Biomedical subjects
Publications and source records attributed to G L Greene.
We describe a neutron radiography technique that can be used to map the distribution of 3He impurities in liquid 4He, providing direct and quantitative access to underlying transport processes. Images reflecting finite normal- and superfluid-component 4He velocity fields are presented.
Many NRs have multiple subtypes that possess distinct expression patterns and that regulate distinct target genes. Antagonists generated through the addition of bulky side chains to agonist scaffolds are limited to being antagonistic on one or more subtypes of a particular NR. The passive antagonism mechanism, as revealed in our studies through direct comparison of the two THC-ER LBD complexes, suggests a new approach to achieving NR antagonism. Compounds could be designed to selectively stabilize the inactive conformations of certain NR subtypes and the active conformations of others. Such ligands are likely to exert novel biological and therapeutic effects.
We report a new measurement of the neutron decay lifetime by the absolute counting of in-beam neutrons and their decay protons. Protons were confined in a quasi-Penning trap and counted with a silicon detector. The neutron beam fluence was measured by capture in a thin 6LiF foil detector with known absolute efficiency. The combination of these simultaneous measurements gives the neutron lifetime: tau(n)=(886.8+/-1.2[stat]+/-3.2[syst]) s. The systematic uncertainty is dominated by uncertainties in the mass of the 6LiF deposit and the 6Li(n,t) cross section. This is the most precise measurement of the neutron lifetime to date using an in-beam method.
Estrogen receptor (ER) binds to estrogen response elements in target genes and recruits a coactivator complex of CBP-pl60 that mediates stimulation of transcription. ER also activates transcription at AP-1 sites that bind the Jun/Fos transcription factors, but not ER. We review the evidence regarding mechanisms whereby ER increases the activity of Jun/Fos and propose two pathways of ER action depending on the ER (alpha or beta) and on the ligand. We propose that estrogen-ERalpha complexes use their activation functions (AF-1 and AF-2) to bind to the p 160 component of the coactivator complex recruited by Jun/Fos and trigger the coactivator to a higher state of activity. We propose that selective estrogen receptor modulator (SERM) complexes with ERbeta and with truncated ERalpha derivatives use their DNA binding domain to titrate histone deacetylase (HDAC)-repressor complexes away from the Jun/Fos coactivator complex, thereby allowing unfettered activity of the coactivators. Finally, we consider the possible physiological significance of ER action at AP-1 sites.
BACKGROUND: Mounting experimental evidence suggests that estrogen treatment protects against neointima formation in response to vascular injury in vivo. Previous studies have suggested that this process includes the activation and migration of adventitial fibroblasts. The present in vitro study was designed to establish a mechanism whereby estrogen attenuates migration of adventitial fibroblasts. METHDS AND RESULTS: Primary cultures of vascular smooth muscle cells (VSMCs) and adventitial fibroblasts were derived from female Sprague-Dawley rats. Reverse transcriptase-polymerase chain reaction and Western blotting were used to determine that expression of the estrogen receptor (ER) was restricted to early-passage VSMCs. Migration of transduced (retrovirally mediated) fibroblasts was determined by counting the number of blue lacZ-expressing cells attached to Boyden-type chambers preconditioned under defined experimental conditions. Compared with growth medium alone, chambers treated with medium conditioned by VSMCs demonstrated a 2-fold increase in fibroblast migration, suggesting that VSMCs release soluble factor(s) competent to bind the Transwell membrane and promote fibroblast migration. In contrast, treatment of VSMCs with 17beta-estradiol (10(-9) to 10(-7) mol/L) before preconditioning of the chamber induced a dose-dependent inhibition of fibroblast migration. Cotreatment of VSMCs with 17beta-estradiol and the ER antagonist ICI-182780 (10(-7) mol/L) blocked the inhibitory effect of estrogen on fibroblast migration. CONCLUSIONS: These observations suggest a novel mechanism of hormonal vasoprotection by which estrogen directly modulates VSMC expression of factor(s) controlling migration of adventitial fibroblasts via an ER-dependent mechanism.
The existence of a putative membrane estrogen receptor (ER) has been supported by studies accomplished over the past 20 yr. However, the origin and functions of this receptor are not well defined. To study the membrane receptor, we transiently transfected cDNAs for ERalpha or ERbeta into Chinese hamster ovary (CHO) cells. Transfection of ERalpha resulted in a single transcript by Northern blot, specific binding of labeled 17beta-estradiol (E2), and expression of ER in both nuclear and membrane cell fractions. Competitive binding studies in both compartments revealed near identical dissociation constants (K(d)S) of 0.283 and 0.287 nM, respectively, but the membrane receptor number was only 3% as great as the nuclear receptor density. Transfection of ERbeta3 also yielded a single transcript and nuclear and membrane receptors with respective Kd values of 1.23 and 1.14 nM; the membrane receptor number was only 2% compared with expressed nuclear receptors. Estradiol binding to CHO-ERalpha or CHO-ERbeta activated Galphaq and G(alpha)s proteins in the membrane and rapidly stimulated corresponding inositol phosphate production and adenylate cyclase activity. Binding by 17-beta-E2 to either expressed receptor comparably enhanced the nuclear incorporation of thymidine, critically dependent upon the activation of the mitogen-activated protein kinase, ERK (extracellular regulated kinase). In contrast, c-Jun N-terminal kinase activity was stimulated by 17-beta-E2 in ERbeta-expressing CHO, but was inhibited in CHO-ERalpha cells. In summary, membrane and nuclear ER can be derived from a single transcript and have near-identical affinities for 17-beta-E2, but there are considerably more nuclear than membrane receptors. This is also the first report that cells can express a membrane ERbeta. Both membrane ERs activate G proteins, ERK, and cell proliferation, but there is novel differential regulation of c-Jun kinase activity by ERbeta and ERalpha.
Cross-interference (squelching) among nuclear receptors has been proposed to reflect the titration of coactivators that bind the receptors in a hormone-dependent manner. We have tested whether the coactivators are the only target titrated during squelching of one receptor by another, or whether proteins needed for coactivator function are titrated as well. That the coactivators are indeed one target of squelching is apparent. The isolated ligand-binding domain of the estrogen receptor (ER-LBD) squelches transcriptional activation by the thyroid hormone receptor (TR) only when the LBD is bound to ligands that promote coactivator interactions and only when regions of the LBD that promote coactivator interactions are undisturbed. Furthermore, the ER-LBD and the TR compete in vitro for the related p160 coactivators, SRC1a and GRIP1 (glucocorticoid receptor interacting protein 1), or the putative corepressor, RIP140. Finally TR action becomes more potent when coactivator levels are raised. Nonetheless, supplying excess SRC1a or GRIP1 does not abolish squelching by the ER. In fact, squelching becomes even more severe when coactivators are abundant. Supplying combinations of coactivators from the p160 class and the CREB-binding protein (CBP)/p300 class makes squelching most severe. Elevated RIP140 inhibits TR action, but also protects the residual TR action from squelching by the ER-LBD. We conclude that ER-LBD squelches TR both by titrating p160-CBP coactivators and additionally by cooperating with the coactivators to titrate a second factor. The second factor would be needed by the TR for coactivator-mediated transcriptional stimulation.
Ligand-dependent activation of transcription by nuclear receptors (NRs) is mediated by interactions with coactivators. Receptor agonists promote coactivator binding, and antagonists block coactivator binding. Here we report the crystal structure of the human estrogen receptor alpha (hER alpha) ligand-binding domain (LBD) bound to both the agonist diethylstilbestrol (DES) and a peptide derived from the NR box II region of the coactivator GRIP1 and the crystal structure of the hER alpha LBD bound to the selective antagonist 4-hydroxytamoxifen (OHT). In the DES-LBD-peptide complex, the peptide binds as a short alpha helix to a hydrophobic groove on the surface of the LBD. In the OHT-LBD complex, helix 12 occludes the coactivator recognition groove by mimicking the interactions of the NR box peptide with the LBD. These structures reveal the two distinct mechanisms by which structural features of OHT promote this "autoinhibitory" helix 12 conformation.
Cylindrical traps made from Alsynite fiberglass were placed in 4 habitats in a confined cattle feedlot environment from 2 May to 30 October 1996 to evaluate abundance, sex ratio, physiological age structure, and blood-fed status of trapped adult stable flies, Stomoxys calcitrans (L.). Significantly more stable flies were caught on the trap located between host cattle and trees. The abundance of stable flies decreased geometrically with increasing distance from the host cattle in the open area. The spatial distribution of adult flies in cattle feedlot habitats became less heterogeneous as the population density increased. Male stable flies clearly dominated the total collection (66.7:33.3, male:female). A higher proportion (38.9%) of female stable flies was found on the trap close to a major immature developing area (manure pile). Most of the trapped flies (64.8%) showed a positive blood-fed status, but only 331 (8.8%) of 3,767 flies had recently fed. A majority (72.6%) of the attracted females was at the early nulliparous stage. Trap positioning did not have any significant influence on specific physiological age groups of the captured flies.
The estrogen receptor (ER) is a ligand-dependent transcription factor that regulates the expression of estrogen-responsive genes. ER-mediated transcriptional changes are brought about by interaction of the ER with the estrogen response element (ERE). In this study, we examined the interaction of the Xenopus laevis ER DNA binding domain (DBD) and the intact ER with the X. laevis vitellogenin A2 ERE and the human pS2 ERE. Using gel mobility shift, DNase I footprinting, and methylation interference assays, we demonstrated that the DBD bound only as a dimer to the A2 ERE. However, the DBD bound as a monomer to the consensus pS2 ERE half site at lower DBD concentrations and then as a homodimer to the consensus and imperfect pS2 ERE half site at higher DBD concentrations. Antibody supershift experiments carried out with partially purified, yeast-expressed full-length ER demonstrated that three ER-specific antibodies interacted differentially with A2 and pS2 ERE-bound ER, indicating that receptor epitopes were differentially exposed. Furthermore, partial digestion of the A2 and pS2 ERE-bound ER with chymotrypsin or trypsin produced distinct protease cleavage patterns. Taken together, these data provide evidence that differential interaction of the DBD with the A2 and pS2 EREs brings about global changes in ER conformation. The conformational changes in ER induced by individual ERE sequences could lead to association of the receptor with different transcription factors and assist in the differential modulation of estrogen-responsive genes in target cells.
Oestrogens are involved in the growth, development and homeostasis of a number of tissues. The physiological effects of these steroids are mediated by a ligand-inducible nuclear transcription factor, the oestrogen receptor (ER). Hormone binding to the ligand-binding domain (LBD) of the ER initiates a series of molecular events culminating in the activation or repression of target genes. Transcriptional regulation arises from the direct interaction of the ER with components of the cellular transcription machinery. Here we report the crystal structures of the LBD of ER in complex with the endogenous oestrogen, 17beta-oestradiol, and the selective antagonist raloxifene, at resolutions of 3.1 and 2.6 A, respectively. The structures provide a molecular basis for the distinctive pharmacophore of the ER and its catholic binding properties. Agonist and antagonist bind at the same site within the core of the LBD but demonstrate different binding modes. In addition, each class of ligand induces a distinct conformation in the transactivation domain of the LBD, providing structural evidence of the mechanism of antagonism.
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BACKGROUND: Premenopausal women have much lower susceptibility to coronary artery disease than do men or postmenopausal women. It has been proposed that estrogen plays a role in cardioprotection, but little information is available regarding the mechanism by which estrogen may help to protect the vasculature. Here, we describe an estrogen receptor (ER) in human coronary artery and umbilical vein endothelial cells. METHODS AND RESULTS: Human umbilical vein endothelial cells and human coronary artery endothelial cells were cultured in hormone-free medium for 48 hours before experiments. Estradiol (3.7 nmol/L) added to cultures promoted proliferation by a mechanism that is inhibited by the specific ER antagonist ICI182,780. Estradiol-treated cells incorporated twice the [3H]thymidine of hormone-free cells; this increase was prevented by ICI182,780. Endothelial cells from both sources stained in a nuclear pattern with an ER-specific antibody. Ribonuclease protection assay detected mRNA for the ER. Ligand-binding studies estimated 2 x 10(4) to 8 x 10(4) receptors per cell and a Kd of approximately 5 nmol/L. Interaction of ERs with a consensus estrogen response element was shown by an electrophoretic mobility shift assay. In addition, an antibody against the ER supershifted the protein-DNA complex. CONCLUSIONS: These studies define the presence of an ER in human coronary artery and umbilical vein endothelial cells. They support the hypothesis that cardioprotective effects of estrogen are mediated, at least in part, through a classic steroid hormone receptor mechanism.
UNLABELLED: Only a few parameters such as tumor grade and stage are of value in prognosticating disease course in endometrial carcinoma. Biochemical steroid hormone receptor assays could also be useful but are difficult to perform and interpret. Immunocytochemical assay (ICA) might be the method of choice for detecting endometrial receptors. METHODS: Frozen tissue from 78 cases of endometrial adenocarcinoma was examined for the presence of estrogen (ER) and progesterone receptors (PgR) with specific monoclonal anti-receptor antibodies and the peroxidase-antiperoxidase method. In over 60 cases, frozen tissue was also assayed for ER and PgR by biochemical means. RESULTS: Fifty-five (71%) of the endometrial carcinomas were ERICA-positive and 55 (71%) PgRICA-positive. Although both ERICA and PgRICA correlated significantly with biochemical ER and PgR only ERICA was predictive of survival. A woman with a negative ERICA was 4 times more likely to die of her disease than if she were ERICA-positive (P = 0.009; mean follow-up, 37.5 months). Three cases ERICA-positive and PgRICA-negative survived while 3 others ERICA-negative and PgRICA-positive died. CONCLUSION: ERICA, a technique easy to perform and interpret at the community hospital level, appears to provide prognostic information independent of tumor stage and grade. Such information might be of value in planning postoperative therapies for women with endometrial cancer.
We have examined the ability of estrogen receptor (ER) to bind and bend DNA fragments containing the Xenopus laevis vitellogenin A2 estrogen response element (ERE), which contains a palindromic, consensus ERE sequence, the X. laevis vitellogenin B1 ERE2, which contains a 1-bp mismatch in the 5'-end of the half-palindrome, and the human pS2 ERE, which contains a 1-bp mismatch in the 3'-end of the half-palindrome. ER binding induced a 65 degrees bend in DNA fragments containing the consensus ERE, the vitellogenin B1 ERE2, or the pS2 ERE. However, ER affinity for the consensus ERE was 2-fold greater than for either the vitellogenin B1 ERE2 or the pS2 ERE. When Chinese hamster ovary (CHO) cells were transfected with reporter plasmids containing either the consensus ERE, the vitellogenin B1 ERE2, or the pS2 ERE separated from the TATA sequence by 26 helical turns, exposure to 10 nm 17 beta-estradiol increased transcription 12.7-, 2.4-, and 3.8-fold, respectively. Increasing the spacing between the ERE and TATA sequence to three helical turns decreased the ability of the consensus ERE to activate transcription by 55% and increased the ability of the pS2 ERE to activate transcription by 35% but had no significant effect on vitellogenin B1 ERE2 activity. Further increasing the distance between the ERE and TATA sequence to 3.6 helical turns restored the activity of promoters containing the consensus ERE and pS2 ERE but decreased the activity of the promoter containing the relatively weak vitellogenin B1 ERE2. These data support the idea that 1) the affinity of ER for the ERE, 2) the location of an ERE within the promoter, and 3) the magnitude and orientation of DNA bends induced by binding of ER or other proteins are important in transcription activation of estrogen-responsive genes.
The structure of the ca. 250 amino acid hormone binding domain of the human estrogen receptor (hER-LBD), expressed in E. coli and purified as a complex with estradiol, has been probed by selective proteolysis, with analysis of the protein fragments both by classical methods (SDS-PAGE and Edman N-terminal sequencing) and by mass spectrometry (HPLC-coupled electrospray ionization mass spectrometry (LC/ESI-MS)). Rapid cleavage by several proteases (trypsin, chymotrypsin, thermolysin, and Asp-N endoproteinase) is observed within a localized region (residues 297-303) at the N-terminus. In contrast, proteolytic scission at the C-terminus is less localized and more progressive; initial cuts by trypsin, chymotrypsin, thermolysin, V8, and Asp-N proteinases are observed to occur in the region 553-571, followed by further cleavage with thermolysin (548) and trypsin (548, 531, and 529). Thus, N304 and K529 define the protease-resistant N- and C-termini of a core structure for this domain that appears to contain the elements sufficient for ligand binding. The remaining segment of this domain (530-553), which is known to embody elements essential for ligand-modulated transcription activation (AF-2), is likely a surface-exposed region that, through these studies, is shown to be accessible to proteases. Only a single region within the 26 kDa ligand-binding core (N304-K529) has been identified as being readily accessible to proteases; rapid proteolysis using the proteases trypsin, chymotrypsin, and thermolysin, is localized to residues 465-468, with cleavage occurring at residues K467, L466, and both T465 and S468, respectively. The flexibility implied by the cuts in this internal 465-468 region suggest that the hER-LBD may actually consist of two subdomains. These proteolysis studies provide a substantially refined view of the conformational nature of the human estrogen receptor ligand binding domain.
The ligand binding domain of the human estrogen receptor (hER-LBD), encompassing the sequence MDPS282AG...V595, has been expressed at high levels in Escherichia coli from a pET-23d vector, and a purified preparation has been characterized both by mass spectrometry and biochemical methods. Inclusion bodies from the bacterial expression were solubilized by sonication and the hER-LBD was purified to near homogeneity by affinity chromatography over an estradiol-Sepharose column in urea-containing buffer. This material ran as a single peak on reversed-phase HPLC, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed a band with apparent molecular mass of 31-32 kilodaltons (kDa), somewhat smaller than that expected from the construct (35.6 kDa). Edman degradation revealed a single sequence of MDPSAGDMRA, consistent with an intact N terminus. Further characterization of this material using low resolution matrix-assisted laser desorption ionization mass spectrometry indicated an apparent single protein species of average mass 33,143 daltons (Da). Detailed molecular analysis by electrospray ionization mass spectrometry, however, revealed that this purified hER-LBD preparation was actually composed of a number of both modified and unmodified LBD protein fragments between 32,900-33,400 Da. The bulk of this 33-kDa protein mixture consisted of three LBD protein fragments with C termini at Ala571 (70%), Ala569 (23%), and Ser575 (4%), with only a trace amount (3%) of the full length expressed LBD (MDPS282...V595; 35, 602 Da). These four protein species also appear to be partially chemically modified by carbamylation. The functional integrity of this hER-LBD preparation was investigated by a ligand-exchange assay, which showed that the hER-LBD retained full estradiol-binding capacity; specific, covalent labeling was also observed using either the electrophilic affinity-labeling ligand tamoxifen aziridine (TAZ) or the photoaffinity-labeling ligand hexestrol diazirine. Thus, this expressed hER-LBD preparation, while appearing nominally pure by conventional biochemical techniques and having the expected ligand-binding capacity, was shown by sensitive high resolution electrospray ionization mass spectrometry techniques to be largely truncated 20-26 amino acids from the expected C terminus and to have a substantial level of covalent modification arising from the urea.(ABSTRACT TRUNCATED AT 400 WORDS)
Despite a wealth of information about the structure and composition of steroid receptors and their functional domains, little is known about the role of accessory proteins as mediators of receptor activity. To better define the role of such proteins in estrogen receptor (ER) function, we have used immunoaffinity, steroid affinity, and site-specific DNA-affinity chromatography to identify and characterize proteins that associate with human ER (hER) in extracts from MCF-7 cells and hER-expressing CHO (CHO-ER) cells. In addition to the expected 66-kDa hER, a 70-kDa protein was obtained and subsequently identified as a member of the heat shock protein family (hsp70). A 55-kDa protein, detected by all three approaches, was identified as a member of the protein disulfide isomerase family (PDI). Two proteins that were preferentially retained by an ER-specific DNA affinity column (p48 and p45) remain unidentified. Maximal interaction of purified hER with the vitellogenin A2 estrogen response element (ERE) occurred in the presence of all four associated proteins isolated by DNA-affinity chromatography. The increased stability of this complex was due primarily to an increase in the association rate of hER with ERE. Thus, accessory proteins may be required for optimal interaction of ER with EREs.