bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractThe 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

AbstractAn 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

AbstractActivated 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

AbstractAn 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

AbstractVarious 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

AbstractPituitary 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

AbstractProgesterone 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

Abstract
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

AbstractActivation 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

AbstractMineralocorticoid 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

AbstractHepatocyte 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

AbstractThe 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

AbstractTo 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.