Pgrmc1 as a regulator of cytochrome P450 enzymes in cholesterol metabolism

 说明(申请人提供)：胆固醇是一种对生命至关重要的化合物，但血清胆固醇水平升高会增加心脏病发作和中风的风险。肝脏是胆固醇动态平衡的关键器官。在肝脏中，胆固醇被合成并转化为胆汁酸。细胞色素P450单加氧酶家族在肝脏胆固醇代谢中起着重要作用。在HEK293细胞中，血红素结合蛋白孕酮受体膜组分1(PGRMC1)是细胞色素P450酶CYP51A1的正调节因子，细胞色素P450酶是合成胆固醇所必需的。PGRMC1基因敲除的细胞损害了胆固醇的合成，并积累了中间羊毛甾醇(已知的是抑制HMG-CoA还原酶)。PGRMC1还与胆汁酸合成所需的另一种细胞色素P450酶--细胞色素P450酶7A1结合。基于这些发现，我推测PGRMC1是一种重要的胆固醇代谢调节器，是哺乳动物全身胆固醇稳态所必需的。为了验证这一假设，我将利用我们实验室现有的PGRMC1基因敲除(Pgrmc1KO)小鼠。在目标1中，我将测试PGRMC1在体内对胆固醇合成的作用。我将首先对Pgrmc1KO小鼠的肝脏进行一般的组织学鉴定，并检查肝脏损伤的标志。然后，我将测量Pgrmc1KO小鼠的肝脏类固醇组成、血清胆固醇水平和血清脂蛋白颗粒分布。我还将测量Pgrmc1KO原代肝细胞的胆固醇合成率和Pgrmc1KO肝微粒体的Cyp51A1酶活性。在目标2中，我将测试PGRMC1在体内对胆汁酸合成的作用。我将测量Pgrmc1KO小鼠的稳态胆汁酸水平和肝脏胆汁酸合成率，以及Pgrmc1KO肝微粒体中Cyp7A1的活性。这些研究将确定PGRMC1在调节胆固醇动态平衡中的作用。血清胆固醇水平高的患者使用他汀类药物(HMG-CoA还原酶抑制剂)和胆汁酸阻滞剂进行治疗。抑制PGRMC1可能是一种通过羊毛甾醇积累来抑制HMG-CoA还原酶的新方法。另外，操纵PGRMC1可能是一种刺激胆汁酸合成的新方法。这一建议将扩大我们对体内如何维持胆固醇稳态的理解，并为高胆固醇治疗药物的发展提供一个候选靶点。治疗高胆固醇将降低患者心脏病发作和中风的风险。此外，对PGRMC1的这些研究也将促进我们对细胞色素P450家族酶的理解。P450酶对药物代谢也很重要，并且没有已知的转录后调节因子。

 DESCRIPTION (provided by applicant): Cholesterol is a compound critical for life, but elevated serum cholesterol levels increase the risk of heart attack and stroke. The liver is a critical organ for cholesterol homeostasis. In the liver, cholesterol is synthesized and converted to bile acids. The cytochrome P450 monooxygenase family of enzymes plays a prominent role in cholesterol metabolism in the liver. In HEK293 cells, the heme-binding protein progesterone receptor membrane component 1 (PGRMC1) is a positive regulator of CYP51A1, a cytochrome P450 enzyme required for cholesterol synthesis. PGRMC1 knockdown cells have impaired cholesterol synthesis and accumulate the intermediate lanosterol (which is known to inhibit HMG-CoA reductase). PGRMC1 also binds another cytochrome P450 enzyme, CYP7A1, required for bile acid synthesis. Based on these findings, I hypothesize that PGRMC1 is an important regulator of cholesterol metabolism and necessary for systemic cholesterol homeostasis in mammals. To test this hypothesis, I will take advantage of the existing Pgrmc1 knockout (Pgrmc1KO) mice generated by our laboratory. In Aim 1, I will test the in vivo role of PGRMC1 in cholesterol synthesis. I will first perform a general histological characterization on the livers of Pgrmc1KO mice and check for markers of liver injury. Then, I will measure hepatic sterol composition, serum cholesterol levels, and serum lipoprotein particle profiles in Pgrmc1KO mice. I will also measure the rate of cholesterol synthesis in Pgrmc1KO primary hepatocytes, and Cyp51A1 enzymatic activity in Pgrmc1KO liver microsomes. In Aim 2, I will test the in vivo role of PGRMC1 in bile acid synthesis. I will measure steady state bile acid levels and hepatic bile acid synthesis rates in Pgrmc1KO mice and Cyp7A1 activity in Pgrmc1KO liver microsomes. These studies will establish the role of PGRMC1 in regulating cholesterol homeostasis. Patients with high serum cholesterol levels are treated with statin drugs (HMG-CoA reductase inhibitors) and bile acid sequestrant drugs. Inhibition of PGRMC1 may prove to be a novel method of inhibiting HMG-CoA reductase by lanosterol accumulation. Alternatively, manipulating PGRMC1 may be a novel method for stimulating bile acid synthesis. This proposal will expand our understanding of how cholesterol homeostasis is maintained in vivo and provide a candidate target for the development of therapeutics for high cholesterol. Treating high cholesterol will reduce the risk of heart attack and stroke in patients. Also, these studies on PGRMC1 will advance our understanding of the cytochrome P450 family of enzymes. P450 enzymes are also important for drug metabolism and have no known post-transcriptional regulators.