MECHANISM OF PH-RESPONSE IN PCK GENE EXPRESSION

描述（改编自申请人的摘要）： 胞浆磷酸烯醇式丙酮酸羧激酶（PCK）mRNA增加4倍 在大鼠肾近曲小管内， 酸中毒 这种适应有助于肾功能的持续增加。 氨生成和氨共生，这是必不可少的部分 补偿慢性酸中毒 PCK活性增加的原因是 增加转录。 整个大鼠PCK基因已被分离， 测序 许多PCK启动子元件和相关因子 介导PCK基因在肝脏中的转录调节， 被很好地描述了。 此外，LLC-PK 1-F+细胞，一种促凋亡细胞， 猪肾近端小管样上皮细胞系，显示 PCK mRNA水平对酸性培养基中生长的适应性变化 (pH= 6.9，[HCO 3-]=10 mM），其模拟了体内观察到的那些。 这些细胞 用于确定PCK的CRE-1和P3（II）元素 启动子介导pH响应和cAMP介导的刺激， 转录。 此外，后者的反应被证明利用C/EBP 和Fos/Jun转录因子，因此与 cAMP刺激肝脏中PCK诱导的机制。 因此，该系统 非常适合表征组织特异性反应， cAMP，并确定肾近端小管细胞如何感知 pH值，并转导这一信息，以影响特定基因的表达。 拟议研究的具体目的是表征该机制 的cAMP激活的PCK基因在肾细胞中;表征 转录因子和激活机制，介导的 肾PCK基因的pH响应性诱导;以确定 细胞最初响应于细胞内或细胞外pH的变化， 以确定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。 的 拟议研究的结果应提供深入了解潜在的 可能刺激肾免疫发生的药理学方法， 在导致代谢性疾病的各种临床条件下， 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.