Signal transduction in neutrophil chemotaxis

说明(申请人提供)：中性粒细胞是循环白细胞中含量最丰富的细胞类型，是宿主抵御入侵细菌和其他病原体的第一道防线。它们通过对趋化物质的梯度做出反应，向感染或炎症部位迁移，这一过程被称为趋化作用。中性粒细胞的趋化作用是由趋化信号转导通路介导的。该项目的长期目标是阐明趋化信号的分子基础。我们对肌醇磷脂Ptdlns(3，4，5)P3介导的信号通路特别感兴趣。这一途径已被证明是介导趋化反应的关键，但其调控机制仍不明确。趋化因子刺激启动了Ptdlns(3，4，5)P3在趋化细胞前沿质膜上的局部聚集。一组含有Pleckstrin Homolog(PH)结构域的蛋白通过与Ptdlns(3，4，5)P3的特异性结合被转移到膜上，随后触发下游信号导致趋化。在此之前，人们认为Ptdlns(3，4，5)P3在Ptdlns(3，4，5)P3中的浓度决定了Ph域膜的转位。最近，我们发现细胞内的一种肌醇磷酸，LNS(1，3，4，5)P4与Ptdlns(3，4，5)P3竞争与PH区结合，并减弱中性粒细胞中PH区的膜转位，为PH区功能的调节提供了一种新的模式。这一有趣的结果使我们假设LNS(1，3，4，5)P4通过抑制PH结构域易位，负向调节中性粒细胞的趋化作用。与此一致，我们的初步数据显示，在趋化过程中，LNS(1，3，4，5)P4水平显著升高。此外，经膜修饰的LNS(1，3，4，5)P4可显著抑制中性粒细胞的趋化运动。为了进一步了解LNS(1，3，4，5)P4在趋化过程中对Ptdlns(3，4，5)P3信号的调节，我们将研究趋化刺激(Aim I)上调LNS(1，3，4，5)P4细胞内水平的分子机制。此外，将使用缺乏LNS(1，3，4，5)P4(AIM II)的中性粒细胞来研究LNS(1，3，4，5)P4在化学诱导剂诱导下的增强的生理后果。最后，将使用小鼠腹膜炎模型和背部气囊模型(Aim III)研究LNS(1，3，4，5)P4对活体动物中性粒细胞趋化的贡献。总之，这些研究将有助于更好地了解LNS(1，3，4，5)P4在中性粒细胞趋化中的作用，最终目标是建立LNS(1，3，4，5)P4及其相关通路作为调节中性粒细胞功能的新的治疗靶点。因此，可以开发更有效和更有效的疗法来治疗各种感染性和炎症性疾病，如哮喘、多发性硬化症和类风湿性关节炎。

DESCRIPTION (provided by applicant): Neutrophils are the most abundant cell type among circulating white blood cells and constitute the first line of host defense against invading bacteria and other pathogens. They migrate toward sites of infection or inflammation by responding to gradients of chemoattractants, a process known as chemotaxis. Neutrophil chemotaxis is mediated by chemotactic signal transduction pathways. The long-term goal of this project is to elucidate the molecular basis of the chemotactic signaling. We are particularly interested in the signal pathway mediated by inositol phospholipid Ptdlns(3,4,5)P3. This pathway has proven to be essential for mediating chemotactic responses, while its regulation remains ill defined. Chemoattractant stimulation initiates localized accumulation of Ptdlns(3,4,5)P3 on the plasma membrane at the leading edge of chemotaxing cells. A set of pleckstrin homolog (PH) domain-containing proteins are then translocated onto the membrane via their specific binding to Ptdlns(3,4,5)P3 and subsequently trigger downstream signals leading to chemotaxis. The PH-domain membrane translocation was previously thought to be dependent solely upon concentrations of Ptdlns(3,4,5)P3 in the membrane. Recently, we discovered that an intracellular inositol phosphate, lns(1,3,4,5)P4, competes with Ptdlns(3,4,5)P3 for binding to the PH domain and attenuates PH-domain membrane translocation in neutrophils, providing a novel mode of regulation for PH domain function. This intriguing result led us to hypothesize that lns(1,3,4,5)P4, by suppressing PH- domain translocation, negatively regulates neutrophil chemotaxis. Consistent with this idea, our preliminary data show that lns(1,3,4,5)P4 level is greatly augmented during chemotaxis. In addition, treatment of neutrophils with membrane-permeant lns(1,3,4,5)P4 significantly inhibits their chemotactic movement. To further understand the regulation of Ptdlns(3,4,5)P3 signal by lns(1,3,4,5)P4 in chemotaxis, we will characterize the molecular mechanisms by which the intracellular level of lns(1,3,4,5)P4 is augmented by Chemoattractant stimulation (Aim I). Moreover, the physiological consequences of the chemoattractant- elicited augmentation of lns(1,3,4,5)P4 will be investigated using neutrophils lacking lns(1,3,4,5)P4 (Aim II). Finally, the contribution of lns(1,3,4,5)P4 to neutrophil chemotaxis in live animals will be investigated using a mouse peritonitis model and a dorsal air pouch model (Aim III). Together, these studies will provide a better understanding of the role of lns(1,3,4,5)P4 in neutrophil chemotaxis, with the ultimate goal of establishing lns(1,3,4,5)P4 and related pathways as novel therapeutic targets for modulating neutrophil functions. Accordingly, more efficient and effective therapies could be developed to treat a variety of infectious and inflammatory diseases, such as asthma, multiple sclerosis, and rheumatoid arthritis.