FIGURE 2.

NF-κB RelA dependent expression of AhR. A, U937-derived DC were treated with 0.1 μg/ml LPS, 1 nm TCDD, and co-treated with TCDD and LPS for 6 h in absence or presence of 200 μm PDTC. PDTC or water was added 30 min prior to the addition of DMSO as vehicle, TCDD, or LPS. AhR, ARNT, and CYP1A1 mRNA levels were analyzed using real-time PCR. B, effect of NF-κB inhibitors on TCDD-induced and LPS-enhanced DRE luciferase reporter activity. U937-derived DC were incubated with carrier solvent alone (1 μl/ml), 0.1 μg/ml LPS, 1 nm TCDD, 200 μm PDTC, 5 μg/ml CAPE, 50 μm CAPS, or the indicated combination. TCDD was dissolved in DMSO, LPS, and PDTC in water, and CAPE and CAPS were dissolved in ethanol. Cells were treated with LPS for 16 h and with TCDD for 4 h. PDTC, CAPE, and CAPS were added 30 min prior to LPS and TCDD. Luciferase activity was determined as described under “Experimental Procedures.” Values are expressed as relative luminescence units/mg protein and represent the mean ± S.D. of triplicate determinations. C, siRNA-mediated RelA gene ablation was performed in U937-derived DC. After transient transfection for 48 h, cells were treated with 1 nm TCDD, 0.1 μg/ml LPS, or 0.1% DMSO (Ctrl; control) for 6 h. mRNA levels of AhR and CYP1A1 were determined using real-time PCR. Values for AhR and CYP1A1 mRNA expression are normalized to the expression of β-actin. Values are the mean ± S.D. of three independent experiments. Single asterisk indicates significantly different from control (p < 0.05); double asterisks indicate significantly higher than only TCDD-treated cells (p < 0.05); triple asterisks indicate significantly lower than only TCDD- or LPS-treated cells (p < 0.05). D, RelA protein ablation was confirmed by Western blot analysis of U937-derived DC transfected with RelA siRNA (siRelA) or scrambled siRNA (siCtrl) for 48 h. 25 μg of whole cell protein was loaded per lane.