A novel SNP in a vitamin D response element of the CYP24A1 promoter reduces protein binding, transactivation, and gene expression

Abstract

The active form of vitamin D (1alpha,25(OH)(2)D(3)) is known to have antiproliferative effects and has been implicated in cancers of the colon, breast, and prostate. These cancers occur more frequently among African Americans than Caucasians, and individuals with African ancestry are known to have approximately twofold lower levels of serum vitamin D (25(OH)D) compared with individuals of European ancestry. However, epidemiological studies of the vitamin D receptor (VDR) have shown inconsistent associations with cancer risk, suggesting that differences in other genes in the pathway may be important. We sought to identify functionally significant polymorphic variants in CYP24A1, a gene that is highly inducible by 1alpha,25(OH)(2)D(3) and that encodes the primary catabolic enzyme in the pathway. Here we report the identification of six novel SNPs in the human CYP24A1 promoter, including one at nucleotide -279 occurring within the distal vitamin D response element (VDRE2). Our experiments demonstrate that the VDRE2 variant results in decreased protein binding and transactivation in vitro, and reduced expression of CYP24A1 in cultured primary human lymphocytes provides evidence for an effect in vivo. This variant was only observed in our African American population, and represents a first step toward understanding differences in disease risk among racial/ethnic groups.

Figure 1

Polymorphisms observed in sequencing the human CYP24A1 promoter. The positions of both VDRE and all eight SNPs are shown relative to the transcription start site. Sequences indicated for each VDRE are on the opposite strand from the one that is transcribed.

Figure 2

Transactivation of wild-type and polymorphic CYP24A1 promoter constructs by 1α,25(OH)₂D₃. (A) H1299 and (B) MCF-7 cells were transfected with either wild-type or polymorphic CYP24A1 promoter luciferase constructs alone or in combination with expression plasmids for VDR and RXRα as shown and as described in Section 2. Following 16 h treatment with 100 nM 1α,25(OH)₂D₃ or vehicle, firefly luciferase activity was measured and normalized to Renilla luciferase. Fold inductions by 1α,25(OH)₂D₃ for each of the reporter constructs are shown above their respective bars. All conditions were done in duplicate, and values are the mean of three independent experiments with standard deviations as shown.

Figure 3

Binding of VDR and RXRα to the wild-type and polymorphic vitamin D response elements. (A) EMSA showing impaired binding of VDR-RXRα heterodimers to the polymorphic VDRE probe (lanes 13 and 14) compared with wild-type (lanes 6 and 7). Where indicated, 100 ng recombinant human VDR and/or RXRα proteins were added in the presence or absence of 1 μM 1α,25(OH)₂D₃. (B) Second EMSA testing the impact of 50 vs. 150 mM salt and competitive binding. Binding to wild-type (lanes 1-4) and polymorphic (lanes 5-8) VDRE probe was assayed under both low and high salt conditions with proteins and 1α,25(OH)₂D₃ added as indicated. Competitive binding reactions were also done with both salt conditions using a 200-fold molar excess of unlabeled competitor probes for the wild-type (lanes 9-10) or polymorphic elements (lanes 11-12).

Figure 4

Quantitative PCR of CYP24A1 and VDR gene expression in cultured primary lymphocytes. As described in the Materials and Methods, RNA was isolated from treated and untreated primary lymphocytes from subjects who were either wild-type or heterozygous polymorphic for the CYP24A1 VDRE2 variant. Following reverse transcription, equal amounts of cDNA from each were used for qRT-PCR of CYP24A1 and VDR. Mean relative expression was determined using the SDS program by normalizing to GAPDH and using the ΔΔCt method to calculate the relative quantification of treated vs. untreated cells from the same person. Fold induction of CYP24A1 expression by 1α,25(OH)₂D₃ is shown for each subject above their respective bars. All reactions were done in triplicate and are the mean of two separate qRT-PCR runs.