160 results on '"DeFranco DB"'
Search Results
152. Differential roles of heat shock protein 70 in the in vitro nuclear import of glucocorticoid receptor and simian virus 40 large tumor antigen.
- Author
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Yang J and DeFranco DB
- Subjects
- Amino Acid Sequence, Animals, Biological Transport, Cell Compartmentation, HeLa Cells, Humans, In Vitro Techniques, Molecular Sequence Data, Rats, Sequence Alignment, Sequence Homology, Amino Acid, Antigens, Polyomavirus Transforming metabolism, Cell Nucleus metabolism, Heat-Shock Proteins metabolism, Receptors, Glucocorticoid metabolism
- Abstract
Nuclear import of glucocorticoid receptors (GRs) was analyzed in vitro with digitonin-permeabilized cells (S. A. Adam, R. Sterne-Marr, and L. Gerace, J. Cell Biol. 111:807-816, 1990). Indirect immunofluorescence methods were used to monitor the transport of GRs from rat hepatoma and fibroblast cell cytosol into HeLa nuclei. In vitro nuclear import of GRs was shown to be hormone dependent and to require ATP and incubation at ambient temperatures (i.e., 30 degrees C). Hormone-dependent dissociation of GR-bound proteins, such as the 90-kDa heat shock protein, hsp90, is part of an activation process that is obligatory for the expression of the receptor's DNA-binding activity. Inhibition of in vitro GR activation by Na2MoO4 blocked hormone-dependent nuclear import, demonstrating that receptor activation is required for nuclear import. The addition to GR-containing cytosol of antiserum directed against the cytosolic 70-kDa heat shock protein, hsp70, while effective in blocking the nuclear import of simian virus 40 large tumor antigen (SV40 TAg), did not affect hormone-dependent nuclear import of endogenous, full-length GRs or an exogenously added truncated GR protein (i.e., XGR556) that lacks a hormone-binding domain but possesses a constitutively active nuclear localization signal sequence (NLS). Depletion of hsp70 from HeLa cell cytosol did not affect the nuclear import of exogenously added XGR556 but led to inhibition of SV40 TAg nuclear import. Thus, two closely related NLSs, one contained within GRs and the other contained within SV40 TAg, are distinguished by their differential requirements for hsp70 in vitro.
- Published
- 1994
- Full Text
- View/download PDF
153. Glucocorticoid receptor-mediated repression of gonadotropin-releasing hormone promoter activity in GT1 hypothalamic cell lines.
- Author
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Chandran UR, Attardi B, Friedman R, Dong KW, Roberts JL, and DeFranco DB
- Subjects
- Animals, Cell Line, Dexamethasone pharmacology, Gonadotropin-Releasing Hormone analysis, Mice, RNA, Messenger analysis, Rats, Receptors, Glucocorticoid analysis, Transcription Factors physiology, Transfection, Gonadotropin-Releasing Hormone genetics, Hypothalamus metabolism, Promoter Regions, Genetic, Receptors, Glucocorticoid physiology
- Abstract
The synthesis and release of GnRH within a specific subset of neurons in the hypothalamus, which serves as the primary drive to the hypothalamic-pituitary-gonadal (HPG) axis, is subject to various levels of control. Although a number of direct synaptic connections to GnRH-containing neurons have been identified, which presumably provide some regulatory inputs, the mechanisms responsible for hormonal regulation of GnRH synthesis and release mediated by either cell surface or intracellular receptors remain controversial. The recent demonstration that a subset of GnRH-containing neurons in the rat hypothalamus possesses immunoreactive glucocorticoid receptors (GR) implies that this class of steroid hormones could exert a direct effect to regulate the functioning of these neurons and perhaps the HPG axis. We used the GT1-3 and GT1-7 cell lines of immortalized GnRH-secreting hypothalamic neurons as a model to study the direct effects of glucocorticoids on GnRH gene expression. We demonstrated that these cell lines possess GR that bind the synthetic glucocorticoid, dexamethasone, in vitro with high affinity (Kd = 2-3 nM). These receptors are functional, as indicated by their ability to activate transcription from exogenously introduced heterologous glucocorticoid-responsive promoters. Furthermore, dexamethasone represses both the endogenous mouse GnRH gene, decreasing steady state levels of GnRH mRNA, and the transcriptional activity of transfected rat GnRH promoter-reporter gene vectors. Glucocorticoid repression of rat GnRH promoter activity appears to be mediated by sequences contained within the promoter proximal 459 basepairs and not be influenced by the relative basal activity of the GnRH promoter. Thus, our results provide the first direct demonstration of glucocorticoid repression of transcription in a hypothalamic cell line and suggest that GR acting directly within GnRH neurons could be at least partly responsible for negative regulation of the HPG axis by glucocorticoids.
- Published
- 1994
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- View/download PDF
154. Modulation of cell signaling pathways can enhance or impair glucocorticoid-induced gene expression without altering the state of receptor phosphorylation.
- Author
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Moyer ML, Borror KC, Bona BJ, DeFranco DB, and Nordeen SK
- Subjects
- 1-Methyl-3-isobutylxanthine pharmacology, 8-Bromo Cyclic Adenosine Monophosphate pharmacology, Breast Neoplasms, Chloramphenicol O-Acetyltransferase metabolism, Colforsin pharmacology, Ethers, Cyclic pharmacology, Female, Gene Expression Regulation, Neoplastic drug effects, Genetic Vectors, Humans, Luciferases metabolism, Mammary Tumor Virus, Mouse genetics, Okadaic Acid, Phosphopeptides isolation & purification, Phosphorylation, Promoter Regions, Genetic, Protein Tyrosine Phosphatases antagonists & inhibitors, Recombinant Proteins metabolism, Tetradecanoylphorbol Acetate pharmacology, Transfection, Tumor Cells, Cultured, Dexamethasone pharmacology, Gene Expression drug effects, Receptors, Glucocorticoid metabolism, Signal Transduction drug effects
- Abstract
We have stably introduced expression vectors for the glucocorticoid receptor and a sensitive, hormone-responsive reporter (mouse mammary tumor virus-luciferase) into a human breast carcinoma-derived cell line. Employing this cell line, we have conducted a detailed examination of the induction of glucocorticoid-regulated genes and the phosphorylation of glucocorticoid receptor following pharmacologic manipulation of cell signaling pathways. The hormone response can be enhanced from 2 to 10-fold by activators of protein kinase A, protein kinase C, and inhibitors of protein phosphatase. Forskolin and 8-bromoadenosine 3':5'-cyclic monophosphate (BrcAMP), but not BrcGMP, enhance the hormone effect, yet surprisingly, phosphodiesterase inhibitors, isobutylmethylxanthine and Ro20-1724, strongly inhibit hormone-mediated induction of the reporter gene. These treatments do not alter cellular receptor content, dexamethasone binding, nor hormone-mediated receptor down-regulation. Tryptic peptide analysis of 32P-labeled receptor reveals that neither BrcAMP, isobutylmethylxanthine, nor the tumor promoter and protein kinase C activator, 12-O-tetradecanoyl-phorbol-13-acetate, detectably alter the state of glucocorticoid receptor phosphorylation. The only agent which alters receptor phosphorylation is the protein phosphatase inhibitor okadaic acid, but only at concentrations higher than required for maximum effects on glucocorticoid receptor transactivation. We propose that these effectors do not modify receptor directly but alter its interaction with transcription complexes.
- Published
- 1993
155. Bidirectional transport of glucocorticoid receptors across the nuclear envelope.
- Author
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Madan AP and DeFranco DB
- Subjects
- 3T3 Cells, Amino Acid Sequence, Animals, Antigens, Polyomavirus Transforming genetics, Antigens, Polyomavirus Transforming metabolism, Cell Line, Chlorocebus aethiops, Cycloheximide pharmacology, Half-Life, Kidney, Liver Neoplasms, Experimental, Methionine metabolism, Mice, Molecular Sequence Data, Rats, Receptors, Glucocorticoid biosynthesis, Receptors, Glucocorticoid genetics, Sequence Homology, Amino Acid, Simian virus 40 genetics, Tumor Cells, Cultured, Cell Nucleus metabolism, Nuclear Envelope metabolism, Receptors, Glucocorticoid metabolism
- Abstract
We have used transient interspecies heterokaryons to analyze nucleocytoplasmic shuttling of glucocorticoid receptor (GR) and demonstrated that receptors that accumulate within nuclei upon ligand binding are not statically confined to that compartment, but rather have the capacity to reversibly transverse the nuclear envelope. The ability of various GR mutants to shuttle between nuclei of heterokaryons excluded transcriptional activation and DNA binding as prerequisites for nucleocytoplasmic shuttling of GR. However, a constitutively nuclear GR derivative that has fused at its amino terminus the simian virus 40 large tumor antigen nuclear localization signal sequence was unable to efficiently export from nuclei unless ligand-bound. These results uncover an unexpected effect of ligand binding to GR--i.e., the overriding of a dominant negative effect on nuclear export of a heterologous nuclear import signal sequence. Furthermore, they demonstrate that a nuclear import signal sequence can influence nuclear processing pathways that culminate in efficient nuclear export.
- Published
- 1993
- Full Text
- View/download PDF
156. Cell cycle regulation of glucocorticoid receptor function.
- Author
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Hsu SC, Qi M, and DeFranco DB
- Subjects
- Animals, Biological Transport drug effects, Cell Nucleus metabolism, Dexamethasone pharmacology, Fluorescent Antibody Technique, L Cells, Mice, Mifepristone pharmacology, Phosphorylation, Receptors, Glucocorticoid isolation & purification, Signal Transduction physiology, Transcriptional Activation, Cell Cycle physiology, Receptors, Glucocorticoid metabolism
- Abstract
Glucocorticoid receptor (GR) nuclear translocation, transactivation and phosphorylation were examined during the cell cycle in mouse L cell fibroblasts. Glucocorticoid-dependent transactivation of the mouse mammary tumor virus promoter was observed in G0 and S phase synchronized L cells, but not in G2 synchronized cells. G2 effects were selective on the glucocorticoid hormone signal transduction pathway, since glucocorticoid but not heavy metal induction of the endogenous Metallothionein-1 gene was also impaired in G2 synchronized cells. GRs that translocate to the nucleus of G2 synchronized cells in response to dexamethasone treatment were not efficiently retained there and redistributed to the cytoplasmic compartment. In contrast, GRs bound by the glucocorticoid antagonist RU486 were efficiently retained within nuclei of G2 synchronized cells. Inefficient nuclear retention was observed for both dexamethasone- and RU486-bound GRs in L cells that actively progress through G2 following release from an S phase arrest. Finally, site-specific alterations in GR phosphorylation were observed in G2 synchronized cells suggesting that cell cycle regulation of specific protein kinases and phosphatases could influence nuclear retention, recycling and transactivation activity of the GR.
- Published
- 1992
- Full Text
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157. Internuclear migration of chicken progesterone receptor, but not simian virus-40 large tumor antigen, in transient heterokaryons.
- Author
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Chandran UR and DeFranco DB
- Subjects
- Animals, Biological Transport, Chickens, Cycloheximide pharmacology, Cytoplasm metabolism, Half-Life, Hybrid Cells, Antigens, Polyomavirus Transforming metabolism, Cell Nucleus metabolism, Receptors, Progesterone metabolism
- Abstract
The chicken progesterone receptor (PR) is a transcriptional regulatory protein that localizes predominantly within the nucleus of hormone-treated and untreated cells. Transient heterokaryons were generated between PR-expressing Cos-1 cells and PR-negative NIH3T3 cells to examine whether PRs are confined to the nucleus or are capable of bidirectionally traversing the nuclear envelope. Migration of PR from Cos-1 to NIH3T3 nuclei was observed in both the presence and absence of hormone. Since de novo PR synthesis was inhibited in heterokaryons with cycloheximide treatment, PRs that localize within NIH3T3 nuclei of heterokaryons must derive from preexisting receptors that were exported from Cos-1 nuclei. Thus, PR, like some nucleolar and heat shock proteins, appears to be capable of shuttling between the nuclear and cytoplasmic compartments. Not all proteins that enter the nucleus exhibit this trait, since simian virus-40 large tumor antigen, endogenously expressed in Cos-1 cells, does not efficiently translocate to NIH3T3 nuclei of heterokaryons, which support internuclear migration of PR. Thus, proteins that may use analogous or identical mechanisms for nuclear import may differentially interact with the nuclear export machinery. Furthermore, the fact that PR and simian virus-40 large tumor antigen localization within nuclei is not identical, as revealed by laser scanning confocal microscopy, supports the notion that nuclear export may be influenced by subnuclear compartmentalization.
- Published
- 1992
- Full Text
- View/download PDF
158. Effects of okadaic acid, a protein phosphatase inhibitor, on glucocorticoid receptor-mediated enhancement.
- Author
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Somers JP and DeFranco DB
- Subjects
- Animals, Cell Line, Enhancer Elements, Genetic drug effects, Enhancer Elements, Genetic genetics, Ethers, Cyclic pharmacology, Mutagenesis, Site-Directed genetics, Mutagenesis, Site-Directed physiology, Okadaic Acid, Phosphoprotein Phosphatases antagonists & inhibitors, Promoter Regions, Genetic physiology, Receptors, Glucocorticoid drug effects, Receptors, Glucocorticoid genetics, Transcription Factors physiology, Transcriptional Activation genetics, Transcriptional Activation physiology, Enhancer Elements, Genetic physiology, Phosphoprotein Phosphatases physiology, Receptors, Glucocorticoid physiology
- Abstract
The effects of okadaic acid (OA), a protein phosphatase inhibitor, on transcriptional enhancement activity of rat glucocorticoid receptor (GR) were examined in transiently transfected cells. In the absence of hormone, GRs expressed in CV-1 and COS-1 fibroblasts were capable of enhancing transcription from cotransfected chloramphenicol acetyltransferase reporter plasmids in response to OA treatment. Synergistic enhancement resulted from combined hormone and OA treatment. The effects of OA on GR-mediated enhancement required the presence of linked glucocorticoid response elements and were observed with reporter plasmids that contained different promoters and glucocorticoid response elements. Since OA did not affect nuclear translocation of the receptor, enhancement mediated by unliganded GR was most likely accounted for by the accumulation of some unliganded GRs within nuclei of transfected CV-1 and COS-1 cells. Deletion of individual GR transactivation domains and point mutations within DNA- and hormone-binding domains severely reduced the response of receptors to OA, although some mutant receptors retained the capacity to elicit a synergistic response when exposed to OA and hormone. The effects of OA on transcriptional enhancement did not appear to correlate with major changes in GR phosphorylation, as visualized by two-dimensional tryptic mapping of in vivo 32P-labeled GRs. Thus, phosphorylation of various components of the GR signal transduction pathway, and not necessarily the receptor itself, may influence its transcriptional enhancement activity.
- Published
- 1992
- Full Text
- View/download PDF
159. Protein phosphatase types 1 and/or 2A regulate nucleocytoplasmic shuttling of glucocorticoid receptors.
- Author
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DeFranco DB, Qi M, Borror KC, Garabedian MJ, and Brautigan DL
- Subjects
- Animals, Biological Transport, Cell Nucleus metabolism, Cell Transformation, Neoplastic, Cytoplasm metabolism, Dexamethasone metabolism, Fibroblasts metabolism, Fluorescent Antibody Technique, Gene Expression Regulation, Okadaic Acid, Oncogenes physiology, Peptide Mapping, Phosphoprotein Phosphatases antagonists & inhibitors, Phosphorylation, Rats, Ethers, Cyclic pharmacology, Phosphoprotein Phosphatases metabolism, RNA, Messenger metabolism, Receptors, Glucocorticoid metabolism
- Abstract
We have used okadaic acid (OA), a cell-permeable inhibitor of serine/threonine protein phosphatase types 1 (PP-1) and 2A (PP-2A), to demonstrate that the subcellular distribution of glucocorticoid receptor (GR) in rat fibroblasts is influenced by its phosphorylation state. Nuclear GRs in OA-treated cells retain transcriptional enhancement activity. Nuclear import or export of hormone agonist-bound GRs is not affected by OA. However, a dose of OA that fully inhibits PP-2A and partially inhibits PP-1, but not a lower dose that only partially inhibits PP-2A, leads to inefficient nuclear retention of agonist-bound GRs, and their redistribution into the cytoplasm. These receptors appear to be trapped in the cytoplasmic compartment and are unable to recycle (i.e. reenter the nucleus). Addition of OA during different steps of GR recycling demonstrates that OA must be present during nuclear export of GRs to block GR recycling. A direct role for PP-1 and/or PP-2A in GR recycling is suggested by site-specific hyperphosphorylation of GRs in vivo during OA inhibition of recycling. These are the same sites that undergo in vitro site-specific dephosphorylation by PP-1 and PP-2A. The block in GR recycling that results from inhibition of PP-1 and/or PP-2A resembles effects previously observed in v-mos-transformed rat fibroblasts. Interestingly, OA inhibition of PP-2A in v-mos-transformed cells leads to the reversal of oncoprotein effects on GR recycling and retention of receptors within the nuclear compartment. We propose that GR recycling is influenced by the activities of distinct protein phosphatases (PP-1 and/or PP-2A), and that the interference of this pathway observed in v-mos-transformed cells may be the result of effects of the oncoprotein on the phosphatases or a specific subset of their targets.
- Published
- 1991
- Full Text
- View/download PDF
160. Recycling and desensitization of glucocorticoid receptors in v-mos transformed cells depend on the ability of nuclear receptors to modulate gene expression.
- Author
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Qi M, Stasenko LJ, and DeFranco DB
- Subjects
- Animals, Cell Line, Transformed, Cell Nucleus metabolism, Cell Nucleus ultrastructure, Cytoplasm metabolism, Cytoplasm ultrastructure, Dexamethasone pharmacology, Fluorescent Antibody Technique, Mifepristone metabolism, Mifepristone pharmacology, Oncogene Proteins v-mos, Rats, Receptors, Glucocorticoid genetics, Receptors, Glucocorticoid physiology, Translocation, Genetic, Gene Expression drug effects, Receptors, Cell Surface genetics, Receptors, Glucocorticoid metabolism, Retroviridae Proteins, Oncogenic physiology
- Abstract
In v-mos transformed cells, glucocorticoid receptor (GR) proteins that bind hormone agonist are not efficiently retained within nuclei and redistribute to the cytoplasmic compartment. These cytoplasmic desensitized receptors cannot be reutilized and may represent trapped intermediates derived from GR recycling. We have used the glucocorticoid antagonist RU486 to examine whether v-mos effects can be exerted on any ligand-bound GR. In the rat 6m2 cell line that expresses a temperature-sensitive p85gag-mos oncoprotein, RU486 is a complete antagonist and suppresses dexamethasone induction of metallothionein-1 mRNA at equimolar concentrations. Using indirect immunofluorescence, we observe efficient nuclear translocation of GR in response to RU486 treatment in either the presence or absence of v-mos oncoproteins. However, in contrast to the redistribution of agonist-bound nuclear receptors to the cytoplasm of v-mos-transformed cells, RU486-bound GRs are efficiently retained within nuclei. Interestingly, withdrawal of RU486 does not lead to efficient depletion of nuclear GR in either nontransformed or v-mos transformed cells. It is only after the addition of hormone agonist to RU486 withdrawn v-mos-transformed cells that GRs are depleted from nuclei and subsequently redistributed to the cytoplasm. Thus, only nuclear GRs that are agonist-bound and capable of modulating gene activity can be subsequently processed and recycled into the cytoplasm.
- Published
- 1990
- Full Text
- View/download PDF
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