HYPOXIA INDUCIBLE FACTOR 1 AND OXYGEN HOMEOSTASIS

描述(改编自《调查者摘要》)：人类和所有人 其他哺乳动物需要氧气来进行各种必要的新陈代谢 流程。全身、局部和细胞内的动态平衡反应 是由缺氧引起的，即氧气需求超过 供给。许多对低氧的适应性反应涉及到基因的变化 在转录水平上发生的表达。HIF-1是一种 转录因子被证明是转录所必需的 促红细胞生成素基因增强子介导的低氧激活 细胞。在多种组织培养细胞中检测到HIF-1活性 处于缺氧状态。HIF-1也被牵连到坐标中 大豆糖酵解酶基因转录调控的研究 低氧细胞，表明这一因素在 对细胞缺氧的转录反应。这项提议将 探讨HIF-1活性的分子机制 根据氧分压的变化进行调节，并建立 从HIF-1的正常情况看其在氧稳态中的作用 以及体内表达缺陷的后果。 研究人员通过蛋白质微测序和cdna克隆显示 HIF-1是一种异二聚体碱性螺旋-环-螺旋转录 HIF-1α和HIF-1β亚基均被诱导 在低氧细胞的稳态RNA和蛋白质水平上，以及 HIF-1的RNA、蛋白质和DNA结合活性在 缺氧后的细胞。GAL4/HIF-1α融合基因产物将 在培养的人类细胞中表达以确定其决定因素 HIF-1αRNA和蛋白质的稳定性以及证明其存在 HIF-1α蛋白中的反式激活结构域。这个 研究人员将分离人类HIF-1α基因，并建立 它的结构和亚染色体定位。调查员将 HIF-1α基因转录受低氧诱导 并测定顺式作用DNA序列和反式作用因子 来协调这一反应。缺氧诱导因子-1在全身性脑缺血再灌注损伤中的作用 细胞生理学也将建立起来。第一，表情 将在小鼠中分析HIF-1 RNA和蛋白质的功能 解剖位置、环境氧分压和缺氧持续时间。 第二，小鼠的零等位基因纯合。 HIF-1α基因座将通过同源重组在 胚胎干细胞。这些实验很可能会提供 对氧稳态的分子机制的洞察将 是了解病理生理学的基本基础 涉及缺氧的过程，如肿瘤进展、脑 血管意外和心肌缺血/梗死。

DESCRIPTION (Adapted from Investigator's Abstract): Humans and all other mammals require oxygen for a variety of essential metabolic processes. Systemic, local and intracellular homeostatic responses are elicited by hypoxia, the state in which oxygen demand exceeds supply. Many adaptive responses to hypoxia involved changes in gene expression that occur at the level of transcription. HIF-1 is a transcription factor shown to be necessary for transcriptional activation mediated by the erythropoietin gene enhancer in hypoxic cells. HIF-1 activity is detected in a variety of tissue culture cells subjected to hypoxia. HIF-1 has also been implicated in the coordinate transcriptional regulation of genes encoding glycolytic enzymes in hypoxic cells, suggesting that this factor plays a key role in transcriptional responses to cellular hypoxia. This proposal will explore the molecular mechanisms by which HIF-1 activity is modulated in response to changes in oxygen tension and establish the role of HIF-1 in oxygen homeostasis by analyzing its normal expression and the consequences of deficient expression in vivo. The investigator has shown by protein microsequencing and cDNA cloning that HIF-1 is a heterodimeric basic helic-loop-helix PAS transcription factor, that both HIF-1alpha and HIF-1beta subunits are induced at the level of steady-state RNA and protein in hypoxic cells, and that HIF-1 RNA, protein, and DNA-binding activity rapidly decay in post-hypoxic cells. GAL4/HIF-1alpha fusion gene products will be expressed in cultured human cells to identify determinants of HIF-1alpha RNA and protein stability and to demonstrate the presence of a transactivation domain in the HIF-1alpha protein. The investigator will isolate the human HIF-1alpha gene, and establish its structure and subchromosomal location. The investigator will demonstrate that HIF-1alpha gene transcription is induced by hypoxia and determine the cis-acting DNA sequences and trans-acting factor that mediate this response. The role of HIF-1 in systemic and cellular physiology will also be established. First, expression of HIF-1 RNAs and proteins will be analyzed in mice as a function of anatomic location, ambient oxygen tension, and duration of hypoxia. Second, mice that are homozygous for a null allele at the HIF-1alpha locus will be generated by homologous recombination in embryonic stem cells. These experiments are likely to provide insights into molecular mechanisms of oxygen homeostasis that will serve as a basic foundation for understanding pathophysiologic processes that involve hypoxia, such as tumor progression, cerebral vascular accidents, and myocardial ischemia/infarction.