Regulation of Inflammatory Response by 3-Methyladenine Involves the Coordinative Actions on Akt and Glycogen Synthase Kinase 3β Rather than Autophagy

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-kappa B activation and production of TNF-alpha, inducible NO synthase (iNOS), cyclooxygenase-2, IL-1 beta, and IL-12. In contrast, 3-MA suppressed LPS-induced IFN-beta 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 3 beta (GSK3 beta) 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-kappa B activation, iNOS, and pro-IL-1 beta expression, and these actions were reversed by either GSK3 beta inhibitors or small interfering GSK3 beta. Lastly, we demonstrated that 3-MA acts as an autophagy inducer in RAW264.7 macrophages, but the stimulating effects on NF-kappa B 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-beta pathways, but also highlight the necessity to check autophagy status upon taking 3-MA as a general autophagy inhibitor. The Journal of Immunology, 2012, 189: 4154-4164.