Molecular Adaptation of Hypoxia in Oxygen Sensing Cells

描述（由申请人提供）：哺乳动物细胞需要恒定的氧气供应来维持能量平衡。缺氧导致氧化磷酸化减少和细胞ATP耗竭，这可导致细胞死亡。已经进化出复杂的机制来保护细胞免受缺氧。我们研究的主要目的是确定调节氧敏感细胞（如颈动脉体中发现的细胞）缺氧耐受性和存活的机制。我们已经广泛使用的氧敏感PC 12细胞系作为模型系统，用于此目的。初步调查表明，预先暴露于轻度缺氧（即，“预调节”）防止由于随后暴露于严重缺氧而导致的细胞死亡。我们发现Bcl-2家族的细胞质蛋白参与调节PC 12细胞的耐缺氧性。Bcl-2家族由两种相反的蛋白质群体组成;促进凋亡性细胞死亡的蛋白质和对抗凋亡的蛋白质。我们的初步研究还表明，p38激酶通路在调节氧敏感PC 12细胞系系统的缺氧耐受性的作用。几乎没有人知道Bcl-2蛋白在缺氧过程中的调节或最终导致缺氧耐受或细胞死亡的遗传决定因素。为了深入了解这一关键的信号传导过程，我们开发了一种独特的实验方法，该方法涉及使用我们的模型细胞系系统和缺氧特异性cDNA文库来鉴定赋予耐缺氧表型的信号转导途径和基因。这些定制的cDNA文库包含超过300个低氧调节基因，其编码受体、转运蛋白、离子通道、代谢和信号转导酶、转录因子、结构蛋白以及参与调节细胞存活和死亡的蛋白质。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。主要目的是确定在缺氧耐受期间激活的p38激酶是否对应于p38 α、p38 β、p38 γ或p38 δ。我们将评估缺氧对紧邻p38上游的蛋白激酶（MKK-3和MKK-6）磷酸化的影响，并且Rac、Ras或Cdc 42在缺氧耐受性的发展过程中参与p38的活化。2)确定p38激酶通路在促凋亡和抗凋亡Bcl-2家族成员激活中的作用。我们的初步研究结果表明，p38激酶途径和Bcl-2家族的促凋亡和抗凋亡蛋白参与赋予PC 12细胞耐缺氧。我们将研究p38激酶通路和Bcl-2超家族的关键成员（Bcl-2，BcI-XL，14-3-3，Bax，Bad和Bnip-3）在缺氧期间的相互作用。3)确定p38激酶和Bcl-2家族成员在缺氧反应基因调节中的作用。我们将使用我们的低氧特异性cDNA文库和定制的微阵列来鉴定受低氧预处理调节的基因，以及p38激酶途径和促凋亡和抗凋亡Bcl-2蛋白在这些基因调节中的作用。RNA干扰将用于评估p38和Bcl-2家族成员在调节这些基因中的作用。这项研究的结果将为氧敏感细胞在缺氧期间适应和生存的机制提供新的见解。这些信息可能有助于开发治疗缺氧相关疾病和损伤的策略和靶点。

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.