Induction of TNFα in macrophages by vanadate is dependent on activation of transcription factor NF-κB and free radical reactions

Vanadium-induced TNF alpha production is believed to play an important role in respiratory disease associated with air pollution and occupational exposure. While vanadium is able to induce TNF alpha in macrophages or airway epithelial cells, the underlying mechanism is not well defined. In the present study, mechanisms of vanadate-induced TNF alpha production were analyzed in the murine Raw264.7 cells. Vanadate induces a significant amount of TNF alpha at both the protein and mRNA levels, and the induction is vanadate dose-dependent. The mechanism analysis was focused on transcriptional regulation of TNF alpha gene by vanadate. Transient transfection studies show that the TNF alpha gene promoter was activated by vanadate and this activation was associated with an increase in DNA binding activity of the nuclear factor-kappa B (NF-kappa B). Mutation of the NF-kappa B binding site in the gene promoter led to a loss of the promoter responsiveness to vanadate, indicating requirement of NF-kappa B. This is supported by evidence that inhibition of NF-kappa B activation by SN50, a specific NF-kappa B inhibitor, resulted in a decrease in the TNF alpha production. A role of reactive oxygen species (ROS) was explored in vanadate activity. The result shows that vanadate-induced TNF alpha production is elevated by NADPH, which enhances vanadate-mediated generation of ROS, but is inhibited by an antioxidant, N-acetyl-L-cysteine (NAC). Modification of TNF alpha production is associated with an enhancement or a repression of NF-kappa B activity by NADPH or NAC, respectively. Taken together, these results indicate that: (a) activation of the TNF alpha gene promoter contributes to the vanadate-induced TNF alpha production; (b) NF-kappa B is required for the vanadate-induced promoter activity of TNF alpha gene; (c) free radical reactions are involved in the vanadate-induced TNF alpha production and NF-kappa B activation.