Objectives Rheumatoid Arthritis (RA) is a chronic inflammatory disease of unclear aetiology, which is associated with inflamed human fibroblast-like synoviocytes (HFLS). Epidemiological studies have identified a positive correlation between tobacco smoking (a rich source of aryl hydrocarbon receptor (AHR) agonists) and aggressive RA phenotype. Thus, we hypothesise that antagonism of AHR activity by a potent AHR antagonist GNF351 can attenuate the inflammatory phenotype of HFLS-RA cells. Methods Quantitative PCR was used to examine IL1B-induced mRNA expression in primary HFLS-RA cells. A structurally diverse AHR antagonist CH223191 and transient AHR repression using AHR small interfering RNA (siRNA) in primary HFLS-RA cells were used to demonstrate that effects observed by GNF351 are AHR-mediated. The levels of PTGS2 were determined by western blot and secretory cytokines such as IL1B and IL6 by ELISA. Chromatin-immunoprecipitation was used to assess occupancy of the AHR on the promoters of IL1B and IL6. Results Many of the chemokine and cytokine genes induced by IL1B in HFLS-RA cells are repressed by co-treatment with GNF351 at both the mRNA and protein level. Pretreatment of HLFS-RA cells with CH223191 or transient gene ablation of AHR by siRNA confirmed that the effects of GNF351 are AHR-mediated. GNF351 inhibited the recruitment of AHR to the promoters of IL1B and IL6 confirming occupancy of AHR at these promoters is required for enhanced inflammatory signalling. Conclusions These data suggest that AHR antagonism may represent a viable adjuvant therapeutic strategy for the amelioration of inflammation associated with RA.

Previous studies in head and neck squamous cell carcinoma (HNSCC) cell lines have revealed that the Ah receptor (AHR) plays a significant role in mediating the ‘aggressive’ phenotype of these cells, which includes enhanced inflammatory signaling (e.g. IL6) and migratory potential. Here we sought to identify putative novel targets of the AHR associated with enhanced tumor invasiveness. Global gene expression analysis identified a number of genes that are repressed upon treatment of OSC-19 or HN30 cells with an AHR antagonist. Three growth factors were targets of AHR activity; amphiregulin (AREG), epiregulin (EREG) and platelet-derived growth factor A (PDGFA) were repressed by an AHR antagonist and further examined. Quantitative PCR analysis, ELISA and siRNA-mediated knock down of AHR revealed an attenuation of basal and/or induced levels of expression of these growth factors in two HNSCC lines, following AHR antagonism. In silico analysis revealed that these growth factors possess dioxin-like response elements. Two other AHR ligands, 6-formylindolo[3,2-b]carbazole and benzo(a)pyrene also elicited similar responses. In conclusion, this study identified AREG, EREG and PDGFA as growth factor targets of AHR activity associated with metastatic phenotype of HNSCC cells, suggesting that attenuation of AHR activity may be a therapeutic strategy.

Rheumatoid arthritis (RA) is a chronic autoimmune disease with high morbidity and mortality. Within the inflammatory milieu, resident fibroblast-like synoviocytes (FLS) in the synovial tissue undergo hyperplasia, which leads to joint destruction. Epidemiologic studies and our previous research suggest that activation of the aryl hydrocarbon receptor (AHR) pathway plays an instrumental role in the inflammatory and destructive RA phenotype. In addition, our recent studies implicate the AHR in the regulation of the expression of several growth factors in established tumor cell lines. Thus, under inflammatory conditions, we hypothesized that the AHR is involved in the constitutive and inducible expression of several growth factors, FLS proliferation and migration, along with protease-dependent invasion in FLS from patients with RA