Siow Yaw L, Au-Yeung Kathy K W, Woo Connie W H, O Karmin
Department of Physiology, Faculty of Medicine, University of Manitoba, 730 William Avenue, Winnipeg, Manitoba, Canada R3E 3J7.
Biochem J. 2006 Aug 15;398(1):73-82. doi: 10.1042/BJ20051810.
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 Hcy (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. Hcy treatment (20-100 microM) 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 p47phox siRNA (small interfering RNA) abolished Hcy-induced superoxide anion production, indicating the involvement of NADPH oxidase. Hcy treatment resulted in phosphorylation and subsequently membrane translocation of p47phox and p67phox subunits leading to NADPH oxidase activation. Pretreatment of cells with PKC (protein kinase C) inhibitors Ro-32-0432 (bisindolylmaleimide XI hydrochloride) (selective for PKCalpha, PKCbeta and PKCgamma) abolished Hcy-induced phosphorylation of p47phox and p67phox subunits in monocytes. Transfection of cells with antisense PKCbeta oligonucleotide, but not antisense PKCalpha oligonucleotide, completely blocked Hcy-induced phosphorylation of p47phox and p67phox subunits as well as superoxide anion production. Pretreatment of cells with LY333531, a PKCbeta 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 p47phox and p67phox subunits of NADPH oxidase. Increased superoxide anion production via NADPH oxidase may play an important role in Hcy-induced inflammatory response during atherogenesis.
高同型半胱氨酸血症是动脉粥样硬化所致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。我们之前的研究[Wang和O（2001年）《生物化学杂志》357卷，233 - 240页]表明，同型半胱氨酸（Hcy）处理导致细胞内超氧阴离子显著升高，致使单核细胞中趋化因子受体表达增加。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，我们探究了Hcy诱导单核细胞产生超氧阴离子的分子机制。Hcy处理（20 - 100微摩尔）导致NADPH氧化酶激活以及单核细胞（THP - 1，一种人单核细胞系）中超氧阴离子水平升高。用p47phox小干扰RNA（siRNA）转染细胞消除了Hcy诱导的超氧阴离子产生，表明NADPH氧化酶参与其中。Hcy处理导致p47phox和p67phox亚基磷酸化并随后发生膜易位，从而导致NADPH氧化酶激活。用蛋白激酶C（PKC）抑制剂Ro - 32 - 0432（双吲哚马来酰亚胺XI盐酸盐）（对PKCalpha、PKCbeta和PKCgamma具有选择性）预处理细胞消除了Hcy诱导的单核细胞中p47phox和p67phox亚基的磷酸化。用反义PKCbeta寡核苷酸而非反义PKCalpha寡核苷酸转染细胞完全阻断了Hcy诱导的p47phox和p67phox亚基磷酸化以及超氧阴离子产生。用PKCbeta抑制剂LY333531预处理细胞消除了Hcy诱导的超氧阴离子产生。综上所述，这些结果表明，Hcy刺激单核细胞产生超氧阴离子是通过PKC依赖的NADPH氧化酶p47phox和p67phox亚基磷酸化来调节的。通过NADPH氧化酶增加超氧阴离子产生可能在动脉粥样硬化形成过程中Hcy诱导的炎症反应中起重要作用。
