Control of angiotensin II receptor gene expression

描述（由申请人提供）：在大鼠中，两种不同的血管紧张素型受体（AT1aR） mrna由单个ATtaR Lene通过选择性剪接合成。这些转录本由外显子1和3 （E1,3）以及外显子1、2和3 （E1,2,3）编码。我们发现，与E1,2,3转录本相比，E1,3变异更有效地翻译，导致细胞膜上更高的AT1R密度和更大的Ang ii诱导的信号转导。我们还发现，盐负荷高血压DahL盐敏感（DS）大鼠的E1、3/E1、2、3的比例更高，尽管总mRNA水平没有显著差异。与正常DS和耐达尔盐（DR）动物相比，这一比例的增加与肾皮质AT1R密度的增加相对应。在这项资助中，我们建议研究这些选择性剪接变体的机制和功能后果。在Aim 1中，我们将确定负责外显子2介导的AT1aR翻译抑制的分子机制。我们将验证以下假设：由于外显子2内的抑制性RNA顺式元件，大鼠E1,2-，3转录本在体内的翻译效率低于E1,3转录本，这导致与E1,3变体相比，AT1R蛋白表达和AT1R介导的功能显着减少；并且，外显子2包含一个通过与外显子2特异性RNA结合蛋白相互作用来调节AT1R翻译速率的调控元件。在Aim 2中，我们将确定在盐负荷高血压DS大鼠中观察到的AT1aR转录本表达改变的细胞和组织特异性，以及钠负荷诱导剪接改变的时间过程。我们将验证一种假设，即在维持HS饮食的DS大鼠中观察到的AT1aR E1、3/E、1、2、3比率的增加，是在肾脏内以细胞特异性方式发生的。此外，我们假设那些E1,3/E1,2-，3比值升高的细胞和肾脏结构也会表现出一氧化氮合酶（NOS）异构体和烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶亚基的异常表达，因为Ang II通过AT1R调节这些途径，因为DS大鼠在肾脏中表现出NOS和NADPH氧化酶的异常。我们还假设盐诱导高血压的时间过程以及NOS和NADPH氧化酶表达和活性的变化与诱导At1R剪接改变有关。在Aim 3中，我们将确定在高血压DS大鼠中观察到的与盐负荷相关的哪些因素诱导了ATlaR剪接的改变。我们将验证与钠负荷相关的rena内因子，而不是升高的MAP，诱导AT1R剪接改变的假设。尽管文献表明选择性剪接发生在所有哺乳动物血管紧张素受体中，但很少有研究者关注选择性剪接在血管紧张素受体基因表达的生理和病理生理控制中的作用。因此，这些拟议的研究将对AT1R的基因调控产生新的见解。AT1Rs抑制剂在治疗高血压及相关心血管和肾脏疾病方面非常有效，这一事实表明，这些研究最终可能导致新的药物靶点来治疗这些与年龄相关的疾病。

DESCRIPTION (provided by applicant): In the rat, two distinct angiotensin type la receptor (AT1aR) mRNAs are synthesized from a single ATtaR Lene by alternative splicing. These transcripts are encoded by exons 1 and 3 (E1,3) and exons 1, 2, and 3 (E1,2,3). We found that the E1,3 variant is more efficiently translated and results in higher AT1R densities in cell membranes and greater Ang II-induced signal transduction than the E1,2,3 transcript. We also found that salt-Loaded hypertensive DahL salt-sensitive (DS) rats had higher ratios of E1,3/E1,2,3, even though no significant differences in the total mRNA Levels were observed. This increased ratio corresponds with higher AT1R densities in the renal cortex compared to normotensive DS and Dahl-salt resistant (DR) animals. In this grant, we propose to investigate the mechanisms and functional consequences of these alternatively spliced variants. In Aim 1, we will determine the molecular mechanisms responsible for exon 2-mediated repression of AT1aR translation. We will test the hypotheses that the rat E1,2-,3 transcript is translated less efficiently in vivo than the E1,3 transcript due to an inhibitory RNA cis element within exon 2, which results in significantly less AT1R protein expression and AT1R-mediated function compared to the E1,3 variant; and, that exon 2 contains a regulatory element that serves to modulate rates of AT1R translation by interacting with exon 2 specific RNA binding proteins. In Aim 2, we will determine the cell and tissue specificity of the altered expression of AT1aR transcripts observed in the salt-Loaded hypertensive DS rat and the time course of the alterations in splicing induced by sodium Loading. We will test the hypothesis that increased AT1aR E1,3/E,1,2,3 ratios observed in DS rats maintained on a HS diet, occurs in a cell specific manner within the kidney. In addition, we hypothesize that those cells and kidney structures exhibiting elevated E1,3/E1,2-,3 ratios will also exhibit abnormal expression of nitric oxide synthase (NOS) isoforms and nicotinamide adenine dinucteotide phosphate (NADPH) oxidase subunits since Ang II modulates these pathways through the AT1R and because DS rats exhibit abnormalities in NOS and NADPH oxidase in the kidney. We also hypothesize that the time course of saltinduced hypertension and changes in NOS and NADPH oxidase expression and activity will correlate with induction of altered At1R splicing. In Aim 3 we will determine which factors associated with salt loading induce the alteration in ATlaR splicing that is observed in the hypertensive DS rat. We will test the hypothesis that intra-rena factors associated with sodium loading, rather than elevated MAP, induce the alteration in AT1R splicing. Even though the Literature suggests alternative splicing occurs in all mammalian angiotensin receptors, few investigators have focused on the role of alternative splicing in physiological and pathophysiological control of angiotensin receptor gene expression. Thus, these proposed studies will yield new insights into gene regulation of the AT1R. The fact that inhibitors of AT1Rs are very effective in treating hypertension and associated cardiovascular and renal disease, suggests that these studies could ultimately lead to new pharmaceutical targets to treat these age-related pathologies.