Homocysteine stimulates phosphorylation of NADPH oxidase p47(phox) and P67(phox) subunits in monocytes via protein kinase C beta activation

摘要

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.