Bacteroides fragilis enterotoxin upregulates heme oxygenase-1 in dendritic cells via reactive oxygen species-, mitogen-activated protein kinase-, and Nrf2-dependent pathway

Figure 3 Effects of AP-1 suppression on heme oxygenase-1 expression in dendritic cells stimulated with Bacteroides fragilis enterotoxin. A and B: Bone marrow-derived dendritic cells (DCs) (A) and DC2.4 cells (B) were treated with Bacteroides fragilis toxin (BFT) (100 ng/mL) for the indicated periods. AP-1 DNA binding activity was assessed by EMSA and expression of phospho-c-jun in nuclear extracts was detected using immunoblot. Results are representative of more than three independent experiments. C: DC2.4 cells were transfected with lentivirus containing dominant-negative c-jun plasmid (dn-c-jun) or control virus (GFP). Transfected cells were stimulated with BFT (100 ng/mL) for 1 h. AP-1 binding activity was assayed by EMSA (top panel). Immunoblot results for concurrent phospho-c-jun and lamin B in nuclear extracts under the same conditions are provided beneath the EMSA (middle panel). Expression of heme oxygenase-1 (HO-1) and actin proteins was analyzed by immunoblot (bottom panel). Results are representative of more than three independent experiments. D: Transfected DC2.4 cells were treated with BFT (100 ng/mL) for the indicated periods. Levels of HO-1 mRNA were analyzed by quantitative RT-PCR using a standard RNA. The values are expressed as mean ± SD (n = 5). The β-actin mRNA levels in each group remained relatively constant throughout the same periods (approximately 10⁶ transcripts/μg total RNA). E: DC2.4 cells were transfected with AP-1 c-jun-specific silencing siRNA or NS-RNA as a control for 48 h, after which cells were combined with BFT (100 ng/mL) for 1 h. Nuclear extracts were analyzed by immunoblotting with the indicated Abs (top panel). Transfected cells were stimulated with BFT (100 ng/mL) for 24 h. Expression of HO-1 and actin was detected by immunoblot (bottom panel). Results shown are representative of more than three independent experiments. HO-1: Heme oxygenase-1; BFT: Bacteroides fragilis toxin.