Lipopolysaccharide Induces Rac1-dependent Reactive Oxygen Species Formation and Coordinates Tumor Necrosis Factor-α Secretion through IKK Regulation of NF-κB

Abstract

Reactive oxygen species (ROS) are important second messengers generated in response to many types of environmental stress. In this setting, changes in intracellular ROS can activate signal transduction pathways that influence how cells react to their environment. In sepsis, a dynamic proinflammatory cellular response to bacterial toxins (e.g. lipopolysaccharide or LPS) leads to widespread organ damage and death. The present study demonstrates for the first time that the activation of Rac1 (a GTP-binding protein), and the subsequent production of ROS, constitutes a major pathway involved in NFkappaB-mediated tumor necrosis factor-alpha (TNFalpha) secretion following LPS challenge in macrophages. Expression of a dominant negative mutant of Rac1 (N17Rac1) reduced Rac1 activation, ROS formation, NFkappaB activation, and TNFalpha secretion following LPS stimulation. In contrast, expression of a dominant active form of Rac1 (V12Rac1) mimicked these effects in the absence of LPS stimulation. IKKalpha and IKKbeta were both required downstream modulators of LPS-activated Rac1, since the expression of either of the IKK dominant mutants (IKKalphaKM or IKKbetaKA) drastically reduced NFkappaB-dependent TNFalpha secretion. Moreover, studies using CD14 blocking antibodies suggest that Rac1 induces TNFalpha secretion through a pathway independent of CD14. However, a maximum therapeutic inhibition of LPS-induced TNFalpha secretion occurred when both CD14 and Rac1 pathways were inhibited. Our results suggest that targeting both Rac1- and CD14-dependent pathways could be a useful therapeutic strategy for attenuating the proinflammatory cytokine response during the course of sepsis.

Retroinfusion of NFκB decoy oligonucleotide extends cardioprotection achieved by CD18 inhibition in a preclinical study of myocardial ischemia and retroinfusion in pigs

“…Moreover, we recently reported 25 that apoptosis induced by ischemia and reperfusion in the heart at least in part depends on Fas-L, TNF␣ and TRAIL, three NF B-regulated death ligands. [26][27][28] In the present study, TNF␣ mRNA, as well as apoptosis, was found to be reduced in NF B decoy ODN-treated hearts in comparison to control hearts, suggesting that NF B decoy ODN application does not result in an increased rate of apoptosis if TNF␣ expression is not up-regulated.…”

Endosomal Nox2 Facilitates Redox-Dependent Induction of NF-κB by TNF-α

“…The following E1-deleted recombinant adenoviral vectors were used: (a) Ad.GPx-1, which encodes glutathione peroxidase 1 and degrades cytoplasmic H 2 O 2 (33); (b) Ad.Dyn(DN), which encodes a dominant-negative mutant (K44A) of dynamin and inhibits endocytosis (13); (c) Ad.NF-kBLuc, which encodes an NF-kB-responsive promoter driving luciferase expression and was used to assess NF-kB transcriptional activation in vivo (51); and (d) Ad.BglII, an empty vector with no insert, as a control for viral infection (33). For NF-kB transcriptional assays using infection with two recombinant adenoviruses, a slightly modified sequential infection method was used (51). In this case, cells were infected with experimental vectors [i.e., Ad.Dyn(DN) or Ad.GPx1] 24 h before infection with Ad.NFkBLuc], and cells were used for experiments at 48 h after initial infection.…”

“…In this context, reactive oxygen species (ROS), such as O 2 -and H 2 O 2 , have been shown to be induced in cells after receptor stimulation with various ligands such as tumor necrosis factor alpha (TNF-a) (12,39), lipopolysaccharide (LPS) (24,51), angiotensin II (18), platelet-derived growth factor (PDGF) (56), insulin (37), epidermal growth factor (EGF) (2), and interleukin-1b (IL-1b) (6,39). Furthermore, the induction of ROS by these ligands has been suggested to be important for activating many of these signaling pathways (2,18,33,56).…”

Inhibition of NF-κB and Oxidative Pathways in Human Dendritic Cells by Antioxidative Vitamins Generates Regulatory T Cells

“…NF-B activity was measured using a NF-B luciferase reporter adenovirus containing four tandem copies of the enhancer element located upstream of the firefly luciferase gene (36). This was provided by P. McCray, Jr. (University of Iowa, Iowa City, IA).…”