Rapamycin Down-Regulates Inducible Nitric Oxide Synthase by Inducing Proteasomal Degradation

Abstract

Rapamycin, a macrolide antibiotic, has a potent anti-proliferative effect in multiple cell types and is currently used in kidney transplantation as a clinical immunosuppressant. 1)Rapamycin interacts with FK506-binding protein (12 kDa, FKBP12), and this complex inhibits the mammalian target of rapamycin (mTOR). Recent findings revealed that mTOR controls a diverse set of downstream effectors, some of which are important for cell growth, such as p70 S6 kinase and eukaryotic translation initiation factor 4E-binding proteins.2,3) It is now believed that p70 S6 kinase participates in regulation of transcription, as well as translation of mRNAs required for the G 1 /S phase transition in the cell cycle. 3) Macrophage activation by lipopolysaccharide (LPS) is regulated by various mechanisms, including the phosphatidylinositol 3 (PI3)-kinase signaling pathway. This pathway has also been implicated in LPS-mediated activation of human monocytes.4) p70 S6 kinase may be one downstream effector of PI3-kinase, and is also thought to be involved in production of nitric oxide (NO) and expression of inducible NO synthase (iNOS) in LPS-activated murine macrophages.5)The precise mechanism of this signaling pathway is unclear. In general, iNOS expression is regulated at the transcriptional level through activation of several transcription factors, such as nuclear factor (NF)-kB, signal transducer and activator of transcription 1 (STAT1), and interferon regulatory factor 1 (IRF-1). These transcription factors bind to the promoter region of the iNOS gene, [6][7][8] thereby inducing iNOS protein expression and NO production. 6,7,9) iNOS expression and NO production lead to the activation of macrophages in response to pro-inflammatory cytokines and bacterial products. [9][10][11] In this study, we examined the effect of rapamycin, a specific inhibitor of mTOR, on the expression of iNOS in RAW 264.7 macrophages in response to LPS. We investigated the molecular mechanisms by which LPS induces iNOS expression through the p70 S6 kinase pathway. Our results show that rapamycin suppresses LPS-induced iNOS expression and subsequent NO production. This might be partly mediated by the inhibition of p70 S6 kinase activity at nanomolar concentrations of rapamycin, and the activation of 20S proteasomal activity at higher concentrations. Therefore, our results provide novel insight into the dual function of rapamycin in the regulation of LPS-induced iNOS expression, which includes mTOR signaling and proteasome activation pathways. MATERIALS AND METHODS Cell Culture and ReagentsThe murine macrophage cell line RAW 264.7 from ATCC (American Type Culture Collection) was grown at 37°C humidified atmosphere containing 5% CO 2 in Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (Hyclone Laboratories) and 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen). Rapamycin and lipopolysaccharide (from Escherichia coli, 011:B4) were obtained from Sigma and lactacystin was from Calbiochem. Measurement of Nitr...

TLR-driven early glycolytic reprogramming via the kinases TBK1-IKKɛ supports the anabolic demands of dendritic cell activation

“…That result was unexpected, given that PI(3)K is essential for the long-term commitment to aerobic glycolysis in response to stimulation via TLRs 4 , an observation consistent with the known relationship of PI(3)K and Akt. We reason that this inconsistency reflects the fact that signaling via PI(3)K through mTORC1 is essential for promoting iNOS expression and NO production 42,43 , which forces inflammatory DCs to commit to glycolysis in the later stages of their activation 6 . In addition, such long-term commitment to glycolysis would be further supported by mTORC1, through the induction of HIF-1α 5 .…”

Both the autophagy and proteasomal pathways facilitate the Ubp3p-dependent depletion of a subset of translation and RNA turnover factors during nitrogen starvation in Saccharomyces cerevisiae

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Positive Feedback Regulation of Human Inducible Nitric-oxide Synthase Expression by Ras Protein S-Nitrosylation

“…ulating protein translation, the activity of mTOR may also regulate iNOS expression through effects on protein stability (68). The specific post-transcriptional processes modulated by NO that augment iNOS protein levels require further study.…”