Background Joint pain and stiffness are major side effects associated with aromatase inhibitor (AI) therapy. In a recent genome-wide association study, we identified 4 SNPs near the T-cell leukemia 1A (TCL1A) gene that were associated with these musculoskeletal adverse events . We demonstrated that the presence of these SNPs does result in changes of the estrogen-dependent expression of TCL1A and additional downstream effects on the expression of IL17, IL17RA, IL12, IL12RB2 and IL1R2 . As these findings suggest that TCL1A exerts key regulatory effects on multiple cytokines and cytokine receptors in response to estrogen, we aimed to clarify whether TCL1A expression may also influence chemokine and chemokine receptor expression.
Objectives To determine whether TCL1A variants, which are associated with AI induced musculoskeletal side effects, influence the expression of chemokines and chemokine receptors in response to estrogen
Methods We genome-wide genotyped 300 human lymphoblastoid cells using the Illumina 550K and 510S SNP BeadChip and the Affymetrix SNP array 6.0. We also generated basal Affymetrix U133 2.0 Plus GeneChip expression array data for all cell lines. Associations between TCL1A expression and chemokine/chemokine receptor expression were validated using siRNA knockdown studies. Functional genomic experiments included determinations of TCL1A, chemokine and chemokine receptor expression in response to estradiol (E2) treatment. To predict putative estrogen receptor binding we queried the TRANSFEC database. The functional relevance of putative binding sites was confirmed using ChIP assays.
Results TCL1A expression was associated with expression of CCR7, CCL20, CCR6, CCR1 and CCL5 (p≤10–8). TCL1A knockdown did demonstrate significant changes in CCR6 and CCL20 expression. No changes were observed for CCR7, CCR1 and CCL5.
Under treatment with E2, CCR6 and CCL20 expression was significantly increased in a dose-dependent manner only in cells with variant genotype.
Using the TRANSFAC database, we predicted that two SNPs, which are in high linkage disequilibrium with the SNPs that where associated with AI induced musculoskeletal pain, create a putative estrogen response element. The ChIP assay demonstrated greater DNA-estrogen receptor α protein binding in cells with the TCL1A SNP variant genotype compared to wild-type cells.
Conclusions TCL1A variants that were associated with AI-dependent musculoskeletal pain, did alter the E2 induced TCL1A expression and it's downstream effects on IL17, CCR6 and CCL20. The CCR6-CCL20–mediated migration of Th17 cells has been suggested as important pathogenetic mechanism in a variety of autoimmune diseases such as rheumatoid arthritis.
Our results provide further insight as to how changes in estrogen levels occurring naturally or as a result of pharmacotherapeutic interventions can modulate various pro-inflammatory pathways.
Future studies will be required to show whether the SNP dependent response of TCL1A to estrogen and its downstream effect on the CCR6/CCL20/IL-17 is of clinical significance in autoimmune diseases that display variation in incidence and disease activity dependent on hormonal status.
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Disclosure of Interest : None declared
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