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D P Edwards

Publications and source records attributed to D P Edwards.

At least 19 recordsLinked to original sources
PubMed1995

The two faces of a steroid antagonist: when an antagonist isn't.

Activation of protein kinase A potentiates the transcriptional response mediated by the glucocorticoid receptor in responsive fibroblasts and in mammary carcinoma cells. This potentiation is ligand-dependent and occurs in responsive fibroblasts and in mammary carcinoma cells. This potentiation is ligand-dependent and occurs without detectable change in the phosphorylation of receptor. The transcriptional response to glucocorticoid or progestin agonists can be blocked by potent antagonists like RU 486. However, upon activation of protein kinase A, the antagonist action of RU 486 on both receptors is blunted. Indeed, RU 486 can itself activate transcription of a hormone-responsive promoter. The conditional agonist activity is observed with type II antagonists, those which recapitulate many of the early steps of ligand-dependent receptor activation, but not type I antagonists, which do not. These studies have now been extended to antimineralocorticoids. In COS-1 cells transfected with a mineralocorticoid receptor expression vector, treatment with 8-BromocAMP potentiates the response to the agonist aldosterone and elicits additional agonist activity in mineralocorticoid antagonists. A model is proposed wherein type II antagonist-receptor complexes occupy receptor binding sites on the genome. The antagonist, however, fails to promote a receptor conformation that can interact productively with a coactivator mediating the communication between receptor and the basal transcription apparatus. Activation of protein kinase A results in the recruitment or activation of a coactivator that permits recovery of receptor-mediated activation function.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line↗
PubMed1995

Contrast sensitivity and spatial frequency response of primate cortical neurons in and around the cytochrome oxidase blobs.

The striate cortex of macaque monkeys contains an array of patches which stain heavily for the enzyme cytochrome oxidase (CO blobs). Cells inside and outside these blobs are often described as belonging to two distinct populations or streams. In order to better understand the function of the CO blobs, we measured the contrast sensitivity and spatial frequency response of single neurons in and around the CO blobs. Density profiles of each blob were assessed using a new quantitative method, and correlations of local CO density with the physiology were noted. We found that the CO density dropped off gradually with distance from blob centers: in a typical blob the CO density dropped from 75% to 25% over 100 microns. Recordings were confined to cortical layers 2/3. Most neurons in these layers have poor contrast sensitivity, similar to that of the parvocellular neurons in the lateral geniculate nucleus. However, in a small proportion of layers 2/3 neurons we found higher contrast sensitivity, similar to that of the magnocellular neurons. These neurons were found to cluster near blob centers. This finding is consistent with (indirect) parvocellular input spread uniformly throughout layers 2/3, and (indirect) magnocellular input focused on CO blobs. We also measured spatial tuning curves for both single units and multiple unit activity. In agreement with other workers we found that the optimal spatial frequencies of cells near blob centers were low (median 2.8 c/deg), while the optimal frequencies of cells in the interblob regions were spread over a wide range of spatial frequencies. The high cut-off spatial frequency of multi-unit activity increased with distance from blob centers. We found no correlation between spatial bandwidth and distance from blob centers. All measured physiological properties varied gradually with distance from CO blob centers. This suggests that the view of blob cells subserving visual functions which are entirely distinct from non-blob cells may have to be reevaluated.

Animals↗
PubMed1995

Progesterone receptor and the mechanism of action of progesterone antagonists.

Currently available progesterone antagonists have been suggested to fall into two categories based on differences in how they interact with and inactivate the progesterone receptor (PR). The anti-progestin ZK98299 (Type I) impairs PR association with DNA, while Type II compounds (RU486, ZK112993, ZK98734) promote PR binding to DNA. Type II agents, therefore, appear to inhibit receptor activity at a step downstream of DNA binding, presumably failing to induce conformational changes in PR structure requird for enhancement of transcription. This paper discusses both published and unpublished data supporting the concept of two types of progestin antagonists. Using PR-mediated induction of reporter genes in breast cancer cells as an assay for biological response, both types of anti-progestins, after correction for difference in steroid binding affinity, inhibit progestin induction substoichiometrically. However, Type II anti-progestins are more potent, inhibiting at lower ratios of antagonist to agonist than ZK98299. This suggests that in addition to behaving by classical competitive mechanisms these compounds (in particular Type II) may exhibit additional activity as transrepressors of PR in the same cell bound to hormone agonist. Transrepression may occur by the combined mechanisms of heterodimerization and competition for binding to DNA. In support of this, mixed ligand dimers form readily in solution between a PR subunit bound to agonist and another bound to either type of anti-progestin, whereas these mixed ligand dimers bind poorly, if at all, to specific progesterone response elements (PREs) in vitro. Additionally, when added as a single ligand, Type II agents increase PR dimerization in solution and PR affinity for PREs as compared with single ligand dimers formed by progestin agonist. This contrasts with ZK98299, when given as a single ligand, which reduces PR affinity for PREs without disrupting solution dimerization. Thus the higher affinity of PR for PREs may account for the greater biological potency of Type II compounds as compared with ZK98299. As a further distinction between types of antiprogestins, ZK98299 minimally stimulates phosphorylation of PR whereas RU486 increases site-specific phosphorylation of PR in a manner indistinguishable from that of hormone agonist. Additionally, ZK98299 is not susceptible in vivo to functional switching to a partial agonist by cross talk with cAMP signal transduction pathways, as occurs with Type II compounds. Thus, ZK98299 under certain conditions may be a more pure antagonist than Type II compounds.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Phosphorylation and progesterone receptor function.

Four phosphorylation sites have been identified in the chicken progesterone receptor. Two of these sites exhibit basal phosphorylation which is enhanced upon treatment with hormone and two of the sites are phosphorylated in response to hormone. Mutation of one of these hormone dependent sites, Ser530 to Ala530, causes a decrease in transcriptional activation at low concentrations of hormone, but the activity is unaffected at high concentrations. However, the hormone binding of the mutant is unaffected suggesting that phosphorylation of Ser530 plays a role in facilitating the response of the receptor to low concentrations of hormone. The chicken progesterone receptor can be activated by modulators of kinases in the absence of hormone. The finding that signals initiated by tyrosine phosphorylation (through treatment with EGF) or through the dopamine receptor suggests that there are multiple means of activating chicken progesterone receptor. In contrast, the human progesterone receptor does not exhibit ligand independent activation; however, its activity in the presence of the agonist R5020 is enhanced by treatment with 8-Br-cAMP, an activator of protein kinase A, and treatment with 8-Br-cAMP causes the antagonist, RU486, to act as an agonist.

Amino Acid Sequence↗
PubMed1995

The 5'-flanking region of the ovine follicle-stimulating hormone-beta gene contains six progesterone response elements: three proximal elements are sufficient to increase transcription in the presence of progesterone.

Progesterone (P4) can alter the synthesis and secretion of FSH from pituitary gonadotropes of sheep. In this study, the 5'-flanking region (4.7 kilobases) of the ovine FSH beta gene was tested for binding by human progesterone receptors (hPR), using an immunoprecipitation technique. Three fragments were bound by hPR. Competition experiments using homologous and heterologous DNA fragments revealed this binding to be specific and of high affinity (Kd = 1.2-47 nM). The fragment sequences were screened for potential P4 response elements (PREs). Six PRE-like elements were found among the three immunoprecipitated fragments. Band shift experiments discerned that each of these PRE-like sequences could be bound by hPR. In functional studies, each of the PRE-like elements could enhance the expression of a reporter gene driven by a heterologous promoter in a hormone-dependent manner. The 5'-flanking region of the ovine FSH beta gene was tested for P4 responsiveness using a luciferase reporter. In the presence of P4, there was a 2- to 3-fold increase in luciferase activity when the entire 4.7 kilobases of the 5'-flanking sequence were present, whereas no increase was seen in a construct that contained only 84 basepairs 5' to the transcription start site. This effect on transcription was dose dependent for P4. Deletion studies revealed that the three PRE-like elements closest to the transcription start site (-250 to -137) were sufficient to create the hormone-dependent enhancement. These results indicate that the 5'-flanking sequence of the ovine FSH beta gene contains sequences capable of being bound by hPR and may be responsible for the effects of P4 on FSH beta synthesis and secretion. This study is the first to show binding and function of PR for a gonadotropin gene.

Animals↗
PubMed1995

Interaction of the progesterone receptor with binding proteins for FK506 and cyclosporin A.

T47D human breast carcinoma cells and the chicken oviduct were used to study the structure of the nonactivated progesterone receptor (PR) complex. Immunoprecipitation of PR (B form) from cytosol extracts was performed using monoclonal antibody PR6, a cross-reactive antibody prepared to chicken PR. Analysis of the PR complex by sodium dodecyl sulfate gels and Western immuno-blotting revealed the presence of several specific copurifying proteins. Consistent with previous reports, the two heat shock proteins, hsp90 and hsp70, were shown to be present. A third 59-kilodalton (kDa) protein observed previously was confirmed to be p59 (also called hsp56 or FKBP52), which has been shown to bind the immunosuppressant drug FK506. Two additional PR-associated proteins were observed that had not been previously recognized with human PR. These have molecular masses of 54-kDa and 23-kDa and have been shown by Western blotting to be related to the proteins p54 and p23 that are associated with chicken PR. P23 is a novel protein of unknown function and p54 or FKBP54 has been recently shown to be another FK506-binding protein related to p59. Finally, the cyclosporin A-binding protein, CyP-40, could be detected in isolated chicken PR complexes and in PR complexes that were reconstituted in vitro, but this protein was not detected in human PR complexes, which are less stable than chicken PR complexes in cytosol extracts. The functional significance of FK506 and cyclosporin A-binding proteins to hormone action was tested using a T47D cell line that contained a progestin reporter gene, MMTV-CAT. Treatment with cyclosporin A had no effect on the basal level of CAT expression, but it caused a dramatic increase in the sensitivity and magnitude of the response to the synthetic progestin, R5020. The enhanced response elicited by drug treatment was blocked by the antiprogestin RU486 indicating that this effect was receptor-mediated. While cyclosporin A enhanced progestin action in T47D cells, it inhibited a PR/reporter gene system in L cells. The drugs FK506 and rapamycin had no effect on progestin action in T47D cells, but they stimulated glucocorticoid action in T47D cells. Thus, the effects of these immunosuppressant drugs vary with the cell type and hormonal system that is tested. Whether these drug effects relate directly to the immunophilins bound in receptor complexes remains unknown.

Animals↗
PubMed1995

Identification of a group of Ser-Pro motif hormone-inducible phosphorylation sites in the human progesterone receptor.

The human progesterone receptor (PR) is a member of the steroid/thyroid hormone superfamily of nuclear receptors. The receptor is expressed as two forms, PR-B and the shorter PR-A, which lacks the NH2-terminal 164 amino acids of PR-B; whereas PR-B seems to be predominantly a transcriptional activator, PR-A also functions as a repressor. Our previous studies of PR expressed in T47D breast cancer cells have shown that PR is a phosphoprotein whose phosphorylation is enhanced in response to hormone. There is an initial rapid (minutes) increase in phosphorylation followed by a slower, less substantial increase, which results in decreased mobility of the receptor on sodium dodecyl sulfate gels. We now report the identification of three phosphorylation sites, which are predominantly phosphorylated during the later phase of the response to hormone. These sites, Ser102, Ser294, and Ser345, are all found in Ser-Pro consensus sequences. Whereas Ser294 and Ser345 are common to PR-A and PR-B, Ser102 is unique to PR-B. Finally, we demonstrate that phosphorylation of Ser345 is associated with the altered mobility on sodium dodecyl sulfate gels.

Amino Acid Sequence↗
PubMed1994

Identification of phosphorylation sites unique to the B form of human progesterone receptor. In vitro phosphorylation by casein kinase II.

The human progesterone receptor (PR), a member of the steroid/thyroid receptor superfamily of ligand-activated transcription factors, is expressed in most tissues as two forms that exhibit differential transcriptional activation potentials, full-length PR-B and NH2-terminally truncated PR-A. In human breast cancer cells (T47D) both forms of PR are constitutively phosphorylated but phosphorylation is increased in response to hormone treatment, suggesting that this modification has a role in regulating the activation state of the receptor. To more directly define the functional role of phosphorylation in the action of A and B receptors requires knowledge of the phosphorylated amino acid residues and the protein kinase(s) involved. Toward this end we have developed a strategy that combines isolation of PR phosphotryptic peptides by reverse phase high performance liquid chromatography, secondary analytical protease digestion, manual Edman degradation, and release of 32P that resulted in identification of two major phosphorylation sites, Ser81 and Ser162. Both sites are located in the amino-terminal region unique to PR-B, and one of these sites (Ser81) is encompassed in a casein kinase II (CKII) consensus sequence. Although human PR contains 11 potential CKII consensus sequences, CKII in vitro phosphorylated purified PR-B only at Ser81 suggesting that this may be an authentic site for CKII in vivo.

Amino Acid Sequence↗
PubMed1994

Nuclear accessory factors enhance the binding of progesterone receptor to specific target DNA.

The human progesterone receptor (PR) is dependent upon hormone and a nuclear accessory factor(s) for maximal binding to progesterone response elements (PRES) in vitro. Recombinant full-length PR, expressed in a baculovirus system and purified to apparent homogeneity, was used as a substrate to isolate and identify the accessory factor(s). The major PRE binding enhancement activity present in nuclear extracts was shown to be associated with the high mobility group chromatin protein HMG-1. Moreover, HMG-1 was equally effective in enhancing the DNA binding of both the A and B isoforms of PR. Enhancement of PRE binding was highly selective for HMG-1 as a single purified protein and was not mimicked by a general protein stabilization effect. In gel mobility shift assays, it appeared that HMG-1 enhanced PRE binding without stably participating as a component of the final DNA-PR complex, suggesting that HMG-1 acts indirectly by modifying the PR protein or the target DNA. HMG-1 is a sequence-independent DNA binding protein that recognizes distorted DNA structures and is also able to promote further distortions by bending DNA. Enhancement of PRE binding was found to be intrinsic to the conserved DNA binding domain of HMG-1 suggesting that HMG-1 acts by promoting a structural alteration in the target PRE-DNA.

Animals↗
PubMed1994

The DNA-bending protein HMG-1 enhances progesterone receptor binding to its target DNA sequences.

Steroid hormone receptors are ligand-dependent transcriptional activators that exert their effects by binding as dimers to cis-acting DNA sequences termed hormone response elements. When human progesterone receptor (PR), expressed as a full-length protein in a baculovirus system, was purified to homogeneity, it retained its ability to bind hormonal ligand and to dimerize but exhibited a dramatic loss in DNA binding activity for specific progesterone response elements (PREs). Addition of nuclear extracts from several cellular sources restored DNA binding activity, suggesting that PR requires a ubiquitous accessory protein for efficient interaction with specific DNA sequences. Here we have demonstrated that the high-mobility-group chromatin protein HMG-1, as a highly purified protein, dramatically enhanced binding of purified PR to PREs in gel mobility shift assays. This effect appeared to be highly selective for HMG-1, since a number of other nonspecific proteins failed to enhance PRE binding. Moreover, HMG-1 was effective when added in stoichiometric amounts with receptor, and it was capable of enhancing the DNA binding of both the A and B amino-terminal variants of PR. The presence of HMG-1 measurably increased the binding affinity of purified PR by 10-fold when a synthetic palindromic PRE was the target DNA. The increase in binding affinity for a partial palindromic PRE present in natural target genes was greater than 10-fold. Coimmunoprecipitation assays using anti-PR or anti-HMG-1 antibodies demonstrated that both PR and HMG-1 are present in the enhanced complex with PRE. HMG-1 protein has two conserved DNA binding domains (A and B), which recognize DNA structure rather than specific sequences. The A- or B-box domain expressed and purified from Escherichia coli independently stimulated the binding of PR to PRE, and the B box was able to functionally substitute for HMG-1 in enhancing PR binding. DNA ligase-mediated ring closure assays demonstrated that both the A and B binding domains mediate DNA flexure. It was also demonstrated in competition binding studies that the intact HMG-1 protein binds to tightly curved covalently closed or relaxed DNA sequences in preference to the same sequence in linear form. The finding that enhanced PRE binding was intrinsic to the HMG-1 box, combined with the demonstration that HMG-1 or its DNA binding boxes can flex DNA, suggests that HMG-1 facilitates the binding of PR by inducing a structural change in the target DNA.

Animals↗
PubMed1993

Modulators of cellular protein phosphorylation alter the trans-activation function of human progesterone receptor and the biological activity of progesterone antagonists.

Addition of progesterone to breast cancer cells in vivo increases phosphorylation of human progesterone receptor (PR), suggesting that phosphorylation has a regulatory role in producing the activated form of receptor. Kinetic analysis indicates that hormone-dependent phosphorylation is sequential and that early stages of phosphorylation(s) are closely associated with enhancement of PR-DNA binding while later stages are associated with a trans-activation function. Various agents that stimulate cellular protein phosphorylation (8-Br cAMP, okadaic acid, TPA) functionally synergize with progesterone to enhance progesterone-dependent PR trans-activation in intact cells. These results suggest that protein phosphorylation does have a role in modulating the trans-activation function of PR in vivo. They also demonstrate cross-talk between second messenger signal transduction pathways and nuclear steroid receptors. Whether the phosphorylated target that provides the link between these two signal transduction pathways is PR itself or another protein involved in PR-mediated gene transcription is not known. Positive cooperative interactions were also observed between cAMP signaling pathways and the progesterone antagonist RU486, that resulted in RU486 exerting substantial agonist activities. This ability of cross-talk between second messenger and steroid receptor signal transduction pathways to override the antagonistic effects of RU486 suggests a novel mechanism to explain the problem of resistance to clinically important steroid antagonists.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Effects of the steroid antagonist RU486 on dimerization of the human progesterone receptor.

We previously reported, using a coimmunoprecipitation assay, that the B form (PR-B) of the human progesterone receptor from T47D human breast cancer cells dimerizes in solution with the A receptor (PR-A) and that the extent of dimerization correlates with receptor binding activity for specific DNA sequences [DeMarzo, A.M., Beck, C.A., Oñate, S.A., & Edwards, D.P. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 72-76]. This suggested that solution dimerization is an intermediate step in the receptor activation process. The present study has tested the effects of the progesterone antagonist RU486 on solution dimerization of progesterone receptors (PR). As determined by the coimmunoprecipitation assay, RU486 binding did not impair dimerization of receptors; rather, the antagonist promoted more efficient solution dimerization than the progestin agonist R5020. This enhanced receptor dimerization correlated with a higher DNA binding activity for transformed receptors bound with RU486. RU486 has been shown previously to produce two other alterations in the human PR when compared with R5020. PR-RU486 complexes in solution exhibit a faster sedimentation rate (6 S) on salt-containing sucrose density gradients than PR-R5020 complexes (4 S), and PR-DNA complexes have a faster electrophoretic mobility on gel-shift assays in the presence of RU486. We presently show that the 6 S PR-RU486 complex is a receptor monomer, not a dimer. The increased sedimentation rate and increased mobility on gel-shift assays promoted by RU486 were also observed with recombinant PR-A and PR-B separately expressed in insect cells from baculovirus vectors. These results suggest that RU486 induces a distinct conformational change both in PR monomers in solution and in dimers bound to DNA. We also examined whether conformational changes in PR induced by RU486 would prevent a PR polypeptide bound to RU486 from heterodimerization with another PR polypeptide bound to R5020. To evaluate this, PR-A and PR-B that were separately bound to R5020 or RU486 in whole cells were mixed in vitro. PR-A-RU486 was capable of dimerization with PR-B-R5020, and this was demonstrated for heterodimers both formed in solution and bound to specific DNA. The capability to form heterodimers in vitro raises the possibility that the antagonist action of RU486 in vivo could in part be imposed in a dominant negative fashion through heterodimerization between one receptor subunit bound to an agonist and another bound to RU486.

Baculoviridae↗
PubMed1992

Hormone and antihormone induce distinct conformational changes which are central to steroid receptor activation.

Antihormones are potent antagonists of hormone action in vivo, but the mechanism underlying this antagonism is not understood. Several steroid hormones transform (activate) their receptors from a cytosolic, non-DNA binding 8 S sedimentation form to a nuclear, DNA binding 4 S form. Transformation is accompanied by the loss of associated heat shock proteins. We have previously demonstrated that an additional hormone-dependent step, separate from heat shock protein removal, is required for activation of the human progesterone receptor. We have devised an assay in which the human progesterone receptor translated in vitro binds to its specific response element in a hormone-dependent manner. As assessed by limited proteolytic digestion, hormone treatment of the nascent receptor induces a dramatic conformational change within the protein. The conformational change occurs in the absence of DNA and renders the entire ligand binding domain resistant to digestion by proteases. A number of antiprogestins, including RU486, induce an equally dramatic, but distinct, structural alteration of the ligand binding domain. The distinction centers upon the final 30 to 40 amino acids at the carboxyl terminus. The conformational change can be induced by ligand prior to dissociation of the 8 S complex and is not induced by heat shock protein removal in the absence of hormone. Remarkably, virtually identical hormone-induced conformational changes were detected following proteolytic analysis of in vitro translated retinoic acid receptors. Our data indicate that the sole necessary event in the activation of steroid receptors is conformational modification by the ligand. Furthermore, we conclude that transcriptional inactivation of steroid receptors by antihormones involves the induction of an inappropriate structural conformation at the extreme carboxyl terminus of the ligand binding domain.

Base Sequence↗
PubMed1992

Heat shock alters the composition of heteromeric steroid receptor complexes and enhances receptor activity in vivo.

Under normal cellular conditions, human progesterone receptors (PR), immune-isolated from cytosols of T47D breast cancer cells, associate with two heat shock proteins (hsps), hsp 90 and hsp 70. Receptors activated by hormone binding in vivo and extracted from nuclei with 0.5 M NaCl no longer associate with hsp 90 but retain association with hsp 70. We have examined the effect of heat shock treatment of cells on hsp-receptor interactions and on receptor function. Heat shock resulted in a partial reduction in cellular levels of PR, but receptors that remained were functional for both steroid and DNA binding activities. By steady-state [35S]methionine labeling prior to heat shock treatment, it was determined that heat shock did not affect the composition or maintenance of preexisting cytosolic PR.hsp 90.hsp 70 complexes. By contrast, immune isolation of PR complexes from cells pulse-labeled with [35S]methionine showed that heat shock altered the composition of newly synthesized hsps associated with PR. After heat shock, both the highly inducible form of hsp 70 (72K hsp) and a 100K hsp were bound to cytosol PR, and inducible 72K hsp remained bound with the nuclear-activated PR. Neither of these hsps were associated in detectable amounts with PR under normal cellular conditions. With respect to receptor function, heat shock treatment substantially enhanced the activity of PR in vivo as determined by measuring hormone-dependent PR-mediated transcription of a target reporter gene (MMTV-CAT) that was stably transfected into T47D cells. Heat shock treatment alone, in the absence of hormone, did not stimulate MMTV-CAT expression nor did it affect transcription from a control reporter gene, pSV2-CAT, suggesting that enhanced receptor activity was due to an effect on PR-mediated processes and not to a general effect on transcription. Induction of the heat shock response by a related chemical stress (sodium arsenite) also enhanced PR activity in vivo. Interestingly, sodium arsenite produced both a greater induction of hsp 90 and hsp 70 synthesis and a greater fold enhancement of PR-mediated gene transcription than did heat shock. This suggests that enhancement of PR activity is related not only to induction of hsp synthesis but also to the severity of the stress response. The present results provide an indication that in certain cells there may exist an interrelationship between the activation pathways by which cells respond to stress and to steroid hormones. Possible mechanisms responsible for heat shock effects on PR activity are discussed.

Arsenic↗
PubMed1992

Ligands induce conformational changes in the carboxyl-terminus of progesterone receptors which are detected by a site-directed antipeptide monoclonal antibody.

We have prepared a monoclonal antibody, C-262, to a synthetic peptide that contains the carboxy-terminal 14 amino acids from progesterone receptors (PR). This sequence is 100% conserved in all species of PRs that have been cloned to date, suggesting that this antibody will recognize all mammalian and avian PR. The C-262 antibody recognizes both native and denatured forms of the receptor. However, it does not recognize PR when they are bound to the hormone agonists progesterone or R5020. Surprisingly the antibody does recognize PR when they are bound to the steroid antagonist RU486. This suggests that progestin agonists induce a conformational change in the receptor that occludes the C-262 epitope in the carboxyl-terminus, whereas unliganded receptors and receptors bound with RU486 assume distinct conformations that leaves the C-terminal tail accessible to the C-262 antibody.

Amino Acid Sequence↗