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.
대표적 면역억제물질인 dexamethasone (DEX), acetylaminofluorene (AAF), 및 $Δ^9$-THC-tetrahydorcannabinol ($Δ^9$-THC)이 macrophage에서 lipopolysaccharide (LPS)에 의해 유도되는 inducible nitric oxide synthase (iNOS) 유전자의 발현에 미치는 영향에 관하여 연구하였다. DEX, AAF 및 $Δ^9$-THC는 macrophage cell line인 RAW 264.7 cell에서 nitric oxide (NO)의 생성을 저해 하였으며, comeptitive RT-PCR 분석결과 iNOS 유전자의 발현도 이 면역억제 물질들에 의해 저해됨을 확인하였다. iNOS 유전자의 promoter (-54 ~ -1126)에는 전사조절제인 NF-ΚB/Rel, AP-1, NF-IL6 및 Oct의 결합부위가 있으며, 그 결합부위는 LPS에 의해 유도되는 iNOS 유전자 발현에 중요하다고 알려져 있으므로, 약물들이 전사조절제의 활성에 미치는 영향을 조사하였다. DEX는 NF-ΚB/Rel와 AP-1에 의해 유도되는 chloramphenicol acetyltransferase 활성을 dose dependent 하게 저해하였으며 NF-IL6와 Oct에는 아무런 영향을 미치지 못하였다. NF-ΚB/Rel와 AP-1의 DNA결합도 DEX에 의해 저해되었고, NF-ΚB/Rel의 component인 p65, p50 및 c-rel의 nuclear translocation도 저해되었으나 AP-1 component인 c-jun과 c-fos의 translocation에는 아무런 영향도 미치지 못하였다. AAF는 NF- B/Rel의 활성만을 저해하였고, p65, p50 및 c-rel의 nuclear translocation도 저해하였다. NF-ΚB/Rel의 저해제인 phyrrolidine dithiocarbamate를 RAW 264.7 cell에 처리한 결과 iNOS 유전자의 발현과 NO생성을 저해하였다. 따라서 NF-ΚB/Rel은 iNOS 유전자의 발현에 매우 중요하며 DEX와 AAF의 molecular target이 될 수 있음을 알 수 있었다. 최근에는 또한 cAMP 경유한 signal이 NF-ΚB/Rel의 활성과 iNOS 유전자의 발현에 중요하다는 주장이 제기됨에 따라, cAMP pathway의 저해제인 $Δ^9$-THC-THC를 처리한 결과 iNOS 유전자의 발현이 저해되었고 NF-ΚB/Rel의 DNA binding activity도 저해되었다. 결론적으로 macrophage에서 NF-ΚB/Rel은 LPS에 의해 활성화되어 iNOS 유전자의 발현을 유도하고 DEX, AAF, 및 $Δ^9$-THC는 이 NF-ΚB/Rel의 활성화를 저해하여 iNOS 유전자의 발현을 감소시키는 것으로 생각된다