title: 6-Mercaptopurine attenuates tumor necrosis factor-α production in microglia through Nur77-mediated transrepression and PI3K/Akt/mTOR signaling-mediated translational regulation.
abstract: 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-α (TNF-α) 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-α 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-α 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-κB was measured by ELISA-based DNA binding analysis and luciferase reporter assay. Chromatin immunoprecipitation (ChIP) analysis was performed to examine NF-κB p65 and coactivator p300 enrichments and histone modifications at the endogenous TNF-α promoter.
  
  
  RESULTS
  Treatment of LPS-activated microglia with 6-MP significantly attenuated TNF-α production. In 6-MP pretreated microglia, LPS-induced MAPK signaling, IκB-α degradation, NF-κB p65 nuclear translocation, and in vitro p65 DNA binding activity were not impaired. However, 6-MP suppressed transactivation activity of NF-κB and TNF-α 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-α promoter, ultimately leading to a decrease in p65/coactivator-mediated transcription of TNF-α 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-α production. Additionally, 6-MP also impeded TNF-α 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.
authors.affiliation: Department of Medical Research, Buddhist Tzu Chi General Hospital, Hualien, Taiwan.
authors.forename: Hsin-Yi
authors.initials: HY
authors.lastname: Huang
authors.affiliation: Department of Medical Research, Buddhist Tzu Chi General Hospital, Hualien, Taiwan.
authors.forename: Hui-Fen
authors.initials: HF
authors.lastname: Chang
authors.affiliation: Department of Emergency Medicine, Ditmanson Medical Foundation Chiayi Christian Hospital, Chiayi, Taiwan.
authors.forename: Ming-Jen
authors.initials: MJ
authors.lastname: Tsai
authors.affiliation: Department of Medical Research, Buddhist Tzu Chi General Hospital, Hualien, Taiwan.
authors.forename: Jhih-Si
authors.initials: JS
authors.lastname: Chen
authors.affiliation: Department of Medical Research, Buddhist Tzu Chi General Hospital, Hualien, Taiwan. mjwang@tzuchi.com.tw.
authors.forename: Mei-Jen
authors.initials: MJ
authors.lastname: Wang
mesh_terms: D000818:Animals; D015153:Blotting, Western; D047369:Chromatin Immunoprecipitation; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D007249:Inflammation / Q000139:chemically induced / Q000276:immunology / Q000378:metabolism; D008070:Lipopolysaccharides / Q000633:toxicity; D015122:Mercaptopurine / Q000494:pharmacology*; D051379:Mice; D017628:Microglia / Q000187:drug effects* / Q000378:metabolism; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1 / Q000276:immunology / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000276:immunology / Q000378:metabolism; D016133:Polymerase Chain Reaction; D051057:Proto-Oncogene Proteins c-akt / Q000276:immunology / Q000378:metabolism; D034741:RNA, Small Interfering; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology; D058570:TOR Serine-Threonine Kinases / Q000276:immunology / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
chemical_list: D007166:Immunosuppressive Agents; D008070:Lipopolysaccharides; D015122:Mercaptopurine; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering; D058570:TOR Serine-Threonine Kinases; D014409:Tumor Necrosis Factor-alpha; C539818:Nr4a1 protein, mouse; C546843:mTOR protein, mouse
keywords: 6-Mercaptopurine; Histone H3 acetylation; Microglia; Nuclear factor-κB; Nur77; PI3K/Akt; TNF-α; mTOR