Intron Retention Generates a Novel Isoform of the Murine Vitamin D Receptor That Acts in a Dominant Negative Way on the Vitamin D Signaling Pathway

Abstract

We identified and characterized a novel rat vitamin D receptor isoform (rVDR1), which retains intron 8 of the canonical VDR (rVDR0) during alternative splicing. In this isoform protein directed by the stop codon in this newly identified exon, a part of the ligand binding domain (86 amino acids) is truncated at the C-terminal end but contains 19 extra amino acids. The rVDR1 transcript was expressed at a level 1/15 to 1/20 of that of rVDR0 in the kidney and intestine in adult rats but not in embryos. The recombinant rVDR1 protein showed no ligand binding activity. Homo-and heterodimers of the recombinant rVDR0 and rVDR1 proteins bound to a consensus vitamin D response element (VDRE) but not to consensus response elements for thyroid hormone and retinoic acid. However, unlike rVDR0, rVDR1 did not form a heterodimeric complex with RXR on the VDRE. A transient expression assay showed that this isoform acted as a dominant negative receptor against rVDR0 transactivation. Interestingly, the dominant negative activities of rVDR1 differed among VDREs. Thus, the present study indicates that this new VDR isoform negatively modulates the vitamin D signaling pathway, through a particular set of target genes.Most of the biological actions of 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ], such as regulating calcium homeostasis and cytodifferentiation, are exerted through gene expression mediated by the nuclear vitamin D receptor (VDR) (for a review, see reference 8). VDR is a member of the nuclear receptor superfamily, which functions as a ligand-inducible transcription factor (for reviews, see references 2, 10, 13, and 39). This family includes nuclear receptors for steroid hormones, thyroid hormone, retinoic acid, and unknown ligands (orphan receptors). Together with the retinoid receptors (RAR␣, -␤, and -␥ and RXR␣, -␤, and -␥) and thyroid hormone receptors (TR ␣ and ␤), VDR forms a subfamily, based on structural and functional similarities, in the nuclear receptor superfamily. Like RARs and TRs, VDR heterodimerizes with RXR. These heterodimers bind distinct but similar target enhancer elements composed of two directly repeated core AGGTCA (or related hexamer core) motifs. The spacer between the two core motifs (3 bp [DR3] for RXR/VDR, 4 bp [DR4] for RXR/TR, and 2 [DR2] and [DR5] 5 bp for RXR/RAR) discriminates among these nuclear receptors for recognizing target enhancer elements (for reviews, see references 12, 19, 22, 41, and 50). In addition to the heterodimeric form of VDR with RXR, VDR forms a homodimer with some vitamin D response elements (VDRE), suggesting the presence of two signaling pathways for vitamin D (5, 48). Irrespective of the functional and structural similarities of these receptors, only one type of VDR protein has been found in sharp contrast to multiple subtypes and isoforms of RAR, RXR, and TR (17, 24, 53). The RAR and TR isoforms are generated by alternative splicing and/or by direction of differential promoters. Functional analysis by a transient-expression assay showed that the transactivational acti...

The vitamin D receptor and its ligand 1α,25-dihydroxyvitamin D3 in Atlantic salmon (Salmo salar)

“…Intron retention has been demonstrated for a mouse VDR once before (Ebihara et al 1996). This is the first report for such a phenomenon outside mammals, in a teleostean VDR.…”

“…Intron retention of both the sVDR1 (intron 7) and sVDR2 (intron 6) occurs in the E/F domain and as a consequence both receptors are predicted to be impaired in retinoid X receptor (RXR) heterodimerization and ligand binding. The splicing of the murine VDR occurs at intron 8, and also in the E/F domain (Ebihara et al 1996), and it was concluded that homodimer-spliced variants may compete at specific VDR responsive elements and by doing so prevent actions of the canonical VDR. We can only speculate on a similar role for the two spliced variants in salmon.…”

“…The canonical salmon VDR and splice variant sVDR1 could function along the model proposed by Norman (2006); the splice variant sVDR2 lacks amino acids R274 and S278, two residues that are crucial for ligand stabilization and their absence could impair functionality of this receptor. As both splice variants are impaired in their heterodimerization with the RXR (Ebihara et al 1996), genomic actions seem excluded, and rapid membrane-bound receptor activity remains a possibility. Indeed, it now appears that the VDR in some cells is associated with caveolae in or near the plasma membrane (Huhtakangas et al 2006), which would give an anatomical argument for plasma membraneassociated VDR activity.…”

The role of vitamin D receptor gene polymorphisms in Turkish infants with urolithiasis

“…As mentioned above, polymorphisms on the VDR gene does not cause a particular amino acid sequence. Ebihara et al 27 performed a study on a novel rat population which retains the intron 8 of VDR during alternative splicing, and showed that this event resulted in negative modulation in the vitamin D signaling pathway through a set of target genes. That the same type of change could occur in the human VDR gene is highly probable, and the relationship between VDR gene polymorphisms and phenotypes could be explained by the differences in the intron.…”

Repression of basal transcription by vitamin D receptor: evidence for interaction of unliganded vitamin D receptor with two receptor interaction domains in RIP13delta1

“…This phenomenon and the possible role of RIP13 1 in repression remains to be fully elucidated. A naturally occurring dominant negative isoform of VDR has been reported (Ebihara et al 1996) and thus basal and hormone induced levels of certain 1,25-(OH) 2 D 3 -responsive genes in different cells may be dependent upon the presence and amount of both negative and positive coregulators.…”