The activity of NF-kappaB in Swiss 3T3 cells exposed to aqueous extracts of cigarette smoke is dependent on thioredoxin

Abstract

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.