Transcriptional Regulation of Angiotensinogen Gene

描述（由申请人提供）：高血压是心肌梗死、心力衰竭、血管疾病、中风和肾衰竭的严重风险因素。肾素-血管紧张素系统在血压调节中起着重要作用。以往的研究表明：（a）血管紧张素原（AGT）基因位点与人类原发性高血压相关，（B）AGT基因的变体-6A与高加索人和日本受试者的高血压相关，（c）AGT基因的过度表达增加转基因小鼠的血压。我们在人类AGT基因启动子-217位点发现了A/G多态性，并表明在非裔美国人高血压患者中等位基因A的频率显著增加。AGT基因主要在肝脏中表达，并且我们已经表明，含有在-217处具有核苷A的AGT基因启动子的报道构建体在人肝细胞中瞬时转染时具有增加的基础和IL-6诱导的启动子活性。虽然hAGT基因在其启动子的1.2Kb区域有7个多态位点，但变异体-217A几乎总是与-532T、-793A和-1074T一起出现，变异体-217G、-532C、-793G和-1074G总是一起出现，形成两种单倍型。由于等位基因-6A在非裔美国人中占优势（频率0.85），AGT基因可分为4种单倍型：-6A：-217A（AA）、-6A：-217G（AG）、-6G：-217A（GA）和-6G：-217G（GG）。然而，单倍型GA和GG是非常罕见的，留下AA和AG作为两个突出的单倍型。我们已经证明：（a）与AG单倍型相比，AA单倍型的频率在高血压患者中增加，和（B）与AG单倍型相比，含有AA单倍型的报道构建体具有增加的基础以及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基因的AA单倍型的转录增加在高血压的发展中起着重要作用。为了证明这一假设，我们已经产生了转基因小鼠含有人肾素基因和AA或AG单倍型的hAGT基因使用敲入策略在HPRT基因座。我们已经表明，AGT的mRNA和蛋白质水平增加，在双转基因小鼠含有AA单倍型的AGT基因相比，AG单倍型。我们还表明，与含有hAGT基因和hRen基因的AG单倍型的转基因小鼠相比，含有hAGT基因和hRen基因的AA单倍型的双转基因小鼠具有升高的血压。我们现在将在含有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研究这些单倍型在体内情况下对血压调节的作用。

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.