Molecular Basis of GM-CSF-induced Neutrophil Chemotaxis

GM-CSF 诱导中性粒细胞趋化的分子基础

• 批准号: 7104775
• 负责人: JULIAN G. CAMBRONERO
• 金额: $ 3.7万
• 依托单位: WRIGHT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2008-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7104775
• 关键词: actins; biological signal transduction; chemotaxis; clinical research; colony stimulating factor; human subject; mass spectrometry; matrix assisted laser desorption ionization; mitogen activated protein kinase; neutrophil; phospholipase D; phosphorylation; protein isoforms; protein protein interaction; proteomics; sirolimus; site directed mutagenesis; tubulin

DESCRIPTION (provided by applicant): The inflammatory process involving neutrophils is critical for host defense and normal healing. However, neutrophils also play a significant role in such pathologic disorders as ischemia-reperfusion injury in the heart, emphysema, asthma, and rheumatoid arthritis. When blood flow is restored after a heart attack, neutrophils can inflict collateral damage on the inflammatory/ischemic reperfusion site and surrounding healthy tissue, because they release powerful lytic enzymes and oxygen radicals. Neutrophils arrive at the target tissue by a process called chemotaxis. The molecular mechanisms leading to chemotaxis in ischemia/reperfusion injury are not well understood. Preliminary data in our laboratory have indicated for the first time that granulocytemacrophage colony-stimulating factor (GM-CSF) is a cytokine that acts as a chemoattractant for neutrophils. GM-CSF-induced chemotaxis seems to involve the activation of the enzyme ribosomal p70S6K kinase (p70S6K). Data from our lab have also indicated that another signaling molecule, mitogen-activated protein kinase (MAPK), is active during cell migration and is spatially associated to p70S6K. Finally, we have found that there is a connection between phospholipase D (PLD), chemotaxis and p70S6K activity. This grant focuses on the molecular mechanisms underlying neutrophil chemotaxis and the crosstalk between the aforementioned molecules. Our central hypothesis is that GM-CSF-induced neutrophil chemotaxis is mediated by the rapamycin-sensitive p70S6K pathway that, upregulated by MAPK and PLD, acts upon tubulin and actin for enhanced functionality. The Specific Aims will test the following three interrelated hypotheses, by using cellular, molecular and proteomic techniques: 1-. MAPK is an upstream regulator of S6K-induced chemotaxis in GM-CSF-stimulated neutrophils. The goal is to test if an interaction between the MAPK and p70S6K pathways, involving covalent modification by phosphorylation, leads to chemotaxis. 2-. There is a molecular crosstalk between PLD and S6K in GM-CSF-stimulated neutrophil chemotaxis. The goal is to test if PLD (PLD 1/2), and at what level (upstream or downstream), is involved in the mechanism of p70S6K activation and subsequent functional chemotaxis. 3. Alternate mechanisms of rapamycin action exist for the inhibition of GM-CSF-induced chemotaxis. The goal is to test which of the following is inhibited by rapamycin: the signaling molecules TOR (target of rapamycin) and p70S6K; nascent protein translation; or the actuator molecules actin and tubulin. Given the devastating effects inflicted by neutrophils during reperfusion, it is reasonable to act on the first event: chemotaxis. This proposal will advance our knowledge of the molecular mechanisms that govern cell movement. Inhibitors can then be designed to prevent leukocyte-mediated tissue injury that occurs during ischemia/reperfusion and in similar host-damaging inflammation pathologies.

描述（由申请人提供）： 涉及中性粒细胞的炎症过程对于宿主防御和正常愈合至关重要。然而，中性粒细胞在心脏缺血再灌注损伤、肺气肿、哮喘和类风湿性关节炎等病理性疾病中也发挥着重要作用。当心脏病发作后血流恢复时，中性粒细胞会对炎症/缺血再灌注部位和周围健康组织造成附带损害，因为它们会释放强大的裂解酶和氧自由基。中性粒细胞通过称为趋化性的过程到达目标组织。导致缺血/再灌注损伤中趋化性的分子机制尚不清楚。我们实验室的初步数据首次表明粒细胞巨噬细胞集落刺激因子（GM-CSF）是一种对中性粒细胞起化学引诱作用的细胞因子。 GM-CSF 诱导的趋化性似乎涉及核糖体 p70S6K 激酶 (p70S6K) 的激活。我们实验室的数据还表明，另一种信号分子丝裂原激活蛋白激酶 (MAPK) 在细胞迁移过程中处于活跃状态，并且与 p70S6K 在空间上相关。最后，我们发现磷脂酶 D (PLD)、趋化性和 p70S6K 活性之间存在联系。该资助重点研究中性粒细胞趋化性的分子机制以及上述分子之间的串扰。我们的中心假设是，GM-CSF 诱导的中性粒细胞趋化性是由雷帕霉素敏感的 p70S6K 途径介导的，该途径受 MAPK 和 PLD 上调，作用于微管蛋白和肌动蛋白以增强功能。具体目标将通过使用细胞、分子和蛋白质组技术来测试以下三个相互关联的假设：1-。 MAPK 是 GM-CSF 刺激的中性粒细胞中 S6K 诱导的趋化性的上游调节因子。目的是测试 MAPK 和 p70S6K 通路之间的相互作用（涉及磷酸化的共价修饰）是否会导致趋化性。 2-.在 GM-CSF 刺激的中性粒细胞趋化作用中，PLD 和 S6K 之间存在分子串扰。目标是测试 PLD (PLD 1/2) 是否以及在什么水平（上游或下游）参与 p70S6K 激活和随后的功能趋化性机制。 3. 雷帕霉素存在抑制 GM-CSF 诱导的趋化作用的替代机制。目的是测试雷帕霉素抑制以下哪些物质：信号分子 TOR（雷帕霉素靶标）和 p70S6K；新生蛋白质翻译；或致动分子肌动蛋白和微管蛋白。考虑到中性粒细胞在再灌注过程中造成的破坏性影响，对第一个事件采取行动是合理的：趋化性。该提议将增进我们对控制细胞运动的分子机制的了解。然后可以设计抑制剂来预防缺血/再灌注期间以及类似的宿主破坏性炎症病理过程中发生的白细胞介导的组织损伤。