A Central Dinucleotide within Vitamin D Response Elements Modulates DNA Binding and Transactivation by the Vitamin D Receptor in Cellular Response to Natural and Synthetic Ligands

Abstract

includes regulating the expression of genes involved in calcium and bone metabolism (1). In addition, the hormone is an important mediator of cell growth and differentiation (2, 3). In a large number of 1,25-(OH) 2 D 3 -dependent target genes, distinct vitamin D response elements (VDREs) have been identified that are composed of two hexameric half-sites separated by three base pairs (DR3-type VDRE). The vitamin D receptor (VDR)/retinoid X receptor (RXR) complex binds with defined polarity to these VDREs with the RXR occupying the distal half-site and the VDR the proximal half-site (4 -7).The spacing of the half-sites plays an important role in specifying the type of receptor pair that interacts with hormone response elements (7). There is considerable divergence in the nucleotide sequence of the distal and proximal half-site, which is attributable at least in part to redundancy in the consensus recognition motifs of the VDR and the RXR (8). The sequences of steroid hormone half-elements also influence the relative affinities for different steroid hormone receptors and thus may support receptor-specific gene activation by discriminating between different receptors (7). In addition, specific nucleotides in the VDRE appear to influence the conformation of bound VDR/RXR heterodimers, reflected by a VDRE-specific alteration in epitope accessibility (6). Furthermore, minor changes in the nucleotide sequence of half-elements can change a negative VDRE that suppresses transcription into a positive VDRE, which transactivates in a 1,25-(OH) 2 D 3 -dependent manner (9). The impact of minor changes in nucleotide sequences on receptor DNA binding is not restricted to VDR/ VDRE interactions, as evidenced by half-element point mutations that have been shown to alter the affinity (10) or identity of the cognate receptor (11).Two major 1,25-(OH) 2 D 3 -regulated genes are the two noncollagenous, osteoblast-derived bone matrix proteins osteocalcin and osteopontin. These genes have sequence variations in their VDREs (Refs. 12-14 and Table I). To gain insight into the biological relevance of nucleotide variation in naturally occurring VDREs for the action of 1,25-(OH) 2 D 3 and its potent analog KH1060 (15-18), we examined the binding and activity of the osteocalcin and osteopontin VDREs. The main finding of our study is that nucleotide differences between osteopontin and osteocalcin VDREs affect DNA binding and transactivation in response to 1,25-(OH) 2 D 3 and its potent analog, KH1060. Our findings show that the primary sequence of VDREs may influence the biological activities of natural and synthetic ligands that bind to the VDR. Finally, the strong biological potency of the analog KH1060 is only reflected by DNA binding of VDR/RXR heterodimers in the environment of nuclear proteins and not by the binding of in vitro synthesized VDR and RXR␣, underscoring the importance of a cellular context for ligand-induced DNA binding.

Identification of an Endogenous Ligand Bound to a Native Orphan Nuclear Receptor

“…It has been well established for other NR-ligand pairs that ligands can have different effects on different promoters [9], [45]–[47]. Furthermore, we hypothesized that LA might have a more pronounced effect on endogenous targets with the appropriate chromatin structure.…”

Direct Regulation of HOXA10 by 1,25-(OH)2D3in Human Myelomonocytic Cells and Human Endometrial Stromal Cells

“…Additionally, specific nucleotides in the VDRE influence the conformation of bound VDR/ RXR heterodimers and determine functional activity. Minor changes in the nucleotide sequence of halfelements can change a negative VDRE that suppresses transcription into a positive VDRE that activates in a 1,25-(OH) 2 D 3 -dependent manner (56,57). Osteocalcin and osteopontin are two well-characterized 1,25-(OH) 2 D 3 -regulated genes in osteoblasts.…”

Osteogenic Differentiation of Stem Cells Alters Vitamin D Receptor Expression

“…VDR and PDIA3 are receptors for 1a,25(OH) 2 D 3 ; osteoprotegerin (OPG) is secreted by differentiated osteoblasts [41]; osteopontin is a 1a,25(OH) 2 D 3 -sensitive extracellular matrix protein that possesses a VDRE [42] and is regulated via PDIA3 [39]; and a2b1 integrin expression is associated with osteoblast differentiation through collagen type 1 recognition [43,44].…”