启动子荧光素酶报告系统检测低浓度过氧化氢激活神经肽PACAP特异受体PAC1-R启动子的作用

Analyzing Activation of the Promoter of Neuropeptide PACAP Preferring Receptor PAC1-R under Low Concentration Hydrogen Peroxide Exposure using Promoter Luciferase Reporter System

垂体腺苷酸环化酶激活多肽(pituitary adenylate cyclase-activating polypeptide,PACAP)特异受体PAC1-R(PACAP receptor 1)是神经系统疾病药物开发的重要靶点。为了研究其表达的生物学机制,本研究克隆了PAC1-R基因转录起始位点上游从-2 500到+26的2 526 bp启动子片段,构建PAC1-R启动子驱动的荧光素酶基因报告载体p GL3-PAC1-Rp,并确证PAC1-R启动子荧光素酶报告系统在小鼠脑神经瘤细胞Neuro-2a和人神经母细胞瘤细胞SH-SY5Y中工作正常。运用此PAC1-R启动子荧光素酶报告系统,首次发现低浓度过氧化氢(hydrogen peroxide,H_2O_2)有效激活PAC1-R启动子,此作用可被转录因子特化蛋白1(specificity protein 1,SP1)抑制剂光神霉素A(mithramycin A)所抑制,提示SP1参与介导H_2O_2对PAC1-R启动子的激活作用。生物信息学分析显示,PAC1-R启动子含有多个SP1结合位点。PAC1-R启动子的荧光素酶报告系统的构建为深入探索PAC1-R高表达的作用与机制奠定了基础,低浓度H_2O_2对PAC1-R启动子激活作用的发现有助于深入诠释低浓度活性氧的生理学作用。

PACAP receptor 1（ PAC1-R） is neuropeptide PACAP（ pituitary adenylate cyclase-activating polypeptide） preferring receptor,which is an important drug development target for the nervous system diseases. In order to clarify the biological mechanism of its expression,we cloned the 2 526 bp promoter fragment from- 2 500 to ＋ 26 of the transcription initiation site and constructed the promoter luciferase reporter construct p GL3-PAC1-Rp,in which the luciferase reporter gene is driven by the PAC1-R promoter. The activity of the PAC1-R promoter luciferase reporter system was further confirmed in the mouse neurocytoma Neuro2 a cells and human neuroblastoma SH-SY5 Y cells. Using PAC1-R promoter luciferase reporter system,we found for the first time that low concentration of hydrogen peroxide（ H2O2） exposure significantly promoted the activity of PAC1-R promoter,which was significantly inhibited by the transcription factor SP1 inhibitor mithramycin A. The result indicated that SP1 wasinvolved in the activation of PAC1-R promoter by low concentration of H2O2,consistent with the bioinformatics analysis showing several SP1 binding sites on the 2 526 bp PAC1-R promoter. The PAC1-R promoter luciferase reporter system lays the pavement for the further research on the physiological and pathological role. The mechanism of PAC1-R＇s high expression and the finding of the activation of PAC1-R promoter by low concentration of H2O2 would help to clarify the physiological effect of low dose reactive oxygen.