Arsenic Inhibits NF-κB-mediated Gene Transcription by Blocking IκB Kinase Activity and IκBα Phosphorylation and Degradation

The inflammatory cytokine, TNF-α, induces IL-8 gene transcription via a mechanism involving proteasome-mediated IκBα degradation and NF-κB activation. Here, we investigated whether arsenic, which has been shown to inhibit the ubiquitin-proteasome pathway, could inhibit TNF-α-mediated increases in IL-8 expression. Using RT-PCR, we show that the addition of TNF-α to human bronchial epithelial (BEAS 2B) or embryonic kidney (HEK293) cells resulted in increased steady-state levels of IL-8 mRNA. This was preceded by a rapid decrease in cellular IκBα levels, as demonstrated by Western analysis, and an increase in nuclear levels of NF-κB, as demonstrated by gel shift analysis. Further demonstrating the activation of NF-κB, TNF-α induced the transcription of a NF-κB-dependent reporter gene. Exposing the cells to 500 μM arsenite, prior to adding TNF-α, completely inhibited IκBα degradation, NF-κB translocation, NF-κB-dependent gene transcription, and transcription of the endogenous gene for IL-8. In comparison with the proteasome inhibitor MG-132, which does not affect the phosphorylation and ubiquitination of IκBα, arsenite inhibited the phosphorylation of IκBα. Furthermore, arsenite directly blocked the activity of IKK, the kinase responsible for IκBα phosphorylation. These studies demonstrate that high levels of arsenic may inhibit NF-κB-mediated gene transcription by specifically blocking IKK activity, thereby limiting the phosphorylation and subsequent degradation of the NF-κB inhibitor, IκBα.