Involvement of RelB in aryl hydrocarbon receptor-mediated induction of chemokines

Abstract

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a well-known immunotoxic compound affecting the expression of inflammatory genes. We found that TCDD induces the expression of the B-cell activating factor of the tumor necrosis factor family (BAFF), B-lymphocyte chemoattractant (BLC), CC-chemokine ligand 1 (CCL1), and the transcription factor interferon gamma responsive factor (IFR3) in U937 macrophages in an aryl hydrocarbon receptor- (AhR) and RelB-dependent manner. The induction was associated with increased binding activity of an AhR/RelB complex without participation of ARNT to a NF-kappaB element that is recognized by the NF-kappaB subunit RelB and localized on promoters of the cytokine and chemokine genes BAFF, BLC, CCL1, and the transcription factor IRF3. The interaction of AhR with RelB binding on a novel type of NF-kappaB binding site represents a new regulatory function of the AhR.

Fig. 1

Induction of BAFF, BLC, CCL1, and IRF3 by TCDD is RelB- and AhR-dependent. (A) Time-course study of mRNA expression in U937 macrophages. Cells were treated for 1 to 24h with 10 nM TCDD or 0.1% Me₂SO (Control). Quantitative detection of mRNA was performed using real time RT-PCR. Values for mRNA expression are normalized to the expression of β-actin. (B) Western blot analysis of AHR, RelB, and ARNT protein levels 48h posttransfection with the indicated siRNAs. (C) Quantitative mRNA expression analyses after treatment with TCDD for 24 h. Total RNA was prepared 72 h post-transfection with either a scrambled siRNA or a specific siRNA targeted against ARNT, AhR, or RelB. *, significantly different from control (p<0.01); **, significantly lower than cells transfected with scrambled or ARNT siRNA.

Fig. 1

Induction of BAFF, BLC, CCL1, and IRF3 by TCDD is RelB- and AhR-dependent. (A) Time-course study of mRNA expression in U937 macrophages. Cells were treated for 1 to 24h with 10 nM TCDD or 0.1% Me₂SO (Control). Quantitative detection of mRNA was performed using real time RT-PCR. Values for mRNA expression are normalized to the expression of β-actin. (B) Western blot analysis of AHR, RelB, and ARNT protein levels 48h posttransfection with the indicated siRNAs. (C) Quantitative mRNA expression analyses after treatment with TCDD for 24 h. Total RNA was prepared 72 h post-transfection with either a scrambled siRNA or a specific siRNA targeted against ARNT, AhR, or RelB. *, significantly different from control (p<0.01); **, significantly lower than cells transfected with scrambled or ARNT siRNA.

Fig. 2

Induction of (A) CCL1 and (B) IRF3 is increased in AhR and RelB overexpressing cells. Cells were transiently transfected with 200 ng/ml of AhR or RelB expression plasmid. Control cells were transfected with an empty control vector. After 48 h cells were treated with 10 nM TCDD for 24 h. Expression of CCL1 and IRF3 mRNA was analyzed by real-time PCR as described above. *, significantly different from control cells (p<0.01); **, significantly higher than non-transfected cells (p<0.01)

Fig. 3

(A) Effect of TCDD on binding activity of RelB/AhR response elements located on promoters of BAFF, BLC, CCL1, IL-8, and IRF3. Nuclear protein extracts of control (C) or TCDD- (T) stimulated U937 macrophages were incubated with oligonucleotides containing the NF-κB-RelB-binding site identified on promoters of the corresponding chemokines. (B-E) Supershift analysis of nuclear protein extracts from control or macrophages treated with 10 nM TCDD for 2h. A possible binding of AhR, ARNT, and NF-κB subunits p50, RelA, RelB, or c-Rel was identified by supershift analyses in control cells and TCDD-treated cells using specific antibodies for the corresponding protein. The binding activity of RelB/AhR response elements located on promoters of (B) BAFF (5′-ACAGTTCTGTGAGATTTGACAAGTGCA-3′), (C) BLC (5′-GATGAGGGAGATTTGGTTCTCTAG-3′), (D) CCL1 (5′-ATCTTGAAAAAGTGCGTGGGAACTGTCCTG-3′), and (E) IRF3 (5′-AGAGCTCTGGGAGATGTAACATATCCT-3′) was analyzed. To confirm specificity a 100-fold excess of the unlabeled oligonucleotides was added.

Fig. 3

(A) Effect of TCDD on binding activity of RelB/AhR response elements located on promoters of BAFF, BLC, CCL1, IL-8, and IRF3. Nuclear protein extracts of control (C) or TCDD- (T) stimulated U937 macrophages were incubated with oligonucleotides containing the NF-κB-RelB-binding site identified on promoters of the corresponding chemokines. (B-E) Supershift analysis of nuclear protein extracts from control or macrophages treated with 10 nM TCDD for 2h. A possible binding of AhR, ARNT, and NF-κB subunits p50, RelA, RelB, or c-Rel was identified by supershift analyses in control cells and TCDD-treated cells using specific antibodies for the corresponding protein. The binding activity of RelB/AhR response elements located on promoters of (B) BAFF (5′-ACAGTTCTGTGAGATTTGACAAGTGCA-3′), (C) BLC (5′-GATGAGGGAGATTTGGTTCTCTAG-3′), (D) CCL1 (5′-ATCTTGAAAAAGTGCGTGGGAACTGTCCTG-3′), and (E) IRF3 (5′-AGAGCTCTGGGAGATGTAACATATCCT-3′) was analyzed. To confirm specificity a 100-fold excess of the unlabeled oligonucleotides was added.

Fig. 3

(A) Effect of TCDD on binding activity of RelB/AhR response elements located on promoters of BAFF, BLC, CCL1, IL-8, and IRF3. Nuclear protein extracts of control (C) or TCDD- (T) stimulated U937 macrophages were incubated with oligonucleotides containing the NF-κB-RelB-binding site identified on promoters of the corresponding chemokines. (B-E) Supershift analysis of nuclear protein extracts from control or macrophages treated with 10 nM TCDD for 2h. A possible binding of AhR, ARNT, and NF-κB subunits p50, RelA, RelB, or c-Rel was identified by supershift analyses in control cells and TCDD-treated cells using specific antibodies for the corresponding protein. The binding activity of RelB/AhR response elements located on promoters of (B) BAFF (5′-ACAGTTCTGTGAGATTTGACAAGTGCA-3′), (C) BLC (5′-GATGAGGGAGATTTGGTTCTCTAG-3′), (D) CCL1 (5′-ATCTTGAAAAAGTGCGTGGGAACTGTCCTG-3′), and (E) IRF3 (5′-AGAGCTCTGGGAGATGTAACATATCCT-3′) was analyzed. To confirm specificity a 100-fold excess of the unlabeled oligonucleotides was added.