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

A genomic DNA clone for 1 alpha,25-dihydroxyvitamin D-3 (1,25-(OH)2D3) 24-hydroxylase was isolated from a human chromosome 20 library. It spans 2.42 kb, containing the first two exons, the first and part of the second introns, and a 1.26 kb 5'-flanking region. Putative transcription cis-elements were revealed throughout the 5'-flanking region, including TATA box, CAAT box, GC boxes, vitamin D-responsive elements (VDRE), AP1, and AP2 sites. In a CAT reporter gene expression assay, the 24-hydroxylase promoter with its 1.2 kb 5'-flanking sequence elicits a 1,25-(OH)2D3-induced transactivation activity. Gel mobility shift assays of those putative DREs have identified that two different elements can form specific complexes with porcine intestinal nuclear extract (PINE). The specificity of VDRE-PINE complexes was verified by supershift assay with VDR-specific monoclonal antibody VXIE10B6. The proximal element VDREp (-172/-143) consists of three direct repeat half-sites, GAGTCAgcgAGGTGAgcgAGGGCG, in anti-sense orientation. The distal element VDREd (-293/-273) consists of two direct repeat half-sites, GCGTTCaccGGGTGT, also in anti-sense orientation. Both VDREs can direct a reporter gene expression using a heterologous herpes simplex virus thymidine kinase (TK) promoter in a 1,25-(OH)2D3-dependent fashion. Further characterization of these VDREs in various constructs with either a native or TK promoter suggests that both VDREs are required for the optimal induction of 24-hydroxylase expression by 1,25-(OH)2D3.
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PMID:Cloning of the human 1 alpha,25-dihydroxyvitamin D-3 24-hydroxylase gene promoter and identification of two vitamin D-responsive elements. 763 26

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

Although several studies have been performed on the biological activities of analogs of 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3) at the whole animal and cellular levels, little work has been done to analyze their transcriptional activation properties. A highly inducible 1,25-(OH)2 D3-responsive promoter composed of three copies of the mouse osteopontin vitamin D3 response element (VDRE3) inserted upstream of a herpes simplex virus thymidine kinase promoter has been constructed, and its transcriptional properties have been analyzed by transient transfection into the monkey kidney cell line COS-7 and the rat osteoblast-like osteosarcoma line ROS 17/2.8. We have studied systematically transcriptional activation by a number of 1,25-(OH)2 D3 analogs, particularly those substituted at positions 16, 23, 26, and 27, sites that are targets for metabolism. Strikingly, except for derivatives that bind the 1,25-(OH)2 D3 receptor (VDR) very weakly, we find no parallel between the potency of action of a derivative as a transcriptional inducer and its affinity for the VDR. Derivatives substituted by multiple bonds at positions 16 and/or 23, although having varying affinities for the VDR, all stimulate transcription more potently than D3, in some cases at 100-fold lower concentrations. The peak transcriptional activity observed varies by only approximately 20% among different active analogs, indicating little difference in the activity of the VDR once bound to ligand. Gel retardation assays with ROS 17/2.8 nuclear extracts suggest that the VDR binds to the mouse osteopontin VDRE predominantly as a heterodimer with retinoid X receptor(s) (RXR(s)). We find that 9-cis-retinoic acid, the cognate ligand for RXRs, does not have a significant effect on the response of the VDRE3 promoter to 1,25-(OH)2 D3 or a number of its derivatives in ROS 17/2.8 or in COS-7 cells, under conditions in which promoters containing retinoid X response elements are activated. This suggests that 9-cis-retinoic acid may not act on the response to 1,25-(OH)2 D3 or its derivatives by directly influencing the transcriptional activity of VDR/RXR heterodimers. This promoter/reporter system should be useful for analyzing the tissue-specific transcriptional activity of 1,25-(OH)2 D3 and its derivatives in any cell type amenable to transient transfection.
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PMID:Highly potent transcriptional activation by 16-ene derivatives of 1,25-dihydroxyvitamin D3. Lack of modulation by 9-cis-retinoic acid of response to 1,25-dihydroxyvitamin D3 or its derivatives. 830 Jun 29

The sequences in the rat osteocalcin gene that lie 3' to the vitamin D response element (VDRE) have been shown to augment transcriptional activation by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. These DNA sequences, however, are unable to bind the VDR or mediate 1,25-(OH)2D3 responsiveness independently of the VDRE. To further characterize this region, the functional properties of a series of mutant oligonucleotides were examined in transiently transfected ROS 17/2.8 cells. When these mutant oligonucleotides were expressed upstream of the heterologous herpes simplex virus thymidine kinase promoter, the bases between -420 and -414 of the rat osteocalcin gene were identified as critical for maximal transactivation by 1,25-(OH)2D3. Furthermore, mutation of these sequences in the context of the native osteocalcin promoter and enhancer totally abolished the ability of the VDRE to mediate 1,25-(OH)2D3 responsiveness. These bases, which are essential for the 1,25-(OH)2D3 responsiveness of the rat osteocalcin gene, are also present in a similar position, relative to the VDRE, in the human osteocalcin gene. To explore whether these sequences could enhance transactivation by other inducible transcription factors, they were examined for their ability to synergize with the chick vitellogenin estrogen response element and the rat somatostatin cAMP response element. When placed upstream to the herpes simplex virus thymidine kinase promoter and transfected into ROS 17/2.8 cells, these sequences were able to enhance transcriptional responsiveness to 17beta-estradiol and forskolin, respectively, demonstrating that they also contribute to transactivation by other inducible transcription factors.
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PMID:DNA sequences downstream from the vitamin D response element of the rat osteocalcin gene are required for ligand-dependent transactivation. 901 68

Crystallographic structures of the ligand-binding domains for the retinoid X (RXR) and estrogen receptors have identified conserved surface residues that participate in dimer formation. Homologous regions have been identified in the human vitamin D receptor (hVDR). Mutating Lys-386 to Ala (K386A) in hVDR significantly reduced binding to glutathione S-transferase-RXRalpha in solution, whereas binding of an I384R/Q385R VDR mutant was almost undetectable. The K386A mutant formed heterodimers with RXRalpha on DR-3 (a direct repeat of AGGTCA spaced by three nucleotides), whereas the I384R/Q385R mutant completely eliminated heterodimer formation. Wild type hVDR effected a 3-fold induction of DR-3-dependent thymidine kinase-luciferase activity in cultured neonatal rat atrial myocytes, an effect that was increased to 8-9-fold by cotransfected hRXRalpha. Induction by K386A, in the presence or absence of RXRalpha, was only slightly lower than that seen with wild type VDR. On the other hand, I384R/Q385R alone displayed no stimulatory activity and less than 2-fold induction in the presence of hRXRalpha. Qualitatively similar findings were observed with the negative regulation of the human atrial natriuretic peptide gene promoter by these mutants. Collectively, these studies identify specific amino acids in hVDR that play a critical role in heterodimer formation and subsequent modulation of gene transcription.
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PMID:Vitamin D-dependent suppression of human atrial natriuretic peptide gene promoter activity requires heterodimer assembly. 1019 14

Farnesoid X receptor (FXR), the receptor for bile acids, including chenodeoxycholic acid (CDCA), is a member of the nuclear receptor superfamily, which also includes the receptors for retinoic acid, vitamin D (D3), thyroid hormone, thiazolidinedione and 22(R)-hydroxycholesterol. Here, we have evaluated the effects of a series of ligands and their receptors on the promoter activity induced by CDCA/FXR. The kidney cell line, CV1, was cotransfected with FXR-expression plasmid and the luciferase-based reporter gene that has a thymidine kinase promoter fused to the canonical FXR-responsive element or the natural promoter for the small heterodimer partner (SHP), bile salt export pump (BSEP), and ileum bile acid (I-BABP) gene. D3 and its receptor (VDR) inhibited the transactivation of all four reporter constructs that are enhanced by CDCA/FXR. The effect of D3 on the expression of the BSEP and SHP genes in HepG2 cells and that of the I-BABP gene in Caco-2 cells were confirmed by reverse transcription (RT)-PCR. Deletion analysis of VDR revealed that its ligand-binding domain (LBD) is responsible for the repression and the DNA-binding domain (DBD) is dispensable. Specific interaction between FXR and VDR was detected with the in vitro pull-down assay using chimeric FXR or VDR fused to glutathione-S-transferase.
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PMID:1,25-dihydroxyvitamin D3 and its receptor inhibit the chenodeoxycholic acid-dependent transactivation by farnesoid X receptor. 1652 42