Effects of mTOR Complex-selective Inhibitors on iNOS Expression in LPS-stimulated RAW264.7 Cells

다국어 초록 (Multilingual Abstract)

Objectives: Phosphoinositide 3-kinase (PI3K) and mechanistic target of rapamycin (mTOR) plays key roles in signaling pathways for cell survival and proliferation. Studies on the role of PI3K/mTOR signaling are somewhat inadequate in relation to the function of innate immune cells as compared to B cells and T cells of acquired immune cells. In this study, I investigated the role of PI3K and two different complexes of mTOR in macrophage, an innate immune cell type. For this purpose, I assessed the effects of selective inhibitors of PI3K and mTOR complexes on nitric oxide (NO) production and inducible NO synthase (iNOS) expression in RAW264.7 mouse macrophage cells.

Methods: Selective inhibitors of PI3K/mTOR were used to determine their effects on NO production and iNOS expression in RAW264.7 macrophage cells. NO production in lipopolysaccharide (LPS)-stimulated RAW264.7 cell was measured by Griess assay. Cell viability in the selective inhibitor-treated RAW264.7 cells was measured by MTT assay. Western blot analysis was performed to examine the effects of the inhibitors on expression of iNOS protein and AKT/MAPKs phosphorylation in LPS-induced RAW264.7 cell. The iNOS mRNA level in LPS-stimulated RAW264.7 cells was determined by reverse transcription-polymerase chain reaction (RT-PCR).

Results: LPS induced NO production in RAW264.7 cells and the level of NO was decreased by pan-PI3K inhibitor (ZSTK474), mTORC1 inhibitor (rapamycin), mTORC1/2 inhibitor (PP242), and PI3K/mTORCs dual inhibitor (BEZ235). No effect was observed in cell viability in any cells treated with each inhibitor. LPS increased iNOS protein expression in RAW264.7 cells, which was decreased by any inhibitor tested in a dose-dependent manner. The mRNA level in LPS-stimulated RAW264.7 cells was decreased by ZSTK474, rapamycin, and PI103, while no effect was observed in PP242- and BEZ235-treated groups. To analyze upstream signals, phosphorylation status of AKT and MAPKs was evaluated. All inhibitors decreased the LPS-induced phosphoylation of AKT, but the phosphorylation of p38 kinase was decreased only by BEZ235. No effect on the phosphorylation of ERK1/2 and JNK was observed in any cells treated with each selective inhibitor.

Conclusion: In LPS-induced RAW264.7 cells, pan-PI3K inhibitors, mTORC1 inhibitors and dual PI3K/mTORs inhibitors suppressed NO production. This inhibitory effect was mediated through the down-regulation of iNOS expression. The level of iNOS mRNA was also suppressed by pan-PI3K inhibitor and mTORC1 inhibitor, but not by mTORC1/2 inhibitor and PI3K/mTORCs dual inhibitor. Any inhibitor tested decreased the LPS-induced phosphorylation of AKT. Only p38 kinase phosphorylation was affected by BEZ235, while no effect was observed in the phosphorylation of other MAPKs, ERK1/2 and JNK. These results shows that NO production and iNOS expression are suppressed by selective inhibitors of PI3K and mTOR complexes but the modes of action are somewhat different.

목차 (Table of Contents)

• LIST OF FIGURES
• ABSTRACT (KOREAN) i
• ABSTRACT (ENGLISH) iv
• I. INTRODUCTION 1
• II. OBJECTIVES 9
• III. MATERIALS AND METHODS 10
• A. RAW264.7 cell culture
• B. Chemicals
• C. Nitrite Assay
• D. MTT Assay
• E. Western Blot
• F. Reverse Transcription-Polymerase Chain Reaction(RT-PCR)
• analysis
• G. Statistics
• IV. RESULTS 14
• A. Effects of PI3K and mTOR inhibitors on Nitric oxide
• production in LPS-induced RAW264.7 cells
• B. Cell viability of PI3K and mTOR inhibitors in RAW264.7
• cells
• C. Effects of Selective PI3K/mTOR inhibitors on the
• expression of iNOS protein, AKT and MAPKs
• phosphorylation
• D. Effects of Selective PI3K/mTOR inhibitors on the
• expression of iNOS mRNA
• V. DISCUSSION 26
• VI. CONCLUSION 30
• REFERENCES 32