Phiel CJ et al. (2001), Histone deacetylase is a direct target of valpr...

XB-ART-8662

J Biol Chem 2001 Sep 28;27639:36734-41. doi: 10.1074/jbc.M101287200.

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Histone deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen.

Phiel CJ , Zhang F , Huang EY , Guenther MG , Lazar MA , Klein PS .

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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 (IC(50) 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.

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Species referenced: Xenopus
Genes referenced: h2ac21 h2bc21 hdac1 hdac3 mapt

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	FIG. 1. Activation of Lef-dependent transcription by VPA and lithium. A, lithium (LiCl) causes a dose-dependent increase in Lefluciferase activity in 293T cells. The OT cell line (light gray boxes) was stably transfected with a reporter (OT-luciferase) containing three wild-type Lef binding sites regulating luciferase expression, whereas OF cells (dark gray boxes) contain a reporter (OF-luciferase) in which the Lef binding sites are mutated. Fold Increase represents luciferase activity in presence of drug relative to no drug. B, VPA treatment of OT and OF cell lines (as in A). C, VPA and lithium synergize to activate the Lef-luciferase reporter in OT cells. Cells were treated with equimolar amounts of VPA and lithium. The activities of VPA and lithium alone (each at 20 mM) from panels A and B are shown for comparison. D, VPA causes a dose-dependent increase in SV-40-Renilla luciferase activity. Neuro2A cells were transiently transfected with SV-40-Renilla luciferase and treated for 24 h with VPA. 50% activation was observed at 0.8 mM VPA. (Experiments were performed in triplicate in at least four independent experiments; panel B repeated five times in triplicate with similar results. Error bars represent standard deviation. Note change in scale of y axis in each panel).
	FIG. 2. Regulation of -catenin expression by VPA. A, response to VPA is slow compared with LiCl. Neuro2A cells were treated with VPA (2 mM) or LiCl; (20 mM) and harvested at the indicated times (shown in hours). -Catenin protein was detected by Western blotting. -Tubulin protein levels are shown as loading controls. B, increase in -catenin due to VPA is dependent on new protein synthesis. Neuro2A cells were treated with cycloheximide (CHX) alone () or with lithium (Li) or VPA (V). Cells were harvested at the indicated times, and -catenin and -tubulin protein levels were detected by Western blotting. C, VPA causes a time- and dose-dependent increase in -catenin mRNA. Neuro2A cells were treated with 0, 2, or 5 mM VPA for 18 h (right panel) or were treated with 5 mM VPA for the times indicated (left panel) and then harvested for Northern blot with -catenin cDNA probe. The lower panel shows 18 S rRNA as control for equal loading.
	FIG. 3. VPA does not inhibit GSK-3 phosphorylation of tau or GS-2 peptide. A, Neuro2A cells were treated with the indicated amounts of VPA, 2-methyl- 2-propylpentenoic acid (2M2P), 4-pentenoic acid (4-PA), or lithium VPA for 24 h. Endogenous phosphorylated tau (phospho-tau) and total tau protein were detected by Western blotting. (Multiple isoforms of endogenous tau are visible). LiCl inhibits tau phosphorylation but VPA does not. B, VPA does not inhibit GSK-3 phosphorylation of a peptide derived from glycogen synthase (GS-2) in vitro. Recombinant GSK-3 was assayed by incorporation of [32P]phosphate into GS-2 peptide in the presence of VPA or LiCl.
	FIG. 4. VPA inhibits HDAC activity. A, human HDAC1 activity was assayed in vitro as release of [3 H]acetate from labeled histones (36) 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 bars represent standard deviation.
	FIG. 5. VPA causes hyperacetylation of endogenous histones in Neuro2A cells. Neuro2A cells were cultured for 24 h in 0–5 mM VPA or 300 nM TSA; nuclear proteins were isolated and immunoblotted with an antibody specific for acetylated histone-H4 (upper panel). Acetylation of H4 was detectable at 0.5 mM VPA. Coomassie Blue-stained gel (lower panel) shows loading of histones. Control lane (con) shows a mixture of purified, non-acetylated histones. H4 is the fastest migrating band in the lower panel, whereas the bracket indicates (in decreasing size) histones H3, H2B, and H2A.
	FIG. 6. HDAC1 overexpression reverses VPA-mediated activation of transcription in vivo. 293T cells were transfected with CMVRenilla and SV-40-SEAP, with or without an HDAC1 expression vector. Activities have been normalized to levels of SEAP in media prior to the addition of the VPA. Renilla activity without HDAC is shown in light bars, whereas activity in the presence of overexpressed HDAC1 is shown as dark bars. Experiments were performed in triplicate in three independent experiments. Error bars represent standard deviation.
	FIG. 7. Inhibition of HDAC correlates with teratogenicity. A, non-teratogenic analogues valpromide (VPM; 5 mM) and 2-methyl-2-propylpentenoic acid (2M2P;5mM) 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.

References :

Stephan, Reviewers are blinkered by bibliometrics. 2017, Pubmed