Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Milk protein gene expression is regulated by the synergistic interactions of several lactogenic hormones, including insulin, PRL, and glucocorticoids. Whey acidic protein (WAP) gene expression is highly dependent on glucocorticoids, and to a lesser extent than casein gene expression, on the presence of PRL. Previous studies have demonstrated that a distal DNase I hypersensitive site in the rat WAP gene 5'-flanking region containing several binding sites for nuclear factor I is required for high level WAP gene expression in transgenic mice. In this study several specific glucocorticoid receptor (GR) binding sites were identified flanking these nuclear factor I sites using an in vitro DNase I footprinting assay with baculovirus-expressed GR. These sites were able to confer dexamethasone inducibility to a heterologous thymidine kinase-chloramphenicol acetyltransferase reporter gene construct in transient cotransfection experiments with GR in CV1 cells. Administration of dexamethasone to adrenal-ectomized mice carrying the +2020 rat WAP transgene during lactation demonstrated that glucocorticoids are required to maintain transgene expression in the mammary gland. Furthermore, glucocorticoid-induced changes in transgene expression were correlated with the appearance of DNase I hypersensitive sites. These results indicate that at least part of glucocorticoid regulation of WAP gene expression is mediated through the direct interaction of GR with glucocorticoid response elements in the distal promoter region resulting in steroid hormone-dependent alterations in chromatin structure.
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PMID:Glucocorticoid regulation of rat whey acidic protein gene expression involves hormone-induced alterations of chromatin structure in the distal promoter region. 785 50

We have established a system in which we observe a synergistic interaction between insulin and glucocorticoids. This includes chimeric genes constructed to contain synthetic glucocorticoid-responsive elements, 5' of the HSV thymidine kinase promoter and the chloramphenicol acetyltransferase reporter gene. The magnitude of induction of gene expression by glucocorticoid was dependent on the number of GREs. Insulin alone had virtually no effect on the expression of any of these genes but together with dexamethasone acted in a synergistic manner. This synergy diminished as the number of GREs in the promoter increased. The synergy is independent of promoter sequences other than the GREs and a functional TATAA box. Three different approaches demonstrate that the effect of insulin is not directly on the glucocorticoid signal transduction pathway. Insulin does not change the dose-response relationship for dexamethasone. The effect of insulin is independent of the intracellular concentration of glucocorticoid receptor. The effect is independent of any specific domain of the glucocorticoid receptor. The target of insulin action is likely to be part of the normal host cell transcriptional initiation complex or a putative adaptor molecule.
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PMID:Insulin enhances glucocorticoid receptor-mediated induction of gene expression independent of a specific insulin response element. 786 47

The somatostatin (SS) gene is transcriptionally regulated via the cyclic AMP (cAMP) response element (CRE), located in the proximal promoter (-41 to -48 bp). We have previously reported that glucocorticoids induce dose-dependent cell-specific alterations in the steady-state SS mRNA level. Here we have investigated direct transcriptional control of the SS gene by glucocorticoids. We have examined transcriptional interaction between glucocorticoids and the cAMP signalling pathway and mapped the 5' upstream regulatory region of the SS gene involved in glucocorticoid transactivation. Transcriptional regulation was determined by analysis of chloramphenicol acetyltransferase (CAT) activity in PC12 rat pheochromocytoma cells and A126-1B2 (protein kinase A-deficient mutant PC12) cells, by acute transfection of 5' flanking SS DNA (- 750, -250 and -71 bp) ligated to the reporter (CAT) gene. Dexamethasone (DEX) induced a dose-dependent 2.2-fold stimulation of SS gene transcription in PC12 cells, but not in A126-1B2 cells. Other steroid and thyroid hormones tested, and retinoic acid, were ineffective, while cAMP and forskolin stimulated gene transcription 4-5-fold in PC12 cells but not in A126-1B2 cells. DEX exerted an additive effect on cAMP-induced gene transcription. Deletion of the promoter from -750 to -71 bp (but not from -750 to -250 bp) abolished all stimulatory effects of DEX without affecting cAMP responsiveness. Mutation of the CRE abrogated both DEX- and cAMP-dependent gene enhancement. Gel electrophoretic mobility shift assays confirmed that the -250 to -71 bp region of the SS promoter (but not the -71 to +55 bp domain) binds specifically to a glucocorticoid response element-sensitive nuclear protein(s) from PC12 cells, suggesting a putative glucocorticoid receptor interaction with SS promoter DNA. We conclude that glucocorticoids regulate SS gene transcription positively. Glucocorticoid-induced transactivation shows dependence on protein kinase. A activity, and may be mediated via protein-protein interaction between the glucocorticoid receptor and the CRE binding protein. DNA sequences upstream from the CRE between -250 and -71 bp in the SS promoter appear to be the target of glucocorticoid action.
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PMID:Glucocorticoids activate somatostatin gene transcription through co-operative interaction with the cyclic AMP signalling pathway. 791 2

To determine the relative potency of synthetic glucocorticoids, glucocorticoid receptor expressing cells were transfected with a hormone-inducible reporter gene, and were cultured in the presence of various glucocorticoid ligands. Hormonal inducibility was determined by means of a chloramphenicol acetyltransferase assay. Dexamethasone and prednisolone, as well as cortisol, induced the expression of the reporter gene in a dose-dependent fashion. The relative potency of each ligand was in this order when inducibility was quantitatively assessed. In conclusion, the transcription assay described here may be a convenient and alternative method to evaluate the relative potency of given glucocorticoids.
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PMID:Relative glucocorticoid potency revisited. 793 39

Estrogenic effects on the proliferation and differentiated cellular functions of bone cells have been described in vivo and in vitro. In particular, stimulatory effects on the growth rate of osteoblasts have been observed, although these are generally small. In an attempt to produce a more sensitive model for the study of estrogen action in bone, HTB 96 human osteoblast-like osteosarcoma cells, which lack endogenous estrogen receptor (ER), were stably transfected with an expression vector coding for the human ER gene. Several HTB 96 sublines expressing ER protein, detected by ligand binding and immunoassay, were isolated. The ability of 17 beta-estradiol (E2) to induce chloramphenicol acetyltransferase (CAT) activity from a cotransfected reporter vector containing the CAT gene linked to the Xenopus vitellogenin A2 gene estrogen response element demonstrated that the expressed ER was functional. ER continued to be expressed over a 30 week culture period. E2 but not other steroids significantly reduced growth rates and produced an altered morphology in HTB 96 sublines expressing higher levels of ER. The antiestrogen 4-hydroxytamoxifen partially reversed the E2 effect on growth rate. Transient transfection of cells expressing ER with a vector containing the CAT gene linked to the mouse mammary tumor virus long terminal repeat sequence, which contains response elements for the glucocorticoid receptor but not the ER, showed that E2 was able to inhibit CAT induction by dexamethasone. This result suggest that in ER-transfected HTB 9 cells the effects of E2 may result not from direct activation of endogenous genes but instead by transcriptional interference.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Overexpression of estrogen receptor in HTB 96 human osteosarcoma cells results in estrogen-induced growth inhibition and receptor cross talk. 797 7

Cytosolic phosphoenolpyruvate carboxykinase (GTP) (PEPCK) is a key glyceroneogenic enzyme in adipose tissue. The regulation of PEPCK gene expression by retinoic acid (RA) and dexamethasone (DEX) was studied in 3T3-F442A adipocytes maintained in a serum-free medium. RA induced whereas DEX reduced PEPCK mRNA steady-state level. RA stimulation was about 4-fold and DEX repression was of 80% in 4 hrs. In addition to reducing basal mRNA level, DEX was able to counteract RA induction in a dominant manner. The use of the glucocorticoid antagonist RU 38486 indicated that the DEX effect was mediated by the glucocorticoid receptor. Stable transfectants bearing the region of the PEPCK promoter from -2100 to +69 fused to the chloramphenicol acetyltransferase (CAT) gene (pPL1-CAT) were used to study PEPCK gene regulation in differentiated adipocytes. In such cells, RA stimulated CAT expression 3 to 5.5 fold. DEX had no effect on basal CAT activity whereas it inhibited the stimulation induced by RA. Thus, in adipocytes, the PEPCK gene regulatory region between -2100 and +69 bp mediates both stimulation by RA and repression by DEX of RA action.
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PMID:Glucocorticoids antagonize retinoic acid stimulation of PEPCK gene transcription in 3T3-F442A adipocytes. 798 26

We have examined the effects of heat shock on glucocorticoid receptor (GR)-mediated gene transcription in an L929 cell line derivative (LMCAT2) stably transfected with the mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter plasmid. Exposure of the LMCAT2 cells to heat or chemical shock resulted in a large increase in dexamethasone (Dex)-induced expression of CAT enzyme activity. This potentiation of hormone-induced MMTV-CAT expression was dependent on the magnitude of the stress event and on the Dex concentration, with maximal increases observed for 1 microM Dex after 2 h at 43 C or 2 h at 200 microM sodium arsenite. Heat shock potentiation of MMTV-CAT expression was not seen in an L929 cell derivative devoid of GR or in LMCAT2 cells treated with RU486 antagonist, suggesting that this effect of stress on CAT gene expression was mediated by the GR. Using a quantitative Western blot procedure, the amount of GR protein in the nucleus of cells subjected to combined heat shock and Dex treatment was no greater than the amount of nuclear GR in cells treated with hormone alone, indicating that the stress potentiation effect was not the result of increased nuclear translocation or retention by the GR. In addition, equally strong potentiations of MMTV-CAT expression were observed for cells subjected to heat shock either before or after Dex-mediated translocation of the GR to the nucleus. Thus, the major effect of stress on GR transcription enhancement activity appears to occur after the GR is bound to its high affinity nuclear acceptor sites. We have used a series of MMTV-CAT reporter constructs containing varying portions of the long terminal repeat regulatory region to show that a putative heat shock transcription factor-binding sequence at position -437 of the long terminal repeat is not required for this effect of heat shock on MMTV-CAT expression. A stress-induced increase in hormone-mediated CAT gene expression was observed for a minimal CAT reporter controlled by two synthetic glucocorticoid response elements and a TATA box sequence. Thus, it is unlikely that any DNA-binding transcription factor, other than GR, is required for this effect of stress on transcription by the hormone-bound GR. Based on these results, a model of heat shock enhancement of GR-mediated gene expression is developed in which stress acts on the DNA-bound GR, on a putative heat shock-activated adaptor, or on components of the RNA-polymerase-II complex.
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PMID:Potentiation of glucocorticoid receptor-mediated gene expression by heat and chemical shock. 805 62

Site-directed mutagenesis was employed to make two single amino acid substitutions for highly conserved amino acid residues near the C-terminus of the 783-amino acid mouse glucocorticoid receptor. Substitution of leucine for histidine-781 caused little or no change in the concentration of dexamethasone required for half-maximal activation of a chloramphenicol acetyltransferase reporter gene expressed from a mouse mammary tumor virus promoter. However, when phenylalanine-780 was changed to alanine, the half-maximal concentrations of various agonists were increased as follows, compared with the wild-type glucocorticoid receptor: triamcinolone acetonide by 7-fold, dexamethasone by 25-fold, and hydrocortisone and deoxycorticosterone by more than 150-fold. Binding of labeled steroids by the mutant receptor in vitro and in vivo was also decreased. In contrast, this mutation caused a small decrease in the concentration of RU486 required for antagonist or partial agonist activity. Thus, the phenyl group of phenylalanine-780 of the mouse glucocorticoid receptor is an important determinant of ligand binding affinity and specificity.
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PMID:Phenylalanine-780 near the C-terminus of the mouse glucocorticoid receptor is important for ligand binding affinity and specificity. 805 63

Glucocorticoids have been shown to increase the levels of cell surface insulin receptors and their mRNA in many different cell types. Previously, we have reported that glucocorticoids induce the transcription of the human insulin receptor (hIR) gene in rat 208F cells and we also identified a putative glucocorticoid response element (GRE) to which the glucocorticoid receptor binds in a specific manner. In this study we have mapped four additional regions of the hIR promoter to which glucocorticoid receptor binds specifically; one residues at -1340 and the others are distributed within a 100 base pair region from -750 to -650. Within each DNase I footprinting region resides at least one putative GRE sequence. They function as GREs to confer glucocorticoid inducibility when fused to a heterologous promoter-chloramphenicol acetyltransferase reporter construct. The functional significance of these putative GREs was further substantiated by mutational analysis. Taken together, our results indicate that these GREs are capable of conferring glucocorticoid-dependent transcriptional induction similar to those observed in vivo. Therefore, the increase of hIR mRNA and insulin binding to surface receptor in response to glucocorticoids is likely mediated by enhancement of transcription. The functional redundancy of the GREs may reflect the biological mechanism which ensures the glucocorticoid regulation of the hIR gene at the transcriptional level.
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PMID:Multiple hormone response elements can confer glucocorticoid regulation on the human insulin receptor gene. 805 70

The glucocorticoid receptor in chicken embryonic neural retina is expressed early in ontogeny, yet the tissue's response to the glucocorticoid hormone, i.e., induction of glutamine synthetase (GS), develops later, only during week 2 of ontogeny. Transient transfection of embryonic day 7 (E7) retinal cells, which are nonresponsive to glucocorticoids, with chimeric plasmids containing the chloramphenicol acetyltransferase reporter gene under the control of glucocorticoid-responsive promoters demonstrated that GR in E7 cells is a functional transactivating factor. We show that the limiting transcription factor that controls the developmental acquisition of responsiveness to glucocorticoids is similar to a CCAAT enhancer-binding protein (C/EBP). This protein recognizes a sequence in the promoter of the chick GS gene, which is required for eliciting the glucocorticoid response. Retinal C/EBP-like protein was not detected in the glucocorticoid-nonresponsive (E7) proliferating glioblasts but was found to be present in the glucocorticoid-responsive (E12) postmitotic cells. Premature expression of C/EBP in the nonresponsive E7 cells by transfection was shown to enhance the developmental acquisition of responsiveness to the glucocorticoid hormone, as deduced from the level of GS inducibility.
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PMID:Involvement of a C/EBP-like protein in the acquisition of responsiveness to glucocorticoid hormones during chick neural retina development. 809 26


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