单核细胞通过消除病原体在先天免疫中起关键作用,释放高水平的细胞因子,分化为几种细胞类型,包括巨噬细胞和树突状细胞。与其他吞噬细胞相似,单核细胞通过NADPH氧化酶复合物产生超氧阴离子,它由两种膜蛋白(p22phox和gp91phox/NOX2)和四种胞浆蛋白(p47phox,p67phox,p40phox和Rac1)。与嗜中性粒细胞相比,单核细胞中NADPH氧化酶激活所涉及的途径鲜为人知。这里,我们显示p22phox与人单核细胞中的Rho相关卷曲螺旋激酶2(ROCK2)相关,而与中性粒细胞无关。这种相互作用发生在p22phox的胞质区(氨基酸132至195)和ROCK2的卷曲螺旋区(氨基酸400至967)之间。有趣的是,ROCK2不磷酸化p22phox,p40phox,p67phox,或体外gp91phox,但在Ser304,Ser315,Ser320和Ser328上磷酸化p47phox。此外,ROCK2的选择性抑制剂KD025抑制单核细胞中的活性氧(ROS)产生和p47phox磷酸化。siRNA对THP1单核细胞系ROCK2表达的特异性抑制抑制了ROS的产生。这些数据表明,ROCK2与p22phox相互作用,磷酸化p47phox,并表明p22phox可能是ROCK2的穿梭,以允许人单核细胞中的p47phox磷酸化和NADPH氧化酶激活。
Monocytes play a key role in innate immunity by eliminating pathogens, releasing high levels of cytokines, and differentiating into several cell types, including macrophages and dendritic cells. Similar to other phagocytes, monocytes produce superoxide anions through the NADPH oxidase complex, which is composed of two membrane proteins (p22phox and gp91phox/NOX2) and four cytosolic proteins (
p47phox, p67phox, p40phox and Rac1). The pathways involved in NADPH oxidase activation in monocytes are less known than those in neutrophils. Here, we show that p22phox is associated with Rho-associated coiled-coil kinase 2 (ROCK2) in human monocytes but not neutrophils. This interaction occurs between the cytosolic region of p22phox (amino acids 132 to 195) and the coiled-coil region of ROCK2 (amino acids 400 to 967). Interestingly, ROCK2 does not phosphorylate p22phox, p40phox, p67phox, or gp91phox in vitro but phosphorylates
p47phox on Ser304, Ser315, Ser320 and Ser328. Furthermore, KD025, a selective inhibitor of ROCK2, inhibited reactive oxygen species (ROS) production and
p47phox phosphorylation in monocytes. Specific inhibition of ROCK2 expression in THP1-monocytic cell line by siRNA inhibited ROS production. These data show that ROCK2 interacts with p22phox and phosphorylates
p47phox, and suggest that p22phox could be a shuttle for ROCK2 to allow
p47phox phosphorylation and NADPH oxidase activation in human monocytes.