rac1 Regulates a Cytokine-Stimulated, Redox-Dependent Pathway Necessary for NF-κB Activation

Abstract

The signal transduction pathway leading to the activation of the transcription factor NF-κB remains incompletely characterized. We demonstrate that in HeLa cells, transient expression of a constitutively active mutant of the small GTP-binding protein rac1 (V12rac1) leads to a significant increase in NF-κB transcriptional activity. In addition, expression of a dominant-negative rac1 mutant (N17rac1) inhibits basal and interleukin 1β-stimulated NF-κB activity. Gel shift analysis using nuclear extract prepared from HeLa cells infected with a recombinant adenovirus encoding N17rac1 (Ad.N17rac1) showed reduced levels of cytokine-stimulated DNA binding to a consensus NF-κB binding site. We demonstrate that rac proteins function downstream of ras proteins in the activation of NF-κB. In addition, V12rac1 stimulation of NF-κB activity is shown to be independent of the ability of rac proteins to activate the family of c-jun amino-terminal kinases. In an effort to further explore how rac proteins might regulate NF-κB activity, we demonstrate that expression of V12rac1 in HeLa cells or stimulation with cytokine results in a significant increase in intracellular reactive oxygen species (ROS). Treatment of cells with either of two chemically unrelated antioxidants inhibits the rise in ROS that occurs following V12rac1 expression as well as the ability of V12rac1 to stimulate NF-κB activity. These results suggest that in HeLa cells, rac1 regulates intracellular ROS production and that rac proteins function as part of a redox-dependent signal transduction pathway leading to NF-κB activation.