RUI: Developmental Regulation of Hepatic Beta2-Adrenergic Receptor Gene Expression

9728212 Baeyens许多激素和药物通过与位于质膜上的特定细胞表面受体结合来启动它们对细胞的作用。对于儿茶酚胺、肾上腺素和去甲肾上腺素，受体被称为肾上腺素能受体（AR），包括两种主要类型，称为α 1-AR和β-AR。肾上腺素和去甲肾上腺素对肝脏代谢包括糖原分解和糖原合成有重要作用。肝细胞表达α-AR和β 2-AR亚型，它们通过不同的第二信使系统发挥作用，将儿茶酚胺刺激转化为生物学效应。α 1-和β 2-AR均通过增加磷酸化酶活性促进糖原分解来调节肝脏代谢。在雄性大鼠肝脏中，葡萄糖代谢的儿茶酚胺控制具有年龄依赖性。在胎儿和年轻的雄性大鼠（小于14天）中，肾上腺素通过β 2-AR激活磷酸化酶，而在成年雄性大鼠中，肾上腺素几乎完全通过α 1-AR增加磷酸化酶活性。受体水平随发育的变化反映在β 2-AR mRNA水平的变化上。由于雄性大鼠肝细胞出生后早期发育过程中β 2-AR基因的转录速率和β 2-AR mRNA的稳定性均下降，因此在发育中的大鼠肝脏中β 2-AR基因表达似乎存在多级调控。这项研究所基于的工作假设是，β 2-AR基因表达在早期肝脏发育过程中在转录和转录后水平上受到动态调节。本研究的目的是确定在早期肝脏发育过程中负责控制β 2-AR基因表达的机制的性质。为了研究在早期发育过程中调节雄性大鼠肝细胞中β 2-AR基因表达的基本转录控制机制，将通过用包含β 2-AR基因调节区不同部分的构建体分离不同年龄的肝细胞来定位关键顺式元件。胎儿和出生后早期肝核提取物中存在的关键顺式元件和相互作用的反式因子的进一步表征将使用标准分子技术进行，包括凝胶迁移率变化和DNA酶足迹分析。为了研究发育过程中β 2-AR基因表达的转录后控制，将检查与β 2-AR mRNA相互作用并改变其稳定性的蛋白质。一种这样的蛋白质，命名为p85，只在出生后的肝细胞质中发现，并可能作为一个不稳定的元素。使用交联技术，将精确鉴定β 2-AR转录物上p85的结合位点。p85调节转录物稳定性的机制将通过突变β 2-AR基因上的碱基来研究，这些碱基在β 2-AR mRNA上产生p85的结合位点。用突变基因转染不同发育年龄的肝细胞后，将测定总mRNA和β 2-AR受体数量。 这项研究的主要目的是确定为什么肝脏β 2-AR基因在出生后早期发育过程中关闭。信息从DNA复制到信使RNA的速率可能在出生后降低，或者mRNA可能在出生后不太稳定。这项研究将有助于更好地了解哺乳动物发育过程中基因表达变化的机制。

9728212 Baeyens Many hormones and drugs initiate their actions on cells by binding to specific cell surface receptors located in the plasma membrane. For the catecholamines, epinephrine and norepinephrine, the receptors are known as adrenergic receptors (ARs) and include two major types termed alpha1- and beta-ARs. Epinephrine and norepinephrine have important effects on liver metabolism including glycogenolysis and gluconeogenesis. Liver cells express both alpha- and beta2-AR subtypes that work by different second messenger systems to transduce catecholamine stimulation into biological effects. Both alpha1- and beta2-ARs regulate liver metabolism by promoting glycogenolysis through increased phosphorylase activity. Catecholamine control of glucose metabolism is age-dependent in male rat liver. In fetal and young male rats (less than 14 days old), epinephrine works through beta2-ARs to activate phosphorylase, while in adult male rats epinephrine acts almost exclusively through alpha1-ARs to increase phosphorylase activity. The changes in receptor levels with development are mirrored by changes in beta2-AR mRNA levels. There appears to be a multilevel regulatory control of beta2-AR gene expression in the developing rat liver since both the transcription rate of the beta2-AR gene and the stability of beta2-AR mRNA decline during early postnatal development in male rat hepatocytes. The working hypothesis upon which this research is based is that beta2-AR gene expression is dynamically regulated during early liver development at both the transcriptional and post-transcriptional levels. The goal of this study is to determine the nature of the mechanisms responsible for controlling beta2-AR gene expression during early hepatic development. To examine the basic transcriptional control mechanisms regulating beta2-AR gene expression in male rat hepatocytes during early development, key cis-element(s) will be located on the beta2-AR gene by transfecting hepatocytes of different ages with constructs cons isting of various parts of the regulatory region of the beta2-AR gene. Further characterization of key cis-elements and interacting trans-factors present in fetal and early postnatal hepatic nuclear extracts will be done using standard molecular techniques including gel mobility shift and DNase footprinting assays. To investigate the post-transcriptional control of beta2-AR gene expression during development, proteins that interact with and alter beta2-AR mRNA stability will be examined. One such protein, designated p85, is found only in postnatal hepatic cytosol and may serve as a destabilizing element. Using cross-linking techniques the binding site for p85 on the beta2-AR transcript will be precisely identified. Mechanisms by which p85 regulates transcript stability will be investigated by mutating bases on the beta2-AR gene which give rise to the binding site of p85 on beta2-AR mRNA. Following transfection of hepatocytes of different developmental ages with the mutated gene, total mRNA and beta2-AR receptor numbers will be determined. The primary purpose of this study is to determine why the liver beta2-AR gene switches off during early postnatal development. The rate at which the information is copied from the DNA into messenger RNA may decrease after birth or the mRNA may be less stable after birth. This study should lead to a better understanding of the mechanisms underlying the changes in gene expression that occur during mammalian development.