Background/Purpose: The rheumatoid arthritis (RA) risk locus CCR6 SNP rs3093024, is in tight linkage disequilibrium with rs3093023 which has been associated with RA risk. RA is more common in females with an increased risk of disease and flares during the postpartum or postmenopausal periods—times of rapidly falling estrogen levels. We previously reported that these two SNPs in CCR6 are associated with changes in the expression of CCR6 and, downstream, those of CCL20, IL17RA and IL17A in a SNP-dependent fashion in response to 17-β-estradiol (E2) treatment. In the present study, we set out to determine the effects of these CCR6 SNPs on the expression of CCR6, CCL20, IL17RA and IL17A during estrogen withdrawal, which has been implicated in RA pathogenesis, as well as mechanism(s) underlying CCR6SNP function and estrogen-dependent gene expression regulation by functional genomic studies.

Methods: “Human Variation Panel” lymphoblastoid cell lines (LCLs) consisting of LCLs from 300 subjects were used to obtain genome-wide mRNA expression and SNP data. A panel of LCLs with six wild-type (WT; homozygous) and six variant (V; homozygous) genotypes for these SNPs, were used for functional genomic study. Cells were treated with E2 and the estrogen receptor α (ERα) antagonist fulvestrant. Changes in mRNA and protein expression after treatment were determined. ChIP assays were used to determine ERα binding to estrogen response elements (EREs) before and after ER blockade.

Results: Knockdown of CCR6 resulted in down-regulation of NF-κB p65 and phosphorylated IκB, indicating that CCR6 plays a role in inflammatory pathways. CCR6, CCL20, IL17RA and IL17A expression was altered in a CCR6 SNP estrogen-dependent fashion. Specifically, CCR6 and IL17RA were significantly up-regulated in response to E2 treatment, but only in cells homozygous for the CCR6 SNP V alleles. Conversely, the expression of CCL20 and IL17A, which are the ligands for CCR6 and IL17RA respectively, were significantly increased in response to E2 treatment, but only in cells homozygous for WT alleles for the CCR6 SNPs. However, this pattern of gene expression could be ‘’reversed’’ in a CCR6SNP-dependent fashion in the presence of the ERα antagonist fulvestrant, compatible with our DNA-ERα protein binding observations for two EREs near the rs3093023 SNP. When E2 was present, cells homozygous for the V allele for rs3093023 displayed elevated ERα binding compared with cells homozygous for the WT allele. However, a reversal of ERα binding was observed in the presence of fulvestrant.

Conclusion: We observed differential ERα binding before and after ER blockade which might be associated with underlying mechanisms for the SNP and estrogen-dependent variation of CCR6 expression and, downstream, the expression of CCL20, IL17RA and IL17A–all of which play roles in the pathogenesis of RA. These observations also suggest that the expression of immune mediators could potentially be pharmacologically manipulated in a SNP-dependent fashion. Our findings suggest a novel molecular mechanism that may play an important role in the interplay between estrogen and immune mediators in RA—a disease that displays striking variation in incidence and disease activity which might be related to hormonal status.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/rheumatoid-arthritis-ra-risk-gene-ccr6-polymorphism-drug-induced-reversal-of-er-binding-and-the-expression-of-inflammatory-mediators-potential-molecular-mechanism-links-between-estrogen-and/