bluuebunny/crossref_metadata_2025_split huggingface dataset:
Antiinflammatory Properties of a Plant-Derived Nonsteroidal, Dissociated Glucocorticoid Receptor Modulator in Experimental Autoimmune Encephalomyelitis
Authors: van Loo Geert , Sze Mozes , Bougarne Nadia , Praet Jelle , McGuire Conor , Ullrich Andrea , Haegeman Guy , Prinz Marco , Beyaert Rudi , De Bosscher Karolien 
Published: January 2014

Abstract
Compound A (CpdA), a plant-derived phenyl aziridine precursor, was recently characterized as a fully dissociated nonsteroidal antiinflammatory agent, acting via activation of the glucocorticoid receptor, thereby down-modulating nuclear factor-κB-mediated transactivation, but not supporting glucocorticoid response element-driven gene expression. The present study demonstrates the effectiveness of CpdA in inhibiting the disease progress in experimental autoimmune encephalomyelitis (EAE), a well-characterized animal model of multiple sclerosis. CpdA treatment of mice, both early and at the peak of the disease, markedly suppressed the clinical symptoms of EAE induced by myelin oligodendrocyte glycoprotein peptide immunization. Attenuation of the clinical symptoms of EAE by CpdA was accompanied by reduced leukocyte infiltration in the spinal cord, reduced expression of inflammatory cytokines and chemokines, and reduced neuronal damage and demyelination. In vivo CpdA therapy suppressed the encephalogenicity of myelin oligodendrocyte glycoprotein peptide-specific T cells. Moreover, CpdA was able to inhibit TNF- and lipopolysaccharide-induced nuclear factor-κB activation in primary microglial cells in vitro, in a differential mechanistic manner as compared with dexamethasone. Finally, in EAE mice the therapeutic effect of CpdA, in contrast to that of dexamethasone, occurred in the absence of hyperinsulinemia and in the absence of a suppressive effect on the hypothalamic-pituitary-adrenal axis. Based on these results, we propose CpdA as a compound with promising antiinflammatory characteristics useful for therapeutic intervention in multiple sclerosis and other neuroinflammatory diseases.
Disruption of the Glucocorticoid Receptor Assembly with Heat Shock Protein 90 by a Peptidic Antiglucocorticoid
Authors: Dao-Phan Hai-Pascal , Formstecher Pierre , Lefebvre Philippe 
Published: January 2014

AbstractAssociation of glucocorticoid (GR) and progesterone (PR) receptors with a set of molecular chaperones, including the 90-kDa heat shock protein (hsp90), is a dynamic process required for proper folding and maintaining these nuclear receptors under a transcriptionally inactive, ligand-responsive state. Mutational studies of the chicken hsp90 complementary DNA suggested that three regions of this protein (A, B, and Z) interact with the hormone-binding domain of GR, whereas region A is dispensable for hsp90 binding to PR. We found that this 69-amino acid region can be narrowed down to a 35-mer α-helical, acidic peptide, which is by itself able to inhibit hsp90 association to GR translated in vitro. The hsp90-free GR did not bind ligand, but was devoid of any specific DNA-binding activity, and higher peptide concentrations specifically inhibited the binding of activated GR to DNA. When overexpressed in cultured cells, this peptide acted as an antiglucocorticoid and inhibited the antiactivating protein-1 activity and the ligand-dependent nuclear transfer of GR. None of these effects, either in vivo and in vitro, was observed for PR. The region from residue 232 to residue 265 of hsp90 is, therefore, a domain critical for its association to GR, an association that is a prerequisite for receptor transcriptional activity. More importantly, these results demonstrate that targeting specific protein/protein interaction interfaces is a powerful means to specifically modulate nuclear receptor signaling pathways in a ligand-independent manner.
Inhibition of Gonadotropin-Releasing Hormone Receptor Signaling by Expression of a Splice Variant of the Human Receptor
Authors: Grosse Robert , Schöneberg Torsten , Schultz Günter , Gudermann Thomas 
Published: January 2014

AbstractGnRH binds to a specific G protein-coupled receptor in the pituitary to regulate synthesis and secretion of gonadotropins. Using RT-PCR and human pituitary poly(A)+ RNA as a template, the full-length GnRH receptor (wild type) and a second truncated cDNA characterized by a 128-bp deletion between nucleotide positions 522 and 651 were cloned. The deletion causes a frame shift in the open reading frame, thus generating new coding sequence for further 75 amino acids. The truncated cDNA arises from alternative splicing by accepting a cryptic splicing acceptor site in exon 2. Distinct translation products of approximately 45–50 and 42 kDa were immunoprecipitated from COS-7 cells transfected with cDNA coding for wild type GnRH receptor and the truncated splice variant, respectively. Immunocytochemical and enzyme-linked immunosorbent assay studies revealed a membranous expression pattern for both receptor isoforms. Expression of the splice variant, however, occurred at a significantly lower cell surface receptor density. In terms of ligand binding and phospholipase C activation, the wild type receptor showed characteristics of a typical GnRH receptor, whereas the splice variant was incapable of ligand binding and signal transduction. Coexpression of wild type and truncated proteins in transiently or stably transfected cells, however, resulted in impaired signaling via the wild type receptor by reducing maximal agonist-induced inositol phosphate accumulation. The inhibitory effect depended on the amount of splice variant cDNA cotransfected and was specific for the GnRH receptor because signaling via other Gq/11-coupled receptors, such as the thromboxane A2, M5 muscarinic, and V1 vasopressin receptors, was not affected. Immunological studies revealed that coexpression of the wild type receptor and the truncated splice variant resulted in impaired insertion of the wild type receptor into the plasma membrane. Thus, expression of truncated receptor proteins may highlight a novel principle of specific functional inhibition of G protein-coupled receptors.
Shc Phosphotyrosine-Binding Domain Dominantly Interacts with Epidermal Growth Factor Receptors and Mediates Ras Activation in Intact Cells
Authors: Sakaguchi Kazuhiko , Okabayashi Yoshinori , Kido Yoshiaki , Kimura Sachiko , Matsumura Yoko , Inushima Koichi , Kasuga Masato 
Published: January 2014

AbstractThe adaptor protein Shc contains a phosphotyrosine binding (PTB) domain and a Src homology 2 (SH2) domain, both of which are known to interact with phosphorylated tyrosines. We have shown previously that tyrosine 1148 of the activated epidermal growth factor (EGF) receptor is a major binding site for Shc while tyrosine 1173 is a secondary binding site in intact cells. In the present study, we investigated the interaction between the PTB and SH2 domains of Shc and the activated human EGF receptor. Mutant 52-kDa Shc with an arginine-to-lysine substitution at residue 175 in the PTB domain (Shc R175K) or 397 in the SH2 domain (Shc R397K) was coexpressed in Chinese hamster ovary cells overexpressing the wild-type or mutant EGF receptors that retained only one of the autophosphorylation sites at tyrosine 1148 (QM1148) or 1173 (QM1173). Shc R397K was coprecipitated with the QM1148 and QM1173 receptors, was tyrosine-phosphorylated, and associated with Grb2 and Sos. In contrast, coprecipitation of Shc R175K with the mutant receptors was barely detectable. In cells expressing the QM1173 receptor, Shc R175K was tyrosine-phosphorylated and associated with Grb2, while association of Sos was barely detectable. In cells expressing the QM1148 receptor, tyrosine phosphorylation of Shc R175K was markedly reduced. When both Shc R175K and 46-kDa Shc R397K were coexpressed with the mutant receptors, p46 Shc R397K was dominantly tyrosine-phosphorylated. In cells expressing the wild-type receptor, Shc R397K, but not Shc R175K, translocated to the membrane in an EGF-dependent manner. In addition, Ras activity stimulated by the immunoprecipitates of Shc R397K was significantly higher than that by the immunoprecipitates of Shc R175K. The present results indicate that tyrosine 1148 of the activated EGF receptor mainly interacts with the Shc PTB domain in intact cells. Tyrosine 1173 interacts with both the PTB and SH2 domains, although the interaction with the PTB domain is dominant. In addition, Shc bound to the activated EGF receptor via the PTB domain dominantly interacts with Grb2-Sos complex and plays a major role in the Ras-signaling pathway.
The Carboxyl-Terminal Region Is a Determinant for the Intracellular Behavior of the Chorionic Gonadotropin β Subunit: Effects on the Processing of the Asn-Linked Oligosaccharides
Authors: Muyan Mesut , Boime Irving 
Published: January 2014

AbstractThe placental hormone human CG (hCG) consists of two noncovalently linked α- and β-subunits similar to the other glycoprotein hormones LH, FSH, and TSH. These heterodimers share a common α subunit but differ in their structurally distinct β subunits. The CGβ subunit is distinguished among the β subunits by the presence of a C-terminal extension with four serine-linked oligosaccharides (carboxyl terminal peptide or CTP). In previous studies we observed that deleting this sequence decreased assembly of the truncated CGβ subunit (CGβ114) with the α-subunit and increased the heterogeneity of the secreted forms of the uncombined subunit synthesized in transfected Chinese hamster ovary (CHO) cells. The latter result was attributed to alterations in the processing of the two N-linked oligosaccharides. To examine at what step this heterogeneity occurs, the CGβ and CGβ114 genes were transfected into wild-type and mutant CHO cell lines that are defective in the late steps of the N-linked carbohydrate-processing pathway. We show here that removal of the CTP alters the processing of the core mannosyl unit of the subunit to complex forms at both glycosylation sites and that the oligosaccharides contain polylactosamine. Although it has been presumed that there is little intramolecular interaction between the CTP and the proximal domains of the subunit, our data suggest that the CTP sequence participates in the folding of the newly synthesized subunit, which is manifest by the posttranslational changes observed here.
Determination of Gab1 (Grb2-Associated Binder-1) Interaction with Insulin Receptor-Signaling Molecules
Authors: Rocchi Stéphane , Tartare-Deckert Sophie , Murdaca Joseph , Holgado-Madruga Marina , Wong Albert J. , Van Obberghen Emmanuel 
Published: January 2014

AbstractThe newly identified insulin receptor (IR) substrate, Gab1 [growth factor receptor bound 2 (Grb2)-associated binder-1] is rapidly phosphorylated on several tyrosine residues by the activated IR. Phosphorylated Gab1 acts as a docking protein for Src homology-2 (SH2) domain-containing proteins. These include the regulatory subunit p85 of phosphatidylinositol 3-kinase and phosphotyrosine phosphatase, SHP-2. In this report, using a modified version of the yeast two-hybrid system, we localized which Gab1 phospho-tyrosine residues are required for its interaction with phosphatidylinositol 3-kinase and with SHP-2. Our results demonstrate that to interact with p85 or SHP-2 SH2 domains, Gab1 must be tyrosine phosphorylated by IR. Further, we found that Gab1 tyrosine 472 is the major site for association with p85, while tyrosines 447 and 589 are participating in this process. Concerning Gab1/SHP-2 interaction, only mutation of tyrosine 627 prevents binding of Gab1 to SHP-2 SH2 domains, suggesting the occurrence of a monovalent binding event. Finally, we examined the role of Gab1 PH (Pleckstrin homology) domain in Gab1/IR interaction and in Gab1 tyrosine phosphorylation by IR. Using the modified two-hybrid system and in vitro experiments, we found that the Gab1 PH domain is not important for IR/Gab1 interaction and for Gab1 tyrosine phosphorylation. In contrast, in intact mammalian cells, Gab1 PH domain appears to be crucial for its tyrosine phosphorylation and association with SHP-2 after insulin stimulation.
Somatostatin Activation of Mitogen-Activated Protein Kinase via Somatostatin Receptor 1 (SSTR1)
Authors: Florio Tullio , Yao Hong , Carey Kendall D. , Dillon Tara J. , Stork Philip J. S. 
Published: January 2014

AbstractHormones and growth factors regulate cell growth via the mitogen-activated protein (MAP) kinase cascade. Here we examine the actions of the hormone somatostatin on the MAP kinase cascade through one of its two major receptor subtypes, the somatostatin receptor 1 (SSTR1) stably expressed in CHO-K1 cells. Somatostatin antagonizes the proliferative effects of fibroblast growth factor in CHO-SSTR1 cells via the SSTR1 receptor. However, in these cells, somatostatin robustly activates MAP kinase (also called extracellular signal regulated kinase; ERK) and augments fibroblast growth factor-stimulated ERK activity. We show that the activation of ERK via SSTR1 is pertussis toxin sensitive and requires the small G protein Ras, phosphatidylinositol 3-kinase, the serine/threonine kinase Raf-1, and the protein tyrosine phosphatase SHP-2. The activation of ERK by SSTR1 increased the expression of the cyclin-dependent protein kinase inhibitor p21cip1/WAF1. Previous studies have suggested that somatostatin-stimulated protein tyrosine phosphatase activity mediates the growth effects of somatostatin. Our data suggest that SHP-2 stimulation by SSTR1 may mediate some of these effects through the activation of the MAP kinase cascade and the expression of p21cip1/WAF1.
Functional Analysis of a Novel Estrogen Receptor-β Isoform
Authors: Hanstein Bettina , Liu Hong , Yancisin Molly C. , Brown Myles 
Published: January 2014

AbstractA new level of complexity has recently been added to estrogen signaling with the identification of a second estrogen receptor, ERβ. By screening a rat prostate cDNA library, we detected ERβ as well as a novel isoform that we termed ERβ2. ERβ2 contains an in-frame inserted exon of 54 nucleotides that results in the predicted insertion of 18 amino acids within the ERβ hormone-binding domain. We also have evidence for the expression of both ERβ1 and ERβ2 in human cell lines. Competition ligand binding analysis of bacterially expressed fusion proteins revealed an 8-fold lower affinity of ERβ2 for 17β-estradiol (E2)[ dissociation constant (Kd ∼ 8 nm)] as compared with ERβ1 (Kd ∼ 1 nm). In vitro transcribed and translated ERβ1 and ERβ2 bind specifically to a consensus estrogen responsive element in a gel mobility shift assay. Furthermore, we show heterodimerization of ERβ1 and ERβ2 with each other as well as with ERα. In affinity interaction assays for proteins that associate specifically with the hormone-binding domain of these receptors, we demonstrate that the steroid receptor coactivator SRC-1 interacts in an estrogen-dependent manner with ERα and ERβ1, but not with ERβ2. In cotransfection experiments with expression plasmids for ERα, ERβ1, and ERβ2 and an estrogen-responsive element-containing luciferase reporter, the dose response of ERβ1 to E2 was similar to that of ERα although the maximal stimulation was approximately 50%. In contrast, ERβ2 required 100- to 1000-fold greater E2 concentrations for maximal activation. Thus, ERβ2 adds yet another facet to the possible cellular responses to estrogen.
A Fusion Protein of the Estrogen Receptor (ER) and Nuclear Receptor Corepressor (NCoR) Strongly Inhibits Estrogen-Dependent Responses in Breast Cancer Cells
Authors: Chien Pei-Yu , Ito Masafumi , Park Youngkyu , Tagami Tetsuya , Gehm Barry D. , Jameson J. Larry 
Published: January 2014

AbstractNuclear receptor corepressor (NCoR) mediates repression (silencing) of basal gene transcription by nuclear receptors for thyroid hormone and retinoic acid. The goal of this study was to create novel estrogen receptor (ER) mutants by fusing transferable repressor domains from the N-terminal region of NCoR to a functional ER fragment. Three chimeric NCoR-ER proteins were created and shown to lack transcriptional activity. These fusion proteins silenced basal transcription of the ERE2-tk-Luc reporter gene and inhibited the activity of cotransfected wild-type ER (wtER), indicating that they possess dominant negative activity. One of the fusion proteins (CDE-RD1), containing the ER DNA-binding and ligand-binding domains linked to the NCoR repressor domain (RD1), was selected for detailed examination. Its hormone affinity, intracellular localization, and level of expression in transfected cells were similar to wtER, and it bound to the estrogen response element (ERE) DNA in gel shift assays. Glutathione-S-transferase pull-down assays showed that CDE-RD1 retains the ability to bind to steroid receptor coactivator-1. Introduction of a DNA-binding domain mutation into the CDE-RD1 fusion protein eliminated silencing and dominant negative activity. Thus, the RD1 repressor domain prevents transcriptional activation despite the apparent ability of CDE-RD1 to bind DNA, ligand, and coactivators. Transcriptional silencing was incompletely reversed by trichostatin A, suggesting a histone deacetylase-independent mechanism for repression. CDE-RD1 inhibited ER-mediated transcription in T47D and MDA-MB-231 breast cancer cells and repressed the growth of T47D cells when delivered to the cells by a retroviral vector. These ER-NCoR fusion proteins provide a novel means for inhibiting ER-mediated cellular responses, and analogous strategies could be used to create dominant negative mutants of other transcription factors.
Alternative Splicing of Vascular Endothelial Growth Factor (VEGF)-R1 (FLT-1) pre-mRNA Is Important for the Regulation of VEGF Activity
Authors: He Yulong , Smith Stephen K. , Day Kate A. , Clark Dawn E. , Licence Diana R. , Charnock-Jones D. Stephen 
Published: January 2014

AbstractAngiogenesis is essential for normal mammalian development and is controlled by the local balance of pro- and antiangiogenic factors. Here we describe a novel mouse cDNA sequence encoding sFLT-1 that is a potent antagonist to vascular endothelial growth factor (VEGF) and show for the first time its in vivo production. In situ hybridization and Northern blot analysis with probes specific for sFLT-1 or FLT-1 showed that the relative abundance of their mRNAs changed markedly in spongiotrophoblast cells in the placenta as gestation progressed. On day 11 of pregnancy, sFLT-1 mRNA was undetectable but FLT-1 readily apparent, and by day 17 sFLT-1 mRNA was abundant but FLT-1 barely detectable. sFLT-1 was identified in conditioned medium of cultured placenta from day 17 pregnant mice and likely to be present in the circulation, as there is a substantial increase of VEGF-binding activity in the serum from day 13 of pregnancy, which coincides with the abundant sFLT-1 expression in placenta. Expression of sFLT-1 was also observed in adult lung, kidney, liver, and uterus. These data suggest a novel mechanism of regulation of angiogenesis by alternative splicing of FLT-1 pre-mRNA. Treatment of pregnant mice with exogenous VEGF from day 9 to 17 of pregnancy, which alters the ratio of VEGF to sFLT-1, resulted in an increase in the number of resorption sites and fibrin deposition in the placenta of ongoing pregnancies. These findings have important implications for understanding placental function and may be relevant in a range of disease states.
Transcriptional Activities of the Orphan Nuclear Receptor ERRα (Estrogen Receptor-Related Receptor-α)
Authors: Vanacker Jean-Marc , Bonnelye Edith , Chopin-Delannoy Sandrine , Delmarre Cateline , Cavaillès Vincent , Laudet Vincent 
Published: January 2014

AbstractEstrogen receptor-related receptor α (ERRα) is an orphan nuclear receptor closely related to the estrogen receptor (ER), whose expression covers various stages of embryonic development and persists in certain adult tissues. We show that ERRα binds as a homodimer on a specific target sequence, the SFRE (SF-1 response element), already known to respond to the orphan nuclear receptor SF-1. Target sequences that are related to the SFRE and that discriminate between ERRα and SF-1 were identified. We have also analyzed the transcriptional properties of the ERRα originating from various species. All ERRα orthologs act as potent transactivators through the consensus SFRE. ERRα activity depends on the putative AF2AD domain, as well as on a serum compound that is withdrawn by charcoal treatment, suggesting the existence of a critical regulating factor brought by serum.
Estradiol Suppresses Phosphorylation of Cyclic Adenosine 3′,5′-Monophosphate Response Element Binding Protein (CREB) in the Pituitary: Evidence for Indirect Action via Gonadotropin-Releasing Hormone
Authors: Duan W. Rachel , Shin Jennifer L. , Jameson J. Larry 
Published: January 2014

AbstractEstradiol acts on the hypothalamus and pituitary gland to modulate the synthesis and secretion of gonadotropins. We recently reported that GnRH-induced transcription of the human gonadotropin α-gene promoter is increased markedly in transfected pituitary cells derived from animals treated with estradiol. Because the cAMP response element binding (CREB) protein plays an important role in the transcriptional regulation of this promoter and is highly regulated by posttranslational phosphorylation, we hypothesized that it might serve as a target for estradiol-induced sensitivity to GnRH. In this study, we assessed the roles of estradiol and GnRH in the regulation of CREB phosphorylation in the rat pituitary. Using an antibody that specifically recognizes phosphorylated CREB (pCREB), we found that the pituitary content of pCREB was inversely related to the level of estradiol during the estrous cycle. Ovariectomy increased the level of pCREB, and treatment with estradiol for 10 days decreased the content of pCREB dramatically (93% inhibition). A similar reduction of pCREB was seen when ovariectomized rats were treated with a GnRH receptor antagonist for 10 days. This result indicates that the ovariectomy-induced increase in pCREB is GnRH-dependent. In αT3 gonadotrope cells, estradiol had no direct effect on CREB phosphorylation, whereas GnRH increased CREB phosphorylation 4- to 5-fold within 5 min. We conclude that estradiol inhibits CREB phosphorylation in the gonadotrope, probably by inhibiting GnRH production. The estradiol-induced decrease in CREB phosphorylation is proposed to lower basalα -promoter activity and increase its responsiveness to GnRH. (Molecular Endocrinology 13: 1338–1352, 1999)