Background Oxidative stress plays a critical role in various diseases including cancer, neuronal degeneration, atherosclerosis and rheumatoid arthritis. Identifying the target proteins for redox modification is crucial in understanding how oxidants mediate the pathological processes. As previously demonstrated activation of T cells with anti CD3 antibody or with phorbol ester results in a rapid production of intracellular reactive oxygen species (ROS). In this paper we further dissect the signalling pathways leading to ROS generation and we propose a possible critical role for the small GTPase Rap1 in antagonising the intracellular generated ROS.
Methods In our experiments we made use of the free radical dye 6-carboxy-2’7’dichlorofluoresceine DCF to measure radical generation at the single cell using flow cytometry. Transfected Jurkat cells were incubated with the dye 20 min 37° before addition of stimuli. Readings were taken every 5-min over a 20 min period. The positive transfected Jurkat cell population was identified by detection of a co-transfected CD20 receptor.
Results T cells as well as Jurkat cells rapidly produce intracellular ROS in a time dependent manner, after stimulation with either CD3 or PMA+ionomycine. The intracellular ROS generated after T cell stimulation seem to be induced through a signalling pathway downstream of the small GTPase Ras, since Jurkat cells transfected with a constitutively active mutant of p21Ras, RasV12, produced large amounts of intracellular ROS, that could not be augmented by an additional stimulus. The observed ROS production after addition of a stimulus was suppressed by the expression of dominant negative mutant of Ras, RasN17. Jurkat cells transfected with the constitutive active mutant of another small GTPase, Rap1, failed to generate ROS after any stimulus tested. Moreover co-transfection of RapV12 and RasV12 showed that RapV12 abolishes the intracellular ROS generated by RasV12. The inhibition of ROS production appears to involve on a PI3K-dependent signalling pathway as is shown by inhibition of ROS production after Jurkat transfection with RapV12C40 (a Rap1 mutation that, analogue with RasV12C40 could affect specific binding to PI3K). Accordingly, pre-incubation with LY 294002, an inhibitor of the lipid kinase activity of PI3K, blocked the observed inhibition of ROS production by Rap1 in RasV12+RapV12 co-transfection experiments. In addition we report that Rap1 activation in T cells after activation with CD3 or PMA proved to be redox sensitive as shown by pretreatment of T cells with N-acetylcysteine and BSO. Furthermore hydrogen peroxide was capable of Rap1 activation in T cells, and pre-incubation with diphenylene iodinium (a flavoproteine inhibitor) completely blocked Rap1 activation.
Conclusion We define a role for ROS in T cell signalling pathways downstream from Ras and we propose a possible pivotal role for the small GTPase Rap1. Rap1 can be activated by ROS and antagonises the intracellular generated ROS in signalling pathways.
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