RAC1 REGULATES A CYTOKINE-STIMULATED, REDOX-DEPENDENT PATHWAY NECESSARY FOR NF-KAPPA-B ACTIVATION

The signal transduction pathway leading to the activation of the trans cription factor NF-kappa B remains incompletely characterized. We demo nstrate that in HeLa cells, transient expression of a constitutively a ctive mutant of the small GTP-binding protein rad (V12rac1) leads to a significant increase in NF-kappa B transcriptional activity, In addit ion, expression of a dominant-negative rad mutant (N17rac1) inhibits b asal and interleukin 1 beta-stimulated NF-kappa B 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-kappa B bi nding site. We demonstrate that rac proteins function downstream of ra s proteins in the activation of NF-kappa B. In addition, V12rac1 stimu lation of NF-kappa B activity is shown to be independent of the abilit y of rac proteins to activate the family of c-jun amino-terminal kinas es, In an effort to further explore how rac proteins might regulate NF -kappa B activity, we demonstrate that expression of V12rac1 in HeLa c ells 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 R OS that occurs following V12rac1 expression as well as the ability of V12rac1 to stimulate NF-kappa B activity. These results suggest that i n HeLa cells, rad regulates intracellular ROS production and that rac proteins function as part of a redox-dependent signal transduction pat hway leading to NF-kappa activation.