Homocysteine stimulates phosphorylation of NADPH oxidase p47phox and P67phox subunits in monocytes via protein kinase Cβ activation
Homocysteine stimulates phosphorylation of NADPH oxidase p47phox and P67phox subunits in monocytes via protein kinase Cβ activation
复制标题
DOI：
10.1042/bj20051810
复制
发表时间：
2006-08-15
影响因子：
4.1
通讯作者：
O, Karmin
中科院分区：
文献类型：
--
作者：
Siow, Yaw L.;Au-Yeung, Kathy K. W.;O, Karmin
高同型半胱氨酸血症是动脉粥样硬化引起的心血管疾病的独立危险因素。动脉粥样硬化的发展涉及活性氧诱导的血管细胞氧化应激。我们先前的研究[Wang和O（2001）Biochem.J.357，233-240]证明了Hey（高半胱氨酸）处理引起细胞内超氧阴离子的显著升高，导致单核细胞中趋化因子受体的表达增加。NADPH氧化酶主要负责单核细胞中超氧阴离子的产生。在本研究中，我们研究了同型半胱氨酸诱导单核细胞产生超氧阴离子的分子机制。Hey处理（20100 μ M）引起NADPH氧化酶的活化和单核细胞（THP-1，人单核细胞系）中超氧阴离子水平的增加。用p47（phox）siRNA（小干扰RNA）转染细胞可抑制Hcy诱导的超氧阴离子产生，表明NADPH氧化酶参与其中。Hey处理导致磷酸化和随后的p47（phox）和p67（phox）亚基的膜转位，导致NADPH氧化酶活化。用PKC（蛋白激酶C）抑制剂Ro-32-0432（双吲哚马来酰亚胺XI盐酸盐）（对PKC α、PKC β和PKC γ具有选择性）预处理细胞，可消除单核细胞中Hcy诱导的p47（phox）和p67（phox）亚基磷酸化。用反义PKC β寡核苷酸转染细胞，而不是反义PKCa寡核苷酸，完全阻断Hcy诱导的p47（phox）和p67（phox）亚基磷酸化以及超氧阴离子的产生。用PKC β抑制剂LY 333531预处理细胞，可消除Hcy诱导的超氧阴离子产生。总之，这些结果表明，Hcy刺激的单核细胞超氧阴离子的产生是通过PKC依赖的磷酸化NADPH氧化酶的p47（phox）和p67（phox）亚基的调节。通过NADPH氧化酶增加超氧阴离子的产生可能在Hcy诱导的动脉粥样硬化形成过程中的炎症反应中起重要作用。
Hyperhomocysteinaemia is an independent risk factor for cardiovascular diseases due to atherosclerosis. The development of atherosclerosis involves reactive oxygen species-induced oxidative stress in vascular cells. Our previous study [Wang and O (2001) Biochem. J. 357, 233-240] demonstrated that Hey (homocysteine) treatment caused a significant elevation of intracellular superoxide anion, leading to increased expression of chemokine receptor in monocytes. NADPH oxidase is primarily responsible for superoxide anion production in monocytes. In the present study, we investigated the molecular mechanism of Hcy-induced superoxide anion production in monocytes. Hey treatment (20100 mu M) caused an activation of NADPH oxidase and an increase in the superoxide anion level in monocytes (THP-1, a human monocytic cell line). Transfection of cells with p47(phox) siRNA (small interfering RNA) abolished Hcy-induced superoxide anion production, indicating the involvement of NADPH oxidase. Hey treatment resulted in phosphorylation and subsequently membrane translocation of p47(phox) and p67(phox) subunits leading to NADPH oxidase activation. Pretreatment of cells with PKC (protein kinase C) inhibitors Ro-32-0432 (bisindolylmaleimide XI hydrochloride) (selective for PKC alpha, PKC beta and PKC gamma) abolished Hcy-induced phosphorylation of p47(phox) and p67(phox) subunits in monocytes. Transfection of cells with antisense PKC beta oligonucleotide, but not antisense PKCa oligonucleotide, completely blocked Hcy-induced phosphorylation of p47(phox) and p67(phox) subunits as well as superoxide anion production. Pretreatment of cells with LY333531, a PKC beta inhibitor, abolished Hcy-induced superoxide anion production. Taken together, these results indicate that Hcy-stimulated superoxide anion production in monocytes is regulated through PKC-dependent phosphorylation of p47(phox) and p67(phox) subunits of NADPH oxidase. Increased superoxide anion production via NADPH oxidase may play an important role in Hcy-induced inflammatory response during atherogenesis.