Transcriptional regulation of proapoptotic kinase PKCδ expression in dopaminergic neurons: relevance to gene-gene and gene-environment interactions in neurodegeneration

Jin, Huajun
Major Professor
Anumantha G. Kanthasamy
Committee Member
Journal Title
Journal ISSN
Volume Title
Research Projects
Organizational Units
Biomedical Sciences
Organizational Unit
Journal Issue
Biomedical Sciences

We investigated the mechanisms of transcriptional regulation of the PKCδ gene. By deletion analysis of the ~1.4 kb (-1448 to +1, relative to transcription start site) 5'-flanking sequence of the mouse PKCδ gene, we have identified a basal promoter region, nucleotide -148 to +1, required for sufficient PKCδ transcription in NIE115, MN9D, and N2a cells. We further identified two NFκB binding sites (κB 1, κB 2) as well as a NERF1a site within the basal promoter as key regulatory elements in the mouse PKCδ TATA-less promoter. Subsequent functional studies using site-directed mutation analysis revealed that κB 1, but not κB 2, is necessary for PKCδ basal expression in both NIE115 and MN9D cells. To further facilitate analysis of the regulation of the PKCδ promoter, we cloned a ~2 kb (-1694 to +289) 5'-promoter segment of the mouse PKCδ gene including the putative PKCδ promoter (1694 bp) as well as the GC-rich sequences of the first, non-coding exon (289 bp). Deletion analysis of this region indicated the non-coding exon1 GC-rich region that contains multiple Sp sites, including four GC boxes and one CACCC box, greatly enhances the basal PKCδ promoter activity and directs the highest levels of transcription in NIE115 and MN9D cells. In addition, an upstream regulatory region containing adjacent repressive and anti-repressive elements with opposing regulatory activities was identified within the region -712 to -560. Detailed mutagenesis revealed that each Sp site made a positive contribution to PKCδ promoter expression. Overexpression of Sp family proteins markedly stimulated PKCδ promoter activity without any synergistic transactivating effect in NIE115 cells. Furthermore, experiments in SL2 fly cells identified the long-isoform Sp3 as the essential activator of PKCδ transcription. Importantly, both PKCδ promoter activity and endogenous PKCδ mRNA in NIE115 cells and primary striatal cultures were inhibited by the Sp protein inhibitor, mithramycin-A. The results from chromatin immunoprecipitation and gel shift assays further confirmed the functional binding of Sp proteins to the PKCδ promoter. Additionally, we demonstrated that overexpression of p300 or CBP increases the PKCδ promoter activity. This stimulatory effect requires intact Sp binding sites and is independent of p300 HAT activity. We also investigated the possible involvement of epigenetic mechanisms, such as DNA methylation and histone acetylation, in regulation of the PKCδ gene. Using bioinformatics method, we found a putative CpG island (+39 to +400) that overlaps with mouse PKCδ promoter. By methylation-specific PCR, we found that the PKCδ promoter is partially methylated in NIE115, MN9D, and N2a cells. Furthermore, administration of DNA methylation inhibitor 5-Aza-deoxycytidine induced hypomethylation of the PKCδ promoter and increased expression of PKCδ mRNA in NIE115 cells, further suggesting that DNA methylation is involved in mouse PKCδ gene expression in these cells. To examine the role of histone acetylation in PKCδ gene expression, we also explored the effects of various histone deacetylase (HDAC) inhibitors both in vitro and in vivo. Treatment with sodium butyrate (NaBu) significantly enhanced the PKCδ protein and mRNA levels in primary striatal and nigral neurons and in NIE115 and MN9D cells. Other HDAC inhibitors, valproic acid (VPA), scriptaid, trichostatin A (TSA), and apicidin, all mimicked the action of NaBu to induce PKCδ. Furthermore, NaBu treatment in the C57 black mouse model caused a time-dependent induction of PKCδ gene expression in the substantial nigra and striatum regions. NaBu-induced PKCδ expression correlated with hyperacetylation of the H4 histone associated with PKCδ promoter, clearly suggesting that acetylation-dependent chromatin remodeling may play a role in PKCδ upregulation. To further explore the molecular basis of histone acetylation-dependent PKCδ upregulation, PKCδ promoter analysis was performed using reporter gene assays. NaBu and other tested HDAC inhibitors all dramatically increased the PKCδ promoter activity in a dose-dependent manner. By using deletion analyses, the minimal fragment of the PKCδ promoter in response to NaBu was mapped to an 81 bp non-coding exon 1 region (+209 to +289). The site-directed mutagenesis studies revealed that multiple GC sites within this region are major elements conferring the responsiveness to NaBu-induced promoter activity. In addition, transcriptional activities of Sp1 and Sp3 were significantly induced by NaBu. Importantly, the ectopic expression of Sp1, Sp3, or Sp4 significantly enhanced NaBu-mediated transactivation of PKCδ promoter, whereas the ectopic expression of dominant negative mutant of Sp1 or Sp3 did not cause this effect. Moreover, the Sp protein inhibitors mithramycin-A and tolfenamic acid dose-dependently blocked NaBu-induced PKCδ promoter activity. In addition, transcriptional activity of Sp1 and Sp3 was significantly induced by NaBu in a one-hybrid system. By utilizing the same assay, we found that the B domain and the glutamine-rich segment of the A domain of Sp1 and Sp3 (amino acids Sp1 146-494; Sp3 81-499) was essential for the NaBu-induced transactivation of the PKCδ promoter. Transient overexpression of p300 or CBP potentiated NaBu-induced transactivation potential of Sp1 or Sp3, whereas transient overexpression of HDACs attenuated this effect, suggesting that p300/CBP and HDACs may act as co-activators or co-repressors in response to NaBu exposure. Next, we evidenced a novel association between α-synuclein, a protein associated with the pathogenesis of Parkinson's diseases (PD), and PKCδ, in which α-synuclein negatively modulates the p300- and NFκB-dependent transactivation to down-regulate proapoptotic kinase PKCδ expression and thereby protects against apoptosis in dopaminergic neuronal cells. Stable-expression human wild-type α-synuclein at physiological levels in dopaminergic neuronal cells resulted in an isoform-dependent transcriptional suppression of PKCδ expression without changes in the stability of mRNA and protein or DNA methylation. The reduction in PKCδ transcription was mediated, in part, through the suppression of constitutive NFκB activity targeted at two proximal PKCδ-promoter κB sites. This occurred independently of NFκB/IκBα nuclear translocation, but was associated with decreased NFκB-p65 acetylation. Also, αsyn reduced p300 levels and its histone acetyl-transferase (HAT) activity, thereby contributing to diminished PKCδ transactivation. Importantly, reduced PKCδ and p300 expression also were observed within nigral dopaminergic neurons in αsyn transgenic mice. Finally, we examined whether environmental neurotoxicant exposure alters PKCδ expression. Manganese exposure potently induced PKCδ levels in primary striatal neurons and NIE115 cells. The use of primary neurons from mice lacking PKCδ subsequently demonstrated that the level of PKCδ plays a critical role in manganese-induced neurodegeneration. Experiments on manganese-exposed mice also confirmed the action of manganese in upregulation of PKCδ. Using NIE115 cells, we further elucidated the mechanisms underlying the manganese-induced up-regulation of PKCδ. We identified that NFκB is essential for the manganese-mediated expression of PKCδ in NIE115 cells. Taken together, our studies show that 1) PKCδ promoter contains multiple positive and negative cis-acting elements, and both Sp family proteins and NFκB function as essential trans-acting factors to regulate PKCδ transcription, 2) epigenetic mechanisms including DNA methylation and histone acetylation appear to have a direct role in PKCδ expression, 3) PKCδ expression can be induced by parkinsonian environmental toxin, manganese, or negatively regulated by the PD-related gene, α-synuclein.