Toxicological Sciences 59, 75-81 (2001)
Copyright © 2001 by the Society of Toxicology
Carcinogenicity |
The Activity of NF-
B in Swiss 3T3 Cells Exposed to Aqueous Extracts of Cigarette Smoke Is Dependent on Thioredoxin
INBIFO Institut für biologische Forschung, Fuggerstr.3, D-51149 Köln, Germany
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 
B (NF-B) is a transcription factor involved in both inflammatory and stress-dependent cell-signaling processes. Here we report on the activity of NF-B 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-B 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 IB-
, as evidenced by the lack of phosphorylation and degradation of IB- in CS-treated cells. However, as demonstrated in immuno-coprecipitation experiments, the kinetics of NF-B DNA binding is strictly paralleled by decreased and increased complex formation between NF-B and thioredoxin (Trx), the reducing catalyst of Cys-62 of NF-B subunit p50, the reduced thiol function of which is essential for efficient NF-B 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-B DNA binding, NF-B/Trx complex formation, and TrxR expression, along with a lack of IB- phosphorylation and degradation, these results suggest that the activity of NF-B 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, IB-.
Key Words: cigarette smoke; NF-B; thioredoxin; thiol oxidation; glutathione.
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