title: The role of the JAK2-STAT3 pathway in pro-inflammatory responses of EMF-stimulated N9 microglial cells.
abstract: BACKGROUND
  In several neuropathological conditions, microglia can become overactivated and cause neurotoxicity by initiating neuronal damage in response to pro-inflammatory stimuli. Our previous studies have shown that exposure to electromagnetic fields (EMF) activates cultured microglia to produce tumor necrosis factor (TNF)-α and nitric oxide (NO) through signal transduction involving the activator of transcription STAT3. Here, we investigated the role of STAT3 signaling in EMF-induced microglial activation and pro-inﬂammatory responses in more detail than the previous study.
  
  
  METHODS
  N9 microglial cells were treated with EMF exposure or a sham treatment, with or without pretreatment with an inhibitor (Pyridone 6, P6) of the Janus family of tyrosine kinases (JAK). The activation state of microglia was assessed via immunoreaction using the microglial marker CD11b. Levels of inducible nitric oxide synthase (iNOS), TNF-α and NO were measured using real-time reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and the nitrate reductase method. Activation of JAKs and STAT3 proteins was evaluated by western blotting for specific tyrosine phosphorylation. The ability of STAT3 to bind to DNA was detected with an electrophoresis mobility shift assay (EMSA).
  
  
  RESULTS
  EMF was found to significantly induce phosphorylation of JAK2 and STAT3, and DNA-binding ability of STAT3 in N9 microglia. In addition, EMF dramatically increased the expression of CD11b, TNF-α and iNOS, and the production of NO. P6 strongly suppressed the phosphorylation of JAK2 and STAT3 and diminished STAT3 activity in EMF-stimulated microglia. Interestingly, expression of CD11b as well as gene expression and production of TNF-α and iNOS were suppressed by P6 at 12 h, but not at 3 h, after EMF exposure.
  
  
  CONCLUSIONS
  EMF exposure directly triggers initial activation of microglia and produces a significant pro-inflammatory response. Our findings confirm that the JAK2-STAT3 pathway may not mediate this initial microglial activation but does promote pro-inflammatory responses in EMF-stimulated microglial cells. Thus, the JAK2-STAT3 pathway might be a therapeutic target for reducing pro-inflammatory responses in EMF-activated microglia.
authors.affiliation: Key Laboratory of Medical Protection for Electromagnetic Radiation Ministry of Education, Third Military Medical University, Chongqing, China.
authors.forename: Xuesen
authors.initials: X
authors.lastname: Yang
authors.forename: Genlin
authors.initials: G
authors.lastname: He
authors.forename: Yutong
authors.initials: Y
authors.lastname: Hao
authors.forename: Chunhai
authors.initials: C
authors.lastname: Chen
authors.forename: Maoquan
authors.initials: M
authors.lastname: Li
authors.forename: Yuan
authors.initials: Y
authors.lastname: Wang
authors.forename: Guangbin
authors.initials: G
authors.lastname: Zhang
authors.forename: Zhengping
authors.initials: Z
authors.lastname: Yu
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D004574:Electromagnetic Fields; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D007249:Inflammation / Q000378:metabolism* / Q000473:pathology; D053614:Janus Kinase 2 / Q000378:metabolism*; D051379:Mice; D017628:Microglia / Q000378:metabolism / Q000473:pathology / Q000528:radiation effects*; D018613:Microscopy, Confocal; D009569:Nitric Oxide / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology / Q000528:radiation effects; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050796:STAT3 Transcription Factor / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology / Q000528:radiation effects*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism
chemical_list: D050796:STAT3 Transcription Factor; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D011505:Protein-Tyrosine Kinases; D053614:Janus Kinase 2