Expression and purification of recombinant human progesterone receptor in baculovirus and bacterial systems.
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Animals↗
Biomedical subjects
D P Edwards
Publications and source records attributed to D P Edwards.
Identification of a phosphorylation site in the hinge region of the human progesterone receptor and additional amino-terminal phosphorylation sites.
We have previously reported the identification of seven in vivo phosphorylation sites in the amino-terminal region of the human progesterone receptor (PR). From our previous in vivo studies, it was evident that several phosphopeptides remained unidentified. In particular, we wished to determine whether human PR contains a phosphorylation site in the hinge region, as do other steroid receptors including chicken PR, human androgen receptor, and mouse estrogen receptor. Previously, problematic trypsin cleavage sites hampered our ability to detect phosphorylation sites in large incomplete tryptic peptides. Using a combination of mass spectrometry and in vitro phosphorylation, we have identified six previously unidentified phosphorylation sites in human PR. Using nanoelectrospray ionization mass spectrometry, we have identified two new in vivo phosphorylation sites, Ser(20) and Ser(676), in baculovirus-expressed human PR. Ser(676) is analogous to the hinge site identified in other steroid receptors. Additionally, precursor ion scans identified another phosphopeptide that contains Ser(130)-Pro(131), a likely candidate for phosphorylation. In vitro phosphorylation of PR with Cdk2 has revealed five additional in vitro Cdk2 phosphorylation sites: Ser(25), Ser(213), Thr(430), Ser(554), and Ser(676). At least two of these, Ser(213) and Ser(676), are authentic in vivo sites. We confirmed the presence of the Cdk2-phosphorylated peptide containing Ser(213) in PR from in vivo labeled T47D cells, indicating that this is an in vivo site. Our combined studies indicate that most, if not all, of the Ser-Pro motifs in human PR are sites for phosphorylation. Taken together, these data indicate that the phosphorylation of PR is highly complex, with at least 14 phosphorylation sites.
Amino Acid Sequence↗
The role of coactivators and corepressors in the biology and mechanism of action of steroid hormone receptors.
Steroid hormone receptors are members of a superfamily of ligand-dependent transcription factors. As such they have a DNA binding domain that recognizes specific target gene sequences along with separate transcriptional activation domains. What sets steroid hormone receptors (and other nuclear hormone receptors) apart from other families of sequence specific transcriptional activators is the presence of a ligand binding domain (LBD) that acts as a molecular switch to turn on transcriptional activity when a hormonal ligand induces a conformational change in the receptor. Upon binding hormone, steroid receptors recruit a novel coactivator protein complex with an essential role in receptor-mediated transcriptional activation. Coactivators function as adaptors in a signaling pathway that transmits transcriptional responses from the DNA bound receptor to the basal transcriptional machinery. Hormone agonists induce a conformational change in the carboxyl-terminal transcriptional activation domain, AF-2, that creates a new protein interaction site on the surface of the LBD that is recognized by LXXLL motifs in the p160 family of coactivators. In contrast, steroid antagonists such as the antiestrogen tamoxifen for the estrogen receptor induce an alternate conformation in AF-2 that occludes the coactivator binding site and recruits corepressors that can actively silence steroid responsive genes. Thus, the cellular availability of coactivators and corepressors is an important determinant in the biological response to both steroid hormone agonists and antagonists. This paper provides an update on the properties and mechanism of action of nuclear receptor coactivators, the nature of the coactivator-binding site, and the structural and mechanistic basis for ligand-dependent binding of coactivators to receptors.
Amino Acid Motifs↗
The place of colostomy after late detection of a colonic injury and faecal peritonitis.
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Adult↗
The opposing transcriptional activities of the two isoforms of the human progesterone receptor are due to differential cofactor binding.
The human progesterone receptor (PR) exists as two functionally distinct isoforms, hPRA and hPRB. hPRB functions as a transcriptional activator in most cell and promoter contexts, while hPRA is transcriptionally inactive and functions as a strong ligand-dependent transdominant repressor of steroid hormone receptor transcriptional activity. Although the precise mechanism of hPRA-mediated transrepression is not fully understood, an inhibitory domain (ID) within human PR, which is necessary for transrepression by hPRA, has been identified. Interestingly, although ID is present within both hPR isoforms, it is functionally active only in the context of hPRA, suggesting that the two receptors adopt distinct conformations within the cell which allow hPRA to interact with a set of cofactors that are different from those recognized by hPRB. In support of this hypothesis, we identified, using phage display technology, hPRA-selective peptides which differentially modulate hPRA and hPRB transcriptional activity. Furthermore, using a combination of in vitro and in vivo methodologies, we demonstrate that the two receptors exhibit different cofactor interactions. Specifically, it was determined that hPRA has a higher affinity for the corepressor SMRT than hPRB and that this interaction is facilitated by ID. Interestingly, inhibition of SMRT activity, by either a dominant negative mutant (C'SMRT) or histone deacetylase inhibitors, reverses hPRA-mediated transrepression but does not convert hPRA to a transcriptional activator. Together, these data indicate that the ability of hPRA to transrepress steroid hormone receptor transcriptional activity and its inability to activate progesterone-responsive promoters occur by distinct mechanisms. To this effect, we observed that hPRA, unlike hPRB, was unable to efficiently recruit the transcriptional coactivators GRIP1 and SRC-1 upon agonist binding. Thus, although both receptors contain sequences within their ligand-binding domains known to be required for coactivator binding, the ability of PR to interact with cofactors in a productive manner is regulated by sequences contained within the amino terminus of the receptors. We propose, therefore, that hPRA is transcriptionally inactive due to its inability to efficiently recruit coactivators. Furthermore, our experiments indicate that hPRA interacts efficiently with the corepressor SMRT and that this activity permits it to function as a transdominant repressor.
Amino Acid Sequence↗
Differential hormone-dependent phosphorylation of progesterone receptor A and B forms revealed by a phosphoserine site-specific monoclonal antibody.
Human progesterone receptor (PR) is phosphorylated on multiple serine residues (at least seven sites) in a manner that involves distinct groups of sites coordinately regulated by hormone and different kinases. Progress on defining a functional role for PR phosphorylation has been hampered both by the complexity of phosphorylation and the lack of simple, nonradioactive methods to detect the influence of ligands and other signaling pathways on specific PR phosphorylation sites in vivo. Toward this end, we have produced monoclonal antibodies (MAbs) that recognize specific phosphorylation sites within human PR including a basal site at Ser 190 (MAb P190) and a hormone-induced site at Ser 294 (MAb P294). Biochemical experiments showed the differential reactivity of the P190 and P294 MAbs for phosphorylated and unphosphorylated forms of PR. Both MAbs recognize specific phosphorylated forms of PR under different experimental conditions including denatured PR protein by Western blots and PR in its native conformation in solution or complexed to specific target DNA. As detected by Western blot of T47D cells treated with hormone for different times, hormone-dependent down-regulation of total PR and the Ser 190 phosphorylation site occurred in parallel, whereas the Ser 294 phosphorylation site was down-regulated more rapidly. This difference in kinetics suggests that the Ser 294 site is more labile than basal sites and is acted upon by distinct phosphatases. A strong preferential hormone-dependent phosphorylation of Ser 294 was observed on PR-B as compared with the amino-terminal truncated A form of PR. This was unexpected because Ser 294 and flanking sequences are identical on both proteins, suggesting that a distinct conformation of the N-terminal domain of PR-A inhibits phosphorylation of this site. That Ser 294 lies within an inhibitory domain that mediates the unique repressive functions of PR-A raises the possibility that differential phosphorylation of Ser 294 is involved in the distinct functional properties of PR-A and PR-B.
Amino Acid Sequence↗
Progesterone regulates proliferation of endothelial cells.
The use of steroid hormones in postmenopausal replacement therapy has been associated with prevention of cardiovascular disease. Although the contribution of estradiol to endothelial cell function has been addressed, little information is available on the effect of progestins on this cell type. Here, we provide direct evidence for the presence of functional nuclear progesterone receptor in endothelial cells and demonstrate that physiological levels of progesterone inhibit proliferation through a nuclear receptor-mediated mechanism. The effects of progesterone were blocked by pretreatment with a progesterone receptor antagonist, and progesterone receptor-deficient endothelial cells failed to respond to the hormone. We evaluated the effect of progesterone by analysis of aorta re-endothelialization experiments in wild-type and progesterone receptor knockout mice. The rate of re-endothelialization was significantly decreased in wild-type mice when in the presence of progesterone, whereas there was no difference between control and progesterone-treated progesterone receptor knockout mice. FACS analysis showed that progestins arrest endothelial cell cycle in G1. The lag in cell cycle progression involved reduction in cyclin-dependent kinase activity, as shown by down-regulation in retinoblastoma protein phosphorylation. In addition, treatment of endothelial cells with progestins altered the expression of cyclin E and A in accordance with G1 arrest. These results have important implications to our current knowledge of the effect of steroids on endothelial cell function and to the overall contribution of progesterone to vascular repair.
Animals↗
Coregulatory proteins in steroid hormone receptor action: the role of chromatin high mobility group proteins HMG-1 and -2.
To directly activate specific gene expression, the progesterone receptor must bind to specific hormone response elements in target promoters. We have previously reported that progesterone receptor requires a nuclear factor, high mobility group 1 or 2 (HMG-1/-2) for high-affinity interaction with DNA in vitro and for full transcriptional activity in vivo. We have also observed that HMG-1/-2 selectively influences the activity of the steroid hormone class of nuclear receptors but does not affect other classes of nuclear receptors. This report is a summary of our published and unpublished studies to determine the effects of HMG-1/-2 on a broad range of nuclear receptor supergene family members and to define the mechanism for the specific effect of HMG-1/-2 on the steroid class of receptors. Our studies and available structural data suggest a model where the DNA binding domains of nonsteroid nuclear receptors contain a minor groove DNA interface, termed the C-terminal extension, that contributes to high-affinity DNA binding. Steroid receptors lack such a minor groove interface and therefore require an additional protein, HMG-1/-2, that functionally substitutes for the C-terminal extension to facilitate high-affinity interactions with DNA.
Base Sequence↗
Progesterone and regulation of the follicle-stimulating hormone (FSH-beta) gene.
Progesterone (P) biphasically modulates follicle-stimulating hormone (FSH) secretion in the rat both in vivo and in vitro with the duration of estrogen priming determining the biphasic nature of the P action, probably through estrogen up-regulation of the anterior pituitary progesterone receptor (PR) levels. P has been also shown to regulate anterior pituitary levels of FSH-beta mRNA in the rat. Although the mechanism of this action has not been determined, steroids may regulate gene expression through the binding of liganded receptors to gene sequences known as hormone response elements (HRE); however, it is not known whether HRE's exist on the rat FSH-beta gene. We have localized a series of progesterone response elements (PRE)-like sequences on the rat FSH-beta gene and have begun testing the hypothesis that P modulates the expression of the rat FSH-beta gene through the direct binding of the P/PR complex to these PRE-like sequences. Electromobility shift assays indicate that these PRE-like sequences bind PR with high affinity and specificity. In addition, when a 361-base pair sequence, which contains the three PRE-like sequences localized in the upstream region of the gene, was cloned into a luciferase expression vector driven by a heterologous promoter and transiently transfected into anterior pituitary cell cultures, progestin stimulation elicited increased luciferase expression. These results indicated that the 361-base pair sequence conferred P-responsiveness to a heterologous promoter. The data further suggest that FSH synthesis in the rat is modulated by direct binding of PR to PRE-like sequences.
Animals↗
Coregulatory proteins in nuclear hormone receptor action.
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Activating Transcription Factor 2↗
Comparison of two closure techniques for the repair of experimental colonic perforations.
BACKGROUND: Primary repair of penetrating wounds of the colon is gaining increasing acceptance in surgical practice. This study compared two techniques for the repair of experimental colonic perforations in the presence of peritonitis. METHODS: Multiple colonic perforations were created in the colon of 24 pigs. Following a 6-h delay the perforations were closed either by local excision and suture or by skin staples applied to the seromuscular layers of the colon. The repairs were assessed biomechanically and histologically for up to 14 days after surgery. RESULTS: All animals had diffuse peritonitis at the time of colonic repair. Stapled repairs were completed significantly faster than sutured repairs (mean(s.d.) 4.8(1.6) versus 30.7(4.0) min, P < 0.001). Bio- mechanical evaluation of repairs revealed no significant differences between the two techniques. Histological examination of repairs closed by staples demonstrated more advanced healing compared with suture closure, on the basis of tissue apposition and inflammatory changes. CONCLUSION: Experimental colonic injuries may be treated successfully by primary repair in the presence of peritonitis. The use of skin staples for repair does not appear to prejudice colonic wound healing.
Animals↗
Acute presentation of bilateral radial artery pseudoaneurysms following arterial cannulation.
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Acute Disease↗
The history of colonic surgery in war.
The history of laparotomy for penetrating abdominal wounds is reviewed from its origins in the sixteenth century to the large series from World War II. It is shown that the mass casualties of World War I allowed the management of organ specific injuries to be studied. This review concentrates upon one of the most contentious topics in war surgery-the management of colonic wounds, and suggests that post-war surgical practice may have been influenced too strongly by experiences from the pre-Penicillin era.
Abdominal Injuries↗
Ganglion of the hip joint--we present a logical approach to the exploration of a mass in the femoral triangle.
Hip joint ganglion is a rare cause of a mass in the femoral triangle. Our patient presented with a swelling in the groin and a history of femoral hernia repair 5 years previously. Pre-operative assessment with ultrasound suggested a possible femoral artery aneurysm. We propose that safe exploration of a mass closely related to the femoral vessels must include vascular control.
Aged↗
Gastrointestinal haemorrhage after abdominal aortic grafting.
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Aged↗
A C619Y mutation in the human androgen receptor causes inactivation and mislocalization of the receptor with concomitant sequestration of SRC-1 (steroid receptor coactivator 1)
Androgen ablation therapy is a primary treatment for advanced prostate cancer, but tumors become refractive to therapy. Consequently, the role of the androgen receptors (ARs) and of mutations in the AR in prostate cancer has been a subject of much concern. In the course of analyzing tumors for mutations, we identified a somatic mutation that substitutes tyrosine for a cysteine at amino acid 619 (C619Y), which is near the cysteines that coordinate zinc in the DNA binding domain in the AR. The mutation was re-created in a wild-type expression vector and functional analyses carried out using transfection assays with androgen-responsive reporters. The mutant is transcriptionally inactive and unable to bind DNA. In response to ligand treatment, AR619Y localizes abnormally in numerous, well circumscribed predominantly nuclear aggregates in the nucleus and cytoplasm. Interestingly, these aggregates also contain the bulk of the coexpressed steroid receptor coactivator SRC-1, suggesting, in analogy to AR in spinal bulbar muscular atrophy, that this mutant may alter cellular physiology through sequestration of critical proteins. Although many inactivating mutations have been identified in androgen insensitivity syndrome patients, to our knowledge, this is the first characterization of an inactivating mutation identified in human prostate cancer.
Cell Nucleus↗
Hormone-dependent interaction between the amino- and carboxyl-terminal domains of progesterone receptor in vitro and in vivo.
Full transcriptional activation by steroid hormone receptors requires functional synergy between two transcriptional activation domains (AF) located in the amino (AF-1) and carboxyl (AF-2) terminal regions. One possible mechanism for achieving this functional synergy is a physical intramolecular association between amino (N-) and carboxyl (C-) domains of the receptor. Human progesterone receptor (PR) is expressed in two forms that have distinct functional activities: full-length PR-B and the amino-terminally truncated PR-A. PR-B is generally a stronger activator than PR-A, whereas under certain conditions PR-A can act as a repressor in trans of other steroid receptors. We have analyzed whether separately expressed N- (PR-A and PR-B) and C-domains [hinge plus ligand-binding domain (hLBD)] of PR can functionally interact within cells by mammalian two-hybrid assay and whether this involves direct protein contact as determined in vitro with purified expressed domains of PR. A hormone agonist-dependent interaction between N-domains and the hLBD was observed functionally by mammalian two-hybrid assay and by direct protein-protein interaction assay in vitro. With both experimental approaches, N-C domain interactions were not induced by the progestin antagonist RU486. However, in the presence of the progestin agonist R5020, the N-domain of PR-B interacted more efficiently with the hLBD than the N-domain of PR-A. Coexpression of steroid receptor coactivator-1 (SRC-1) and the CREB binding protein (CBP), enhanced functional interaction between N- and C-domains by mammalian two-hybrid assay. However, addition of SRC-1 and CBP in vitro had no influence on direct interaction between purified N- and C-domains. These results suggest that the interaction between N- and C-domains of PR is direct and requires a hormone agonist-induced conformational change in the LBD that is not allowed by antagonists. Additionally, coactivators are not required for physical association between the N- and C-domains but are capable of enhancing a functionally productive interaction. In addition, the more efficient interaction of the hLBD with the N-domain of PR-B, compared with that of PR-A, suggests that distinct interactions between N- and C-terminal regions contribute to functional differences between PR-A and PR-B.
Animals↗
Trans-axillary first rib excision for thoracic outlet syndrome.
AIM: Thoracic outlet syndrome (TOS) is a clinical diagnosis treatable by excision of the first rib. This study was undertaken to assess the alleviation of symptoms following trans-axillary rib excision in patients with a diagnosis of TOS suggested by a positive Elevated Arm Stress Test and, by inference, to estimate the prevalence of the syndrome. METHOD: A retrospective review of trans-axillary first rib excision was performed. No referrals were accepted from outwith the catchment area of our hospital. Post-operative symptomatic improvement was accepted as a confirmation of correct pre-operative diagnosis. RESULTS: Over six years, 52 rib resections were performed in 46 patients, mean age (+/- S.D.) 38.8 (+/- 10.6) years. The indications for surgery were principally neurological symptoms (n = 42 excisions), arterial compromise (n = 5) and venous compromise (n = 5). Symptoms had been present for a mean of 15.8 months prior to surgery. The median follow-up after surgery was 33 months. 42 patients (48 resections) showed immediate improvement in symptoms following surgery, although symptoms recurred in three patients (4 resections) between 6 and 8 months post-operatively. In the final two years of this study, 20 resections resulting in symptomatic improvement were performed, suggesting a prevalence for TOS of at least 10 per 100,000 per year. CONCLUSIONS: Thoracic outlet decompression was performed more frequently in this series than many previous reports, suggesting that TOS may be under-diagnosed.
Adult↗