title: Homocysteine stimulates phosphorylation of NADPH oxidase p47phox and p67phox subunits in monocytes via protein kinase Cbeta activation.
abstract: 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.
authors.affiliation: Department of Physiology, Faculty of Medicine, University of Manitoba, 730 William Avenue, Winnipeg, Manitoba, Canada R3E 3J7.
authors.forename: Yaw L
authors.initials: YL
authors.lastname: Siow
authors.forename: Kathy K W
authors.initials: KK
authors.lastname: Au-Yeung
authors.forename: Connie W H
authors.initials: CW
authors.lastname: Woo
authors.forename: Karmin
authors.initials: K
authors.lastname: O
mesh_terms: D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D006710:Homocysteine / Q000494:pharmacology*; D006801:Humans; D009000:Monocytes / Q000187:drug effects* / Q000201:enzymology*; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D064546:Protein Kinase C beta; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D013481:Superoxides / Q000378:metabolism
chemical_list: D002121:Calcium Channel Blockers; D016376:Oligonucleotides, Antisense; D010750:Phosphoproteins; D021122:Protein Subunits; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C079789:neutrophil cytosol factor 67K; D006710:Homocysteine; D013481:Superoxides; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D011493:Protein Kinase C; D064546:Protein Kinase C beta