Downregulation of TrkB Expression and Signaling by Valproic Acid and Other Histone Deacetylase Inhibitors

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.

Valproic acid upregulates the expression of the p75NTR/sortilin receptor complex to induce neuronal apoptosis

“…Thus, the VPA-induced changes observed in the present study may have clinical relevance when the drug is used to treat extra-cranial tumours, such as neuroblastoma. Moreover, within the same concentration range VPA was previously found to cause hyperacetylation of H3 histone and downregulation of TrkB in SH-SY5Y cells [ 57 ], indicating that the drug can induce chromatin remodelling and altered expression of neurotrophin receptors with similar potencies.…”

“…Relatively little is known on the control of SORT1 gene expression and the precise molecular mechanisms linking HDAC inhibition and sortilin upregulation remain to be elucidated. Nonetheless, the observation that the HDAC6 inhibitor tubacin and the HDAC8 inhibitor PCI-34,051, which do not induce histone hyperacetylation [38,39,57], had no effects on either sortilin or p75NTR expression indicates that both responses are not induced by generic HDAC inhibition but involve specific HDAC isoforms capable of producing chromatin remodelling. Immunoprecipitation experiments indicated that in VPA-treated SH-SY5Y cells the upregulation of p75NTR and sortilin expression was accompanied by an enhancement of proNGF-induced association of the two receptor proteins and potentiation of JNK activation, as indicated by the increased phosphorylation of JNK and c-Jun.…”

“…Moreover, cell treatment with the EZH2 depleting agent DZNep or the EZH2 inhibitor TZM upregulated p75NTR. Interestingly, VPA-induced downregulation of TrkB was recently found to involve depletion of EZH2 and derepression of RUNX3 [57], a negative regulator of the NTRK2 gene. Thus, a common epigenetic mechanism based on inhibition of PRC2 transcriptional repression appears to mediate the VPA opposite effects on p75NTR and TrkB expression.…”

“…Relatively little is known on the control of SORT1 gene expression and the precise molecular mechanisms linking HDAC inhibition and sortilin upregulation remain to be elucidated. Nonetheless, the observation that the HDAC6 inhibitor tubacin and the HDAC8 inhibitor PCI-34,051, which do not induce histone hyperacetylation [ 38 , 39 , 57 ], had no effects on either sortilin or p75NTR expression indicates that both responses are not induced by generic HDAC inhibition but involve specific HDAC isoforms capable of producing chromatin remodelling.…”

Inhibition of EZH2 by chidamide exerts antileukemia activity and increases chemosensitivity through Smo/Gli-1 pathway in acute myeloid leukemia

“…It is reported that inhibition of enhancer of zeste homolog 2 (EZH2) contributes to the antitumor effect of HDAC inhibitor in neuroblastoma cells and lung cancer cells [28,29]. EZH2 is the functional core subunit of the polycomb repressive complex 2 (PRC2), and plays a pivotal role in catalyzing the methylation of the lysine 27 of histone H3 (H3K27) [30][31][32][33].…”