MECHANISM OF PH-RESPONSE IN PCK GENE EXPRESSION

描述(改编自申请人的摘要)： 胞浆中磷酸烯醇式丙酮酸羧酸激酶(PCK)的mRNA增加了4倍 大鼠肾脏近曲小管对代谢的反应 酸中毒。这种适应有助于肾脏的持续增加。 氨化作用和糖异生作用是部分 弥补慢性酸中毒。PCK活性增加的原因是 增加了转录。整个大鼠PCK基因已经被分离出来并 已排序。多个PCK启动子元件及其相关因素 在肝脏中介导PCK基因转录调控的有哪些 被很好地刻画出来了。此外，LLC-PK1-F+细胞是一种糖异生细胞 猪肾近端小管样上皮细胞系，展示 酸性介质中PCK基因表达水平对生长的适应性变化 (pH=6.9，[HCO3-]=10 mm)，模拟体内观察到的情况。这些细胞 被用来确定PCK的Cre-1和P3(II)元件 启动子介导pH反应和cAMP介导的刺激 抄写。此外，后者的反应被显示为利用C/EBP 和Fos/Jun转录因子，因此与 CAMP刺激肝脏PCK诱导的机制。因此，这个系统 非常适合用来描述组织对 CAMP，并确定肾近端小管细胞感觉如何变化。 PH并传递这些信息来影响特定基因的表达。 建议的研究的具体目的是描述这种机制。 肾细胞中PCK基因的cAMP激活；以表征 转录因子及其介导的激活机制 PH反应诱导肾脏PCK基因；以确定是否 细胞最初对细胞内或细胞外pH的变化做出反应 确定相关的信号转导途径；并识别和 鉴定PCK信使核糖核酸中包含的不稳定元件。这个 拟议研究的结果应提供对潜力的洞察 可能刺激肾脏免疫生成和 导致代谢的不同临床条件下的糖异生 酸中毒。

DESCRIPTION (Adapted from the Applicant's Abstract): The level of the cytosolic phosphoenolpyruvate carboxykinase (PCK) mRNA is increased 4-fold within the rat renal proximal convoluted tubule in response to metabolic acidosis. This adaptation contributes to the sustained increase in renal ammoniagenesis and gluconeogenesis which are essential to partially compensate a chronic acidosis. The increased PCK activity results from increased transcription. The entire rat PCK gene has been isolated and sequenced. Many of the PCK promoter elements and the associated factors which mediate the transcriptional regulation of the PCK gene in liver have been well-characterized. Furthermore, LLC-PK1-F+ cells, a gluconeogenic line of porcine renal proximal tubule-like epithelial cells, exhibit adaptive changes in PCK mRNA levels in response to growth in acidic medium (pH = 6.9, [HCO3-]=10 mM) which model those observed in vivo. These cells were used to establish that the CRE-1 and P3(II) elements of the PCK promoter mediate both the pH-responsive and cAMP-mediated stimulations of transcription. Furthermore, the latter response was shown to utilize C/EBP and Fos/Jun transcription factors and thus differ significantly from the mechanism of cAMP stimulation of PCK induction in liver. Thus, this system is extremely well-suited to characterize the tissue specific response to cAMP and to determine how the renal proximal tubule cell senses changes in pH and transduces this information to effect expression of specific genes. The Specific Aims of the proposed research are to characterize the mechanism of cAMP activation of the PCK gene in kidney cells; to characterize the transcription factors and the mechanism of activation which mediate the pH-responsive induction of the renal PCK gene; to determine whether the cells initially respond to changes in intracellular or extracellular pH and to define the associated signal transduction pathway; and to identify and characterize the instability elements contained in the PCK mRNA. The results of the proposed studies should provide insight into potential pharmacological approaches that may stimulate renal immunogenesis and gluconeogenesis in various clinical conditions which lead to metabolic acidosis.