Modulation of IL-12 p35 and p40 promoter activity by smokeless tobacco extract is associated with an effect upon activation of NF-κB but not IRF transcription factors

Interleukin (IL)-12(p70), composed of p35 and p40 subunits, stimulates cellular immunity and inflammation. Stimulation of IL-12 production by smokeless tobacco extract (STE) could increase the chances of oral inflammatory disease. However, p40 forms homodimers and is part of IL-23 heterodimers. Expression of p35 and p40 in response to lipopolysaccharide (LPS) and interferon (IFN)-gamma requires activation of nuclear factor-kappa-B (NF-kappaB) and interferon regulatory factor (IRF) transcription factors. To determine the impact of STE on expression of p35 and p40, the activities of p35 and p40 promoter reporter plasmids in RAW264.7 cells stimulated with STE alone or in the presence of IFN-gamma and LPS were assessed. In addition, nuclear localizations of NF-kappaB p50, p65 and IRF-1, -2 and -8 in RAW264.7 cells treated with STE were evaluated. The results show that STE alone stimulates p40 and p35 promoter activity and enhances IFN-gamma-induced p40 and p35 promoter activity. In contrast, STE had no effect on LPS-induced p35 and p40 promoter activity and diminished IFN-gamma/LPS-induced p35 promoter activity. STE had little effect upon nuclear localization of IRFs, but it stimulated nuclear localization of both NF-kappaB p5O and p65. STE also stimulated IFN-gamma-induced activation of NF-kappaB p50 but reduced nuclear localization of IFN-gamma- and IFN-gamma/LPS-induced NF-kappaB p65. SN50, an inhibitor of NF-kappaB nuclear localization, significantly lowered STE-induced p35 and p40 promoter activity. These results suggest that STE stimulation of bioactive IL-12 production is correlated with its impact upon both p35 and p40 and can be attributed in part through an effect upon NF-kappaB p50 nuclear localization. (C) 2003 Elsevier Science B.V. All rights reserved.