Rapamycin inhibits lipopolysaccharide induction of granulocyte‐colony stimulating factor and inducible nitric oxide synthase expression in macrophages by reducing the levels of octamer‐binding factor‐2

• Published in: The FEBS journal 2011-01, Vol.278 (1), p.85-96
• Main Authors: Chou, Yuan‐Yi, Gao, Jhen‐I, Chang, Shwu‐Fen, Chang, Po‐Yuan, Lu, Shao‐Chun
• Format: Article
• Language: English
• Subjects: Animals; Binding sites; Cell Line; chromatin; Down-Regulation; Gene expression; Gene Expression Regulation, Enzymologic - drug effects; genes; granulocyte colony-stimulating factor; Granulocyte Colony-Stimulating Factor - antagonists & inhibitors; Granulocyte Colony-Stimulating Factor - genetics; Granulocyte Colony-Stimulating Factor - metabolism; granulocyte‐colony stimulating factor (G‐CSF); Humans; inducible nitric oxide synthase; inducible nitric oxide synthase (iNOS); lipopolysaccharide (LPS); Lipopolysaccharides; macrophage; macrophages; Macrophages - drug effects; mammalian target of rapamycin (mTOR); messenger RNA; Mice; Nitric Oxide Synthase Type II - antagonists & inhibitors; Nitric Oxide Synthase Type II - genetics; Nitric Oxide Synthase Type II - metabolism; Octamer Transcription Factor-2 - metabolism; octamer‐binding factor‐2 (Oct‐2); plasmids; precipitin tests; Promoter Regions, Genetic; Proteins; rapamycin; RNA interference; Sirolimus - pharmacology
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2010.07929.x

This article reports an inhibitory effect of rapamycin on the lipopolysaccharide (LPS)‐induced expression of both inducible nitric oxide synthase (iNOS) and granulocyte‐colony stimulating factor (G‐CSF) in macrophages and its underlying mechanism. The study arose from an observation that rapamycin inhibited the LPS‐induced increase in octamer‐binding factor‐2 (Oct‐2) protein levels through a mammalian target of rapamycin (mTOR)‐dependent pathway in mouse RAW264.7 macrophages. As both iNOS and G‐CSF are potential Oct‐2 target genes, we tested the effect of rapamycin on their expression and found that it reduced the LPS‐induced increase in iNOS and G‐CSF mRNA levels and iNOS and G‐CSF protein levels. Blocking of mTOR‐signaling using a dominant‐negative mTOR expression plasmid resulted in inhibition of the LPS‐induced increase in iNOS and G‐CSF protein levels, supporting the essential role of mTOR. Forced expression of Oct‐2 using the pCG-Oct‐2 plasmid overcame the inhibitory effect of rapamycin on the LPS‐induced increase in iNOS and G‐CSF mRNA levels. Chromatin immunoprecipitation assays showed that LPS enhanced the binding of Oct‐2 to the iNOS and G‐CSF promoters and that this effect was inhibited by pretreatment with rapamycin. Moreover, RNA interference knockdown of Oct‐2 reduced iNOS and G‐CSF expression in LPS‐treated cells. The inhibitory effect of rapamycin on the LPS‐induced increase in Oct‐2 protein levels and on the iNOS and G‐CSF mRNA levels was also detected in human THP‐1 monocyte‐derived macrophages. This study demonstrates that rapamycin reduces iNOS and G‐CSF expression at the transcription level in LPS‐treated macrophages by inhibiting Oct‐2 expression.