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Alternative Exon 3 Inclusion in the Vitamin D Receptor (VDR) Modulates Transcriptional Activity for Target Genes in a Ligand-Independent Manner

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  • Xenobiotic exposures may induce alternative splicing in the pre-mRNA of nuclear hormone receptor (NHR) genes, yielding unpredictable patterns of gene expression that may promote adverse drug events and promote environmental disease. Informed by observations in the androgen receptor (AR; class I NHR), whereby alternatively-spliced AR transcripts induce ligand-independent hormone signaling via interactions with the wild-type (WT) AR, we designed an analogous system for probing similar phenomenon in the vitamin D receptor (VDR). The VDR is a prototype, class II NHR that regulates transcription of itself, and several cytochrome P450 (CYP) genes (e.g. CYP24A1, CYP3A4 and CYP3A5) linked to vitamin D metabolism. We hypothesized that deletion of exon 3, which encodes the DNA-binding domain (DBD) in all NHRs, would create a dominant negative VDR capable of modulating WT VDR function in a ligand-independent manner. To explore this concept, a synthetic, exon 3 deleted VDR (DEx3) construct, which remains in-frame, was prepared and stably-integrated into Caco-2 (TC7) cells expressing WT VDR. Baseline transcription levels for VDR, CYP24A1, CYP3A4 and CYP3A5 were measured by quantitative RT-PCR (qPCR), and compared to levels from cells stably-integrated with the DEx3 construct. In the absence of calcitriol, DEx3 derepresses VDR transcriptional activity, potentiating a > 4000X increase in CYP24A1 transcript levels. In the presence of calcitriol (500 nM), DEx3 VDR is a dominant negative at the promoters of the VDR and CYP3A4, but not CYP24A1 or CYP3A5. Furthermore, DEx3 VDR interacts differentially with the CYP3A4 promoter in the presence of calcitriol, unlike the CYP3A5 promoter, suggesting a more complex steroidogenic response, possibly linked to interactions with the pregnane X receptor (PXR). Because transcript variants of NHR genes are increasingly encountered via RNAseq methods, the discrete phenotypic changes associated with their expression warrant further attention.
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  • This research was sponsored by the Department of Environmental & Molecular Toxicology in the College of Agricultural Sciences.
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