bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression Cloning of a Novel Estrogenic Mouse 17β-Hydroxysteroid Dehydrogenase/ 17-Ketosteroid Reductase (m17HSD7), Previously Described as a Prolactin Receptor-Associated Protein (PRAP) in Rat
Authors: Nokelainen Pasi , Peltoketo Hellevi , Vihko Reijo , Vihko Pirkko 
Published: January 2014

Abstract17β-Hydroxysteroid dehydrogenases/17-ketosteroid reductases (17HSDs) modulate the biological activity of certain estrogens and androgens by catalyzing reductase or dehydrogenase reactions between 17-keto- and 17β-hydroxysteroids. In the present study, we demonstrate expression cloning of a novel type of 17HSD, chronologically named 17HSD type 7, from the HC11 cell line derived from mouse mammary gland. The cloned cDNA, 1.7 kb in size, encodes a protein of 334 amino acids with a calculated molecular mass of 37,317 Da. The primary structure contains segments characteristic of enzymes belonging to the short-chain dehydrogenase/reductase superfamily. Strikingly, mouse 17HSD type 7 (m17HSD7) shows 89% identity with a recently cloned rat protein called PRL receptor-associated protein (PRAP). The function of PRAP has not yet been demonstrated.The enzymatic characteristics of m17HSD7 and RT-PCR-cloned rat PRAP (rPRAP) were analyzed in cultured HEK-293 cells, where both of the enzymes efficiently catalyzed conversion of estrone (E1) to estradiol (E2). With other substrates tested no detectable 17HSD or 20α-hydroxysteroid dehydrogenase activities were found. Kinetic parameters for m17HSD7 further indicate that E1 is a preferred substrate for this enzyme. Relative catalytic efficiencies (Vmax/Km values) for E1 and E2 are 244 and 48, respectively. As it is the case with rPRAP, m17HSD7 is most abundantly expressed in the ovaries of pregnant animals. Further studies show that the rat enzyme is primarily expressed in the middle and second half of pregnancy, in parallel with E2 secretion from the corpus luteum. The mRNA for m17HSD7 is also apparent in the placenta, and a slight signal for m17HSD7 is found in the ovaries of adult nonpregnant mice, in the mammary gland, liver, kidney, and testis.Altogether, because of their similar primary structures, enzymatic characteristics, and the tissue distribution of m17HSD7 and rPRAP, we suggest that rPRAP is rat 17HSD type 7. Furthermore, the results indicate that 17HSD7 is an enzyme of E2 biosynthesis, which is predominantly expressed in the corpus luteum of the pregnant animal.
Positive and Negative Discrimination of Estrogen Receptor Agonists and Antagonists Using Site-Specific DNA Recombinase Fusion Proteins
Authors: Logie Colin , Nichols Mark , Myles Kathy , Funder John W. , Stewart A. Francis 
Published: January 2014

AbstractActivation of the estrogen receptor (ER) by hormone involves at least two steps. First, hormone binding initially relieves repression, a property imposed on ER in cis by its ligand-binding domain (EBD). Subsequently, the derepressed ER binds specific genomic sites and regulates transcription. In addition to the natural hormone, ER binds a broad range of ligands that evoke a spectrum of responses ranging from full ER activation by agonists to partial activation and inhibition by partial or complete antagonists. How these different ligands evoke different ER responses remains unclear. To address this issue, we have developed a nontranscriptional assay for ER ligand responsiveness based on Flp recombinase/human EBD protein chimeras. These fusion proteins transduce the transient event of ligand binding into a permanent DNA change in a human cell line system. A fusion protein including ER D, E, and F domains was activated by all the ER ligands tested, demonstrating that both agonists and antagonists serve to relieve initial repression, and that differences between them lie downstream in the activation pathway. Mutant variants of the Flp-ER protein that distinguish between agonists and antagonists, and a mutant EBD that selectively lost the ability to respond to 17β-estradiol but not to other ligands, were also identified. Thus, agonists and antagonists can be functionally distinguished in a nontranscriptional assay.
Dual Signaling of Human Mel1a Melatonin Receptors via Gi2, Gi3, and Gq/11 Proteins
Authors: Brydon Lena , Roka Florian , Petit Laurence , de Coppet Pierre , Tissot Michèle , Barrett Perry , Morgan Peter J. , Nanoff Christian , Strosberg A. Donny , Jockers Ralf 
Published: January 2014

AbstractMel 1a melatonin receptors belong to the superfamily of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. So far, interest in Mel 1a receptor signaling has focused mainly on the modulation of the adenylyl cyclase pathway via pertussis toxin (PTX)-sensitive G proteins. To further investigate signaling of the human Mel 1a receptor, we have developed an antibody directed against the C terminus of this receptor. This antibody detected the Mel 1a receptor as a protein with an apparent molecular mass of approximately 60 kDa in immunoblots after separation by SDS-PAGE. It also specifically precipitated the 2-[125I]iodomelatonin (125I-Mel)-labeled receptor from Mel 1a-transfected HEK 293 cells. Coprecipitation experiments showed that Gi2, Gi3, and Gq/11 proteins couple to the Mel 1a receptor in an agonist-dependent and guanine nucleotide-sensitive manner. Coupling was selective since other G proteins present in HEK 293 cells, (Gi1, Go, Gs, Gz, and G12) were not detected in receptor complexes. Coupling of the Mel 1a receptor to Gi and Gq was confirmed by inhibition of high-affinity 125I-Mel binding to receptors with subtype-selective G protein α-subunit antibodies. Gi2 and/or Gi3 mediated adenylyl cyclase inhibition while Gq/11 induced a transient elevation in cytosolic calcium concentrations in HEK 293 cells stably expressing Mel 1a receptors. Melatonin-induced cytosolic calcium mobilization via PTX-insensitive G proteins was confirmed in primary cultures of ovine pars tuberalis cells endogenously expressing Mel 1a receptors. In conclusion, we report the development of the first antibody recognizing the cloned human Mel 1a melatonin receptor protein. We show that Mel 1a receptors functionally couple to both PTX-sensitive and PTX-insensitive G proteins. The previously unknown signaling of Mel 1a receptors through Gq/11 widens the spectrum of potential targets for melatonin.
Estrogen Receptor β Activates the Human Retinoic Acid Receptorα -1 Promoter in Response to Tamoxifen and Other Estrogen Receptor Antagonists, but Not in Response to Estrogen
Authors: Zou Aihua , Marschke Keith B. , Arnold Katharine E. , Berger Elaine M. , Fitzgerald Patrick , Mais Dale E. , Allegretto Elizabeth A. 
Published: January 2014

AbstractHuman estrogen receptor-α (hERα) or -β (hERβ) transfected into Hep G2 or COS1 cells each responded to estrogen to increase transcription from an estrogen-responsive element (ERE)-driven reporter vector with similar fold induction through a classical mechanism involving direct receptor binding to DNA. ER antagonists inhibited this estrogen induction through both hERα and hERβ, although raloxifene was more potent through ERα than ERβ, and tamoxifen was more potent via ERβ than ERα. We have shown previously that estrogen stimulated the human retinoic acid receptor-α-1 (hRARα-1) promoter through nonclassical EREs by a mechanism that was ERα dependent, but that did not involve direct receptor binding to DNA. We show here that in contrast to hERα, hERβ did not induce reporter activity driven by the hRARα-1 promoter in the presence of estrogen. While hERβ did not confer estrogen responsiveness on this promoter, it did elicit transcriptional activation in the presence of 4-hydroxytamoxifen (4-OH-Tam). Additionally, this 4-OH-Tam agonist activity via ERβ was completely blocked by estrogen. Like ERα, transcriptional activation of this promoter by ERβ was not mediated by direct receptor binding to DNA. While hERα was shown to act through two estrogen-responsive sequences within the promoter, hERβ acted only at the 3′-region, through two Sp1 sites, in response to 4-OH-Tam. Other ER antagonists including raloxifene, ICI-164,384 and ICI-182,780 also acted as agonists through ERβ via the hRARα-1 promoter. Through the use of mutant and chimeric receptors, it was shown that the 4-OH-Tam activity via ERβ from the hRARα-1 promoter in Hep G2 cells required the amino-terminal region of ERβ, a region that was not necessary for estrogen-induced ERβ activity from an ERE in Hep G2 cells. Additionally, the progesterone receptor (PR) antagonist RU486 acted as a weak (IC50 &gt;1 μm) antagonist via hERα and as a fairly potent (IC50 ∼200 nm) antagonist via hERβ from an ERE-driven reporter in cells that do not express PR. Although RU486 bound only weakly to ERα or ERβ in vitro, it did bind to ERβ in whole-cell binding assays, and therefore, it is likely metabolized to an ERβ-interacting compound in the cell. Interestingly, RU486 acted as an agonist through ERβ to stimulate the hRARα-1 promoter in Hep G2 cells. These findings may have ramifications in breast cancer treatment regimens utilizing tamoxifen or other ER antagonists and may explain some of the known estrogenic or antiestrogenic biological actions of RU486.
Early Growth Response Protein 1 Binds to the Luteinizing Hormone-β Promoter and Mediates Gonadotropin-Releasing Hormone-Stimulated Gene Expression
Authors: Wolfe Michael W. , Call Gerald B. 
Published: January 2014

AbstractThe hypothalamic neuropeptide, GnRH, regulates the synthesis and secretion of LH from pituitary gonadotropes. Furthermore, it has been shown that the LH β-subunit gene is regulated by the transcription factors steroidogenic factor-1 (SF-1) and early growth response protein 1 (Egr1) in vitro and in vivo. The present study investigated the roles played by Egr1 and SF-1 in regulating activity of the equine LHβ-subunit promoter in the gonadotrope cell line, αT3–1, and the importance of these factors and cis-acting elements in regulation of the promoter by GnRH. All four members of the Egr family were found to induce activity of the equine promoter. The region responsible for induction by Egr was localized to the proximal 185 bp of the promoter, which contained two Egr response elements. Coexpression of Egr1 and SF-1 led to a synergistic activation of the equine (e)LHβ promoter. Mutation of any of the Egr or SF-1 response elements attenuated this synergism. Endogenous expression of Egr1 in αT3–1 cells was not detectable under basal conditions, but was rapidly induced after GnRH stimulation. Reexamination of the promoter constructs harboring mutant Egr or SF-1 sites indicated that these sites were required for GnRH induction. In fact, mutation of both Egr sites within the eLHβ promoter completely attenuated its induction by GnRH. Thus, GnRH induces expression of Egr1, which subsequently activates the eLHβ promoter. Finally, GnRH not only induced expression of Egr1, but also its corepressor, NGFI-A (Egr1) binding protein (Nab1), which can repress Egr1- induced transcription of the eLHβ promoter.
Adenovirus-Directed Expression of a Nonphosphorylatable Mutant of CREB (cAMP Response Element-Binding Protein) Adversely Affects the Survival, but Not the Differentiation, of Rat Granulosa Cells
Authors: Somers Jeremy P. , DeLoia Julie A. , Zeleznik Anthony J. 
Published: January 2014

Abstract
Although usually considered to be a constitutively expressed protein, in the primate ovary the expression of CREB (cAMP response element-binding protein) is extinguished after ovulation, and its loss is temporally associated with the cessation of proliferation of luteal cells and the ultimate commitment of the corpus luteum to undergo regression. To determine the cellular consequences of the loss of CREB expression, we expressed a nonphosphorylatable mutant of CREB (CREB M1) in primary cultures of rat granulosa cells using a replication-defective adenovirus vector. Expression of CREB M1 did not block granulosa cell differentiation as assessed by acquisition of the ability to produce estrogen and progesterone in response to FSH or forskolin. However, granulosa cells expressing CREB M1, but not adenovirus-directed β-galactosidase or enhanced green fluorescent protein, exhibited a 35% reduction in viability that was further reduced to 65% after stimulation with 10 μm forskolin. These results demonstrate that the trophic effects of cAMP (proliferation and survival) on ovarian granulosa cells are functionally separate from the effects of cAMP on differentiation and provide novel evidence that CREB may function as a cell survival factor in the ovary. The separation of signaling pathways that govern differentiation and survival in the ovary thereby provides a mechanism by which progesterone production, which is absolutely essential for the maintenance of pregnancy, can continue despite the cessation of proliferation of luteal cells and their commitment to cell death (luteolysis).
Transcription Factor GATA-4 Enhances Müllerian Inhibiting Substance Gene Transcription through a Direct Interaction with the Nuclear Receptor SF-1
Authors: Tremblay Jacques J. , Viger Robert S. 
Published: January 2014

AbstractSecretion of Müllerian-inhibiting substance (MIS) by Sertoli cells of the fetal testis and subsequent regression of the Müllerian ducts in the male embryo is a crucial event that contributes to proper sex differentiation. The zinc finger transcription factor GATA-4 and nuclear receptor SF-1 are early markers of Sertoli cells that have been shown to regulate MIS transcription. The fact that the GATA and SF-1 binding sites are adjacent to one another in the MIS promoter raised the possibility that both factors might transcriptionally cooperate to regulate MIS expression. Indeed, coexpression of both factors resulted in a strong synergistic activation of the MIS promoter. GATA-4/SF-1 synergism was the result of a direct protein-protein interaction mediated through the zinc finger region of GATA-4. Remarkably, synergy between GATA-4 and SF-1 on a variety of different SF-1 targets did not absolutely require GATA binding to DNA. Moreover, synergy with SF-1 was also observed with other GATA family members. Thus, these data not only provide a clearer understanding of the molecular mechanisms that control the sex-specific expression of the MIS gene but also reveal a potentially novel mechanism for the regulation of SF-1-dependent genes in tissues where SF-1 and GATA factors are coexpressed.
Alteration of a Single Amino Acid in Peroxisome Proliferator-Activated Receptor-α (PPARα) Generates a PPARδ Phenotype
Authors: Takada Ichiro , Yu Ruth T. , Xu H. Eric , Lambert Millard H. , Montana Valerie G. , Kliewer Steven A. , Evans Ronald M. , Umesono Kazuhiko 
Published: January 2014

Abstract
Three pharmacologically important nuclear receptors, the peroxisome proliferator-activated receptors (PPARs α,γ , and δ), mediate key transcriptional responses involved in lipid homeostasis. The PPARα and γ subtypes are well conserved from Xenopus to man, but the β/δ subtypes display substantial species variations in both structure and ligand activation profiles. Characterization of the avian cognates revealed a close relationship between chick (c) α and γ subtypes to their mammalian counterparts, whereas the third chicken subtype was intermediate to Xenopus (x) β and mammalian δ, establishing that β and δ are orthologs. Like xPPARβ, cPPARβ responded efficiently to hypolipidemic compounds that fail to activate the human counterpart. This provided the opportunity to address the pharmacological problem as to how drug selectivity is achieved and the more global evolutionary question as to the minimal changes needed to generate a new class of receptor. X-ray crystallography and chimeric analyses combined with site-directed mutagenesis of avian and mammalian cognates revealed that a Met to Val change at residue 417 was sufficient to switch the human and chick phenotype. These results establish that the genetic drive to evolve a novel and functionally selectable receptor can be modulated by a single amino acid change and suggest how nuclear receptors can accommodate natural variation in species physiology.
Human Androgen Receptor Mutation Disrupts Ternary Interactions between Ligand, Receptor Domains, and the Coactivator TIF2 (Transcription Intermediary Factor 2)
Authors: Lim Joyce , Ghadessy Farid J. , Abdullah Abdullah A. R. , Pinsky Leonard , Trifiro Mark , Yong E. L. 
Published: January 2014

AbstractThe androgen receptor (AR) is a ligand-dependent X-linked nuclear transcription factor regulating male sexual development and spermatogenesis. The receptor is activated when androgen binds to the C-terminal ligand-binding domain (LBD), triggering a cascade of molecular events, including interactions between the LBD and the N-terminal transactivation domain (TAD), and the recruitment of transcriptional coactivators. A nonconservative asparagine to lysine substitution in AR residue 727 was encountered in a phenotypically normal man with subfertility and depressed spermatogenesis. This N727K mutation, although located in the LBD, did not alter any ligand-binding characteristic of the AR in the patient’s fibroblasts or when expressed in heterologous cells. Nonetheless, the mutant AR displayed only half of wild-type transactivation capacity when exposed to physiological or synthetic androgens. This transactivation defect was consistently present when examined with two different reporter systems in three cell lines, using three androgen-driven promoters (including the complex human prostate-specific antigen promoter), confirming the pathogenicity of the mutation. In mammalian two-hybrid assays, N727K disrupted LBD interactions with the AR TAD and with the coactivator, transcription intermediary factor 2 (TIF2). Strikingly, the transactivation defect of the mutant AR can be rectified in vitro with mesterolone, consistent with the ability of this androgen analog to restore sperm production in vivo. Mesterolone, but not the physiological androgen dihydrotestosterone, restored mutant LBD interactions with the TAD and with TIF2, when expressed as fusion proteins in the two-hybrid assay. Our data support an emerging paradigm with respect to AR mutations in the LBD and male infertility: pathogenicity is transmitted through reduced interdomain and coactivator interactions, and androgen analogs that are corrective in vitro may indicate hormonal therapy.
A Novel Cyclic Adenosine 3′,5′-Monophosphate-Responsive Element Involved In the Transcriptional Regulation of the Lutropin Receptor Gene in Granulosa Cells
Authors: Chen Shiyou , Liu Xuebo , Segaloff Deborah L. 
Published: January 2014

AbstractThe induction of the lutropin receptor (LHR) in granulosa cells by FSH is mediated, at least in part, by cAMP. However, the classic cAMP-responsive element (CRE) is not present in the 5′-flanking region of the rat LHR gene. Previous studies from our laboratory had shown that three Sp1 sites within the promoter region of the rat LHR (rLHR) bind Sp1 and Sp3 and are involved in the basal and cAMP-mediated transcription of the rLHR gene. In the present studies we show that the rLHR promoter region forms a complex (designated complex A) with nuclear extracts from rat granulosa cells, and the abundance of complex A is markedly increased when using cells that had been pretreated with 8-bromo (Br)-cAMP. We have localized the binding of the protein(s) in complex A to a DNA sequence immediately upstream and partially overlapping with the Sp1c binding site. The core site (designated SAS for Sp1c adjacent sequence) is localized to nucleotide (nt) −146 to −142 and contains the sequence GGGGG. The consensus sequence for the core portion of this element appears to be (G/T)GGGG. Mutations of the SAS site, but not SP1c site, abolish complex A formation. Experiments utilizing rat granulosa cells transfected with luciferase reporter genes driven by the 5′-flanking region of the rLHR gene demonstrate a functional role for the SAS site in the cAMP responsiveness of the rLHR gene.
New Androgen Response Elements in the Murine Pem Promoter Mediate Selective Transactivation
Authors: Barbulescu Karina , Geserick Christoph , Schüttke Iris , Schleuning Wolf-Dieter , Haendler Bernard 
Published: January 2014

AbstractThe Pem homeobox transcription factor is expressed under androgen control in the testis and epididymis. It is also transcribed in the ovary, muscle, and placenta. The mouse Pem gene promoter was cloned and sequenced. It was analyzed in transactivation tests using CV-1 and PC-3 cells expressing the AR and found to be strongly stimulated by androgens. EMSAs and mutational analysis of the Pem promoter allowed the identification of two functional androgen response elements named ARE-1 and ARE-2. They both differed from the consensus semipalindromic steroid response element and exhibited characteristics of direct repeats of the TGTTCT half-site. Unlike the steroid response element, both Pem androgen response elements were selectively responsive to androgen stimulation. Specific mutations in the left half-site of Pem ARE-1 and ARE-2, but not of the steroid response element, were still compatible with AR binding in the EMSA. In addition, Pem ARE-1, but not ARE-2 or the steroid response element, showed some flexibility with regard to spacing between half-sites. These results strongly suggest that the AR interacts differently with direct repeats than with inverted repeats, potentially leading to cis element-driven selective properties. Thus, the existence of several classes of DNA response elements might be an essential feature of differential androgen regulation.
Cooperative Effects of STAT5 (Signal Transducer and Activator of Transcription 5) and C/EBP β (CCAAT/Enhancer-Binding Protein-β) onβ -Casein Gene Transcription Are Mediated by the Glucocorticoid Receptor
Authors: Wyszomierski Shannon L. , Rosen Jeffrey M. 
Published: January 2014

Abstractβ-Casein gene transcription is controlled primarily by a composite response element (CoRE) that integrates signaling from the lactogenic hormones, PRL, insulin, and hydrocortisone, in mammary epithelial cells. This CoRE contains binding sites for STAT5 (signal transducer and activator of transcription 5) and C/EBPβ (CCAAT/enhancer-binding protein-β) and several half-sites for glucocorticoid receptor (GR). To examine how interactions among these three transcription factors might regulateβ -casein gene transcription, a COS cell reconstitution system was employed. Cooperative transactivation was observed when all three factors were expressed, but unexpectedly was not seen between STAT5 and C/EBPβ in the absence of full-length, transcriptionally active GR. Cooperativity required the amino-terminal transactivation domain of C/EBPβ, and neither C/EBPα nor C/EBPδ was able to substitute for C/EBPβ when cotransfected with STAT5 and GR. Different GR determinants were needed for transcriptional cooperation between STAT5 and GR as compared with those required for all three transcription factors. These studies provide some new insights into the mechanisms responsible for high level, tissue-specific expression conferred by theβ -casein CoRE.