ROLE OF GLUCOSE IN hAT1 GENE EXPRESSION

描述(申请人提供)：高血压和糖尿病是糖尿病肾病发病机制中的两个主要危险因素。血管紧张素转换酶抑制剂治疗糖尿病肾病广泛有效，提示肾素血管紧张素系统在糖尿病肾病的进展中起重要作用。血管紧张素II是肾素血管紧张素系统的活性成分，主要通过血管紧张素1型受体发挥作用。与大鼠编码AT1受体的两个基因(AT1A、AT1B)相比，在人类中，只有一个基因编码血管紧张素1型(HAT1)，提示该受体的物种特异性表达和差异调节。在糖尿病中，从发病到终末期肾病，hAT1受体在肾脏不同区域的表达有所不同，但近端小管除外，在近端小管上，该受体一直下调，提示该受体在小管上具有特异性调节。血管紧张素转换酶抑制剂不能逆转hAT1受体的下调，说明该受体的下调不是通过血管紧张素Ⅱ的上调来实现的。糖尿病患者hAT1受体下调的分子机制尚不清楚。近端肾小管AT1基因表达的任何改变(增加/减少)都具有显著的病理生理后果。我们假设在正常生理学中，hAT1受体的表达是通过葡萄糖和胰岛素在hAT1基因转录上的归一化相互作用实现的。另一方面，在糖尿病中，当细胞外葡萄糖水平高而胰岛素水平低时，葡萄糖和胰岛素之间的平衡相互作用将发生改变，最终结果将是hAT1基因的表达减少。最近，我们已经在hAT1基因启动子中确定了其基础转录所需的特定序列，并作为胰岛素反应(增强子)元件发挥作用。进一步的研究发现，增强子上游有一个抑制元件，可以对正常/高水平的胞外葡萄糖做出反应。我们的观察是，在有葡萄糖存在的情况下(正常/高血糖)，胰岛素对hAT1转录抑制没有增强作用，而在没有葡萄糖或存在低糖的情况下，胰岛素增强了hAT1基因的转录。此外，我们有证据表明，这些调控元件识别葡萄糖和胰岛素诱导的特定核交易因子。我们的观察首次证明hAT1基因转录的生理水平受抑制元件控制，可能是通过葡萄糖和胰岛素之间的相互作用。虽然我们认识到葡萄糖和胰岛素都是hAT1转录的重要调节因子，但在本提案中，我们将主要关注葡萄糖介导的hAT1基因转录。因此，本应用的总体目标是确定葡萄糖在控制hAT1基因转录中的功能意义，并确定与葡萄糖信号相关的特定调控因子的作用，以了解hAT1基因在糖尿病和高血压等路径生理中参与调控的分子和生化机制。

DESCRIPTION (provided by applicant): Hypertension and diabetes are two major risk factors in the pathogenesis of diabetic nephropathy. Angiotensin converting enzyme inhibitor therapy is broadly effective in patients with diabetic nephropathy suggesting an important role for renin angiotensin system in the progression of this disorder. Angiotensin II, the active component of the renin angiotensin system, acts primarily through angiotensin type 1 (AT1) receptors. Compared to rat in which two genes (AT1A, AT1B) code for AT1 receptor, in humans, a single gene codes for angiotensin type 1 (hAT1), suggesting species-specific expression and differential regulation of the receptor. In diabetes, from the onset to end-stage nephropathy, hAT1 receptor expression varies in different regions of the kidney with one exception, the proximal tubule in which, this receptor is down regulated at all times suggesting tubule specific regulation of the receptor. Reduction in hAT1 receptors could not be reversed by ACE inhibitors demonstrating that the receptor downregulation was not mediated by the up-regulation of angiotensin II. The molecular mechanisms leading to hAT1 receptor down-regulation in diabetes are not known. Any alterations (increase/decrease) in AT1gene expression in proximal tubule have significant pathophysiological consequences. We hypothesize that in normal physiology, expression of the hAT1 receptor is achieved by normalized interactions between glucose and insulin on hAT1 gene transcription. Alternatively, in diabetes, when extracellular glucose levels are high and insulin levels are low, the equilibrium interaction between glucose and insulin will shift and the end-result will be decreased expression of hAT1 gene. Recently, we have identified a specific sequence in the hAT1 gene promoter required for its basal transcription and functions as an insulin response (enhancer) element. Additional studies revealed a repressor element upstream of the enhancer that can respond to normal/high levels of extracellular glucose. Our observation is that in the presence of glucose (normal/high), insulin has no enhancer effect on hAT1 transcriptional repression, where as in the absence of glucose or presence of low glucose insulin enhances the h AT1 gene transcription. In addition, we have evidence that these regulatory elements recognize specific nuclear transacting factors induced by glucose and insulin. Our observation is the first evidence that physiological levels of hAT1 gene transcription is controlled by a repressor element perhaps through an interplay between glucose and insulin. Although we recognize that both glucose and insulin are important regulators of hAT1 transcription, in this proposal we will focus primarily on glucose mediated hAT1 gene transcription. Therefore the overall goal of this application is to determine the functional significance of glucose in the control of hAT1 gene transcription and identify the role of specific transacting factors associated with glucose signaling in order to understand the molecular and biochemical mechanisms involved in the regulation of hAT1 gene in path physiology such as diabetes and hypertension.