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

RUI：肝脏 β2 肾上腺素能受体基因表达的发育调控

• 批准号: 9728212
• 负责人: Dennis Baeyens
• 金额: $ 20万
• 依托单位: University of Arkansas Little Rock
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-11-01 至 2002-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9728212&HistoricalAwards=false
• 关键词: RUI; Developmental; Regulation; Hepatic; Beta2

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.

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对细胞的作用。对于儿茶酚胺，肾上腺素和去甲肾上腺素的受体被称为肾上腺素能受体(AR)，包括两种主要类型，称为α-AR和β-AR。肾上腺素和去甲肾上腺素对肝脏代谢有重要影响，包括糖原分解和糖异生。肝细胞同时表达α-和β2-AR亚型，它们通过不同的第二信使系统将儿茶酚胺刺激转化为生物效应。α-1和β2-Ars均通过提高磷酸酶活性促进糖原分解，从而调节肝脏代谢。儿茶酚胺对雄性大鼠肝脏糖代谢的调控是年龄依赖性的。在胎鼠和年轻雄性大鼠(出生不到14天)中，肾上腺素通过β2-Ars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α1-Ars作用于提高磷酸化酶活性。随着发育，受体水平的变化反映在β2-AR mRNA水平的变化上。在发育中的大鼠肝脏中，β2-AR基因的转录速率和β2-AR基因的稳定性在出生后早期大鼠肝细胞中的转录速率和稳定性都下降，因此，在发育中的大鼠肝脏中似乎存在多水平的调控。这项研究的工作假设是，在肝脏早期发育过程中，Beta2-AR基因的表达在转录和转录后水平上都受到动态调节。本研究的目的是确定在肝脏发育早期控制β2-AR基因表达的机制的性质。为了研究雄性大鼠肝细胞在发育早期调控β2-AR基因表达的基本转录调控机制，将关键顺式元件(S)用含有β2-AR基因调控区不同部分的结构物导入不同年龄的大鼠肝细胞，定位于β2-AR基因。对胎儿和出生后早期肝细胞核提取液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，包括凝胶迁移率变化和DNA酶足迹分析。为了研究在发育过程中Beta2-AR基因表达的转录后控制，将检测与Beta2-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，只在出生后的肝脏胞浆中发现，可能是一个不稳定的因素。通过使用交联技术，将准确地确定P85在Beta2-AR转录本上的结合位点。P85调节转录物稳定性的机制将通过突变β2-AR基因的碱基来研究，该突变导致P85与β2-AR基因的结合位点。将突变基因导入不同发育年龄段的肝细胞，测定总mRNA和β2-AR受体的数量。这项研究的主要目的是确定为什么肝脏β2-AR基因在出生后早期发育期间关闭。信息从DNA复制到信使RNA的速度在出生后可能会降低，或者在出生后信使RNA可能不太稳定。这项研究应该有助于更好地理解哺乳动物发育过程中基因表达变化的潜在机制。