Transcriptional Regulation of Angiotensinogen Gene

描述(申请人提供)：高血压是心肌梗死、心力衰竭、血管疾病、中风和肾功能衰竭的严重危险因素。肾素-血管紧张素系统在血压调节中起着重要作用。先前的研究表明：(A)血管紧张素原(AGT)基因与人类高血压有关，(B)AGT基因-6A变异与高加索人和日本人的高血压有关，(C)AGT基因的过度表达会增加转基因小鼠的血压。我们在人类AGT基因启动子-217处发现了A/G多态，并发现等位基因A在-217处的频率在非裔美国人高血压患者中显著增加。AGT基因主要在肝脏中表达，我们已经证明，含有AGT基因启动子和核苷A at-217的报告载体在瞬时转染人肝细胞时可以增加基础启动子和IL-6诱导的启动子的活性。虽然hagt基因启动子1.2Kb区域有7个多态位点，但-217A变异体几乎总是与-532T、-793A和-1074T一起出现，变异体-217G、-532C、-793G和-1074G总是同时出现，形成两种单倍型。由于-6A等位基因在非裔美国人中占主导地位(频率为0.85)，因此AGT基因可细分为四种单倍型-6A：-217A(AA)；-6A：-217G(AG)；-6G：-217A(GA)和-6G：-217G(GG)。然而，GA和GG的单倍型非常罕见，AA和AG是两个突出的单倍型。我们发现：(A)与AG单倍型相比，高血压患者AA单倍型的频率增加；(B)与AG单倍型相比，含有AA单倍型的报告结构具有更高的基础启动子活性和IL-6诱导的启动子活性。由于炎症在高血压中起着重要作用，我们的假设是AGT基因AA单倍型转录增加在高血压的发生发展中起重要作用。为了验证这一假设，我们利用HPRT基因座的敲入策略产生了含有人肾素基因和HAGT基因AA或AG单倍型的转基因小鼠。我们已经证明，与AG单倍型相比，含有AGT基因AA单倍型的双转基因小鼠的AGT mRNA和蛋白水平增加。我们还发现，与含有aG单倍型hagt基因和hren基因的转基因小鼠相比，含有hagt基因和hren基因AA单倍型的双转基因小鼠血压升高。我们现在将在体内情况下研究这些单倍型在含有Hagt基因和hren基因的AA或AG单倍型但缺乏mAGT基因的转基因小鼠中对血压调节的作用。公共卫生相关性：高血压是心肌梗死、心力衰竭、血管疾病、中风和肾功能衰竭的严重危险因素。据估计，高血压影响着5000万美国人，在成年高加索人口中的患病率为25%-30%。在非裔美国人中，高血压的发病率和由高血压引起的并发症甚至更多。我们的研究将有助于我们了解高血压的分子机制。

DESCRIPTION (provided by applicant): Hypertension is a serious risk factor for myocardial infarction, heart failure, vascular disease, stroke, and renal failure. The renin-angiotensin system plays an important role in the regulation of blood pressure. Previous studies have suggested that: (a) angiotensinogen (AGT) gene locus is associated with human essential hypertension, (b) variant -6A of the AGT gene is associated with hypertension in Caucasian and Japanese subjects and (c) over-expression of the AGT gene increases blood pressure in transgenic mice. We have found an A/G polymorphism at -217 in the human AGT gene promoter and have shown that frequency of allele A at -217 is significantly increased in African-American hypertensive patients. AGT gene is primarily expressed in the liver and we have shown that reporter constructs containing AGT gene promoter with nucleoside A at -217 have increased basal and IL-6 induced promoter activity on transient transfection in human liver cells. Although hAGT gene has seven polymorphic sites in 1.2Kb region of its promoter, variants -217A almost always occurs with -532T, -793A, and -1074T and variants -217G, -532C, -793G, and -1074G always occur together forming two haplotypes. Since allele - 6A is the predominant allele (frequency 0.85) in African-Americans, AGT gene can be subdivided into four haplotypes -6A:-217A (AA); -6A:-217G (AG); -6G:-217A (GA) and -6G:-217G (GG). However, haplotypes GA and GG are very rare leaving AA and AG as two prominent haplotypes. We have shown that: (a) frequency of AA haplotype is increased in hypertensive patients as compared to the AG haplotype and (b) reporter constructs containing AA haplotype have increased basal as well as IL-6 induced promoter activity as compared to AG haplotype. Since inflammation plays an important role in hypertension, our hypothesis is that increased transcription of AA haplotype of the AGT gene plays an important role in the development of hypertension. In order to prove this hypothesis, we have generated transgenic mice containing human renin gene and either AA or AG haplotype of the hAGT gene using knock-in strategy at the HPRT locus. We have shown that AGT mRNA and protein level is increased in double transgenic mice containing AA haplotype of the AGT gene as compared to the AG haplotype. We have also shown that double transgenic mice containing AA haplotype of the hAGT gene and hRen gene have increased blood pressure as compared to transgenic mice containing AG haplotype of the hAGT gene and hRen gene. We will now study the role of these haplotypes on blood pressure regulation in an in vivo situation in transgenic mice containing either AA or AG haplotype of the hAGT gene and hRen gene but devoid of mAGT gene. PUBLIC HEALTH RELEVANCE: Hypertension is a serious risk factor for myocardial infarction, heart failure, vascular disease, stroke, and renal failure. It is estimated that hypertension affects 50 million Americans with a prevalence rate of 25-30% in the adult Caucasian population. The incidence of hypertension and complications due to hypertension are even greater in the African American population. Our studies will help us understand molecular mechanism involved in hypertension.