The activity of NF-kappaB in Swiss 3T3 cells exposed to aqueous extracts of cigarette smoke is dependent on thioredoxin.
Multiple studies in vitro have demonstrated that aqueous extracts of mainstream cigarette smoke (CS) [smoke-bubbled phosphate-buffered saline (PBS)] induce a distinct pattern of stress response in cultured cells, which may be related to the reported pro-inflammatory activities of CS in vitro and in vivo. Nuclear factor kappaB (NF-kappaB) is a transcription factor involved in both inflammatory and stress-dependent cell-signaling processes. Here we report on the activity of NF-kappaB in cells exposed to subcytotoxic concentrations of smoke-bubbled PBS. Using electrophoretic mobility shift assay (EMSA) techniques, we observed a decreased DNA binding of NF-kappaB during the first 2 h of exposure, which was followed by a more than 2-fold increase over controls after 4 to 6 h of exposure. This type of kinetics is not regulated by IkappaB-alpha, as evidenced by the lack of phosphorylation and degradation of IkappaB-alpha in CS-treated cells. However, as demonstrated in immuno-coprecipitation experiments, the kinetics of NF-kappaB DNA binding is strictly paralleled by decreased and increased complex formation between NF-kappaB and thioredoxin (Trx), the reducing catalyst of Cys-62 of NF-kappaB subunit p50, the reduced thiol function of which is essential for efficient NF-kappaB DNA binding. Monitoring the expression of the gene encoding thioredoxin reductase (TrxR), which is required to keep Trx in a functional reduced state, we observed a significant increase in TrxR mRNA after 2 to 6 h of exposure. Based on the correspondence between the kinetics of NF-kappaB DNA binding, NF-kappaB/Trx complex formation, and TrxR expression, along with a lack of IkappaB-alpha phosphorylation and degradation, these results suggest that the activity of NF-kappaB in CS-treated cells is subject mainly to a redox-controlled mechanism dependent on the availability of reduced Trx rather than being controlled by its normal regulator, IkappaB-alpha.
Gebel
S
S
INBIFO Institut für biologische Forschung, Fuggerstr.3, D-51149 Köln, Germany.
Müller
T
T
D016475:3T3 Cells / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D017931:DNA Primers / Q000032:analysis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000378:metabolism; D020672:I-kappa B Proteins; D017403:In Situ Hybridization; D051379:Mice; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010947:Plants, Toxic; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012906:Smoke / Q000009:adverse effects*; D013880:Thioredoxin-Disulfide Reductase / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana; D014158:Transcription, Genetic / Q000187:drug effects*
D017931:DNA Primers; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606205:Nfkbia protein, mouse; D012906:Smoke; D000072000:NF-KappaB Inhibitor alpha; D013879:Thioredoxins; D012313:RNA; D004247:DNA; D013880:Thioredoxin-Disulfide Reductase; D005978:Glutathione