Abstract
Activation of the aryl hydrocarbon receptor (AHR) by endogenous ligands has been implicated in a variety of physiological processes such as cell cycle regulation, cell differentiation and immune responses. It is reported that tryptophan metabolites, such as kynurenine (Kyn) and 6-formylindolo(3,2-b)carbazole (FICZ), are endogenous ligands for AHR (Stockinger et al., 2014). This protocol is designed for treatment with Kyn or FICZ in mouse embryonic fibroblasts (MEFs) or primary peripheral monocytes.
Keywords: Aryl hydrocarbon receptor, Kynurenine, 6-formylindolo(3,2-b)carbazole, Tryptophan, TCDD-inducible poly(ADP-ribose)polymerase
Background
Tryptophan metabolites such as Kyn and FICZ are endogenous ligands for AHR under physiological conditions. Kyn is generated by tryptophan-2,3-dioxygenase (TDO) and/or indoleamine-2,3-dioxygenases 1 and 2 (IDO1/2) and contributes to the suppression of antitumor response and malignant progression (Stockinger et al., 2014). FICZ is produced by exposure of L-tryptophan to ultraviolet B irradiation and is involved in many biological processes (Smirnova et al., 2016). In the adaptive immune system, FICZ is shown to promote Th17 cell response (Stockinger et al., 2014). It has also been shown that innate interferon response during viral infection is suppressed by treatment with these endogenous AHR ligands (Yamada et al., 2016). In order to evaluate the effect of AHR activation by treatment with these ligands, tryptophan-free culture medium and dialyzed FBS are used to cultivate cells under tryptophan-free conditions (Opitz et al., 2011). The detection of TCDD-inducible poly(ADP-ribose)polymerase (TIPARP) (Ma, 2002), one of the AHR-inducible genes, is analyzed to verify ligand-induced AHR activation.
Materials and Reagents
Equipment
Procedure
Data analysis
Recipes
Acknowledgments
This protocol, which was used in Figure 3b and Supplementary Figure 2g of Yamada et al. (2016), is based on the earlier work by Opitz et al. (2011). This was supported by a grant from the Ministry of Health, Labour and Welfare of Japan, the Ministry of Education, Culture, Sports, Science and Technology of Japan (grant-in-aid for scientific research (A) [25253030] and grant-in-aid for scientific research on innovative areas [25115502, 23112701]), the Kato Memorial Bioscience Foundation, the Yasuda Medical Foundation, the Takeda Science Foundation and the Waksman Foundation of Japan to A.T.
References
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