Figure 2
AHR directly represses Saa3 transcription in a dose-responsive and ligand-dependent manner. (a and b) AHR represses Saa3 induction by various cytokines. Hepa1c1c7 cells were treated with 10 nM TCDD or vehicle control. After 30 min, cells were treated with 2 ng/ml IL1B, IL6, TNFA or a combination of IL1B and IL6 for an additional 6 h. (c and d) Various classes of AHR ligands can suppress Saa3. Hep1c1c7 cells were treated with different AHR ligands at the described doses for 30 min before interleukin (IL1B + IL6, 2 ng/ml each) treatment. Saa3 mRNA (c) and Cyp1a1 mRNA (d) were measured by real-time PCR. (e and f) Analysis of TCDD dose–response of AHR-mediated Saa3 repression. Hepa1c1c7 cells treated with increasing doses of TCDD (0.2 nM to 10 nM) for 30 min before interleukin (IL1B + IL6, 2 ng/ml each) treatment. (e) Closed triangles represent repression of IL-induced Saa3 mRNA by various doses of TCDD, as determined by real-time PCR. Closed squares represent uninduced Saa3 mRNA levels, as a control. (f) TCDD-driven Cyp1a1 mRNA induction, as measured by real-time PCR. (g) AHR-mediated Saa3 repression is because of direct transcriptional inhibition. Real-time PCR on RNA from Hepa1c1c7 cells treated first with (black bars), or without (open bars), the translational inhibitor—cycloheximide (10 μg/ml) for 30 min, then with TCDD (10 nM) for 30 min and finally with TNFA for 6 h. Data represent mean induction±s.e.m. (n=3/treatment group) and were analyzed to determine significance (*P<0.05).
