Histone Deacetylase Is a Direct Target of Valproic Acid, a Potent Anticonvulsant, Mood Stabilizer, and Teratogen

Valproic acid is widely used to treat epilepsy and bipolar disorder and is also a potent teratogen, but its mechanisms of action in any of these settings are unknown. We report that valproic acid activates Wntdependent gene expression, similar to lithium, the mainstay of therapy for bipolar disorder. Valproic acid, however, acts through a distinct pathway that involves direct inhibition of histone deacetylase (IC50 for HDAC1 = 0.4 mm). At therapeutic levels, valproic acid mimics the histone deacetylase inhibitor trichostatin A, causing hyperacetylation of histones in cultured cells. Valproic acid, like trichostatin A, also activates transcription from diverse exogenous and endogenous promoters. Furthermore, valproic acid and trichostatin A have remarkably similar teratogenic effects in vertebrate embryos, while non-teratogenic analogues of valproic acid do not inhibit histone deacetylase and do not activate transcription. Based on these observations, we propose that inhibition of histone deacetylase provides a mechanism for valproic acid-induced birth defects and could also explain the efficacy of valproic acid in the treatment of bipolar disorder. Valproic acid is widely used to treat epilepsy and bipolar disorder and is also a potent teratogen, but its mechanisms of action in any of these settings are unknown. We report that valproic acid activates Wntdependent gene expression, similar to lithium, the mainstay of therapy for bipolar disorder. Valproic acid, however, acts through a distinct pathway that involves direct inhibition of histone deacetylase (IC50 for HDAC1 = 0.4 mm). At therapeutic levels, valproic acid mimics the histone deacetylase inhibitor trichostatin A, causing hyperacetylation of histones in cultured cells. Valproic acid, like trichostatin A, also activates transcription from diverse exogenous and endogenous promoters. Furthermore, valproic acid and trichostatin A have remarkably similar teratogenic effects in vertebrate embryos, while non-teratogenic analogues of valproic acid do not inhibit histone deacetylase and do not activate transcription. Based on these observations, we propose that inhibition of histone deacetylase provides a mechanism for valproic acid-induced birth defects and could also explain the efficacy of valproic acid in the treatment of bipolar disorder. valproic acid γ-aminobutyric acid valpromide 2-methyl-2-propylpentenoic acid protein kinase C glycogen synthase kinase-3β histone deacetylase cytomegalovirus cycloheximide polyacrylamide gel electrophoresis secreted alkaline phosphatase midblastula transition trichostatin A modified Marc's Ringer's activator protein-1 Valproic acid (VPA)1 is a short-chained fatty acid widely used in humans as an anticonvulsant and as a mood stabilizer (1Johannessen C.U. Neurochem. Int. 2000; 37: 103-110Crossref PubMed Scopus (333) Google Scholar, 2Tunnicliff G. J. Physiol. Pharmacol. 1999; 50: 347-365PubMed Google Scholar). The effectiveness of VPA as an anticonvulsant was discovered serendipitously when other compounds were dissolved in VPA for administration to animals used in experimental models of epilepsy (1Johannessen C.U. Neurochem. Int. 2000; 37: 103-110Crossref PubMed Scopus (333) Google Scholar, 2Tunnicliff G. J. Physiol. Pharmacol. 1999; 50: 347-365PubMed Google Scholar, 3Meunier H. Carraz G. Meunier Y. et al.Therapie. 1963; 18: 435-438PubMed Google Scholar). Since then, VPA has been used to control a variety of seizures, including generalized and partial seizures (1Johannessen C.U. Neurochem. Int. 2000; 37: 103-110Crossref PubMed Scopus (333) Google Scholar). Several hypotheses have been put forth to explain the anticonvulsant activity of VPA, and, given the efficacy of VPA in diverse forms of epilepsy, it may act through more than one target (1Johannessen C.U. Neurochem. Int. 2000; 37: 103-110Crossref PubMed Scopus (333) Google Scholar). VPA increases the level of the inhibitory neurotransmitter γ-aminobutyric acid (GABA), with acute administration causing a 15–45% increase in GABA in the brains of rodents (cited in Ref. 1Johannessen C.U. Neurochem. Int. 2000; 37: 103-110Crossref PubMed Scopus (333) Google Scholar). Because inhibition of GABAergic signaling can cause seizures and potentiation of GABA signaling can prevent seizures, this effect of VPA on GABA levels has been proposed as a mechanism for the anticonvulsant activity of VPA. However, the target(s) of VPA in this setting has not been definitively identified; VPA can stimulate GABA biosynthetic enzymes and inhibit enzymes involved in GABA degradation in vitro, but it is not clear whether these are important in vivo targets of VPA (1Johannessen C.U. Neurochem. Int. 2000; 37: 103-110Crossref PubMed Scopus (333) Google Scholar,2Tunnicliff G. J. Physiol. Pharmacol. 1999; 50: 347-365PubMed Google Scholar, 4Davies J.A. Seizure. 1995; 4: 267-271Abstract Full Text PDF PubMed Scopus (82) Google Scholar). VPA is a potent teratogen in humans (5Robert E. Guibaud P. Lancet. 1982; 2: 937Abstract PubMed Scopus (457) Google Scholar) and is widely studied as a model teratogen in rodents. Although the target of VPA in this setting is unknown, strict structural requirements have been defined for the teratogenic activity of VPA and VPA-related compounds. Thus, potently teratogenic analogues of VPA contain a tetrahedral α-carbon bound to a free carboxyl group, a hydrogen, and two alkyl groups (6Lampen A. Siehler S. Ellerbeck U. Göttlicher M. Nau H. Toxicol. Appl. Pharmacol. 1999; 160: 238-249Crossref PubMed Scopus (57) Google Scholar, 7Nau H. Hauck R.S. Ehlers K. Pharmacol. Toxicol. 1991; 69: 310-321Crossref PubMed Scopus (255) Google Scholar). In contrast, analogues such as valpromide (VPM), in which the carboxyl group is modified to an amide, and 2-methyl-2-propylpentenoic acid (2M2P), in which a methyl group is added to the α-carbon, do not cause neural tube defects in mouse embryos. These analogues can still protect against chemically induced seizures in mice (6Lampen A. Siehler S. Ellerbeck U. Göttlicher M. Nau H. Toxicol. Appl. Pharmacol. 1999; 160: 238-249Crossref PubMed Scopus (57) Google Scholar, 7Nau H. Hauck R.S. Ehlers K. Pharmacol. Toxicol. 1991; 69: 310-321Crossref PubMed Scopus (255) Google Scholar), suggesting that at least some of the clinically observed effects of VPA involve distinct molecular targets. In the treatment of bipolar disorder, VPA is effective both in acute mania and as a prophylaxis for recurrent mania and depression, similar to lithium (1Johannessen C.U. Neurochem. Int. 2000; 37: 103-110Crossref PubMed Scopus (333) Google Scholar, 2Tunnicliff G. J. Physiol. Pharmacol. 1999; 50: 347-365PubMed Google Scholar). However, as with lithium, the mechanism of VPA action in bipolar disorder remains unknown. A number of interesting mechanisms have been proposed, but in each case, the direct target of VPA has not been defined. The characteristic delay in response to lithium or VPA has led to the proposal that both drugs act through modulation of gene expression, and this is supported by data fromin vitro as well as in vivo systems (8Wang J.F. Bown C. Young L.T. Mol. Pharmacol. 1999; 55: 521-527Crossref PubMed Scopus (15) Google Scholar, 9Manji H.K. McNamara R. Chen G. Lenox R.H. Aust. N. Z. J. Psychiatry. 1999; 33 (suppl.): S65-S83Crossref PubMed Google Scholar, 10Lenox R.H. McNamara R.K. Papke R.L. Manji H.K. J. Clin. Psychiatry. 1998; 59: 37-47PubMed Google Scholar, 11Chen G. Yuan X. Jiang Y.M. Huang L.D. Manji H.K. Brain Res. Mol. Brain Res. 1999; 64: 52-58Crossref PubMed Scopus (94) Google Scholar, 12Asghari V. Wang J.F. Reiach J.S. Young L.T. Brain Res. Mol. Brain Res. 1998; 58: 95-102Crossref PubMed Scopus (85) Google Scholar). Furthermore, lithium and VPA can down-regulate expression of protein kinase C isoforms PKCα and PKCε, induce expression of the anti-apoptotic gene bcl-2, and activate AP-1-dependent transcription (through a direct effect on c-jun activity and by increasing expression of c-Jun (11Chen G. Yuan X. Jiang Y.M. Huang L.D. Manji H.K. Brain Res. Mol. Brain Res. 1999; 64: 52-58Crossref PubMed Scopus (94) Google Scholar, 12Asghari V. Wang J.F. Reiach J.S. Young L.T. Brain Res. Mol. Brain Res. 1998; 58: 95-102Crossref PubMed Scopus (85) Google Scholar)). Both VPA and lithium also stimulate glutamate release and inositol 1,4,5-trisphosphate accumulation in mouse cerebral cortex slices, although apparently through distinct mechanisms (14Dixon J.F. Hokin L.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4757-4760Crossref PubMed Scopus (75) Google Scholar). Furthermore, both VPA and lithium have been shown to confer protection from neurotoxic agents (15Mora A. Gonzalez-Polo R.A. Fuentes J.M. Soler G. Centeno F. Eur. J. Biochem. 1999; 266: 886-891Crossref PubMed Scopus (101) Google Scholar, 16Nonaka S. Katsube N. Chuang D.M. J. Pharmacol. Exp. Ther. 1998; 286: 539-547PubMed Google Scholar, 17Nonaka S. Hough C.J. Chuang D.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2642-2647Crossref PubMed Scopus (403) Google Scholar). In each of these settings, there is a delay in the response, similar to that observed clinically; thus the direct targets of VPA in these settings have not been determined. Several direct targets of lithium have been identified (reviewed in Ref. 18Phiel C.J. Klein P.S. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 789-813Crossref PubMed Scopus (455) Google Scholar), including inositol monophosphatase (19Hallcher L.M. Sherman W.R. J. Biol. Chem. 1980; 255: 10896-10901Abstract Full Text PDF PubMed Google Scholar, 20Berridge M.J. Downes C.P. Hanley M.R. Cell. 1989; 59: 411-419Abstract Full Text PDF PubMed Scopus (890) Google Scholar), a family of related phosphomonoesterases (21York J.D. Ponder J.W. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5149-5153Crossref PubMed Scopus (156) Google Scholar), and glycogen synthase kinase-3β (GSK-3β (22Klein P.S. Melton D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8455-8459Crossref PubMed Scopus (2098) Google Scholar)). GSK-3β is a negative regulator of the Wnt signaling pathway, which regulates numerous processes, including axonal remodeling, cellular proliferation, embryonic patterning, and organogenesis (23Salinas P.C. Biochem. Soc. Symp. 1999; 65: 101-109PubMed Google Scholar, 24Dickinson M.E. Krumlauf R. McMahon A.P. Development. 1994; 120: 1453-1471PubMed Google Scholar, 25Morin P.J. Bioessays. 1999; 21: 1021-1030Crossref PubMed Scopus (817) Google Scholar, 26Miller J.R. Hocking A.M. Brown J.D. Moon R.T. Oncogene. 1999; 18: 7860-7872Crossref PubMed Scopus (607) Google Scholar). Because GSK-3β phosphorylates β-catenin, leading to its rapid degradation, inhibition of GSK-3β by either lithium or Wnt signaling leads to stabilization and accumulation of β-catenin protein (27Hedgepeth C.M. Conrad L.J. Zhang J. Huang H.C. Lee V.M. Klein P.S. Dev. Biol. 1997; 185: 82-91Crossref PubMed Scopus (564) Google Scholar, 28Stambolic V. Ruel L. Woodgett J. Curr. Biol. 1996; 6: 1664-1668Abstract Full Text Full Text PDF PubMed Google Scholar); β-catenin then translocates to the nucleus where it activates transcription of Wnt-dependent genes by binding to factors of the Tcf/Lef family. Activation of Wnt signaling by lithium has been proposed to explain the similarity between lithium and Wnts in a variety of settings (22Klein P.S. Melton D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8455-8459Crossref PubMed Scopus (2098) Google Scholar), but a role for this pathway in bipolar disorder has not been demonstrated. Although VPA does not inhibit inositol monophosphatase (29Murray M. Greenberg M.L. Mol. Microbiol. 2000; 36: 651-661Crossref PubMed Scopus (61) Google Scholar, 30Vadnal R. Parthasarathy R. Neuropsychopharmacology. 1995; 12: 277-285Crossref PubMed Scopus (55) Google Scholar), it has also been reported to inhibit GSK-3β-mediated phosphorylation of a peptide derived from the CREB protein in vitro, and exposure of SH-SY5Y cells to VPA can also cause an increase in β-catenin protein levels (31Chen G. Huang L.D. Jiang Y.M. Manji H.K. J. Neurochem. 1999; 72: 1327-1330Crossref PubMed Scopus (421) Google Scholar), raising the interesting possibility that VPA and lithium both act through inhibition of GSK-3β. However, VPA has not yet been shown to inhibit GSK-3β in vivo nor to activate Wnt-dependent gene expression. We have further investigated whether VPA activates Wnt signaling and find that VPA can indeed activate Wnt-dependent gene expression, similar to lithium, but through a distinct mechanism that involves direct activation of transcription. We show that VPA potently inhibits histone deacetylase (HDAC), a negative regulator of gene expression in multiple settings, at therapeutically relevant levels. Furthermore, the teratogenicity of VPA in vertebrate embryos is mimicked by the HDAC inhibitor trichostatin A, whereas non-teratogenic analogues of VPA do not inhibit HDAC. These findings lead us to propose that HDAC is an important target of VPA in the pathogenesis of birth defects. HDAC also offers a plausible novel target for VPA action in the treatment of bipolar disorder. Luciferase constructs containing three wild-type (Lef-OT) or three mutated (Lef-OF) Lef binding sites were gifts from Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD) and have been described elsewhere (32Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers Science. 1997; 275: 1784-1789Crossref PubMed Scopus (2950) Google Scholar). Renilla luciferase plasmids pRL-SV40 and pRL-CMV were purchased from Promega. Plasmids encoding secreted alkaline phosphatase (pSEAP) and enhanced green fluorescence protein were purchased from CLONTECH. Human HDAC1 in pcDNA3.1-myc/His-A (33Miska E.A. Karlsson C. Langley E. Nielsen S.J. Pines J. Kouzarides T. EMBO J. 1999; 18: 5099-5107Crossref PubMed Scopus (472) Google Scholar) was a gift of Dr. T. Kouzarides (The Wellcome/Cancer Research Campaign Institute, Cambridge, UK). 293T and Neuro2A cells were obtained from American Type Culture Center. 293T cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS). Neuro2A cells were grown in Eagle's Basal Medium in 10% FBS. Stable 293T cell lines were prepared by transfecting Lef-OT or Lef-OF together with pcDNA3.1. Cells were selected and maintained in Dulbecco's modified Eagle's medium with 10% FBS and 400 mg/liter G418. For all transfections, cells were plated in 6-well dishes at a density of 2.75 × 105 cells per well. Plasmids were transfected as follows: 1 μg of Lef-OT and Lef-OF reporter plasmids; 10 ng of pRL-CMV or pRL-SV40; and 0.5 μg of pSEAP. Firefly andRenilla luciferase activities for each sample were measured on a Monolight 3010 luminometer (Turner Designs) using the Dual Luciferase assay kit (Promega). All transfections included 0.5 μg of enhanced green fluorescence protein to assess transfection efficiency. In some cases (e.g. overexpression of HDAC1), pSEAP was also transfected, and SEAP activity in culture medium was used as an independent measure of transfection efficiency. Thus, 24 h after transfection, an aliquot of media was removed for SEAP assay, and VPA or LiCl was then added to the cells. After an additional 24 h, cells were harvested for luciferase assay. Valproic acid (sodium salt; Sigma Chemical Co.) and lithium chloride (Sigma) were prepared in sterile water as concentrated stocks and added to the final concentrations as indicated in the figures. Valpromide (kind gift of Katwijk Chemie B.V.) was prepared in Me2SO. 2-Methyl-2-propylpentenoic acid and 4-pentenoic acid were purchased from Alfa Aesar. For cycloheximide experiments, Neuro2A cells, plated at 5 × 105 cells per well, were treated 24 h later with the following compounds: 10 μg/ml cycloheximide (CHX), 20 mm LiCl, or 2 mm VPA. At the indicated time points, cells were harvested using Reporter lysis buffer (Promega) supplemented with a mixture of protease and phosphatase inhibitors (1:100, Sigma). Samples were centrifuged at 14,000 rpm for 10 min at 4 °C, and the supernatants were added to Laemmli sample buffer and boiled for 2 min. Samples were separated by electrophoresis on 7.5% polyacrylamide gels (SDS-PAGE), immunoblotted using β-catenin antibody (1:1000; Transduction Laboratories), or β-tubulin antibody (TUJ1, 1:1000, BabCO), and visualized by enhanced chemiluminescent detection (Amersham Pharmacia Biotech). For detection of tau protein, Neuro2A cells were exposed to VPA, 2-methyl-2-propylpentenoic acid, 4-pentenoic acid, or LiCl for 24 h and then harvested in TNE buffer (10 mm Tris, pH 7.8, 150 mm NaCl, 1 mm EDTA, 1% Nonidet P-40) supplemented with 50 mm NaF and Sigma protease inhibitor mixture (1:100). Samples were electrophoresed on 7.5% SDS-PAGE and immunoblotted with PHF-1 antibody (1:250, provided by Peter Davies (34Greenberg S.G. Davies P. Schein J.D. Binder L.I. J. Biol. Chem. 1992; 267: 564-569Abstract Full Text PDF PubMed Google Scholar)) or Tau antibody (17026, 1:1000, provided by Virginia Lee) and visualized by enhanced chemiluminescent detection (Amersham Pharmacia Biotech). To detect acetylation of endogenous histone H4, Neuro2A cells (1.6 × 106 cells per sample) were exposed to VPA or TSA at concentrations indicated in figure; nuclear extracts were prepared as described previously (35Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3918) Google Scholar) and then adjusted to 0.4n H2SO4. Precipitated proteins were collected by centrifugation and resuspended in 2 ml of 20 mm HEPES, 1 mm EDTA, 1 mm EGTA, followed by centrifugation through a Centricon 10 membrane to concentrate protein (final volume 150 μl). SDS sample buffer was added, and the proteins were separated by electrophoresis on 12.5% acrylamide gels (SDS-PAGE). Gels were either stained with Coomassie Blue or immunoblotted with acetyl-histone H4 antibody (1:1000; Upstate Biotechnology Inc.). GSK-3β assay was performed as described previously (22Klein P.S. Melton D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8455-8459Crossref PubMed Scopus (2098) Google Scholar), except that MgCl2was 1 mm. VPA was added at concentrations indicated in Fig.3B. For in vitro HDAC assays, myc epitope-tagged HDAC1 was transfected into HeLa cells and immunoprecipitated (36Huang E.Y. Zhang J. Miska E.A. Guenther M.G. Kouzarides T. Lazar M.A. Genes Dev. 2000; 14: 45-54PubMed Google Scholar). Immunoprecipitates were washed, resuspended in HD buffer (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 10% glycerol), and stored as frozen aliquots. HDAC1 was then added to a tube containing 40,000 cpm 3H-labeled acetylated histones (purified from HeLa cells) in 200 μl of HD buffer ± VPA, trichostatin A (Sigma), valpromide, or 2-methyl-2-propylpentenoic acid (at concentrations described in Figs. 3, 4, and 7). After rotation for 2 h at 37 °C, the reaction was stopped by the addition of 50 μl of stop solution (1 m HCl, 0.16 m acetic acid) and released 3H-labeled acetic acid was extracted and analyzed by scintillation counting. To assay total nuclear HDAC activity, nuclear extracts from HeLa cells (30 μg) were used as a source of HDAC activity in place of immunoprecipitated HDAC1, as described (36Huang E.Y. Zhang J. Miska E.A. Guenther M.G. Kouzarides T. Lazar M.A. Genes Dev. 2000; 14: 45-54PubMed Google Scholar). HDAC assay was otherwise as described above for HDAC1.Figure 4VPA inhibits HDAC activity.A, human HDAC1 activity was assayed in vitro as release of [3H]acetate from labeled histones (36Huang E.Y. Zhang J. Miska E.A. Guenther M.G. Kouzarides T. Lazar M.A. Genes Dev. 2000; 14: 45-54PubMed Google Scholar) in the presence of 0–20 mm VPA. VPA inhibited HDAC1 with an IC50 of 0.4 mm, well within the therapeutic range of VPA in humans. Percent HDAC activity is shown with respect to the activity of HDAC alone (100%). B, VPA inhibits endogenous HDACs present in HeLa cell nuclear extracts. Nuclear extracts from untransfected HeLa cells were isolated and added to HDAC assay as described in A. Percent HDAC activity is shown with respect to the activity of HDAC alone (100%). Error barsrepresent standard deviation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Inhibition of HDAC correlates with teratogenicity. A, non-teratogenic analogues valpromide (VPM; 5 mm) and 2-methyl-2-propylpentenoic acid (2M2P; 5 mm) do not inhibit HDAC1, whereas VPA (5 mm) and the established HDAC inhibitor TSA (300 nm) do inhibit HDAC1. Assay conditions are as in Fig. 4A. B–E,Xenopus embryos were treated from stage 8 until neurula stage with buffer, VPA, VPM, or TSA, and then scored at tadpole stages.B, control Xenopus tadpole. C, tadpole after exposure to VPA is shorter and lacks anterior structures.D, tadpole after exposure to VPM with normal anterior development. E, tadpole after exposure to TSA is shorter and lacks anterior structures, similar to VPA-treated tadpole.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Xenopus eggs and embryos were maintained in 0.1× MMR according to standard protocols (37Peng H.B. Methods Cell Biol. 1991; 36: ???Google Scholar). Stage 8 embryos were incubated in 0.1× MMR containing valproic acid or valpromide (1.0, 2.5, or 5.0 mm) or trichostatin A (25, 50, or 100 nm) for 24 h. Embryos were then transferred to fresh 0.1× MMR and cultured until tadpole stages. VPA stock (2m) was prepared in water, whereas valpromide (2m) and TSA (100 μm) stocks were prepared in Me2SO. Control Me2SO-treated embryos developed normally. To test whether VPA can activate Wntdependent gene expression, we generated stable cell lines in human embryonic kidney cells (293T) transfected with firefly luciferase reporters containing either three wild-type Lef binding sites (Lef-OT) or three mutant Lef binding sites (Lef-OF) (32Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers Science. 1997; 275: 1784-1789Crossref PubMed Scopus (2950) Google Scholar). These two stable cell lines were treated with VPA or lithium chloride (LiCl) for 24 h and then harvested to measure luciferase activity. Cells treated with lithium show a dosedependent increase in Lef-luciferase activity (over 70-fold) for the reporter containing wild-type, but not mutated, Lef sites (Fig.1A), as reported for transiently transfected C57MG cells (38Staal F.J. Burgering B.M. van de Wetering M. Clevers H.C. Int. Immunol. 1999; 11: 317-323Crossref PubMed Scopus (67) Google Scholar). Similarly, VPA also induces Lef-dependent luciferase activity over 20-fold (Fig.1B). Interestingly, the addition of both drugs to the 293T stable lines resulted in marked synergistic activation of reporter activity (Fig. 1C), with up to 315-fold activation, far exceeding additive effects. This synergy raises the possibility that lithium and VPA act through independent mechanisms in this assay. This could also explain the efficacy of combining lithium and valproate in bipolar disorder patients that are resistant to single drug therapy. To test whether neuronal cells may respond to VPA in a similar manner, Neuro2A cells were transiently transfected with Lef-OT or Lef-OF, together with a control reporter (pRL-SV40) encoding Renilla luciferase driven by the SV-40 promoter, and firefly and Renillaluciferase activities were measured after 24 h. As in 293T cells, VPA activated OT-Lef up to 6-fold in Neuro2A cells (not shown). Surprisingly, VPA also consistently activated the control reporter up to 10-fold (Fig. 1D), with half-maximal activation at 0.8 mm VPA. Transfection efficiency, assessed by frequency of green fluorescence protein-positive cells or by expression of secreted alkaline phosphatase (SEAP), was similar in each group prior to addition of VPA. Lithium did not stimulate this or other control reporters (not shown). VPA can activate AP-1-dependent transcription, and an increase in the activity of the SV-40 promoter has been proposed to be due to the presence of AP-1 sites within the SV-40 promoter (39Manji H.K. Lenox R.H. Biol. Psychiatry. 1999; 46: 1328-1351Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). However, VPA also induces Renilla expression driven by the cytomegalovirus (CMV) promoter (pRL-CMV), which does not contain an AP-1 site (Fig. 4B), suggesting a more general mechanism of activation. VPA has also been reported to activate the Rous sarcoma virus promoter and peroxisomal proliferator-activated receptor-δ-dependent transcription in F9 teratocarcinoma cells (6Lampen A. Siehler S. Ellerbeck U. Göttlicher M. Nau H. Toxicol. Appl. Pharmacol. 1999; 160: 238-249Crossref PubMed Scopus (57) Google Scholar). The effect of VPA on diverse promoters suggests that VPA acts through a mechanism distinct from lithium and may involve direct activation of transcription. Wnt signaling, or exposure to lithium, causes stabilization and accumulation of β-catenin protein (26Miller J.R. Hocking A.M. Brown J.D. Moon R.T. Oncogene. 1999; 18: 7860-7872Crossref PubMed Scopus (607) Google Scholar). We therefore examined the effect of VPA on levels of β-catenin protein by Western blotting. In Neuro2A cells, both lithium (20 mm) and VPA (2 mm) caused accumulation of β-catenin protein, similar to published work on VPA in SY5Y cells (31Chen G. Huang L.D. Jiang Y.M. Manji H.K. J. Neurochem. 1999; 72: 1327-1330Crossref PubMed Scopus (421) Google Scholar). However, the rate of β-catenin accumulation differed with the two drugs. Lithium caused β-catenin accumulation within 30 min of treatment, whereas the effect of VPA was not evident until 10 h after treatment (Fig.2A). Although this could reflect differences in the access of VPA and lithium, VPA has been shown to cross the blood-brain barrier within 1 min after intravenous injection and similarly is rapidly taken up by cells in culture (40Hammond E.J. Perchalski R.J. Villarreal H.J. Wilder B.J. Brain Res. 1982; 240: 195-198Crossref PubMed Scopus (13) Google Scholar). An alternative possibility to explain the delay in β-catenin accumulation after VPA exposure is that VPA acts by increasing the expression of β-catenin rather than stabilizing the protein. To distinguish between these two possibilities, Neuro2A cells were cultured in the presence of cycloheximide (CHX), an inhibitor of protein synthesis. Agents, such as lithium, that stabilize β-catenin should slow its degradation, but no new protein will accumulate. Agents that induce new transcription or translation of β-catenin should have no effect on β-catenin protein levels in the presence of CHX. Under these conditions, β-catenin is rapidly degraded and is almost undetectable after 30 min of CHX treatment (Fig. 2B,lane 2). In the presence of CHX, lithium stabilized existing β-catenin protein, slowing the rate of degradation so that β-catenin protein was readily detectable at 30 min, 5 h, and 10 h (Fig. 2B, lanes 3, 6, and9). Conversely, VPA treatment did not stabilize β-catenin; the protein was rapidly degraded within 30 min and was barely detectable at 5 or 10 h (Fig. 2B, lanes 4,7, and 10), as in cells treated with CHX alone. These observations suggest that VPA acts at the level of transcription or translation. Northern blot analysis confirmed that VPA increases the level of β-catenin mRNA in Neuro2A cells in a dose- and time-dependent manner (Fig. 2C), with increased β-catenin mRNA detected as early as 4.5 h. These data strongly support that VPA induces β-catenin at the level of transcription (or message stability) rather than through post-translational regulation. VPA can inhibit GSK-3β-mediated phosphorylation of a CREB peptidein vitro, providing an intriguing potential mechanism for VPA action, but the effect of VPA on GSK-3β activity in vivo has not been studied. In vivo inhibition of GSK-3β can be followed by examining phosphorylation of the microtubule-associated protein tau, which is phosphorylated by GSK-3βin vivo at specific sites recognized by the PHF-1 antibody (34Greenberg S.G. Davies P. Schein J.D. Binder L.I. J. Biol. Chem. 1992; 267: 564-569Abstract Full Text PDF PubMed Google Scholar, 41Mandelkow E.M. Biernat J. Drewes G. Steiner B. Lichtenberg K.B. Wille H. Gustke N. Mandelkow E. Ann. N. Y. Acad. Sci. 1993; 695: 209-216Crossref PubMed Scopus (47) Google Scholar). This phosphorylation is inhibited by lithium in vitro (22Klein P.S. Melton D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8455-8459Crossref PubMed Scopus (2098) Google Scholar) and in vivo (27Hedgepeth C.M. Conrad L.J. Zhang J. Huang H.C. Lee V.M. Klein P.S. Dev. Biol. 1997; 185: 82-91Crossref PubMed Scopus (564) Google Scholar, 28Stambolic V. Ruel L. Woodgett J. Curr. Biol. 1996; 6: 1664-1668Abstract Full Text Full Text PDF PubMed Google Scholar), as well as by other GSK-3β inhibitors (18Phiel C.J. Klein P.S. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 789-813Crossref PubMed Scopus (455) Google Scholar). We therefore examined the levels of tau phosphorylation in mouse Neuro2A cells treated with VPA. VPA from 0.5 to 20 mm did not inhibit tau phosphorylation even after 24 h of exposure (Fig.3A). Rather, VPA caused a modest increase in the level of tau protein (phosphorylated and unphosphorylated forms). The non-teratogenic analogues of VPA, 2-methyl-2-propylpentenoic acid (2M2P) and 4-pentenoic acid (both at 2 mm) had no effect on levels of tau protein or tau phosphorylation. Furthermore, lithium inhibited tau phosphorylation in the presence of VPA (Fig. 3A), indicating that the tau phosphorylation under these conditions depends on GSK-3β activity. VPA also did not inhibit tau phosphorylation in Xenopusooc