Arsenic inhibits NF-κB-mediated gene transcription by blocking IκB kinase activity and IκBα phosphorylation and degradation

The inflammatory cytokine, TNF-alpha, induces IL-8 gene transcription via a mechanism involving proteasome-mediated I kappa B alpha degradation and NF-kappa B activation. Here, we investigated whether arsenic, which has been shown to inhibit the ubiquitin-proteasome pathway, could inhibit TNF-alpha-mediated increases in IL-8 expression. Using RT-PCR, we show that the addition of TNF-alpha 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 I kappa B alpha levels, as demonstrated by Western analysis, and an increase in nuclear levels of NF-kappa B, as demonstrated by gel shift analysis. Further demonstrating the activation of NF-kappa B, TNF-alpha induced the transcription of a NF-kappa B-dependent reporter gene. Exposing the cells to 500 mu M arsenite, prior to adding TNF-alpha; completely inhibited I kappa B alpha degradation, NF-kappa B translocation, NF-kappa B-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 I kappa B alpha; arsenite inhibited the phosphorylation of I kappa B alpha. Furthermore, arsenite directly blocked the activity of IKK, the kinase responsible for I kappa B alpha phosphorylation. These studies demonstrate that high levels of arsenic may inhibit NF-kappa B-mediated gene transcription by specifically blocking IKK activity, thereby limiting the phosphorylation and subsequent degradation of the NF-kappa B inhibitor, I kappa B alpha. (C) 2000 Academic Press.