Aryl Hydrocarbon Receptor Negatively Regulates LPS-induced Osteopontin Expression By Disrupting DNA Looping

Aryl hydrocarbon receptor (AhR) mediates crucial immunoregulatory functions by regulating target genes transcription. However, the molecular mechanisms that AhR regulates gene expression, especially at the chromatin level, remain largely unknown. We have previously demonstrated that osteopontin (OPN) expression was upregulated through the formation of DNA loop in the OPN promoter between NF-κB binding site and AP-1binding site following LPS activation in macrophages.In the present study, we showed that AhR agonist2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibits LPS-induced OPN production in an AhR-dependent manner. siRNA knockdown of AhR expression in peritoneal macrophages greatly increased LPS-induced OPN expression.Objectives:1. Investigate the function of AhR in LPS-induced osteopontin expression.2. Investigate the mechanism of AhR in LPS-induced osteopontin expression。Methods:1. LPS-induced AhR expression in macrophages. 1.1Peritoneal macrophages were treated with LPS for indicated time periods. AhR mRNA expression was examined by both RT-PCR and quantitative PCR. AhR protein expression was detected by Western blot1.2AhR expression in mouse primary peritoneal macrophages, mouse splenocytes, and macrophage cell line RAW264.7was detected by RT-PCR. Similar results were obtained in three independent experiments.2. AhR negatively regulates LPS-induced OPN expression.2.1. Mouse peritoneal macrophages (upper panel) or RAW264.7macrophages (bottom panel) were pretreated with DMSO or10nM TCDD for40min and then stimulated with LPS for the indicated periods. OPN expression was examined by Western blot.2.4. Mouse peritoneal macrophages were transfected with control siRNA or AhR siRNA for36h. AhR expression was examined by Western blot.2.5. Mouse peritoneal macrophages were transfected with control siRNA or AhR siRNA for36h, pretreated with DMSO or10nM TCDD for40min and then stimulated with LPS. OPN expression was examined by Western blot.2.6. Mouse peritoneal macrophages were transfected with control siRNA or AhR siRNA for36h and then stimulated with LPS. OPN expression was examined by Western blot. Similar results were obtained in three independent experiments.3. AhR binding to OPN promoter.3.1Mouse peritoneal macrophages were pretreated with DMSO or10nM TCDD for40min and then stimulated with LPS, and the ChIP assay was used to assess the binding of AhR to the AhR binding sites within the-2407to-2230region of the murine OPN promoter. Total extract was used as a loading control, and immunoprecipitation with irrelevant antibody (anti-actin) was used as a negative control. PCR products from the amplication of a AhR site-free region within-1926to-1759of the murine OPN promoter were used as specificity controls. Similar results were obtained in three independent experiments.4. AhR disrupted NF-κB and AP-1mediated DNA looping. 4.1Mouse peritoneal macrophages were pretreated with DMSO or10nM TCDD for40min and then stimulated with LPS. The3C assay was performed for OPN promoter using primer A and B.4.2Mouse peritoneal macrophages were pretreated with DMSO or10nM TCDD for40min and then stimulated with LPS.ChIP assay was used to assess the binding of p300, p65, and c-Jun to the NF-kB binding sites within the-1926to-1759region and to the AP-1binding sites within the-127to+42region of the murine OPN promoter. Total extract was used as a loading control, and immunoprecipitation with irrelevant Ab (anti-actin) was used as a negative control. Similar results were obtained in three independent experiments.Results and Conclusion:We demonstrated that AhR binds to a distal region in the OPN promoter and disrupts NF-κB and AP-1mediated DNA looping following TCDD treatment. Therefore, our study revealed a novel mechanism for AhR to remodel the local chromatin structure and spatial conformation to regulate gene expression.