Valproic acid induces functional heat-shock protein 70 via Class I histone deacetylase inhibition in cortical neurons: a potential role of Sp1 acetylation

Neuroprotective properties of the mood stabilizer valproic acid (VPA) are implicated in its therapeutic efficacy. Heat-shock protein 70 (HSP70) is a molecular chaperone, neuroprotective and anti-inflammatory agent. This study aimed to investigate underlying mechanisms and functional significance of HSP70 induction by VPA in rat cortical neurons. VPA treatment markedly up-regulated HSP70 protein levels, and this was accompanied by increased HSP70 mRNA levels and promoter hyperacetylation and activity. Other HDAC inhibitors – sodium butyrate, trichostatin A, and Class I HDAC-specific inhibitors MS-275 and apicidin, – all mimicked the ability of VPA to induce HSP70. Pre-treatment with phosphatidylinositol 3-kinase inhibitors or an Akt inhibitor attenuated HSP70 induction by VPA and other HDAC inhibitors. VPA treatment increased Sp1 acetylation, and a Sp1 inhibitor, mithramycin, abolished the induction of HSP70 by HDAC inhibitors. Moreover, VPA promoted the association of Sp1 with the histone acetyltransferases p300 and recruitment of p300 to the HSP70 promoter. Further, VPA-induced neuroprotection against glutamate excitotoxicity was prevented by blocking HSP70 induction. Taken together, the data suggest that the phosphatidylinositol 3-kinase/Akt pathway and Sp1 are likely involved in HSP70 induction by HDAC inhibitors, and induction of HSP70 by VPA in cortical neurons may contribute to its neuroprotective and therapeutic effects.     

PKCepsilon is essential for gelsolin expression by histone deacetylase inhibitor apicidin in human cervix cancer cells

Down-regulation of gelsolin expression is associated with cellular transformation and induction of gelsolin exerts antitumorigenic effects. In this study, we show that protein kinase C (PKC) signaling pathway is required for the induction of gelsolin by the histone deacetylase inhibitor apicidin in HeLa cells. Apicidin induces gelsolin mRNA independently of the de novo protein synthesis. Inhibitor study has revealed that the PKC signaling pathway is involved in the gelsolin expression. Furthermore, inhibition of PKCepsilon by either siRNA or dominant-negative mutant completely abrogates the expression of gelsolin by apicidin, indicating that PKCepsilon is the major isoform for this process. In parallel, apicidin induction of gelsolin is antagonized by the inhibition of Sp1 using dominant-negative Sp1 or specific Sp1 inhibitor mithramycin, and inhibition of PKC leads to suppression of Sp1 promoter activity. Our results provide mechanistic insights into molecular mechanisms of gelsolin induction by apicidin.     

Endothelin-1 stimulates cyclin D1 expression in rat cultured astrocytes via activation of Sp1

Endothelins (ETs), a family of vasoconstrictor peptides, are up-regulated in several pathological conditions in the brain, and induce astrocytic proliferation. We previously observed that ET-1 increased the expression of cyclin D1 protein. Thus, we confirmed the intracellular up-regulation of cyclin D1 by ET-1 in rat cultured astrocytes. Real-time PCR analysis indicated that ET-1 (100 nM) and Ala^1,3,11,15-ET-1 (100 nM), a selective agonist of the ET_B receptor, induced a time-dependent and transient increase in cyclin D1 mRNA. The effect of ET-1 was diminished by an ET_B antagonist (1 μM BQ788) or inhibitors of Sp1 (500 nM mithramycin), ERK (50 μM PD98059), p38 (20 μM SB203580) and JNK (1 μM SP600125), but not inhibitors of NF-κB (10 μM SN50 and 100 μM pyrrolidine dithiocarbamate). The binding assay for Sp1 indicated that ET-1 increased the binding activity of Sp1 to consensus sequences, and two oligonucleotides of the cyclin D1 promoter including the Sp1-binding sites diminished the effect of ET-1. Western blot analysis showed that ET-1 induced time-dependent and transient phosphorylation of Sp1 on Thr453 and Thr739 via the ET_B receptor. ET-1-induced phosphorylation of Sp1 was attenuated by PD98059 and SP600125. Additionally, ET-1 increased the incorporation of bromodeoxyuridine (BrdU) in cultured astrocytes and the number of BrdU-positive cells decreased in the presence of PD98059, SP600125 and mithramycin. These results suggest that ET-1 increases the expression of cyclin D1 via activation of Sp1 and induces astrocytic proliferation.     

PKC-delta-dependent pathways contribute to PDGF-stimulated ERK1/2 activation in vascular smooth muscle

Platelet-derived growth factor (PDGF) is an important regulator of vascular smooth muscle (VSM) cell growth and migration and has been identified as a key mediator of neointima formation resulting from vascular injury. PDGF exerts its effects, in part, through activation of ERK1/2. Previously, we reported that PKC-delta, specifically compared with PKC-alpha, mediated phorbol ester- and ATP-dependent activation of ERK1/2 in VSM cells. The purpose of this study was to determine whether PKC-delta was involved in PDGF-dependent activation of ERK1/2 in VSM cells. The addition of PDGF resulted in the activation, and Src family kinase-dependent tyrosine phosphorylation, of PKC-delta. Treatment with rottlerin (0.1-10 microM), a selective PKC-delta inhibitor, or adenoviral overexpression of kinase-negative PKC-delta significantly attenuated PDGF-induced activation of ERK1/2. The effects of the PKC-delta inhibitors decreased with increasing concentrations of activator PDGF. Interestingly, treatment with Go6976 (0.1-3 microM), a selective inhibitor of cPKCs, or adenoviral overexpression of kinase-negative PKC-alpha also inhibited PDGF-stimulated ERK1/2. Furthermore, inhibition of cPKC activity with Go6976 or overexpression of kinase-negative PKC-alpha attenuated PKC-delta activation and tyrosine phosphorylation in response to PDGF. These studies indicate involvement of both PKC-delta and PKC-alpha isozymes in PDGF-stimulated signaling in VSM and suggest an unexpected role for PKC-alpha in the regulation of PKC-delta activity.