NoxR1, a regulator of Nox4-dependent cytoskeletal remodeling in vascular cells

近年来，活性氧簇(ROS)在正常血管中起着关键作用。 血管疾病的功能和发病机制。这些分子对血管有深远的影响 平滑肌细胞(VSMC)的生长、迁移和分化。血管细胞中ROS的一个主要来源是 NADPH氧化酶(NOX)家族。大动脉血管平滑肌细胞(VSMCs) 表达两种类型的NADPH氧化酶，NOX1和NOX4。这两种氧化酶在体内定位不同 细胞，具有明显的激动剂特异性，并调节特定的细胞功能。然而，不太清楚的是， 这两种酶的活性是如何被具体调控的。而Nox1的监管机制是 与吞噬细胞氧化酶类似，NOX4不需要任何先前发现的胞浆 酶活性的氧化酶调节亚基。我们已经鉴定并克隆了一种新的蛋白质NoxR1， 在物理和功能上与NOX4相互作用。初步实验表明，NoxR1 VSMCs的过度表达导致NADPH氧化酶活性显著增加，局灶性增加 粘连，增加应力纤维的形成，而敲除NoxR1导致深刻的改变 细胞骨架改变并损害VSMC的迁移。因此，这个项目的总体目标是定义生理学 研究NoxR1对NOX4的调节作用，并确定其在体内迁移模型中的作用。在第一个 具体地说，我们计划确定NoxR1在调节局部黏附转换和细胞 迁移，而Aim 2的设计是为了确定NoxR1在体内新生内膜形成中的作用 创建了NoxR1基因敲除小鼠。因为NOXR1是已知的第一个NOX4的调节器，NOX4是NOX家族成员 它调节衰老、分化和存活等基本细胞过程，这些研究 可能对许多血管和非血管疾病产生深远影响。

In recent years, reactive oxygen species (ROS) have been shown to have critical roles in normal vascular function and the pathogenesis of vascular disease. These molecules have profound effects on vascular smooth muscle cell (VSMC) growth, migration and differentiation. A major source of ROS in vascular cells is the NADPH oxidase (Nox) family of enzymes. Vascular smooth muscle cells (VSMCs) from large arteries express two types of NADPH oxidases, Nox1 and Nox4. These two oxidases are differentially localized within the cell, have distinct agonist specificity, and regulate specific cellular functions. What is less clear, however, is how the activity of these two enzymes is specifically regulated. While Nox1 regulatory mechanisms are similar to those of the phagocyte oxidase, Nox4 does not require any of the previously identified cytosolic oxidase regulatory subunits for its enzymatic activity. We have identified and cloned a new protein, NoxR1, that physically and functionally interacts with Nox4. Preliminary experiments indicate that NoxR1 overexpression in VSMCs causes a significant increase in NADPH oxidase activity, an increase in focal adhesions, and an increase stress fiber formation, while knockdown of NoxR1 induces a profound alteration of the cytoskeleton and impairs VSMC migration. The overall goal of this project is thus to define the physiological function of NoxR1 regulation of Nox4 and to determine its role in an in vivo model of migration. In the first specific aim, we plan to determine the role of NoxR1 in the regulation of focal adhesion turnover and cell migration, while Aim 2 is designed to determine the role of NoxR1 in neointimal formation in vivo using a newly created NoxR1 knockout mouse. Because NoxR1 is the first known regulator of Nox4, a Nox family member that regulates such basic cellular processes as senescence, differentiation and survival, these investigations are likely to have far-reaching implications for a number of vascular and nonvascular diseases.