Regulation of Renal PEPCK Gene Expression

描述（由申请方提供）：代谢性酸中毒发作激活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、HCO 3合成和重吸收以及再生的适应性增加。这种反应的一个充分表征的例子是在大鼠肾近曲小管内发生的磷酸烯醇式丙酮酸羧激酶（PEPCK）mRNA水平增加6倍。全长PEPCK cDNA和基因组已被分离和测序。许多启动子元件和相关的转录因子，介导其在肝脏，肾脏和脂肪组织中的转录调控的特点。此外，PEPCK启动子和3 '-非翻译区的各种片段和特定突变已经表达为报告构建体或转基因。此外，LLC-PK 1-F+细胞（一种猪肾近端小管样细胞系）在转移至酸性培养基[pH 6.9，10 mM HCO 3-]时，内源性PEPCK基因或CRC 362 PEPCK转基因的表达增加3-4倍。PEPCK mRNA还含有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基因和该细胞系作为一个有效的范例，以研究pH值的变化激活近端小管内的特定基因的转录的机制。本研究的具体目的是：利用CRC 362转基因定位和表征调节PEPCK基因转录的pH响应元件;表征p38 a SAPK/ATF-2信号通路在PEPCK基因pH响应诱导中的作用;以及鉴定介导PEPCK mRNA周转的结合蛋白和机制。拟议的实验的结果应显着增加的分子机制，调节这种基本的适应性反应的理解，并提供洞察力，可能会导致改善慢性酸中毒的临床治疗。

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.