rac1 regulates a cytokine-stimulated, redox-dependent pathway necessary for NF-kappaB activation.
The signal transduction pathway leading to the activation of the transcription factor NF-kappaB 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-kappaB transcriptional activity. In addition, expression of a dominant-negative rac1 mutant (N17rac1) inhibits basal and interleukin 1beta-stimulated NF-kappaB activity. Gel shift analysis using nuclear extract prepared from HeLa cells infected with a recombinant adenovirus encoding N17rac1 (Ad.N17racl) showed reduced levels of cytokine-stimulated DNA binding to a consensus NF-kappaB binding site. We demonstrate that rac proteins function downstream of ras proteins in the activation of NF-kappaB. In addition, V12rac1 stimulation of NF-kappaB 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-kappaB 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-kappaB 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-kappaB activation.
Sulciner
D J
DJ
Cardiology Branch, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892, USA.
Irani
K
K
Yu
Z X
ZX
Ferrans
V J
VJ
Goldschmidt-Clermont
P
P
Finkel
T
T
D016207:Cytokines / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects; D020744:rac GTP-Binding Proteins
D016207:Cytokines; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins