Abstract
Valproic acid (VPA) has been shown to regulate the levels of brain-derived neurotrophic factor (BDNF), but it is not known whether this drug can affect the neuronal responses to BDNF. In the present study, we show that in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells, prolonged exposure to VPA reduces the expression of the BDNF receptor TrkB at the protein and mRNA levels and inhibits the intracellular signaling, neurotrophic activity, and prosurvival function of BDNF. VPA downregulates TrkB and curtails BDNF-induced signaling also in differentiated Kelly and LAN-1 neuroblastoma cells and primary mouse cortical neurons. The VPA effect is mimicked by several histone deacetylase (HDAC) inhibitors, including the class I HDAC inhibitors entinostat and romidepsin. Conversely, the class II HDAC inhibitor MC1568, the HDAC6 inhibitor tubacin, the HDAC8 inhibitor PCI-34051, and the VPA derivative valpromide have no effect. In neuroblastoma cells and primary neurons both VPA and entinostat increase the cellular levels of the transcription factor RUNX3, which negatively regulates TrkB gene expression. Treatment with RUNX3 siRNA attenuates VPA-induced RUNX3 elevation and TrkB downregulation. VPA, entinostat, HDAC1 depletion by siRNA, and 3-deazaneplanocin A (DZNep), an inhibitor of the polycomb repressor complex 2 (PRC2), decrease the PRC2 core component EZH2, a RUNX3 suppressor. Like VPA, HDAC1 depletion and DZNep increase RUNX3 and decrease TrkB expression. These results indicate that VPA downregulates TrkB through epigenetic mechanisms involving the EZH2/RUNX3 axis and provide evidence that this effect implicates relevant consequences with regard to BDNF efficacy in stimulating intracellular signaling and functional responses. SIGNIFICANCE STATEMENT: The tropomyosin-related kinase receptor B (TrkB) mediates the stimulatory effects of brain-derived neurotrophic factor (BDNF) on neuronal growth, differentiation, and survival and is highly expressed in aggressive neuroblastoma and other tumors. Here we show that exposure to valproic acid (VPA) downregulates TrkB expression and functional activity in retinoic acid-differentiated human neuroblastoma cell lines and primary mouse cortical neurons. The effects of VPA are mimicked by other histone deacetylase (HDAC) inhibitors and HDAC1 knockdown and appear to be mediated by an epigenetic mechanism involving the upregulation of RUNX3, a suppressor of TrkB gene expression. TrkB downregulation may have relevance for the use of VPA as a potential therapeutic agent in neuroblastoma and other pathologies characterized by an excessive BDNF/TrkB signaling.
Highlights
The short-chain branched fatty acid valproic acid (VPA) is commonly used in the clinic as an anticonvulsant for the treatment of various forms of epilepsy, as a mood stabilizer for the management of bipolar mood disorder, and as an analgesic for migraine headaches (Loscher, 2002)
In agreement with previous studies (Kaplan et al, 1993; Encinas et al, 1999; Dedoni et al, 2012), Western blot analysis of tropomyosin-related kinase receptor B (TrkB) expression in RAdifferentiated SH-SY5Y cells detected the presence of an immunoreactive band of 145 kDa representing the catalytic tyrosine kinase full-length isoform of TrkB (TrkB-FL) and an additional broader band of ∼95 kDa, which corresponded to the molecular mass of the truncated TrkB (TrkB-T) isoforms TrkB.T1 and TrkB.Shc lacking the tyrosine kinase domain (Fig. 1A) (Klein et al, 1990; Stoilov et al, 2002)
The downregulation of TrkB occurred at VPA concentrations (0.6–1.0 mM) that are within the plasma concentrations associated with therapeutic effects in epilepsy (40–100 mg/ml, ∼0.3–0.7 mM) (Loscher, 2002)
Summary
The short-chain branched fatty acid valproic acid (VPA) is commonly used in the clinic as an anticonvulsant for the treatment of various forms of epilepsy, as a mood stabilizer for the management of bipolar mood disorder, and as an analgesic for migraine headaches (Loscher, 2002). In clinical trials VPA has been investigated as a potential anticancer agent for both hematologic and solid tumors (Chateauvieux et al, 2010). This wide array of pharmacological properties likely stems from the ability of VPA to display multiple. Mechanisms of action, including inhibition of voltage-gated sodium and calcium channels, potentiation of GABAergic transmission, attenuation of glutamatergic function, and alteration of the activity of distinct intracellular signaling mediators (Loscher, 2002). Among the four distinct classes of mammalian HDACs so far identified, VPA preferentially inhibits HDACs belonging to class I and IIa (Bolden et al, 2006)
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