Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

High affinity receptors (VDR) for 1,25-dihydroxycholecalciferol (calcitriol) are expressed in HL60 human leukemia cells and in low numbers in peripheral blood lymphocytes (PBL). HL60 cells, expressing some characteristics of promyelocytes, can be induced to monocytoid differentiation by calcitriol. Specific nuclear translocation of [3H]calcitriol/VDR was examined after exposure of whole cells to 10(-9) M/l calcitriol in the presence and absence of a 500-fold excess of unlabeled ligand and subsequent isolation of nuclei. Specific nuclear translocation of [3H]calcitriol/VDR was found to be time dependent reaching a maximum of approximately 2100 binding sites/nucleus after 3 h of incubation in HL60 cells, whereas a maximum of approximately 310 binding sites/nucleus was found after 3 h in PBL. Pulse exposure of HL60 to radiolabeled hormone for 3 h followed by culture in medium without serum and calcitriol lead to nuclear retention of approximately 1600 radiolabeled VDR by 8 h and approximately 1000 VDR by 24 h. Radiolabeled VDR disappeared from the nuclear compartment with a halflife of approximately 30 min if cells were cultured with identical concentrations of unlabeled hormone after the pulse (pulse/chase-experiments). No difference of VDR retention in pulse and pulse/chase-experiments was seen in PBL, where VDR halflife was approximately 30 min. No specific translocation into the nuclear compartment was seen when isolated nuclei were incubated in [3H]calcitriol. Radiolabeled hormone/receptor complexes of nuclei isolated from cells exposed for 3 h to radiolabeled hormone--in contrast to identical experiments with intact cells--did not disappear from the nuclear compartment upon incubation of nuclei with identical concentrations of the unlabeled compound. The activity of DNA relaxing enzymes (e.g. topoisomerases I and II) in nuclear extracts was measured using a PBR 322-relaxation-assay. Enhanced overall enzyme activity was found in nuclear extracts by 1 h after incubation with calcitriol (final ethanol concentration 0.0001% v/v) in HL60 and PBL. The enhanced activity disappeared after 2 h in PBL, whereas it was still enhanced by 4 h in HL60. No effect was seen in ethanol treated controls. We conclude that a specific nuclear translocation mechanism exists for calcitriol in both cell types examined, most likely due to translocation of receptor proteins after hormone binding. Translocated hormone/receptor complexes compete for a limited number of specific nuclear binding sites. Enhanced activity of topoisomerases in nuclear extracts upon translocation of VDR might reflect interaction of both within the nuclear compartment, thus initiating DNA-unwinding, a prerequisite of transcription initiation.
J Steroid Biochem Mol Biol 1992 Mar
PMID:Kinetics of nuclear translocation and turnover of the vitamin D receptor in human HL60 leukemia cells and peripheral blood lymphocytes--coincident rise of DNA-relaxing activity in nuclear extracts. 131 93

Rabbit and chicken antibodies were raised against two peptides synthesized according to the structure of human 1,25-dihydroxyvitamin D3 receptor (hVDR): rabbit alpha hVDR-103 against the N-terminal amino acids 5-18 and alpha hVDR-104 against the amino acids 172-186 in the hinge region and chicken alpha hVDR-cab11 against the amino acids 172-186, respectively. The specificity of the antibodies was tested by peptide saturation, SDS-PAGE immunoblotting, gel shift assay and sucrose gradient centrifugation. Immunoblotting of a soluble extract (cytosol) from osteosarcoma cell line MG-63 showed a single band with an M(r) of about 48,000 and human intestine cytosol a broad band (50-63,000) for both antibodies. The antibodies recognized activated (3.2S) hVDR by shifting the centrifugation sedimentation profile to 5-6S. The antibodies showed nuclear immunostaining of unoccupied VDR in human osteosarcoma cells MG-63, U2-Os and SaOs-2. The immunoreaction could be saturated with the corresponding synthetic peptide. In immunoblot alpha hVDR-103 reacted with human and rat VDR, whereas alpha hVDR-104 recognized human VDR only. Similarly in immunohistochemistry, alpha hVDR-103 showed staining with hVDR and rVDR, whereas alpha hVDR-104 reacted only with hVDR. All antibodies recognized the native hVDR as verified with sucrose gradient centrifugation or immunoprecipitation but only alpha hVDR-103 and alpha hVDR-cab11 in gel shift assay of hVDR associated with the vitamin D-responsive element of human osteocalcin gene promoter.
J Steroid Biochem Mol Biol 1992 Dec
PMID:Characterization of human 1,25-dihydroxyvitamin D3 receptor anti-peptide antibodies. 147 57

The metabolism of dihydrotachysterol (DHT), a hydrogenated analogue of vitamin D, has been studied in vivo using man and rat and in vitro using the perfused rat kidney, and hepatoma (3B) and osteosarcoma (UMR-106) cell lines. In vivo a large number of metabolites appeared in the plasma of rats given DHT2 and DHT3. Of particular interest was a compound more polar than 25-hydroxy-DHT, which has been designated compound H. Further study of this compound showed that it was composed of two components, one (Ha) being in much lower concentration than the other (Hb). The production of T2/H (peak H from DHT2) was demonstrated in human plasma after administration of oral DHT2. Comparison of the metabolites formed in vivo with those isolated from the rat kidney perfused with 25-hydroxy-DHT3 in vitro showed that 25-hydroxy-DHT3 was metabolized along two metabolic pathways previously described for vitamin D, culminating in the production of 25-hydroxy-DHT3-23,26-lactone and 23,25-dihydroxy-24-oxo-DHT3. The osteosarcoma cell line metabolized 25-OH-DHT3 in vitro along the same two metabolic pathways already demonstrated in the perfused rat kidney. More polar metabolites than compound H seen in rat plasma in vivo were shown to be metabolites of compound H and similar metabolites were also produced in the osteosarcoma cell line from chemically synthesized 1 alpha,25-dihydroxy-DHT3. The hepatoma cell line 25-hydroxylated DHT and no feed-back inhibition was observed. Use of the hepatoma cell to 25-hydroxylate a number of chemically synthesized 1-hydroxy-DHTs indicated that compound Ha was indistinguishable from 1 alpha,25-dihydroxy-DHT whereas compound Hb is possibly 1 beta,25-dihydroxy-DHT. Studies with the VDR in both chick gut and calf thymus indicated that 1 alpha,25-dihydroxy-DHT is very effective in displacing radiolabelled 1 alpha,25-dihydroxyvitamin-D3 and is thus most likely to be the calcaemic metabolite of DHT.
J Steroid Biochem Mol Biol 1992 Mar
PMID:The metabolism of dihydrotachysterols: renal side chain and non-renal nuclear hydroxylations in vivo and in vitro. 156 63

The abundance of 1,25-dihydroxyvitamin D3 receptors (VDR) in cultured cells has been shown to vary in direct relation to the rate of cell proliferation. This study examines the question of whether the growth-factor mediated up-regulation of VDR is due to direct modulation of VDR gene expression or is secondary to the stimulation of cell cycle events. Mitogenic agents, such as basic fibroblast growth factor and phorbol esters, were found to cause significant decreases in VDR abundance, while substantially stimulating proliferation of NIH-3T3 cells. Potent phorbol esters, such as phorbol myristate acetate (PMA) and phorbol-12,13-dibutyrate, whose biological actions have been shown to be mediated through the activation of protein kinase-C, down-regulated VDR in a time- and dose-dependent manner. An inactive phorbol ester, 4 alpha-phorbol-12,13-didecanoate, which does not activate protein kinase-C, did not alter VDR levels. Desensitization of protein kinase-C by prolonged exposure of cells to phorbol esters eliminated the PMA-mediated down-regulation of VDR. Staurosporine, an inhibitor of protein kinase-C, blocked the actions of PMA. Oleoyl acetyl glycerol, a synthetic diacyl glycerol, and A23187, a calcium ionophore, were both able to suppress VDR abundance alone and were additive in combination. The results suggest that activation of the protein kinase-C pathway and elevation of intracellular Ca2+ lead to significant down-regulation of VDR. The inhibitory effect of PMA appears to be exerted at the level of VDR mRNA expression. Northern blot analysis revealed significant decreases in steady state levels of VDR mRNA species that qualitatively corresponded to the decrease in VDR protein concentration seen on a Western blot.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1991 Apr
PMID:Activation of protein kinase-C inhibits vitamin D receptor gene expression. 165 39

The human vitamin D3 receptor (hVDR) cDNA was cloned into the E1 region of the adenovirus genome to generate recombinant viruses which were used to infect 293 (adenovirus-transformed human fetal kidney) cells. High salt extracts from cells infected with the recombinant viruses were subjected to immunoblot analysis using a monoclonal antibody to chicken VDR and were shown to contain large quantities of a protein of approximately 50 kDa with a migration identical to that of the hVDR in T47D (human mammary adenocarcinoma) cells. Scatchard analysis showed that the infected cells express approximately 100-fold more receptor than T47D cells and that this receptor binds 1,25-dihydroxyvitamin D3 with high affinity. The overexpressed hVDR also binds to DNA-cellulose and is eluted with a KCl concentration similar to that determined for fully active endogenous VDR. Nuclear extracts from cells infected with the hVDR-expressing adenoviruses contain an activity that specifically binds an oligonucleotide with sequences from the rat osteocalcin vitamin D3 response element, as determined by gel mobility shift. This interaction can be inhibited by the presence of an anti-VDR antibody, but not by nonspecific immunoglobulins. We conclude, therefore, that the overexpressed receptor has the ligand- and DNA-binding characteristics defined for endogenous VDR and that adenoviruses can be used to efficiently express large quantities of functional hVDR in a human cell line. Finally, a second binding activity, specific for the vitamin D response element, but distinct from the VDR, has been identified in extracts from uninfected cells.
Mol Endocrinol 1991 Jun
PMID:Overexpression of the human vitamin D3 receptor in mammalian cells using recombinant adenovirus vectors. 165 44

The human 1,25-dihydroxyvitamin D3 receptor (hVDR) has been recently shown to be phosphorylated in vitro by casein kinase-II. Most of the residues phosphorylated by this enzyme were shown to reside between Asn160 and Asp232, a region near the N-terminal boundary of the hormone-binding domain. We report here that the hVDR is also phosphorylated in vivo after transfection into ROS 17/2.8 cells. In addition to testing full-length hVDR, we analyzed several internally deleted hVDR mutants. The expression and phosphorylation of full-length and mutated hVDRs were monitored in transfected cells by metabolic labeling with either [35S]methionine or [32P]orthophosphate, followed by immunopurification using monoclonal anti-VDR antibody linked to agarose beads. Transfected hVDR is distinguishable from the endogenous rat VDR when the immunoprecipitated proteins are resolved on sodium dodecyl sulfate-polyacrylamide gels. Significant phosphorylation of transfected full-length hVDR was observed in ROS 17/2.8 cells, and it was less dependent on the presence of 1,25-dihydroxyvitamin D3 than that of the endogenous rat receptor. Most importantly, the region of in vivo phosphorylation, as defined by internal deletion mutants, resides between Met197 and Val234. Therefore, we have localized the major site of phosphorylation of hVDR to residues in the N-terminal region of the hormone-binding domain. The boundaries of this region fall within the amino acid segment defined for phosphorylation of hVDR by casein kinase-II in vitro, suggesting that VDR is an in vivo substrate for casein kinase-II or a related protein kinase.
Mol Endocrinol 1991 Aug
PMID:Vitamin D receptor phosphorylation in transfected ROS 17/2.8 cells is localized to the N-terminal region of the hormone-binding domain. 165 37

Lymphocyte cell lines were established from five patients with vitamin D-dependent rickets, type II (VDDR-II). These lines were established by infection with human T-lymphotrophic virus type I (HTLV-I). Binding of [3H]1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) to its receptor in these cell lines was compared to binding studies using a T-lymphocyte cell line (S-LB1) from a normal individual. The 1,25(OH)2D3 receptor of S-LB1 was comparable to the well-characterized chick intestinal 1,25(OH)2D3 receptor in terms of its ligand binding affinity and capacity, its mobility on 5-20% sucrose gradients, and its adsorption to and elution properties from DNA-cellulose. Three cell lines established from patients with VDDR-II (Rh-VDR, Sh-VDR, and Ab-VDR) showed no specific binding of 1,25(OH)2D3 to a receptor and treatment of the cultured cells with 1,25(OH)2D3 did not stimulate production of 24,25-dihydroxy-vitamin D3 (24,25(OH)2D3), a response which is diagnostic of the presence of a functional 1,25(OH)2D3 receptor. In a fourth cell line, A1-VDR, the receptor for 1,25(OH)2D3 had a low binding capacity and 25(OH)D3-24-hydroxylase activity was not detectable. Induction of 24,25-(OH)2D3 synthesis by 1,25(OH)2D3 was observed in the fifth cell line, designated Ro-VDR, although the sensitivity to hormone treatment was lower than in the control cell line from a normal donor. The capacity of the receptor for 1,25(OH)2D3 was low in Ro-VDR. In all cell lines where 1,25(OH)2D3 binding to a receptor was detectable, the receptor had the typical sedimentation coefficient of 3.7 S on sucrose density gradient analysis. Binding and elution properties to DNA-cellulose, however, differed from normal in both Ro-VDR and A1-VDR cells where elution from DNA-cellulose occurred at a lower salt concentration than is typical of the 1,25(OH)2D3 receptor. While Ro-VDR cells showed typical nuclear localization of the unoccupied 1,25(OH)2D3 receptor, neither the unoccupied nor the occupied receptor from A1-VDR cells was completely localized in the nucleus. In a series of functional studies we found that modulation of the level of the mRNAs coding for both the c-myc oncogene and the growth factor known as granulocyte-monocyte colony stimulating activity by 1,25(OH)2D3 correlated with the 1,25(OH)2D3 receptor status of these cells. Use of these cell lines will facilitate further study of the molecular defect(s) in the receptor for 1,25(OH)2D3 in vitamin D-dependent rickets type II and will allow a correlation with impairment of cellular functions.
Mol Cell Endocrinol 1990 Mar 26
PMID:Lymphocyte cell lines from vitamin D-dependent rickets type II show functional defects in the 1 alpha,25-dihydroxyvitamin D3 receptor. 216 Mar 80

Residues located between amino acids 244 and 263 in the human vitamin D receptor (hVDR) show extensive homology with other members of the steroid/thyroid/retinoid hormone receptor superfamily. The corresponding region of the glucocorticoid receptor has been shown to interact with the 90-kilodalton heat shock protein (hsp90), yet hVDR does not appear to bind to hsp90. Herein we report a study of hVDR in which the functional role of five conserved residues was tested by replacing Phe-244, Lys-246, Leu-254, Gln-259, and Leu-262 with glycines by site-directed mutagenesis. Initial screening of these mutants indicated that all were significantly impaired in their ability to activate transcription from a vitamin D-responsive reporter construct when expressed in transfected VDR-deficient COS-7 cells. Further characterization revealed two classes of mutants: the predominant class binds the 1,25-dihydroxyvitamin D3 ligand normally but is defective in its ability to form a heterodimeric complex with the retinoid X receptor (RXR) on a vitamin D responsive element (VDRE). A second unique class, represented by a single mutant at Lys-246, is normal both with respect to ligand binding and complex formation but still very impaired in transactivation ability. The distinction between these two classes was confirmed by the demonstration that a member of the first class, with a mutation at Gln-259, could be restored to near wild type transactivation ability by supplying excess RXR, while the Lys-246 mutant could not be so rescued. We therefore conclude that the primary function of this conserved domain in hVDR is the mediation of heterodimerization with RXR, leading to VDRE binding and transactivation. The possibility also exists that the Lys-246 mutant may be impaired in a step of transactivation that is distal to complex formation with RXR on the VDRE, perhaps in interactions with the transcriptional machinery itself.
Mol Endocrinol 1995 Sep
PMID:A highly conserved region in the hormone-binding domain of the human vitamin D receptor contains residues vital for heterodimerization with retinoid X receptor and for transcriptional activation. 749 Nov 9

Three regions of the human placental lactogen (hPL) promoter that contain several half-site motifs that closely resemble the responsive elements for thyroid hormone (TR), all trans retinoic acid (RAR) and 1,25 dihydroxyvitamin D3 (VDR) have been identified and characterized. Transfection studies in BeWo choriocarcinoma cells indicate that site A (nt -979 to -954) is responsive to RAR alpha but not TR beta. Site B (nt -1140 to -1170) is responsive to both RAR alpha and TR beta, and site C (nt -550 to -580) is not responsive to either RAR alpha or TR. These findings, together with the observation that placental cells express retinoid receptors and TRs, strongly suggest a role for these receptors in the regulation of the hPL gene. Site B on the hPL promoter is able to integrate the responses to RA and T3 through a single element.
Mol Cell Endocrinol 1995 Jul
PMID:Identification of a composite steroid hormone response element on the human placental lactogen promoter. 758 79

Members of the steroid/thyroid hormone receptor superfamily such as TR, RAR, RXR and VDR are known to play important roles in regulation of gene expression during development, differentiation and homeostasis. COUP-TFs are orphan members of this superfamily of nuclear receptors and have been shown to negatively regulate the ability of these nuclear receptors to transactivate target genes. Two different mechanisms are implicated in this repression. First, COUP-TFs bind to AGGTCA direct repeats and palindromes with various spacings, which include response elements for TR, RAR, RXR and VDR, allowing for direct competition of COUP-TFs for the response elements. Second, COUP-TFs can heterodimerize with RXRs, the essential cofactor for effective binding of VDR, TRs and RARs to their cognate response elements. The physiological significance of this negative effect of COUP-TF on the activity of these receptors has been analyzed. Detection of COUP-TF transcripts during mouse development reveal discrete spatial and temporal expression domains consistent with COUP-TFs being involved in regulation of gene expression during embryogenesis. Transcripts are localized within discrete regions of the central and peripheral nervous system including the inner ear. In addition, COUP-TFs are found in many tissues including testes, ovary, prostate, skin, kidney, lung, stomach, intestine, pancreas and salivary gland. Some of these expression domains colocalize with those of TR, RAR, and RXR. The simultaneous expression of these genes raise the possibility that COUP-TFs can act as negative regulatory factors during development and differentiation.
J Steroid Biochem Mol Biol 1995 Jun
PMID:Chicken ovalbumin upstream promoter transcription factor (COUP-TF): expression during mouse embryogenesis. 762 1


1 2 3 4 5 6 7 8 9 10 Next >>