6-Mercaptopurine attenuates tumor necrosis factor-α production in microglia through Nur77-mediated transrepression and PI3K/Akt/mTOR signaling-mediated translational regulation

Abstract

BackgroundThe 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.MethodsLipopolysaccharide (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.ResultsTreatment 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.ConclusionsThese results suggest that 6-MP might have a therapeutic potential in neuroinflammation-related neurodegenerative disorders through downregulation of microglia-mediated inflammatory processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0543-5) contains supplementary material, which is available to authorized users.

Microglia Mediated Neuroinflammation: Focus on PI3K Modulation

“…In this study, in which once again LPS was used to induce inflammatory responses in cultured primary microglia or murine BV-2 microglial cells, it was reported that 6-MP was able to adverse TNF-α mRNA translation through prevention of LPS activated PI3K/Akt/mTOR signaling cascades. These results corroborated the idea that the modulation of PI3K/Akt/mTOR signaling might contribute to the downregulation of microglia mediated inflammation, suggesting its possible therapeutic application in neuroinflammation related neurodegenerative disorders [72].…”

“…In addition, it was demonstrated that well established immunosuppressive drug 6 mercaptopurine (6-MP) was able to downregulate microglial inflammatory responses by modulating microglial TNF-α production, suggesting the possible mechanism of this modulation [72]. In this study, in which once again LPS was used to induce inflammatory responses in cultured primary microglia or murine BV-2 microglial cells, it was reported that 6-MP was able to adverse TNF-α mRNA translation through prevention of LPS activated PI3K/Akt/mTOR signaling cascades.…”

Novel Phosphodiesterase 4 Inhibitor FCPR03 Alleviates Lipopolysaccharide-Induced Neuroinflammation by Regulation of the cAMP/PKA/CREB Signaling Pathway and NF-κB Inhibition

“…LPS induces inflammation by activating microglia and produces various proinflammatory cytokines (Smith et al, 2012). Based on these properties, microglial cells treated with LPS are widely used as an in vitro cell model of neuroinflammation, as well as a tool for the evaluation of potential antineuroinflammatory compounds (Gupta and Kaur, 2016;Huang et al, 2016;Pfluger et al, 2016). Thus, this cell model was used in the present study to evaluate the antineuroinflammatory effect of FCPR03.…”

Nur77 exacerbates PC12 cellular injury in vitro by aggravating mitochondrial impairment and endoplasmic reticulum stress

“…Nur77, a transcription factor of the nuclear receptor, plays important roles in neuroinflammation, cell proliferation, differentiation, apoptosis, metabolism and development 2 7 8 9 10 . In humans, three family members have been identified: nerve growth factor IB (also known as Nur77, NR4A1, or TR3), Nuclear receptor related 1 (also known as Nurr1, or NR4A2), and neuron-derived orphan receptor 1 (Nor-1, NR4A3).…”