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转运蛋白的基因的转录，这些酶和转运蛋白维持肾氨排泄、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大鼠肾近曲小管内磷酸烯醇丙酮酸羧激酶（PEPCK） mRNA水平增加6倍。对PEPCK全长cDNA和基因组进行了分离和测序。许多在肝脏、肾脏和脂肪组织中介导其转录调控的启动子元件和相关转录因子已经被表征。此外，PEPCK启动子和3'-非翻译区的各种片段和特定突变已被表达为报告基因或转基因。此外，猪肾近端小管样细胞lc - pk1 -f +细胞在转入酸性培养基[pH 6.9, 10 mM HCO3-]时，内源性PEPCK基因或CRC362 PEPCK转基因的表达增加了3-4倍。PEPCK mRNA还包含一个独特的不稳定元件，该元件可解释其快速周转和camp依赖性稳定。因此，PEPCK基因和该细胞系可以作为研究pH变化激活近端小管内特定基因转录的机制的有效范例。提出的研究的具体目的是：使用CRC362转基因来绘制和表征调节PEPCK基因转录的ph反应元件；表征p38a SAPK/ATF-2信号通路在ph响应性诱导PEPCK基因中的作用；鉴定介导PEPCK mRNA周转的结合蛋白和机制。这些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见解。

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.