Korean J Physiol Pharmacol.  2017 Sep;21(5):547-554. 10.4196/kjpp.2017.21.5.547.

Hydroquinone suppresses IFN-β expression by targeting AKT/IRF3 pathway

• Kim Y
• Kim HG
• Han SY
• Jeong D
• Yang WS
• Kim JI
• Kim JH
• Yi YS
• Cho JY

• KMID: 2388699
• DOI: http://doi.org/10.4196/kjpp.2017.21.5.547

Abstract

Previous studies have demonstrated the role of hydroquinone (HQ), a hydroxylated benzene metabolite, in modulating various immune responses; however, its role in macrophage-mediated inflammatory responses is not fully understood. In this study, the role of HQ in inflammatory responses and the underlying molecular mechanism were explored in macrophages. HQ down-regulated the expression of interferon (IFN)-β mRNA in LPS-stimulated RAW264.7 cells without any cytotoxicity and suppressed interferon regulatory factor (IRF)-3-mediated luciferase activity induced by TIR-domain-containing adapter-inducing interferon-β (TRIF) and TANK-binding kinase 1 (TBK1). A mechanism study revealed that HQ inhibited IRF-3 phosphorylation induced by lipopolysaccharide (LPS), TRIF, and AKT by suppressing phosphorylation of AKT, an upstream kinase of the IRF-3 signaling pathway. IRF-3 phosphorylation is highly induced by wild-type AKT and poorly induced by an AKT mutant, AKT C310A, which is mutated at an inhibitory target site of HQ. We also showed that HQ inhibited IRF-3 phosphorylation by targeting all three AKT isoforms (AKT1, AKT2, and AKT3) in RAW264.7 cells and suppressed IRF-3-mediated luciferase activities induced by AKT in HEK293 cells. Taken together, these results strongly suggest that HQ inhibits the production of a type I IFN, IFN-β, by targeting AKTs in the IRF-3 signaling pathway during macrophage-mediated inflammation.

Keyword

AKT; Hydroquinone; IFN-β; Inflammation; IRF-3; Macrophage

MeSH Terms

Benzene
HEK293 Cells
Inflammation
Interferons
Luciferases
Macrophages
Phosphorylation
Phosphotransferases
Protein Isoforms
RNA, Messenger
Benzene
Interferons
Luciferases
Phosphotransferases
Protein Isoforms
RNA, Messenger

Figure

• 

  Fig. 1 Chemical structure of hydroquinone (HQ).

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  Fig. 2 Effect of HQ on the mRNA expression of IFN-β(A) RAW264.7 cells (1×106 cells/ml) pretreated with the indicated doses of HQ for 30 min were incubated with LPS (1 µg/ml) for 6 h. Expression levels of IFN-β and GAPDH mRNA were determined by semi-quantitative PCR. (B) RAW264.7 cells (1×106 cells/ml) were treated with the indicated doses of HQ for 24 h, and cell viability was determined by MTT assay. (C) HEK293 cells (1×106 cells/ml) cotransfected with IRF-3-Luc (1.0 µg/ml), β-gal (0.1 µg/ml), and either (Left panel) TRIF (1.0 µg/ml) or TBK1 (1.0 µg/ml) for 24 h were treated with the indicated doses of HQ for another 24 h. Luciferase activity was measured with a luminometer. All luciferase activities were normalized to β-galactosidase activity. *p<0.05, **p<0.01 compared to the control.

• 

  Fig. 3 Effect of HQ on the activation of IRF-3(A) RAW264.7 cells (1×106 cells/ml) pretreated with either HQ (50 µM) or vehicle for 30 min were incubated with LPS (1.0 µg/ml) for the indicated time. Phosphorylated and total levels of IRF-3 in total cell lysates were determined by Western blot analysis. (B) RAW264.7 cells (1×106 cells/ml) transfected with TRIF (1.0 µg/ml) for 24 h were treated with the indicated doses of HQ for 24 h, and phosphorylated and total levels of IRF-3 in total cell lysates were determined by Western blot analysis.

• 

  Fig. 4 Effect of HQ on AKT activation in IRF3 pathway(A) RAW264.7 cells (1×106 cells/ml) pretreated with either HQ (50 µM) or vehicle for 30 min were incubated with LPS (1.0 µg/ml) for the indicated time. Phosphorylated and total levels of TBK1 and AKT in total cell lysates were determined by Western blot analysis. (B) RAW264.7 cells (1×106 cells/ml) transfected with HA-AKT (1.0 µg/ml) for 24 h were treated with the indicated doses of HQ for another 24 h, and phosphorylated and total levels of IRF-3 and HA in total cell lysates were determined by Western blot analysis. (C) RAW264.7 cells (1×106 cells/ml) were transfected with either HA-AKT (1.0 µg/ml) or HA-AKT C310A (1.0 µg/ml) for 48 h. Phosphorylated and total levels of IRF-3, AKT, and HA in total cell lysates were determined by Western blot analysis. (D) RAW264.7 cells (1×106 cells/ml) transfected with either HA-AKT1 (1.0 µg/ml), HA-AKT2 (1.0 µg/ml), or HA-AKT3 (1.0 µg/ml) for 24 h were treated with either HQ (50 µM) or vehicle for another 24 h. Phosphorylated and total levels of IRF-3, AKT, and HA in the total cell lysates were determined by Western blot analysis. (E) HEK293 cells (1×106 cells/ml) cotransfected with TBK1 (1.0 µg/ml), IRF-3-Luc (1.0 µg/ml), β-gal (0.1 µg/ml), and HA-AKT1 (1.0 µg/ml), HA-AKT2 (1.0 µg/ml), HA-AKT3 (1.0 µg/ml), or HA-AKT KD (kinase domain deletion mutant) for 24 h were treated with either HQ (50 µM) or vehicle for another 24 h. Luciferase activity was measured with a luminometer. All luciferase reporter gene activities were normalized to β-galactosidase activity. *p<0.05, **p<0.01 compared to the control.

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  Fig. 5 Proposed model for HQ-mediated suppression of IFN-β production by targeting AKT kinases in the IRF-3 signaling pathway during macrophage-mediated inflammatory responsesBold denotes a signaling molecule targeted by HQ in macrophages during inflammatory responses.

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