CerS6 Is a Novel Transcriptional Target of p53 Protein Activated by Non-genotoxic Stress
CerS6 Is a Novel Transcriptional Target of p53 Protein Activated by Non-genotoxic Stress
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DOI：
10.1074/jbc.m116.716902
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发表时间：
2016-08-05
影响因子：
4.8
通讯作者：
Krupenko, Natalia I.
中科院分区：
文献类型：
--
作者：
Fekry, Baharan;Jeffries, Kristen A.;Krupenko, Natalia I.
我们之前的研究表明，神经酰胺合酶 6 (CerS6)（鞘脂生物合成中的一种酶）受 p53 调节：在几种细胞系中，CerS6 在响应 p53 的瞬时表达或响应叶酸应激（已知叶酸应激会激活 p53）时升高。然而，尚不清楚 CerS6 基因是否是 p53 的直接转录靶标，或者这是否是通过其他调控因子的间接作用。在本研究中，我们发现 CerS6 启动子在荧光素酶测定中被 p53 激活，而转录失活的 R175H p53 突变体未能诱导该启动子的荧光素酶表达。体外免疫沉淀测定和凝胶位移分析进一步证明，纯化的 p53 结合在跨越转录起始位点上游 91 bp 和下游 60 bp 的 CerS6 启动子序列内。用于预测转录因子结合位点的 Promo 3.0.2 在线工具表明 CerS6 启动子中存在许多推定的非规范 p53 结合基序。荧光素酶测定和凝胶位移分析已鉴定出转录起始上游的单个基序作为关键的 p53 响应元件。用 Nutlin-3 或低浓度放线菌素 D 处理细胞会导致 CerS6 mRNA 和蛋白质显着升高，从而证明 CerS6 是非基因毒性 p53 依赖性细胞应激反应的一个组成部分。这项研究表明，通过直接转录激活 CerS6，p53 可以调节特定的神经酰胺生物合成，从而有助于促凋亡细胞反应。
Our previous study suggested that ceramide synthase 6 (CerS6), an enzyme in sphingolipid biosynthesis, is regulated by p53: CerS6 was elevated in several cell lines in response to transient expression of p53 or in response to folate stress, which is known to activate p53. It was not clear, however, whether CerS6 gene is a direct transcriptional target of p53 or whether this was an indirect effect through additional regulatory factors. In the present study, we have shown that the CerS6 promoter is activated by p53 in luciferase assays, whereas transcriptionally inactive R175H p53 mutant failed to induce the luciferase expression from this promoter. In vitro immunoprecipitation assays and gel shift analyses have further demonstrated that purified p53 binds within the CerS6 promoter sequence spanning 91 bp upstream and 60 bp downstream of the transcription start site. The Promo 3.0.2 online tool for the prediction of transcription factor binding sites indicated the presence of numerous putative non-canonical p53 binding motifs in the CerS6 promoter. Luciferase assays and gel shift analysis have identified a single motif upstream of the transcription start as a key p53 response element. Treatment of cells with Nutlin-3 or low concentrations of actinomycin D resulted in a strong elevation of CerS6 mRNA and protein, thus demonstrating that CerS6 is a component of the non-genotoxic p53-dependent cellular stress response. This study has shown that by direct transcriptional activation of CerS6, p53 can regulate specific ceramide biosynthesis, which contributes to the pro-apoptotic cellular response.