Osteocalcin Vitamin D Response Element Research Articles

The effect of microgravity on 1,25-dihydroxyvitamin d3 signalling in osteoblasts

Microgravity encountered during space flight induces bone loss, as seen in both humans and rats. This type of bone loss is mainly caused by decreased bone formation due to reduced osteoblast proliferation and differentiation. Yet, the molecular alterations induced by microgravity during osteoblast differentiation are still enigmatic. Therefore, the effect of microgravity on the intracellular signalling pathway of 1,25-dihydroxyvita-min D3 was investigated during the Odissea Mission. The ligand 1,25-dihydroxyvitamin D3 interacts with the vitamin D receptor (VDR) and this complex binds to vitamin D response elements (VDRE) in the promoter region of target genes to stimulate or suppress gene transcription. To investigate the interaction of liganded VDR with VDRE, the mouse osteoblastic cell line, MC3T3, was stable transfected with a construct containing multiple VDREs of the rat osteocalcin promoter fused to growth hormone as reporter gene. Treatment of these transfectants with 1,25-dihydroxyvitamin D3 resulted in a time- and dose-dependent release of growth hormone in the culture medium. Space flight cultures responded to 1,25-dihydroxyvitamin D3 treatment with increased growth hormone production that was comparable with the induction observed in ground cultures. No 1g centrifuge was available during the space flight. These data indicate that microgravity for 5 days did not alter the interaction of VDR with the osteocalcin VDRE or the subsequent gene transcription.

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

There is considerable divergence in the sequences of steroid receptor response elements, including the vitamin D response elements (VDREs). Two major VDRE-containing and thus 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3))-regulated genes are the two non-collagenous, osteoblast-derived bone matrix proteins osteocalcin and osteopontin. We observed a stronger induction of osteopontin than osteocalcin mRNA expression by 1,25-(OH)(2)D(3). Subsequently, we have shown that vitamin D receptor/retinoid X receptor alpha (VDR/RXRalpha) heterodimers bind more tightly to the osteopontin VDRE than to the osteocalcin VDRE. Studies using point mutants revealed that the internal dinucleotide at positions 3 and 4 of the proximal steroid half-element are most important for modulating the strength of receptor binding. In addition, studies with VDRE-driven luciferase reporter gene constructs revealed that the central dinucleotide influences the transactivation potential of VDR/RXRalpha with the same order of magnitude as that observed in the DNA binding studies. The synthetic vitamin D analog KH1060 is a more potent stimulator of transcription and inducer of VDRE binding of VDR/RXR in the presence of nuclear factors isolated from ROS 17/2.8 osteoblast-like cells than the natural ligand 1,25-(OH)(2)D(3). Interestingly, however, KH1060 is comparable or even less potent than 1,25-(OH)(2)D(3) in stimulating VDRE binding of in vitro synthesized VDR/RXRalpha. Thus, the extent of 1,25-(OH)(2)D(3)- and KH1060-dependent binding of VDR/RXRalpha is specified by a central dinucleotide in the VDRE, and the ligand-induced effects on DNA binding are in part controlled by the cellular context of nuclear proteins.

25-Dehydro-1alpha-hydroxyvitamin D3-26,23S-lactone antagonizes the nuclear vitamin D receptor by mediating a unique noncovalent conformational change.

(23S)-25-dehydro-1alpha-Dihydroxyvitamin D3-26,23-lactone (TEI-9647; MK) has been reported to antagonize the 1alpha,25-dihydroxyvitamin D3 nuclear receptor (VDR)- mediated increase in transcriptional activity. Using a transient transfection system incorporating the osteocalcin VDRE (vitamin D response element) in Cos-1 cells, we found that 20 nM MK antagonizes VDR-mediated transcription by 50% when driven by 1 nM 1alpha,25(OH)2D3. Four analogs of 1alpha,25(OH)2D3, also at 1 nM, were antagonized 25 to 39% by 20 nM MK. However, analogs with 16-ene/23-yne or 20-epi modifications, which have a significantly lower agonist ED50 for the VDR than 1alpha,25(OH)2D3, were antagonized by 20 nM MK only at 100 pM or 10 pM, respectively. One possible mechanism for antagonism is that the 25-dehydro alkene of MK might covalently bind the ligand-binding site of the VDR rendering it inactive. Utilization of a ligand exchange assay, however, demonstrated that MK bound to VDR is freely exchanged with 1alpha,25(OH)2D3 in vitro. These data support the apparent correlation between VDR transcriptional activation by agonists and the effective range of MK antagonism by competition. Furthermore, protease sensitivity analysis of MK bound to VDR indicates the presence of a unique conformational change in the VDR ligand-binding domain, showing a novel doublet of VDR fragments centered at 34 kDa, whereas 1alpha,25(OH)2D3 as a ligand produces only a single 34-kDa fragment. In comparison, the natural metabolite 1alpha,25-dihydroxyvitamin D3-26,23-lactone yields only the 30-kDa fragment that is produced by all ligands to varying degrees. Collectively, these results support that MK is a potent partial antagonist of the VDR for 1alpha,25(OH)2D3 and its analogs when in appropriate excess of the agonist.

Turning a negative into a positive: vitamin D receptor interactions with the avian parathyroid hormone response element.

1 ,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] negatively regulates expression of the avian PTH (aPTH) gene transcript, and a vitamin D response element (VDRE) near the promoter of the aPTH gene had previously been identified. The present report assessed whether the negative activity imparted by the aPTH VDRE could be converted to a positive transcriptional response through selective mutations introduced into the element. The tested sequences were derived from individual and combined mutations to 2 bp in the 3'-half of the direct repeat element, GGGTCAggaGGGTGT. Cold competition experiments using mutant and wild-type oligonucleotides in the mobility shift assay revealed minor differences in the ability of any of these sequences to compete for binding to a heterodimer complex comprised of recombinant proteins. Ethylation interference footprint analysis for each of the mutants produced unique patterns over the 3'-half-sites that were distinct from the weak, wild-type footprint. Transcriptional outcomes evaluated from a chloramphenicol acetyltransferase reporter construct utilizing the aPTH promoter found that the individual T-->A mutant produced an attenuated negative transcriptional response while the G-->C mutant resulted in a reproducibly weak positive transcriptional outcome. The double mutant, however, yielded a 4-fold increase in transcription, similar to the 7-fold increase observed from an analogous construct using the human osteocalcin VDRE. UV light crosslinking to gapped oligonucleotides assessed the polarity of heterodimer binding to the wild-type and double mutant sequences and was consistent with the vitamin D receptor preferentially binding to the 5'-half of both elements. Finally, DNA affinity chromatography was used to immobilize heterodimer complexes bound to the wild-type and double mutant sequences as bait to identify proteins that may preferentially interact with these DNA-bound heterodimers. This analysis revealed the presence of a p160 protein that specifically interacted with the heterodimer bound to the wild-type VDRE, but was absent from complexes bound to response elements associated with positive transcriptional activity. Thus, the sequence of the individual VDRE appears to play an active role in dictating transcriptional responses that may be mediated by altering the ability of a vitamin D receptor heterodimer to interact with accessory factor proteins.

Thyroid Hormone Receptor Does Not Heterodimerize with the Vitamin D Receptor but Represses Vitamin D Receptor-Mediated Transactivation

The 9,000 Mr calcium-binding protein calbindin-D9k (CaBP9k) is markedly induced by 1,25-dihydroxyvitamin D3[ 1,25-(OH)2D3] in mammalian intestine. However, although a vitamin D response element (VDRE) has been reported in the promoter of the rat CaBP9k gene (at −490/−472), the CaBP9k promoter is weakly transactivated by 1,25-(OH)2D3. Previous studies indicated that when MCF-7 cells are transfected with the rat CaBP9k VDRE ligated to the thymidine kinase promoter and treated with both 1,25-(OH)2D3 and T3 there is an enhancement of the response observed with 1,25-(OH)2D3 alone, suggesting direct cross-talk between thyroid hormone and the vitamin D endocrine system and activation via the formation of vitamin D receptor (VDR)-thyroid hormone receptor (TR) heterodimers. To determine whether the weak response of the rat CaBP9k natural promoter to 1,25-(OH)2D3 could be enhanced by T3, CaBP9k promoter/reporter chloramphenicol acetyltransferase constructs were transfected in MCF-7 cells, and the cells were treated with the two hormones alone or in combination. No induction with T3 alone and no enhancement of reporter activity in the presence of both hormones was observed. To determine whether a lack of effect by T3 was specific for the CaBP9k promoter and to further examine the possibility of cross-talk between the TR- and VDR-signaling pathways, the 1,25-(OH)2D3-responsive rat 24 hydroxylase [24(OH)ase] promoter and the rat osteocalcin VDRE (−457/−430), both fused to reporter genes were similarly examined in MCF-7 cells. Again, no enhancement of the response to 1,25-(OH)2D3 was observed in the presence of T3. In addition, a similar lack of response to T3 but responsiveness to 1,25-(OH)2D3 was observed when UMR106–01 osteosarcoma cells [which, like MCF-7 cells, express VDR, TR, and the retinoid X receptor (RXR) endogenously] were transfected with a 1,25-(OH)2D3 responsive mouse osteopontin promoter reporter. In vitro DNA binding assays were carried out using purified human VDR, human RXRα, and chick T3Rα and 24(OH)ase, osteocalcin, osteopontin, and CaBP9k VDRE oligonucleotide probes. No VDR-TR heterodimer binding on any of these VDREs was observed, although, as expected, there was binding by the VDR-RXR complex and strong TR-RXR binding to a consensus thyroid hormone response element. Simultaneous gel retardation assays using similar and lower concentrations of TR with RXR showed strong binding of TR-RXR on a 32P-labeled thyroid response element. Studies using the yeast two-hybrid system also did not provide evidence for the formation of a VDR-TR protein-protein interaction. In addition, in vivo data showed that transfection of TR, in fact, repressed VDR-mediated transcription and that the repression could be reversed by the addition of RXR. Thus, in vitro and in vivo experiments do not support ligand-sensitive transactivation mediated by VDR-TR heterodimer formation but rather suggest that TR expression can repress 1,25-(OH)2D3-induced transcription predominantly by sequestering RXR.

Thyroid hormone receptor does not heterodimerize with the vitamin D receptor but represses vitamin D receptor-mediated transactivation.

The 9,000 Mr calcium-binding protein calbindin-D9k (CaBP9k) is markedly induced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in mammalian intestine. However, although a vitamin D response element (VDRE) has been reported in the promoter of the rat CaBP9k gene (at -490/-472), the CaBP9k promoter is weakly transactivated by 1,25-(OH)2D3. Previous studies indicated that when MCF-7 cells are transfected with the rat CaBP9k VDRE ligated to the thymidine kinase promoter and treated with both 1,25-(OH)2D3 and T3 there is an enhancement of the response observed with 1,25-(OH)2D3 alone, suggesting direct cross-talk between thyroid hormone and the vitamin D endocrine system and activation via the formation of vitamin D receptor (VDR)-thyroid hormone receptor (TR) heterodimers. To determine whether the weak response of the rat CaBP9k natural promoter to 1,25-(OH)2D3 could be enhanced by T3, CaBP9k promoter/reporter chloramphenicol acetyltransferase constructs were transfected in MCF-7 cells, and the cells were treated with the two hormones alone or in combination. No induction with T3 alone and no enhancement of reporter activity in the presence of both hormones was observed. To determine whether a lack of effect by T3 was specific for the CaBP9k promoter and to further examine the possibility of cross-talk between the TR- and VDR-signaling pathways, the 1,25-(OH)2D3-responsive rat 24 hydroxylase [24(OH)ase] promoter and the rat osteocalcin VDRE (-457/-430), both fused to reporter genes were similarly examined in MCF-7 cells. Again, no enhancement of the response to 1,25-(OH)2D3 was observed in the presence of T3. In addition, a similar lack of response to T3 but responsiveness to 1,25-(OH)2D3 was observed when UMR106-01 osteosarcoma cells [which, like MCF-7 cells, express VDR, TR, and the retinoid X receptor (RXR) endogenously] were transfected with a 1,25-(OH)2D3 responsive mouse osteopontin promoter reporter. In vitro DNA binding assays were carried out using purified human VDR, human RXRalpha, and chick T3Ralpha and 24(OH)ase, osteocalcin, osteopontin, and CaBP9k VDRE oligonucleotide probes. No VDR-TR heterodimer binding on any of these VDREs was observed, although, as expected, there was binding by the VDR-RXR complex and strong TR-RXR binding to a consensus thyroid hormone response element. Simultaneous gel retardation assays using similar and lower concentrations of TR with RXR showed strong binding of TR-RXR on a 32P-labeled thyroid response element. Studies using the yeast two-hybrid system also did not provide evidence for the formation of a VDR-TR protein-protein interaction. In addition, in vivo data showed that transfection of TR, in fact, repressed VDR-mediated transcription and that the repression could be reversed by the addition of RXR. Thus, in vitro and in vivo experiments do not support ligand-sensitive transactivation mediated by VDR-TR heterodimer formation but rather suggest that TR expression can repress 1,25-(OH)2D3-induced transcription predominantly by sequestering RXR.

Characteristics of Vitamin D3 Receptor (VDR) binding to the Vitamin D Response Element (VDRE) in rat bone sialoprotein gene promoter

Bone sialoprotein (BSP) is a mineralised tissue-specific protein that is highly expressed during the initial formation of bone and cementum. Expression of BSP is suppressed by the osteotropic hormone, 1,25-dihydroxyvitamin D3 (vitamin D3), which regulates bone remodelling. In previous studies, we have identified a vitamin D response element (VDRE) that is integrated with a novel inverted TATA box in the rat BSP promoter which mediates the suppression of BSP transcription (1). Although the nucleotide sequences of VDREs in different genes conform to a direct (hexamer) repeat, spaced by three nucleotides, the precise sequences are unique for each VDRE. To determine whether the nucleotide differences in the VDRE influence VDR binding, we have compared interactions of VDR proteins with various VDREs using gel mobility shift analysis. Both natural and recombinant VDRs bound to rat BSP and both mouse and porcine osteopontin (OPN) VDRE oligonucleotides in a concentration-dependent manner with a strong preference for dimer formation, whereas equal amounts of dimer and monomer were bound to the human osteocalcin VDRE. However, whereas a truncated VDR comprising the DNA binding domain alone bound the mouse osteopontin VDRE, it failed to interact with the porcine OPN and rat BSP VDREs. VDR binding to the BSP was sequence specific, as shown by mutagenesis analysis, and could be abolished by heat and VDR antibody. These studies demonstrate that subtle differences in the nucleotide sequence of VDREs affect VDR binding, which mediates the vitamin D3 response.

Effect of glyoxylate on the function of the calcitriol receptor and vitamin D metabolism

The biological action of calcitriol is mostly mediated through the interaction of the calcitriol receptor (VDR) with vitamin D response elements (VDREs) of target genes. These interactions produce special proteins that carry out the biological activities of calcitriol. Recently, we showed that the interaction of VDRs with VDREs is inhibited by uremic toxins. We hypothesize that uremic toxins that contain aldehyde or ketone groups potentially could form Schiff bases with lysine residues of the VDR DNA binding domain and inhibit VDR interaction with VDREs. We therefore chose glyoxylate, a compound which has an aldehyde group, to test this hypothesis. In vitro glyoxylate inhibited VDR binding to the osteocalcin and osteopontin VDREs as assessed by electrophoretic mobility shift assay and the inhibition was reversed when glyoxylate was preincubated with lysine. Further, this chemical compound also blocked the induction of chloramphenicol acetyltransferase (CAT) enzyme induced by calcitriol in cells transfected with a calcitriol responsive CAT reporter gene. Since induction of 24-hydroxylase synthesis is a VDR regulated process, we also studied the effect of glyoxylate on the activity of intestinal 24-hydroxylase in rats. This enzyme activity was suppressed in rats infused with glyoxylate. Taken together, our study suggests that glyoxylate could inhibit the interaction of VDR with VDREs and alter the biological action of calcitriol.

Effect of glucose on the function of the calcitriol receptor and vitamin D metabolism

The genomic action of calcitriol is mediated through the interaction of the calcitriol receptor (VDR) with vitamin D response elements (VDREs) of the target genes. It has been proposed that chemicals capable of Schiff base formation with the VDR potentially could alter the physiological function of VDR and calcitriol metabolism. Since glucose has been shown to form Schiff bases with proteins, we tested the hypothesis that glucose could influence the function of VDR and thereby alter calcitriol metabolism. Glucose 6-phosphate inhibited VDR binding to the osteocalcin VDRE and chemically modified the DNA binding domain or the dimerization domain of the VDR in vitro. Further, glucose also blocked the production of chloramphenicol acetyltransferase (CAT) enzyme induced by calcitriol in cells transfected with a constructed VDRE attached to a CAT reporter gene. Hyperglycemia induced by glucose infusion or by streptozotocin in normal rats significantly reduced intestinal 1 alpha, 25-dihydroxyvitamin D-24-hydroxylase activity. Taken together, these findings are consistent with the hypothesis that glucose could interact with the VDR to impair its DNA binding and function within cells.

Identification and Characterization of 1,25-Dihydroxyvitamin D3-responsive Repressor Sequences in the Rat Parathyroid Hormone-related Peptide Gene

Parathyroid hormone-related peptide (PTHRP) gene transcription is suppressed by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D3. In the present report, we examined 1, 25(OH)2D3-mediated repression of PTHRP expression by transfection of PTHRP promoter/reporter constructs in normal human keratinocytes and by DNA binding. We localized an element conferring 1, 25(OH)2D3-mediated repression in vivo to a 47-base pair (bp) region located -1121 to -1075 from the transcriptional start site. Mobility shift analysis revealed that this vitamin D response element (VDRE) forms DNA-protein complexes. The addition of a monoclonal antibody that recognizes the DNA binding region of the vitamin D receptor (VDR) attenuated binding of the receptor to the 47-bp sequence, whereas the addition of monoclonal antibody raised against the retinoid X receptor (RXR) further retarded the mobility of the protein-DNA complex. Consequently, the PTHRP promoter element binds a VDR.RXR heterodimer. Examination of this VDRE revealed complete sequence homology with a half-site of the human and rat osteocalcin VDRE (GGGTGA). Furthermore, mutation analysis suggests that a 16-bp domain consisting of an almost perfect repeat separated by a 3-base pair "spacer" GGGTGGAGAGGGGTGA is responsible for the DNA-protein interaction within this 47-bp sequence. Our results therefore indicate the existence of an inhibitory VDRE within the PTHRP promoter that is similar in sequence composition and cellular factor requirement to classical up-regulatory VDREs.

Vitamin D receptor binding to the negative human parathyroid hormone vitamin D response element does not require the retinoid x receptor.

An important physiological control of PTH gene expression is its transcriptional repression by 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)]. The mechanism of this 1,25-(OH)(2)D(3)-mediated transcriptional repression is poorly understood. Previous investigations have identified a DNA sequence in the 5'-regulatory region of the human PTH (hPTH) gene that binds the vitamin D receptor (VDR) and mediates transcription repression in response to 1,25(OH)(2)D(3) in GH4CI cells. The hPTH gene sequence does not mediate transcriptional repression in ROS 17/2.8 cells, even though up-regulatory vitamin D response elements (VDREs) are active in these cells. The hPTH DNA sequence differs from the upregulatory VDREs in that it contains a single copy of a hexameric motif (AGGUC) homologous to those repeated in the up-regulatory VDREs. The protein-DNA interactions of this sequence were examined using nuclear extracts from bovine parathyroid, GH4CI, and ROS 17/2.8 cells. In bovine parathyroid nuclear extracts, the VDR binds the down-regulatory hPTH DNA sequence independently of the retinoid X receptor (RXR). In GH4C1 nuclear extracts, two VDR-containing complexes are observed: one lacking RXR and one containing RXR. In ROS 17/2.8 nuclear extracts, a single VDRdependent complex containing RXR is observed. When the up-regulatory rat osteocalcin VDRE is used as a probe, only VDR-RXR-containing complexes are generated using nuclear extracts from all three cell types. These results demonstrate that the sequence that mediates transcriptional repression in response to 1 ,25-(OH)(2)D(3) differs from the up-regulatory response elements both in sequence composition and in its ability to bind VDR independently of RXR.

The T-Box near the Zinc Fingers of the Human Vitamin D Receptor Is Required for Heterodimeric DNA Binding and Transactivation

The T-box mediates binding of retinoid X receptor (RXR) homodimers to DNA while the P- and D-box in the zinc fingers of steroid hormone receptors play roles in DNA-binding specificity and homodimerization, respectively. We investigated the function of these elements in the human vitamin D receptor (hVDR) by mutating a Lys-Glu pair of amino acids in the T-box, and by altering the P- and D-boxes to the corresponding residues of the glucocorticoid receptor (GR). The T-box mutant hVDR displayed attenuated vitamin D responsive element (VDRE) binding in the presence of RXR and was severely compromised in transcriptional activation. In contrast, GR P/D-box mutant hVDRs bound to the rat osteocalcin VDRE and elicited near normal transcriptional activation. The T-box mutant uniquely exhibited dominant negative properties, highlighting the significance of this region of hVDR for heterodimeric transcriptional activation.

Inhibition of calcitriol receptor binding to vitamin D response elements by uremic toxins.

The genomic action of calcitriol (1,25-dihydroxy-vitamin D3) is mediated through the interaction of the calcitriol receptor (VDR) with vitamin D response elements (VDREs). Although renal failure is associated with resistance to the action of calcitriol, the mechanism of this resistance is not well understood. Therefore, we used the electrophoretic mobility shift assay to compare the ability of VDRs from normal and renal failure rats to bind to the osteocalcin gene VDRE. The results indicate that VDRs from renal failure rats have only half the DNA binding capacity as VDRs from control rats, despite identical calcitriol binding. Furthermore, incubation of normal VDRs with a uremic plasma ultrafiltrate resulted in a loss of > 50% of the binding sites for the osteocalcin VDRE. When VDRs bound to DNA as heterodimers with retinoid X receptors, the inhibitory effect of the uremic ultrafiltrate was due to a specific interaction with the VDR, not retinoid X receptors. In addition, uremic ultrafiltrate blocked calcitriol-induced reporter gene activity in transfected JEG-3 cells. Taken together, the results indicate that an inhibitory effect of a uremic toxin(s) on VDR-VDRE binding could underlie the calcitriol resistance of renal failure.

Specific binding to vitamin D response elements of chicken intestinal DNA-binding activity is not related to the vitamin D receptor.

In this report we confirm that the putative vitamin D response element (VDRE), located between -320 and -306 in the chicken calbindin-D28K gene, is not a binding site for the vitamin D3 receptor (VDR). In examining the ability of chicken intestinal nuclear extracts (CINE) to bind known VDREs, we observed a specific VDRE-binding activity, which is distinct from VDR. In fact, VDR-depleted CINE retains the ability to bind the rat osteocalcin VDRE. The VDRE-binding activity binds DNA with high affinity and contacts it at the same guanine residues as VDR. Its specificity in binding structural variants of the AGGTCA repeat is broader than that of VDR, as direct repeats spaced by 3, 4, and 5 base pairs are almost equally effective competitors when added to the probe in molar excess. Palindromic arrangements of the same motif are lower affinity competitors. The retinoid-X receptor is involved in the binding complex, as incubation of CINE with antibody to retinoid-X receptor results in a quantitative supershift. Antibodies to retinoic acid receptors (RAR alpha and -beta), T3 receptor, or chicken ovalbumin up-stream promoter-transcription factor had no apparent effect. These data suggest that species specificity is a relevant aspect of VDR/VDRE recognition, and that a novel factor(s), different from VDR, might be involved in the effect of vitamin D on gene expression.

Difference and similarity of DNA sequence recognized by VDR homodimer and VDR/RXR heterodimer.

Nuclear receptors for the thyroid hormone and vitamin A and D cooperate with the retinoid X receptor (RXR) in activating the transcription. Although the hormone response elements for these receptors have been proposed in which spacing of the direct repeated motifs determine the specificity (so called 3-4-5 rule), vitamin D response elements (VDREs) in the natural context consist of often imperfect direct repeats. Vitamin D receptor (VDR) alone can bind to the mouse osteopontin (mSPP-1) VDRE, which contains a direct repeat separated by 3 nucleotides, but not to the rat osteocalcin (rOST) VDRE having inexact direct repeat. The presence of RXR not only allows the VDR to bind to the rOST VDRE, but also increases the binding affinity for the mSPP-1 VDRE. The RXR/VDR heterodimer exhibits the similar affinity constants for the mSPP-1 VDRE and the rOST VDRE, in spite of the apparently different affinities for two VDREs of the VDR homodimer. A random oligonucleotide selection procedure revealed that the consensus sequence selected by the RXR homodimer is the direct repeat spaced by one A residue. In contrast, the sequences preferentially selected by the VDR homodimer and the VDR/RXR heterodimer are similar, which are the direct repeats spaced by 3 nucleotides. The difference and similarity of DNA sequence recognition are discussed.

Retinoid X receptors stimulate and 9-cis retinoic acid inhibits 1,25-dihydroxyvitamin D3-activated expression of the rat osteocalcin gene.

The vitamin D receptor (VDR) binds the vitamin D-responsive element (VDRE) as a heterodimer with an unidentified receptor auxiliary factor (RAF) present in mammalian cell nuclear extracts. VDR also interacts with the retinoid X receptors (RXRs), implying that RAF may be related to the RXRs. Here we demonstrate that highly purified HeLa cell RAF contained RXR beta immunoreactivity and that both activities copurified and precisely coeluted in high-resolution hydroxylapatite chromatography. Furthermore, an RXR beta-specific antibody disrupted VDR-RAF-VDRE complexes in mobility shift assays. These data strongly indicate that HeLa RAF is highly related to or is identical to RXR beta. Consequently, the effect of the 9-cis retinoic acid ligand for RXRs was examined in 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-activated gene expression systems. Increasing concentrations of 9-cis retinoic acid (1 nM to 1 microM) markedly reduced 1,25(OH)2D3-dependent accumulation of osteocalcin mRNA in osteoblast-like ROS 17/2.8 cells. All-trans retinoic acid also interfered with vitamin D responsiveness, but it was consistently less potent than the 9-cis isomer. Transient transfection studies revealed that attenuation by 9-cis retinoic acid was at the transcriptional level and was mediated through interactions at the osteocalcin VDRE. Furthermore, overexpression of both RXR beta and RXR alpha augmented 1,25(OH)2D3 responsiveness in transient expression studies. Direct analysis of VDRE binding in mobility shift assays demonstrated that heteromeric interactions between VDR and RXR were enhanced by 1,25(OH)2D3 and were not affected appreciably by 9-cis retinoic acid, except that inhibition was observed at high retinoid concentrations. These data suggest a regulatory mechanism for osteocalcin gene expression that involves 1,25(OH)2D3-induced heterodimerization of VDR and unliganded RXR. 9-cis retinoic acid may attenuate 1,25(OH)2D3 responsiveness by diverting RXRs away from VDR-mediated transcription and towards other RXR-dependent transcriptional pathways.

Retinoid X receptors stimulate and 9-cis retinoic acid inhibits 1,25-dihydroxyvitamin D3-activated expression of the rat osteocalcin gene.

The vitamin D receptor (VDR) binds the vitamin D-responsive element (VDRE) as a heterodimer with an unidentified receptor auxiliary factor (RAF) present in mammalian cell nuclear extracts. VDR also interacts with the retinoid X receptors (RXRs), implying that RAF may be related to the RXRs. Here we demonstrate that highly purified HeLa cell RAF contained RXR beta immunoreactivity and that both activities copurified and precisely coeluted in high-resolution hydroxylapatite chromatography. Furthermore, an RXR beta-specific antibody disrupted VDR-RAF-VDRE complexes in mobility shift assays. These data strongly indicate that HeLa RAF is highly related to or is identical to RXR beta. Consequently, the effect of the 9-cis retinoic acid ligand for RXRs was examined in 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-activated gene expression systems. Increasing concentrations of 9-cis retinoic acid (1 nM to 1 microM) markedly reduced 1,25(OH)2D3-dependent accumulation of osteocalcin mRNA in osteoblast-like ROS 17/2.8 cells. All-trans retinoic acid also interfered with vitamin D responsiveness, but it was consistently less potent than the 9-cis isomer. Transient transfection studies revealed that attenuation by 9-cis retinoic acid was at the transcriptional level and was mediated through interactions at the osteocalcin VDRE. Furthermore, overexpression of both RXR beta and RXR alpha augmented 1,25(OH)2D3 responsiveness in transient expression studies. Direct analysis of VDRE binding in mobility shift assays demonstrated that heteromeric interactions between VDR and RXR were enhanced by 1,25(OH)2D3 and were not affected appreciably by 9-cis retinoic acid, except that inhibition was observed at high retinoid concentrations. These data suggest a regulatory mechanism for osteocalcin gene expression that involves 1,25(OH)2D3-induced heterodimerization of VDR and unliganded RXR. 9-cis retinoic acid may attenuate 1,25(OH)2D3 responsiveness by diverting RXRs away from VDR-mediated transcription and towards other RXR-dependent transcriptional pathways.

Baculovirus-mediated expression of the human vitamin D receptor. Functional characterization, vitamin D response element interactions, and evidence for a receptor auxiliary factor.

A baculovirus expression vector system (BEVS) was used to overproduce the full-length human vitamin D receptor (hVDR) in Spodoptera frugiperda ovarian cells. hVDR was expressed to a level of 0.5% of the total soluble protein in this system. Western analysis demonstrated that the baculovirus-generated protein had electrophoretic and immunologic properties equivalent to those of hVDR expressed in mammalian cells. The BEVS-derived receptor displayed specificity and high affinity (apparent Kd = 0.7 nM) for the 1,25(OH)2D3 ligand. Recombinant hVDR generated a specific protein-DNA complex with a duplex oligomer containing a vitamin D-responsive element (VDRE) in gel mobility shift assays. The intensity of the VDR.VDRE complex was not affected by 1,25(OH)2D3. However, the complex exhibited increased mobility in the presence of hormone, possibly the result of a 1,25(OH)2D3-dependent conformational change. A nuclear extract obtained from CV-1 cells markedly enhanced the intensity of this VDR.VDRE complex and produced an additional distinct VDR-dependent complex, thus implicating a role for nuclear auxiliary factors in multiple high affinity VDR.VDRE interactions. Finally, methylation interference studies defined the guanine residues contacted when the putative VDR-auxiliary factor complex associates with the rat osteocalcin VDRE; specifically, all of the GC base pairs in the sequence GGGTGAATGAGGACA. Therefore, these results show that the BEV system elicits high level expression of hVDR with critical functional characteristics being preserved.

The vitamin D-responsive element in the human osteocalcin gene. Association with a nuclear proto-oncogene enhancer.

A vitamin D-responsive element (VDRE) locus within the 5' region of the human osteocalcin gene promoter contains a steroid response-like half-site immediately proximal to a consensus site for the AP-1 nuclear oncogene family. In the studies described here, internal mutagenesis of the osteocalcin promoter coupled to functional assays reveal that the interaction of the vitamin D receptor is limited to the proximal region of the VDRE locus. Mutations within the distal AP-1 consensus site reduce the basal activity of the promoter but have little effect on vitamin D inducibility. The absolute level of promoter activity induced by hormone, however, is dramatically reduced in the absence of an intact AP-1 site suggesting a functional synergism between the receptor and AP-1-related proteins. In vitro receptor-DNA binding studies confirm the lack of requirement for the distal component in receptor binding. These results suggest that the osteocalcin VDRE is limited to 15 nucleotides closely juxtaposed to a distal functional AP-1 site. The close association of the two sites may lead to proto-oncogene and steroid receptor interactions that result in interesting functional consequences.