bluuebunny/crossref_metadata_2025_split huggingface dataset:
Visualization of Pit-1 Transcription Factor Interactions in the Living Cell Nucleus by Fluorescence Resonance Energy Transfer Microscopy
Authors: Day Richard N. 
Published: January 2014

AbstractThe pituitary-specific transcription factor Pit-1 forms dimers when interacting with specific DNA elements and has been shown to associate with several other nuclear proteins. Recently, techniques have become available that allow visualization of protein-protein interactions as they occur in single living cells. In this study, the technique of fluorescence resonance energy transfer (FRET) microscopy was used to visualize the physical interactions of Pit-1 proteins fused to spectral variants of the jellyfish green fluorescent protein (GFP) that emit green or blue light [blue fluorescent protein (BFP)]. An optimized imaging system was used to discriminate fluorescence signals from single cells coexpressing the BFP- and GFP-fusion proteins, and the contribution of spectral overlap to background fluorescence detected in the FRET images was established. Energy transfer signals from living cells expressing a fusion protein in which GFP was tethered to BFP by short protein linker was used to demonstrate acquisition of FRET signals. Genetic vectors encoding GFP- and BFP-Pit-1 proteins were prepared, and biological function of the fusion proteins was confirmed. FRET microscopy of HeLa cells coexpressing the GFP- and BFP-Pit-1 demonstrated energy transfer, which required the two fluorophores to be separated by less than 100 Å. Biochemical studies previously demonstrated that Pit-1 physically interacts with both c-Ets-1 and the estrogen receptor. FRET imaging of cells coexpressing BFP-Pit-1 and GFP-Ets-1 demonstrated energy transfer between these fusion proteins, a result consistent with their association in the nucleus of these living cells. In contrast, there was no evidence for energy transfer between the BFP-Pit-1 and an estrogen receptor-GFP fusion proteins. It is likely that the FRET imaging approach described here can be applied to many different protein-partner pairs in a variety of cellular contexts.
The Protein Kinase C System Acts through the Early Growth Response Protein 1 to Increase LHβ Gene Expression in Synergy with Steroidogenic Factor-1
Authors: Halvorson Lisa M. , Kaiser Ursula B. , Chin William W. 
Published: January 2014

AbstractExpression of the LHβ gene has been shown to be modulated by both the orphan nuclear receptor, steroidogenic factor-1 (SF-1), and the early growth response protein 1, Egr-1. It is also well known that LHβ mRNA levels are increased after hormonal activation of the protein kinase C (PKC) signaling system, for example by GnRH; however, the mechanisms by which the PKC system exerts this effect has not been fully characterized. By transient transfection of the GH3 cell line, we demonstrate that activation of the PKC system with the phorbol ester, phorbol 12-myristate 13-acetate (PMA), increases activity of region −207/+5 of the rat LHβ gene promoter (∼2-fold) and markedly augments SF-1-induced stimulation (95-fold in the presence of both factors vs. 13-fold for SF-1 alone). Mutation of the two previously identified Egr-1 sites not only prevents Egr-1 effects on the LHβ gene promoter, but also eliminates the synergistic response to PMA and SF-1 together, findings that were confirmed in a longer construct spanning region −797/+5. In the gonadotrope-derived cell line,α T3–1, these mutations eliminate the GnRH responsiveness of the− 207/+5 LHβ promoter construct. We next show that PMA treatment (GH3 and αT3–1 cells) or GnRH treatment (αT3–1 cells) induces expression of Egr-1, as detected by Egr-1 interaction with Egr-1 DNA-binding sites in the rat LHβ gene promoter sequence. Furthermore, we demonstrate that PMA increases steady-state Egr-1 mRNA levels via increased Egr-1 transcription. We conclude that PMA-induced stimulation of LHβ gene expression is achieved, at least in part, by induction of Egr-1 expression.
New Natural Inactivating Mutations of the Follicle-Stimulating Hormone Receptor: Correlations between Receptor Function and Phenotype
Authors: Touraine P. , Beau I. , Gougeon A. , Meduri G. , Desroches A. , Pichard C. , Detoeuf M. , Paniel B. , Prieur M. , Zorn J-R , Milgrom E. , Kuttenn F. , Misrahi M. 
Published: January 2014

Abstract
Premature ovarian failure occurs in almost 1% of women under age 40. Molecular alterations of the FSH receptor (FSHR) have recently been described. A first homozygous mutation of the FSHR was identified in Finland. More recently, we described two new mutations of the FSHR in a woman presenting a partial FSH-resistance syndrome (patient 1). We now report new molecular alterations of the FSHR in another woman (patient 2) who presented at the age of 19 with primary amenorrhea contrasting with normal pubertal development. She had high plasma FSH, and numerous ovarian follicles up to 3 mm in size were evidenced by ultrasonography. Histological and immunohistochemical examination of ovarian biopsies revealed the presence of a normal follicular development up to the antral stage and disruption at further stages.
DNA sequencing showed two heterozygous mutations: Asp224Val in the extracellular domain and Leu601Val in the third extracellular loop of FSHR. Cells transfected with expression vectors encoding the wild type or the mutated Leu601Val receptors bound hormone with similar affinity, whereas binding was barely detectable with the Asp224Val mutant. Confocal microscopy showed the latter to have an impaired targeting to the cell membrane. This was confirmed by its accumulation as a mannose-rich precursor. Adenylate cyclase stimulation by FSH of the Leu601Val mutant receptor showed a 12 ± 3% residual activity, whereas in patient 1 a 24 ± 4% residual activity was detected for the Arg573Cys mutant receptor. These results are in keeping with the fact that estradiol and inhibin B levels were higher in patient 1 and that stimulation with recombinant FSH did not increase follicular size, estradiol, or inhibin B levels in patient 2 in contrast to what was observed for patient 1. Thus, differences in the residual activity of mutated FSHR led to differences in the clinical, biological, and histological phenotypes of the patient.
Cyclic Adenosine-3′,5′-Monophosphate-Mediated Activation of a Glutamine Synthetase Composite Glucocorticoid Response Element
Authors: Richardson Jan , Vinson Charles , Bodwell Jack 
Published: January 2014

AbstractThe glutamate synthetase gene (GS) contains a composite glucocorticoid response element (cGRE) comprised of a GRE and an adjacent element with features of both a cAMP-response element (CRE) and a 12-O-tetradecanoylphorbol 13-acetate (TPA) response element (TRE). The CRE/TRE element of the cGRE contributed to two modes of transcriptional activation: 1) enhancement of the response to cortisol and 2) a synergistic response to cortisol and increased cAMP. COS-7 cells transfected with a cGRE-luciferase construct show minimal expression under basal conditions or forskolin treatment. After cortisol treatment, luciferase activity from the cGRE is enhanced 4- to 8-fold greater than the GRE portion of the cGRE or a GRE from the tyrosine aminotransferase gene. Treatment with both forskolin and cortisol produced a 2- to 4-fold synergistic response over cortisol alone. Synergy is also seen with 8-bromo-cAMP, is specific for the cGRE, and occurs in a number of established cell lines. Elimination of the GRE or CRE/TRE reduces the synergy by 70–100%. Altering the CRE/TRE to GRE spacing changed both enhancement and synergy. Moving the elements 3 bp closer or extending 15 bp reduced enhancement. Synergy was markedly reduced when elements were one half of a helical turn out of phase. Western blots verified that CREB (cAMP-responsive binding protein) and ATF-1 (activating transcription factor-1) binds to the cGRE sequence. A specific dominant negative inhibitor of the CREB family, A-CREB, reduced synergy by 50%. These results suggest that the GS cGRE can potentially integrate signaling from both the cAMP and glucocorticoid receptor transduction pathways and that CREB/ATF-1 may play an important role in this process.
Targeted Disruption of the Insl3 Gene Causes Bilateral Cryptorchidism
Authors: Zimmermann Stephan , Steding Gerd , Emmen Judith M. A. , Brinkmann Albert O. , Nayernia Karim , Holstein Adolf F. , Engel Wolfgang , Adham Ibrahim M. 
Published: January 2014

AbstractThe sexual dimorphic position of the gonads in mammals is dependent on differential development of two ligaments, the cranial suspensory ligament (CSL) and the gubernaculum. During male embryogenesis, outgrowth of the gubernaculum and regression of the CSL result in transabdominal descent of the testes, whereas in the female, development of the CSL in conjunction with failure of the gubernaculum development holds the ovaries in a position lateral to the kidneys. Several lines of evidence suggest that regression of the CSL and induction of gubernaculum development are mediated by testosterone and a yet unidentified testicular factor, respectively. The Insl3 gene (orginally designated Ley I-L), a member of the insulin-like superfamily, is specifically expressed in Leydig cells of the fetal and postnatal testis and in theca cells of the postnatal ovary. Here we show that male mice homozygous for a targeted deletion of the Insl3 locus exhibit bilateral cryptorchidism with free moving testes and genital ducts. These malformations are due to failure of gubernaculum development during embryogenesis. In double-mutant male mice for Insl3 and androgen receptor genes, testes are positioned adjacent to the kidneys and steadied in the abdomen by the CSL. These findings demonstrate, that the Insl3 induces gubernaculum development in an androgen-independent way, while androgen-mediated regression of the CSL occurs independently from Insl3.
A Dominant Role for the Raf-MEK Pathway in Forskolin, 12-O-Tetradecanoyl-phorbol Acetate, and Platelet-Derived Growth Factor-Induced CREB (cAMP-Responsive Element-Binding Protein) Activation, Uncoupled from Serine 133 Phosphorylation in NIH 3T3 Cells
Authors: Seternes Ole Morten , Johansen Bjarne , Moens Ugo 
Published: January 2014

AbstractIn this study we describe that platelet-derived growth factor (PDGF), 12-0-tetradecanoyl-phorbol-acetate (TPA), and forskolin induced CREB (cAMP-responsive element-binding protein) Ser-133 phosphorylation with comparable magnitude and kinetics in NIH 3T3 cells. While forskolin was the most potent activator of CREB, TPA or PDGF modestly increased CREB activity. The role of protein kinase C, protein kinase A, and the Raf-MEK kinase pathway in the activation and Ser-133 phosphorylation of CREB by these three stimuli was investigated. We found that inhibition of the Raf-MEK kinase pathway efficiently blocks transcriptional activation of CREB by all three stimuli. This dominant involvement of Raf-MEK in CREB transcriptional activation seems to be uncoupled from CREB Ser-133 phosphorylation. We further demonstrate that although inhibition of Raf-MEK represses forskolin-induced CREB activation, forskolin by itself failed to activate ERK1/2 and Elk-1 mediated transcription. These results suggest that a basal level of Raf-MEK activity is necessary for both PDGF- and forskolin-induced CREB activation, independent of CREB Ser-133 phosphorylation.
ARIP3 (Androgen Receptor-Interacting Protein 3) and Other PIAS (Protein Inhibitor of Activated STAT) Proteins Differ in Their Ability to Modulate Steroid Receptor-Dependent Transcriptional Activation
Authors: Kotaja Noora , Aittomäki Saara , Silvennoinen Olli , Palvimo Jorma J. , Jänne Olli A. 
Published: January 2014

AbstractSteroid receptors mediate their actions by using various coregulatory proteins. We have recently characterized ARIP3/PIASxα as an androgen receptor (AR)-interacting protein (ARIP) that belongs to the PIAS [protein inhibitor of activated STAT (signal transducer and activator of transcription)] protein family implicated in the inhibition of cytokine signaling. We have analyzed herein the roles that four different PIAS proteins (ARIP3/PIASxα, Miz1/PIASxβ, GBP/PIAS1, and PIAS3) play in the regulation of steroid receptor- or STAT-mediated transcriptional activation. All PIAS proteins are able to coactivate steroid receptor-dependent transcription but to a differential degree, depending on the receptor, the promoter, and the cell type. Miz1 and PIAS1 are more potent than ARIP3 in activating AR function on minimal promoters. With the natural probasin promoter, PIAS proteins influence AR function more divergently, in that ARIP3 represses, but Miz1 and PIAS1 activate it. Miz1 and PIAS1 possess inherent transcription activating function, whereas ARIP3 and PIAS3 are devoid of this feature. ARIP3 enhances glucocorticoid receptor-dependent transcription more efficiently than Miz1 or PIAS1, and all PIAS proteins also activate estrogen receptor- and progesterone receptor-dependent transcription but to a dissimilar degree. The same amounts of PIAS proteins that modulate steroid receptor-dependent transcription influence only marginally transactivation mediated by various STAT proteins. It remains to be established whether the PIAS proteins play a more significant physiological role in steroid receptor than in cytokine signaling.
Activation of Peroxisome Proliferator-Activated Receptors (PPARs) by Their Ligands and Protein Kinase A Activators
Authors: Lazennec Gwendal , Canaple Laurence , Saugy Damien , Wahli Walter 
Published: January 2014

AbstractThe nuclear peroxisome proliferator-activated receptors (PPARs) α, β, and γ activate the transcription of multiple genes involved in lipid metabolism. Several natural and synthetic ligands have been identified for each PPAR isotype but little is known about the phosphorylation state of these receptors. We show here that activators of protein kinase A (PKA) can enhance mouse PPAR activity in the absence and the presence of exogenous ligands in transient transfection experiments. Activation function 1 (AF-1) of PPARs was dispensable for transcriptional enhancement, whereas activation function 2 (AF-2) was required for this effect. We also show that several domains of PPAR can be phosphorylated by PKA in vitro. Moreover, gel retardation experiments suggest that PKA stabilizes binding of the liganded PPAR to DNA. PKA inhibitors decreased not only the kinase-dependent induction of PPARs but also their ligand-dependent induction, suggesting an interaction between both pathways that leads to maximal transcriptional induction by PPARs. Moreover, comparing PPARα knockout (KO) with PPARα WT mice, we show that the expression of the acyl CoA oxidase (ACO) gene can be regulated by PKA-activated PPARα in liver. These data demonstrate that the PKA pathway is an important modulator of PPAR activity, and we propose a model associating this pathway in the control of fatty acidβ -oxidation under conditions of fasting, stress, and exercise.
Novel Role for the Nuclear Phosphoprotein SET in Transcriptional Activation of P450c17 and Initiation of Neurosteroidogenesis
Authors: Compagnone Nathalie A. , Zhang Peilin , Vigne Jean-Louis , Mellon Synthia H. 
Published: January 2014

AbstractNeurosteroids are important endogenous regulators of γ-aminobutryic acid (GABAA) and N-methyl-d-aspartate (NMDA) receptors and also influence neuronal morphology and function. Neurosteroids are produced in the brain using many of the same enzymes found in the adrenal and gonad. The crucial enzyme for the synthesis of DHEA (dehydroepiandrosterone) in the brain is cytochrome P450c17. The transcriptional strategy for the expression of P450c17 is clearly different in the brain from that in the adrenal or gonad. We previously characterized a novel transcriptional regulator from Leydig MA-10 cells, termed StF-IT-1, that binds at bases −447/−399 of the rat P450c17 promoter, along with the known transcription factors COUP-TF (chicken ovalbumin upstream promoter transcription factor), NGF-IB (nerve growth factor inducible protein B), and SF-1 (steroidogenic factor-1). We have now purified and sequenced this protein from immature porcine testes, identifying it as the nuclear phosphoprotein SET; a role for SET in transcription was not established previously. Binding of bacterially expressed human and rat SET to the DNA site at −418/−399 of the rat P450c17 gene transactivates P450c17 in neuronal and in testicular Leydig cells. We also found SET expressed in human NT2 neuronal precursor cells, implicating a role in neurosteroidogenesis. Immunocytochemistry and in situ hybridization in the mouse fetus show that the ontogeny and distribution of SET in the developing nervous system are consistent with SET being crucial for initiating P450c17 transcription. SET’s developmental pattern of expression suggests it may participate in the early ontogenesis of the nervous, as well as the skeletal and hematopoietic, systems. These studies delineate an important new factor in the transcriptional regulation of P450c17 and consequently, in the production of DHEA and sex steroids.
Requirement of Bmp8b for the Generation of Primordial Germ Cells in the Mouse
Authors: Ying Ying , Liu Xiao-Ming , Marble Amy , Lawson Kirstie A. , Zhao Guang-Quan 
Published: January 2014

AbstractIn the mouse embryo, the generation of primordial germ cells (PGCs) from the epiblast requires a bone morphogenetic protein-4 (BMP4) signal from the adjacent extraembryonic ectoderm. In this study, we report that Bmp8b, a member of the Gbb-60A class of the BMP superfamily, is expressed in the extraembryonic ectoderm in pregastrula and gastrula stage mouse embryos and is required for PGC generation. A mutation in Bmp8b on a mixed genetic background results in the absence of PGCs in 43% null mutant embryos and severe reduction in PGC number in the remainder. The heterozygotes are unaffected. On a largely C57BL/6 background, Bmp8b null mutants completely lack PGCs, and Bmp8b heterozygotes have a reduced number of PGCs. In addition, Bmp8b homozygous null embryos on both genetic backgrounds have a short allantois, and this organ is missing in some more severe mutants. Since Bmp4 heterozygote embryos have reduced numbers of PGCs, we used a genetic approach to generate double-mutant embryos to study interactions of Bmp8b and Bmp4. Embryos that are double heterozygotes for the Bmp8b and Bmp4 mutations have similar defects in PGC number as Bmp4 heterozygotes, indicating that the effects of the two BMPs are not additive. These findings suggest that BMP4 and BMP8B function as heterodimers and homodimers in PGC specification in the mouse.
Unliganded and Liganded Estrogen Receptors Protect against Cancer Invasion via Different Mechanisms
Authors: Platet Nadine , Cunat Séverine , Chalbos Dany , Rochefort Henri , Garcia Marcel 
Published: January 2014

AbstractWhile estrogens are mitogenic in breast cancer cells, the presence of estrogen receptor α (ERα) clinically indicates a favorable prognosis in breast carcinoma. To improve our understanding of ERα action in breast cancer, we used an original in vitro method, which combines transient transfection and Matrigel invasion assays to examine its effects on cell invasiveness. ERα expression in MDA-MB-231 breast cancer cells reduced their invasiveness by 3-fold in the absence of hormone and by 7-fold in its presence. Integrity of hormone and DNA-binding domains and activating function 2 were required for estradiol-induced inhibition, suggesting that transcriptional activation of estrogen target genes was involved. In contrast, these domains were dispensable for hormone-independent inhibition. Analysis of deletion mutants of ERα indicated that amino acids 179–215, containing the N-terminal zinc finger of the DNA-binding domain, were required for ligand-independent receptor action. Among different members of the nuclear receptor family, only unliganded ERα and ERβ reduced invasion. Calreticulin, a Ca2+-binding protein that could interact with amino acids 206–211 of ERα, reversed hormone-independent ERα inhibition of invasion. However, since calreticulin alone also inhibited invasion, we propose that this protein probably prevents ERα interaction with another unidentified invasion-regulating factor. The inhibitor role of the unliganded ER was also suggested in three ERα-positive cell lines, where ERα content was inversely correlated with cell migration. We conclude that ERα protects against cancer invasion in its unliganded form, probably by protein-protein interactions with the N-terminal zinc finger region, and after hormone binding by activation of specific gene transcription.
Conditional Deletion Of Brain-Derived Neurotrophic Factor in the Postnatal Brain Leads to Obesity and Hyperactivity
Authors: Rios Maribel , Fan Guoping , Fekete Csaba , Kelly Joseph , Bates Brian , Kuehn Ralf , Lechan Ronald M. , Jaenisch Rudolf 
Published: January 2014

Abstract
Brain-derived neurotrophic factor has been associated previously with the regulation of food intake. To help elucidate the role of this neurotrophin in weight regulation, we have generated conditional mutants in which brain-derived neurotrophic factor has been eliminated from the brain after birth through the use of the cre-loxP recombination system. Brain-derived neurotrophic factor conditional mutants were hyperactive after exposure to stressors and had higher levels of anxiety when evaluated in the light/dark exploration test. They also had mature onset obesity characterized by a dramatic 80–150% increase in body weight, increased linear growth, and elevated serum levels of leptin, insulin, glucose, and cholesterol. In addition, the mutants had an abnormal starvation response and elevated basal levels of POMC, an anorexigenic factor and the precursor forα -MSH. Our results demonstrate that brain derived neurotrophic factor has an essential maintenance function in the regulation of anxiety-related behavior and in food intake through central mediators in both the basal and fasted state.
ERα Gene Expression in Human Primary Osteoblasts: Evidence for the Expression of Two Receptor Proteins
Authors: Denger Stefanie , Reid George , Koš Martin , Flouriot Gilles , Parsch Dominik , Brand Heike , Korach Kenneth S. , Sonntag-Buck Vera , Gannon Frank 
Published: January 2014

Abstract
The beneficial influence of E2 in the maintenance of healthy bone is well recognized. However, the way in which the actions of this hormone are mediated is less clearly understood. Western blot analysis of ERα in osteoblasts clearly demonstrated that the well characterized 66-kDa ERα was only one of the ERα isoforms present. Here we describe a 46-kDa isoform of ERα, expressed at a level similar to the 66-kDa isoform, that is also present in human primary osteoblasts. This shorter isoform is generated by alternative splicing of an ERα gene product, which results in exon 1 being skipped with a start codon in exon 2 used to initiate translation of the protein. Consequently, the transactivation domain AF-1 of this ERα isoform is absent. Functional analysis revealed that human (h)ERα46 is able to heterodimerize with the full-length ERα and also with ERβ. Further, a DNA-binding complex that corresponds to hERα46 is detectable in human osteoblasts. We have shown that hERα46 is a strong inhibitor of hERα66 when they are coexpressed in the human osteosarcoma cell line SaOs. As a functional consequence, proliferation of the transfected cells is inhibited when increasing amounts of hERα46 are cotransfected with hERα66. In addition to human bone, the expression of the alternatively spliced ERα mRNA variant is also detectable in bone of ERα knockout mice.
These data suggest that, in osteoblasts, E2 can act in part through an ERα isoform that is markedly different from the 66-kDa receptor. The expression of two ERα protein isoforms may account, in part, for the differential action that estrogens and estrogen analogs have in different tissues. In particular, the current models of the action of estrogens should be reevaluated to take account of the presence of at least two ERα protein isoforms in bone and perhaps in other tissues.