PMID- 27075886 OWN - NLM STAT- MEDLINE DCOM- 20161219 LR - 20211203 IS - 1742-2094 (Electronic) IS - 1742-2094 (Linking) VI - 13 IP - 1 DP - 2016 Apr 13 TI - 6-Mercaptopurine attenuates tumor necrosis factor-alpha production in microglia through Nur77-mediated transrepression and PI3K/Akt/mTOR signaling-mediated translational regulation. PG - 78 LID - 10.1186/s12974-016-0543-5 [doi] LID - 78 AB - BACKGROUND: The pathogenesis of several neurodegenerative diseases often involves the microglial activation and associated inflammatory processes. Activated microglia release pro-inflammatory factors that may be neurotoxic. 6-Mercaptopurine (6-MP) is a well-established immunosuppressive drug. Common understanding of their immunosuppressive properties is largely limited to peripheral immune cells. However, the effect of 6-MP in the central nervous system, especially in microglia in the context of neuroinflammation is, as yet, unclear. Tumor necrosis factor-alpha (TNF-alpha) is a key cytokine of the immune system that initiates and promotes neuroinflammation. The present study aimed to investigate the effect of 6-MP on TNF-alpha production by microglia to discern the molecular mechanisms of this modulation. METHODS: Lipopolysaccharide (LPS) was used to induce an inflammatory response in cultured primary microglia or murine BV-2 microglial cells. Released TNF-alpha was measured by enzyme-linked immunosorbent assay (ELISA). Gene expression was determined by real-time reverse transcription polymerase chain reaction (RT-PCR). Signaling molecules were analyzed by western blotting, and activation of NF-kappaB was measured by ELISA-based DNA binding analysis and luciferase reporter assay. Chromatin immunoprecipitation (ChIP) analysis was performed to examine NF-kappaB p65 and coactivator p300 enrichments and histone modifications at the endogenous TNF-alpha promoter. RESULTS: Treatment of LPS-activated microglia with 6-MP significantly attenuated TNF-alpha production. In 6-MP pretreated microglia, LPS-induced MAPK signaling, IkappaB-alpha degradation, NF-kappaB p65 nuclear translocation, and in vitro p65 DNA binding activity were not impaired. However, 6-MP suppressed transactivation activity of NF-kappaB and TNF-alpha promoter by inhibiting phosphorylation and acetylation of p65 on Ser276 and Lys310, respectively. ChIP analyses revealed that 6-MP dampened LPS-induced histone H3 acetylation of chromatin surrounding the TNF-alpha promoter, ultimately leading to a decrease in p65/coactivator-mediated transcription of TNF-alpha gene. Furthermore, 6-MP enhanced orphan nuclear receptor Nur77 expression. Using RNA interference approach, we further demonstrated that Nur77 upregulation contribute to 6-MP-mediated inhibitory effect on TNF-alpha production. Additionally, 6-MP also impeded TNF-alpha mRNA translation through prevention of LPS-activated PI3K/Akt/mTOR signaling cascades. CONCLUSIONS: These results suggest that 6-MP might have a therapeutic potential in neuroinflammation-related neurodegenerative disorders through downregulation of microglia-mediated inflammatory processes. FAU - Huang, Hsin-Yi AU - Huang HY AD - Department of Medical Research, Buddhist Tzu Chi General Hospital, Hualien, Taiwan. FAU - Chang, Hui-Fen AU - Chang HF AD - Department of Medical Research, Buddhist Tzu Chi General Hospital, Hualien, Taiwan. FAU - Tsai, Ming-Jen AU - Tsai MJ AD - Department of Emergency Medicine, Ditmanson Medical Foundation Chiayi Christian Hospital, Chiayi, Taiwan. FAU - Chen, Jhih-Si AU - Chen JS AD - Department of Medical Research, Buddhist Tzu Chi General Hospital, Hualien, Taiwan. FAU - Wang, Mei-Jen AU - Wang MJ AD - Department of Medical Research, Buddhist Tzu Chi General Hospital, Hualien, Taiwan. mjwang@tzuchi.com.tw. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20160413 PL - England TA - J Neuroinflammation JT - Journal of neuroinflammation JID - 101222974 RN - 0 (Immunosuppressive Agents) RN - 0 (Lipopolysaccharides) RN - 0 (Nr4a1 protein, mouse) RN - 0 (Nuclear Receptor Subfamily 4, Group A, Member 1) RN - 0 (RNA, Small Interfering) RN - 0 (Tumor Necrosis Factor-alpha) RN - E7WED276I5 (Mercaptopurine) RN - EC 2.7.1.1 (mTOR protein, mouse) RN - EC 2.7.11.1 (Proto-Oncogene Proteins c-akt) RN - EC 2.7.11.1 (TOR Serine-Threonine Kinases) SB - IM MH - Animals MH - Blotting, Western MH - Chromatin Immunoprecipitation MH - Enzyme-Linked Immunosorbent Assay MH - Gene Expression Regulation/drug effects/immunology MH - Immunosuppressive Agents/*pharmacology MH - Inflammation/chemically induced/immunology/metabolism MH - Lipopolysaccharides/toxicity MH - Mercaptopurine/*pharmacology MH - Mice MH - Microglia/*drug effects/metabolism MH - Nuclear Receptor Subfamily 4, Group A, Member 1/immunology/*metabolism MH - Phosphatidylinositol 3-Kinases/immunology/metabolism MH - Polymerase Chain Reaction MH - Proto-Oncogene Proteins c-akt/immunology/metabolism MH - RNA, Small Interfering MH - Signal Transduction/drug effects/immunology MH - TOR Serine-Threonine Kinases/immunology/metabolism MH - Transfection MH - Tumor Necrosis Factor-alpha/*biosynthesis PMC - PMC4831152 OTO - NOTNLM OT - 6-Mercaptopurine OT - Histone H3 acetylation OT - Microglia OT - Nuclear factor-kappaB OT - Nur77 OT - PI3K/Akt OT - TNF-alpha OT - mTOR EDAT- 2016/04/15 06:00 MHDA- 2016/12/20 06:00 PMCR- 2016/04/13 CRDT- 2016/04/15 06:00 PHST- 2015/12/05 00:00 [received] PHST- 2016/04/07 00:00 [accepted] PHST- 2016/04/15 06:00 [entrez] PHST- 2016/04/15 06:00 [pubmed] PHST- 2016/12/20 06:00 [medline] PHST- 2016/04/13 00:00 [pmc-release] AID - 10.1186/s12974-016-0543-5 [pii] AID - 543 [pii] AID - 10.1186/s12974-016-0543-5 [doi] PST - epublish SO - J Neuroinflammation. 2016 Apr 13;13(1):78. doi: 10.1186/s12974-016-0543-5.