Suppression of CSE-induced COX-2 protein expression and PGE₂ production does not require DNA binding activity of the AhR.

<p>Lung fibroblasts were generated from <i>AhR^DBD/DBD</i> or <i>AhR^DBD/B6</i> mice and treated with AhR ligands (TCDD, ITE and B[<i>a</i>]P) or 1% CSE and cellular RNA or protein was collected for qPCR/western blot analysis. Cell culture supernatant was assayed for PGE₂ production. (A) <i>AhR^DBD/DBD</i> and <i>AhR^DBD/B6</i> lung fibroblasts exposed to AhR ligands (TCDD, B[<i>a</i>]P or ITE) induce less CYP1B1 protein expression compared to lung fibroblasts from mice expressing one copy of the wild-type AhR. TCDD-exposed AhR^+/+ and AhR^−/− cells are included for comparison; note the lack of induction in the AhR^−/− fibroblasts. (B) <i>Cox-2</i> mRNA- There was a slight but not statistically significant increase in <i>Cox-2</i> mRNA in <i>AhR^DBD/DBD</i> cells (fold-change was 1.97±0.54; p = 0.5 compared to media-only). There was a significant increase in <i>Cox-2</i> mRNA in <i>AhR^DBD/B6</i> fibroblasts in response to CSE (4.3±0.8; ***p<0.001 compared to media-only; ^$$p<0.01 compared to CSE-exposed <i>AhR^DBD/DBD</i> cells). Results are expressed as mean ± SEM of 5 independent experiments. (C) There was a slight, but not significant, induction of COX-2 protein in response to 1% CSE. The relative level of induction in COX-2 protein was similar between <i>AhR^DBD/DBD</i> and <i>AhR^DBD/B6</i> cells. Results are expressed as mean ± SEM of 5 independent experiments. Representative western blot is shown. (D) Baseline PGE₂ levels did not differ between <i>AhR^DBD/DBD</i> (235±73 pg/ml) and <i>AhR^DBD/B6</i> (247±99 pg/ml) cells. Exposure to 1% CSE did not significantly increase the concentration of PGE₂ in either <i>AhR^DBD/DBD</i> or <i>AhR^DBD/B6</i> lung fibroblasts. Samples were run in duplicate and the results are expressed as mean ± SEM of 3–6 independent experiments.