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Clinically relevant concentrations of valproic acid modulate melatonin MT1 receptor, HDAC and MeCP2 mRNA expression in C6 glioma cells

https://doi.org/10.1016/j.ejphar.2008.04.058Get rights and content

Abstract

C6 glioma cells were treated with clinically relevant concentrations of valproic acid (0.5 or 1.0 mM) for 1–7 days and RT-PCR used to examine expression of the melatonin MT1 receptor and selected epigenetic modulators. Valproic acid caused significant time-dependent changes in the mRNA expression of the melatonin MT1 receptor, histone deacetylase (HDAC) 1, 2 and 3, and methyl CpG binding protein 2 (MeCP2). A structurally distinct HDAC inhibitor, trichostatin A, also caused a significant concentration-dependent induction of melatonin MT1 receptor mRNA expression, suggesting involvement of an epigenetic mechanism. The ability of clinical concentrations of valproic acid to significantly alter melatonin MT1 receptor expression, suggests a role for this receptor in the diverse neuropharmacological and oncostatic effects of this agent.

Introduction

Valproic acid (2-propylpentanoic acid), a short-chain branched fatty acid, is widely used clinically as an anticonvulsant and mood stabilizer (Qiao et al., 2006). Various mechanisms including enhancement of GABAergic activity (Perucca, 2002) and the modulation of multiple signaling cascades such as the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and WNT signaling pathways (Hao et al., 2004, Wiltse, 2005) are thought to underlie the clinical and neuropharmacological properties of valproic acid. In addition, it is now known that valproic acid inhibits histone deacetylase (HDAC) enzyme activity resulting in chromatin decondensation (Marchion et al., 2005), with consequent changes in gene transcription (Rosenberg, 2007).

In mammals, melatonin signals via two G protein-coupled MT1 and MT2 receptors, which are involved in its modulation of diverse physiological activities including circadian rhythmicity, neuroendocrine and immune function (von Gall et al., 2002, Dubocovich and Markowska, 2005). This indoleamine hormone also exerts antioxidant and neuroprotective effects in the CNS (Rodriguez et al., 2004, Sharma et al., 2006), and it may play a role in mood modulation, as melatonin MT1 receptor knockout mice exhibit depression-like behaviour (Weil et al., 2006). Moreover, the antidepressant effect of the melatonin MT1/MT2 agonist, agomelatine (Pandi-Perumal et al., 2006, Ghosh and Hellewell, 2007), implicates the MT1 receptor in this therapeutic action, given its predominant and widespread expression in the CNS (Liu et al., 1997, Mazzucchelli et al., 1996, Uz et al., 2005). We have reported that valproic acid, at high (supraclinical) concentrations of 3 and 5 mM, induces melatonin MT1 receptor mRNA and protein expression after treatment for 24 or 48 h (Castro et al., 2005). In addition, these concentrations of valproic acid caused a significant increase in HDAC1 mRNA expression, suggesting involvement of an epigenetic mechanism in melatonin MT1 receptor induction (Castro et al., 2005). In order to determine whether, after longer treatment periods, lower clinically relevant concentrations (Bowden et al., 1994, Cloyd et al., 2003) of valproic acid (0.5 and 1 mM), can similarly modulate these and other related gene targets, the mRNA expression of the melatonin MT1 receptor, HDAC1, HDAC2, HDAC3 and methyl CpG binding protein 2 (MeCP2) was examined over 1 to 7 days. In addition, the effect of a structurally different and more potent HDAC inhibitor, trichostatin A, on melatonin MT1 receptor expression was examined for the first time.

Section snippets

Cell culture

Rat C6 glioma cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), penicillin/streptomycin (100 IU/mL/100 μL/mL), and fungizone (1.25 μg/mL; Gibco, Burlington, ON, Canada), at 37 °C under 5% CO2/air. After three days, the medium was changed to DMEM with 1% FBS. Cells from passages 41 to 44 were used for valproic acid treatments. For 1 and 3-day treatments, cells at 55–70% confluence were used while those at 20–30% confluence were used for 5 and

Effects of valproic acid and trichostatin A on MT1 mRNA expression in C6 cells

Two-way ANOVA indicated a significant treatment × time interaction (F(6,23) = 2.77, P < 0.035) for MT1 mRNA data from C6 cells treated with valproic acid (0.5 and 1 mM) for 1, 3, 5 or 7 days. Further analysis of data at each treatment time, using one-way ANOVA and Neuman–Keuls tests, revealed significant increases in melatonin MT1 receptor mRNA following treatment with 0.5 mM valproic acid for 1 or 7 days (P < 0.05 and P < 0.01, respectively), as shown in Fig. 1A and D. A stronger time-dependent

Discussion

In earlier studies, we observed that valproic acid upregulates expression of melatonin MT1 receptor mRNA and protein in C6 cells, especially at higher concentrations (3 and 5 mM), after treatment for 24 or 48 h (Castro et al., 2005). We now report that treatment of C6 cells with lower clinically relevant concentrations of valproic acid (0.5 mM or 1 mM) caused significant time-dependent increases in MT1 mRNA expression (Fig. 1A–D). After 7 days, cells treated with 0.5 mM valproic acid still

Acknowledgements

This work was supported by NSERC Canada via a research grant to LPN and an undergraduate student research award to BK.

References (28)

  • BowdenC.L. et al.

    Efficacy of divalproex vs lithium and placebo in the treatment of mania. The Depakote Mania Study Group

    JAMA

    (1994)
  • CastroL.M. et al.

    Novel targets for valproic acid: up-regulation of melatonin receptors and neurotrophic factors in C6 glioma cells

    J. Neurochem.

    (2005)
  • ChenP.S. et al.

    Valproate protects dopaminergic neurons in midbrain neuron/glia cultures by stimulating the release of neurotrophic factors from astrocytes

    Mol. Psychiatry

    (2006)
  • DongE. et al.

    Histone hyperacetylation induces demethylation of reelin and 67-kDa glutamic acid decarboxylase promoters

    Proc. Natl. Acad. Sci. U. S. A.

    (2007)
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