Effects of immunosuppressants on iNOS gene expression in macrophages

Abstract

In the present study, the mechanism by which dexamethasone (DEX), acetylaminofluorene (AAF), and $Δ^9$-tetrahydrocannabinol ($Δ^9$-THC) inhibited iNOS gene expression in bacterial lipopolysaccharide (LPS)-activated RAW 264.7 cells was investigated. The decrease in NO, as demonstrated by measurement of nitrite was found to correlate well with a decrease in inducible nitric oxide synthase (iNOS) mRNA. Since the promoter in iNOS gene contains binding motifs for NF-ΚB/Rel, AP-1, NF-IL6, and Oct, which appear to be important in LPS-mediated iNOS induction, the effect of the chemicals on the activation of these transcription factors were determined. Treatment of DEX to RAW 264.7 cells induced a dose-related inhibitions of NF-ΚB/Rel and AP-1 in chloramphenicol acetyltransferase (CAT) activity, while neither NF-IL6 nor Oct activation was affected by DEX. Treatment of RAW 264.7 cells with DEX inhibited DNA binding of NF-ΚB/Rel proteins to their cognate DNA sites as measured by electrophoretic mobility shift assay. In addition, DEX treatment caused a significant inhibition of nuclear translocation of c-rel, p65, and p50 proteins. Treatment of AAF to RAW 264.7 cells inhibited NF-ΚB/Rel activation, nuclear translocation, and DNA binding. It has recently been shown that iNOS transcription is under the control of the cAMP signaling cascade. We demonstrate that $Δ^9$-THC, an inhibitor of cAMP signaling, inhibits the activation of NF-ΚB/Rel in response to LPS stimulation. LPS treatment of RAW 264.7 cells also induced the activation of the cAMP cascade as indicated by an increase in binding of nuclear factors to the cAMP response element (CRE), and the activation of CRE binding proteins was inhibited by $Δ^9$-THC. Collectively, this series of experiments indicates that NF-ΚB/Rel is positively regulated by LPS to help initiate iNOS gene expression in macrophages. This activation of iNOS is attenuated by DEX, AAF, and $Δ^9$-THC through the inhibition of NF-ΚB/Rel.