Molecular Basis of GM-CSF-induced Neutrophil Chemotaxis

描述(由申请人提供)： 中性粒细胞参与的炎症过程对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，中性粒细胞会释放强大的裂解酶和氧自由基，从而对炎症/缺血再灌注部位和周围健康组织造成附带损害。中性粒细胞通过一种叫做趋化的过程到达靶组织。在缺血/再灌注损伤中导致趋化作用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趋化作用的细胞因子。GM-CSF诱导的趋化作用似乎涉及核糖体p70S6K激酶(P70S6K)的激活。我们实验室的数据也表明，另一种信号分子，丝裂原活化蛋白激酶(MAPK)，在细胞迁移过程中是活跃的，并在空间上与p70S6K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。这项资助的重点是中性粒细胞趋化的分子机制和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是由雷帕霉素敏感的p70S6K通路介导的，该通路被MAPK和PLD上调，作用于微管蛋白和肌动蛋白以增强功能。具体的目标将通过使用细胞、分子和蛋白质组学技术来检验以下三个相互关联的假设：1-。MAPK是S6K诱导GM-CSF刺激的中性粒细胞趋化的上游调节因子。目的是测试MAPK和p70S6K通路之间的相互作用，包括通过磷酸化进行的共价修饰，是否导致趋化。2-。在GM-CSF刺激的中性粒细胞趋化中，PLD和S6K之间存在分子串扰。我们的目标是测试PLD(PLD 1/2)是否以及在什么水平(上游或下游)参与p70S6K激活和随后的功能性趋化机制。3.雷帕霉素抑制GM-CSF诱导的趋化作用存在不同的作用机制。我们的目标是测试雷帕霉素抑制以下哪些基因：信号分子TOR(雷帕霉素的靶标)和p70S6K；新生蛋白翻译；或激活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。这一提议将促进我们对控制细胞运动的分子机制的了解。然后，可以设计抑制剂来防止在缺血/再灌注期间和类似的宿主损伤性炎症病理中发生的白细胞介导的组织损伤。

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.