Transcriptional Regulation of Pro-apoptotic Protein Kinase Cδ IMPLICATIONS FOR OXIDATIVE STRESS-INDUCED NEURONAL CELL DEATH
Transcriptional Regulation of Pro-apoptotic Protein Kinase Cδ IMPLICATIONS FOR OXIDATIVE STRESS-INDUCED NEURONAL CELL DEATH
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DOI：
10.1074/jbc.m110.203687
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发表时间：
2011-06-03
影响因子：
4.8
通讯作者：
Kanthasamy, Anumantha G.
中科院分区：
文献类型：
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作者：
Jin, Huajun;Kanthasamy, Arthi;Kanthasamy, Anumantha G.
我们以前证明，蛋白激酶C δ（PKC δ; PKC δ）是一种氧化应激敏感性激酶，在神经元细胞的细胞凋亡中起着因果作用。虽然PKC δ的激活已被广泛研究，但对控制PKC δ表达的分子机制知之甚少。为了表征PKC δ表达的调控，我们克隆了与小鼠Prkcd基因的2-kbp 5 ' -启动子片段相似的片段。缺失分析表明，非编码外显子1区域包含多个Sp位点，包括4个GC盒和1个CACCC盒，其指导神经元细胞中的最高水平的转录。此外，在-712至-560区域内鉴定了含有具有相反调节活性的相邻阻遏和反阻遏元件的上游调节区域。详细的诱变研究表明，每个Sp位点的PKC δ启动子表达作出了积极的贡献。Sp家族蛋白的过表达显著刺激PKC δ启动子活性，而没有任何协同反式激活作用。此外，在SP缺陷型SL 2细胞中的实验表明长同种型Sp3是PKC δ转录的必要激活剂。重要的是，光神霉素A抑制NIE 115细胞和原代纹状体培养物中PKC δ启动子活性和内源性PKC δ表达。染色质免疫沉淀和凝胶迁移试验的结果进一步证实了Sp蛋白与PKC δ启动子的功能结合。此外，我们还证明了p300或CREB结合蛋白的过表达增加了PKC δ启动子的活性。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深刻地改变了氧化损伤诱导的细胞死亡，证明了Sp依赖性PKC delta基因表达的功能意义。总的来说，我们的研究结果可能对开发新的抗氧化损伤的翻译策略有影响。
We previously demonstrated that protein kinase C delta (PKC delta; PKC delta) is an oxidative stress-sensitive kinase that plays a causal role in apoptotic cell death in neuronal cells. Although PKC delta activation has been extensively studied, relatively little is known about the molecular mechanisms controlling PKC delta expression. To characterize the regulation of PKC delta expression, we cloned an similar to 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 PKC delta promoter expression. Overexpression of Sp family proteins markedly stimulated PKC delta promoter activity without any synergistic transactivating effect. Furthermore, experiments in Sp-deficient SL2 cells indicated long isoform Sp3 as the essential activator of PKC delta transcription. Importantly, both PKC delta promoter activity and endogenous PKC delta 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 PKC delta promoter. Additionally, we demonstrated that overexpression of p300 or CREB-binding protein increases the PKC delta 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 PKC delta gene expression. Collectively, our findings may have implications for development of new translational strategies against oxidative damage.