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.