Regulation of phagocyte function by Rac2

Rac2 对吞噬细胞功能的调节

• 批准号: 6595706
• 负责人: Mary C Dinauer
• 金额: $ 21.74万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-22 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6595706
• 关键词: NAD(P)H dehydrogenase; chemoattractants; enzyme activity; gene targeting; genetically modified animals; guanine nucleotide binding protein; guanosinetriphosphatases; hematopoiesis; isozymes; laboratory mouse; phagocytes; phagocytosis; protein localization; protein structure function; superoxides

The Rho family GTPase Rac plays a key role in regulating a variety of phagocyte functions, including NADPH oxidase-catalyzed superoxide generation, Fcgamma receptor-mediated phagocytosis, and cytoskeletal changes during membrane ruffling and cell movement. To examine the role of the hematopoietic specific Rac2 GTPase, which is approximately 90% identical to the more widely expressed Rac1 and Rac3 isoforms, mice with a targeted disruption of the Rac2 gene were generated. NADPH oxidase activation in Rac2-deficient neutrophils )which still express Rac1) was substantially reduced in response to fMLP, IgG- opsonized particles, and phorbol ester, but normal in response to complement-coated zymosan. Other neutrophil defects observed in initial studies include impaired actin polymerization, chemotaxis, and poor L-selectin-dependent adhesion. In addition, mice exhibited decreased exudate formulation in vivo and an increased susceptibility to invasive Aspergillosis. An important role for Rac in neutrophil function has been further established by the identification of a dominant-negative mutant of Rac2 in an infant who presented with recurrent pyogenic infections and functional neutrophil defects similar to those found in Rc2-/-. These data suggest that Rac2 regulates multiple cellular responses in phagocytes downstream of specific receptor-activated pathways, and has non-overlapping functions with Rac1 and Rac3 in the host infection and inflammation. Project 3 will focus on testing this hypothesis, particular as it relates to activation of the NADPH oxidase, and explore underlying biochemical mechanisms. There are four Specific Aims, which will take advantage of the Rac2-/- mouse. These propose to (1) further define defects in Rac2-/- phagocytes to examine macrophage superoxide production, phagocytosis, and chemoattractant-induced movement; (2) examine whether functional defects in Rac2-/- phagocytes, and chemoattractant-induced movement; (2) examine whether functional defects in Rac2-/- phagocytes reflect selective activation, localization, or usage of Rac2, and if specific Rac2 effector sequences are required; (3) investigate upstream and downstream signaling events involved in Rac-dependent assembly of the NADPH oxidase complex and (4) determine the impact of Rac2 deficiency on host defense and inflammation. This work will provide further insight into how Rho-GTPases regulate phagocytic leukocyte functions and the biochemical mechanisms that mediate specific agonist-induced cellular responses. These studies may also result in new approaches to modulating phagocyte function in inflammation and host defense.

Rho家族GTdR ac在调节多种吞噬细胞功能中起关键作用，包括NADPH氧化酶催化的超氧化物生成、Fc γ受体介导的吞噬作用以及膜皱褶和细胞运动期间的细胞骨架变化。为了检查造血特异性Rac 2 GTdR的作用，其与更广泛表达的Rac 1和Rac 3同种型约90%相同，产生了具有Rac 2基因靶向破坏的小鼠。在仍然表达Rac 1的Rac 2缺陷型嗜中性粒细胞中，NADPH氧化酶的活化在对fMLP、IgG调理颗粒和佛波酯的应答中显著降低，但在对补体包被的酵母聚糖的应答中正常。在最初的研究中观察到的其他中性粒细胞缺陷包括受损的肌动蛋白聚合、趋化性和较差的L-选择素依赖性粘附。此外，小鼠体内渗出液减少，对侵袭性铜绿假单胞菌病的易感性增加。Rac在中性粒细胞功能中的重要作用已通过鉴定Rac 2的显性阴性突变体而进一步确定，该突变体表现为复发性化脓性感染和与Rc 2-/-中发现的类似的功能性中性粒细胞缺陷。这些数据表明，Rac 2调节吞噬细胞下游的特异性受体激活途径的多种细胞反应，并在宿主感染和炎症中与Rac 1和Rac 3具有非重叠的功能。项目3将重点测试这一假设，特别是因为它涉及到NADPH氧化酶的激活，并探索潜在的生化机制。有四个特定目标，这将利用Rac 2-/-鼠标。这些建议（1）进一步定义Rac 2-/-吞噬细胞中的缺陷，以检查巨噬细胞超氧化物的产生、吞噬作用和化学引诱剂诱导的运动;（2）检查Rac 2-/-吞噬细胞中是否存在功能缺陷，以及化学引诱剂诱导的运动;（2）检查Rac 2-/-吞噬细胞中的功能缺陷是否反映Rac 2的选择性激活、定位或使用，以及是否需要特异性Rac 2效应子序列;（3）研究参与NADPH氧化酶复合物的Rac依赖性组装的上游和下游信号传导事件和（4）确定Rac 2缺陷对宿主防御和炎症的影响。这项工作将提供进一步了解Rho-GTPases如何调节吞噬白细胞功能和介导特定激动剂诱导的细胞反应的生化机制。这些研究也可能导致在炎症和宿主防御中调节吞噬细胞功能的新方法。