Molecular Adaptation of Hypoxia in Oxygen Sensing Cells

氧敏感细胞缺氧的分子适应

• 批准号: 7215689
• 负责人: DAVID E MILLHORN
• 金额: $ 29.11万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-10 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7215689
• 关键词: Acute; Apoptosis; Apoptotic; Biological Models; Carotid Body; Cell Death; Cell Fate Control; Cell Line; Cell Survival; Cells; Cessation of life; Complementary DNA; Condition; Custom; Cytoplasmic Protein; Databases; Development; Disease; Enzymes; Event; Exposure to; Family; Family member; Gene Expression; Gene Expression Regulation; Gene Silencing; Genes; Genetic Determinism; Homeostasis; Hypoxia; Injury; Investigation; Ion Channel; Laboratories; Lead; Libraries; MAPK11 gene; MAPK12 gene; MAPK13 gene; MAPK14 gene; Mammalian Cell; Mediating; Metabolic; Mitochondria; Modeling; Molecular; Nature; Numbers; Oxidative Phosphorylation; Oxygen; PC12 Cells; Pathway interactions; Pattern; Phenotype; Pheochromocytoma; Phosphotransferases; Population; Protein Kinase; Proteins; Purpose; RNA Interference; Regulation; Research; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Role; Sequence Analysis; Signal Transduction; Signal Transduction Pathway; Somatotype; Stimulus; Stress; Structural Protein; System; Techniques; Time; Type I Epithelial Receptor Cell; base; cDNA Library; computerized data processing; conditioning; energy balance; insight; member; novel; prevent; pro-apoptotic protein; programs; receptor; transcription factor

DESCRIPTION (provided by applicant): Mammalian cells require a constant supply of oxygen to maintain energy balance. Hypoxia leads to reduced oxidative phosphorylation and depletion of cellular ATP, which can result in cell death. Sophisticated mechanisms have evolved which protect cells against hypoxia. A primary objective of our research is to identify the mechanisms that regulate hypoxia tolerance and survival in oxygen-sensing cells (such as those found in the carotid body). We have made extensive use of the oxygen-sensing PC12 cell line as a model system for this purpose. Preliminary investigations indicated that pre-exposure to mild hypoxia (i.e., "pre-conditioning") prevents cell death due to a subsequent exposure to severe hypoxia. We found that the Bcl-2 family of cytoplasmic proteins is involved in regulating this hypoxia tolerance in PC12 cells. The Bcl-2 family is composed of two opposing populations of proteins; those that promote apoptotic cell death, and those that oppose apoptosis. Our preliminary studies also indicated a role for the p38 kinase pathway in regulating hypoxia tolerance in the oxygen-sensing PC12 cell line system. Virtually nothing is known about the regulation of the Bcl-2 proteins during hypoxia or the genetic determinants that ultimately lead to hypoxia tolerance or cell death. In order to gain insights into this critical signaling process, we have developed a unique experimental approach that involves the use of our model cell line system and hypoxia-specific cDNA libraries to identify signal transduction pathways and genes that confer the hypoxia tolerant phenotype. These custom cDNA libraries contain more that 300 hypoxia-regulated genes that encode receptors, transporters, ion channels, metabolic and signal transduction enzymes, transcription factors, structural proteins, and proteins involved in regulating cell survival and death. The specific aims of the proposed study are: 1) Delineate the mechanism(s) by which the stress-activated p38 pathway is activated by hypoxia. A primary objective is to determine if the p38 kinase that is activated during hypoxia tolerance corresponds to p38alpha, p38beta, p38gamma, or p38delta. We will evaluate the effect of hypoxia on phosphorylation of the protein kinases immediately upstream of p38 (MKK-3 and MKK-6), and Rac, Ras, or Cdc42 are involved in the activation of p38 during development of tolerance to hypoxia. 2) Determine the role of the p38 kinase pathway in activation of pro-apoptotic and anti-apoptotic Bcl-2 family members. Our preliminary results indicate that both the p38 kinase pathway and the Bcl-2 family of pro-apoptotic and anti-apoptotic proteins are involved in conferring hypoxia tolerance to PC12 cells. We will examine interactions between the p38 kinase pathway and key members (Bcl-2, BcI-XL, 14-3-3, Bax, Bad, and Bnip-3) of the Bcl-2 superfamily during hypoxia. 3) Determine the role of p38 kinase and Bcl-2 family members in the regulation of hypoxia-responsive genes. We will use our hypoxia-specific cDNA libraries and custom microarrays to identify the genes that are regulated by hypoxia pre-conditioning and the role of the p38 kinase pathway and pro- and anti-apoptotic Bcl-2 proteins in the regulation of these genes. RNA interference will be used to evaluate the role of p38 and members of the Bcl-2 family in regulation of these genes. Results from this study will provide new insights into the mechanisms by which O2-sensing cells adapt and survive during hypoxia. This information may be helpful for developing strategies and targets for treating hypoxia-associated diseases and injury.

描述(申请人提供)：哺乳动物细胞需要持续的氧气供应来维持能量平衡。低氧导致氧化磷酸化减少和细胞内ATP耗尽，从而导致细胞死亡。已经进化出保护细胞免受缺氧的复杂机制。我们研究的一个主要目标是确定氧敏感细胞(如在颈动脉小体中发现的细胞)耐缺氧和存活的调节机制。为此，我们广泛使用了氧敏感PC12细胞系作为模型系统。初步研究表明，预先暴露于轻度低氧(即“预适应”)可防止因随后暴露于严重低氧而导致的细胞死亡。我们发现，胞浆蛋白Bcl2家族参与了PC12细胞耐缺氧的调节。Bcl2家族由两种截然相反的蛋白质组成：促进细胞凋亡的蛋白质和反对细胞凋亡的蛋白质。我们的初步研究还表明，p38激酶通路在氧敏感的PC12细胞系系统中调节低氧耐受性中起作用。事实上，对于在缺氧过程中对Bcl2蛋白的调节，或者最终导致耐缺氧或细胞死亡的遗传决定因素，我们一无所知。为了深入了解这一关键的信号转导过程，我们开发了一种独特的实验方法，包括使用我们的模型细胞系系统和低氧特异的cDNA文库来确定信号转导途径和赋予耐低氧表型的基因。这些定制的cDNA文库包含300多个低氧调节基因，编码受体、转运体、离子通道、代谢和信号转导酶、转录因子、结构蛋白和参与调节细胞生存和死亡的蛋白。本研究的具体目的是：1)阐明低氧激活应激激活p38通路的机制(S)。一个主要的目标是确定在低氧耐受期间被激活的p38激酶是否对应于p38α、p38β、p38伽马或p38增量。我们将评估缺氧对p38上游蛋白激酶(MKK-3和MKK-6)磷酸化的影响，以及RAC、RAS或CDC42参与p38在耐缺氧形成过程中的激活。2)确定p38激酶通路在促凋亡和抗凋亡的Bcl2家族成员激活中的作用。我们的初步结果表明，p38激酶通路和促凋亡和抗凋亡蛋白的Bcl2家族都参与了PC12细胞耐缺氧的过程。我们将研究p38激酶通路与Bcl2超家族关键成员(Bcl2、Bci-xl、14-3-3、Bax、Bad和Bnip-3)在低氧条件下的相互作用。3)确定p38激酶和Bcl2家族成员在低氧反应基因调控中的作用。我们将使用我们的低氧特异的cDNA文库和定制的微阵列来确定受低氧预适应调控的基因，以及p38激酶途径和促凋亡和抗凋亡的Bcl-2蛋白在这些基因调控中的作用。RNA干扰将被用来评估p38和Bcl-2家族成员在这些基因调控中的作用。这项研究的结果将为氧气敏感细胞在低氧条件下适应和生存的机制提供新的见解。这些信息可能有助于制定治疗低氧相关疾病和损伤的策略和目标。