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Role of TNF Receptor-Associated Factor 3 in the CD40 Signaling by Production of Reactive Oxygen Species through Association with p40^phox, a Cytosolic Subunit of Nicotinamide Adenine Dinucleotide Phosphate Oxidase ¹

Yun Jung Ha^* and Jong Ran Lee^2,*,

^* Division of Molecular Life Sciences and Center for Cell Signaling Research, and Department of Life Science, College of Natural Sciences, Ewha Womans University, Seoul, Korea

To extend our previous report, which showed the production of the reactive oxygen species (ROS) after the CD40 ligation in the B cells, we further examined the possible mechanisms for ROS production and the involvement of CD40-induced ROS in p38 activation. Our research shows that the stimulation of WEHI 231 B lymphomas with anti-CD40 induced ROS production and p38 activation. An antioxidant N-acetyl-L-cysteine or an inhibitor for NADPH oxidase blocked both of these, but the inhibitors for 5-lipoxygenase did not. We also show that the treatment of cells with inhibitors for the phosphatidylinositol 3-kinase (PI3-K) interfered with the CD40-induced ROS production and p38 activation. In addition, when overexpressed with a dominant negative form of either Rac1 (N17Rac1) or the TNFR-associated factor (TRAF) 3, the WEHI 231 B cells did not show a full response to the CD40 stimulation to produce ROS. Molecular association studies further revealed that the TRAF3 association with p40^phox, a cytosolic subunit of NADPH oxidase and p85 (a subunit of PI3-K), may possibly be responsible for the production of ROS by CD40 stimulation in WEHI 231 B cells. Collectively, these data suggest that the CD40-induced ROS production by NADPH oxidase in WEHI 231 requires the role of TRAF3, as well as activities of PI3-K and Rac1.

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