Transcriptional Regulation of Pro-apoptotic Protein Kinase Cδ: IMPLICATIONS FOR OXIDATIVE STRESS-INDUCED NEURONAL CELL DEATH

Huajun Jin,Arthi Kanthasamy,Vellareddy Anantharam,Ajay Rana,Anumantha G Kanthasamy

doi:10.1074/jbc.m110.203687

Huajun Jin, Arthi Kanthasamy + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m110.203687

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Abstract

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.

Highlights

We previously demonstrated that protein kinase C␦ (PKC␦; PKC delta) is an oxidative stress-sensitive kinase that plays a causal role in apoptotic cell death in neuronal cells
Examination of the PKC␦ promoter did not reveal the classic initiator elements or the downstream promoter elements, which are located at various distances downstream of the transcription start site (TSS) and are utilized by most TATA-less promoters to initiate transcription, suggesting that there might be other promoter motifs involved in the regulation of PKC␦ gene transcription
Inhibition of PKC␦ Transcription Ameliorates Oxidative Stress-induced Neurodegeneration—As described above, we have demonstrated that the Sp family of transcription factors, the long Sp3 isoform, induces PKC␦ gene activity in neuronal cells and primary neurons via multiple Sp-binding elements and that high levels of PKC␦ protein are associated with an exacerbated sensitivity to oxidative stress

Summary

Introduction

We previously demonstrated that protein kinase C␦ (PKC␦; PKC delta) is an oxidative stress-sensitive kinase that plays a causal role in apoptotic cell death in neuronal cells. Given the great enhancing effect of the crucial GC-rich motif from ϩ2 to ϩ289 bp on the transcriptional activity of the PKC␦ basal promoter region Ϫ147 to ϩ2 (Fig. 1), we investigated whether this GC-rich domain is sufficient to function as an enhancer element in NIE115 cells.

Results

Conclusion