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 μM) 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 PKCα, PKCβ and PKCγ) abolished Hcy-induced phosphorylation of p47phox and p67phox subunits in monocytes. Transfection of cells with antisense PKCβ oligonucleotide, but not antisense PKCα oligonucleotide, completely blocked Hcy-induced phosphorylation of p47phox and p67phox subunits as well as superoxide anion production. Pretreatment of cells with LY333531, a PKCβ 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.
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Research Article|
July 27 2006
Homocysteine stimulates phosphorylation of NADPH oxidase p47phox and p67phox subunits in monocytes via protein kinase Cβ activation
Yaw L. Siow;
Yaw L. Siow
1
*Department of Physiology, Faculty of Medicine, University of Manitoba, 730 William Avenue, Winnipeg, Manitoba, Canada R3E 3J7
†Laboratory of Integrative Biology, Canadian Centre for Agri-food Research in Health and Medicine, St. Boniface Hospital Research Centre, 351 Taché Avenue, Winnipeg, Manitoba, Canada R2H 2A6
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Kathy K. W. Au-Yeung;
Kathy K. W. Au-Yeung
1
†Laboratory of Integrative Biology, Canadian Centre for Agri-food Research in Health and Medicine, St. Boniface Hospital Research Centre, 351 Taché Avenue, Winnipeg, Manitoba, Canada R2H 2A6
‡Department of Animal Science, Faculty of Agriculture and Food Sciences, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
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Connie W. H. Woo;
Connie W. H. Woo
*Department of Physiology, Faculty of Medicine, University of Manitoba, 730 William Avenue, Winnipeg, Manitoba, Canada R3E 3J7
†Laboratory of Integrative Biology, Canadian Centre for Agri-food Research in Health and Medicine, St. Boniface Hospital Research Centre, 351 Taché Avenue, Winnipeg, Manitoba, Canada R2H 2A6
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Karmin O
Karmin O
2
*Department of Physiology, Faculty of Medicine, University of Manitoba, 730 William Avenue, Winnipeg, Manitoba, Canada R3E 3J7
†Laboratory of Integrative Biology, Canadian Centre for Agri-food Research in Health and Medicine, St. Boniface Hospital Research Centre, 351 Taché Avenue, Winnipeg, Manitoba, Canada R2H 2A6
‡Department of Animal Science, Faculty of Agriculture and Food Sciences, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
2To whom correspondence should be addressed, at Laboratory of Integrative Biology, St. Boniface Hospital Research Centre (email karmino@sbrc.ca).
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Publisher: Portland Press Ltd
Received:
November 10 2005
Revision Received:
April 17 2006
Accepted:
April 21 2006
Accepted Manuscript online:
April 21 2006
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 398 (1): 73–82.
Article history
Received:
November 10 2005
Revision Received:
April 17 2006
Accepted:
April 21 2006
Accepted Manuscript online:
April 21 2006
Citation
Yaw L. Siow, Kathy K. W. Au-Yeung, Connie W. H. Woo, Karmin O; Homocysteine stimulates phosphorylation of NADPH oxidase p47phox and p67phox subunits in monocytes via protein kinase Cβ activation. Biochem J 15 August 2006; 398 (1): 73–82. doi: https://doi.org/10.1042/BJ20051810
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