bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of a Functional Androgen-Response Element in the Exon 1-Coding Sequence of the Cystatin-Related Protein Gene crp2
Authors: Devos A. , Claessens F. , Alen P. , Winderickx J. , Heyns W. , Rombauts W. , Peeters B. 
Published: January 2014

AbstractTwo hormone-responsive segments, one in the region of the promoter and one in intron 1, are identified in two homologous androgen-regulated and differentially expressed rat genes encoding the cystatin-related proteins (CRPs). Footprint analysis with the androgen receptor (AR) DNA-binding domain on the promoter-containing fragments reveals an AR-binding site downstream of the transcription start point in the crp2 gene (ARBSd/crp2,+ 40/+63). It displays an androgen response element-like sequence motif 5′-AGAAGAaaaTGTACA-3′ and overlaps with the ATG translation start codon. A double-stranded oligonucleotide containing this sequence forms a DNA-protein complex with the full-length AR synthesized by vaccinia, as seen in band shift assays. Additional AR-binding sites, ARBSu/crp1 and ARBSu/crp2, occur 5′ upstream of the transcription start point and are located at an identical position (−142/−120) in crp1 and crp2. The AR affinity for these two slightly different sequence motifs is relatively weak. The biological function of all three AR-binding sites as transcription control elements has been studied. The ARBSd/crp2 element clearly shows androgen-response element characteristics. The contribution of the common upstream element to the androgen-dependent control of reporter gene transcription is less clear. The transcription of a reporter gene construct containing the crp2 footprint fragment crp2F (−273/+88) is hormonally regulated as determined by transfection into the human breast cancer cell line T-47D. Androgens, but also glucocorticoids, efficiently stimulate steroid-dependent transcription of the chloramphenicol acetyltransferase gene. Mutation of the 5′-TGTACA-3′ sequence in ARBSd/crp2 destroys the AR binding and abolishes the androgen-dependent synthesis of chloramphenicol acetyltransferase. A large fragment derived from intron 1 of the crp1 and crp2 gene can also provide the androgen-dependent transcription of chimeric constructs in T-47D cells. However, the induction measured is less than the one observed with crp2F (−273/+88), and this activity seems to reside in several subfragments that each display a low but consistent androgen responsiveness.
Identification of Transcripts Initiated from an Internal Promoter in the c-erbAα Locus That Encode Inhibitors of Retinoic Acid Receptor-α and Triiodothyronine Receptor Activities
Authors: Chassande Olivier , Fraichard Alexandre , Gauthier Karine , Flamant Frédéric , Legrand Claude , Savatier Pierre , Laudet Vincent , Samarut Jacques 
Published: January 2014

AbstractThe thyroid hormone receptor-coding locus, c-erbAα, generates several mRNAs originating from a single primary transcript that undergoes alternative splicing. We have identified for the first time two new transcripts, called TRΔα1 and TRΔα2[ mRNA for isoform α1 and α2 of the T3 receptor (TR), respectively], whose transcription is initiated from an internal promoter located within intron 7 of the c-erbAα gene. These two new transcripts exhibit tissue-specific patterns of expression in the mouse. These two patterns are in sharp contrast with the expression patterns of the full-length transcripts generated from the c-erbAα locus. TRΔα1 and TRΔα2 mRNAs encode N-terminally truncated isoforms of T3Rα1 and T3Rα2, respectively. The protein product of TRΔα1 antagonizes the transcriptional activation elicited by T3 and retinoic acid. This protein inhibits the ligand-induced activating functions of T3Rα1 and 9-cis-retinoic acid receptor-α but does not affect the retinoic acid-dependent activating function of retinoic acid receptor-α. We predict that these truncated proteins may work as down-regulators of transcriptional activity of nuclear hormone receptors in vivo.
Dissociation of Early Folding Events from Assembly of the Human Lutropin β-Subunit
Authors: Muyan Mesut , Ruddon Raymond W. , Norton Sheila E. , Boime Irving , Bedows Elliott 
Published: January 2014

AbstractThe human LH of the anterior pituitary is a member of the glycoprotein hormone family that includes FSH, TSH, and placental CG. All are noncovalently bound heterodimers that share a common α-subunit and β-subunits that confer biological specificity. LHβ and CGβ share more than 80% amino acid sequence identity; however, in transfected Chinese hamster ovary (CHO) cells, LHβ assembles with the α-subunit more slowly than does hCGβ, and only a fraction of the LHβ synthesized is secreted, whereas CGβ is secreted efficiently. To understand why the assembly and secretion of these related β-subunits differ, we studied the folding of LHβ in CHO cells transfected with either the LHβ gene alone, or in cells cotransfected with the gene expressing the common α-subunit, and compared our findings to those previously seen for CG. We found that the rate of conversion of the earliest detectable folding intermediate of LH, pβ1, to the second major folding form, pβ2, did not differ significantly from the pβ1-to-pβ2 conversion of CGβ, suggesting that variations between the intracellular fates of the two β-subunits cannot be explained by differences in the rates of their early folding steps. Rather, we discovered that unlike CGβ, where the folding to pβ2 results in an assembly-competent product, apparently greater than 90% of the LH pβ2 recovered from LHβ-transfected CHO cells was assembly incompetent, accounting for inefficient LHβ assembly with the α-subunit. Using the formation of disulfide (S-S) bonds as an index, we observed that, in contrast to CGβ, all 12 LHβ cysteine residues formed S-S linkages as soon as pβ2 was detected. Attempts to facilitate LH assembly with protein disulfide isomerase in vitro using LH pβ2 and excess urinary α-subunit as substrate were unsuccessful, although protein disulfide isomerase did facilitate CG assembly in this assay. Moreover, unlike CGβ, LHβ homodimers were recovered from transfected CHO cells. Taken together, these data suggest that differences seen in the rate and extent of LH assembly and secretion, as compared to those of CG, reflect conformational differences between the folding intermediates of the respectiveβ -subunits.
Cell-Specific Expression of the Mouse Glycoprotein Hormone α-Subunit Gene Requires Multiple Interacting DNA Elements in Transgenic Mice and Cultured Cells
Authors: Brinkmeier Michelle L. , Gordon David F. , Dowding Janet M. , Saunders Thomas L. , Kendall Susan K. , Sarapura Virginia D. , Wood William M. , Ridgway E. Chester , Camper Sally A. 
Published: January 2014

AbstractThe glycoprotein hormone α-subunit gene is expressed and differentially regulated in pituitary gonadotropes and thyrotropes. Previous gene expression studies suggested that cell specificity may be regulated by distinct DNA elements. We have identified an enhancer region between −4.6 and −3.7 kb that is critical for high level expression in both gonadotrope and thyrotrope cells of transgenic mice. Fusion of the enhancer to −341/+43 mouseα -subunit promoter results in appropriate pituitary cell specificity and transgene expression levels that are similar to levels observed with the intact −4.6 kb/+43 construct. Deletion of sequences between− 341 and −297 resulted in a loss of high level expression and cell specificity, exhibited by ectopic transgene activation in GH-, ACTH-, and PRL-producing pituitary cells as well as in other peripheral tissues. Consistent with these results, transient cell transfection studies demonstrated that the enhancer stimulated activity of a− 341/+43 α-promoter in both αTSH and αT3 cells, but it did not enhance α-promoter activity significantly in CV-1 cells. Removal of sequences between −341 and −297 allowed the enhancer to function in heterologous cells. Loss of high level expression and cell specificity may be due to loss of sequences required for binding of the LIM homeoproteins or the α-basal element 1. These data demonstrate that the enhancer requires participation by both proximal and distal sequences for high level expression and suggests that sequences from− 341 to −297 are critical for restricting expression to the anterior pituitary.
Calcitonin Receptor-Mediated Growth Suppression of HEK-293 Cells Is Accompanied by Induction of p21WAF1/CIP1 and G2/M Arrest
Authors: Evdokiou Andreas , Raggatt Liza-Jane , Atkins Gerald J. , Findlay David M. 
Published: January 2014

Abstract
We investigated the mechanisms by which calcitonin (CT) suppresses cellular proliferation, using HEK-293 cells stably transfected with either the rat C1a CT receptor (CTR) or the insert-negative form of the human CTR. CT treatment of clonal cell lines expressing either receptor type, but not untransfected HEK-293 cells, strongly suppressed cell growth in a concentration-dependent manner. The reduction in cell growth with CT treatment could not be attributed to cellular necrosis or apoptotic cell death, the latter assessed by both DNA fragmentation analysis and caspase 3 (CPP-32) assay. Growth inhibition was associated with an accumulation of cells in the G2 phase of the cell cycle. CT treatment of the human and rat CTR-expressing cell lines resulted in a rapid and sustained induction of mRNA encoding the cyclin-dependent kinase inhibitor, p21WAF1/CIP1, increased levels of which were maintained at least 48 h after initiation of treatment. Western blot analysis showed a rapid corresponding increase in p21WAF1/CIP1 protein, whereas protein levels of another member of the cyclin-dependent kinase inhibitor family, p27kip1, were unchanged. In parallel with the induction of p21, CT treatment reduced levels of p53 mRNA and protein. CT treatment resulted in a specific cell cycle block in G2, which was associated with inhibition of Cdc2/cyclin B kinase activity as measured by histone H1 phosphorylation. There was no evidence for p21 association with this complex despite the inhibition of Cdc2 activity. Evidence that p21 induction was causative of cell growth suppression was obtained from p21 antisense oligonucleotide experiments. Treatment with a p21 antisense oligonucleotide blocked induction of p21 expression and significantly reduced the CT-mediated growth inhibition. These observations suggest that p21 is required for the G2 arrest in response to CT, but argue against a direct role of p21 in the inhibition of Cdc2 activity. These studies suggest a novel regulation of cell cycle progression by CT and will provide a basis for detailed examination of the molecular mechanisms involved.
Signals from the AT2 (Angiotensin Type 2) Receptor of Angiotensin II Inhibit p21ras and Activate MAPK (Mitogen-Activated Protein Kinase) to Induce Morphological Neuronal Differentiation in NG108–15 Cells
Authors: Gendron∗ Louis , Laflamme∗ Liette , Rivard Nathalie , Asselin Claude , Payet Marcel D. , Gallo-Payet Nicole 
Published: January 2014

AbstractIn a previous study, we had shown that activation of the AT2 (angiotensin type 2) receptor of angiotensin II (Ang II) induced morphological differentiation of the neuronal cell line NG108–15. In the present study, we investigated the nature of the possible intracellular mediators involved in the AT2 effect. We found that stimulation of AT2 receptors in NG108–15 cells resulted in time-dependent modulation of tyrosine phosphorylation of a number of cytoplasmic proteins. Stimulation of NG108–15 cells with Ang II induced a decrease in GTP-bound p21ras but a sustained increase in the activity of p42mapk and p44mapk as well as neurite outgrowth. Similarly, neurite elongation, increased polymerized tubulin levels, and increased mitogen-activated protein kinase (MAPK) activity were also observed in a stably transfected NG108–15 cell line expressing the dominant-negative mutant of p21ras, RasN17. These results support the observation that inhibition of p21ras did not impair the effect of Ang II on its ability to stimulate MAPK activity. While 10 μm of the MEK inhibitor, PD98059, only moderately affected elongation, 50 μm PD98059 completely blocked the Ang II- and the RasN17-mediated induction of neurite outgrowth. These results demonstrate that some of the events associated with the AT2 receptor-induced neuronal morphological differentiation of NG108–15 cells not only include inhibition of p21ras but an increase in MAPK activity as well, which is essential for neurite outgrowth.
Cellular Distribution of Constitutively Active Mutant Parathyroid Hormone (PTH)/PTH-Related Protein Receptors and Regulation of Cyclic Adenosine 3′,5′-Monophosphate Signaling by β-Arrestin2
Authors: Ferrari Serge L. , Bisello Alessandro 
Published: January 2014

AbstractPTH promotes endocytosis of human PTH receptor 1 (PTH1Rc) by activating protein kinase C and recruitingβ -arrestin2. We examined the role of β-arrestin2 in regulating the cellular distribution and cAMP signaling of two constitutively active PTH1Rc mutants, H223R and T410P. Overexpression of aβ -arrestin2-green fluorescent protein (GFP) conjugate in COS-7 cells inhibited constitutive cAMP accumulation by H223R and T410P in a dose-dependent manner, as well as the response to PTH of both mutant and wild-type PTH1Rcs. The cellular distribution of PTH1Rc-GFP conjugates, fluorescent ligands, and βarrestin2-GFP was analyzed by fluorescence microscopy in HEK-293T cells. In cells expressing either receptor mutant, a ligand-independent mobilization ofβ -arrestin2 to the cell membrane was observed. In the absence of ligand, H223R and wild-type PTH1Rcs were mainly localized on the cell membrane, whereas intracellular trafficking of T410P was also observed. While agonists promoted β-arrestin2-mediated endocytosis of both PTH1Rc mutants, antagonists were rapidly internalized only with T410P. The protein kinases inhibitor, staurosporine, significantly decreased internalization of ligand-PTH1Rc mutant complexes, although the recruitment of β-arrestin2 to the cell membrane was unaffected. Moreover, in cells expressing a truncated wild-type PTH1Rc lacking the C-terminal cytoplasmic domain, agonists stimulated translocation ofβ -arrestin2 to the cell membrane followed by ligand-receptor complex internalization without associated β-arrestin2. In conclusion, cAMP signaling by constitutively active mutant and wild-type PTH1Rcs is inhibited by a receptor interaction withβ -arrestin2 on the cell membrane, possibly leading to uncoupling from Gsα. This phenomenon is independent from protein kinases activity and the receptor C-terminal cytoplasmic domain. In addition, there are differences in the cellular localization and internalization features of constitutively active PTH1Rc mutants H223R and T410P.
Calcium Responses to Thyrotropin-Releasing Hormone, Gonadotropin-Releasing Hormone and Somatostatin in Phospholipase Cβ3 Knockout Mice
Authors: Romoser Valerie A. , Graves Thomas K. , Wu Dianqing , Jiang Huiping , Hinkle Patricia M. 
Published: January 2014

AbstractThese studies examined the importance of phospholipase Cβ (PLCβ) in the calcium responses of pituitary cells using PLCβ3 knockout mice. Pituitary tissue from wild-type mice contained PLCβ1 and PLCβ3 but not PLCβ2 or PLCβ4. Both Gαq/11 and Gβγ can activate PLCβ3, whereas only Gαq/11 activates PLCβ1 effectively. In knockout mice, PLCβ3 was absent, PLCβ1 was not up-regulated, and PLCβ2 and PLCβ4 were not expressed. Since somatostatin inhibited influx of extracellular calcium in pituitary cells from wild-type and PLCβ3 knockout mice, the somatostatin signal pathway was intact. However, somatostatin failed to increase intracellular calcium in pituitary cells from either wild-type or knockout mice under a variety of conditions, indicating that it did not stimulate PLCβ3. In contrast, somatostatin increased intracellular calcium in aortic smooth muscle cells from wild-type mice, although it evoked no calcium response in cells from PLCβ3 knockout animals. These results show that somatostatin, like other Gi/Go-linked hormones, can stimulate a calcium transient by activating PLCβ3 through Gβγ, but this response does not normally occur in pituitary cells. The densities of Gi and Go, as well as the relative concentrations of PLCβ1 and PLCβ3, were similar in cells that responded to somatostatin with an increase in calcium and pituitary cells. Calcium responses to 1 nm and 1μ m TRH and GnRH were identical in pituitary cells from wild-type and PLCβ3 knockout mice, as were responses to other Gq-linked agonists. These results show that in pituitary cells, PLCβ1 is sufficient to transmit signals from Gq-coupled hormones, whereas PLCβ3 is required for the calcium-mobilizing actions of somatostatin observed in smooth muscle cells.
Association of β-Arrestin 1 with the Type 1A Angiotensin II Receptor Involves Phosphorylation of the Receptor Carboxyl Terminus and Correlates with Receptor Internalization
Authors: Qian Hongwei , Pipolo Luisa , Thomas Walter G. 
Published: January 2014

AbstractArrestins bind to phosphorylated G protein-coupled receptors and participate in receptor desensitization and endocytosis. Although arrestins traffic with activated type 1 (AT1A) angiotensin II (AngII) receptors, the contribution of arrestins to AT1A receptor internalization is controversial, and the physical association of arrestins with the AT1A receptor has not been established. In this study, by coimmunoprecipitating AT1A receptors and β-arrestin 1, we provide direct evidence for an association between arrestins and the AT1A receptor that was agonist- and time-dependent and contingent upon the level ofβ -arrestin 1 expression. Serial truncation of the receptor carboxyl terminus resulted in a graded loss of β-arrestin 1 association, which correlated with decreases in receptor phosphorylation. Truncation of the AT1A receptor to lysine325 prevented AngII-induced phosphorylation and β-arrestin 1 association as well as markedly inhibiting receptor internalization, indicating a close correlation between these receptor parameters. AngII-induced association was also dramatically reduced in a phosphorylation- and internalization-impaired receptor mutant in which four serine and threonine residues in the central portion of the AT1A receptor carboxyl terminus (Thr332, Ser335, Thr336, Ser338) were substituted with alanine. In contrast, substitutions in another serine/threonine-rich region (Ser346, Ser347, Ser348) and at three PKC phosphorylation sites (Ser331, Ser338, Ser348) had no effect on AngII-inducedβ -arrestin 1 association or receptor internalization. While AT1A receptor internalization could be inhibited by a dominant-negative β-arrestin 1 mutant (βarr1319–418), treatment with hyperosmotic sucrose to inhibit internalization did not abrogate the differences in arrestin association observed between the wild-type and mutant receptors, indicating that arrestin binding precedes, and is not dependent upon, receptor internalization. Interestingly, a substituted analog of AngII,[ Sar1Ile4Ile8]-AngII, which promotes robust phosphorylation of the receptor but does not activate receptor signaling, stimulated strong β-arrestin 1 association with the full-length AT1A receptor. These results identify the central portion of the AT1A receptor carboxyl terminus as the important determinant for β-arrestin 1 binding and internalization and indicate that AT1A receptor phosphorylation is crucial for β-arrestin docking.
EAR2 and EAR3/COUP-TFI Regulate Transcription of the Rat LH Receptor
Authors: Zhang Ying , Dufau Maria L. 
Published: January 2014

AbstractOur previous studies demonstrated regulation of the human LH receptor (hLHR) promoter by nuclear orphan receptors EAR2, EAR3/COUP-TFI (repression), and TR4 (activation) through a direct-repeat motif (hDR). The current studies investigated the differential binding of orphan receptors to rat (rLHR) and hLHR promoters, and their modulation of rLHR gene transcription in rat granulosa cells. The rLHR DR with one nucleotide difference from hDR at its core sequence mediated inhibition of the rLHR transcription, to which EAR2 and EAR3/COUP-TFI but not TR4 bound. The A/C mismatch was responsible for the lack of TR4 binding and function, but had no effect on EAR2 and EAR3/COUP-TFI. EAR2 and EAR3/COUP-TF bound to the rLHR DR with lower affinity than to the hDR, and exhibited lesser inhibitory capacity. This difference resulted from the lack of a guanine in the rDR, which is present 3′ next to the hDR core. These studies have identified sequence-specific requirements for the binding of EAR2, EAR3/COUP-TFI, and TR4 to the DRs that explain their differential regulation of rat and human LHR genes. In addition, hCG treatment significantly reduced the inhibition of rLHR gene in granulosa cells and also decreased EAR2 and EAR3/COUP-TFI protein levels. These results indicate that hormonally regulated expression of EAR2 and EAR3/COUP-TFI contributes to gonadotropin-induced derepression of LHR promoter activity in granulosa cells.
Serine Phosphorylation of Insulin Receptor Substrate-1: A Novel Target for the Reversal of Insulin Resistance
Authors: Sykiotis Gerasimos P. , Papavassiliou Athanasios G. 
Published: January 2014

Abstract
Insulin resistance, the failure to respond to normal circulating concentrations of insulin, is a common state associated with obesity, aging, and a sedentary lifestyle. Compelling evidence implicates TNFα as the cause and link between obesity and insulin resistance. Serine phosphorylation of insulin receptor substrate-1 seems prominent among the mechanisms of TNFα-induced insulin resistance. Recent advances indicate that serine kinases may phosphorylate and thus inhibit the tyrosine phosphorylation of insulin receptor substrate-1, revealing an integration point of TNFα and insulin signaling pathways. Selective targeting of the molecular scenery whereby this key phosphorylation occurs/operates represents a rich area for the development of rationally designed new antidiabetic drugs. In relation to efficacy and side effects, this prospect should permit a more precise and perhaps individualized approach to therapeutic intervention, allowing clinicians to focus the attack where the problem lies.
Stage-Sensitive Blockade of Pituitary Somatomammotrope Development by Targeted Expression of a Dominant Negative Epidermal Growth Factor Receptor in Transgenic Mice
Authors: Roh Meejeon , Paterson Andrew J. , Asa Sylvia L. , Chin Edward , Kudlow Jeffrey E. 
Published: January 2014

AbstractThe epidermal growth factor receptor (EGFR) and its ligands EGF and transforming growth factor-α (TGFα) are expressed in the anterior pituitary, and overexpression of TGFα in the lactotrope cells of the pituitary gland in transgenic mice results in lactotrope hyperplasia and adenomata, suggesting a role for EGFR signaling in pituitary cell proliferation. To address the role of EGFR signaling in pituitary development in vivo, we blocked EGFR signaling in transgenic mice using the dominant negative properties of a mutant EGFR lacking an intracellular protein kinase domain (EGFR-tr). We directed EGFR-tr expression to GH- and PRL- producing cells using GH and PRL promoters, and a tetracycline-inducible gene expression system, to allow temporal control of gene expression. EGFR-tr overexpression in GH-producing cells during embryogenesis resulted in dwarf mice with pituitary hypoplasia. Both somatotrope and lactotrope development were blocked. However, when EGFR-tr overexpression was delayed to the postnatal period either by directing its expression with the PRL promoter or by delaying the onset of induction with tetracycline in the GH cells, no specific phenotype was observed. Lactotrope hyperplasia during pregnancy also occurred normally in the PRL-EGFR-tr mice. These data suggest that EGFR signaling is required for the differentiation and/or maintenance of somatomammotropes early in pituitary organogenesis but not later in life. (Molecular Endocrinology 15: 600–613, 2001)