A 55-kilodalton accessory factor facilitates vitamin D receptor DNA binding.

The interaction of the vitamin D receptor with a vitamin D-responsive element (VDRE) derived from the human osteocalcin promoter in vitro has been shown to require a nuclear accessory factor (NAF) derived from monkey kidney cells. In this report we show that this factor is widely distributed in cells and tissues, including those that do not express the vitamin D receptor (VDR). NAF is required for VDR binding to a variety of known VDREs. VDR and NAF independently bind the VDRE weakly, as assessed by elution profiles generated during VDRE affinity chromatography. Together, however, both proteins coelute from this column with a profile that indicates a tighter strength of interaction. Analogous chromatography of the VDR derived from ROS 17/2.8 cells treated with 1,25-dihydroxyvitamin D3 in culture also reveals a dual profile of weak and strong binding, suggesting that in vivo modifications are unlikely to alter receptor DNA binding. NAF is a protein of 55 kDa, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and cross-linking experiments suggest that the VDR and NAF together form a heterodimer on a single VDRE with a mol wt of 103 kDa. These data demonstrate that NAF is required for VDR binding to specific DNA in vitro and suggest the possibility that NAF may be required for the transactivation capability of the VDR in vivo.     

In vivo function of VDR in gene expression-VDR knock-out mice

Vitamin D exerts many biological actions through nuclear vitamin D receptor (VDR)-mediated gene expression. The transactivation function of VDR is activated by binding 1,25-dihydroxyvitamin D₃[1,25(OH)₂D₃], an active form of vitamin D. Conversion from 25(OH)D₃ is finely regulated in kidney by 25(OH)D₃ 1-hydroxylase[25(OH)D 1-hydroxylase], keeping serum levels of 1,25(OH)₂D₃ constant. Deficiency of vitamin D and mutations in the genes like VDR (type II genetic rickets) are known to cause rickets like lowered serum calcium, alopecia and impaired bone formation. However, the molecular basis of vitamin D–VDR system in the vitamin D action in intact animals remained to be established. In addition, the 1-hydroxylase gene from any species had not yet been cloned, irrespective of its biological significance and putative link to the type I genetic rickets. We generated VDR-deficient mice (VDR KO mice). VDR KO mice grew up normally until weaning, but after weaning they developed abnormality like the type II rickets patients. These results demonstrated indispensability of vitamin D–VDR system in mineral and bone metabolism only in post-weaning life. Using a newly developed cloning system, we cloned the cDNA encoding a novel P450 enzyme, mouse and human 1-hydroxylase. The study in VDR KO mice demonstrated the function of liganded VDR in the negative feed-back regulation of 1,25(OH)₂D₃ production. Finally, from the analysis of type I rickets patients, we found missense genetic mutations in 1-hydroxylase, leading to the conclusion that this gene is responsible for the type I rickets.     

DNA bending is induced by binding of vitamin D receptor-retinoid X receptor heterodimers to vitamin D response elements.

The ability of vitamin D receptor-retinoid X receptor (VDR-RXR) heterodimers to induce a DNA bend upon binding to various vitamin D response elements (VDRE) has been investigated by circular permutation and phasing analysis. Recombinant rat VDR expressed in the baculovirus system and purified recombinant human RXR beta have been used. The VDREs were from 1,25-dihydroxyvitamin D3 (1,25-[OH]2D3) enhanced genes (rat osteocalcin, rOC; mouse osteopontin, mOP, and rat 1,25-dihydroxyvitamin D3-24-hydroxylase, r24-OHase), and a 1,25-(OH)2D3 repressed gene (human parathyroid hormone, hPTH). As shown by circular permutation analysis, VDR-RXR induced a distortion in DNA fragments containing various VDREs. Calculated distortion angles were similar in magnitude (57 degrees, 56 degrees, 61 degrees, and 59 degrees, respectively for rOC, mOP, r24-Ohase, and hPTH). The distortions took place with or without a 1,25-(OH)2D3 ligand. The centers of the apparent bend were found in the vicinity of the midpoint of all VDREs, except for rOC VDRE which was found 4 bp upstream. Phasing analysis was performed with DNA fragments containing mOP VDRE and revealed that VDR-RXR heterodimers induced a directed bend of 26 degrees, not influenced by the presence of hormone. In this study we report that similar to other members of the steroid and thyroid nuclear receptor superfamily, VDR-RXR heterodimers induce DNA bending.     

Vitamin D receptor interactions with the murine osteopontin response element

The nature of the DNA binding interactions of the human vitamin D receptor (hVDR) with the murine osteopontin vitamin D response element (mOP VDRE) was examined. Both recombinant hVDR and human retinoid X receptor β (hRXRβ) proteins were obtained from baculovirus-infected Sf9 insect cells. Mixing extracts of the two recombinant proteins resulted in the strong, specific formation of a slower migrating complex in the electrophoretic mobility shift assay. Crude extracts of the expressed hVDR alone were also capable of binding with high affinity to the mOP sequence, and this binding was enhanced in the presence of 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃). Competition experiments confirmed the specificity of this interaction and revealed that the human osteocalcin VDRE was a poor competitor for this binding. Ethylation interference footprint analyses of hVDR/hRXRβ and hVDR complexes revealed only subtle differences in how these two different VDR-containing complexes interacted with the mOP VDRE. The footprints displayed contact points in both halves of the direct repeat format, confirming the dimeric and major groove interactions of both types of complexes. DNA affinity chromatography of labelled hVDR extracts revealed a peak eluting at ca. 290 mM KCl that was capable of rebinding to the mOP sequence in gel shift experiments. Ultraviolet (UV) light-crosslinking experiments of hVDR extracts alone to radiolabelled DNA were consistent with the existence of a homodimeric hVDR interaction. Additionally, these experiments confirmed the direct interaction of a hVDR/hRXRβ heterodimer when mixed extracts were utilized. From these results we infer that homodimers of the hVDR which respond with enhanced DNA binding to particular vitamin D response elements when exposed to 1,25-(OH)₂D₃ are possible. This may be of functional significance when RXR proteins are limiting or RXR ligand is present within a cell.     

Vitamin D receptor interaction with specific DNA. Association as a 1,25-dihydroxyvitamin D3-modulated heterodimer.

The vitamin D receptor (VDR) is a member of the steroid receptor gene family. In this report, we examine the nature of specific VDR DNA binding utilizing the vitamin D-responsive element derived from the human osteocalcin promoter. Association of the VDR with the human osteocalcin 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) responsive element (VDRE) in vitro was characterized on VDRE affinity columns by both weak and strong interactions. Weak interaction was a property of the VDR itself, monomeric in nature, and determined exclusively by the VDR's DNA-binding domain. Strong interaction, in contrast, was dependent upon an intact receptor molecule as well as a heterologous mammalian cell nuclear accessory factor (NAF). Heteromeric interaction between VDR and NAF was independent of the VDR DNA-binding domain, suggesting the presence of a functional dimerization domain separate from that for DNA binding. Direct association of NAF with immobilized VDR revealed that the interaction does not require the presence of DNA. Most importantly, while occupancy of the VDR by 1,25(OH)2D3 was not required for VDR interactions with either DNA or NAF, the presence of hormone increased the apparent relative affinity of the VDR for NAF approximately 10-fold. These studies suggest that high affinity association of the VDR with DNA requires both the DNA-binding domain as well as an additional independent structure located within the steroid-binding region. This protein subdomain interacts with NAF and is regulated by 1,25(OH)2D3.     

Vitamin D receptor interactions with positive and negative DNA response elements: an interference footprint comparison

Interference footprinting protocols were utilized to examine the interactions of the vitamin D receptor (VDR) with either a positive or a negative vitamin D response element (VDRE). A sequence from the human osteocalcin (hOC) gene was chosen for the prototypical positive DR+3 VDRE, while an analogous sequence linked to the avian parathyroid hormone gene (aPTH) was used as the negative VDRE. Both types of response elements were examined for phosphate backbone contacts, as well as base-specific interactions with guanine and thymine residues. Sources of VDR included partially purified canine intestinal preparations, as well as extracts of recombinant human VDR and retinoid X receptor alpha prepared from baculovirus-infected Sf9 insect cells. Cold competition experiments using variable amounts of these oligonucleotides in the mobility shift assay revealed that the hOC element was a five-fold better competitor for heterodimer complex binding than the negative VDRE. Interference footprints revealed extensive strong contacts to the phosphate backbone and individual guanine and thymine nucleotides of the hOC element. The composite hOC footprint was asymmetric for the number and strength of interactions observed over each of the respective direct repeat half-sites. In contrast, the aPTH VDRE footprints revealed fewer points of DNA contact that were limited to the hexanucleotide repeat regions and were strikingly weaker in nature. The alignment of DNA contact points for both elements produced a 5' stagger that was indicative of successive major groove interactions, and consistent with dimer binding. DNA helical representations indicate that the heterodimer contacts to these response elements are substantially different and provide insight into functional aspects of each complex.     

Circulating vitamin D3 and 25-hydroxyvitamin D in humans: An important tool to define adequate nutritional vitamin D status

Circulating 25-hydroxyvitamin D [25(OH)D] is generally considered the means by which we define nutritional vitamin D status. There is much debate, however, with respect to what a healthy minimum level of circulation 25(OH)D should be. Recent data using various biomarkers such as intact parathyroid hormone (PTH), intestinal calcium absorption, and skeletal density measurements suggest this minimum level to be 80 nmol (32 ng/mL). Surprisingly, the relationship between circulating vitamin D₃ and its metabolic product—25(OH)D₃ has not been studied. We investigated this relationship in two separate populations: the first, individuals from Hawaii who received significant sun exposure; the second, subjects from a lactation study who received up to 6400 IU vitamin D₃/day for 6 months.

Results (1) the relationship between circulating vitamin D₃ and 25(OH)D in both groups was not linear, but appeared saturable and controlled; (2) optimal nutritional vitamin D status appeared to occur when molar ratios of circulating vitamin D₃ and 25(OH)D exceeded 0.3; at this point, the V_max of the 25-hydroxylase appeared to be achieved. This was achieved when circulating 25(OH)D exceeded 100 nmol.

We hypothesize that as humans live today, the 25-hydroxylase operates well below its V_max because of chronic substrate deficiency, namely vitamin D₃. When humans are sun (or dietary) replete, the vitamin D endocrine system will function in a fashion as do these other steroid synthetic pathways, not limited by substrate. Thus, the relationship between circulating vitamin D and 25(OH)D may represent what “normal” vitamin D status should be.     

Identification of a functional vitamin D response element in the human insulin-like growth factor binding protein-3 promoter

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] plays a critical role in maintaining calcium and phosphate homeostasis and bone formation but also exhibits antiproliferative activity on many cancer cells, including prostate cancer. We have shown that the antiproliferative actions of 1,25-(OH)2D3 in the LNCaP human prostate cancer cell line are mediated in part by induction of IGF binding protein-3 (IGFBP-3). The purpose of this study was to determine the molecular mechanism involved in 1,25-(OH)2D3 regulation of IGFBP-3 expression and to identify the putative vitamin D response element (VDRE) in the IGFBP-3 promoter. We cloned approximately 6 kb of the IGFBP-3 promoter sequence and demonstrated its responsiveness to 1,25-(OH)2D3 in transactivation assays. Computer analysis identified a putative VDRE between -3296/-3282 containing the direct repeat motif GGTTCA ccg GGTGCA that is 92% identical with the rat 24-hydroxylase distal VDRE. In EMSAs, the vitamin D receptor (VDR) showed strong binding to the putative IGFBP-3 VDRE in the presence of 1,25-(OH)2D3. Supershift assays confirmed the presence of VDR in the IGFBP-3 VDRE complex. Chromatin immunoprecipitation assay demonstrated that 1,25-(OH)2D3 recruited the VDR/retinoid X receptor heterodimer to the VDRE site in the natural IGFBP-3 promoter in intact cells. In transactivation assays, the putative VDRE coupled to a heterologous simian virus 40 promoter construct was induced 2-fold by 1,25-(OH)2D3. Mutations in the VDRE resulted in a loss of inducibility confirming the critical hexameric sequence. In conclusion, we have identified a functional VDRE in the distal region of the human IGFBP-3 promoter. The induction of IGFBP-3 by 1,25-(OH)2D3 appears to be directly mediated via VDR interaction with this VDRE.