title: Regulation of inflammatory response by 3-methyladenine involves the coordinative actions on Akt and glycogen synthase kinase 3β rather than autophagy.
abstract: 3-Methyladenine (3-MA) is one of the most commonly used inhibitors in autophagy research today. However, rather than inhibiting class III PI3K that is involved in autophagy suppression, 3-MA might also interfere with class I PI3K and consequently augment autophagy flux. In this study, we aim to get a thorough understanding on the action mechanisms of 3-MA in TLR4-mediated inflammatory responses in RAW264.7 macrophages and, moreover, to decipher the action of 3-MA in modulation of autophagy. We found that 3-MA could enhance LPS-induced NF-κB activation and production of TNF-α, inducible NO synthase (iNOS), cyclooxygenase-2, IL-1β, and IL-12. In contrast, 3-MA suppressed LPS-induced IFN-β production and STAT signaling. Studies revealed that 3-MA can, through inhibition of Akt as a result of class I PI3K interference, positively regulate p38, JNK, and p65, but negatively regulate TANK-binding kinase 1 and IFN regulatory factor 3 mediated by TLR4. As glycogen synthase kinase 3β (GSK3β) is an important Akt substrate, we further explored its involvement in the actions of 3-MA. 3-MA was found to enhance LPS-induced NF-κB activation, iNOS, and pro-IL-1β expression, and these actions were reversed by either GSK3β inhibitors or small interfering GSK3β. Lastly, we demonstrated that 3-MA acts as an autophagy inducer in RAW264.7 macrophages, but the stimulating effects on NF-κB activation and iNOS and cyclooxygenase-2 expression were not affected in LPS-stimulated macrophages with small interfering autophagy protein-5 treatment. These results not only shed new light on the action mechanisms of 3-MA to differentially regulate inflammatory outcomes derived from TLR4-mediated MyD88 and Toll/IL-1R domain-containing adapter inducing IFN-β pathways, but also highlight the necessity to check autophagy status upon taking 3-MA as a general autophagy inhibitor.
authors.affiliation: Department of Pharmacology, College of Medicine, National Taiwan University, Taipei 10051, Taiwan.
authors.forename: Yi-Chieh
authors.initials: YC
authors.lastname: Lin
authors.forename: Hsuan-Cheng
authors.initials: HC
authors.lastname: Kuo
authors.forename: Jang-Shiun
authors.initials: JS
authors.lastname: Wang
authors.forename: Wan-Wan
authors.initials: WW
authors.lastname: Lin
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000502:physiology; D000818:Animals; D001343:Autophagy / Q000187:drug effects / Q000276:immunology*; D002460:Cell Line; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D018836:Inflammation Mediators / Q000494:pharmacology / Q000502:physiology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology / Q000276:immunology; D051379:Mice; D061251:Primary Cell Culture; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology; D015854:Up-Regulation / Q000187:drug effects / Q000276:immunology
chemical_list: D018836:Inflammation Mediators; D008070:Lipopolysaccharides; C025946:3-methyladenine; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3; D000225:Adenine