| Summary: | 博士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 99 === The study consists of two parts. The first part reports an inhibitory effect of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), 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 pre-treatment with rapamycin or transfection with dominant-negative (DN)-mTOR expression plasmid inhibited the LPS-induced increase in octamer-binding factor-2 (Oct-2) protein levels through an 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, iNOS and G-CSF protein levels, NO production and G-CSF promoter activity. Blocking of mTOR-signaling using a DN-mTOR expression plasmid resulted in inhibition of the LPS-induced increase in iNOS and G-CSF protein levels and NO production, supporting the essential role of mTOR. The involvement of Oct-2 in LPS-induced iNOS and G-CSF expression was further supported by the finding that 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 (ChIP) assays showed that LPS enhanced the binding of Oct-2 to the iNOS and G-CSF promoters and that this effect was inhibited by pre-treatment with rapamycin. Moreover, RNA interference knockdown of Oct-2 expression 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. The second part investigated the effect of rapamycin on the lipoteichoic acid (LTA)-induced expression of G-CSF in macrophages and its underlying mechanism. Our data showed that LTA treatment induced G-CSF expression in RAW264.7 and bone marrow-derived macrophages in a dose- and time-dependent manner and that this effect was inhibited by pre-treatment with rapamycin. Analysis of the G-CSF 5’ flanking sequence revealed that the −283 to +35 fragment, which contains CSF and octamer elements, was required for maximal promoter activity in response to LTA stimulation. When the effects of rapamycin and LTA on levels of proteins that bind to the CSF and octamer elements were investigated, Western blot analyses and electrophoretic mobility shift assays (EMSAs) showed that LTA induced nuclear translocation of NF-κB p50 and p65, increased protein levels of phospho-IκB-α, C/EBPβ and Oct-2, and enhanced the DNA binding of NF-κB and C/EBPβ, and that rapamycin inhibited the LTA-induced increase in Oct-2 protein levels, but not the other effects. Neither rapamycin nor LTA altered the expression of Oct-1. We then investigated the critical role of Oct-2 in LTA-induced G-CSF expression. Knockdown of Oct-2 expression by RNA interference resulted in a decrease in LTA-induced G-CSF mRNA levels. Moreover, forced expression of Oct-2 by transfection with the pCG-Oct-2 plasmid overcame the inhibitory effect of rapamycin on the LTA-induced increase in G-CSF mRNA levels and promoter activity. This study demonstrates that rapamycin reduces G-CSF expression in LTA-treated macrophages by inhibiting Oct-2 expression.
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