Induction of TNFalpha in macrophages by vanadate is dependent on activation of transcription factor NF-kappaB and free radical reactions.

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