Regulation of inflammatory response by 3-methyladenine involves the coordinative actions on akt and glycogen synthase kinase 3β rather than autophagy

Yi Chieh Lin, Hsuan Cheng Kuo, Jang Shiun Wang, Wan Wan Lin

Research output: Contribution to journalArticle

23 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)4154-4164
Number of pages11
JournalJournal of Immunology
Volume189
Issue number8
DOIs
Publication statusPublished - Oct 15 2012

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Glycogen Synthase Kinase 3
Autophagy
Phosphatidylinositol 3-Kinases
Nitric Oxide Synthase
Macrophages
Cyclooxygenase 2
Interleukin-1
3-methyladenine
Interferon Regulatory Factor-3
Interleukin-12

ASJC Scopus subject areas

  • Immunology

Cite this

Regulation of inflammatory response by 3-methyladenine involves the coordinative actions on akt and glycogen synthase kinase 3β rather than autophagy. / Lin, Yi Chieh; Kuo, Hsuan Cheng; Wang, Jang Shiun; Lin, Wan Wan.

In: Journal of Immunology, Vol. 189, No. 8, 15.10.2012, p. 4154-4164.

Research output: Contribution to journalArticle

Lin, Yi Chieh ; Kuo, Hsuan Cheng ; Wang, Jang Shiun ; Lin, Wan Wan. / Regulation of inflammatory response by 3-methyladenine involves the coordinative actions on akt and glycogen synthase kinase 3β rather than autophagy. In: Journal of Immunology. 2012 ; Vol. 189, No. 8. pp. 4154-4164.
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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.",
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