Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.1.21 (thymidine kinase)
 7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A primary response of the avian intestine to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is increased synthesis of a 28-kD calcium-binding protein, calbindin-D28k (CaBP). This study examined whether 1,25-(OH)2D3 regulates CaBP gene transcription by an interaction of the vitamin D receptor (VDR) with a vitamin D-responsive element (VDRE) in the CaBP promoter. A genomic clone of CaBP containing about 1 kb of 5'-flanking DNA and 13 kb of the structural gene was isolated. 5'-Flanking DNA from -320 to -306 had considerable sequence similarity to VDREs identified in other genes. Indeed, a portion of the CaBP gene containing this region (-743 to +47) linked to a growth hormone reporter construct elicited a 1,25-(OH)2D3-dependent, VDR-dependent increase in reporter expression in transiently transfected chicken embryo fibroblasts. However, deletion analysis demonstrated that the sequences responsible for this induction reside 3' to -133 and the putative VDRE at -320 to -306 was not involved in the response. Furthermore, transfection of heterologous promoter constructs consisting of a Ban I fragment (-354 to -252) linked to the Herpes simplex thymidine kinase promoter revealed no effect of this region on reporter expression. Gel mobility shift analysis confirmed that this putative VDRE in the CaBP promoter was not a high-affinity binding site for VDR. Consequently, functional significance with respect to the primary induction of CaBP by 1,25-(OH)2D3 cannot be ascribed to this region of the CaBP promoter.
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PMID:Evaluation of a putative vitamin D response element in the avian calcium binding protein gene. 131 20

1 alpha-25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3], together with vitamin D receptor (VDR), directly activates human osteocalcin (hOC) gene expression through a vitamin D-responsive element (VDRE) located in the promoter of the hOC gene. We investigated the effect of 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] on the regulation of the hOC gene promoter and compared it with that of 1 alpha,25(OH)2D3. 24R,25(OH)2D3 did not activate the natural promoter in VDR-negative CV-1 cells. 24R,25(OH)2D3, however, induced the activation of this promoter following cotransfection with an hVDR expression vector. In VDR-positive MC3T3-E1 cells, 24R,25(OH)2D3 activated not only the natural hOC promoter but also a chimeric promoter composed of a synthetic hOC VDRE sequence linked to the thymidine kinase promoter. In combination with 1 alpha-25(OH)2D3, 24R,25(OH)2D3 did not exhibit any antagonist activity on the hOC promoter. These results suggest that under conditions of high 24R,25(OH)2D3 levels in vivo, this metabolite of vitamin D3 may activate hOC gene expression through receptor mechanisms identical to that for 1 alpha,25(OH)2D3.
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PMID:Activation of the human osteocalcin gene by 24R,25-dihydroxyvitamin D3 occurs through the vitamin D receptor and the vitamin D-responsive element. 787 65

Mitochondrial cytochrome P450(24) expression in the vitamin D-degradation pathway is induced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. The molecular basis of this enzyme regulation was investigated by isolating the rat P450(24) gene and examining the 5'-flanking region for possible cis-acting regulatory elements involved in the induction process. Constructs containing different lengths of 5'-flanking region of the gene were linked to a luciferase reporter gene and transiently co-transfected with a human vitamin D receptor (hVDR) expression vector (pRSV-hVDR) into COS-1 cells. These experiments showed that the flanking region from -298 to -122 directed a 24-fold increase in luciferase activity in response to 1,25-(OH)2D3 provided that the cells were co-transfected with pRSV-hVDR. Within this region, the sequence from position -171 to -123 conferred 1,25-(OH)2D3 responsiveness to both the native P450(24) promoter and the heterologous thymidine kinase promoter. Mutagenesis revealed that the sequence from position -150 to -136 is required for induction by 1,25-(OH)2D3 and that this sequence shares similarity to other vitamin D responsive elements (VDREs) reported for other genes. Gel shift mobility assays showed this region specifically bound a nuclear protein complex from 1,25-(OH)2D3 treated COS-1 cells that had been co-transfected with pRSV-hVDR. The retarded band was specifically competed with the well characterized VDRE from the mouse osteopontin gene. A VDRE at position -150 to -136 in the promoter of the rat P450(24) gene is identified in this study and found to be important in mediating the enhanced expression of the gene by 1,25-(OH)2D3.
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PMID:Identification of a vitamin D responsive element in the promoter of the rat cytochrome P450(24) gene. 803 72

The 5'-flanking region of the rat vitamin D3 24-hydroxylase (P450cc24) gene was examined and a vitamin D-responsive element (VDRE) responsible for the 1 alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3) enhancement was identified. Unidirectional deletion analyses of the 5'-flanking region indicated that the region [-167/-102] is involved in vitamin D responsiveness. Further functional analyses showed that the segment [-204/-129] conferred the hormone responsiveness in an orientation-independent manner when it was placed upstream to the heterologous thymidine kinase promoter or the rabbit beta-globin promoter. The segment [-204/-129] contained two direct repeat motifs homologous to other VDREs found in the osteocalcin and osteopontin genes. Synthetic oligonucleotides containing the putative VDRE were used for functional analyses and gel mobility shift assays. The proximal [-151/-137], but not the distal [-169/-155] direct repeat activated the transcription in response to 1,25-(OH)2D3 through the beta-globin promoter. Furthermore, the proximal direct repeat formed a complex with the vitamin D receptor and a nuclear accessory factor(s) from COS cells (or retinoid X receptor) in the presence of 1,25-(OH)2D3. These results indicate that a direct repeat motif, AGGTGAgt-gAGGGCG, located at -151 base pairs upstream in the antisense strand binds to a heterologous dimer consisting of the VDR occupied with 1,25-(OH)2D3 and the nuclear accessory factor and that it plays a critical role in mediating the vitamin D enhancement of the rat P450cc24 gene expression.
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PMID:Identification of a vitamin D-responsive element in the 5'-flanking region of the rat 25-hydroxyvitamin D3 24-hydroxylase gene. 814 41

We have examined the 5' flanking region of the mouse calbindin-D28k gene and identified a 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-responsive element by deletion mutant analysis of the native promoter as well as by studies with a heterologous thymidine kinase (TK) promoter. The segment between residues -200 and -169 was found to confer a dose-dependent 1,25-(OH)2D3 responsiveness through the TK promoter in Ros 17/2.8 cells as well as in CV-1 cells cotransfected with pAV-hVDR (human vitamin D receptor expression vector). This region contains sequences homologous to the rat osteocalcin vitamin D response element (VDRE). Incubation of this element with nuclear extracts from 1,25-(OH)2D3-treated Ros 17/2.8 cells or from 1,25-(OH)2D3-treated COS cells that had been transfected with pAV-hVDR resulted in a specific protein-DNA interaction. In addition to 1,25-(OH)2D3, sodium butyrate, a differentiating agent, has also been found to modulate expression of calbindin-D28k. Deletion analysis of the mouse calbindin-D28k promoter as well as studies with a heterologous TK promoter resulted in identification of a butyrate-responsive element between -180 and -150 that was found to bind specifically to nuclear factors from butyrate-treated Ros 17/2.8 cells. This butyrate-responsive element may represent a genetic element acted upon by enhancer binding proteins. In summary, the 5' flanking region of the mouse calbindin-D28k gene contains responsive elements that interact with nuclear factors and may mediate, at least in part, the enhanced expression of this gene by 1,25-(OH)2D3 and butyrate.
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PMID:Identification of sequence elements in mouse calbindin-D28k gene that confer 1,25-dihydroxyvitamin D3- and butyrate-inducible responses. 846 15

Transcription of the CYP24 gene is induced by 1,25-(OH)2D3 through a vitamin D receptor-dependent process. The functional activities of three possible vitamin D response elements (VDREs), located on the antisense strand of the rat CYP24 promoter, were investigated by transient expression of native and mutant promoter constructs in COS-1, JTC-12, and ROS 17/2.8 cells. A putative VDRE with a half-site spacing of 6 base pairs at -249/-232 (VDRE-3) did not contribute to 1,25-(OH)2D3 induced expression in the native promoter, although activity has been reported when the element was fused to the heterologous thymidine kinase promoter. Two VDREs with half-site spacings of 3 base pairs at -150/-136 and -258/-244 (VDRE-1 and VDRE-2, respectively), showed transcriptional synergism in COS-1 cells when treated with 1,25-(OH)2D3 (10(-7) to 10(-11) M). The contribution of both VDREs was hormone-concentration dependent from 10(-10) to 10(-12) M, with VDRE-1 demonstrating greatest sensitivity to 1,25-(OH)2D3. Transactivation by VDRE-1 was always greater than VDRE-2, but the converse was observed for the binding of vitamin D receptor-retinoid X receptor complex by each VDRE in gel mobility shift assays. The synergy observed between VDRE-1 and VDRE-2 may have important implications in cellular responses to different circulating levels of 1,25-(OH)2D3.
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PMID:Transcriptional synergism between vitamin D-responsive elements in the rat 25-hydroxyvitamin D3 24-hydroxylase (CYP24) promoter. 893 5

Vitamin D is an important regulator of phosphate homeostasis. The effects of vitamin D on the expression of renal Na+-dependent inorganic phosphate (Pi) transporters (types I and II) were investigated. In vitamin D-deficient rats, the amounts of type II Na+-dependent Pi transporter (NaPi-2) protein and mRNA were decreased in the juxtamedullary kidney cortex, but not in the superficial cortex, compared with control rats. The administration of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) to vitamin D-deficient rats increased the initial rate of Pi uptake as well as the amounts of NaPi-2 mRNA and protein in the juxtamedullary cortex. The transcriptional activity of a luciferase reporter plasmid containing the promoter region of the human type II Na+-dependent Pi transporter NaPi-3 gene was increased markedly by 1,25-(OH)2D3 in COS-7 cells expressing the human vitamin D receptor. A deletion and mutation analysis of the NaPi-3 gene promoter identified the vitamin D-responsive element as the sequence 5'-GGGGCAGCAAGGGCA-3' nucleotides -1977 to -1963 relative to the transcription start site. This element bound a heterodimer of the vitamin D receptor and retinoid X receptor, and it enhanced the basal transcriptional activity of the promoter of the herpes simplex virus thymidine kinase gene in an orientation-independent manner. Thus, one mechanism by which vitamin D regulates Pi homeostasis is through the modulation of the expression of type II Na+-dependent Pi transporter genes in the juxtamedullary kidney cortex.
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PMID:Regulation of type II renal Na+-dependent inorganic phosphate transporters by 1,25-dihydroxyvitamin D3. Identification of a vitamin D-responsive element in the human NAPi-3 gene. 960 73

Repression of basal transcription of a 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) responsive 25-hydroxyvitamin D3-24-hydroxylase (CYP24) promoter construct as observed in kidney cells in the absence of ligand and this repression was dependent on a functional vitamin D response element (VDRE). Basal repression was also seen with a construct where a consensus DR-3-type VDRE was fused to the thymidine kinase promoter. Expression of a dominant negative vitamin D receptor (VDR) isoform that strongly bound to the VDRE motif in the CYP24 promoter ablated basal repression. This VDR isoform lacked sequence in the hinge- and ligand-binding domains implicating one or both of these domains in basal repression. It is well known that thyroid hormone and retinoic acid receptors silence basal transcription of target genes in the absence of ligands and this repressor function can be mediated by the nuclear receptor corepressor N-CoR. Two variants of N-CoR have been described, RIP13a and RIP13delta1. N-CoR and the variants contain two receptor interaction domains, ID-I and ID-II, which are identical except region ID-II in RIP13delta1 has an internal deletion. We have used the mammalian two hybrid system to investigate whether VDR, in the absence of ligand 1,25-(OH)2D3, can interact with these domains. The data showed that unliganded VDR does not interact with either ID-I or ID-II from RIP13a and RIP13delta1, but does interact strongly with a composite domain of ID-I and ID-II from RIP13delta1 (but not from RIP13a) and this strong interaction is abrogated in the presence of ligand. This finding implicates RIP13delta1 in VDR-dependent basal repression of the promoter constructs under investigation. However, over-expression of RIP13delta1 in kidney cell lines did not alter basal expression of the CYP24 promoter construct. It is concluded that either the level of endogenous RIP13delta1 in these kidney cells permits maximal repression or that repression occurs by a mechanism that is independent of RIP13delta1. Alternatively, repression may be dependent on RIP13delta1 but requires an additional cofactor that is limiting in these cells.
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PMID:Repression of basal transcription by vitamin D receptor: evidence for interaction of unliganded vitamin D receptor with two receptor interaction domains in RIP13delta1. 968 55

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.
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PMID:Thyroid hormone receptor does not heterodimerize with the vitamin D receptor but represses vitamin D receptor-mediated transactivation. 973 5

The actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3) are mediated through the nuclear vitamin D receptor (VDR). The regulation of VDR abundance plays an important role in determining the magnitude of the target cell response to 1,25(OH)2D3. The major physiological activity of 1,25(OH)2D3 is the regulation of calcium absorption in the small intestine, and the level of VDR is an important factor in this regulation. However, the characterization of VDR gene expression in the small intestine remains unknown. In the present study, we investigated the regulation of the human VDR (hVDR) gene expression in the small intestine. The 4.0 kb of the 5'-flanking region of the hVDR gene promoter was cloned and characterized by the measurement of luciferase activity and an electrophoretic mobility-shift assay (EMSA). With the EMSA, we found that Cdx-2 (a homeodomain protein-related caudal) binds to the sequence 5'-ATAAAAACTTAT-3' at -3731 to -3720 bp (hVD-SIF1) relative to the transcription start site of the hVDR promoter. This sequence is very similar to the human sucrase-isomaltase footprint 1 (SIF1) element. With a competition analysis and specific antibodies for Cdx-2, we demonstrated that Cdx-2 is able to activate VDR gene transcription by binding to this element. The mutation of the hVD-SIF1 sequence in the hVDR gene promoter markedly suppressed the transactivation of the reporter gene in Caco-2 cells. In addition, the DNA fragment (-3996 to -3286) containing the hVD-SIF1 binding site increased transcription when placed upstream of the herpes simplex virus thymidine kinase promoter. These findings suggest that Cdx-2 plays an important role in the intestine-specific transcription of the hVDR gene.
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PMID:The caudal-related homeodomain protein Cdx-2 regulates vitamin D receptor gene expression in the small intestine. 993 78


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