Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Small changes in the concentrations and/or combinations of trans-acting factors can result in profound alterations in gene expression. Synergistic interaction between different classes of transcription factors bound to distinct sites within a promoter/enhancer region is one mechanism by which this can occur. Reflecting this, hormone response elements, DNA recognition sites for steroid/nuclear receptors, are often found in promoter regions organized as multiple copies or are clustered among binding sites for other trans-acting factors. To systematically examine the potential interactions between one such receptor, the vitamin D3 receptor (VDR), and other nonreceptor transcription factors, we constructed a series of reporter plasmids containing one copy of the osteopontin (Spp1) vitamin D response element (VDRE), consisting of two direct repeats spaced by 3 base pairs, and one binding site for the transcription factors SP1, NF-1, Oct-1, or AP-1. We also generated reporters either under the control of two copies of Spp1 VDRE, or a distinct VDRE from the human osteocalcin gene promoter. The various reporters were used to transiently transfect HeLa or CV-1 cells in the presence and absence of 1,25-dihydroxyvitamin D3. Our results show that VDR transactivates 12-20 times more strongly from two Spp1-VDREs than from one, indicating that VDR synergizes with itself. VDR also synergizes with the other nonreceptor factors, since we observe a 6- to 12-fold degree of synergistic induction after ligand addition, depending on the particular factor. The functional basis for the transcriptional synergism appears to be at the level of cooperative DNA binding, at least for VDR alone and VDR-Oct-1, as demonstrated in vitro by gel mobility shift assays using purified factors. Consistent with this, we show that the minimal requirement for transcriptional synergism in vivo by VDR is its DNA-binding domain.
Mol Endocrinol 1994 Dec
PMID:Transcriptional synergism between the vitamin D3 receptor and other nonreceptor transcription factors. 770 50

Noradrenaline and ATP evokes a transient increase in the intracellular calcium concentration ([Ca2+]i) in FRTL-5 cells. In a previous study, we showed that 1,25-dihydroxyvitamin-D3 (1,25(OH)2-D3) increases the ATP evoked changes in [Ca2+]i. In the present paper, we found that pre-incubating the cells with 10 nM 1,25(OH)2-D3 for 48 h did not affect the noradrenaline-evoked increase in [Ca2+]i. We subsequently examined if this could be due to an effect of 1,25(OH)2-D3 on alpha 1-adrenergic receptor number, or receptor affinity. Pretreatment with 10 nM 1,25(OH)2-D3 for 48 h decreased the binding of the alpha 1-adrenergic specific antagonist [3H]prazosin by 55% (Bmax for 1,25(OH)2-D3 treated = 27.6 +/- 5.0 fmol/mg protein, untreated = 61.7 +/- 5.4 fmol/mg protein). No effect of 1,25(OH)2-D3 on the affinity for [3H]prazosin was observed. The effect of 1,25(OH)2-D3 on the [3H]prazosin binding was both time- and dose-dependent and could first be seen after 8-12 h of 1,25(OH)2-D3 treatment, indicating a genomic effect. The effect of 1,25(OH)2-D3 could be abolished with the protein synthesis inhibitor cycloheximide. No effect on the [3H]prazosin binding could be seen after a 48 h preincubation with 100 nM of either 24,25-dihydroxyvitamin D3 and 25-dihydroxyvitamin D3, indicating that the effect of 1,25(OH)2-D3 was specific. The cellular cAMP concentration was decreased after 48 h treatment with 10 nM 1,25(OH)2-D3. When TSH was replaced with dibutyryl cAMP or forskolin the [3H]prazosin binding increased. 1,25(OH)2-D3 also reduced the dibutyryl cAMP and forskolin stimulated [3H]prazosin binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Endocrinol 1995 Feb 27
PMID:1,25-Dihydroxyvitamin D3 reduces the number of alpha 1-adrenergic receptors in FRTL-5 rat thyroid cells. 775 27

Sarcoidosis is characterized by the accumulation of activated lymphocytes in the lungs and other organs and by the spontaneous production of the active form of vitamin D, calcitriol. We hypothesized that calcitriol may modulate the responsiveness of human lymphocytes to the relevant biologic mediator, interleukin-2 (IL-2). After culture for 48 h with phytohemagglutinin, human peripheral blood lymphocytes migrated through nitrocellulose filters, secured in microchemotaxis chambers, in response to IL-2. When calcitriol at 1 nM was included in the cultures, the migratory response to IL-2 was completely abrogated. This inhibitory effect was seen despite the fact that cultured lymphocytes continued to express the IL-2 receptor and other activation markers. A similar but more rapid effect could be demonstrated by including calcitriol in the lower well during our 3-h chemokinesis assay. Calcitriol blocked IL-2-induced lymphocyte migration in a dose-dependent fashion. The synthetic noncalcemic vitamin D analogue MC 903 was equally effective in this assay. IL-2-induced migration could also be prevented by the protein kinase C inhibitor H-7, but calcitriol appeared to be at least 1,000 times more potent. Our studies suggest that calcitriol is a potent natural immunomodulator with rapid suppressive effects that may be mediated through protein kinase C. Synthetic analogues such as MC 903 may offer exciting therapeutic opportunities.
Am J Respir Cell Mol Biol 1995 Jun
PMID:Calcitriol and its synthetic analogue MC 903 inhibit the interleukin-2-induced migration of human lymphocytes. 776 30

Although steroid hormone receptor activation has been known to be dependent on ligand binding, we report here ligand-independent transcriptional activation of the vitamin D receptor and retinoid receptors. In these studies, CV1 cells were transiently transfected with a human vitamin D receptor (VDR) expression vector and a reporter plasmid that contains multiple copies of the rat osteocalcin vitamin D response element up-stream of the bacterial chloramphenicol acetyltransferase (CAT) gene [osteocalcin (OC)VDREtkCAT]. Treatment of cells with 10(-8) M 1,25-dihydroxyvitamin D3 resulted in a 25-fold induction of CAT activity. When cells were treated with 5-50 nM okadaic acid (OA), an inhibitor of protein phosphatase-1 and -2A, significant inductions of CAT activity (18- to 57-fold) were observed. As VDR and dopamine receptors are colocalized in certain brain regions, we also examined whether VDR-mediated transcription can be activated by dopamine. VDR was found to activate CAT gene expression in cells treated with 200-500 microM dopamine (3- to 11-fold induction) or the selective D1 agonist SKF38393 (20-fold induction). Cells were also transfected with retinoic acid receptor (RAR) or retinoid-X receptor (RXR) expression vectors and reporter plasmids that contain either a retinoic acid response element or an RXR-specific response element. OA alone induced chloramphenicol acetyltransferase (CAT) activity in cells transfected with RAR alpha, RAR beta, RXR alpha, RXR beta, or RXR gamma (3- to 18-fold induction). However, OA did not affect transcription by RAR gamma, suggesting specificity of activation by OA among the retinoid receptors. Although the retinoid receptors have been detected in brain, maximum stimulation of transcription was not greater than 1.6-fold in the presence of 100-500 microM dopamine or 100 microM SKF38393 treatment. These data suggest specificity for dopamine activation among steroid hormone receptors and that phosphorylation alone, in the absence of ligand, can activate VDR- and retinoid receptor-mediated transcription.
Mol Endocrinol 1995 Feb
PMID:Ligand occupancy is not required for vitamin D receptor and retinoid receptor-mediated transcriptional activation. 777 73

The purpose of this study was to investigate whether vitamin D3 deficiency and 1,25-dihydroxyvitamin D3 treatment affect some aspects of heart metabolism in the rat. To this end, five experimental groups were studied: (1) the control group of the vitamin D3 supplemented rats (Group A); (2) rachitic rats (Group B); (3) rachitic rats treated with 1,25-dihydroxyvitamin D3 (Group C); (4) rats fed a vitamin D-deficient diet (Group D); (5) rats fed a vitamin D-deficient diet and treated with 1,25-dihydroxyvitamin D3 (Group E). The five groups were compared by checking in the heart some metabolic parameters, i.e. citrate content, and enzyme activities in cytosol and mitochondria. Citrate content was higher in the heart of treated animals when compared with the control. As regards the enzymatic activities in heart mitochondria, NAD(+)-dependent isocitrate dehydrogenase remarkably decreased in Group B rats and 1,25-dihydroxyvitamin D3 restored quite normal values. NADP(+)-dependent isocitrate dehydrogenase decreased in Group B and Group D animals, and 1,25-dihydroxyvitamin D3 treatment was effective in restoring control values. Cytochrome c oxidase activity did not change, while citrate synthase showed an increase in all the treated rats. As regards the cytosolic enzymes, fructose-6-phosphate kinase increased in the two groups of vitamin D-deplete rats in comparison with the control. Glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase showed a similar trend: an increase in all the treated animals. In heart homogenate, acylphosphatase and acid phosphatase activities were also determined. Acylphosphatase increased in the treated rats, while acid phosphatase decreased in the rats injected with 1,25-dihydroxyvitamin D3. These results support the hypothesis of a participation of 1,25-dihydroxyvitamin D3 in some aspects of heart metabolism.
J Mol Cell Cardiol 1994 Nov
PMID:Effect of vitamin D deficiency and 1,25-dihydroxyvitamin D3 on rat heart metabolism. 789 66

The transcription of vitamin D (VD) responsive genes is regulated by three different nuclear signalling pathways mediated by homodimers of VD receptors (VDRs), heterodimers of VDRs and retinoid X receptors (RXRs) and heterodimers of VDRs with retinoic acid receptors (RARs), Here, the in vitro DNA-binding affinity of all three receptor complexes was shown to be enhanced by the presence of VD. However, the specificity of the three pathways was dictated by the differential affinities of the receptor complexes for VD response elements. Potential response elements were distinguished by the sequence, the separation and the relative orientation of the hexameric core binding motifs. It was found that both VDR-RAR and VDR-RXR heterodimers act functionally on all three response element configurations: direct repeats, palindromes and inverted palindromes. With direct repeats, neither heterodimer type showed a preference for any of the three principal core motifs, (A/G)GGTGA, (A/G)GGTCA and (A/G)GTTCA. However, while they did exhibit preferences for core motifs in palindromes, the spacing requirements were identical for both complexes. Inverted palindromes, however, formed the most specific response elements. A simple model explains a steric link between the optimal spacing of direct repeats and that of inverted palindromes. Taken together, the experimental data and the model provide further criteria for the screening of VD responsive genes.
J Mol Endocrinol 1994 Jun
PMID:Response element selectivity for heterodimerization of vitamin D receptors with retinoic acid and retinoid X receptors. 791 71

An expanded role for vitamin D (1 alpha,25-(OH)2D3) in mammalian systems has been suggested by recent evidence that its receptor (vitamin D receptor [VDR]) is present not only in classical target organs, but in a variety of normal tissues and organs, tumor tissues, and cancer cell lines. Vitamin D is involved not only in the regulation of calcium homeostasis and bone metabolism, but in the regulation of cell proliferation, differentiation, and immune responses. The role vitamin D may play in normal lung growth, development, and maintenance is unknown. Likewise, its part in lung tumorigenesis is unclear. The present study examined VDR binding activity and VDR expression in normal mouse lung and ethylnitrosourea-induced lung adenomas. Binding of 1 alpha,25-(OH)2D3 was specific and saturable over the concentration range of 0.01 to 0.50 nM, with an affinity (Kd) of 0.93 x 10(-10) M and a total binding capacity (Bmax) of 22 fmol/mg of protein. Scatchard analysis yielded a convex curve, which suggests positive receptor cooperativity. The calculated Hill coefficient equals 1.69, at a receptor concentration of 0.4 nM, consistent with dimerization of the receptor. Western blot analysis showed the presence of 60 kD VDR protein in tumor homogenates, while Northern blot analysis detected the 4.4 kb VDR mRNA in tumor tissue preparations. Immunohistochemistry and in situ hybridization revealed that both adenomatous Clara cells and normal bronchiolar epithelial Clara cells expressed VDR, with the receptor protein present in their nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1994 Oct
PMID:Vitamin D3 receptor expression in N-ethylnitrosourea-induced mouse pulmonary adenomas. 791 16

DNA binding site discrimination within a subgroup of nuclear receptors, including the human vitamin D3 receptor (hVDR), appears to be influenced primarily by spacing and orientation differences of response element half-sites, since many receptors recognize and bind to the same hexameric half-site sequence, AGGTCA. Small sequence differences within half-sites, however, may also play an important role in distinguishing between different receptor complexes. Several laboratories have reported that the AGGTCA element in a direct repeat (DR) configuration appears to be a high affinity recognition site for only nuclear receptor-9 retinoid X receptor (RXR) heterodimers. However, we have previously shown that a closely related, but distinct, element (AGTTCA; essentially the mouse osteopontin [Spp-1] vitamin D response element) acts as a high affinity target for purified hVDR in the absence of RXR. This suggests that some half-site sequences could be targets for hVDR alone while others serve as recognition elements for hVDR-RXR complexes. In this report, we test this hypothesis by selecting, using purified hVDR only, for high affinity receptor binding sites in a complex DNA mixture which should by chance contain such sequences. We find that the purified receptor selects a heptameric sequence resembling a half-site of the osteopontin vitamin D response element, consistent with osteopontin-like sequences acting as high affinity targets for hVDR in the absence of RXR. We directly test this by comparing the in vitro DNA binding activity of purified hVDR to DR+3 elements comprised of osteopontin-like AGTTCA or AGGTCA half-sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1994 Mar
PMID:DNA sequences that act as high affinity targets for the vitamin D3 receptor in the absence of the retinoid X receptor. 801 45

The brain isozyme of creatine kinase (CKB) is a major component of the estrogen-induced proteins in the rat uterus. Hormonal specificity of this response was studied in cotransfection assays using the rat CKB promoter linked to the bacterial chloramphenicol acetyltransferase gene. Response was specific for estrogen as 17 beta-estradiol in the presence of estrogen receptor dramatically stimulated the CKB promoter. This induction was completely blocked by the estrogen antagonist ICI 164,384. Nuclear receptors for progesterone, androgen, glucocorticoid and vitamin D did not significantly activate the CKB promoter in the presence of their respective ligands. Creatine kinase (CK) activity was analyzed in decidualized mouse uterus to assess estrogenic activity in vivo. Upon oil stimulation, uterine horns of day 4 pseudopregnant mice underwent a dramatic outgrowth in response to endogenous progesterone. This response was accompanied by a significant decrease in CK activity from a control value of 1.44 +/- 0.25 to 0.38 +/- 0.08 IU/mg protein (P < 0.001), indicating that the action of estrogen was suppressed. Treatment of females one day prior to oil-stimulation with progesterone receptor antagonists, RU486 (Mifepristone) or ZK299 (Onapristone), or with a monoclonal antibody to progesterone (DB3), abolished decidualization, and also restored the CK activity to the control value. These results suggest that CK can be used as a specific cellular marker to detect unopposed estrogen action in the mouse uterus associated with progesterone withdrawal or receptor blockade.
J Steroid Biochem Mol Biol 1994 Jun
PMID:Creatine kinase activity as an indicator of unopposed estrogen action in the mouse uterus associated with anti-progesterone treatment. 803 8

The physicochemical principle of "die and coin" complementarity proffered by Pauling and Delbruck and exemplified in Watson and Crick DNA was used to design new antineoplastic compounds. In search of an explanation for why certain molecules and not others are present in nature, biologically active small molecules were discovered to exhibit complementarity when inserted into cavities between base pairs in DNA. Ligands in the steroid/thyroid hormone/vitamin D family fit particularly well into the site 5'-dTdG-3'.5'-dCdA-3'. Degree of fit of various candidate compounds in the manner of a given hormone correlated with degree of hormonal activity. Hormone antagonists fit into the same site but in a different manner than the agonists. Computer graphics and energy calculations confirmed salient observations including the remarkable complementarity of estradiol and DNA. Using the above criteria, a new candidate antiestrogen, para-hydroxyphenyl-acetylamino-2,6-piperidinedione was successfully designed. Taken as a whole, these results coupled with recent independent findings raise the possibility that the mode of action of certain hormones and hormone antagonists may involve direct insertion into DNA mediated by classical protein receptors and other transcription factors.
J Steroid Biochem Mol Biol 1994 Jun
PMID:Design of novel antiestrogens. 804 89


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