Transcriptional regulation of human angiotensin receptor

描述（由申请人提供）：八肽血管紧张素-II (Ang II) 是已知最有效的血管活性物质之一，调节多种生理反应，包括体液稳态、醛固酮产生、肾功能和血管平滑肌 (VSM) 收缩。血管紧张素受体 1 (AT1R) 基因的过度表达会产生高血压，尤其是在雌性转基因小鼠中。这些研究表明 hAT1 R 基因转录增加可能导致高血压。因此，我们分析了 hAT1R 基因 5' 侧翼区域的单核苷酸多态性 (SNP) 在高血压中的作用。我们发现hAT1 R基因启动子具有至少5个SNP的单倍型块，由-777处的T/A、-680处的T/G、-214处的A/C、-213处的G/C和-119处的A/G组成。我们的研究表明，变体-777T、-680T、-214A、-213G和-119A总是一起出现（分别创建包含TTAGA的单倍型-1和包含AGCCG的单倍型-II）。我们的研究表明，hAT1R 基因的单倍型-1 与白种人女性的高血压相关，并且与单倍型-II 相比，瞬时转染含有 hAT1R 基因的单倍型-1 的报告构建体增加了肾上腺皮质细胞和 VSMC 中的启动子活性。我们的凝胶位移测定显示：(a)与-119G(单倍型-II)相比，转录因子C/EBP与含有-119A(单倍型-1)的寡核苷酸更强地结合，并且(b)与含有-119G(单倍型-II)的相同寡核苷酸相比，转录因子USF(其与E-盒基序CANNTG结合)与在-214和-213处含有核苷A和G(单倍型-1)的寡核苷酸强力结合。 -213 和 -214 处的核苷 C（单倍型-II）。因此，我们将使用肾上腺皮质和血管平滑肌细胞在体外系统中分析单倍型-I和II对hAT1R基因转录调控的影响，使用雄性和雌性转基因小鼠检查单倍型-I和II对hAT1R mRNA水平和血压的影响，并检查IL-6、糖皮质激素和雌激素对转基因小鼠中hAT1R mRNA水平的影响hAT1 R 基因的单倍型 I 和 II，并将转基因小鼠中 mRNA 水平的潜在变化与血压相关联。

DESCRIPTION (provided by applicant): The octapeptide, angiotensin-ll (Ang II) is one of the most potent vasoactive substances known and regulates a variety of physiological responses, including fluid homeostasis, aldosterone production, renal function and contraction of vascular smooth muscle (VSM). Over-expression of the angiotensin receptor-1 (AT1R) gene produces hypertension especially in female transgenic mice. These studies have suggested that increased transcription of the hAT1 R gene may lead to hypertension. We have therefore analyzed the role of single nucleotide polymorphisms (SNPs) in the 5' flanking region of the hAT1R gene in hypertension. We have found that hAT1 R gene promoter has a haplotype block of at least five SNPs consisting of T/A at - 777, T/G at -680, A/C at -214, G/C at -213, and A/G at -119. Our studies have shown that variants -777T, - 680T, -214A, -213G, and -119A always occur together (creating haplotype-l containing TTAGA and haplotype-ll containing AGCCG respectively). Our studies have shown that haplotype-l of the hAT1R gene is associated with hypertension in Caucasian women and transient transfection of reporter construct containing haplotype-l of the hAT1R gene has increased promoter activity in adrenal cortical cells and VSMC as compared to haplotype-ll. Our gel shift assays have shown that: (a) transcription factor C/EBP binds more strongly to an oligonucleotide containing -119A (haplotype-l) as compared to -119G (haplotype- ll) and (b) transcription factor USF (which binds to an E-box motif CANNTG) binds strongly to an oligonucleotide containing nucleoside A and G at -214 and -213 (haplotype-l) as compared to the same oligonucleotide containing nucleoside C at -213 and -214 (haplotype-ll). We will therefore analyze the effect of haplotypes -I and II on transcriptional regulation of the hAT1R gene in an in-vitro system using adrenocortical and vascular smooth muscle cells, examine the effect of haplotypes-l and II on hAT1 R mRNA level and on blood pressure using male and female transgenic mice, and examine the effect of IL-6, glucocorticoids, and estrogens on hAT1R mRNA level in transgenic mice containing haplotype I and II of the hAT1 R gene, and to correlate potential changes in mRNA levels with blood pressure in transgenic mice.