bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative Genomic Hybridization Analysis of Adrenocortical Tumors
Authors: Sidhu Stan , Marsh Deborah J. , Theodosopoulos George , Philips Jeanette , Bambach Christopher P. , Campbell Peter , Magarey Christopher J. , Russell Colin F. J. , Schulte Klaus-Martin , Röher Hans-Dietrich , Delbridge Leigh , Robinson Bruce G.
Published: January 2014

Comparative genomic hybridization (CGH) is a molecular cytogenetic technique that allows the entire genome of a tumor to be surveyed for gains and losses of DNA copy sequences. A limited number of studies reporting the use of this technique in adult adrenocortical tumors have yielded conflicting results.

In this study we performed CGH analysis on 13 malignant, 18 benign, and 1 tumor of indeterminate malignant potential with the aim of identifying genetic loci consistently implicated in the development and progression of adrenocortical tumors. Tissue samples from 32 patients with histologically proven adrenocortical tumors were available for CGH analysis. CGH changes were seen in all cancers, 11 of 18 (61%) adenomas, and the 1 tumor of indeterminate malignant potential. Of the adrenal cancers, the most common gains were seen on chromosomes 5 (46%), 12 (38%), 19 (31%), and 4 (31%). Losses were most frequently seen at 1p (62%), 17p (54%), 22 (38%), 2q (31%), and 11q (31%). Of the benign adenomas, the most common change was gain of 4q (22%).

Mann-Whitney analysis showed a highly significant difference between the cancer group (mean changes, 7.6) and the adenoma group (mean changes, 1.1) for the number of observed CGH changes (P < 0.01). Logistic regression analysis showed that the number of CGH changes was highly predictive of tumor type (P < 0.01).

This study has identified several chromosomal loci implicated in adrenocortical tumorigenesis. Activation of a protooncogene(s) on chromosome 4 may be an early event, with progression from adenoma to carcinoma involving activation of oncogenes on chromosomes 5 and 12 and inactivation of tumor suppressor genes on chromosome arms 1p and 17p.
Follicle Stimulating Hormone-Regulated Expression of Serum/Glucocorticoid-Inducible Kinase in Rat Ovarian Granulosa Cells: A Functional Role for the Sp1 Family in Promoter Activity
Authors: Alliston Tamara N. , Maiyar Anita C. , Buse Patricia , Firestone Gary L. , Richards JoAnne S.
Published: January 2014

Recently, a family of novel, serine/threonine protein kinases has been identified. One of these transcriptionally inducible, immediate-early genes encodes serum/glucocorticoid inducible-protein kinase, sgk. By in situ hybridization, we show that sgk expression in the rat ovary is selectively localized to granulosa cells. In culture, FSH or forskolin, activators of the protein kinase A (PKA) pathway, rapidly (2 h) and transiently increased sgk mRNA levels in undifferentiated granulosa cells. Sgk mRNA exhibited a biphasic expression pattern, with maximal levels observed at 48 h of FSH/forskolin as granulosa cells differentiate to the preovulatory phenotype. Deletion analyses using sgk promoter-reporter constructs (−4.0 kb to −35 bp) identified a region between −63 and− 43 bp that mediated FSH and forskolin-responsive transcription in undifferentiated and differentiated granulosa cells. This G/C-rich region 1) conferred both basal and inducible transcription to the minimal −35 sgk promoter chloramphenicol acetyltransferase reporter construct, 2) specifically bound Sp1 and Sp3 present in granulosa cell extracts, and 3) bound recombinant Sp1. Mutation of 2 bp in this region not only prevented Sp1 and Sp3 binding, but also abolished the PKA-mediated transactivation observed when using the wild type construct. Sp1 and Sp3 DNA-binding activity and protein levels did not change significantly during sgk induction. Collectively, these data indicate that Sp1/Sp3 transactivation of the sgk promoter likely involves regulated, phosphorylation-dependent interaction with other factors. Thus the novel, biphasic induction of sgk that correlates with granulosa cell progression from proliferation to differentiation appears to involve sequential, coordinated actions of FSH, PKA, and transcription factors, including Sp1 and Sp3.
A Functional Sp1 Binding Site Is Essential for the Activity of the Adult Liver-Specific Human Insulin-Like Growth Factor II Promoter
Authors: Rodenburg Richard J. T. , Holthuizen P. Elly , Sussenbach John S.
Published: January 2014

The human gene encoding insulin-like growth factor II contains four promoters (P1–P4) that are differentially activated in various tissues during development. Expression of insulin-like growth factor II in adult liver tissue is directed by P1, which is activated by liver-enriched members of the CCAAT/enhancer binding protein family of transcription factors. In the present report we show that the region around −48 relative to the transcription start site contains a high affinity Sp1 binding site. This was demonstrated by electrophoretic mobility shift assays using nuclear extracts from Hep3B hepatoma cells and with specific antibodies directed against Sp1. Competition electrophoretic mobility shift assays revealed that the Sp1 binding site of P1 and a consensus Sp1 binding site bind Sp1 with comparable efficiencies. Mutation of the Sp1 binding site results in an 85% decrease in P1 promoter activity in transient transfection assays using two different cell lines, COS-7 and Hep3B. Investigation of P1 mutants in which the spacing of the Sp1 binding site and the transcription start site was increased showed that the role of the Sp1 binding site in regulation of P1 is position dependent. Interestingly, the Sp1-responsive element cannot be exchanged by a functional TATA box. Activation of P1 by transactivators CCAAT/enhancer binding protein-β and hepatocyte nuclear factor-3β is strongly impaired after mutation of the Sp1 binding site. These results demonstrate that the specific presence of a binding site for the ubiquitously expressed transcription factor Sp1 is of eminent importance for efficient activation of P1 by liver-enriched transactivators.
Coupling Specificity between Somatostatin Receptor sst2A and G Proteins: Isolation of the Receptor-G Protein Complex with a Receptor Antibody
Authors: Gu Yi-Zhong , Schonbrunn Agnes
Published: January 2014

Somatostatin initiates its actions via a family of seven-transmembrane domain receptors. Of the five somatostatin receptor genes cloned, sst2 exists as two splice variants with the sst2A isoform being predominantly expressed. This receptor is widely distributed in endocrine, exocrine, and neuronal cells, as well as in hormonally responsive tumors, and leads to inhibition of secretion, electrical excitability, and cell proliferation. To investigate the specificity of signal transduction by the sst2A receptor, we developed antibodies against two overlapping peptides located within the C terminus of the receptor protein: peptide 2CSG, containing amino acids 334–348, and peptide 2CER, containing amino acids 339–359. Although antibodies to both peptides bound the inducing antigen with high affinity, only the antibodies against peptide 2CER precipitated the receptor. The best antibody, R2–88, precipitated about 80% of the sst2A receptor-ligand complex solubilized from transfected CHO cells and was specific for the sst2A receptor isotype. Addition of GTPγS (10μ m) to the immunoprecipitated ligand-sst2A receptor complex markedly accelerated ligand dissociation, indicating that G proteins remained functionally associated with the receptor in the immuno-precipitate. Analysis of the G proteins coprecipitated with the sst2A receptor by immunoblotting with G protein antibodies showed that both Gα and Gβ subunits were bound to the hormone-receptor complex. Immunoprecipitation of the receptor was not affected by the presence of bound ligand. However, G protein subunits were coprecipitated only with the hormone-occupied receptor. Thus, the unoccupied receptor has low affinity for G proteins, and hormone binding stabilizes the receptor-G protein complex. Use of subtype-specific G protein antisera further showed that Gαi1, Gαi2, and Gαi3 were complexed with the sst2A receptor whereas Gαo, Gαz, and Gαq were not. Together, these studies demonstrate that the sst2A receptor interacts selectively with Gαi proteins in a hormone-dependent manner. The finding that this receptor couples to all three Gαi subunits may help explain how somatostatin can regulate multiple signaling pathways.
Oct-1 Binds Promoter Elements Required for Transcription of the GnRH Gene
Authors: Eraly Satish A. , Nelson Shelley B. , Huang Karen M. , Mellon Pamela L.
Published: January 2014

The GnRH gene is exclusively expressed in a discrete population of neurons in the hypothalamus. The promoter-proximal 173 bp of the rat GnRH gene are highly conserved through evolution and are bound by multiple nuclear proteins found in the neuronal cell line, GT1–7, a model for the GnRH-expressing hypothalamic neuron. To explore the protein-DNA interactions that occur within this promoter and the role of these interactions in targeting GnRH gene expression, we have mutagenized individual binding sites in this region. Deoxyribonuclease I protection experiments reveal that footprint 2, a 51-bp sequence that confers a 20-fold induction of the GnRH gene, is comprised of at least three independent protein-binding sites. Transfections of the GnRH promoter-reporter plasmid containing a series of block mutations of footprint 2 into GT1–7 neurons indicate that each of the three putative component sites contributes to transcriptional activity. Mutations in footprint 4 also decrease GnRH gene expression. Footprint 4 and the promoter-proximal site in footprint 2 contain octamer-like motifs, an element that is also present in the neuron-specific enhancer of the rat GnRH gene located approximately 1.6 kb upstream of the promoter. Previous studies in our laboratory have demonstrated that two enhancer octamer sites are bound by the POU-homeodomain transcription factor Oct-1 in GT1–7 cells. We now show that Oct-1 binds the octamer motifs within footprints 2 and 4. Thus, Oct-1 plays a critical role in the regulation of GnRH transcription, binding functional elements in both the distal enhancer and the promoter-proximal conserved region.
The High Mobility Group Protein 1 Enhances Binding of the Estrogen Receptor DNA Binding Domain to the Estrogen Response Element
Authors: Romine Lorene E. , Wood Jennifer R. , Lamia LuAnne A. , Prendergast Paul , Edwards Dean P. , Nardulli Ann M.
Published: January 2014

We have examined the ability of the high-mobility group protein 1 (HMG1) to alter binding of the estrogen receptor DNA-binding domain (DBD) to the estrogen response element (ERE). HMG1 dramatically enhanced binding of purified, bacterially expressed DBD to the consensus vitellogenin A2 ERE in a dose-dependent manner. The ability of HMG1 to stabilize the DBD-ERE complex resulted in part from a decrease in the dissociation rate of the DBD from the ERE. Antibody supershift experiments demonstrated that HMG1 was also capable of forming a ternary complex with the ERE-bound DBD in the presence of HMG1-specific antibody. HMG1 did not substantially affect DBD-ERE contacts as assessed by methylation interference assays, nor did it alter the ability of the DBD to induce distortion in ERE-containing DNA fragments. Because HMG1 dramatically enhanced estrogen receptor DBD binding to the ERE, and the DBD is the most highly conserved region among the nuclear receptor superfamily members, HMG1 may function to enhance binding of other nuclear receptors to their respective response elements and act in concert with coactivator proteins to regulate expression of hormone-responsive genes.
Reconstitution of the Protein Kinase A Response of the Rat Prolactin Promoter: Differential Effects of Distinct Pit-1 Isoforms and Functional Interaction with Oct-1
Authors: Diamond Scott E. , Chiono Matt , Gutierrez-Hartmann Arthur
Published: January 2014

PRL gene transcription is primarily regulated by dopamine, which lowers cAMP levels and inhibits protein kinase A (PKA) activity. Current data indicate that the cAMP/PKA response maps to the most proximal Pit-1/Pit-1β binding site footprint I (FP I) on the rat PRL (rPRL) promoter. Pit-1, a POU-homeo domain transcription factor, is specifically expressed in the anterior pituitary and is required both for the normal development of anterior pituitary cell types, somatotrophs, lactotrophs, and thyrotrophs, and for the expression of their hormones: GH, PRL, and TSHβ. Pit-1 has been shown to functionally interact, via FP I, with several transcription factors, including Oct-1, a ubiquitous homeobox protein, and thyrotroph embryonic factor, which is found in lactotrophs, to activate basal rPRL promoter activity. Pit-1β/GHF-2, a distinct splice isoform of Pit-1, acts to inhibit Ras-activated transcription from the rPRL promoter, which is mediated by a functional interaction between Pit-1 and Ets-1 at the most distal Pit-1 binding site (FP IV). In this manuscript we show 1) that the Pit-1β isoform not only fails to block PKA activation, but is, in fact, a superior mediator of the PKA response; 2) that the PKA response requires intact POU-specific and POU-homeo domains of Pit-1; and 3) that Oct-1, but not thyrotroph embryonic factor, functions as a Pit-1-interacting factor to mediate an optimal PKA response.
Transcriptional and Posttranscriptional Regulation of Insulin-Like Growth Factor Binding Protein-3 by Cyclic Adenosine 3′,5′-Monophosphate: Messenger RNA Stabilization Is Accompanied by Decreased Binding of a 42-kDa Protein to a Uridine-Rich Domain in the 3′-Untranslated Region
Authors: Erondu N. E. , Nwankwo J. , Zhong Y. , Boes M. , Dake B. , Bar R. S.
Published: January 2014

The Madin Darby bovine kidney (MDBK) cell line was used to investigate the mechanisms underlying the cAMP regulation of insulin-like growth factor binding protein-3 (IGFBP-3) gene expression. Treatment of confluent monolayers either with forskolin or cAMP produced a 60- to 75-fold induction of IGFBP-3 mRNA and protein levels. This effect did not require new protein synthesis as inhibition of translation by cycloheximide actually caused a 2-fold increase in the cAMP induction. The rates of IGFBP-3 gene transcription, assessed by nuclear run-on assays, increased approximately 15-fold in cells exposed to cAMP. In addition, the half-life of the IGFBP-3 mRNA transcript was increased ∼3-fold in the presence of cAMP. Gel mobility shift and competition experiments revealed the specific binding of an approximately 42-kDa cytoplasmic protein factor to the 3′-untranslated region (3′-UTR) of the IGFBP-3 mRNA. A 21-nucleotide uridine-rich segment that contained no AUUUA motif was sufficient for the specific binding. The binding activity of this protein was reduced after cAMP treatment but was increased by phosphatase treatment. In conclusion, the cAMP induction of IGFBP-3 mRNA in MDBK cells occurred at both the transcriptional and posttranscriptional levels. The IGFBP-3 mRNA stabilization in MDBK cells probably involved the phosphorylation of a member of the family of U-rich region mRNA-binding proteins and is the first reported member whose RNA-binding activity is reduced by cAMP.
Potential Regulation of Membrane Trafficking by Estrogen Receptorα via Induction of Rab11 in Uterine Glands during Implantation
Authors: Chen Dahu , Ganapathy Padmaja , Zhu Li-Ji , Xu Xueping , Li Quanxi , Bagchi Indrani C. , Bagchi Milan K.
Published: January 2014

The steroid hormone estrogen profoundly influences the early events in the uterus leading to embryo implantation. It is thought that estrogen triggers the expression of a unique set of genes in the preimplantation endometrium that in turn control implantation. To identify these estrogen-induced genes, we used a delayed implantation model system in which embryo attachment to endometrium is dependent on estrogen administration. Using a mRNA differential display (DD) method, we isolated a number of cDNAs representing mRNAs whose expression is either turned on or turned off in response to an implantation-inducing dose of estrogen. We identified one of these cDNAs as that encoding rab11, a p21ras-like GTP-binding protein (G protein), which functions in the targeting of transport vesicles to the plasma membrane. In normal pregnant rats, rab11 mRNA was expressed at low levels on days 1–2 of pregnancy, but its expression was markedly enhanced (∼6- to 8-fold) between days 3–5 immediately before implantation. In situ hybridization and immunocytochemistry revealed that rab11 expression in the uterus was predominantly in the glandular epithelium. In ovariectomized rats, the expression of rab11 mRNA was induced in the endometrium in response to estrogen. To determine whether this effect of estrogen was mediated through its nuclear receptors, we examined rab11 expression in a transformed endometrial cell line, Ishikawa. In transient transfection experiments, we observed that overexpression of estrogen receptor (ER) α or β induced endogenous rab11 mRNA in a hormone-dependent manner. ER bound to an antagonist, ICI 182,780, failed to activate this gene expression. These findings, together with the observation that ERα but not ERβ is detected in the glands of the preimplantation uterus, indicate that rab11 is one of the proteins that are specifically induced by estrogen-complexed ERα in rat endometrium at the onset of implantation. Our results imply that estrogen, which induces the synthesis of many growth factors and their receptors and other secretory proteins that are thought to be critical for implantation, may also facilitate their transport to the membrane and/or secretion by stimulating the expression of rab11, a component of the membrane-trafficking pathway. This study therefore provides novel insights into the diverse cellular mechanisms by which estrogen, acting via its nuclear receptors, may influence blastocyst implantation.
Cloning of Interferon-Stimulated Gene 17: The Promoter and Nuclear Proteins That Regulate Transcription
Authors: Perry David J. , Austin Kathy J. , Hansen Thomas R.
Published: January 2014

A member of the interferon-stimulated gene (ISG) family encodes a 17-kDa ubiquitin homolog called ISG17 that is induced in the bovine uterine endometrium by interferon-τ (IFN-τ) during early pregnancy. The bovine (b) ISG17 cDNA shares 30% identity with a tandem ubiquitin repeat and 70% identity with human (h) ISG15. The present experiments were designed to sequence the bISG17 gene, compare general structure with the hISG15 gene, and to identify transcription factors that were induced by IFN-τ in bovine endometrial (BEND) cells. The promoter of the bISG17 gene was similar to the hISG15 gene in placement of a tandem IFN-stimulatory response element (ISRE) at position −90, but unique in the presence of three additional ISREs at positions −123, −332, and −525. IFN-τ (25 nm) induced nuclear proteins in BEND cells that interacted with a tandem bISG17 ISRE in electrophoretic mobility shift assay (EMSA). IFN-regulatory factor-1 (IRF-1) bound to this ISRE based upon supershift EMSA using antiserum against IRF-1. IFN-τ activated STAT-1 (signal transducer and activator of transcription-1) and -2 by 0.5 h, and IRF-1 by 2 h in BEND cells. It is concluded that the bISG17 gene is similar to the hISG15 gene, retains an ISRE that interacts with IRF-1, and is possibly induced initially by the STATs and later by IRF-1 in response to IFN-τ during early pregnancy.
The Promoter of the Rat 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Gene Contains a Tissue-Specific Estrogen-Responsive Region
Authors: Di Croce Luciano , Vicent Guillermo P. , Pecci Adali , Bruscalupi Giovannella , Trentalance Anna , Beato Miguel
Published: January 2014

The isoprenoid metabolic pathway is mainly regulated at the level of conversion of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) to mevalonate, catalyzed by HMG CoA reductase. As estrogens are known to influence cholesterol metabolism, we have explored the potential regulation of the HMG CoA reductase gene promoter by estrogens. The promoter contains an estrogen-responsive element-like sequence at position −93 (termed Red-ERE), which differs from the ERE consensus by one mismatch in each half of the palindrome. A Red-ERE oligonucleotide specifically bound estrogen receptor in vitro and conferred receptor-dependent estrogen responsiveness to a heterologous promoter in all cell lines tested. However, expression of a reporter driven by the rat HMG CoA reductase promoter was induced by estrogen treatment after transient transfection into the breast cancer cell line MCF-7 cells but not in hepatic cell lines expressing estrogen receptor. Estrogen induction in MCF-7 cells was dependent on the Red-ERE and was strongly inhibited by the antiestrogen ICI 164,384. A functional cAMP-responsive element is located immediately upstream of the Red-ERE, but cAMP and estrogens inhibit each other in terms of transactivation of the promoter. Similarly, induction by estrogens was inhibited by micromolar concentrations of cholesterol, likely acting via changes in occupancy of the sterol-responsive element located 70 bp upstream of the Red-ERE. Thus, within its natural context, Red-ERE is able to mediate hormonal regulation of the HMG CoA reductase gene in tissues that respond to estrogens with enhanced cell proliferation, while it is not operative in liver cells. We postulate that this tissue-specific regulation of HMG CoA reductase by estrogens could partially explain the protective effect of estrogens against heart disease.
Differential Hormone-Dependent Phosphorylation of Progesterone Receptor A and B Forms Revealed by a Phosphoserine Site-Specific Monoclonal Antibody
Authors: Clemm David L. , Sherman Lori , Boonyaratanakornkit Viroj , Schrader William T. , Weigel Nancy L. , Edwards Dean P.
Published: January 2014

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.
Human ERRγ, a Third Member of the Estrogen Receptor-Related Receptor (ERR) Subfamily of Orphan Nuclear Receptors: Tissue-Specific Isoforms Are Expressed during Development and in the Adult
Authors: Heard David J. , Norby Peder L. , Holloway Jim , Vissing Henrik
Published: January 2014

The nuclear receptor protein superfamily is a large group of transcription factors involved in many aspects of animal development, tissue differentiation, and homeostasis in the higher eukaryotes. A subfamily of receptors, ERRα and β (estrogen receptor-related receptor α and β), closely related to the ER, were among the first orphan nuclear receptors identified. These receptors can bind DNA as monomers and are thought to activate transcription constitutively, unaffected by β-estradiol. Studies of the expression patterns of ERRα and gene disruption experiments of ERRβ indicate that they play an important role in the development and differentiation of specific tissues in the mouse. In this work we demonstrate the existence in humans of a third member of this subfamily of receptors, termed ERRγ, which is highly expressed in a number of diverse fetal and adult tissues including brain, kidney, pancreas, and placenta. The ERRγ mRNA is highly alternatively spliced at the 5′-end, giving rise to a number of tissue-specific RNA species, some of which code for protein isoforms differing in the N-terminal region. Like ERRα andβ , ERRγ binds as a monomer to an ERRE. A GAL4-ERRγ fusion protein activates transcription in a ligand-independent manner in transfected HEK293 cells to a greater degree than either the GAL4-ERRα or -β fusion proteins.
AgRP(83–132) Acts as an Inverse Agonist on the Human-Melanocortin-4 Receptor
Authors: Nijenhuis Wouter A. J. , Oosterom Julia , Adan Roger A. H.
Published: January 2014

The central melanocortin (MC) system has been demonstrated to act downstream of leptin in the regulation of body weight. The system comprises α-MSH, which acts as agonist, and agouti-related protein (AgRP), which acts as antagonist at the MC3 and MC4 receptors (MC3R and MC4R). This property suggests that MCR activity is tightly regulated and that opposing signals are integrated at the receptor level. We here propose another level of regulation within the melanocortin system by showing that the human (h) MC4R displays constitutive activity in vitro as assayed by adenylyl cyclase (AC) activity. Furthermore, human AgRP(83–132) acts as an inverse agonist for the hMC4R since it was able to suppress constitutive activity of the hMC4R both in intact B16/G4F melanoma cells and membrane preparations. The effect of AgRP(83–132) on the hMC4R was blocked by the MC4R ligand SHU9119. Also the hMC3R and the mouse(m)MC5R were shown to be constitutively active. AgRP(83–132) acted as an inverse agonist on the hMC3R but not on the mMC5R. Thus, AgRP is able to regulate MCR activity independently of α-MSH. These findings form a basis to further investigate the relevance of constitutive activity of the MC4R and of inverse agonism of AgRP for the regulation of body weight.
Glucocorticoid Suppression of IGF I Transcription in Osteoblasts
Authors: Delany Anne M. , Durant Deena , Canalis Ernesto
Published: January 2014

Glucocorticoids have profound effects on bone formation, decreasing IGF I transcription in osteoblasts, but the mechanisms involved are poorly understood. We previously showed that the bp +34 to+ 192 region of the rat IGF I exon 1 promoter was responsible for repression of IGF I transcription by cortisol in cultures of osteoblasts from fetal rat calvariae (Ob cells). Here, site-directed mutagenesis was used to show that a binding site for members of the CAAT/enhancer binding protein family of transcription factors, within the +132 to +158 region of the promoter, mediates this glucocorticoid effect. EMSAs demonstrated that cortisol increased binding of osteoblast nuclear proteins to the +132 to +158 region of the IGF I promoter. Supershift assays showed that CAAT/enhancer binding protein α, β, and δ interact with this sequence, and binding of CAAT/enhancer binding protein δ, in particular, was increased in the presence of cortisol. Northern blot analysis showed that CAAT/enhancer binding protein δ and β transcripts were increased by cortisol in Ob cells. Further, cortisol increased the transcription of these genes and increased the stability of CAAT/enhancer binding protein δ mRNA. In conclusion, cortisol represses IGF I transcription in osteoblasts, and CAAT/enhancer binding proteins appear to play a role in this effect.
The Activation of the Phosphotyrosine Phosphatase η (r-PTPη) Is Responsible for the Somatostatin Inhibition of PC Cl3 Thyroid Cell Proliferation
Authors: Florio Tullio , Arena Sara , Thellung Stefano , Iuliano Rodolfo , Corsaro Alessandro , Massa Alessandro , Pattarozzi Alessandra , Bajetto Adriana , Trapasso Francesco , Fusco Alfredo , Schettini Gennaro
Published: January 2014

The aim of this study was the characterization of the intracellular effectors of the antiproliferative activity of somatostatin in PC Cl3 thyroid cells. Somatostatin inhibited PC Cl3 cell proliferation through the activation of a membrane phosphotyrosine phosphatase. Conversely, PC Cl3 cells stably expressing the v-mos oncogene (PC mos) were completely insensitive to the somatostatin antiproliferative effects since somatostatin was unable to stimulate a phosphotyrosine phosphatase activity. In PC mos cells basal phosphotyrosine phosphatase activity was also reduced, suggesting that the expression of a specific phosphotyrosine phosphatase was impaired in these transformed cells. We suggested that this phosphotyrosine phosphatase could be r-PTPη whose expression was abolished in the PC mos cells. To directly prove the involvement of r-PTPη in somatostatin’s effect, we stably transfected this phosphatase in PC mos cells. This new cell line (PC mos/PTPη) recovered somatostatin’s ability to inhibit cell proliferation, showing dose-dependence and time course similar to those observed in PC Cl3 cells. Conversely, the transfection of a catalytically inactive mutant of r-PTPη did not restore the antiproliferative effects of somatostatin. PC mos/PTPη cells showed a high basal phosphotyrosine phosphatase activity which, similarly to PC Cl3 cells, was further increased after somatostatin treatment. The specificity of the role of r-PTPη in somatostatin receptor signal transduction was demonstrated by measuring its specific activity after somatostatin treatment in an immunocomplex assay. Somatostatin highly increased r-PTPη activity in PCCl3 and PC mos/PTPη (+300%, P < 0.01) but not in PCmos cells. Conversely, no differences in somatostatin-stimulated SHP-2 activity, (∼ +50%, P < 0.05), were observed among all the cell lines. The activation of r-PTPη by somatostatin caused, acting downstream of MAPK kinase, an inhibition of insulin-induced ERK1/2 activation with the subsequent blockade of the phosphorylation, ubiquitination, and proteasome degradation of the cyclin-dependent kinase inhibitor p27kip1. Ultimately, high levels of p27kip1 lead to cell proliferation arrest. In conclusion, somatostatin inhibition of PC Cl3 cell proliferation requires the activation of r-PTPη which, through the inhibition of MAPK activity, causes the stabilization of the cell cycle inhibitor p27kip1.
Proximal cis-Acting Elements, Including Steroidogenic Factor 1, Mediate the Efficiency of a Distal Enhancer in the Promoter of the Rat Gonadotropin-Releasing Hormone Receptor Gene
Authors: Pincas Hanna , Amoyel Karine , Counis Raymond , Laverrière Jean-Noël
Published: January 2014

The gonadotrope-specific and regulated expression of the GnRH receptor (GnRH-R) gene is dependent on multiple transcription factors that interact with the noncanonical GnRH-R activating sequence (GRAS), the activator protein-1 (AP-1) element, and the steroidogenic factor-1 (SF-1) binding site. However, these three elements are not sufficient to mediate the complete cell-specific expression of the rat GnRH-R gene. In the present study, we demonstrate, by transient transfection in gonadotrope-derived αT3–1 and LβT2 cell lines, the existence of a distal enhancer [GnRH-R- specific enhancer (GnSE)] that is highly active in the context of the GnRH-R gene promoter. We show that the GnSE activity (–1,135/–753) is mediated through a functional interaction with a proximal region (–275/–226) that includes the SF-1 response element. Regions of similar length containing either the AP-1 or GRAS elements are less active or inactive. Transfection assays using an artificial promoter containing two SF-1 elements fused to a minimal PRL promoter indicate that SF-1 is crucial in this interaction. In addition, by altering the promoter with deletion and block- replacement mutations, we have identified the active elements of GnSE within two distinct sequences at positions –983/–962 and –871/–862. Sequence analysis and electrophoretic mobility shift experiments suggest that GnSE response elements interact, in these two regions, with GATA- and LIM-related factors, respectively. Altogether, these data establish the importance of the GnSE in the GnRH-R gene expression and reveal a novel role for SF-1 as a mediator of enhancer activity, a mechanism that might regulate other SF-1 target genes.
A Single Pitx1 Binding Site Is Essential for Activity of the LHβ Promoter in Transgenic Mice
Authors: Quirk Christine C. , Lozada Kristen L. , Keri Ruth A. , Nilson John H.
Published: January 2014

Reproduction depends on regulated expression of the LHβ gene. Tandem copies of regulatory elements that bind early growth response protein 1 (Egr-1) and steroidogenic factor 1 (SF-1) are located in the proximal region of the LHβ promoter and make essential contributions to its activity as well as mediate responsiveness to GnRH. Located between these tandem elements is a single site capable of binding the homeodomain protein Pitx1. From studies that employ overexpression paradigms performed in heterologous cell lines, it appears that Egr-1, SF-1, and Pitx1 interact cooperatively through a mechanism that does not require the binding of Pitx1 to its site. Since the physiological ramifications of these overexpression studies remain unclear, we reassessed the requirement for a Pitx1 element in the promoter of the LHβ gene using homologous cell lines and transgenic mice, both of which obviate the need for overexpression of transcription factors. Our analysis indicated a striking requirement for the Pitx1 regulatory element. When assayed by transient transfection using a gonadotrope-derived cell line (LβT2), an LHβ promoter construct harboring a mutant Pitx1 element displayed attenuated transcriptional activity but retained responsiveness to GnRH. In contrast, analysis of wild-type and mutant expression vectors in transgenic mice indicated that LHβ promoter activity is completely dependent on the presence of a functional Pitx1 binding site. Indeed, the dependence on an intact Pitx1 binding site in transgenic mice is so strict that responsiveness to GnRH is also lost, suggesting that the mutant promoter is inactive. Collectively, our data reinforce the concept that activity of the LHβ promoter is determined, in part, through highly cooperative interactions between SF-1, Egr-1, and Pitx1. While Egr-1 can be regarded as a key downstream effector of GnRH, and Pitx1 as a critical partner that activates SF-1, our data firmly establish that the Pitx1 element plays a vital role in permitting these functions to occur in vivo.
Cloning and Characterization of a Rat Ortholog of MMP-23 (Matrix Metalloproteinase-23), a Unique Type of Membrane-Anchored Matrix Metalloproteinase and Conditioned Switching of Its Expression during the Ovarian Follicular Development
Authors: Ohnishi Junji , Ohnishi Eriko , Jin Mulan , Hirano Wakako , Nakane Dai , Matsui Hitoshi , Kimura Atsushi , Sawa Hirofumi , Nakayama Kazuhisa , Shibuya Hiroshi , Nagashima Kazuo , Takahashi Takayuki
Published: January 2014

In our attempt to study the role of matrix metalloproteinases (MMPs) in the process of mammalian ovulation, we isolated a rat ortholog of the recently reported human MMP-23 from gonadotropin-primed immature rat ovaries. Transient expression of epitope-tagged rat and human MMP-23 in COS-1 cells revealed that they were synthesized as a membrane-anchored glycoprotein with type II topology. Indirect immunofluorescent analysis showed that subcellular localization of MMP-23 was predominantly in the perinuclear regions. The transfected human MMP-23 protein was processed endogenously to the soluble form in COS-1 cells. However, cotransfection of MMP-23 with the mouse furin cDNA did not enhance this processing, indicating that furin may not be involved in this event. Notably, in situ hybridization analysis revealed a dramatic switching of MMP-23 mRNA localization from granulosa cells to theca-externa/fibroblasts and ovarian surface epithelium during the follicular development. In serum-free primary culture of rat granulosa cells, a drastic diminution of MMP-23 mRNA expression was observed in response to FSH action between 24 h and 48 h of culture. The observed effect of FSH on MMP-23 expression was mimicked by treatment of granulosa cells with forskolin or 8-bromo (Br)-cAMP. In contrast, MMP-23 mRNA levels increased in theca-interstitial cells regardless of the presence of LH in the culture. However, treatment of theca-interstitial cells with forskolin or 8-Br-cAMP markedly reduced the expression of MMP-23 with a concomitant increase in progesterone production. These results indicate that the MMP-23 gene is spatially and temporally regulated in a cell type-specific manner in ovary via the cAMP signaling pathway.
Phosphorylation of the Receptor for PTH and PTHrP Is Required for Internalization and Regulates Receptor Signaling
Authors: Tawfeek Hesham A. W. , Qian Fang , Abou-Samra Abdul B.
Published: January 2014

We have previously shown that agonist-dependent phosphorylation of the PTH/PTHrP receptor occurs on its carboxyl-terminal tail. Using site- directed mutagenesis, phosphopeptide mapping, and direct sequencing of cyanogen bromide-cleaved fragments of phosphoreceptors, we report here that PTH-dependent phosphorylation occurs on the serine residues at positions 491, 492, 493, 495, 501, and 504, and that the serine residue at position 489 is required for phosphorylation. When these seven sites were mutated to alanine residues, the mutant receptor was no longer phosphorylated after PTH stimulation. The phosphorylation-deficient receptor, stably expressed in LLCPK-1 cells, was impaired in PTH-dependent internalization and showed an increased sensitivity to PTH stimulation; the EC50 for PTH-stimulated cAMP accumulation was decreased by 7-fold. Furthermore, PTH stimulation of the phosphorylation-deficient PTH/PTHrP receptor caused a sustained elevation in intracellular cAMP levels. These data indicate that agonist-dependent phosphorylation of the PTH/PTHrP receptor plays an important role in receptor function.
The mRNA Structure Has Potent Regulatory Effects on Type 2 Iodothyronine Deiodinase Expression
Authors: Gereben Balázs , Kollár Anna , Harney John W. , Larsen P. Reed
Published: January 2014

Type 2 deiodinase (D2) is a selenoenzyme catalyzing the activation of T4 to T3. D2 activity/mRNA ratios are often low, suggesting that there is significant posttranscriptional regulation. The D2 mRNA in higher vertebrates is more than 6 kb, containing long 5′ and 3′ untranslated regions (UTRs). The D2 5′UTRs are greater than 600 nucleotides and contain 3–5 short open reading frames. These full-length 5′UTRs reduce the D2 translation efficiency approximately 5-fold. The inhibition by human D2 5′UTR is localized to a region containing the first short open reading frame encoding a tripeptide—MKG. This inhibition was abolished by mutating the AUG start codon and weakened by modification of the essential purine of the Kozak consensus. Deletion of the 3.7-kb 3′UTR of the chicken D2 mRNA increased D2 activity approximately 3.8-fold due to an increase in D2 mRNA half-life. In addition, alternatively spliced D2 mRNA transcripts similar in size to the major 6- to 7-kb D2 mRNAs but not encoding an active enzyme are present in both human and chicken tissues. Our results indicate that a number of factors reduce the D2 protein levels. These mechanisms, together with the short half-life of the protein, ensure limited expression of this key regulator of T4 activation.
Frequency of Impaired Fibroblast Growth Factor Receptor 1 Signaling as a Cause of Normosmic Idiopathic Hypogonadotropic Hypogonadism
Authors: Raivio Taneli , Sidis Yisrael , Plummer Lacey , Chen Huaibin , Ma Jinghong , Mukherjee Abir , Jacobson-Dickman Elka , Quinton Richard , Van Vliet Guy , Lavoie Helene , Hughes Virginia A. , Dwyer Andrew , Hayes Frances J. , Xu Shuyun , Sparks Susan , Kaiser Ursula B. , Mohammadi Moosa , Pitteloud Nelly
Published: January 2014

Context

FGFR1 mutations have been identified in about 10% of patients with Kallmann syndrome. Recently cases of idiopathic hypogonadotropic hypogonadism (IHH) with a normal sense of smell (nIHH) have been reported.

Aims

The objective of the study was to define the frequency of FGFR1 mutations in a large cohort of nIHH, delineate the spectrum of reproductive phenotypes, assess functionality of the FGFR1 mutant alleles in vitro, and investigate genotype-phenotype relationships.

Design

FGFR1 sequencing of 134 well-characterized nIHH patients (112 men and 22 women) and 270 healthy controls was performed. The impact of the identified mutations on FGFR1 function was assessed using structural prediction and in vitro studies.

Results

Nine nIHH subjects (five males and four females; 7%) harbor a heterozygous mutation in FGFR1 and exhibit a wide spectrum of pubertal development, ranging from absent puberty to reversal of IHH in both sexes. All mutations impair receptor function. The Y99C, Y228D, and I239T mutants impair the tertiary folding, resulting in incomplete glycosylation and reduced cell surface expression. The R250Q mutant reduces receptor affinity for FGF. The K618N, A671P, and Q680X mutants impair tyrosine kinase activity. However, the degree of functional impairment of the mutant receptors did not always correlate with the reproductive phenotype, and variable expressivity of the disease was noted within family members carrying the same FGFR1 mutation. These discrepancies were partially explained by additional mutations in known IHH loci.

Conclusions

Loss-of-function mutations in FGFR1 underlie 7% of nIHH with different degrees of impairment in vitro. These mutations act in concert with other gene defects in several cases, consistent with oligogenicity.
Melatonin Sensitizes Human Myometrial Cells to Oxytocin in a Protein Kinase Cα/Extracellularly Regulated Kinase-Dependent Manner
Authors: Sharkey James T. , Cable Casey , Olcese James
Published: January 2014

abstract

Context: Studies have shown that labor occurs primarily in the night/morning hours. Recently, we identified the human myometrium as a target for melatonin (MEL), the neuroendocrine output signal coding for circadian night.

Objective: The purpose of this study was to determine the signaling pathway underlying the effects of MEL on contractility and the contractile machinery in immortalized human myometrial cells.

Design: To ascertain the signaling pathway of MEL leading to its effects on myometrial contractility in vitro, we performed gel retraction assays with cells exposed to iodo-MEL (I-MEL) with or without oxytocin and the Rho kinase inhibitor Y27632. I-MEL effects on inositol trisphosphate (IP3)/diacylglycerol (DAG)/protein kinase C (PKC) signaling were also investigated. Additionally, we assayed for caldesmon phosphorylation and ERK1/2 activation.

Results: I-MEL was found to activate PKCα via the phospholipase C/IP3/DAG signaling pathway, which was confirmed by PKC enzyme assay. I-MEL did not affect myosin light chain phosphatase activity, and its effects on contractility were insensitive to Rho kinase inhibition. I-MEL did increase phosphorylation of ERK1/2 and caldesmon, which was inhibited by the MAPK kinase inhibitor PD98059 or the PKC inhibitor C1.

Conclusions: MEL sensitizes myometrial cells to subsequent procontractile signals in vitro through activation of the phospholipase C/IP3/DAG signaling pathway, resulting in specific activation of PKCα and ERK1/2, thereby phosphorylating caldesmon, which increases actin availability for myosin binding and cross-bridging. In vivo, this sensitization would provide a mechanism for the increased nocturnal uterine contractility and labor that has been observed in late-term human pregnancy.
Novel Activating Mutations of the Calcium-Sensing Receptor: The Calcilytic NPS-2143 Mitigates Excessive Signal Transduction of Mutant Receptors
Authors: Letz Saskia , Rus Ramona , Haag Christine , Dörr Helmuth-Günther , Schnabel Dirk , Möhlig Matthias , Schulze Egbert , Frank-Raue Karin , Raue Friedhelm , Mayr Bernhard , Schöfl Christof
Published: January 2014

Context and Objective: Activating mutations in the calcium-sensing receptor (CaSR) gene cause autosomal dominant hypocalcemia (ADH). The aims of the present study were the functional characterization of novel mutations of the CaSR found in patients, the comparison of in vitro receptor function with clinical parameters, and the effect of the allosteric calcilytic NPS-2143 on the signaling of mutant receptors as a potential new treatment for ADH patients.

Methods: Wild-type and mutant CaSR (T151R, P221L, E767Q, G830S, and A844T) were expressed in human embryonic kidney cells (HEK 293T). Receptor signaling was studied by measuring intracellular free calcium in response to different concentrations of extracellular calcium ([Ca2+]o) in the presence or absence of NPS-2143.

Results: All ADH patients had lowered serum calcium ranging from 1.7 to 2.0 mm and inadequate immunoreactive PTH and urinary calcium excretion. In vitro testing of CaSR mutations from these patients revealed exaggerated [Ca2+]o-induced cytosolic Ca2+ responses with EC50 values for [Ca2+]o ranging from 1.56 to 3.15 mm, which was lower than for the wild-type receptor (4.27 mm). The calcilytic NPS-2143 diminished the responsiveness to [Ca2+]o in the CaSR mutants T151R, E767Q, G830S, and A844T. The mutant P221L, however, was only responsive when coexpressed with the wild-type CaSR.

Conclusion: Calcilytics might offer medical treatment for patients with autosomal dominant hypocalcemia caused by calcilytic-sensitive CaSR mutants.
Low Carbon Economy Policy in Poland: an Example of the Impact of Europeanisation
Authors: Grosse Tomasz Grzegorz
Published: January 2014

Poland has made a commitment to reduce the emission of greenhouse gases by ratifying the Kyoto Protocol and by participating in the climate policy of the European Union (EU). EUClimate and Energy Package (CEP), which was negotiated in 2008 and has been successively introduced into the EUlegal system. The CEP introduces much stricter require­ments for the reduction of greenhouse gases emissions and imposes higher costs on the adjust­ment of Polish energy sector and other sectors of Polish economy to the requirements of the EUlaw. The influence of the EUon the Member States during the course of European integra­tion is described in the literature of the subject as europeanisation. In this study Iwill analyse the influence on the policy of the Polish government with respect to stimulating the develop­ment of low carbon economy (including industry).