Lipopolysaccharide induces Rac1-dependent reactive oxygen species formation and coordinates tumor necrosis factor-alpha secretion through IKK regulation of NF-kappa B.
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.
Sanlioglu
S
S
Department of Internal Medicine-Division of Pulmonary and Critical Care, the Center for Gene Therapy, the University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Williams
C M
CM
Samavati
L
L
Butler
N S
NS
Wang
G
G
McCray
P B
PB
Ritchie
T C
TC
Hunninghake
G W
GW
Zandi
E
E
Engelhardt
J F
JF
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D002374:Catalase / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D004996:Ethidium / Q000031:analogs & derivatives* / Q000494:pharmacology; D005786:Gene Expression Regulation; D005799:Genes, Dominant; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D051550:I-kappa B Kinase; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013481:Superoxides / Q000378:metabolism; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D015854:Up-Regulation; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D016207:Cytokines; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D016328:NF-kappa B; D020033:Protein Isoforms; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; C067883:dihydroethidium; D013481:Superoxides; D002374:Catalase; D008156:Luciferases; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; C496564:Chuk protein, mouse; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; C496562:Ikbkb protein, mouse; C496566:Ikbke protein, mouse; D020830:rac1 GTP-Binding Protein; D004996:Ethidium