Transglutaminase 2 decreases ischemic insult by attenuating hypoxic signaling.

转谷氨酰胺酶 2 通过减弱缺氧信号来减少缺血性损伤。

• 批准号: 7609278
• 负责人: Anthony J Filiano
• 金额: $ 1.78万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-30 至 2009-05-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7609278
• 关键词: ARNT protein; Adenoviruses; Affect; Alteplase; Animals; Apoptosis; Apoptotic; Architecture; Asses; Attenuated; BCL2/Adenovirus E1B 19kd Interacting Protein 3-Like; Binding; Biological Assay; Biological Models; Blood - brain barrier anatomy; C57BL/6 Mouse; Calcium; Cause of Death; Cell Death; Cell Line; Cell Nucleus; Cells; Cessation of life; Condition; Cultured Cells; Data; Development; Effectiveness; Environment; Enzymes; Event; Genes; Genetic; Genetic Transcription; Glucose; Goals; Green Fluorescent Proteins; Guanosine Triphosphate Phosphohydrolases; HIF1alpha protein; Hour; Human; Hypoxia; Hypoxia Inducible Factor; Immunoblot Analysis; Immunohistochemistry; In Situ; Infarction; Infection Control; Injury; Ischemia; Knockout Mice; LacZ Genes; Lactate Dehydrogenase; Lactate Dehydrogenases; Lead; Life; Ligation; Luciferases; Magnetic Resonance Imaging; Measures; Mediating; Messenger RNA; Modeling; Mus; Nerve Degeneration; Neuraxis; Neurites; Neuroblastoma; Neurons; Oxygen; Pathway interactions; Pattern; Permeability; Play; Point Mutation; Polymerase Chain Reaction; Predisposition; Property; Protein Overexpression; Proteins; Quality of life; Rattus; Reporter; Response Elements; Role; Scaffolding Protein; Signal Transduction; Stroke; Stroke Volume; Techniques; Testing; Therapeutic; Thrombolytic Therapy; Time; Transgenic Organisms; United States Food and Drug Administration; Up-Regulation; Vascular Endothelial Growth Factors; Weight; attenuation; base; caspase-3; deprivation; disability; hypoxia inducible factor 1; improved; middle cerebral artery; mouse model; mutant; neuron loss; novel; post stroke; protein expression; response; scaffold; size; small molecule; success; transcription factor; transglutaminase 2; vector

DESCRIPTION (provided by applicant): Stroke is the third leading cause of death and a major cause of long term disability in the US. Inadequate success of current thrombolytic therapy demonstrates a critical need for new targets to improve quality of life post-stroke. Neurons adapt to decreased oxygen delivery subsequent to stroke by up-regulating genes controlled by the hypoxia inducible factor (HIF). This transcription factor is comprised of two subunits: HIF1 alpha and HIF1 beta. Recent studies show that increased HIF1 during ischemia results in upregulation of apoptotic genes. Our preliminary data indicates that Transglutaminase 2 (TG2) binds to HIF1 beta resulting in attenuation of HIF activation and decreased cell death during ischemia. Also, TG2 inhibits the upregulation of the HIF controlled proapoptotic gene Bnip3, but not VEGF. Our hypothesis is that TG2 will facilitate neuronal protection following oxygen and glucose deprivation (OGD) by attenuating apoptotic genes induced by HIF1. We propose to delineate the effects of TG2 on HIF signaling and ischemic cell death with 3 aims. 1) To test the hypothesis that TG2 is upregulated during OGD and decreases HIF-dependent pro-apoptotic signaling. We will use a neuroblastoma cell line (SH-SY5Y) that overexpresses wild type TG2 and two TG2 constructs with point mutations that differentially affect 2 unique functions (transamidating and GTPase) of the enzyme. Using a HIF signaling reporter assay and analysis of mRNA and protein expression patterns of HIF dependent genes, we will distinguish which properties of TG2 are important for regulating HIF signaling. 2) To test the hypothesis that TG2 protects neuronal cells from ischemic insult and which functions of TG2 are important. Using SH-SY5Y cells and primary cortical neurons we will measure OGD-induced cell death and upstream apoptotic events. 3) To test the hypothesis that TG2 leads to overall protection in a mouse model of stroke. We will use a permanent Middle Cerebral Artery ligation model in C57BL/6 mice that express endogenous TG2, overexpress TG2, or lack TG2. We will measure infarct volumes using T2 weighted MRI as well as identifying genes regulated by TG2 with appropriate techniques. Our preliminary data show that overexpression of TG2 decreases stroke infarct volumes by 33%. We hypothesize that TG2 will lead to overall protection against stroke.

描述（由申请人提供）：中风是美国第三大死亡原因和长期残疾的主要原因。目前溶栓治疗的不成功表明迫切需要新的目标来改善卒中后的生活质量。神经元通过上调由缺氧诱导因子（HIF）控制的基因来适应脑卒中后氧气输送的减少。该转录因子由两个亚基组成：HIF1 α和HIF1 β。最近的研究表明，缺血时HIF1升高导致凋亡基因上调。我们的初步数据表明，转谷氨酰胺酶2 （TG2）与HIF1 β结合，导致缺血时HIF激活的衰减和细胞死亡的减少。此外，TG2抑制HIF控制的促凋亡基因Bnip3的上调，但不抑制VEGF。我们的假设是TG2通过减弱HIF1诱导的凋亡基因来促进氧和葡萄糖剥夺（OGD）后的神经元保护。我们提出描述TG2对HIF信号传导和缺血性细胞死亡的影响有三个目的。1)验证TG2在OGD过程中上调并降低hif依赖性促凋亡信号通路的假设。我们将使用一种神经母细胞瘤细胞系（SH-SY5Y），该细胞系过表达野生型TG2和两种TG2构建体，其点突变不同地影响该酶的两种独特功能（转氨酶和GTPase）。通过HIF信号报告者实验和HIF依赖基因的mRNA和蛋白表达模式分析，我们将区分TG2的哪些特性对HIF信号的调节是重要的。2)验证TG2保护神经元细胞免受缺血性损伤的假说，以及TG2的哪些重要功能。使用SH-SY5Y细胞和原代皮质神经元，我们将测量ogd诱导的细胞死亡和上游凋亡事件。3)在脑卒中小鼠模型中验证TG2具有全面保护作用的假设。我们将在表达内源性TG2、过表达TG2或缺乏TG2的C57BL/6小鼠中使用永久性大脑中动脉结扎模型。我们将使用T2加权MRI测量梗死体积，并使用适当的技术识别由TG2调节的基因。我们的初步数据显示，过表达TG2可使脑卒中梗死体积减少33%。我们假设TG2将导致对中风的全面保护。