Aryl hydrocarbon receptor antagonism alters phenotype of primary human fibroblast-like synoviocytes from rheumatoid arthritis patients.
Open Access
- Author:
- Lahoti, Tejas Suresh
- Graduate Program:
- Molecular Toxicology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 16, 2014
- Committee Members:
- Gary H Perdew, Dissertation Advisor/Co-Advisor
Christopher Albert Mullin, Committee Member
Connie Jo Rogers, Committee Member
Andrew David Patterson, Special Member - Keywords:
- Ah receptor
GNF351
AHR antagonism
rheumatoid arthritis
fibroblast-like synoviocytes
HFLS-RA - Abstract:
- Aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that belongs to the family of basic helix-loop-helix PER/ARNT/SIM proteins. The AHR was originally identified based on its ability to bind to environmental contaminants, such as polycyclic aromatic hydrocarbons (PAH) or halogenated polycyclic aromatic hydrocarbons (HPAH). Persistant ligand-mediated AHR activation has shown toxic effects via binding of AHR to the dioxin response elements (DREs) present in the promoters of various functionally diverse target genes, including genes involved in xenobiotic metabolism (eg. CYP1A1). Evolutionary conservation of the receptor insinuates its role beyond toxicity. Epidemiological studies have identified a positive correlation between exposure to environmental contaminants and/ or tobacco smoking (rich sources of AHR agonists) and aggressive rheumatoid arthritis (RA). RA is a chronic systemic autoimmune disease of unknown etiology, which is prevalent in females. It is the leading musculo-skeletal disorder, affecting more than 1% of the world’s population. RA commonly affects the articular joints of the hands and feet leading to systemic joint inflammation. Under the inflammatory milieu in the RA-synovium, resident fibroblast-like synoviocytes (FLS) undergo hyperplasia, a hallmark event in RA. Sustained joint inflammation and further activation of FLS leads to joint destruction. The studies presented here build upon our previously published data in cancer cell lines suggesting activation of AHR can induce expression of pro-inflammatory genes, and several growth factors that are critical in cellular hyperplasia. We have also characterized the instrumental role of AHR in migratory and invasive phenotypes. Since higher levels of AHR protein have been observed in synovial tissues from RA patients, and the phenotype of RA-FLS is reminiscent to that of metastatic cancer cells; we hypothesized that antagonism of AHR by the potent AHR antagonist GNF351 could mitigate cytokine-induced expression of pro-inflammatory genes and growth factors. We also wished to study the role of AHR in primary RA-FLS proliferation, migration, and RA-FLS-mediated protease-dependent matrix degradation. Microarray analysis on RA-FLS exposed to proinflammatory interleukin 1B (IL1B) suggests the involvement of genes in multiple pathways responsible for RA pathogenesis. Based on the microarray data, certain cytokines, chemokines and growth factors that possess DREs in their promoter regions were picked using SCOPE v 2.1.0. Subsequent analysis of mRNA levels suggests AHR antagonist GNF351 can significantly repress such proinflammatory mediators as IL1B, IL6, prostaglandin-endoperoxide synthase 2 (PTGS2), C-C chemokine ligand 20 (CCL20) and growth factors such as vascular endothelial growth factor (VEGF)-A, epiregulin (EREG), amphiregulin (AREG) and basic fibroblast growth factor (FGF)-2. We further identified that the suppressive effects of GNF351 were AHR-dependent. In order to elucidate the molecular mechanism(s) behind GNF351-mediated mitigation, we performed promoter analysis and chromatin-immunoprecipitation (ChIP) assays. Our mechanistic data demonstrate that the occupancy of AHR on the promoters of IL1B, VEGF-A, EREG and AREG facilitates cytokine-mediated induction of these genes. AHR antagonist GNF351 treatment can inhibit AHR translocation into the nucleus and thus prevent promoter occupancy by the AHR/ARNT heterodimer and thus attenuating the cytokine-mediated gene transcription. Due to enhanced levels of proinflammatory mediators and growth factors, FLS in RA-synovium can undergo transformation resulting in FLS-hyperplasia and pannus formation. GNF351 suppressed cytokine-induced RA-FLS hyperplasia. Once activated, these FLS become highly invasive and migratory, and the AHR plays a critical role in such phenotypes. GNF351 not only inhibited migration of RA-FLS but also suppressed mRNA expression of matrix metalloproteinases (MMP-2 and 9) and gelatin matrix degradation. Numerous attempts have been made in RA research to block these aforementioned pathways individually with limited success. However, a suppressive effect of GNF351 on all these pathways collectively reveals that targeting the AHR is a viable combinatorial therapeutic strategy for the amelioration of RA.