bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Interaction between the Estrogen Receptor and CTF1: Analysis of the Vitellogenin Gene B1 Promoter in Yeast
Authors: Tsai-Pflugfelder Monika , Gasser Susan M. , Wahli Walter
Published: January 2014

Eukaryotic gene expression depends on a complex interplay between the transcriptional apparatus and chromatin structure. We report here a yeast model system for investigating the functional interaction between the human estrogen receptor (hER) and CTF1, a member of the CTF/NFI transcription factor family. We show that a CTF1-fusion protein and the hER transactivate a synthetic promoter in yeast in a synergistic manner. This interaction requires the proline-rich transactivation domain of CTF1. When the natural estrogen-dependent vitellogenin B1 promoter is tested in yeast, CTF1 and CTF1-fusion proteins are unable to activate transcription, and no synergy is observed between hER, which activates the B1 promoter, and these factors. Chromatin structure analysis on this promoter reveals positioned nucleosomes at −430 to −270 (±20 bp) and at −270 to− 100 (±20 bp) relative to the start site of transcription. The positions of the nucleosomes remain unchanged upon hormone-dependent transcriptional activation of the promoter, and the more proximal nucleosome appears to mask the CTF/NFI site located at −101 to −114. We conclude that a functional interaction of hER with the estrogen response element located upstream of a basal promoter occurs in yeast despite the nucleosomal organization of this promoter, whereas the interaction of CTF1 with its target site is apparently precluded by a nucleosome.
Functional Activities of the A and B Forms of the Human Androgen Receptor in Response to Androgen Receptor Agonists and Antagonists
Authors: Gao Tianshu , McPhaul Michael J.
Published: January 2014

The androgen receptor (AR) is present in many cells in two forms. The B form migrates with an apparent mass of 110 kDa and constitutes more than 80% of the immunoreactive receptor in most cell types. The A form of the AR migrates with an apparent mass of 87 kDa, appears to derive from internal translation initiation at methionine-188 in the AR open-reading frame, and usually constitutes 20% or less of the immunoreactive AR present. Previous experiments designed to examine the functional capacity of the A and B forms of the AR have been hampered by marked differences in the expression levels of the two isoforms, as the nucleotide sequence surrounding the codon encoding methionine-188 causes it to be used inefficiently as a translation initiation site. To circumvent this, we altered the nucleotide sequence surrounding methionine-188 to render it more similar to that surrounding the codon encoding methionine-1. Transfection of a cDNA containing these changes resulted in similar levels of expression of A and B forms of the AR as assessed by immunoblot assays using antibodies directed at an epitope preserved in both. Functional activities of these cDNAs were assessed using cotransfection assays that employed two model androgen-responsive genes (MMTV-luciferase and PRE2-tk-luciferase) in response to mibolerone, a potent androgen agonist, in three different cell lines. These studies demonstrated subtle differences in the activities of the A and B isoforms, which depended on the promoter and cell context. Additional studies failed to reveal any major differences in the responses of the AR-A and AR-B isoforms to a variety of androgen agonists and antagonists, suggesting that the previously reported functional defect of the AR-A is due principally to its level of expression. When assays of AR function are performed under conditions in which levels of expression of the two isoforms are equivalent, the AR-A and AR-B possess similar functional activities.
Structure, Expression, and Function of Human Pituitary Tumor-Transforming Gene (PTTG)
Authors: Zhang Xun , Horwitz Gregory A. , Prezant Toni R. , Valentini Alberto , Nakashima Masahiro , Bronstein Marcello D. , Melmed Shlomo
Published: January 2014

Despite advances in characterizing the pathophysiology and genetics of pituitary tumors, molecular mechanisms of their pathogenesis are poorly understood. Recently, we isolated a transforming gene [pituitary tumor-transforming gene (PTTG)] from rat pituitary tumor cells. Here we describe the cloning of human PTTG, which is located on chromosome 5q33 and shares striking sequence homology with its rat counterpart. Northern analysis revealed PTTG expression in normal adult testis, thymus, colon, small intestine, brain, lung, and fetal liver, but most abundant levels of PTTG mRNA were observed in several carcinoma cell lines. Stable transfection of NIH 3T3 cells with human PTTG cDNA caused anchorage-independent transformation in vitro and induced in vivo tumor formation when transfectants were injected into athymic mice. Overexpression of PTTG in transfected NIH 3T3 cells also stimulated expression and secretion of basic fibroblast growth factor, a human pituitary tumor growth-regulating factor. A proline-rich region, which contains two PXXP motifs for the SH3 domain-binding site, was detected in the PTTG protein sequence. When these proline residues were changed by site-directed mutagenesis, PTTG in vitro transforming and in vivo tumor-inducing activity, as well as stimulation of basic fibroblast growth factor, was abrogated. These results indicate that human PTTG, a novel oncogene, may function through SH3-mediated signal transduction pathways and activation of growth factor(s).
Role of the Third Intracellular Loop for the Activation of Gonadotropin Receptors
Authors: Schulz Angela , Schöneberg Torsten , Paschke Ralf , Schultz Günter , Gudermann Thomas
Published: January 2014

Hyperfunctional endocrine thyroid and testicular disorders can frequently be traced back to gain-of-function mutations in glycoprotein hormone receptor genes. Deletion mutations in the third intracellular (i3) loop of the TSH receptor have recently been identified as a cause of constitutive receptor activity. To examine whether the underlying mechanism of receptor activation applies to all glycoprotein hormone receptors, we created deletion mutations in the LH and FSH receptors. In analogy to the situation with the TSH receptor, a deletion of nine amino acids resulted in constitutive activity irrespective of the location of deletions within the i3 loop of the LH receptor. In contrast, only one (Δ563–566) of four different 4-amino acid deletion mutants displayed agonist-independent activity. Systematic examination of the structural requirements for this effect in the Δ563–566 mutant revealed that only deletions including D564 resulted in constitutive receptor activity. Replacement of D564 by G, K, and N led to agonist-independent cAMP formation while introduction of a negatively charged E silenced constitutive receptor activity, indicating that an anionic amino acid at this position may be required to maintain an inactive receptor conformation. Insertion of A residues up- and downstream of D564 did not perturb receptor quiescence, showing that a certain degree of spatial freedom of the negatively charged amino acid within the context of the i3 loop is well tolerated. In contrast to the results obtained with the LH receptor, deletion of the corresponding D567 from the i3 loop of the FSH receptor did not cause constitutive receptor activation, highlighting significant differences in the activation mechanism of gonadotropin receptors.
Ligand-Activated Retinoic Acid Receptor Inhibits AP-1 Transactivation by Disrupting c-Jun/c-Fos Dimerization
Authors: Zhou Xiao-Feng , Shen Xi-Qiang , Shemshedini Lirim
Published: January 2014

In the presence of retinoic acid (RA), the retinoid receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR), are able to up-regulate transcription directly by binding to RA-responsive elements on the promoters of responsive genes. Liganded RARs and RXRs are also capable of down-regulating transcription, but, by contrast, this is an indirect effect, mediated by the interaction of these nuclear receptors not with DNA but the transcription factor activating protein-1 (AP-1). AP-1 is a dimeric complex of the protooncoproteins c-Jun and c-Fos and directly regulates transcription of genes important for cellular growth. Previous in vitro results have suggested that RARs can block AP-1 DNA binding. Using a mammalian two-hybrid system, we report here that human RARα (hRARα) can disrupt in a RA-dependent manner the homo- and heterodimerization properties of c-Jun and c-Fos. This inhibition of dimerization is cell specific, occurring only in those cells that exhibit RA-induced repression of AP-1 transcriptional activity. Furthermore, this mechanism appears to be specific for the RARs, since another potent inhibitor of AP-1 activity, the glucocorticoid receptor, does not affect AP-1 dimerization. Our data argue for a novel mechanism by which RARs can repress AP-1 DNA binding, in which liganded RARs are able to interfere with c-Jun/c-Jun homodimerization and c-Jun/c-Fos heterodimerization and, in this way, may prevent the formation of AP-1 complexes capable of DNA binding.
Cell Membrane and Nuclear Estrogen Receptors (ERs) Originate from a Single Transcript: Studies of ERα and ERβ Expressed in Chinese Hamster Ovary Cells
Authors: Razandi Mahnaz , Pedram Ali , Greene Geoffrey L. , Levin Ellis R.
Published: January 2014

The existence of a putative membrane estrogen receptor (ER) has been supported by studies accomplished over the past 20 yr. However, the origin and functions of this receptor are not well defined. To study the membrane receptor, we transiently transfected cDNAs for ERα or ERβ into Chinese hamster ovary (CHO) cells. Transfection of ERα resulted in a single transcript by Northern blot, specific binding of labeled 17β-estradiol (E2), and expression of ER in both nuclear and membrane cell fractions. Competitive binding studies in both compartments revealed near identical dissociation constants (Kds) of 0.283 and 0.287 nm, respectively, but the membrane receptor number was only 3% as great as the nuclear receptor density. Transfection of ERβ also yielded a single transcript and nuclear and membrane receptors with respective Kd values of 1.23 and 1.14 nm; the membrane receptor number was only 2% compared with expressed nuclear receptors. Estradiol binding to CHO-ERα or CHO-ERβ activated Gαq and Gαs proteins in the membrane and rapidly stimulated corresponding inositol phosphate production and adenylate cyclase activity. Binding by 17-β-E2 to either expressed receptor comparably enhanced the nuclear incorporation of thymidine, critically dependent upon the activation of the mitogen-activated protein kinase, ERK (extracellular regulated kinase). In contrast, c-Jun N-terminal kinase activity was stimulated by 17-β-E2 in ERβ-expressing CHO, but was inhibited in CHO-ERα cells. In summary, membrane and nuclear ER can be derived from a single transcript and have near-identical affinities for 17-β-E2, but there are considerably more nuclear than membrane receptors. This is also the first report that cells can express a membrane ERβ. Both membrane ERs activate G proteins, ERK, and cell proliferation, but there is novel differential regulation of c-Jun kinase activity by ERβ and ERα.
Identification of a Placental-Specific Enhancer in the Rat Placental Lactogen II Gene That Contains Binding Sites for Members of the Ets and AP-1 (Activator Protein 1) Families of Transcription Factors
Authors: Sun Yuxiang , Duckworth Mary Lynn
Published: January 2014

We previously identified a 3-kb proximal 5′-flanking region of the rat placental lactogen (rPLII) gene1 that is important for reporter gene transcription in the rat trophoblast cell line, Rcho, and targets expression to the placentas of transgenic mice. In our current studies we have used further deletion analysis and transfection studies in Rcho and GC cells to map more precisely the locations of regulatory elements involved in this placental expression. We show that sequences between −1435 and− 765 are necessary for minimal expression in Rcho cells and that there are negative regulatory elements between −3031 to −2838 and −1729 to− 1435. Most importantly, we have identified a fragment between −1793 to −1729 that is essential for expression levels characteristic of the complete 3-kb 5′-region. When linked to the herpes simplex thymidine kinase minimal promoter, this fragment acts as an enhancing element in Rcho but not GC cells. Deoxyribonuclease I (DNAse I) protection and electrophoretic mobility shift assays with nuclear extracts and in vitro translated proteins identify binding sites for members of the activator protein-1 (AP-1) and Ets families of transcription factors. Site-directed mutagenesis of the individual AP-1- and Ets-binding sites leads to a partial loss of the enhancing activity; a double AP-1/Ets mutation leads to a complete loss of activity, demonstrating the functional importance of these sites. By these criteria, putative GATA-binding sites located within the enhancing fragment are not active. These new data suggest an important role for this enhancing fragment in rPLII placental giant cell expression and are the first to implicate a member of the Ets family in the regulation of this gene family.
Chromatin Recycling of Glucocorticoid Receptors: Implications for Multiple Roles of Heat Shock Protein 90
Authors: Liu Jimin , DeFranco Donald B.
Published: January 2014

Unliganded glucocorticoid receptors (GRs) released from chromatin after hormone withdrawal remain associated with the nucleus within a novel subnuclear compartment that serves as a nuclear export staging area. We set out to examine whether unliganded nuclear receptors cycle between distinct subnuclear compartments or require cytoplasmic transit to regain hormone and chromatin-binding capacity. Hormone-withdrawn rat GrH2 hepatoma cells were permeabilized with digitonin to deplete cytoplasmic factors, and then hormone-binding and chromatin-binding properties of the recycled nuclear GRs were measured. We found that recycled nuclear GRs do not require cytosolic factors or ATP to rebind hormone. Nuclear GRs that rebind hormone in permeabilized cells target to high-affinity chromatin-binding sites at 30 C, but not 0 C, in the presence of ATP. Since geldanamycin, a heat shock protein-90 (hsp90)-binding drug, inhibits hormone binding to recycled nuclear GRs, hsp90 may be required to reassemble the receptor into a form capable of productive interactions with hormone. Geldanamycin also inhibits GR release from chromatin during hormone withdrawal, suggesting that hsp90 chaperone function may play multiple roles to facilitate chromatin recycling of GR.
Transcriptional Activation of E2F1 Gene Expression by 17β-Estradiol in MCF-7 Cells Is Regulated by NF-Y-Sp1/Estrogen Receptor Interactions
Authors: Wang Weili , Dong Lian , Saville Brad , Safe Stephen
Published: January 2014

17β-Estradiol (E2) stimulated proliferation and DNA synthesis in MCF-7 human breast cancer cells, and this was accompanied by induction of E2F1 mRNA and protein levels. Analysis of the E2F1 gene promoter showed that the −146 to− 54 region was required for E2-responsiveness in transient transfection assays, and subsequent deletion/mutation analysis showed that a single upstream GC-rich and two downstream CCAAT-binding sites were required for transactivation by E2. Gel mobility shift assays with multiple oligonucleotides and protein antibodies (for supershifts) showed that the −146 to −54 region of the E2F1 gene promoter bound Sp1 and NF-Y proteins in MCF-7 cells. The estrogen receptor (ER) protein enhanced Sp1 interactions with upstream GC-rich sites, and interactions of ER, Sp1, and ER/Sp1 with downstream DNA bound-NF-Y was investigated by kinetic analysis for protein-DNA binding (on- and off-rates), coimmunoprecipitation, and pulldown assays using wild-type and truncated glutathione S-transferase (GST)-Sp1 chimeric proteins. The results showed that Sp1 protein enhanced the Bmax of NF-Y-DNA binding by more than 5-fold (on-rate); in addition, the Sp1-enhanced NF-Y-DNA complex was further stabilized by coincubation with ER and the rate of dissociation (t1/2) was decreased by approximately 50%. Sp1 antibodies immunoprecipitated [35S]NF-YA after coincubation with unlabeled Sp1 protein. Thus, transcriptional activation of E2F1 gene expression in MCF-7 cells by E2 is regulated by multiprotein ER/Sp1-NF-Y interactions at GC-rich and two CCAAT elements in the proximal region of the E2F1 gene promoter. This represents a unique trans-acting protein complex in which ligand-dependent transactivation by the ER is independent of direct ER interactions with promoter elements.
Stat5b Inhibits NFκB-Mediated Signaling
Authors: Luo Guoyang , Yu-Lee Li-yuan
Published: January 2014

Signal transducers and activators of transcription (Stat) are latent transcription factors that participate in cytokine signaling by regulating the expression of early response genes. Our previous studies showed that Stat5 functions not only as a transcriptional activator but also as a transcriptional inhibitor, depending on the target promoter. This report further investigates the mechanism of Stat5b-mediated inhibition and demonstrates that PRL-inducible Stat5b inhibits nuclear factorκB (NFκB) signaling to both the interferon regulatory factor-1 promoter and to the thymidine kinase promoter containing multimerized NFκB elements (NFκB-TK). Further, PRL-inducible Stat5b inhibits tumor necrosis factor-α signaling presumably by inhibiting endogenous NFκB. This Stat5b-mediated inhibitory effect on NFκB signaling is independent of Stat5b-DNA interactions but requires the carboxyl terminus of Stat5b as well as Stat5b nuclear translocation and/or accumulation, suggesting that Stat5b is competing for a nuclear factor(s) necessary for NFκB-mediated activation of target promoters. Increasing concentrations of the coactivator p300/CBP reverses Stat5b inhibition at both the interferon-regulatory factor-1 and NFκB-TK promoters, suggesting that Stat5b may be squelching limiting coactivators via protein-protein interactions as one mechanism of promoter inhibition. These results further substantiate our observation that Stat factors can function as transcriptional inhibitors. Our studies reveal cross-talk between the Stat5b and NFκB signal transduction pathways and suggest that Stat5b-mediated inhibition of target promoters occurs at the level of protein-protein interactions and involves competition for limiting coactivators.
Endocrine Disrupting Chemicals, Phthalic Acid and Nonylphenol, Activate Pregnane X Receptor-Mediated Transcription
Authors: Masuyama Hisashi , Hiramatsu Yuji , Kunitomi Mamoru , Kudo Takafumi , MacDonald Paul N.
Published: January 2014

Recently, Pregnane X receptor (PXR), a new member of the nuclear receptor superfamily, was shown to mediate the effects of several steroid hormones, such as progesterone, glucocorticoid, pregnenolone, and xenobiotics on cytochrome P450 3A genes (CYP3A) through the specific DNA sequence for CYP3A, suggesting that PXR may play a role in steroid hormone metabolism. In this paper, we demonstrated that phthalic acid and nonylphenol, endocrine-disrupting chemicals (EDCs), stimulated PXR-mediated transcription at concentrations comparable to those at which they activate estrogen receptor-mediated transcription using a transient reporter gene expression assay in COS-7 cells. However, bisphenol A, another EDC, had no effect on PXR-mediated transcription, although this chemical significantly enhanced ER-mediated transcription. In the yeast two-hybrid protein interaction assay, PXR interacted with two nuclear receptor coactivator proteins, steroid hormone receptor coactivator-1 and receptor interacting protein 140, in the presence of phthalic acid or nonylphenol. Thus, EDC-occupied PXR may regulate its specific gene expression through the receptor-coactivator interaction. In contrast, these EDCs had no effect on the interaction between PXR and suppressor for gal 1, a component of proteasome. Finally, the expression of CYP3A1 mRNA in the liver of rats exposed to phthalic acid or nonylphenol markedly increased compared with that in rats treated with estradiol, bisphenol A, or ethanol as assessed by competitive RT-PCR. These data suggest that EDCs may affect endocrine functions by altering steroid hormone metabolism through PXR.
Molecular Cloning, Expression, and Characterization of Rat Homolog of Human AP-2α That Stimulates Neuropeptide Y Transcription Activity in Response to Nerve Growth Factor
Authors: Li Bing-Sheng , Kramer Phillip R. , Zhao Weiqin , Ma Wu , Stenger David A. , Zhang Lei
Published: January 2014

Neuropeptide Y (NPY) plays an important role in the central regulation of neuronal activity, endocrine and sexual behavior, and food intake. Although transcription activity of the NPY gene in PC12 cells is regulated by a number of agents such as nerve growth factor (NGF), the mechanism responsible for the NGF-elicited increase in the transcription of the NPY gene remains to be explored. In this study, we isolated and characterized a nuclear protein that is bound to NGF-response elements (NGFRE) that lie between nucleotide −87 and −33 of the rat NPY promoter gene. This nuclear protein is identical to the rat homolog of human transcription factor AP-2α. We further demonstrated that rat AP-2α promotes efficient NPY transcription activity in response to NGF. Finally, we provide direct evidence that the mice lacking transcription factor AP-2α exhibit reduced expression of NPY mRNA compared with wild-type mice, further supporting the hypothesis that AP-2α is an important transcription factor in regulating NPY transcription activity.
The C-Terminal Tail of the Rat Lutropin/Choriogonadotropin (CG) Receptor Independently Modulates Human (h)CG-Induced Internalization of the Cell Surface Receptor and the Lysosomal Targeting of the Internalized hCG-Receptor Complex
Authors: Kishi Mikiko , Ascoli Mario
Published: January 2014

The analysis of 21 progressive truncations of the C-terminal tail of the rat LH/CG receptor (rLHR) revealed the presence of a region delineated by residues 628–649 that, when removed, enhanced the degradation of the internalized human (h)CG. The analysis of these truncations also revealed the presence of a region delineated by residues 624–631 that, when removed, enhanced the rate of internalization of hCG. Since there is little overlap between these two regions, we conclude that the structural features of the rLHR that mediate internalization and degradation of the internalized hormone are different. Detailed analyses of cells expressing a truncation at Y637 (designated rLHR-t637) showed that the enhanced degradation of hCG observed in the these cells is due to an increase in the rate of transfer of the internalized hCG-rLHR complex from the endosomes to the lysosomes rather than to the enhanced dissociation of the hCG-rLHR complex in the lysosomes.
Identification of an Ovarian Voltage-Activated Na+-Channel Type: Hints to Involvement in Luteolysis
Authors: Bulling Andreas , Berg Frank D. , Berg Ulrike , Duffy Diane M. , Stouffer Richard L. , Ojeda Sergio R. , Gratzl Manfred , Mayerhofer Artur
Published: January 2014

An endocrine type of voltage-activated sodium channel (eNaCh) was identified in the human ovary and human luteinized granulosa cells (GC). Whole-cell patch-clamp studies showed that the eNaCh in GC is functional and tetrodotoxin (TTX) sensitive. The luteotrophic hormone human CG (hCG) was found to decrease the peak amplitude of the sodium current within seconds. Treatment with hCG for 24–48 h suppressed not only eNaCh mRNA levels, but also mean Na+ peak currents and resting membrane potentials. An unexpected role for eNaChs in regulating cell morphology and function was indicated after pharmacological modulation of presumed eNaCh steady-state activity in GC cultures for 24–48 h using TTX (NaCh blocker) and veratridine (NaCh activator). TTX preserved a highly differentiated cellular phenotype. Veratridine not only increased the number of secondary lysosomes but also led to a significantly reduced progesterone production. Importantly, endocrine cells of the nonhuman primate corpus luteum (CL), which represent in vivo counterparts of luteinized GC, also contain eNaCh mRNA. Although the mechanism of channel activity under physiological conditions is not clear, it may include persistent Na+ currents. As observed in GC in culture, abundant secondary lysosomes were particularly evident in the regressing CL, suggesting a functional link between eNaCh activity and this form of cellular regression in vivo. Our results identify eNaCh in ovarian endocrine cells and demonstrate that their expression is under the inhibitory control of hCG. Activation of eNaChs in luteal cells, due to loss of gonadotropin support, may initiate a cascade of events leading to decreased CL function, a process that involves lysosomal activation and autophagy. These results imply that ovarian eNaChs are involved in the physiological demise of the temporary endocrine organ CL in the primate ovary during the menstrual cycle. Because commonly used drugs, including phenytoin, target NaChs, these results may be of clinical relevance.
Characterization of Transiently and Constitutively Expressed Progesterone Receptors: Evidence for Two Functional States
Authors: Smith Catharine L. , Wolford Ronald G. , O’Neill Tara B. , Hager Gordon L.
Published: January 2014

Activated steroid receptors induce chromatin remodeling events in the promoters of some target genes. We previously reported that transiently expressed progesterone receptor (PR) cannot activate mouse mammary tumor virus (MMTV) promoter when it adopts the form of ordered chromatin. However, when expressed continuously, the PR acquires this ability. In this study we explored whether this gain of function occurs through alterations in nucleoprotein structure at the MMTV promoter or through changes in receptor status. We observed no major structural differences at the MMTV promoter in the presence of constitutively expressed PR and found its mechanism of activation to be very similar to that of the glucocorticoid receptor (GR). However, a systematic comparison of the functional behavior of the transiently and constitutively expressed PR elucidated significant differences. The transiently expressed PR is activated in the absence of ligand by cAMP and by components in FBS and has significantly increased sensitivity to progestins. In contrast, the constitutively expressed PR is refractory to activation by cAMP and serum and has normal sensitivity to its ligand. In addition, while the PR is localized to the nucleus in both cases, a significant fraction of the transiently expressed PR is tightly bound to the nucleus even in the absence of ligand, while the majority of constitutively expressed PR is not. These results strongly suggest that the PR undergoes processing in the cell subsequent to its initial expression and that this processing is important for various aspects of its function, including its ability to productively interact with target genes that require chromatin remodeling for activation.
Properties of the Glucocorticoid Modulatory Element Binding Proteins GMEB-1 and -2: Potential New Modifiers of Glucocorticoid Receptor Transactivation and Members of the Family of KDWK Proteins
Authors: Kaul Sunil , Blackford John A. , Chen Jun , Ogryzko Vasily V. , Simons S. Stoney
Published: January 2014

An important component of glucocorticoid steroid induction of tyrosine aminotransferase (TAT) gene expression is the glucocorticoid modulatory element (GME), which is located at −3.6 kb of the rat TAT gene. The GME both mediates a greater sensitivity to hormone, due to a left shift in the dose-response curve of agonists, and increases the partial agonist activity of antiglucocorticoids. These properties of the GME are intimately related to the binding of a heteromeric complex of two proteins (GMEB-1 and -2). We previously cloned the rat GMEB-2 as a 67-kDa protein. We now report the cloning of the other member of the GME binding complex, the 88-kDa human GMEB-1, and various properties of both proteins. GMEB-1 and -2 each possess an intrinsic transactivation activity in mammalian one-hybrid assays, consistent with our proposed model in which they modify glucocorticoid receptor (GR)-regulated gene induction. This hypothesis is supported by interactions between GR and both GMEB-1 and -2 in mammalian two-hybrid and in pull-down assays. Furthermore, overexpression of GMEB-1 and -2, either alone or in combination, results in a reversible right shift in the dose-response curve, and decreased agonist activity of antisteroids, as expected from the squelching of other limiting factors. Additional mechanistic details that are compatible with the model of GME action are suggested by the interactions in a two-hybrid assay of both GMEBs with CREB-binding protein (CBP) and the absence of histone acetyl transferase (HAT) activity in both proteins. GMEB-1 and -2 share a sequence of 90 amino acids that is 80% identical. This region also displays homology to several other proteins containing a core sequence of KDWK. Thus, the GMEBs may be members of a new family of factors with interesting transcriptional properties.
Dysregulation of EGF Family of Growth Factors and COX-2 in the Uterus during the Preattachment and Attachment Reactions of the Blastocyst with the Luminal Epithelium Correlates with Implantation Failure in LIF- Deficient Mice
Authors: Song Haengseok , Lim Hyunjung , Das Sanjoy K. , Paria Bibhash C. , Dey Sudhansu K.
Published: January 2014

Various mediators, including cytokines, growth factors, homeotic gene products, and prostaglandins (PGs), participate in the implantation process in an autocrine, paracrine, or juxtacrine manner. However, interactions among these factors that result in successful implantation are not clearly understood. Leukemia inhibitory factor (LIF), a pleiotropic cytokine, was shown to be expressed in uterine glands on day 4 morning before implantation and is critical to this process in mice. However, the mechanism by which LIF executes its effects in implantation remains unknown. Moreover, interactions of LIF with other implantation-specific molecules have not yet been defined. Using normal and delayed implantation models, we herein show that LIF is not only expressed in progesterone (P4)-primed uterine glands before implantation in response to nidatory estrogen, it is also induced in stromal cells surrounding the active blastocyst at the time of the attachment reaction. This suggests that LIF has biphasic effects: first in the preparation of the receptive uterus and subsequently in the attachment reaction. The mechanism by which LIF participates in these events was addressed using LIF-deficient mice. We observed that while uterine cell-specific proliferation, steroid hormone responsiveness, and expression patterns of several genes are normal, specific members of the EGF family of growth factors, such as amphiregulin (Ar), heparin-binding EGF-like growth factor (HB-EGF), and epiregulin, are not expressed in LIF(−/−) uteri before and during the anticipated time of implantation, although EGF receptor family members (erbBs) are expressed correctly. Furthermore, cyclooxygenase-2 (COX-2), an inducible rate-limiting enzyme for PG synthesis and essential for implantation, is aberrantly expressed in the uterus surrounding the blastocyst in LIF(−/−) mice. These results suggest that dysregulation of specific EGF-like growth factors and COX-2 in the uterus contributes, at least partially, to implantation failure in LIF(−/−) mice. Since estrogen is essential for uterine receptivity, LIF induction, and blastocyst activation, it is possible that the nidatory estrogen effects in the P4-primed uterus for implantation are mediated via LIF signaling. However, we observed that LIF can only partially resume implantation in P4-primed, delayed implanting mice in the absence of estrogen, suggesting LIF induction is one of many functions that are executed by estrogen for implantation.
Targeted Disruption of the Pituitary Adenylate Cyclase-Activating Polypeptide Gene Results in Early Postnatal Death Associated with Dysfunction of Lipid and Carbohydrate Metabolism
Authors: Gray Sarah L. , Cummings Kevin J. , Jirik Frank R. , Sherwood Nancy M.
Published: January 2014

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a hormone belonging to the glucagon superfamily of hormones. These hormones are known to play important roles in metabolism and growth. PACAP is a neuropeptide that causes accumulation of cAMP in a number of tissues and affects the secretion of other hormones, vasodilation, neural and immune functions, as well as the cell cycle. To determine whether PACAP is essential for survival and to evaluate its function(s), we have generated mice lacking the PACAP gene via homologous recombination. We found that most PACAP null mice died in the second postnatal week in a wasted state with microvesicular fat accumulation in liver, skeletal muscle, and heart. Gas chromatography-mass spectrometry showed that fatty acid β-oxidation in liver mitochondria of PACAP−/− mice was not blocked based on the distribution of 3-hydroxy-fatty acids (C6–16) in the plasma. Instead, increased metabolic flux through the β-oxidation pathway was suggested by the presence of ketosis. Also, serum triglycerides and cholesterol were significantly higher (2- to 3-fold) in PACAP null mice than littermates. In the fed state, both serum insulin and blood glucose were normal in 5-d-old null mice compared with their littermates. In contrast, fasted PACAP null pups had a significant increase in insulin, but a decrease in blood glucose compared with littermates. Glycogen in the liver was reduced. These results suggest PACAP is a critical hormonal regulator of lipid and carbohydrate metabolism.
Stromal PRs Mediate Induction of 17β-Hydroxysteroid Dehydrogenase Type 2 Expression in Human Endometrial Epithelium: A Paracrine Mechanism for Inactivation Of E2
Authors: Yang Sijun , Fang Zongjuan , Gurates Bilgin , Tamura Mitsutoshi , Miller Josephine , Ferrer Karen , Bulun Serdar E.
Published: January 2014

Progesterone stimulates the expression of 17β-hydroxysteroid dehydrogenase (HSD) type 2, which catalyzes the conversion of the potent estrogen, E2, to an inactive form, estrone, in epithelial cells of human endometrial tissue. Various effects of progesterone on uterine epithelium have recently been shown to be mediated by stromal PRs in mice. We describe herein a critical paracrine mechanism whereby progesterone induction of 17β-HSD type 2 enzyme activity, transcript levels, and promoter activity in human endometrial epithelial cells are mediated primarily by PR in endometrial stromal cells. Medium conditioned with progestin-pretreated human endometrial stromal cells robustly increased 17β-HSD type 2 enzyme activity (2-fold) and mRNA levels (13.2-fold) in Ishikawa malignant endometrial epithelial cells. In contrast, direct progestin treatment of Ishikawa epithelial cells gave rise to much smaller increases in enzyme activity (1.2-fold) and mRNA levels (4-fold). These results suggest that progesterone- dependent paracrine factors arising from stromal cells are primarily responsible for the induction of epithelial 17β-HSD type 2 expression in the endometrium. We transfected serial deletion mutants of the −1,244 bp 5′-flanking region of the 17β-HSD type 2 gene into Ishikawa cells. No progesterone response elements could be identified upstream of the 17β-HSD type 2 promoter. Stromal PR-dependent induction of the 17β-HSD type 2 promoter was mediated by a critical regulatory region mapped to the −200/−100 bp sequence. Direct treatment of Ishikawa cells with progestin gave rise to a maximal increase in the activity of −200 bp/Luciferase construct only by 1.2-fold, whereas medium conditioned by progestin-pretreated endometrial stromal cells increased promoter activity up to 2.4-fold in a time- and concentration-dependent manner. The stimulatory effect of medium conditioned by progestin-pretreated stromal cells was enhanced strikingly by increasing stromal cell PR levels with the addition of estrogen. This epithelial-stromal interaction was specific for endometrial epithelial cells, since 17β-HSD type 2 could not be induced in malignant breast epithelial cells by media conditioned with progestin-treated breast or endometrial stromal cells. In conclusion, progesterone regulates the conversion of biologically active E2 to estrone by inducing the 17β-HSD type 2 enzyme in human endometrial epithelium primarily via PR in stromal cells, which secrete factors that induce transcription mediated primarily by the −200/−100 bp 5′-regulatory region of the 17β-HSD type 2 promoter.
Ligands Specify Coactivator Nuclear Receptor (NR) Box Affinity for Estrogen Receptor Subtypes
Authors: Bramlett Kelli S. , Wu Yifei , Burris Thomas P.
Published: January 2014

Nuclear receptors (NRs) require coactivators to efficiently activate transcription of their target genes. Many coactivators including the p160 proteins utilize a short NR box motif to recognize the ligand-binding domain of the NR when it is activated by ligand. To investigate the ability of various ligands to specify the affinity of NR boxes for a ligand-bound NR, we compared the capacity of p160 NR boxes to be recruited to estrogen receptor (ERα) and ERβ in the presence of 17β-estradiol, diethylstilbestrol, and genestein. A time-resolved fluorescence-based binding assay was used to determine the dissociation constants for the 10 NR boxes derived from the three p160 coactivators for both ER subtypes in the presence of the each of the agonists. While the affinity of some NR boxes for ER was independent of the agonist, we identified several NR boxes that had significantly different affinities for ER depending on which agonist was bound to the receptor. Therefore, an agonist may specify the affinity of an NR for various NR boxes and thus regulate the coactivator selectivity of the receptor.
Mammary Gland Involution Is Delayed by Activated Akt in Transgenic Mice
Authors: Schwertfeger Kathryn L. , Richert Monica M. , Anderson Steven M.
Published: January 2014

Activation of the antiapoptotic protein kinase Akt is induced by a number of growth factors that regulate mammary gland development. Akt is expressed during mammary gland development, and expression decreases at the onset of involution. To address Akt actions in mammary gland development, transgenic mice were generated expressing constitutively active Akt in the mammary gland under the control of the mouse mammary tumor virus (MMTV) promoter. Analysis of mammary glands from these mice reveals a delay in both involution and the onset of apoptosis. Expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), an inhibitor of matrix metalloproteinases (MMPs), is prolonged and increased in the transgenic mice, suggesting that disruption of the MMP:TIMP ratio may contribute to the delayed mammary gland involution observed in the transgenic mice.
Dynamic Changes in Subcellular Localization of Mineralocorticoid Receptor in Living Cells: In Comparison with Glucocorticoid Receptor using Dual-Color Labeling with Green Fluorescent Protein Spectral Variants
Authors: Nishi Mayumi , Ogawa Hiroshi , Ito Takao , Matsuda Ken-Ichi , Kawata Mitsuhiro
Published: January 2014

Mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) are ligand-dependent transcription factors. Although it is generally accepted that GR is translocated into the nucleus from the cytoplasm only after ligand binding, the subcellular localization of MR is still quite controversial. We examined the intracellular trafficking of MR in living neurons and nonneural cells using a fusion protein of green fluorescent protein (GFP) and rat MR (GFP-MR). Corticosterone (CORT) induced a rapid nuclear accumulation of GFP-MR, whereas in the absence of ligand, GFP-MR was distributed in both cytoplasm and nucleus in the majority of transfected cells. Given the differential action of MR and GR in the central nervous system, it is important to elucidate how the trafficking of these receptors between cytoplasm and nucleus is regulated by ligand. To examine the simultaneous trafficking of MR and GR within single living cells, we use different spectral variants of GFP, yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP), linked to MR and GR, respectively. In COS-1 cells, expressing no endogenous corticosteroid receptors, the YFP-MR chimera was accumulated in the nucleus faster than the CFP-GR chimera in the presence of 10−9m CORT, while there was no significant difference in the nuclear accumulation rates in the presence of 10−6m CORT. On the other hand, in primary cultured hippocampal neurons expressing endogenous receptors, the nuclear accumulation rates of the YFP-MR chimera and CFP-GR chimera were nearly the same in the presence of both concentrations of CORT. These results suggest that CORT-induced nuclear translocation of MR and GR exhibits differential patterns depending on ligand concentrations or cell types.
Maturity-Onset Diabetes of the Young Type 1 (MODY1)-Associated Mutations R154X and E276Q in Hepatocyte Nuclear Factor 4α (HNF4α) Gene Impair Recruitment of p300, a Key Transcriptional Coactivator
Authors: Eeckhoute Jérôme , Formstecher Pierre , Laine Bernard
Published: January 2014

Hepatocyte nuclear factor 4α (HNF4α) is a nuclear receptor involved in glucose homeostasis and is required for normal β-cell function. Mutations in the HNF4α gene are associated with maturity-onset diabetes of the young type 1. E276Q and R154X mutations were previously shown to impair intrinsic transcriptional activity (without exogenously supplied coactivators) of HNF4α. Given that transcriptional partners of HNF4α modulate its intrinsic transcriptional activity and play crucial roles in HNF4α function, we investigated the effects of these mutations on potentiation of HNF4α activity by p300, a key coactivator for HNF4α. We show here that loss of HNF4α function by both mutations is increased through impaired physical interaction and functional cooperation between HNF4α and p300. Impairment of p300-mediated potentiation of HNF4α transcriptional activity is of particular importance for the E276Q mutant since its intrinsic transcriptional activity is moderately affected. Together with previous results obtained with chicken ovalbumin upstream promoter-transcription factor II, our results highlight that impairment of recruitment of transcriptional partners represents an important mechanism leading to abnormal HNF4α function resulting from the MODY1 E276Q mutation. The impaired potentiations of HNF4α activity were observed on the promoter of HNF1α, a transcription factor involved in a transcriptional network and required for β-cell function. Given its involvement in a regulatory signaling cascade, loss of HNF4α function may cause reduced β-cell function secondary to defective HNF1α expression. Our results also shed light on a better structure-function relationship of HNF4α and on p300 sequences involved in the interaction with HNF4α.
The Synergistic Activity of Thyroid Transcription Factor 1 and Pax 8 Relies on the Promoter/Enhancer Interplay
Authors: Miccadei Stefania , De Leo Rossana , Zammarchi Enrico , Natali Pier Giorgio , Civitareale Donato
Published: January 2014

The transcription factors, thyroid transcription factor 1 (TTF-1) and Pax 8, play a pivotal role in the transcriptional regulation of the thyroid differentiation marker genes and in the differentiation of the thyroid follicular cells. They have a very restricted tissue distribution, and the thyrocyte is the only cell type with the simultaneous expression of these factors. Here we show that TTF-1 and Pax 8 cooperatively activate their target genes and that their synergistic activity requires the cross-talk between enhancer and gene promoter. We have characterized the cis and trans requirements of the TTF1/Pax 8 synergistic activity on the thyroperoxidase gene. We show that their synergy is also important for thyroglobulin gene transcription.
Activity of the GR in G2 and Mitosis
Authors: Abel G. Alexander , Wochnik Gabriela M. , Rüegg Joëlle , Rouyer Audrey , Holsboer Florian , Rein Theo
Published: January 2014

To elucidate the mechanisms mediating the reported transient physiological glucocorticoid resistance in G2/M cell cycle phase, we sought to establish a model system of glucocorticoid-resistant cells in G2. We synchronized various cell lines in G2 to measure dexamethasone (DEX)-induced transactivation of either two endogenous promoters (rat tyrosine aminotransferase and mouse metallothionein I) or the mouse mammary tumor virus (MMTV) promoter stably or transiently transfected. To circumvent the need for synchronization drugs, we stably transfected an MMTV-driven green fluorescent protein to directly correlate DEX-induced transactivation with the cell cycle position for each cell of an asynchronous population using flow cytometry. Surprisingly, all promoters tested were DEX-inducible in G2. Even in mitotic cells, only the stably transfected MMTV promoter was repressed, whereas the same promoter transiently transfected was inducible. The use of Hoechst 33342 for synchronization in previous studies probably caused a misinterpretation, because we detected interference of this drug with GR-dependent transcription independent of the cell cycle. Finally, GR activated a simple promoter in G2, excluding a functional effect of cell cycle-dependent phosphorylation of GR, as implied previously. We conclude that GR itself is fully functional throughout the entire cell cycle, but GR responsiveness is repressed in mitosis due to chromatin condensation rather than to specific modification of GR.
Cis- and Trans-Activation of Hormone Receptors: the LH Receptor
Authors: Ji Inhae , Lee ChangWoo , Song YongSang , Conn P. Michael , Ji Tae H.
Published: January 2014

G protein-coupled receptors (GPCRs) accommodate a wide spectrum of activators from ions to glycoprotein hormones. The mechanism of activation for this large and clinically important family of receptors is poorly understood. Although initially thought to function as monomers, there is a growing body of evidence that GPCR dimers form, and in some cases that these dimers are essential for signal transduction. Here we describe a novel mechanism of intermolecular GPCR activation, which we refer to as trans-activation, in the LH receptor, a GPCR that does not form stable dimers. The LH receptor consists of a 350-amino acid amino-terminal domain, which is responsible for high-affinity binding to human CG, followed by seven-transmembrane domains and connecting loops. This seven-transmembrane domain bundle transmits the signal from the extracellular amino terminus to intracellular G proteins and adenylyl cyclase. Here, we show that binding of hormone to one receptor can activate adenylyl cyclase through its transmembrane bundle, intramolecular activation (cis-activation), as well as trans-activation through the transmembrane bundle of an adjacent receptor, without forming a stable receptor dimer. Coexpression of a mutant receptor defective in hormone binding and another mutant defective in signal generation rescues hormone-activated cAMP production. Our observations provide new insights into the mechanism of receptor activation mechanisms and have implications for the treatment of inherited disorders of glycoprotein hormone receptors.