodent depression model, administration of FGF2 relieved the depression-like behavior in animal (Turner et al. 2008). Interestingly, treatments with anti-depressants increase the expression of FGF2 (Maragnoli et al. 2004). Based on the amino acid sequence, structure and receptorbinding property similarity of FGF1 and FGF2, it is reasonable to hypothesize that FGF1 is involved in pharmacological action of mood stabilizer. It has been reported that FGF1 enhances the proliferation of NSPCs (Lee et al. 2009; Ma et al. 2009). Expression of FGF1 in NSPCs is tightly regulated by FGF-1B promoter (Hsu et al. 2009). The


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activity-induced adult neurogenesis, produces a significant increase in FGF-1B promoter activity in dentate gyrus (Ma et al. 2009). Moreover, F1BGFP reporter has been applied to isolate NSPCs (Chiu et al. 2000; Hsu et al. 2009; Lee et al. 2009). In this study, we used flow cytometry to analyze the effects of VPA treatment on the percentage of F1BGFP(+) cells in U-1240 MG/F1BGFP(+), U-1240 MG/F1BGFP( ), U-1242 MG/F1BGFP( ), and CD133/F1BGFP( ) cells (Fig. 1). The increase in F1BGFP(+) cells is more significant in U-1240 MG/F1BGFP( ), U-1242 MG/F1BGFP( ), and CD133/F1BGFP( ) cells than U-1240 MG/F1BGFP(+) cells by VPA treatment. This is because of that the percentage of F1BGFP(+) cells in U-1240 MG/F1BGFP(+) cells was already 80% high. Nonetheless, VPA treatment still significant increased the percentage of F1BGFP(+) cells in U-1240 MG/F1BGFP(+) cells to near 90% (Fig. 1). Interestingly, by promoter analyses, we observed that RFX binding sites exist in other FGF gene promoters (such as FGF2 and other FGFs); however, whether the effects of HDAC inhibitors on other FGFs through RFX transcription factors require further investigation. It has been shown that FGF-1B promoter is regulated by RFX transcription factors and Gadd45b (Ma et al. 2009; Hsu et al. 2012). RFX2/3 heterodimer binds to the 18-bp region and acts as a transcriptional activator in FGF-1B positive cells (Hsu et al. 2012). FGF-1B promoter is also regulated through epigenetic manner. In adult dentate gyrus, Gadd45b promotes the demethylation of FGF-1B promoter during activity-induced adult neurogenesis, and results in promoter activation. Activation of FGF-1B promoter by HDAC inhibitors suggested that the HDACs are involved in epigenetic control of FGF-1B promoter. We have previously shown that RFX1, RFX2, RFX3 bind the 18-bp cis-elements of FGF-1B promoter by EMSA and chromatin immunoprecipitation (Hsu et al. 2010, 2012). Here, we performed EMSA and showed that treatment of VPA affected the binding properties of RFX transcription factors. The binding of RFX1/1, RFX1/2, and RFX1/3 on FGF-1B promoter were greatly reduced by VPA, indicating that these complexes act as negative regulators in FGF-1B promoter activation (Fig. 2). Interestingly, RFX1 has been shown to preferably interact with HDAC1 and mammalian transcriptional repressor, mSin3A. HDAC1 enhances inhibition of collagen promoter activity by RFX1. Furthermore, TSA stimulates the acetylation of RFX proteins and activates the collagen promoter activity (Xu et al. 2006). Our results are in all agreement with this report that HDAC inhibition will reduce RFX1-mediated transcriptional repression. Importantly, treatment of VPA decreased the RFX1-related complexes and increased the binding of RFX2/3 on FGF-1B promoter (Fig. 2). This result is consistent with the notion that RFX2/3 complex is required for FGF-1B expression. Furthermore, the modified RFX2/3 complex generated from VPA-treated cells shows different mobility from the original FGF-1B(+)

cells, reflecting that the post-translational modification might play a role in the regulation of RFX2/3 complex formation, and requires further investigation (Fig. 2). We also studied the mRNA expression of RFX2 and RFX3 after treatment of VPA or LiCl. A significant increase in RFX2 mRNA expression level was observed in VPA- or LiCl-treated cells. Administration of the RFX2-siRNA also attenuated the VPA-induced FGF-1B promoter activity (Fig. 3). In addition to promoter-binding properties being affected, our data showed a positive correlation between VPA treatment and RFX2 expression. Although our data suggested that another HDAC inhibitor, TSA also activated FGF-1B promoter. Knockdown of either RFX2 or RFX3 did not affect the induction level of FGF-1B promoter, suggesting that RFX2 and RFX3 are not likely to involve the TSA-induced FGF1B promoter activation. The knockdown efficiency of RFX2 and RFX3 siRNAs have been shown previously (Hsu et al. 2012). Using EMSA, we demonstrated that TSA treatment could affect RFX1 binding complex on the 18-bp rather than RFX2 and RFX3 (data not shown). TSA may function through HDAC inhibition and reduce RFX1-mediated transcriptional repression (Xu et al. 2006). HDAC inhibition has been implicated in promoting neuronal differentiation (Hsieh et al. 2004; Yu et al. 2009). VPA appears to activate the expression of pro-neurogenic transcription factors, resulting in neuronal differentiation and neurite outgrowth of neural progenitor cells (Hao et al. 2004; Yu et al. 2009). In this study, our result indicates that VPA could induce FGF-1B promoter activity in ESCs (Fig. 5), suggesting that VPA could promote neuronal differentiation of ESCs through up-regulation of FGF-1. To further explore the effect of VPA on different populations within NSPCs, we use fluorescent activated cell sorting to separate NSPCs into F1BGFP(+) and GFP( ) populations. Our recent reports suggested that F1BGFP(+) NSPCs exhibits high self-renewal and neuronal differentiation potency than GFP( ) cells (Hsu et al. 2009; Lee et al. 2009). Consistent with our previous results, the F1BGFP(+) cells express more MAP2 than GFP ( ) cells in differentiation condition (Fig. 6a). We found that VPA-induced neuronal differentiation is prominent in F1BGFP(+) NSPCs. It has been shown that these two factors are involved in the reprogramming of human fibroblast to induced neurons with functional excitatory activities (Pang et al. 2011). Our data here show that VPA can induce NEUROD1 and MYT1L in F1BGFP(+) cells (Fig. 6d), therefore implying the potential applications of VPA and F1BGFP in the generation of induced neurons. As a result of the induction of neuronal marker and pro-neurogenic transcription factors in F1BGFP(+) NSPCs, we postulated that F1BGFP(+) NSPCs is the major cell population responsive for VPA treatment. Increased GSK-3b inhibition by VPA treatment was not shown in previous reports (Hsieh et al. 2004; Yasuda et al. 2007). This may be because of different source of NSPCs and the use of different protocols of



neuronal differentiation. From our results, we showed that VPA increased the phosphorylation at inhibitory site of GSK-3a (Fig. 4a), suggesting that VPA, at least in part, GSK-3a in our cell system. LiCl treatment did not show any induction effect of neuronal differentiation in our studies. Previous studies have shown that the activity of GSK-3 is actually repressed in neural progenitor cells and the mature neuron cells exhibit high GSK-3 activity (Jin et al. 2005). This can explain why we didn`t see the same effect of neuronal induction in LiCl treatment. Notably, FGF-1B promoter is not only activated in neuronal induction but also in selfrenewal of neural progenitor cells. However, the dual role of FGF-1 during neurogenesis and specific stage remains to be elucidated. In conclusion, our results suggest that the mainstream mood stabilizer, VPA, induced the activation of FGF-1B promoter. Using the HDAC inhibitors and GSK-3 inhibitor, we demonstrated that activation of FGF-1B promoter is through HDACs and GSK-3 inhibition. We also demonstrated that VPA activated the FGF-1B promoter through influencing the promoter-binding properties and gene expression of RFX transcription factors. In addition, our data suggested that the F1BGFP(+) NSPCs were the major cell population responsive for VPA-induced neuronal differentiation than GFP( ) cells. These results indicated the crucial role of FGF1 in the therapeutic effects of VPA, thus providing valuable implications for clinical application.

Acknowledgement We thank Su-Liang Chen, Mei-Su Chen, and Hua-Kuo Lin for excellent technical assistance and discussions. This study was supported by the National Science Council, Taiwan, and the National Health Research Institutes, Taiwan. This study was conducted under the Graduate Program of Biotechnology in Medicine sponsored by the National Tsing Hua University and the National Health Research Institutes. Y.C.H and I.M.C contribute to conception and design. C.Y.K, Y.C.H, J.W.L, D.C.L., and Y.F.C performed all the experiments. C.Y.K, Y.C.H, and I.M.C wrote the manuscript.

Conflict of interest None declared.

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