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 GTPase Rac 在调节多种吞噬细胞功能中发挥着关键作用，包括 NADPH 氧化酶催化的超氧化物生成、Fcgamma 受体介导的吞噬作用以及膜扰乱和细胞运动过程中的细胞骨架变化。为了检查造血特异性 Rac2 GTPase（与更广泛表达的 Rac1 和 Rac3 亚型大约 90% 相同）的作用，我们生成了 Rac2 基因有针对性破坏的小鼠。 Rac2 缺陷的嗜中性粒细胞（仍表达 Rac1）中的 NADPH 氧化酶活性在对 fMLP、IgG 调理颗粒和佛波酯的反应中显着降低，但在对补体包被的酵母聚糖的反应中正常。初步研究中观察到的其他中性粒细胞缺陷包括肌动蛋白聚合受损、趋化性和 L-选择素依赖性粘附不良。此外，小鼠体内渗出液的形成减少，对侵袭性曲霉病的易感性增加。通过在一名婴儿中鉴定出 Rac2 的显性失活突变体，进一步确定了 Rac 在中性粒细胞功能中的重要作用，该婴儿出现反复性化脓性感染和与 Rc2-/- 中发现的类似的功能性中性粒细胞缺陷。这些数据表明，Rac2 调节特定受体激活途径下游吞噬细胞的多种细胞反应，并且在宿主感染和炎症中与 Rac1 和 Rac3 具有不重叠的功能。项目 3 将重点测试这一假设，特别是它与 NADPH 氧化酶的激活有关，并探索潜在的生化机制。有四个特定目标，将利用 Rac2-/- 鼠标。这些建议（1）进一步定义 Rac2-/- 吞噬细胞的缺陷，以检查巨噬细胞超氧化物的产生、吞噬作用和趋化剂诱导的运动； (2)检查Rac2-/-吞噬细胞是否存在功能缺陷，以及趋化剂诱导的运动； (2) 检查 Rac2-/- 吞噬细胞的功能缺陷是否反映了 Rac2 的选择性激活、定位或使用，以及是否需要特定的 Rac2 效应序列； (3) 研究参与 NADPH 氧化酶复合物 Rac 依赖性组装的上游和下游信号转导事件，以及 (4) 确定 Rac2 缺陷对宿主防御和炎症的影响。这项工作将进一步深入了解 Rho-GTP 酶如何调节吞噬白细胞功能以及介导特定激动剂诱导的细胞反应的生化机制。这些研究还可能产生调节炎症和宿主防御中吞噬细胞功能的新方法。