TRPM2, Mitochondria, and Cell Survival

TRPM2、线粒体和细胞存活

• 批准号: 9265474
• 负责人: BARBARA A. MILLER
• 金额: $ 30万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9265474
• 关键词: Adenosine Diphosphate Ribose; Bioenergetics; Brain; CREB1 gene; CRISPR/Cas technology; Calcium; Cations; Cell Death; Cell Proliferation; Cell Survival; Cell physiology; Cells; Clinical; Data; Disease; Dominant-Negative Mutation; Doxorubicin; Endotoxins; Enzymes; Family member; Generations; Grant; Growth; Heart; Hematopoietic; Hydrogen Peroxide; Hypoxia; In Vitro; Injury; Ischemia; Length; Lung; MAPK3 gene; Malignant Neoplasms; Mediating; Membrane Potentials; Mitochondria; Mitochondrial Proteins; Neuroblastoma; Oxidants; Oxidative Stress; PTK2B gene; Permeability; Phagocytes; Phosphorylation; Phosphotransferases; Physiological; Physiological Processes; Play; Predisposition; Process; Production; Proteins; RNA Splicing; Reactive Oxygen Species; Regulation; Reperfusion Therapy; Research; Role; SOD2 gene; Subgroup; Technology; Therapeutic; Tissues; Variant; Work; Xenograft procedure; cancer cell; gain of function; hypoxia inducible factor 1; in vivo; loss of function; member; mitochondrial membrane; mutant; neuroblastoma cell; novel strategies; oxidative damage; public health relevance; receptor; response; response to injury; small hairpin RNA; transcription factor; tumor; tumor growth; uptake

 DESCRIPTION (provided by applicant): Transient Receptor Potential (TRP) channels are involved in many fundamental cell functions. Transient Receptor Potential Melastatin 2 (TRPM2), the second member of the TRPM subfamily to be cloned, is a widely expressed calcium-permeable channel activated by ADP-ribose (ADPR) and oxidative stress. A short physiological splice variant (TRPM2-S) inhibits Ca2+ influx through full length TRPM2 (L), functioning as a dominant negative. Preliminary data show that neuroblastoma xenografts expressing TRPM2-L have enhanced tumor growth and reduced sensitivity to doxorubicin compared to those expressing TRPM2-S, through modulation of hypoxia-inducible transcription factors (HIF-1/2α), mitochondrial function, and ROS. These fundamental observations form the basis of our Overall Hypothesis that TRPM2-L sustains cell proliferation and protects viability through moderate Ca2+ influx, which mediates expression of HIF-1/2α, maintains mitochondrial function, reduces ROS production and activates Pyk2. Understanding the function of TRPM2 channels in proliferation and survival and determination of the underlying mechanisms has high significance and clinical impact for many pathophysiological processes. Our aims are: Specific Aim 1: Is cell proliferation or viability reduced by inhibition of TRPM2 mediated Ca2+ influx? We will determine the role of TRPM2-mediated Ca2+ influx in modulation of cell viability, in vivo tumor formation, doxorubicin sensitivity, and ROS generation using TRPM2 loss and gain of function mutants. Specific Aim 2: Does TRPM2-mediated Ca2+ influx modulate mitochondrial function in neuroblastoma? Mitochondrial function including mitochondrial membrane potential, Ca2+ uptake, ATP production, and expression of BNIP3 and NDUFA4L2 are significantly reduced in cells expressing TRPM2-S, and ROS production is increased. This strongly supports the hypothesis that Ca2+ entry through TRPM2-L is important for normal mitochondrial function. (1) We will determine whether loss of Ca2+ influx in TRPM2 loss of function mutants inhibits mitochondrial function. (2) We will examine whether this is mediated through modulation of HIF-1α/2α and target mitochondrial proteins. (3) We will determine if TRPM2-L modulates mitophagy through BNIP3 expression. (4) We will examine the mechanism of regulation of ROS production by TRPM2. Specific Aim 3: Does TRPM2 modulate cell proliferation or viability through Ca2+-dependent Pyk2 activation? Our preliminary data show that proline-rich tyrosine kinase 2 (Pyk2), a Ca2+-sensitive, non-receptor tyrosine kinase, and its downstream targets ERK1/2 and CREB have reduced activation in TRPM2-S expressing cells. We will silence Pyk2, and use loss or gain of Pyk2 function mutants to assess the role of Pyk2 in TRPM2 modulation of proliferation, viability, CREB activation, and mitochondrial Ca2+ uptake. Understanding TRPM2 function has high significance and clinical impact because TRPM2 has an important role in cell survival after oxidative stress. Inhibition is a novel strategy to reduce tumor growth and enhance response to cancer therapeutics.

 描述(申请人提供)：瞬时受体电位(Trp)通道参与许多基本的细胞功能。瞬时受体潜能Melastatin 2(TRPM2)是被克隆的TRPM亚家族中的第二个成员，是一种被ADP核糖(ADPR)和氧化应激激活的广泛表达的钙离子通透通道。一个短的生理剪接变异体(TRPM2-S)通过全长TRPM2(L)抑制钙离子内流，起显性负值作用。初步数据显示，表达TRPM2-L的神经母细胞瘤移植瘤与表达TRPM2-S的神经母细胞瘤移植瘤相比，通过缺氧诱导转录因子(HIF-1/2α)、线粒体功能和ROS的调节，促进了肿瘤的生长，降低了对阿霉素的敏感性。这些基本观察结果构成了我们总体假设的基础，即TRPM2-L通过适度的钙内流维持细胞增殖和保护活性，介导HIF-1/2α的表达，维持线粒体功能，减少ROS产生并激活PYK2。了解TRPM2通道在细胞增殖和存活中的作用并确定其潜在的机制，对于许多病理生理过程具有重要的意义和临床意义。我们的目标是：特定的目标1：抑制TRPM2介导的钙内流是否会降低细胞的增殖或活力？我们将通过TRPM2功能突变的缺失和获得来确定TRPM2介导的钙内流在调节细胞活力、体内肿瘤形成、对阿霉素的敏感性和ROS产生中的作用。特定目标2：TRPM2介导的钙内流是否调节神经母细胞瘤的线粒体功能？表达TRPM2-S的细胞线粒体膜电位、钙摄取、三磷酸腺苷生成、BNIP3和NDUFA4L2表达显著降低，ROS生成增加。这有力地支持了钙离子通过TRPM2-L进入对正常线粒体功能很重要的假说。(1)我们将确定在TRPM2功能缺失突变体中失去钙内流是否会抑制线粒体的功能。(2)我们将研究这是否通过调节HIF-1α/2α和靶线粒体蛋白来实现。(3)我们将确定TRPM2-L是否通过表达BNIP3来调节有丝分裂吞噬。(4)研究TRPM2对ROS产生的调控机制。具体目标3：TRPM2是否通过钙依赖的Pyk2激活来调节细胞的增殖或活性？我们的初步数据表明，富含脯氨酸的酪氨酸激酶2(PYK2)是一种钙敏感的非受体酪氨酸激酶，其下游靶标ERK1/2和CREB降低了TRPM2-S表达细胞的活性。我们将沉默PYK2，并使用PYK2功能突变的缺失或获得来评估PYK2在TRPM2调控增殖、活性、CREB激活和线粒体钙摄取中的作用。了解TRPM2的功能具有重要的意义和临床意义，因为TRPM2在氧化应激后的细胞生存中起着重要作用。抑制肿瘤生长是一种减少肿瘤生长、增强对癌症治疗反应的新策略。