Regulation of Renal PEPCK Gene Expression

描述(由申请人提供)：代谢性酸中毒的发作激活了编码酶和运输蛋白的基因的转录，这些酶和运输蛋白维持肾氨排泄、HCO3合成和重吸收以及糖异生的适应性增加。这种反应的一个典型例子是发生在大鼠肾脏近曲小管内的磷酸烯醇式丙酮酸羧酸激酶(PEPCK)mRNA水平增加6倍。PEPCK的全长cDNA和基因组已被分离和测序。在肝脏、肾脏和脂肪组织中，许多介导其转录调控的启动子元件和相关的转录因子已经被鉴定。此外，PEPCK启动子和3‘-非翻译区的不同片段和特定突变已经以报告构建或转基因的形式表达。此外，猪肾近端小管样细胞系LLC-PK1-F+细胞在酸性介质[pH 6.9，10 mM HCO3-]中，内源性PEPCK基因或CRC362 PEPCK转基因基因的表达增加了3-4倍。PEPCK mRNA还包含一个独特的不稳定元件，这是其快速周转和cAMP依赖的稳定的原因。因此，PEPCK基因和该细胞系是研究pH变化激活近端小管内特定基因转录的机制的有效范例。这项研究的具体目的是：利用CRC362转基因定位和鉴定调节PEPCK基因转录的pH反应元件；鉴定P38A SAPK/ATF-2信号通路在pH反应诱导PEPCK基因中的作用；以及鉴定介导PEPCK mRNA周转的结合蛋白和机制。拟议的实验结果将显著增加对调节这种基本适应性反应的分子机制的理解，并提供可能导致改善慢性酸中毒临床治疗的洞察力。

DESCRIPTION (provided by applicant): Onset of metabolic acidosis activates transcription of the genes that encode the enzymes and transport proteins that sustain the adaptive increases in renal ammonia excretion, HCO3- synthesis and reabsorption, and gluconeogenesis. A well-characterized example of this response is the 6-fold increase in the level of the phosphoenolpyruvate carboxykinase (PEPCK) mRNA that occurs within the rat renal proximal convoluted tubule. The full-length PEPCK cDNA and genome have been isolated and sequenced. Many of the promoter elements and associated transcription factors that mediate its transcriptional regulation in liver, kidney, and adipose tissues have been characterized. In addition, various segments and specific mutations of the PEPCK promoter and 3'-untranslated region have been expressed as reporter constructs or transgenes. Furthermore, LLC-PK1-F+ cells, a porcine line of renal proximal tubule-like cells, exhibit a 3-4-fold increase in expression of the endogenous PEPCK gene or the CRC362 PEPCK transgene when transferred to acidic medium [pH 6.9, 10 mM HCO3-]. The PEPCK mRNA also contains a unique instability element that accounts for its rapid turnover and cAMP-dependent stabilization. Thus, the PEPCK gene and this cell line serve as an effective paradigm to investigate the mechanism by which changes in pH activate transcription of specific genes within the proximal tubule. The specific aims of the proposed research are: to use the CRC362 transgene to map and characterize the pH-response element that regulates transcription of the PEPCK gene; to characterize the role of the p38a SAPK/ATF-2 signaling pathway in the pH-responsive induction of the PEPCK gene; and to identify the binding proteins and the mechanism that mediate the turnover of PEPCK mRNA. The results of the proposed experiments should significantly increase understanding of the molecular mechanism that regulates this essential adaptive response and provide insight that may lead to improved clinical treatment of chronic acidosis.