Transcriptional Regulation of Pro-apoptotic Protein Kinase Cdelta: Implications for Oxidative Stress-induced Neuronal Cell Death

We previously demonstrated that protein kinase Cδ (PKCδ; PKC delta) is an oxidative stress-sensitive kinase that plays a causal role in apoptotic cell death in neuronal cells. Although PKCδ activation has been extensively studied, relatively little is known about the molecular mechanisms controlling PKCδ expression. To characterize the regulation of PKCδ expression, we cloned an ∼2-kbp 5'-promoter segment of the mouse Prkcd gene. Deletion analysis indicated that the noncoding exon 1 region contained multiple Sp sites, including four GC boxes and one CACCC box, which directed the highest levels of transcription in neuronal 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 studies 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. Furthermore, experiments in Sp-deficient SL2 cells indicated long isoform Sp3 as the essential activator of PKCδ transcription. Importantly, both PKCδ promoter activity and endogenous PKCδ expression in NIE115 cells and primary striatal cultures were inhibited by 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 CREB-binding protein increases the PKCδ promoter activity. This stimulatory effect requires intact Sp-binding sites and is independent of p300 histone acetyltransferase activity. Finally, modulation of Sp transcriptional activity or protein level profoundly altered the cell death induced by oxidative insult, demonstrating the functional significance of Sp-dependent PKCδ gene expression. Collectively, our findings may have implications for development of new translational strategies against oxidative damage.