Molecular cloning of cDNA and genomic DNA for human 25-hydroxyvitamin D3 1 alpha-hydroxylase.

The 25-hydroxyvitamin D3 1 alpha-hydroxylase (1 alpha-hydroxylase) is a cytochrome P450 enzyme that catalyzes the conversion of 25-hydroxyvitamin D3 to 1 alpha,25-dihydroxyvitamin D3. This enzyme plays an important role in calcium homeostasis. Here we report the molecular cloning of cDNA and gene for human 1 alpha-hydroxylase.

Vitamin D and multiple sclerosis.

Recently, it has been clearly demonstrated that exogenous 1,25-dihydroxyvitamin D3, the hormonal form of vitamin D3, can completely prevent experimental autoimmune encephalomyelitis (EAE), a widely accepted mouse model of human multiple sclerosis (MS). This finding has focused attention on the possible relationship of this disease to vitamin D. Although genetic traits certainly contribute to MS susceptibility, an environmental factor is also clearly involved.

Cloning and characterization of the mouse vitamin D receptor promoter.

The gene encoding the mouse vitamin D receptor has been cloned. A new exon 1 has been found that changes the numbering established for the human VDR gene. Exons 2 and 3 in the human VDR gene (coding for the zinc fingers 1 and 2, respectively) are named exons 3 and 4 in the mouse vitamin D receptor.

The vitamin D receptor--structure and transcriptional activation.

The vitamin D receptor (VDR) is an integral part of the body's calcium regulatory system. In this review, recent advances in the understanding of VDR structure and function are discussed. Both direct mutagenesis studies on the VDR and structural studies of related receptors have been reviewed.

Association between intestinal vitamin D receptor, calcium absorption, and serum 1,25 dihydroxyvitamin D in normal young and elderly women.

The exact mechanism for the decrease in intestinal calcium absorption with age is not yet understood. A decrease with age in serum 1,25-dihydroxyvitamin D (1,25(OH)2D) or a decrease in the intestinal vitamin D receptor (VDR) protein concentration are possible causes. The objective of this study was to examine the effect of age on these factors.

Effect of vitamin D receptor genotypes on calcium absorption, duodenal vitamin D receptor concentration, and serum 1,25 dihydroxyvitamin D levels in normal women.

It is well established that bone mineral density is under strong genetic control. Recently it was reported that the Bsm I restriction fragment length polymorphism of the vitamin D receptor (VDR) gene could account for up to 75% of the genetic variance in bone mineral density. However, the physiological basis for such an effect has not been established.

Salt concentration determines 1,25-dihydroxyvitamin D3 dependency of vitamin D receptor-retinoid X receptor--vitamin D-responsive element complex formation.

The electrophoretic mobility shift assay was used to determine in vitro formation of the vitamin D receptor-retinoid X receptor beta (VDR-RXR beta) heterodimer complex on vitamin D-response elements (VDREs) from rat osteocalcin, mouse osteopontin, rat 25-hydroxyvitamin D3 24-hydroxylase, and human parathyroid hormone (PTH) genes. Baculovirus-expressed rat VDR was used as VDR and the binding reactions were performed at salt concentrations ranging from 50 to 170 mM KCI. Without ligand, optimum complex formation was observed at 50 mM KCI and markedly decreased with increasing KCI for all VDREs.

Cloning and origin of the two forms of chicken vitamin D receptor.

The cDNA for the chicken vitamin D receptor (VDR) has been cloned in full length by screening cDNA libraries from chicken intestine and chicken kidney. The chicken kidney cDNA library constructed by both oligo(dT) and random primer methods yielded several full-length clones. These contained a 66-bp additional coding sequence at the 5' end in comparison to the cDNAs of human, rat, and mouse VDR.

YY1 regulates vitamin D receptor/retinoid X receptor mediated transactivation of the vitamin D responsive osteocalcin gene.

The responsiveness of genes to steroid hormones is principally mediated by functional interactions between DNA-bound hormone receptors and components of the transcriptional initiation machinery, including TATA-binding protein, TFIIB, or other RNA polymerase II associated factors. This interaction can be physiologically modulated by promoter context-specific transcription factors to facilitate optimal responsiveness of gene expression to hormone stimulation. One postulated regulatory mechanism involves the functional antagonism between hormone receptors and nonreceptor transcription factors interacting at the same hormone response element.

Retinol is specifically required during midgestation for neonatal survival.

Previous work has demonstrated that vitamin A-deficient, retinoic acid-supplemented pregnant rats cannot complete gestation without the administration of retinol. As little as 2 micrograms administered on day 10 of gestation is sufficient to prevent the characteristic fetal resorption that begins at day 15 of gestation. This single dose of retinol supports continued development through day 20 of gestation.

An enzyme-linked immunoassay for the 1,25-dihydroxyvitamin D3 receptor protein.

In this paper, we detail an enzyme-linked immunoassay for the 1,25-dihydroxyvitamin D3 receptor protein. The receptor protein of cell and tissue homogenates is bound between two monoclonal antibodies specific for different epitopes on the receptor protein. The first antibody is bound to the well of an ELISA plate and the second is biotinylated.

Distinct conformations of vitamin D receptor/retinoid X receptor-alpha heterodimers are specified by dinucleotide differences in the vitamin D-responsive elements of the osteocalcin and osteopontin genes.

The 1 alpha,25-dihydroxyvitamin D3 (VD3)-dependent stimulation of osteocalcin (OC) and osteopontin (OP) gene transcription in bone tissue is mediated by interactions of trans-activating factors with distinct VD3-responsive elements (VDREs). Sequence variation between the OC- and OP-VDRE steroid hormone half-elements provides the potential for recognition by distinct hormone receptor homo- and heterodimers. However, the exact composition of endogenous VD3- induced complexes recognizing the OC- and OP-VDREs in osteoblasts has not been definitively established.

Synthesis and biological activity of 1 alpha, 25-dihydroxy-18-norvitamin D3 and 1 alpha, 25-dihydroxy-18,19-dinorvitamin D3.

1 alpha, 25-dihydroxy-18-norvitamin D3 and 1 alpha, 25-dihydroxy-18,19-dinorvitamin D3 were prepared via Wittig-Horner coupling of 25-hydroxy-18-nor Grundmann type ketone with the corresponding A-ring phosphine oxides. Configuration at C-13 in the 18-nor Grundmann type alcohol (C,D-ring synthon), obtained by oxidative degradation of vitamin D3, was determined by 1H NMR spectroscopy and molecular mechanics calculations. Additional proof of the assigned trans-C/D-junction of the key intermediate 18-nor Grundmann type ketone follows from its chiroptical properties (circular dichroism data) and further chemical transformations.

Analysis of binding of the 1,25-dihydroxyvitamin D3 receptor to positive and negative vitamin D response elements.

The binding of the 1,25-dihydroxyvitamin D3 receptor to the vitamin D response elements (VDREs) in the rat osteocalcin (OSC-DRE), mouse osteopontin (MOP-DRE), rat calbindin D-9k (CaBP-DRE), and human parathyroid hormone genes (PTH-DRE) was studied. Binding of VDR to the three positive VDREs is cooperative. The degree of cooperativity is highest with the calbindin VDRE compared with either the OSC-DRE or the MOP-DRE.

1,25-Dihydroxyvitamin D3 reversibly blocks the progression of relapsing encephalomyelitis, a model of multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease believed to be a model for the human disease multiple sclerosis (MS). Induced by immunizing B10.PL mice with myelin basic protein (MBP), EAE was completely prevented by the administration of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3].

Conformationally restricted analogs of 1 alpha, 25-dihydroxyvitamin D3 and its 20-epimer: compounds for study of the three-dimensional structure of vitamin D responsible for binding to the receptor.

Two proteins play important roles in the expression of vitamin D function: the specific nuclear receptor protein (vitamin D receptor, VDR) and the transport protein (vitamin D binding protein, DBP). This study was conducted to clarify the conformation of vitamin D responsible for binding to those proteins. For the purpose, the side chain mobility of 1,25(OH)2D3 (1) and its 20-epimer, 20-epi-1,25(OH)2D3 (2), was analyzed by a systematic conformational search.

Human 25-hydroxyvitamin D3-24-hydroxylase, a multicatalytic enzyme.

Human 25-hydroxyvitamin D-24-hydroxylase has been expressed in Spodoptera frugiperda (Sf21) insect cells using the previously cloned cDNA in baculovirus (AcNPV-P450cc24). The activity of recombinant h-P450cc24 required adrenodoxin, adrenodoxin reductase, and NADPH. Incubation of this reconstituted system with 25-OH-[26,27-(3)H]D3 substrate produced several metabolites that were resolved on a normal-phase cyano HPLC system.

Metabolites of all-trans-retinol in day 10 conceptuses of vitamin A-deficient rats.

It has been previously demonstrated that vitamin A-deficient rats supplemented with retinoic acid cannot complete gestation. Resorption of fetuses invariably occurs in these animals beginning Day 15 of gestation. Retinol must be administered on or before Day 10 of gestation in order to prevent this phenomenon.

Antagonistic effects of transforming growth factor-beta on vitamin D3 enhancement of osteocalcin and osteopontin transcription: reduced interactions of vitamin D receptor/retinoid X receptor complexes with vitamin E response elements.

Osteocalcin and osteopontin are noncollagenous proteins secreted by osteoblasts and regulated by a complex interplay of systemic and locally produced factors, including growth factors and steroid hormones. We investigated the mechanism by which transforming growth factor-beta (TGF beta) inhibits 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-enhanced expression of the osteocalcin (OC) and osteopontin (OP) genes. ROS 17/2.

Retinoid X receptor isotype identity directs human vitamin D receptor heterodimer transactivation from the 24-hydroxylase vitamin D response elements in yeast.

Unlike estrogen and progesterone receptors that operate as homodimers on response elements, retinoid X receptors (RXRs) and vitamin D receptors (VDRs) can function as heterodimers. Studies concerning the significance of heterodimeric partnerships are usually performed utilizing mammalian or insect cells. These cells express endogenous nuclear receptors, making it impossible to assign a role for one receptor subtype over another while studying the function of transfected receptor(s).