Estrogen, SNP-Dependent Chemokine Expression and Selective Estrogen Receptor Modulator Regulation

Abstract

We previously reported, on the basis of a genome-wide association study for aromatase inhibitor-induced musculoskeletal symptoms, that single-nucleotide polymorphisms (SNPs) near the T-cell leukemia/lymphoma 1A (TCL1A) gene were associated with aromatase inhibitor-induced musculoskeletal pain and with estradiol (E2)-induced TCL1A expression. Furthermore, variation in TCL1A expression influenced the downstream expression of proinflammatory cytokines and cytokine receptors. Specifically, the top hit genome-wide association study SNP, rs11849538, created a functional estrogen response element (ERE) that displayed estrogen receptor (ER) binding and increased E2 induction of TCL1A expression only for the variant SNP genotype. In the present study, we pursued mechanisms underlying the E2-SNP-dependent regulation of TCL1A expression and, in parallel, our subsequent observations that SNPs at a distance from EREs can regulate ERα binding and that ER antagonists can reverse phenotypes associated with those SNPs. Specifically, we performed a series of functional genomic studies using a large panel of lymphoblastoid cell lines with dense genomic data that demonstrated that TCL1A SNPs at a distance from EREs can modulate ERα binding and expression of TCL1A as well as the expression of downstream immune mediators. Furthermore, 4-hydroxytamoxifen or fulvestrant could reverse these SNP-genotype effects. Similar results were found for SNPs in the IL17A cytokine and CCR6 chemokine receptor genes. These observations greatly expand our previous results and support the existence of a novel molecular mechanism that contributes to the complex interplay between estrogens and immune systems. They also raise the possibility of the pharmacological manipulation of the expression of proinflammatory cytokines and chemokines in a SNP genotype-dependent fashion.

Figure 1.

Locus Zoom plot showing the association of SNPs identified during the MA.27 musculoskeletal adverse event GWAS within the TCL gene cluster on chromosome 14.

Figure 2.

TCL1A SNPs regulate ERα binding to ERE motifs 3′ of TCL1A. A, Schematic diagrams of two SNP, rs7359033 and rs7160302, in tight LD with rs11849538, the top hit signal from the MA.27 GWAS. Locations of EREs are shown as boxes for these three SNPs that map between the 3′-termini of TCL1A and TCL1B. Distances between the SNPs and EREs are shown and blue arrows indicate the locations of primers used to conduct the ChIP amplifications. B and C, SNP- and estrogen-related variation in TCL1A mRNA expression in LCLs. The cells were treated with varying concentrations of E2 or with 0.1 nM E2 plus increasing concentrations of ICI-182780 (ICI) or 4-OH-TAM (4OH) for an additional 24 hours. Eight cell lines homozygous for the variant (V) genotypes for all three of these SNPs and eight cell lines homozygous for WT genotypes were used in these experiments. D–F, ChIP assays showing the effect of ERα binding to EREs (note that the WT sequence for the rs11849538 SNP did not encode an ERE, so there was no binding; see panel D) in or near the SNPs in response to E2 (0.1 nM), E2 (0.1 nM) plus 4-OH-TAM (10⁻⁷ μM), or ICI (10⁻⁷ μM) treatment of LCLs homozygous for either WT or V genotypes for the rs11849538, rs7359033, and rs7160302 SNPs. Percentage of ChIP DNA/input was determined by qPCR. The level of enrichment was expressed as relative enrichment above background (enrichment relative to IgG control). Data are represented as percentage input (enrichment relative to IgG control) = percentage input (ERα antibody) − percentage input (IgG). *, P < .05, **, P < .005 comparing WT and V SNP genotype cell lines at the same concentrations of E2 and ICI. Three independent experiments were performed (n = 8 for each genotype). G, TCL1A luciferase activity as a function of TCL1A SNP genotypes. HEK293T cells were transfected with the PGL3 basic vector containing the TCL1A promoter and a 548-bp fragment of DNA containing homozygous WT sequences for the rs11849538 and rs7359033 SNPs was subcloned into SaIl and BamHl sites and to create a PGL3-TCL1A-WT reporter construct (referred to as PGL3-TCL1A-WT). This construct was used to perform site-directed mutagenesis and was mutated to variant sequences for rs11849538 and rs7359033 SNPs (referred as PGL3-TCL1A-V). The rs7160302 SNP was 7173 bp distant from TCL1A, so we were unable to include this SNP in the reporter gene construct. Twenty-four hours after transfection, cells were treated with either vehicle or 0.1 nM E2 for 24 hours, followed by 4-OH-TAM or ICI for an additional 24 hours. Luciferase activity was measured 72 hours after transfection. The firefly luciferase activity was normalized by the use of Renilla luciferase to correct for possible variation in transfection efficiency. Results were expressed as fold change in relative luciferase units (RLU) compared with the PGL3 basic vector. Experiments were repeated three times in triplicate.

Figure 3.

E2 induction of TCL1A expression is associated with the expression of CCR6 (A) CCL20 (B), IL-17RA (C), and IL-17A (D). *, P < .05 comparing cell lines homozygous for TCL1A WT and V SNP genotypes at the same concentrations of E2 and 4-OH-TAM. Three independent experiments were performed. Eight cell lines of each genotype were used in each experiment.

Figure 4.

TCL1A is associated with the expression of CCR6 and CCL20. A, Relative mRNA expression of TCL1A, TCL1B, TCL6, CCR6, CCL20, CCR1, CCL5, CCRT, IL-17RA, and IL-17A after knockdown of TCL1A using pooled siRNA in lymphoblastoid cell lines. Eight cell lines of each genotype were used in the experiment. *, P < .05. B, Western blot analysis of TCL1A, IL-17A, IL-17RA, CCR6, CCL20, TCL1B, and ACTB in LCLs after TCL1A was knocked down using pooled TCL1A siRNA. Three independent experiments were performed.

Figure 5.

TCL1A SNP- and estrogen-dependent variation in TCL1B (A) and TCL6 (B) mRNA expression in LCLs. The figure shows the effect of 24-hour incubations with varying concentrations of E2 or 0.1 nM E2 plus increasing concentrations of 4-OH-TAM for an additional 24 hours. A panel of eight homozygous WT and eight homozygous variant (V) LCLs was used to perform the study. *, P < .05 comparing WT and V SNP genotype cell lines at the same concentrations of E2 and 4-OH-TAM. Data represent mean ± SEM. Three independent experiments were performed. Eight cell lines of each genotype were used in the experiment. C, Western blot analysis of TCL1A, IL-17A, IL-17RA, CCR6, CCL20, TCL1B, and ACTB in LCLs after TCL1B was knocked down.

Figure 6.

IL17A SNPs are associated with ERα binding to ERE motifs. A, Schematic diagram of the IL17A promoter rs2275913 SNP. The locations of three EREs (referred to as ERE1, ERE2, and ERE3) that mapped near the SNP are indicated. Distances between the SNP and EREs are listed under the ERE boxes, and blue arrows indicate the locations of primers used to perform the ChIP amplifications. B, ChIP assays showing ERα binding to EREs near the SNP in response to E2 (0.1 nM), E2 (0.1 nM) plus 4-OH-TAM (4OH in the figure) (10⁻⁷ μM), or ICI (10⁻⁷ μM) treatment of LCLs homozygous for either WT or V SNP genotypes for rs2275913. C–F, Effects of E2 and E2 plus 4-OH-TAM or ICI on the expression of IL-17A (C and E) and IL-17RA (D and F). A panel of eight homozygous WT and eight homozygous variant (V) LCLs was used to perform this study. All LCLs were homozygous for WT genotypes for the SNPs 3′ to TCL1A. *, P < .05, **, P < .005 comparing WT and V SNP genotype cell lines at the same concentrations of E2 and 4-OH-TAM or ICI. G, Western blot analysis of IL-17A, IL-17RA, and ACTB in LCLs after IL-17A was knocked down using two individual siRNAs. Three independent experiments were performed.

Figure 7.

CCR6 SNPs are associated with ERα binding to ERE motifs. A, Schematic diagram of the two EREs near the CCR6 rs3093023 SNP. Distances between the SNP and the EREs are listed under the ERE boxes, and blue arrows indicate the locations of primers used to conduct the ChIP amplifications. B and C, ChIP assays showing ERα binding to the two EREs (referred to as ERE1 and ERE2) near the SNP in response to E2 (0.1 nM), E2 (0.1 nM) plus 4-OH-TAM (10⁻⁷ μM), or ICI (10⁻⁷ μM) treatment of LCLs homozygous for either WT or V SNP sequences for rs3093023. D–G, Effects of E2 and E2 plus 4-OH-TAM or fulvestrant (ICI-182780) on the expression of CCR6 (C and F) and CCL20 (D and G). A panel of eight homozygous WT and eight homozygous variant (V) LCLs was used to perform the study. All LCLs were homozygous for WT genotypes for the SNPs 3′ to TCL1A. *, P < .05, **, P < .005 comparing WT and V SNP genotype cell lines at the same concentrations of E2 and 4-OH-TAM or ICI. H, Western blot analysis of CCR6, CCL20, and ACTB in LCLs after CCR6 was knocked down using two individual siRNAs. Three independent experiments were performed.