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J. Biol. Chem., Vol. 276, Issue 34, 31674-31683, August 24, 2001

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The Mood Stabilizer Valproic Acid Activates Mitogen-activated Protein Kinases and Promotes Neurite Growth^*

Pei-Xiong Yuan, Li-Dong Huang, Yi-Ming Jiang, J. Silvio Gutkind^§, Husseini K. Manji^¶, and Guang Chen^¶

From the  Laboratory of Molecular Pathophysiology, Department of Psychiatry and Behavioral Neurosciences, Wayne State University School of Medicine, Detroit, Michigan 48201, the ^§ Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892, and the ^¶ Laboratory of Molecular Pathophysiology, National Institute of Mental Health, Bethesda, Maryland 20892

Received for publication, May 11, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The mood-stabilizing agents lithium and valproic acid (VPA) increase DNA binding activity and transactivation activity of AP-1 transcription factors, as well as the expression of genes regulated by AP-1, in cultured cells and brain regions involved in mood regulation. In the present study, we found that VPA activated extracellular signal-regulated kinase (ERK), a kinase known to regulate AP-1 function and utilized by neurotrophins to mediate their diverse effects, including neuronal differentiation, neuronal survival, long term neuroplasticity, and potentially learning and memory. VPA-induced activation of ERK was blocked by the mitogen-activated protein kinase/ERK kinase inhibitor PD098059 and dominant-negative Ras and Raf mutants but not by dominant-negative stress-activated protein kinase/ERK kinase and mitogen-activated protein kinase kinase 6 mutants. VPA also increased the expression of genes regulated by the ERK pathway, including growth cone-associated protein 43 and Bcl-2, promoted neurite growth and cell survival, and enhanced norepinephrine uptake and release. These data demonstrate that VPA is an ERK pathway activator and produces neurotrophic effects.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mood-stabilizing agents are a very small group of pharmacologic agents utilized in the treatment of manic-depressive illness; they include lithium, valproic acid (VPA)¹ (2-propylpentanoic acid), and carbamazepine (1). Despite their highly dissimilar structures, both lithium (a monovalent cation) and VPA (a branched fatty acid) increase the DNA binding activity of AP-1 transcription factor (2-4) and expression of genes known to be regulated by AP-1, tyrosine hydroxylase (5-7), and c-Jun (8, 9). These effects have been observed both in cultured cells and in regions of rat brain known to be involved in mood regulation. VPA and lithium also increase reporter gene expression driven by a promoter containing consensus TPA response element (TRE) sequences, but not mutant TRE, demonstrating that both agents enhance the transactivation activity of AP-1 (10, 11).

Four mammalian mitogen-activated protein (MAP) kinase pathways, ERK, JNK, p38, and ERK5, are known to regulate AP-1 transcription factors (for reviews, see Refs. 12 and 13). Lithium increases the levels of the activated forms of JNKs but not total JNKs in rat frontal cortex and hippocampus, as well as in human neuroblastoma SH-SY5Y cell (9). Lithium also increases JNK activity in intact cells, as detected with c-Jun PathDetect trans-reporting system (14); these effects are blocked by a dominant-negative SEK1 mutant (9). Furthermore, lithium increases the levels of phosphorylated c-Jun and total c-Jun (9). Taken together, the data indicate that lithium activates the SEK/JNK pathway, an effect that ultimately results in increased c-Jun expression and increases in AP-1 DNA binding activity and transactivation activity (9).

To date, the molecular mechanisms by which VPA regulates AP-1 function have remained completely unknown. In view of the role of MAP kinases in regulating AP-1 function, we have undertaken the present study to determine whether VPA affects the MAP kinase pathways. We have found that VPA at therapeutic concentrations (50-150 µg/ml or 0.41-1.04 mM) (15) stimulates ERK pathway, increases growth cone-associated protein 43 (GAP-43) and Bcl-2 levels, promotes neurite growth and cell survival, and increases norepinephrine (NE) uptake and release. Thus, activation of the ERK pathway by VPA may underlie the effects of VPA on AP-1 transcription factor and on the expression of genes known to be regulated by AP-1. Interestingly, these results are similar to those elicited by several neurotrophic factors in the same cell line (16-19), suggesting that VPA exerts neurotrophic actions.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- pUSE Ha-Ras S17N and pUSE Raf-1 K375M were from Upstate Biotechnology (Lake Placid, NY). The pEBG SEK KR and pCEFL MKK6 KR were previously described (20). The Elk1 PathDetect trans-reporting system and LipoTAXI were purchased from Stratagene (La Jolla, CA). GAP-43 antibody was from Roche Molecular Biochemicals. AP-1 oligo nucleotide, pJNK, JNK, Bcl-2, and Bax antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). pERK, ERK, phospho-p38 (p-p38), and p38 antibodies were from New England Biolabs (Beverly, MA). [³H]NE, [-³²P]ATP, secondary antibodies, and chemiluminescence reagents were from Amersham Pharmacia Biotech. PD098059 and SB203580 were from Calbiochem (San Diego, CA). T4 kinase and all culture reagents were from Life Technologies, Inc. VPA (2-propylpentanoic acid), protease inhibitor mixture, phosphatase inhibitor mixture I and II, and all other standard chemicals were obtained from Sigma.

Cell Culture and AP-1DNA Binding Assay-- Human neuroblastoma SH-SY5Y cell culture and AP-1 DNA binding assay were conducted as previously described (3, 10, 11). In brief, SH-SY5Y cells were cultured in a humidified atmosphere of 95% air/5% CO₂ at 37 °C in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum, 100 IU/ml of penicillin, and 50 µg/ml of streptomycin. When the cultures reached 80% confluence, the cells were treated with VPA (1 mM), PD098059 (50 µM), and SB203580 (10 µM) in serum-free media for 24 h. For AP-1 DNA binding assay, the consensus oligo (5'-CGC TTG ATG ACT CAG CCG GAA-3') was labeled with [-³²P]ATP using T4 kinase according to the manufacturer's specifications. Free [-³²P]ATP was separated from labeled oligos using a pushing column from Stratagene. The washed cells were lysed, and the lysates were sonicated for 10 s and then centrifuged at 14,000 × g for 15 min to remove residual debris. The whole cell extracts were incubated with ³²P-labeled oligos at 37 °C for 15 min. The DNA binding reaction was terminated by the addition of 5× loading buffer, and the reaction mixtures were then subjected to gel electrophoresis using a 6% DNA retardation gel. The specificity of the DNA binding assay was confirmed by demonstrating that the addition of 50-fold excess of unlabeled TRE oligos but not mutant AP-1 oligos completely blocked AP-1 binding; furthermore, an antibody against the AP-1 family proteins supershifted the AP-1 binding band.

Detection of MAP Kinase Activation in Intact Cells-- The Elk1 PathDetect trans-reporting system was used to detect activation of MAP kinases in intact SH-SY5Y cell (14). Transient transfection and luciferase reporter gene activity assays were conducted as previously described (9-11). In brief, trypsinized SH-SY5Y cells were suspended in growth media and subcultured into six-well plates. After an overnight incubation, the cells were transfected with pFR-luc plasmid (encoding a luciferase gene driven by a promoter containing GAL4 binding sites) and pFA-Elk1 plasmid (encoding a fusion protein comprising of a GAL4 DNA binding domain and an Elk1 activation domain) in the media without serum and antibiotics. LipoTAXI reagent was used as the carrier. To block the ERK, JNK, and p38 pathways individually, the cells were cotransfected with pUSE Ha-Ras S17N and pUSE Raf-1 K375M, pEBG SEK KR, or pCEFL MKK6 KR. After 6 h of transfection, an equal volume of medium containing 1% fetal bovine serum was added, and the cells were incubated overnight, prior to the experimental studies. Transfected cells were incubated with VPA at the concentrations and for the time periods indicated in the figures. The incubations were also carried out in the presence of PD098059 (50 µM) or SB203580 (10 µM). Luciferase activity was measured using an assay kit from Promega (Madison, WI).

Immunoblotting-- Immunoblotting was conducted as previously described, with modifications (5, 21, 22). In brief, the cells were washed with phosphate-buffered saline and homogenized by brief sonication in an extraction buffer. The buffer contains 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton-100, 2.5 mM sodium pyrophosphate, 1 mM -glycerophosphate, protease inhibitor mixture (Sigma), and phosphatase inhibitor mixture I and II (Sigma). The homogenates were centrifuged at 14,000 × g for 15 s to remove undissolved debris. Subsequent immunoblotting was performed using amounts of protein demonstrated to be within the linear range for immunoblotting. The antibodies for immunoblots were diluted according to the manufacturer's recommendations. The immunocomplex was detected with an Amersham Pharmacia Biotech ECL or ECL plus kit. Quantitation of the immunoblots was performed by densitometric scanning of the film using an Image Analysis system with NIH Image 1.55 software.

Neurite Growth-- Neurite growth of human neuroblastoma SH-SY5Y cell was set up based on described methods (18, 19). Confluent SH-SY5Y cells were trypsinized and suspended in the growth media. The cell suspension was then diluted 1:120 and transferred to gas plasma-treated polystyrene tissue culture dishes or six-well plates (Becton Dickinson, Franklin Lakes, NJ). The cells were incubated for 2-3 h to allow the cells to attach. The dish or plate was then washed twice with DMEM to remove the residual serum and unattached cells. The cells were then incubated in DMEM in the absence or presence of VPA at varying concentrations and periods of time. The images of the cells were captured and relative neurite length was measured using NIH Image 1.55 software.

NE Uptake and Release-- Uptake and release of NE were determined using previously reported methods (23). SH-SY5Y cells were treated with 1.0 mM VPA in serum-free DMEM for 1 or 5 days in six-well dishes. For uptake, the cells were incubated with [³H]NE in Hepes-buffered saline (HBS) at 37 °C for 1 h, and the cells were then washed three times with HBS. HBS contains 10 mM Hepes (pH 7.4), 135 mM NaCl, 5 mM KCl, 6 mM glucose, 2.5 mM CaCl₂, and 0.6 mM MgSO₄. Desipramine (3 ng/ml) was used in one set of wells to determine the nonspecific incorporation. Release was induced by depolarization with potassium using K⁺-HBS. K⁺-HBS contains 10 mM Hepes (pH 7.4), 40 mM NaCl, 100 mM KCl, 6 mM glucose, 2.5 mM CaCl₂, and 0.6 mM MgSO₄. Ascorbic acid (0.02%) and Clorgyline (0.3 µg/ml) were added to HBS and K⁺-HBS before used. The specific uptake and release were calculated and expressed as cpm/µg of protein.

Statistics-- Statistical analysis was performed by analysis of variance, followed by Fisher's paired least significant difference or Scheffe's tests. p < 0.05 was considered significant. Data are expressed as mean ± S.E.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A MEK Inhibitor, but Not a p38 Inhibitor, Blocks VPA-induced Increase in DNA Binding Activity of AP-1-- As we have previously observed (3), VPA induced a 2-fold increase in the binding activity of AP-1 in serum-deprived human neuroblastoma SH-SY5Y (Fig. 1A). To begin addressing the mechanisms by which VPA affects gene expression, we took advantage of the availability of inhibitors of the MAP kinase pathways. PD098059, a MEK inhibitor (24), blocked VPA-induced increases of AP-1 binding (Fig. 1B); conversely, SB203580, a p38 inhibitor (25), was without significant effect (Fig. 1C), suggesting that the ERK pathway is required for the effects of VPA.

View larger version (41K): [in this window] [in a new window]   Fig. 1.   Differential effects of MAP kinase pathway inhibitors on the induction of AP-1 DNA binding activity by VPA. SH-SY5Y cells were grown in DMEM plus 10% fetal bovine serum, serum-deprived for 16 h, and then treated with 1.0 mM VPA for 24 h in the presence of Me2SO (0.1%, the solvent for PD098059 and SB203580) (A), PD098059 (50 µM) (B), or SB203580 (10 µM) (C). AP-1 binding assays were conducted using 10 µg of whole cell protein extracts. Autoradiograms shown are from a representative experiment, which was repeated three times with similar results. Data are mean ± S.E. of four independent experiments, expressed as percentage change with respect to the control (non-VPA-treated cells). *, p < 0.05 compared with non-VPA-treated cells.

VPA Activates the MAP Kinase Downstream Target Elk1 in a Time- and Concentration-dependent Manner-- To further explore the potential activation of MAP kinases by VPA, the activation of Elk1, a common target for MAP kinases, was investigated using the PathDetect trans-reporting system, in which the luciferase reporter gene activity reflects the activation of Elk1 by MAP kinases in intact cells (14). SH-SY5Y cells were transfected with Elk1 PathDetect trans-reporting system and then treated with VPA. VPA (1 mM) increased luciferase activity by 0.5-fold at 4 h, 2-fold at 8 h, and 13-fold at 24 h (Fig. 2A). One-hour VPA incubation did not produce any significant changes in luciferase activity (Fig. 2A). Concentration dependence studies were conducted with the 8-h incubation and showed that 0.25 mM VPA was without significant effect, whereas 0.7-, 2.5-, and 6-fold increases in luciferase activity were observed with 0.5, 1.0, and 2.0 mM VPA, respectively (Fig. 2B).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Time- and concentration-dependent activation of MAP kinases by VPA and the blockage of VPA-induced MAP kinase activation by MEK inhibitor. SH-SY5Y cells were transfected with Elk1 trans-reporting system and incubated with VPA at 1.0 mM for the times indicated (A), for 8 h at the concentrations indicated (B), and at 1.0 mM for 8 h in the presence of Me2SO (0.1%) or PD098059 (50 µM) (C). Whole cell extracts were prepared, and luciferase activity was determined. Data are mean ± S.E. from four to six independent experiments, expressed as percentage change with respect to non-VPA-treated cells. *, p < 0.05 compared with non-VPA-treated cells.

Ras/Raf/MEK Are Required for the Activation of Elk1 by VPA-- Similar to the results observed with AP-1 DNA binding, SB203580 (10 µM) did not significantly affect VPA-induced luciferase increases (data not shown), whereas PD098059 reduced the effects of VPA (Fig. 2C). To more specifically identify the upstream components in the ERK pathway required in the actions of VPA and reveal the role of other MAP kinase pathways in these actions, the SH-SY5Y cells were co-transfected with the Elk1 trans-reporting system and either pUSE Ha-Ras S17N (22), pUSE Raf-1 K375M, pEBG SEK KR, and pCEFL MKK6 KR, which encode dominant negative Ha-Ras, Raf-1, SEK1, and MKK6 mutants, respectively. The effects of VPA on Elk1 were significantly reduced by dominant negative Ras (Fig. 3A) and Raf (Fig. 3B) mutants but not by SEK (Fig. 3C) and MKK6 (Fig. 3D) mutants.

View larger version (65K): [in this window] [in a new window]   Fig. 3.   Ras and Raf are involved in the VPA-induced MAP kinase activation. SH-SY5Y cells were co-transfected with a fixed amount of Elk1 trans-reporting system and increasing amount of expression vectors carried Ha-Ras S17N (A), Raf-1 K375M (B), SEK KR (C), and MKK6 KR (D). pTATA was used to reach the 2 µg of DNA/well transfection. The cells were then incubated with 1 mM VPA for 8 h. Whole cell extracts were prepared, and luciferase activity was determined. Data are mean ± S.E. from four to six independent experiments, expressed as percentage change with respect to non-VPA-treated cells. *, p < 0.05 compared with non-VPA-treated cells.

VPA Increases Phosphorylation of ERK-- To further confirm activation of MAP kinases by VPA, the levels of the phosphorylated activated forms, as well as total ERKs, were quantitated using immunoblotting. The levels of pERK44 and pERK42 increased 2- and 1-fold by 1 day of VPA incubation and 13- and 7-fold by 5 days of VPA incubation (Fig. 4). Neither the 1-day nor the 5-day VPA incubation significantly altered total ERK44 or ERK42 levels (Fig. 4A). Taken together, the data (Figs. 1-4) demonstrate that VPA activates the ERK pathway, and the activation persists for several days.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   VPA increases phospho-ERK44/42 but not total ERK44/42 levels. SH-SY5Y cells were treated with 1.0 mM VPA in serum-free DMEM for 1 and 5 days. Whole cell extracts were prepared, and phospho-ERK44/42 immunoblotting was conducted using 20 µg of whole cell protein extract; the same blot was then reprobed for ERK44/42. The levels were obtained using an image analysis system with NIH Image 1.55 software. Immunoblots shown are from a representative experiment, which was repeated three times with similar results. Phospho-ERK42 (B) and phospho-ERK44 (C) data are mean ± S.E. of four independent experiments, expressed as percentage change with respect to non-VPA-treated cells. *, p < 0.05 compared with non-VPA-treated cells. VPA did not significantly change total ERK44/42 levels (data not shown).

Effects of VPA on the Levels of Phosphorylation-activated Forms and Total JNK-- One-day VPA incubation did not significantly alter the levels of pJNK54 and pJNK46 (24) (Fig. 5), which is consistent with a lack of significant effect of dominant-negative SEK mutant on VPA induction of Elk1 (Fig. 3C). Five-day VPA incubation significantly increased levels of pJNK54 and pJNK46 (Fig. 5). One- and 5-day VPA incubations did not significantly change the levels of total JNK54 and JNK46 (Fig. 5A). Taken together, the increased levels of pJNKs after the 5-day VPA incubation are likely due to the changes in phosphorylation or dephosphorylation of JNKs, but not the changes in JNK protein levels.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Effects of VPA on phospho-JNK54/46 and total JNK54/46 levels. SH-SY5Y cells were treated with 1.0 mM VPA in serum-free DMEM for 1 and 5 days. Whole cell extracts were prepared, and phospho-JNK54/46 immunoblotting was conducted using 20 µg of whole cell protein extract; the same blot was then reprobed for JNK54/46. The levels were obtained using an image analysis system with NIH Image 1.55 software. Immunoblots shown are from a representative experiment, which was repeated three times with similar results. Phospho-JNK46 (B) and phospho-JNK54 (C) data are mean ± S.E. of four independent experiments, expressed as percentage change with respect to non-VPA-treated cells. *, p < 0.05 compared with non-VPA-treated cells. VPA did not significantly change total JNK54/46 levels (data not shown).

Effects of VPA on the Levels of Phosphorylation-activated Forms and Total p38-- The levels of p-p38 were not detectable in 1-day samples (Fig. 6A). Five-day VPA incubation significantly increased p-p38 levels (Fig. 6, A and B). One- and 5-day VPA incubations significantly increased p38 levels (Fig. 6), suggesting that chronic VPA either activates p38 gene expression or stabilizes p38 protein. Taking the increase in total p38 protein into account, the increases in p-p38 levels after 5-day VPA incubation could be due to changes in total p38, phosphorylated p38, or both.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Effects of VPA on phospho-p38 and total p38 levels. SH-SY5Y cells were treated with 1.0 mM VPA in serum-free DMEM for 1 and 5 days. Whole cell extracts were prepared, and phospho-p38 immunoblotting was conducted using 50 µg of whole cell protein extract; the same blot was then reprobed for p38. The levels were obtained using an image analysis system with NIH Image 1.55 software. Immunoblots (A) shown are from a representative experiment, which was repeated three times with similar results. N and P are negative and positive controls (C6 cell lysates with or without anisomycin treatment) from cell signaling. Phospho-p38 (B) and total p38 (C) data are mean ± S.E. of four independent experiments, expressed as percentage change with respect to non-VPA-treated cells. *, p < 0.05 compared with non-VPA-treated cells. Phospho-p38 levels of 1-day incubation samples could not be reliably detected.

VPA Promotes Neurite Growth in a Time- and Concentration-dependent Manner and Enhances Cell Survival in Serum-free Media-- Because the ERK pathway is known to play a role in neurite growth (18, 24, 26, 27), the effects of VPA on the morphology of the SH-SY5Y cells was investigated. SH-SY5Y cells were seeded at a very low density and treated with 0, 0.25, 0.5, or 1.0 mM VPA in serum-free media for 1-8 days in serum-free media (Fig. 7). In the media without VPA, SH-SY5Y cells displayed very short neurites or spines (<2 × diameter of cell body); and the neurites/spines did not elongate appreciably over time (Fig. 7). In addition, the cells gradually detached and died over the time period in dishes without VPA. By contrast, much longer neurites were seen in the VPA-treated cells (Fig. 7), and more cells remained. The quantitative image analysis showed that treatment of the cells with VPA produced a concentration- and time-dependent increase in neurite length, resulting in an 8.6-, 14.2-, and 23.0-fold increases in neurite length following 8-day incubation with 0.25, 0.5, and 1.0 mM VPA, respectively (Fig. 7).

View larger version (53K): [in this window] [in a new window]   Fig. 7.   VPA promotes neurite growth in a time- and concentration-dependent manner. The neurite growth experiment was conducted as described under "Experimental Procedures." The photographs were taken after 1-, 4-, and 8-day treatments with VPA using a Zeiss Axiovert 35 microscope with a × 10 objective. The lengths of neurites were measured using NIH Image 1.55 software. Photographs shown are from a representative experiment. Data shown in bar graph are mean ± S.E. of 30-100 cells measured in the representative photographs. Similar results were obtained from two additional independent experiments.

ERK Is Required for VPA-induced Neurite Growth and Cell Survival-- To investigate the role of MAP kinases in the effects of VPA on neurite elongation, SH-SY5Y cells were incubated with 1.0 mM VPA in the absence or presence of PD098059 (50 µM). Me₂SO was used as a solvent for PD098059 and was therefore used as the vehicle control. The cells gradually died over the 11-day period in dishes containing PD098059, in either the absence or presence of VPA (Fig. 8). PD098059 did not have marked effects on short neurites (Fig. 8). By contrast, long neurites were only observed in VPA treated cells at days 4, 7, and 11, but not in cells co-treated with VPA and PD098059 at any of these time points (Fig. 8). These results suggest that activation of the ERK pathway is involved in the effects of VPA on neurite growth and cell survival.

View larger version (104K): [in this window] [in a new window]   Fig. 8.   MEK inhibitor blocks VPA-induced neurite growth. Neurite growth was induced by VPA as described in the Fig. 7 legend in the presence of Me2SO (0.1%) or PD098059 (50 µM). The photographs were taken after 4-, 7-, and 11-day treatments using a Zeiss Axiovert 35 microscope with a × 20 objective. Most cells were detached after day 11 in Me2SO, PD098059 or PD098059 plus VPA treatment dishes but not in Me2SO plus 1 mM VPA treatment dishes, and therefore the experiments were stopped at day 11. Similar results were obtained from two or more additional independent experiments.

VPA Increases the Levels of GAP-43 and Bcl-2 but Not of Bax-- GAP-43 and Bcl-2 are proteins involved in neurite growth during development or regeneration (28-33) and are known to be transcriptionally regulated by MAP kinases (34-37). One-day VPA incubation resulted in ~70% increase in GAP-43 levels, whereas a greater than 3-fold increase in GAP-43 levels was observed after 5-day VPA exposure (Fig. 9, B and C). One-day VPA incubation did not produce significant increases in Bcl-2 levels, whereas 5-day VPA exposure produced >5-fold increase in Bcl-2 levels (Fig. 10, A and B). Neither 1-day nor 5-day VPA incubation produced significant changes in levels of Bax (Fig. 10, A and C), the counterpart of Bcl-2 in apoptosis (34).

View larger version (26K): [in this window] [in a new window]   Fig. 9.   VPA increases GAP-43 levels. SH-SY5Y cells were treated with 1.0 mM VPA in serum-free DMEM for 1 and 5 days. GAP-43 immunoblotting was conducted with 20 µg of whole cell protein extracts. The levels were obtained using an image analysis system with NIH Image 1.55 software. A, photograph of 5-day VPA-treated SH-SY5Y cell; the growth cone is indicated by the arrow. Immunoblots (B) are from a representative experiment, which was repeated three more times with similar results. Data in bar graph (C) are mean ± S.E. from four independent experiments, expressed as percentage change with respect to non-VPA-treated cells. *, p < 0.05 compared with non-VPA-treated cells.

View larger version (10K): [in this window] [in a new window]   Fig. 10.   VPA increases Bcl-2 but not Bax levels. SH-SY5Y cells were treated with 1.0 mM VPA in serum-free DMEM for 1 or 5 days. Whole cell extracts were prepared, and Bcl-2 and Bax immunoblotting was conducted with 20 µg of whole cell protein extracts. The levels were obtained using an image analysis system with NIH Image 1.55 software. Immunoblots of Bcl-2 and Bax shown are from a representative experiment, which was repeated three more times with similar results. Bcl-2 (B) and Bax (C) data are mean ± S.E. of four independent experiments, expressed as percentage change with respect to non-VPA-treated cells. *, p < 0.05 compared with non-VPA-treated cells. Five-day VPA treatment significantly increased Bcl-2 levels. By contrast, 1- and 5-day VPA treatment significantly decreased Bax levels.

VPA Enhances NE Uptake and Release-- SH-SY5Y cells possess characteristics of catecholaminergic neuron (23). Therefore, the uptake and release of NE were investigated. Incubation of SH-SY5Y cells with VPA (1.0 mM) increased [³H]NE uptake 1-fold after 1-day incubation and 3.5-fold after 5-day incubation (Fig. 11A). Furthermore, VPA increased [³H]NE release 4-fold after a 1-day incubation and 16-fold after a 5-day incubation (Fig. 11B). These data suggest that VPA treatment was associated with enhanced functional properties.

View larger version (35K): [in this window] [in a new window]   Fig. 11.   VPA increases NE uptake and release. SH-SY5Y cells were treated with 1.0 mM VPA in serum-free DMEM for 1 or 5 days. For uptake, the cells were incubated with [3H]NE in HBS at 37 °C for 1 h, and the cells were then washed three times with HBS. Desipramine (3 ng/ml) was used in one set of wells to determine the nonspecific incorporation. Release was induced by depolarization with potassium (100 mM). The specific uptake and release were expressed as cpm/µg of protein. A, NE uptake; B, NE release. Data are mean ± S.E. from three or more independent experiments. *, p < 0.05 compared with control cells.

VPA Promotes Extended Neurite Growth and Cell Survival-- Finally, we investigated the life span of SH-SY5Y cells with elongated neurites in the presence of VPA. During the extended period (~13-40 days) (Fig. 12), web-like neurite networks developed, in which long neurites projected from clusters of cell bodies and ended in contact with either another neurite or another cluster of cell bodies (Fig. 12). After about day 40, the neurites and cell bodies began to detach, and by day 55, more than 50% of cells had detached. Interestingly, even when detached and suspended in the medium, some of the neurite networks remained. At present, 55 days of treatment is the longest VPA incubation that we have conducted. Without VPA, almost all of the cells detached and died after 12 days in serum-free medium.

View larger version (76K): [in this window] [in a new window]   Fig. 12.   Induction of web-like neurite networks by extended VPA treatment. SH-SY5Y cells were cultured for extended periods with VPA. Neurite growth was induced by VPA as described in the Fig. 5 legend, and the cultures were grown for extended periods of time. The photographs were taken after 13, 20, 40, and 55 days. Without VPA, nearly all cells were detached from the dish after 12 days of the incubation in serum-free media (photograph not show). Similar results were obtained from two additional experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Antimanic action of VPA appears at blood level above 50 µg/ml, and clinical toxicity appears at blood level above 200 µg/ml (15). 150 µg/ml (1.041 mM) was used in the VPA antimanic efficacy trial (35). Therefore, 1 mM VPA was used in most of experiments described in this paper, except that lower and higher concentrations were used in concentration-dependent experiments.

In the present study, we have shown that VPA at therapeutic concentration produces effects similar to those of neurotrophic factors, namely activation of the ERK pathway and promotion of neurite growth and cell survival. VPA also increases GAP-43 and Bcl-2 levels and enhances the neuronal function.

VPA stimulates DNA binding activity, transactivation activity of AP-1 transcription factor, and expression of genes regulated by AP-1 (2, 3, 6, 8, 10). In the present study, we found that VPA activated ERK, the upstream modulator of AP-1. Blockade of ERK pathway by PD098059 blocked VPA-induced increases in AP-1 binding activity. These data indicate that the ERK pathway is required for VPA-induced increases in AP-1 binding. There are at least two pathways through which ERK regulates AP-1 (12). ERK phosphorylates and activates Elk1, thereby enhancing expression of c-Fos, a key member of the AP-1 family, resulting in an increase of AP-1 function (12). It has been reported that VPA increases c-Fos expression (2, 8), but this effect may be transient. ERK activation has also been shown to result in increases in phosphorylation of c-Jun at serine 63 and serine 73 and, secondarily, increases in c-Jun expression (36-39). c-Jun has been implicated in several long lasting AP-1 effects, including neurite outgrowth (27). In our preliminary study, we found that VPA increased c-Jun levels and phosphorylated c-Jun in VPA-treated SH-SY5Y cells. These data suggest that the VPA-induced increases in AP-1 binding and function are likely due, at least in part, to activation of ERK followed by phosphorylation and increase in expression of c-Jun.

Previous studies have found a conservative AP-1 site in the GAP-43 promoter, and AP-1 is involved in enhanced GAP-43 expression (32). In the present study, we also found that VPA increased expression of GAP-43, perhaps through an ERK-AP-1 mechanism. The cAMP-response element site is present in the Bcl-2 promoter and phosphorylation of cAMP-response element-binding protein is associated with increases in Bcl-2 expression upon PKC activation (40, 41, 44, 45). Activation of MAP kinases by neurotrophin results in increases in cAMP-response element-binding protein phosphorylation and enhanced Bcl-2 expression (42-44) through ribosome S6 kinase (45). Thus, the VPA-induced increases in Bcl-2 expression may be mediated through ribosome S6 kinase and cAMP-response element-binding protein.

The precise mechanisms by which VPA brings about these effects are currently unknown, but they likely involve the activation of intracellular signaling pathways because there are no known cell surface "VPA receptors." Furthermore, the time course for the VPA-induced activation of ERK was much slower than that provoked by insulin-like growth factor-I or brain-derived neurotrophic factor in SH-SY5Y cells (16, 18). Thus, it is unlikely that the activation of ERK pathways by VPA is initiated by direct interactions of VPA with a cell surface receptor, such as insulin-like growth factor-I receptor. VPA is not known to bind to any G protein-coupled receptors that activate MAP kinases (46). However, it is still possible that VPA might stimulate synthesis and secretion of a neurotrophic factor, which in turn activates the ERK pathway through cell surface receptor tyrosine kinase or other mechanisms.

Cells have been demonstrated to gradually take up VPA over a time course from minutes to hours (47, 48). In cells, VPA is incorporated into larger phospholipid molecule(s), perhaps phosphatidylcholine (48). From phosphatidylcholine, lysophosphatidylcholine is generated intracellularly by phospholipase A2. Lysophosphatidylcholine activates ERK through the phosphatidylinositol 3-kinase/Janus kinase 2/MEK-1-dependent pathway in endothelial cells (49) and through the tyrosine kinase-Ras-dependent pathway in mesangial cells (50). Thus, a parsimonious working hypothesis is that VPA in neuronal cells affects lysophosphatidylcholine accumulation or valproyl-lysophosphatidylcholine is a potent or long lasting inducer of ERK pathway.

Similar to nerve growth factor in PC-12 cell (24) and insulin-like growth factor-I in SH-SY5Y cell (18), ERK is required for VPA to promote neurite growth. Studies have shown that neurite growth can be induced using constitutively activated ERK pathway components or constitutively activated c-Jun mutants in PC-12 cells (26, 27), demonstrating that stimulation of ERK pathway at steps distal to cell surface receptor is sufficient for these neurobiological effects. In our studies, VPA induced persistent activation of ERK, suggesting that VPA (or a metabolite) behaves like an ERK pathway activator, producing persistent activation of ERK, which results in extended neurite growth.

Although mood disorders have traditionally been conceptualized as neurochemical disorders, recent brain imaging studies in living patients and three-dimensional morphometric studies in postmortem brains have shown that patients with mood disorders display morphometric changes suggestive of cell loss and/or atrophy (51-54). We found that the classic mood stabilizer lithium induced increases in Bcl-2 level and neurogenesis in adult rodent brain (22, 55). We also found that lithium induced increases in N-acetyl-aspartate levels (a putative marker of neuronal viability and function) (56) and total gray matter in human brain (57). In view of the reported glial and neuronal cell loss and atrophy in mood disorders and the neurotrophic actions of the classic mood stabilizer lithium, these neurotrophic effects of VPA may play an important role in its long term therapeutic actions. Furthermore, these results suggest this simple fatty acid may be of utility in the treatment of a variety of neuropsychiatric disorders, a possibility that warrants further investigation.

	FOOTNOTES

^* This work was supported by National Institute of Health Grant MH57743 (to H. K. M.), the Joe Young Senior Research Fund, and the Theodore and Vada Stanley Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Unit on Molecular Neurotherapeutics, Laboratory of Molecular Pathophysiology, NIMH, NIH, Bldg. 49, Rm. B1EE16, 49 Convent Drive, MSC 4405, Bethesda, MD 20892-4405. Tel.: 301-496-9802; Fax: 301-480-0123; E-mail: gchen@codon.nih.gov.

Published, JBC Papers in Press, June 19, 2001, DOI 10.1074/jbc.M104309200

	ABBREVIATIONS

The abbreviations used are: VPA, valproic acid; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular signal-regulated kinase; GAP, growth cone-associated protein; JNK, c-Jun NH₂-terminal kinase; MAP, mitogen-activated protein; MEK, MAP kinase/ERK kinase; NE, norepinephrine; SEK, stress-activated protein kinase/ERK kinase; MKK6, MAP kinase kinase 6; TRE, TPA response element; TPA, 12-O-tetradecanoylphorbol-13-acetate.

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