Technical Reference #1979

1979.

Suppression mechanisms of flavonoids on aryl hydrocarbon receptor-mediated signal transduction Rie Mukai; Yasuhito Shirai; Naoaki Saito; Itsuko Fukuda; Shin Nishiumi; Ken-ichi Yoshida; Hitoshi Ashida, Kobe University, Archives of Biochemistry and Biophysics, 501(1979), (September)
Link To Paper

Abstract:
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates biological and toxicological effects by binding to its agonists such as 2378-tetrachlorodibenzo-p-dioxin (TCDD). Previously we demonstrated that flavonoids suppressed the TCDD-induced DNA-binding activity of the AhR in a structure-dependent manner. In this study we investigated the mechanisms by which flavonoids suppressed the AhR-mediated signal transduction in mouse hepatoma Hepa- 1c1c7 cells. Flavones and flavonols suppressed the TCDD-induced nuclear translocation of the AhR and dissociation of its partner proteins heat shock protein 90 and X-associated protein 2 whereas flavanones and catechins did not. Flavonoids of all these four subclasses suppressed the phosphorylation of both AhR and Arnt and the formation of a heterodimer consisting of these proteins. Since certain flavonoids are known to inhibit mitogen-activated protein kinases (MAPKs) we confirmed the contribution of MAPK/ERK kinase (MEK) to the AhR-mediated signal transduction by using U0126 an inhibitor of MEK1/2. U0126 suppressed TCDD-induced phosphorylation of the AhR and Arnt followed by the DNA-binding activity of the AhR. Flavanones and catechins suppressed the TCDD-induced phosphorylation of ERK1/2. The inhibition of MEK/ERK phosphorylation is one of the mechanisms by which flavanones and catechins suppress the AhR-mediated signal transduction in Hepa-1c1c7 cells.

Keywords:
Flavonoid; AhR; TCDD; ERK; Phosphorylation

Materials & Methods:
Hepa-1c1c7 cells (1 105 cells/dish) were seeded onto a 35- mm glass-bottomed culture dish (MatTek Corp. Ashland MA) and incubated for 24 h before transfection. Transfection mixtures were supplemented with 200 ll of Opti-MEM (Invitrogen Carlsbad CA) 5 ll of FuGENE™ 6 Transfection Reagent (Roche Basel Switzerland) and 4 lg of DNA. The cells were incubated with the transfection mixtures for 4 h at 37 °C.

Microscopic Technique
Confocal Microscopy

Cell Type(s)
Hepa-1c1c7 cells