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.
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.
Department of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Yuan-Yi
YY
Chou
Jhen-I
JI
Gao
Shwu-Fen
SF
Chang
Po-Yuan
PY
Chang
Shao-Chun
SC
Lu
D000818:Animals; D002460:Cell Line; D015536:Down-Regulation; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D016179:Granulocyte Colony-Stimulating Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008070:Lipopolysaccharides; D008264:Macrophages / Q000187:drug effects*; D051379:Mice; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D050813:Octamer Transcription Factor-2 / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D020123:Sirolimus / Q000494:pharmacology*
D008070:Lipopolysaccharides; D050813:Octamer Transcription Factor-2; D016179:Granulocyte Colony-Stimulating Factor; D052247:Nitric Oxide Synthase Type II; D020123:Sirolimus