Med25作为共激活因子对糖皮质激素受体GRα介导的CYP2C9的调控机制研究

CYP2C9 is an important hepatic metabolizing enzyme playing vital roles in the metabolism of xenobiotics as well as endogenous compounds. Mediator complex (Med) acts as a molecular bridge interacts with nuclear receptor and RNA polymerase II (Pol-II), which plays an important role in the gene transcription. Our preliminary work shows that Med25, a subunit of Mediator complex, interacts with glucocorticoid receptor α (GRα) and promotes the transcriptional regulation of CYP2C9 expression by glucocorticoid stimulation. This study will focus on the pivotal point whether Med25 is an essential coactivator of GRα involved in the transcriptional regulation of CYP2C9 expression, and investigate the role and mechanism of Med25 in the regulation of CYP2C9 expression and liver metabolism. We will employ many methods such as immunoprecipitation, chromatin immunoprecipitation (ChIP) assay and confocal microscope technologies to demonstrate Med25 interacts with GRα and recruits Pol-II. By gene reporter assay and silencing Med25, we will determine the role of Med25 in the regulation of CYP2C9 expression and liver metabolism. We will further determine the phosphorylation sites of Med25 through mass spectrometric analysis, and clarify the role of phosphorylation of Med25 in the gene regulation. This study will clarify that Med25 is a vital coactivator of GRα involved in the regulation of CYP2C9 expression, and provide a new possible molecular target of prevention and clinical therapy of some related metabolism diseases.

CYP2C9是重要的肝代谢酶，对内、外源物的代谢具有重要作用。中介体(Med) 是连接核受体与RNA聚合酶II (Pol-II)的桥梁，在基因调控中发挥重要作用。前期研究发现糖皮质激素诱导下，中介体亚基Med25与糖皮质激素受体GRα结合，促进CYP2C9的转录表达。本项目针对Med25是否是GRα的必要共激活因子参与对CYP2C9的调控这一关键问题，研究Med25对GRα介导的CYP2C9的调控机制及其在肝脏代谢中的作用。通过免疫沉淀、ChIP分析、共聚焦显微镜观察等阐明Med25与GRα相互结合并招募Pol-II；利用基因转录活性分析与基因沉默技术等明确Med25在调控CYP2C9表达及肝脏代谢中的作用；质谱分析确定Med25磷酸化位点并阐明Med25磷酸化的作用。明确Med25是GRα介导的调控CYP2C9过程中必要的共激活因子，为相关代谢疾病的预防与治疗提供可能的药物新靶点。

CYP2C9是人体重要的药物代谢酶，对内源物、外源物的代谢具有重要作用。中介体（Med）与核受体相互作用在基因调控过程中发挥着重要作用。本项目发现糖皮质激素受体GRα或雌激素受体ERα通过CYP2C9启动子领域内糖皮质激素受体的识别位点GRE或雌激素受体的识别位点ERE调控CYP2C9的转录表达，中介体亚基Med25参与了GRα或ERα介导的CYP2C9的调控过程。通过免疫共沉淀和基因位点突变实验，确定了Med25可与GRα或ERα相互结合，并明确了Med25序列中的结合位点LXXLL。通过双荧光素酶活性检测并结合Med25的LXXLL位点突变质粒，在糖皮质激素、雌激素、环境拟雌激素双酚A的分别诱导下， 明确了Med25通过LXXLL位点与GRα或ERα共同调控CYP2C9的启动子转录活性。通过荧光定量PCR实验，发现Med25参与并显著增加了GRα或ERα介导的CYP2C9的基因表达。通过CYP2C9 Glo Assay实验，发现Med25参与并显著促进了GRα或ERα介导的CYP2C9的酶活性。本项目以中介体Med25为突破点，证明了Med25是GRα或ERα的必要共激活因子，参与GRα或ERα介导的CYP2C9调控过程，阐明了CYP2C9的基因转录调控机制，为CYP2C亚家族基因的调控机制建立了新的研究模式，为核受体介导的药物代谢酶的调控机制研究提供了新思路和新方法，为脂肪酸等内源物、药物等外源物的代谢机制研究提供了新途径，为相关代谢疾病、药物不良反应、药物中毒等临床治疗提供可能的新分子靶标。