MECHANISM OF PH-RESPONSE IN PCK GENE EXPRESSION

PCK 基因表达中的 PH 响应机制

• 批准号: 2444056
• 负责人: NORMAN P. CURTHOYS
• 金额: $ 17.06万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2444056
• 关键词: acidity /alkalinity; acidosis; enzyme induction /repression; enzyme mechanism; gene expression; gene induction /repression; genetic regulatory element; gluconeogenesis; kidney metabolism; laboratory rat; messenger RNA; molecular cloning; phosphoenolpyruvate carboxylase; renal tubular transport; tissue /cell culture; transcription factor

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.

描述（改编自申请人的摘要）： 胞质磷酸烯醇丙酮酸羧激酶 (PCK) mRNA 增加 4 倍 大鼠肾近曲小管内对代谢的反应 酸中毒。 这种适应有助于肾功能的持续增加 氨生成和糖异生，这对于部分 补偿慢性酸中毒。 PCK 活性增加的原因是 转录增加。 整个大鼠 PCK 基因已被分离并 已测序。 许多 PCK 启动子元件和相关因子 介导肝脏中 PCK 基因的转录调控 得到了很好的表征。 此外，LLC-PK1-F+ 细胞（一种糖异生细胞） 猪肾近曲小管样上皮细胞系，表现出 PCK mRNA 水平响应酸性介质中生长的适应性变化 （pH = 6.9，[HCO3-]=10 mM）模拟体内观察到的情况。 这些细胞 用于确定 PCK 的 CRE-1 和 P3(II) 元件 启动子介导 pH 响应和 cAMP 介导的刺激 转录。 此外，后一种反应被证明利用了 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 非常适合表征组织的特异性反应 cAMP 并确定肾近曲小管细胞如何感知 cAMP 的变化 pH 值并转导该信息以影响特定基因的表达。 拟议研究的具体目标是表征该机制 肾细胞中 PCK 基因的 cAMP 激活；来表征 转录因子及其介导的激活机制 肾脏 PCK 基因的 pH 响应性诱导；以确定是否 细胞最初对细胞内或细胞外 pH 值的变化做出反应 定义相关的信号转导途径；并识别和 表征 PCK mRNA 中包含的不稳定元件。 这 拟议研究的结果应能深入了解潜在的 可能刺激肾脏免疫发生的药理学方法 各种临床情况下的糖异生导致代谢 酸中毒。