Parkinson's Disorder Research Laboratory, Iowa Center for Advanced Neurotoxicology, Department of Biomedical Sciences, Iowa State University, Ames, Iowa 50011, USA.
J Biol Chem. 2011 Jun 3;286(22):19840-59. doi: 10.1074/jbc.M110.203687. Epub 2011 Apr 5.
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.
我们之前已经证明蛋白激酶 Cδ（PKCδ；PKC 德尔塔）是一种对氧化应激敏感的激酶，在神经元细胞凋亡性细胞死亡中起因果作用。虽然已经广泛研究了 PKCδ 的激活，但对于控制 PKCδ 表达的分子机制知之甚少。为了表征 PKCδ 表达的调控，我们克隆了小鼠 Prkcd 基因的约 2kbp5'启动子片段。缺失分析表明，非编码外显子 1 区含有多个 Sp 位点，包括四个 GC 盒和一个 CACCC 盒，它们在神经元细胞中指导转录的最高水平。此外，在 -712 到 -560 区域内鉴定出一个包含相邻抑制和反抑制元件的上游调节区，它们具有相反的调节活性。详细的诱变研究表明，每个 Sp 位点对 PKCδ 启动子表达都有积极贡献。Sp 家族蛋白的过表达显著刺激 PKCδ 启动子活性，而没有任何协同的转录激活作用。此外，在 Sp 缺陷型 SL2 细胞中的实验表明，长型异构体 Sp3 是 PKCδ 转录的必需激活剂。重要的是，NIE115 细胞和原代纹状体培养物中的 PKCδ 启动子活性和内源性 PKCδ 表达均被米托蒽醌 A 抑制。染色质免疫沉淀和凝胶迁移实验的结果进一步证实了 Sp 蛋白与 PKCδ 启动子的功能结合。此外，我们证明 p300 或 CREB 结合蛋白的过表达增加了 PKCδ 启动子活性。这种刺激作用需要完整的 Sp 结合位点，并且独立于 p300 组蛋白乙酰转移酶活性。最后，Sp 转录活性或蛋白水平的调节极大地改变了氧化应激引起的细胞死亡，证明了 Sp 依赖性 PKCδ 基因表达的功能意义。总的来说，我们的发现可能对开发针对氧化损伤的新转化策略具有重要意义。
