Valproic acid induces differentiation and inhibition of proliferation in neural progenitor cells via the beta-catenin-Ras-ERK-p21Cip/WAF1 pathway
© Jung et al; licensee BioMed Central Ltd. 2008
Received: 22 October 2008
Accepted: 09 December 2008
Published: 09 December 2008
Valproic acid (VPA), a commonly used mood stabilizer that promotes neuronal differentiation, regulates multiple signaling pathways involving extracellular signal-regulated kinase (ERK) and glycogen synthase kinase3β (GSK3β). However, the mechanism by which VPA promotes differentiation is not understood.
We report here that 1 mM VPA simultaneously induces differentiation and reduces proliferation of basic fibroblast growth factor (bFGF)-treated embryonic day 14 (E14) rat cerebral cortex neural progenitor cells (NPCs). The effects of VPA on the regulation of differentiation and inhibition of proliferation occur via the ERK-p21Cip/WAF1 pathway. These effects, however, are not mediated by the pathway involving the epidermal growth factor receptor (EGFR) but via the pathway which stabilizes Ras through β-catenin signaling. Stimulation of differentiation and inhibition of proliferation in NPCs by VPA occur independently and the β-catenin-Ras-ERK-p21Cip/WAF1 pathway is involved in both processes. The independent regulation of differentiation and proliferation in NPCs by VPA was also demonstrated in vivo in the cerebral cortex of developing rat embryos.
We propose that this mechanism of VPA action may contribute to an explanation of its anti-tumor and neuroprotective effects, as well as elucidate its role in the independent regulation of differentiation and inhibition of proliferation in the cerebral cortex of developing rat embryos.
Valproic acid (VPA; 2-propyl-pentanoic acid) has been used for mood stabilization and the treatment of epilepsy for several decades . VPA also exhibits potent in vitro and in vivo anti-tumor effects in leukemic cells, neuroblastomas, and gliomas [2–7]. VPA is a histone deacetylase (HDAC) inhibitor and plays a role in modifying chromatin structure and gene expression [8, 9]. VPA has also been found to affect various signaling systems, including the extracellular signal-regulated kinase (ERK), protein kinase C (PKC), and the Wnt/β-catenin pathways [3, 10, 11]. VPA alters the Wnt/β-catenin signaling by directly or indirectly [12, 13] inhibiting the activity of glycogen synthase kinase 3β (GSK3β).
VPA has been reported to regulate the differentiation and proliferation of various cells, including mesenchymal and hematopoietic stem cells, neuroblastoma cells, primary neurons, and neural progenitor cells (NPCs) [8, 14–17]. VPA can also reduce the proliferation of neuroblastoma cells by induction of the cell cycle regulator p21Cip/WAF1 [5, 6], which is also known to be involved in the VPA-induced differentiation of hematopoietic cells . However, the mechanism by which VPA regulates differentiation and proliferation is not understood.
We report here that 1 mM VPA induces differentiation and inhibits proliferation of NPCs by overcoming the effect of basic fibroblast growth factor (bFGF), a factor which inhibits the differentiation of NPCs [19, 20]. VPA-induced activation of the ERK- p21Cip/WAF1 pathway did not occur via the common pathway involving epidermal growth factor receptor (EGFR), an upstream component of the ERK pathway, as indicated by significant reduction in the level of EGFR in the presence of VPA. The level of Ras protein, another upstream component of the ERK pathway, was significantly increased by VPA treatment. This observation led us to conclude that VPA-induced ERK pathway activation occurs via an increase in the stability of Ras, mediated by Wnt/β-catenin signaling [21, 22]. We also found that the common Ras-ERK-p21Cip//WAF1 pathway is involved in generating the mutually exclusive phenotypes of differentiation and proliferation in NPCs and in brain tissue of the cerebral cortex of developing embryos.
VPA overcomes the effects of bFGF on differentiation and proliferation in multipotent NPCs
VPA induces both p21Cip/WAF1 and Tuj1 via the Raf-MEK-ERK cascade in NPCs grown in the presence of bFGF
To ascertain whether the ERK pathway mediates p21Cip/WAF1 induction and neuronal differentiation in response to VPA, we measured the effect of inhibition of the Ras-ERK pathway on the induction of p21Cip/WAF1 and Tuj1 by VPA. Induction of p21Cip/WAF1 by VPA was significantly reduced when ERK activation was inhibited by the MEK inhibitor PD98059 (Figure 2E). Induction of Tuj1 by VPA was also reduced by PD98059. The VPA-induced activation of the ERK pathway in the inhibition of growth of NPCs in the presence of bFGF was confirmed by the observation of re-increase of cell numbers by treatment with PD98059 in the presence of VPA (Figure 2F). We did not observe any growth-stimulatory effect of PD98059 when NPCs were grown in the absence of bFGF; rather, cell number was somewhat reduced by PD98059 treatment (see Additional file 3). In summary, VPA induces differentiation and inhibition of proliferation via the Ras→MEK→ERK pathway in NPCs grown in the presence of bFGF.
p21Cip/WAF1 mediates VPA-induced differentiation and inhibition of proliferation in NPCs grown in the presence of bFGF
VPA activates the ERK-p21Cip/WAF1 pathway and induces differentiation and inhibition of proliferation via β-catenin-mediated accumulation of Ras in NPCs grown in the presence of bFGF
To identify the role of β-catenin in the regulation of the Ras-ERK-p21Cip/WAF1 pathway and subsequent stimulation of differentiation and inhibition of proliferation induced by VPA in NPCs, we measured the effect of overexpression of β-catenin. Overexpression of β-catenin increased ERK activity and Ras protein level and induced expression of p21Cip/WAF1 and Tuj1 (Figure 4C). Cells overexpressing β-catenin were morphologically differentiated and BrdU-negative (Figure 4D). Therefore, β-catenin is an important factor for both differentiation and inhibition of proliferation in cortical E14 NPCs and those physiological responses are simultaneously and independently controlled by β-catenin.
VPA independently induces differentiation and inhibition of proliferation in NPCs in the developing rat brain
In the present study, we found that NPCs undergo neuronal differentiation and inhibition of proliferation following treatment with 1 mM of the commonly prescribed antiepileptic drug VPA . We chose 1 mM VPA because this concentration is non-toxic to the NPCs in our current study and to hippocampal neuronal progenitors . The VPA amount applied to the animals, 200 mg/kg, is identical to that used to achieve whole-brain levels of 1.0–1.5 mM by chronic application .
We investigated the mechanism by which VPA regulates differentiation and inhibition of proliferation. The role of VPA in the exclusive in vivo regulation of differentiation and proliferation of NPCs suggest an application for VPA in the production of functional neurons for therapeutic use in patients. Our study also provides a mechanism that may aid in validating the proposed use of VPA as an anti-tumor and neuroprotective agent [3, 4, 33]. VPA induced both differentiation and inhibition of proliferation in NPCs by overcoming the effect of bFGF, a factor which promotes growth/proliferation and suppresses differentiation, through the common ERK-p21Cip/WAF1 pathway. The VPA-induced inhibition of proliferation was suppressed by the MEK inhibitor PD98059, indicating a role for activation of the ERK pathway in the inhibition of proliferation by VPA. Participation of the ERK pathway in inhibition of proliferation is frequently accompanied by induction of the cell cycle inhibitory factor p21Cip/WAF1 [6, 31, 34, 35]; p21Cip/WAF1 was also induced in the VPA-treated NPCs. The role of p21Cip/WAF1 in inhibition of proliferation by VPA is also shown by release of the VPA-induced inhibition of proliferation by p21Cip/WAF1 siRNA. The role of p21Cip/WAF1 as a potent anti-proliferation factor was further shown by loss of BrdU incorporation in most cells in which p21Cip/WAF1 had been induced by VPA.
A significant decrease in the level of Tuj1 following siRNA-mediated p21Cip/WAF1 knockdown, demonstrated by both biochemical and immunocytochemical analyses, suggests that p21Cip/WAF1 may also be involved in the regulation of differentiation and inhibition of proliferation. It is clear that both inhibition of proliferation and differentiation of NPCs stimulated by VPA occur through the ERK pathway-dependent induction of p21Cip/WAF1.
We have shown that activation of the ERK-p21Cip/WAF1 pathway by VPA did not occur via the segment of the pathway involving EGFR but via the segment involving β-catenin. Although EGFR has been identified as a target of Wnt/β-catenin in liver , EGFR was reduced by VPA in NPCs. The mechanism by which EGFR transcription is inhibited by VPA is unknown; however, it has been established that EGFR transcription is repressed by bone morphogenetic protein 4 (BMP4), an alternative transcription target of β-catenin , in NPCs [38, 39]. We observed significant induction of BMP4 in the VPA-treated NPCs grown in the presence of bFGF (data not shown). These data suggest that the VPA-induced decrease in EGFR in NPCs may be acquired through induction of BMP4.
VPA directly inhibits GSK3β resulting in activation of the β-catenin signaling pathway [33, 40, 41] and β-catenin is, in turn, involved in regulation of the ERK pathway [21, 22]. Evidence for the role of β-catenin in VPA-induced activation of the ERK-p21Cip/WAF1 pathway, and subsequent effects on differentiation and inhibition of proliferation in NPCs, was seen in the reduction of the effects of VPA, including ERK activation and induction of p21Cip/WAF1 and Tuj1, following siRNA-mediated β-catenin knockdown. The β-catenin-mediated activation of the ERK-p21Cip/WAF1 pathway following VPA treatment may be attributed to upregulation of Ras, suggested by the increase in the level of Ras seen after β-catenin overexpression. The VPA-induced increase in the level of Ras may be due to the stabilization of β-catenin as a result of inhibition of GSK3?. Increases in Ras following modulation of the Wnt/β-catenin signaling pathway have been demonstrated in various cell types, including primary hepatocytes, and β-catenin has been identified as an important mediator of that process [21, 22]. Regulation of Ras protein levels by the Wnt/β-catenin system is mediated by polyubiquitination and proteasomal degradation [Kim et al., 2008, Journal of Cell Science, In print]. Differentiation and proliferation occur independently in the cerebellar cortex of the developing embryo [42–44]; however, the mechanism(s) underlying the differential regulation of the two processes has not been described. In this study, we found that differentiation and proliferation occurred independently in regions of the developing brain of embryos treated with VPA. We saw no BrdU-positive cells among Tuj1-positive NPCs following VPA treatment. We also did not observe any proliferating cells among differentiated NPCs stimulated to differentiate by β-catenin overexpression or by VPA treatment to express increased levels of p21Cip/WAF1. These results indicate that mutually exclusive patterns of differentiation and proliferation during neuronal differentiation and development may be regulated via the common Ras-ERK-p21Cip/WAF1 pathway involving β-catenin.
However, the effects of VPA, particularly its effect on proliferation, were modest or only partially inhibited by increases in p21Cip/WAF1 or siRNA-mediated β-catenin knockdown in several different cases. These results indicate the possibility that VPA-induced differentiation and inhibition of proliferation occur in part via different routes, including, e.g., the pathway affected by inhibition of HDAC . Although we improved the efficiency of transfection by making modifications to the standard method, the limited effectiveness of siRNAs in general may also be a contributing factor in the weak effects seen on differentiation and inhibition of proliferation (see Additional file 7). The concomitant stimulation of differentiation and inhibition of proliferation in NPCs and the developing rat embryo by VPA treatment indicate potential utility for VPA in the treatment of neuroblastomas  and/or in neuronal regeneration.
Our results suggest that VPA induces neuronal differentiation and inhibition of proliferation of cortical NPCs at least partly via the Ras-ERK-p21Cip/WAF1-pathway mediated by β-catenin. We propose that this mechanism of VPA action may contribute to an explanation of its anti-tumor and neuroprotective effects, as well as elucidate its role in the independent regulation of differentiation and proliferation in the cerebral cortex of developing rat embryos. Regulation of Ras stability in neuronal differentiation and inhibition of proliferation may indicate a new role of the protooncoprotein Ras in the differentiation of stem cells and in development.
Primary cerebral cortical progenitor cell culture, transfection, and VPA treatment
Sprague-Dawley (SD) rats were purchased from KOATECH (Gyeonggi Do, Korea). All animal procedures were approved by the Institutional Review Board of Severance Hospital, Yonsei University College of Medicine. NPCs were isolated from the cerebral cortex of E14 SD rats as described previously . The isolated cortical cells were plated in dishes coated with 15 μg/ml poly-L-ornithine and 10 μg/ml fibronectin (Sigma-Aldrich, St. Louis, MO) and cultured in N2 medium [DMEM:F12 (1:1) (Invitrogen) containing 100 μM putrescine, 30 nM selenite, 20 nM progesterone, 1.55 mg/ml D-(+)-glucose, 25 μg/ml insulin, 0.1 μg/ml apo-transferrin (Sigma-Aldrich), 0.5 mM Glutamax, 100 IU/ml penicillin, and 100 μg/ml streptomycin] containing 10 ng/ml bFGF (basic Fibroblast Growth Factor; Invitrogen, Carlsbad, CA) in a humidified atmosphere of 95% air/5% CO2 at 37°C. NPCs were plated at 4 – 5 × 105 cells per dish in a 60-mm culture dish and grown to 40% confluence, cells were treated with 0.5 – 10 mM (most often 1 mM) VPA for 48 h. To observe the effect of inhibition of MEK, some cultures were co-treated with 20 μM PD98059 (2'-amino-3'-methoxyflavone) (A G. Scientific Inc, San Diego, CA) in addition to VPA. To test the effects of overexpression or knockdown, vectors or siRNAs were transfected into NPCs using Lipofectamine TM 2000 (Invitrogen). Transfections were performed in N2 medium without antibiotics, followed by growth in N2 medium containing 50 IU/ml penicillin and 50 μg/ml streptomycin, prior to treatment with VPA. Cultures were photographed using the ECLIPS TE2000-U Fluorescent microscope (Nikon, Tokyo, Japan) equipped with a digital CCD camera (Diagnostic Instruments, Inc., Sterling Heights, MI).
VPA Treatment of embryos
Beginning on embryonic day 13.5 (E13.5) of gestation, rats were intravenously injected with 200 mg/kg VPA (Acros Organics, Belgium) or phosphate-buffered saline (PBS) twice at 24 h intervals, and provided with water containing 15 g/l VPA for 48 h. On day15.5 embryos were collected for subsequent immunohistochemical analyses.
Cells were rinsed with ice-cold PBS and lysed in 1× Laemmli buffer (0.14 M Tris, pH 6.8, containing 2.4 M glycerol, 0.21 M sodium dodecyl sulfate, and 0.3 mM bromophenol blue). Cell lysates were heated at 100°C for 10 min and separated by electrophoresis through 8 – 12% SDS-polyacrylamide gels. Following electrophoretic transfer of proteins to nitrocellulose, membranes were blocked in 5% nonfat dry milk in PBS containing 0.1% (v/v) Tween 20 for 30 min at 25°C followed by incubation with anti-p-ERK, anti-p-MEK, anti-p-GSK3β (Ser-9) (Cell Signaling Biotechnology, Beverly, MA), anti-β-catenin (rabbit polyclonal antibody produced in this laboratory), anti-p21Cip/WAF1, anti-EGFR (Santa Cruz Biotechnology, Santa Cruz, CA), anti-p-Raf-1 (Ser-338), anti-pan-Ras (Upstate Biotechnology, Lake Placid, NY), anti-Tuj1 (Covance, Princeton, NJ), or anti-β-actin (Abcam Ltd, MA) antibody followed by horseradish peroxidase-conjugated secondary anti-rabbit, anti-rat (Calbiochem, La Jolla, CA), or anti-mouse IgG (Bio-Rad Laboratories, Hercules, CA). Protein bands were visualized using enhanced chemiluminescence (ECL; Amersham, Inc, UK) and the LAS-3000 imaging system (Fujifilm, Tokyo, Japan).
Immunocytochemistry and Immunohistochemistry
For immunocytochemistry, NPCs were plated on coverslips coated with poly-L-ornithine and fibronectin in 24-well plates at a density of 2 – 2.5 × 104 cells/well. When cultures reached 30% confluence VPA was added to the medium. Where indicated, the cells had been transfected with 100 nM p21Cip/WAF1 siRNA or β-catenin siRNA for 12 h before VPA treatment. For the proliferation assay, cultures were incubated with 25 μM BrdU (Sigma-Aldrich) for 2.5 h before fixation. Cells were fixed in 70% ethanol for 30 min, washed three times with PBS, and permeabilized by incubation in 0.1% Triton X-100 in PBS for 30 min. To measure BrdU incorporation, cells were incubated in 2 M HCl for 30 min and washed three times with PBS. The cells were incubated in blocking solution (10% normal goat serum in PBS) for 30 min followed by incubation with anti-Tuj1, anti-BrdU (Dako Co., Carpinteria, CA), anti-Flag (Sigma-Aldrich), or anti-p21Cip/WAF1 in blocking solution at 4°C overnight. Cells were washed three times in PBS and incubated with Alexa Fluor 488- or Alexa Fluor 555-conjugated IgG secondary antibody (Molecular Probes, Eugene, OR) at room temperature for 1 h. Nuclei were counterstained by incubation in 1 μg/ml DAPI (4',6-diamidino-2-phenylindole; Boehringer Mannheim, Mannheim, Germany) for 10 min followed by exhaustive washing in distilled water. Coverslips were mounted in GelMount (Biomeda, Foster City, CA). Fluorescent labeling was observed using a Radiance 2100 multi-photon imaging system (Bio-Rad Laboratories) and LSM510META (Carl Zeiss, Germany) at excitation wavelengths of 488 nm (Alexa Fluor 488), 543 nm (Alexa Fluor 555), or 405 nm (DAPI). Approximately 300–400 cells were counted in each of three independent experiments to quantify relative p21Cip/WAF1-, Tuj1-, or BrdU- positive cells. For histological analyses, embryos were fixed in 10% neutral buffered formalin for 48 h, dehydrated by serial immersion in alcohols, cleared in xylene, and embedded in paraffin. Four-micron sections were cut using a RM2245 microtome (Leica Microsystems Wetzlar, Germany). Immunohistochemical analysis was performed using Alexa Fluor 555- and 488-conjugated IgG secondary antibodies. Antigen retrieval was performed using citrate buffer, pH 6.0. All incubations were carried out in humidified chambers in the dark. Immunofluorescent labeling of tissue sections was performed using the staining procedure described above.
Vector and siRNAs
Flag-β-catenin-pcDNA3.0 was obtained from Dr. Eric R. Fearon of the University of Michigan School of Medicine. Small interfering RNAs (siRNAs) targeting rat p21Cip/WAF1 (GenBank accession number NM_080782) and β-catenin (GenBank accession number NM_053357) mRNAs were designed using the template design tool (Ambion, Austin, TX). The p21 Cip/WAF1 target sequences are 5'-AGACCAGCCUAACAGATTTTT-3' (431–459) and 5'-GAACGGTGGAACTTTGACTTT-3' (136–154). β-catenin target sequences are 5'-AAGGCTTTTCCCAGTCCTTCA-3' (203–223) and 5'-AAGATGATGGTGTGCCAAGTG-3' (1303–1323). β-catenin and p21Cip/WAF1siRNAs were synthesized by Samchully Pharm Co., LTD (Gyeonggi do, Korea).
basic fibroblast growth factor
bone morphogenetic protein 4
extracellular signal-regulated kinase
glycogen synthase kinase3β
neural progenitor cells
class III β-tubulin
proliferating cell nuclear antigen
small interfering RNA
valproic acid, 2-propyl-pentanoic acid.
This work was supported by a Korea Science and Engineering Foundation (KOSEF) grant funded by Ministry of Education, Science and Technology (MIST) of Korea (No. 2005-01564; 2006-02681; R112000078010020). JY Yoon, BS Moon, DW Yang, and HY Kim were supported by a BK21 studentship from the MEST.
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