bluuebunny/crossref_metadata_2025 huggingface dataset:
An X-Linked NDI Mutation Reveals a Requirement for Cell Surface V2R Expression
Authors: Sadeghi Hamid M. , Innamorati Giulio , Birnbaumer Mariel
Published: January 2014

Function and biochemical properties of the V2 vasopressin receptor (V2R) mutant R337ter, identified in patients suffering from X-linked recessive nephrogenic diabetes insipidus, were investigated by expression in COS.M6 or HEK293 cells. Binding assays and measurements of adenylyl cyclase activity failed to detect function for the truncated receptor, although metabolic labeling demonstrated normal levels of protein synthesis. ELISA assays performed on cells expressing the receptors tagged at the amino terminus with the HA epitope failed to detect V2R R337ter on the plasma membrane. Treatment with endoglycosidase H revealed that the receptor was present only as a precursor form because the mature R337ter V2R, resistant to endoglycosidase H treatment, was not detected. The precursor of V2R-R337ter had a longer half-life than that of the wild type V2R, suggesting that arrested maturation may slow the degradation of the precursor. Unrelated experiments had demonstrated that V2R-G345ter, containing eight additional amino acids, was expressed on the plasma membrane and functioned normally. Receptor truncations longer than 337ter revealed that four of the eight amino acids identified initially provided the minimum length required for the protein to acquire cell surface expression. This was shown by the production of mature receptor (V2R-341ter) detectable in SDS-PAGE, which mediated arginine vasopressin stimulation of adenylyl cyclase activity and bound ligand. In addition, the identity of amino acid 340 was found to play a role in this phenomenon. In conclusion, these data demonstrate that the V2R R337ter is nonfunctional because it does not reach the plasma membrane and that the minimal protein length required for translocation of the V2R to the cell surface is sufficient to confer function to the receptor protein. They also suggest the existence of a protein quality control in the endoplasmic reticulum independent of glycosylation.
Progesterone Receptor (PR) Inhibits Expression of Insulin-Like Growth Factor-Binding Protein-1 (IGFBP-1) in Human Endometrial Cell Line HEC-1B: Characterization of the Inhibitory Effect of PR on the Distal Promoter Region of the IGFBP-1 Gene
Authors: Gao Jiaguo , Tseng Linda
Published: January 2014

Progestin has been shown to have both stimulatory and inhibitory effects on the expression of insulin-like growth factor binding protein-1 (IGFBP-1) in human endometrial cells. In this study, progestin was found to reduce levels of secreted IGFBP-1 and IGFBP-1 messenger RNA and IGFBP-1 promoter activity after stably transfecting a progesterone receptor (PR; B form) expression vector into HEC-1B cells. Deletion analysis of the IGFBP-1 promoter revealed that PR specifically inhibited promoter activity derived from a 59-bp distal BsaHI/RsaI fragment. It was concluded that PR inhibited the promoter activity through protein-protein interactions based on the facts that 1) no progesterone-responsive element was revealed by a series block mutation in the BsaHI/RsaI fragment; 2) PR bound by the antiprogesterone ZK98299 inhibited IGFBP-1 promoter activity; 3) a DNA-binding mutant of PR inhibited the IGFBP-1 promoter activity; and 4) in an in vivo competition assay, the DNA-binding domain of PR did not release the inhibitory effect of intact PR. Analysis of PR deletion mutants indicated that both transcriptional activation domains of PR (TAF-1 and TAF-2) were involved in the inhibition of IGFBP-1 expression. Thus, our data may explain the superinduction of IGFBP-1 in human endometrial cells after progestin withdrawal or progestin replacement with antiprogestin.
Growth Hormone Stimulates Transcription of the Gene Encoding the Acid-Labile Subunit (ALS) of the Circulating Insulin-Like Growth Factor-Binding Protein Complex and ALS Promoter Activity in Rat Liver
Authors: Ooi Guck T. , Cohen Fredric J. , Tseng Lucy Y.-H. , Rechler Matthew M. , Boisclair Yves R.
Published: January 2014

The growth-promoting activity of GH, the principal hormonal determinant of body size, is mediated by insulin-like growth factor I (IGF-I). Most of the IGF-I in plasma circulates in a 150-kDa complex that contains IGF-binding protein-3 (IGFBP-3) and an acid-labile subunit (ALS). The 150-kDa complex serves as a reservoir of IGF-I and determines its bioavailability to the tissues. Formation of the 150-kDa complex depends upon the synthesis of ALS, which is synthesized primarily in liver and is regulated by GH. The present study demonstrates that GH stimulates ALS gene transcription in rat liver and ALS promoter activity in a rat hepatoma cell line. ALS messenger RNA (mRNA) and ALS nuclear transcripts were decreased to similar extents in the livers of GH-deficient hypophysectomized rats. GH increased hepatic ALS mRNA within 3–4 h to about 65% of the levels seen in sham-operated control rats. To confirm that GH stimulated ALS gene transcription, we transiently transfected an ALS promoter-luciferase reporter gene construct into H4-II-E rat hepatoma cells and primary rat hepatocytes. Recombinant human GH (hGH) stimulated promoter activity about 3-fold. In contrast, basal promoter activity was lower, and GH stimulation was absent when the ALS reporter construct was transfected into GH-responsive 3T3-F442A mouse preadipocyte fibroblasts. GH stimulation of ALS promoter activity in H4-II-E cells was mediated by functional GH receptors; nonprimate (rat and bovine) GH gave identical stimulation to hGH, and stimulation by hGH occurred at physiological concentrations. Reverse transcriptase-PCR analysis indicated that GH receptor mRNA was present in H4-II-E cells at approximately 40% of the level seen in rat liver. GH also induced the expression of the endogenous c-fos gene, indicating that the signaling pathway necessary for the activation of gene expression by GH was intact in H4-II-E cells. Thus, H4-II-E cells are a GH-responsive liver cell line that should provide a useful system in which to study the molecular mechanism of transcriptional regulation by GH of ALS and other hepatic genes.
A 6-kb Promoter Fragment Mimics in Transgenic Mice the Prostate-Specific and Androgen-Regulated Expression of the Endogenous Prostate-Specific Antigen Gene in Humans
Authors: Cleutjens Kitty B. J .M. , van der Korput Hetty A. G. M. , Ehren-van Eekelen Conny C. , Sikes Robert A. , Fasciana Claudia , Chung Leland W. , Trapman Jan
Published: January 2014

Prostate-specific antigen (PSA) is a kallikrein-like serine protease, which is almost exclusively synthesized in the luminal epithelial cells of the human prostate. PSA expression is androgen regulated. Previously, we characterized in vitro the proximal promoter, and a strong enhancer region, approximately 4 kb upstream of the PSA gene. Both regions are needed for high, androgen-regulated activity of the PSA promoter in LNCaP cells. The goal of the present study is the in vivo characterization of the PSA promoter. Three transgenic mouse lines carrying the Escherichia coli LacZ gene, driven by the 632-bp proximal PSA promoter, and three lines with LacZ, driven by the 6-kb PSA promoter, were generated. Expression of the LacZ reporter gene was analyzed in a large series of tissues. Transgene expression could not be demonstrated in any of the transgenic animals carrying the proximal PSA promoter. All three lines carrying the 6-kb PSA promoter showed lateral prostate-specific β-galactosidase activity. Transgene expression was undetectable until 8 weeks after birth. Upon castration,β -galactosidase activity rapidly declined. It could be restored by subsequent androgen administration. A search for mouse PSA-related kallikrein genes expressed in the prostate led to the identification of mGK22, which was previously demonstrated to be expressed in the submandibular salivary gland. Therefore, the 6-kb PSA-LacZ transgene followed the expression pattern of the PSA gene in humans, which is almost completely prostate-specific, rather than that of mGK22 in mice. In conclusion, the 6-kb promoter fragment appears to contain most, if not all, information for androgen regulation and prostate specificity of the PSA gene.
Multiple Promoter Elements Contribute to Activity of the Follicle-Stimulating Hormone Receptor (FSHR) Gene in Testicular Sertoli Cells
Authors: Heckert Leslie L. , Daggett Melissa A. F. , Chen Jiangkai
Published: January 2014

The FSH receptor (FSHR) is expressed only in granulosa cells of the ovary and Sertoli cells of the testis. This highly specific pattern of gene expression asserts that transcriptional events unique to these two cell types are responsible for activation of the FSHR gene. We have characterized the promoter elements required for activity of the rat FSHR gene in a Sertoli cell line MSC-1, primary cultures of rat Sertoli cells, and two non-Sertoli cell lines. Transient transfection analysis of deletion and block replacement mutants identified several elements, both 5′ and 3′ to the transcriptional start sites, that are essential for full promoter activity in Sertoli cells. These studies confirmed the use of an important E box element (CACGTG), which had the single greatest impact on promoter function. Bases within the core CACGTG of the E box, as well as flanking sequences, were shown to be essential for its function. Electrophoretic mobility shift assays identified both upstream stimulatory factor 1 (USF1) and USF2 as primary components of the complexes binding the E box. Sequence requirements for USF binding in vitro modestly diverged from the sequence requirements for in vivo function of the element. Comparison of the E box binding proteins in different cell types revealed that similar proteins bind the E box in Sertoli and non-Sertoli cell lines. Extracts from primary cultures of rat and mouse Sertoli cells have a second E box-binding complex that cross-reacts with USF antibodies that is not present in the cell lines.
Inhibition of Estrogen Receptor Action by the Orphan Receptor SHP (Short Heterodimer Partner)
Authors: Seol Wongi , Hanstein Bettina , Brown Myles , Moore David D.
Published: January 2014

SHP (short heterodimer partner) is an unusual orphan receptor that lacks a conventional DNA-binding domain. Previous results have shown that it interacts with several other nuclear hormone receptors, including the retinoid and thyroid hormone receptors, and inhibits their ligand-dependent transcriptional activation. Here we show that SHP also interacts with estrogen receptors and inhibits their function. In mammalian and yeast two-hybrid systems as well as glutathione-S-transferase pull-down assays, SHP interacts specifically with estrogen receptor-α (ERα) in an agonist-dependent manner. The same assay systems using various deletion mutants of SHP map the interaction domain with ERα to the same SHP sequences required for interaction with the nonsteroid hormone receptors such as retinoid X receptor and thyroid hormone receptor. In transient cotransfection assays, SHP inhibits estradiol -dependent activation by ERα by about 5-fold. In contrast, SHP interacts with ERβ independent of ligand and reduces its ability to activate transcription by only 50%. These data suggest that SHP functions to regulate estrogen signaling through a direct interaction with ERα.
Structure and Regulation of the Human Growth Hormone-Releasing Hormone Receptor Gene
Authors: Petersenn Stephan , Rasch Anja C. , Heyens Maria , Schulte Heinrich M.
Published: January 2014

The GHRH receptor (GHRH-R) acts as a critical molecule for proliferation and differentiation of somatotrophic pituitary cells. A role in the pathogenesis of GH hypersecretion and GH deficiency has been implicated. We investigated structure and regulation of the human GHRH-R gene. A genomic clone including approximately 12 kb of 5′-flanking region was isolated. The gene is of complex structure consisting of more than 10 exons. Two kilobase pairs of the promoter were sequenced, and putative transcription factor binding sites were identified. The transcription start site was defined by ribonuclease protection assay. Transcriptional regulation was investigated by transient transfections using promoter fragments ranging in size from 108-1456 bp. GHRH-R promoter (1456 bp) directed high levels of luciferase expression in GH4 rat pituitary cells whereas no activity was detected in JEG3 chorion carcinoma cells or COS-7 monkey kidney cells. A minimal 202-bp promoter allowed pituitary-specific expression. Its activity in COS-7 cells is enhanced by cotransfection of the pituitary-specific transcription factor Pit-1. We did not find any regulation of the GHRH-R promoter by forskolin, phorbol-myristate-acetate, or T3. Glucocorticoids lead to a significant stimulation, and estrogen leads to a significant inhibition. Further mapping suggests a glucocorticoid-responsive element between −1456 and −1181 and an estrogen-responsive element between −202 and −108. These studies demonstrate the complex nature of the human GHRH-R gene and identify its 5′-flanking region. Furthermore, specific activity of the promoter and regulation by various hormones are demonstrated.
Enhanced Binding to the Molecular Chaperone BiP Slows Thyroglobulin Export from the Endoplasmic Reticulum
Authors: Muresan Zoia , Arvan Peter
Published: January 2014

To examine how binding of BiP (a molecular chaperone of the hsp70 family that resides in the endoplasmic reticulum) influences the conformational maturation of thyroglobulin (Tg, the precursor for thyroid hormone synthesis), we have developed a system of recombinant Tg stably expressed in wild-type Chinese hamster ovary (CHO) cells and CHO-B cells genetically manipulated for selectively increased BiP expression. The elevation of immunoreactive BiP in CHO-B cells is comparable to that seen during the unfolded protein response in the thyrocytes of certain human patients and animals suffering from congenital hypothyroid goiter with defective Tg. However, in CHO-B cells, we expressed Tg containing no mutations that induce misfolding (i.e. no unfolded protein response), so that levels of all other endoplasmic reticulum chaperones were normal. Increased availability of BiP did not accelerate Tg secretion; rather, the export of newly synthesized Tg was delayed. Tg detained intracellularly was concentrated in the endoplasmic reticulum. By coimmunoprecipitation, BiP exhibited enhanced binding to Tg in CHO-B cells. Moreover, two-dimensional gel analysis showed that BiP associated especially well with intracellular Tg containing mispaired disulfide bonds, thought to represent early Tg folding intermediates. An endoplasmic reticulum chaperone of the hsp90 family, GRP94, was also associated in Tg-chaperone complexes. The results suggest that increased binding of BiP to Tg leads to its delayed conformational maturation in the endoplasmic reticulum.
Transcription of the Rat Serine Protease Inhibitor 2.1 Gene in Vivo: Correlation with GAGA Box Promoter Occupancy and Mechanism of Cytokine-Mediated Down-Regulation
Authors: Simar-Blanchet Anne Emmanuelle , Legraverend Catherine , Thissen Jean Paul , Le Cam Alphonse
Published: January 2014

Two GH-response elements (GHREs) and a single glucocorticoid (GC)-response element were found to regulate activity of the rat serine protease inhibitor 2.1 gene (spi 2.1) promoter in vitro. To assess the physiological relevance of these observations, we have investigated the relationship existing between the level of spi 2.1 gene transcription, structural modifications of the chromatin, and in vivo nuclear protein-promoter interactions monitored by genomic footprinting, in control, hypophysectomized, and inflamed rats. We also addressed the mechanism of inflammation-mediated gene down-regulation. We found that a high level of spi 2.1 gene transcription correlates with hypersensitivity of the promoter to deoxyribonuclease I (DNase I) and maximal occupancy of the GAGA box (GHRE-I). The failure of GAGA-box binding proteins (GAGA-BPs) to interact with the GAGA box appears to result from an impairment in GH action due to its absence (i.e. hypophysectomized animals) or to the appearance of a cytokine-mediated GH-resistant state (i.e. inflamed rats) in liver. Unlike the GAGA box, signal transducer and activator of transcription (STAT) factor-binding sites included in the GHRE-II were never found to be protected against DNase I attack but displayed a differential DNase I reactivity depending on the level of gene transcription. Alterations in DNase I reactivity of the GC-response element region suggest that GC receptor-GC complexes may associate, in a transient manner, with the promoter in the actively transcribing control state. Taken together, our studies suggest a mechanism of spi 2.1 gene activation in vivo whereby the GH-dependent chromatin remodeling caused by or concomitant to the recruitment of GAGA-box binding proteins is the first compulsory and presumably predominant step.
A Nuclear Receptor Corepressor Modulates Transcriptional Activity of Antagonist-Occupied Steroid Hormone Receptor
Authors: Zhang Xun , Jeyakumar M. , Petukhov Sergei , Bagchi Milan K.
Published: January 2014

Synthetic steroid hormone antagonists are clinically important compounds that regulate physiological responses to steroid hormones. The antagonists bind to the hormone receptors, which are ligand-inducible transcription factors, and modulate their gene-regulatory activities. In most instances, a steroid receptor, such as progesterone receptor (PR) or estrogen receptor (ER), is transcriptionally inactive when complexed with an antagonist and competitively inhibits transactivation of a target steroid-responsive gene by the cognate hormone-occupied receptor. In certain cellular and promoter contexts, however, antagonist-occupied PR or ER acquires paradoxical agonist-like activity. The cellular mechanisms that determine the switch from the negative to the positive mode of transcriptional regulation by an antagonist-bound steroid receptor are unknown. We now provide strong evidence supporting the existence of a cellular inhibitory cofactor that interacts with the B form of human PR (PR-B) complexed with the antiprogestin RU486 to maintain it in a transcriptionally inactive state. In the presence of unliganded thyroid hormone receptor (TR) or ER complexed with the antiestrogen 4-hydroxytamoxifen, which presumably sequesters a limiting pool of the inhibitory cofactor, RU486-PR-B functions as a transcriptional activator of a progesterone-responsive gene even in the absence of hormone agonist. In contrast, hormone-occupied TR or ER fails to induce transactivation by RU486-PR-B. Recent studies revealed that a transcriptional corepressor, NCoR (nuclear receptor corepressor), interacts with unliganded TR but not with liganded TR. Interestingly, coexpression of NCoR efficiently suppresses the partial agonistic activity of antagonist-occupied PR-B but fails to affect transactivation by agonist-bound PR-B. We further demonstrate that RU486-PR-B interacts physically with NCoR in vitro. These novel observations suggest that the inhibitory cofactor that associates with RU486-PR-B and represses its transcriptional activity is either identical or structurally related to the corepressor NCoR. We propose that cellular mechanisms that determine the switch from the antagonistic to the agonistic activity of RU486-PR-B involve removal of the corepressor from the antagonist-bound receptor so that it can effect partial but significant gene activation.
The Basic Helix-Loop-Helix, Leucine Zipper Transcription Factor, USF (Upstream Stimulatory Factor), Is a Key Regulator of SF-1 (Steroidogenic Factor-1) Gene Expression in Pituitary Gonadotrope and Steroidogenic Cells
Authors: Harris Adrienne N. , Mellon Pamela L.
Published: January 2014

Tissue-specific expression of the mammalian FTZ-F1 gene is essential for adrenal and gonadal development and sexual differentiation. The FTZ-F1 gene encodes an orphan nuclear receptor, termed SF-1 (steroidogenic factor-1) or Ad4BP, which is a primary transcriptional regulator of several hormone and steroidogenic enzyme genes that are critical for normal physiological function of the hypothalamic-pituitary-gonadal axis in reproduction. The objective of the current study is to understand the molecular mechanisms underlying transcriptional regulation of SF-1 gene expression in the pituitary. We have studied a series of deletion and point mutations in the SF-1 promoter region for transcriptional activity in αT3–1 and LβT2 (pituitary gonadotrope), CV-1, JEG-3, and Y1 (adrenocortical) cell lines. Our results indicate that maximal expression of the SF-1 promoter in all cell types requires an E box element at −82/−77. This E box sequence (CACGTG) is identical to the binding element for USF (upstream stimulatory factor), a member of the helix-loop-helix family of transcription factors. Studies of the SF-1 gene E box element using gel mobility shift and antibody supershift assays indicate that USF may be a key transcriptional regulator of SF-1 gene expression.
Atrial Natriuretic Peptide Inhibits Calcium-Induced Steroidogenic Acute Regulatory Protein Gene Transcription in Adrenal Glomerulosa Cells
Authors: Cherradi Nadia , Brandenburger Yves , Rossier Michel F. , Vallotton Michel B. , Stocco Douglas M. , Capponi Alessandro M.
Published: January 2014

Atrial natriuretic peptide (ANP) is a potent inhibitor of mineralocorticoid synthesis induced in adrenal glomerulosa cells by physiological agonists activating the calcium messenger system, such as angiotensin II (Ang II) and potassium ion (K+). While the role of calcium in mediating Ang II- and K+-induced aldosterone production is clearly established, the mechanisms leading to blockade of this steroidogenic response by ANP remain obscure. We have used bovine adrenal zona glomerulosa cells in primary culture, in which an activation of the calcium messenger system was mimicked by a 2-h exposure to an intracellular high-calcium clamp. The effect of ANP was studied on the following parameters of the steroidogenic pathway: 1) pregnenolone and aldosterone production; 2) changes in cytosolic ([Ca2+]c) and mitochondrial ([Ca2+]m) Ca2+ concentrations, as assessed with targeted recombinant aequorin; 3) cholesterol content in outer mitochondrial membranes (OM), contact sites (CS), and inner membranes (IM); 4) steroidogenic acute regulatory (StAR) protein import into mitochondria by Western blot analysis; 5) StAR protein synthesis, as determined by[ 35S]methionine incorporation, immunoprecipitation, and SDS-PAGE; 6) StAR mRNA levels by Northern blot analysis with a StAR cDNA; 7) StAR gene transcription by nuclear run-on analysis.

While clamping Ca2+ at 950 nm raised pregnenolone output 3.5-fold and aldosterone output 3-fold, ANP prevented these responses with an IC50 of 1 nm and a maximal effect of 90% inhibition at 10 nm. In contrast, ANP did not affect the[ Ca2+]c or[ Ca2+]m changes occurring under Ca2+ clamp or Ang II stimulation in glomerulosa cells. The accumulation of cholesterol content in CS (139.7 ± 10.7% of control) observed under high-Ca2+ clamp was prevented by 10 nm ANP (92.4 ± 4% of control). Similarly, while Ca2+ induced a marked accumulation of StAR protein in mitochondria of glomerulosa cells to 218 ± 44% (n = 3) of controls, the presence of ANP led to a blockade of StAR protein mitochondrial import (113.3 ± 15.0%). This effect was due to a complete suppression of the increased[ 35S]methionine incorporation into StAR protein that occurred under Ca2+ clamp (94.5 ± 12.8% vs. 167.5 ± 17.3%, n = 3). Furthermore, while the high-Ca2+ clamp significantly increased StAR mRNA levels to 188.5 ± 8.4 of controls (n = 4), ANP completely prevented this response. Nuclear run-on analysis showed that increases in intracellular Ca2+ resulted in transcriptional induction of the StAR gene and that ANP inhibited this process.

These results demonstrate that Ca2+ exerts a transcriptional control on StAR protein expression and that ANP appears to elicit its inhibitory effect on aldosterone biosynthesis by acting as a negative physiological regulator of StAR gene expression.
Roles of Pathway-Specific and Inhibitory Smads in Activin Receptor Signaling
Authors: Lebrun Jean-Jacques , Takabe Kazuaki , Chen Yan , Vale Wylie
Published: January 2014

Activins and other members of the transforming growth factor-β-like superfamily of growth factors transduce their signals by interacting with two types of receptor serine/threonine kinases. The Smad proteins, a new family of intracellular mediators are involved in the signaling pathways of these receptors, but the initial stages of their activation as well as their specific functions remain to be defined. We report here that the pathway-specific Smad2 and 3 can form a complex with the activin receptor in a ligand-dependent manner. This complex formation is rapid but also transient. Indeed, soon after their association with the activin receptor, Smad2 and Smad3 are released into the cytoplasm where they interact with the common partner Smad4. These Smad complexes then mediate activin-induced transcription. Finally, we show that the inhibitory Smad7 can prevent the association of the two pathway-specific Smads with the activin receptor complex, thereby blocking the activin signal.
Human Insulin Receptor Substrate-2 (IRS-2) Is a Primary Progesterone Response Gene
Authors: Vaßen Lothar , Wegrzyn Wojciech , Klein-Hitpass Ludger
Published: January 2014

Elevated cAMP has been shown to unmask agonist activity of antiprogestin/antiglucocorticoid RU486. In our search for cellular target genes induced through this cross-talk mechanism, we identified human insulin receptor substrate-2 (IRS-2), a cytoplasmic signaling molecule that mediates effects of insulin, insulin-like growth factor-1 (IGF-I), and other cytokines by acting as a molecular adaptor between diverse receptor tyrosine kinases and downstream effectors. Our analysis of the regulation of IRS-2 in HeLa cell models shows that synergistic induction of IRS-2 by cAMP and RU486 can be mediated by progesterone receptors (PR) and glucocorticoid receptors (GR) and occurs through a relative slow mechanism that requires ongoing protein synthesis. Importantly, we demonstrate that IRS-2 mRNA is also inducible by progesterone, while glucocorticoid effects are only observed in the presence of cAMP. Up-regulation of IRS-2 by progesterone depends strictly on the presence of PR and occurs through a rapid mechanism, suggesting that it represents a primary transcriptional response. Furthermore, we show that expression of IRS-1, which also binds to receptors of insulin, IGF-I, and cytokines, is unaffected by progesterone. Thus, our results demonstrate that progesterone alters the ratio of IRS-1 and IRS-2 in PR-positive cells and implicate a mechanism through which progesterone can modulate the effects of insulin, IGF-I, and cytokines on cell proliferation, differentiation, and homeostasis.
Ternary Complex Factors Elk-1 and Sap-1a Mediate Growth Hormone-Induced Transcription of Egr-1 (Early Growth Response Factor-1) in 3T3-F442A Preadipocytes
Authors: Clarkson Richard W. E. , Shang Catherine A. , Levitt Linda K. , Howard Tammy , Waters Michael J.
Published: January 2014

In our search for transcription factors induced by GH, we have analyzed immediate early gene activation in a model of GH-dependent differentiation. Here we describe the activation of early growth response factor-1 (egr-1) in GH-stimulated 3T3-F442A preadipocytes and the transcription factors responsible for its transactivation. Binding activity of egr-1 in electrophoretic mobility shift assay (EMSA) increased transiently 1 h after GH stimulation, accompanied by a concomitant increase in egr-1 mRNA. egr-1 induction appeared not to be related to proliferation since it was amplified in quiescent preadipocytes at a time when cells were refractive to GH-stimulated DNA synthesis.

Truncations of the proximal 1 kb of the egr-1 promoter revealed that a 374-bp region (−624 to −250) contributes about 80% of GH inducibility in 3T3-F442A cells and approximately 90% inducibility in CHO-K1 cells. This region contains three juxtaposed SRE (serum response element)/Ets site pairs known to be important for egr-1 activity in response to exogenous stimuli. Site-specific mutations of individual SRE and Ets sites within this region each reduced GH inducibility of the promoter. Use of these site-specific mutations in EMSA showed that disruption of either Ets or SRE sites abrogated ternary complex formation at the composite sites. DNA binding of ternary complexes, but not binary complexes, in EMSA was rapidly and transiently increased by GH. EMSA supershifts indicated these ternary complexes contained serum response factor (SRF) and the Ets factors Elk-1 and Sap-1a. Coexpression of Sap-1a and Elk-1 resulted in a marked increase in GH induction of egr-1 promoter activity, although transfection with expression vectors for either Ets factor alone did not significantly enhance the GH response. We conclude that GH stimulates transcription of egr-1 primarily through activation of these Ets factors at multiple sites on the promoter and that stabilization of ternary complexes with SRF at these sites maximizes this response.
Determination of Peptide Contact Points in the Human Angiotensin II Type I Receptor (AT1) with Photosensitive Analogs of Angiotensin II
Authors: Laporte Stéphane A. , Boucard Antony A. , Servant Guy , Guillemette Gaétan , Leduc Richard , Escher Emanuel
Published: January 2014

To identify ligand-binding domains of Angiotensin II (AngII) type 1 receptor (AT1), two different radiolabeled photoreactive AngII analogs were prepared by replacing either the first or the last amino acid of the octapeptide by p-benzoyl-l-phenylalanine (Bpa). High yield, specific labeling of the AT1 receptor was obtained with the 125I-[Sar1,Bpa8]AngII analog. Digestion of the covalent 125I-[Sar1,Bpa8]AngII-AT1 complex with V8 protease generated two major fragments of 15.8 kDa and 17.8 kDa, as determined by SDS-PAGE. Treatment of the[ Sar1,Bpa8]AngII-AT1 complex with cyanogen bromide produced a major fragment of 7.5 kDa which, upon further digestion with endoproteinase Lys-C, generated a fragment of 3.6 kDa. Since the 7.5-kDa fragment was sensitive to hydrolysis by 2-nitro-5-thiocyanobenzoic acid, we circumscribed the labeling site of 125I-[Sar1,Bpa8]AngII within amino acids 285 and 295 of the AT1 receptor. When the AT1 receptor was photolabeled with 125I-[Bpa1]AngII, a poor incorporation yield was obtained. Cleavage of the labeled receptor with endoproteinase Lys-C produced a glycopeptide of 31 kDa, which upon deglycosylation showed an apparent molecular mass of 7.5 kDa, delimiting the labeling site of 125I-[Bpa1]AngII within amino acids 147 and 199 of the AT1 receptor. CNBr digestion of the hAT1 I165M mutant receptor narrowed down the labeling site to the fragment 166–199. Taken together, these results indicate that the seventh transmembrane domain of the AT1 receptor interacts strongly with the C-terminal amino acid of[ Sar1, Bpa8]AngII, whereas the N-terminal amino acid of[ Bpa1]AngII interacts with the second extracellular loop of the AT1 receptor.
SF-1 (Steroidogenic Factor-1) and C/EBPβ (CCAAT/Enhancer Binding Protein-β) Cooperate to Regulate the Murine StAR (Steroidogenic Acute Regulatory) Promoter
Authors: Reinhart Adam J. , Williams Simon C. , Clark Barbara J. , Stocco Douglas M.
Published: January 2014

The steroidogenic acute regulatory (StAR) protein mediates the rate-limiting step of steroidogenesis, which is the transfer of cholesterol to the inner mitochondrial membrane. In steroidogenic tissues, StAR expression is acutely regulated by trophic hormones through a cAMP second messenger pathway, leading to increased StAR mRNA levels within 30 min, reaching maximal levels after 4–6 h of stimulation. The molecular mechanisms underlying such regulation remain unknown. We have examined the StAR promoter for putative transcription factor-binding sites that may regulate transcription in a developmental and/or hormone-induced context. Through sequence analysis, deoxyribonuclease I (DNAse I) footprinting and electrophoretic mobility shift assays (EMSAs), we have identified two putative CCAAT/enhancer binding protein (C/EBP) DNA elements at −113 (C1) and −87 (C2) in the mouse StAR promoter. Characterization of these sites by EMSA indicated that C/EBPβ bound with high affinity to C1 and C2 was a low-affinity C/EBP site. Functional analysis of these sites in the murine StAR promoter showed that mutation of one or both of these binding sites decreases both basal and (Bu)2cAMP-stimulated StAR promoter activity in MA-10 Leydig tumor cells, without affecting the fold activation[ (Bu)2cAMP-stimulated/basal] of the promoter. Furthermore, we have demonstrated that these two C/EBP binding sites are required for steroidogenic factor-1 (SF-1)-dependent transactivation of the StAR promoter in a nonsteroidogenic cell line. These data indicate that in addition to SF-1, C/EBPβ is involved in the transcriptional regulation of the StAR gene and may play an important role in developmental and hormone-responsive regulation of steroidogenesis.
Hormone-Dependent Interaction between the Amino- and Carboxyl-Terminal Domains of Progesterone Receptor in Vitro and in Vivo
Authors: Tetel Marc J. , Giangrande Paloma H. , Leonhardt Susan A. , McDonnell Donald P. , Edwards Dean P.
Published: January 2014

Full transcriptional activation by steroid hormone receptors requires functional synergy between two transcriptional activation domains (AF) located in the amino (AF-1) and carboxyl (AF-2) terminal regions. One possible mechanism for achieving this functional synergy is a physical intramolecular association between amino (N-) and carboxyl (C-) domains of the receptor. Human progesterone receptor (PR) is expressed in two forms that have distinct functional activities: full-length PR-B and the amino-terminally truncated PR-A. PR-B is generally a stronger activator than PR-A, whereas under certain conditions PR-A can act as a repressor in trans of other steroid receptors. We have analyzed whether separately expressed N- (PR-A and PR-B) and C-domains [hinge plus ligand-binding domain (hLBD)] of PR can functionally interact within cells by mammalian two-hybrid assay and whether this involves direct protein contact as determined in vitro with purified expressed domains of PR. A hormone agonist-dependent interaction between N-domains and the hLBD was observed functionally by mammalian two-hybrid assay and by direct protein-protein interaction assay in vitro. With both experimental approaches, N-C domain interactions were not induced by the progestin antagonist RU486. However, in the presence of the progestin agonist R5020, the N-domain of PR-B interacted more efficiently with the hLBD than the N-domain of PR-A. Coexpression of steroid receptor coactivator-1 (SRC-1) and the CREB binding protein (CBP), enhanced functional interaction between N- and C-domains by mammalian two-hybrid assay. However, addition of SRC-1 and CBP in vitro had no influence on direct interaction between purified N- and C-domains. These results suggest that the interaction between N- and C-domains of PR is direct and requires a hormone agonist-induced conformational change in the LBD that is not allowed by antagonists. Additionally, coactivators are not required for physical association between the N- and C-domains but are capable of enhancing a functionally productive interaction. In addition, the more efficient interaction of the hLBD with the N-domain of PR-B, compared with that of PR-A, suggests that distinct interactions between N- and C-terminal regions contribute to functional differences between PR-A and PR-B.
Targeted Expression of Bcl-2 in Mouse Oocytes Inhibits Ovarian Follicle Atresia and Prevents Spontaneous and Chemotherapy-Induced Oocyte Apoptosis In Vitro
Authors: Morita Yutaka , Perez Gloria I. , Maravei Daniel V. , Tilly Kim I. , Tilly Jonathan L.
Published: January 2014

Members of the Bcl-2 family serve as central checkpoints for cell death regulation, and overexpression of Bcl-2 is known to inhibit apoptosis in many cell types. To determine whether targeted expression of Bcl-2 could be used to protect female germ cells from apoptosis, we generated transgenic mice expressing fully functional human Bcl-2 protein only in oocytes. Transgenic mice were produced using a previously characterized 480-bp fragment of the mouse zona pellucida protein-3 (ZP3) gene 5′-flanking region to direct oocyte-specific expression of a human bcl-2 complementary DNA. Immunohistochemical analyses using a human Bcl-2-specific antibody showed that transgene expression was restricted to growing oocytes and was not observed in the surrounding ovarian somatic cells or in any other nonovarian tissues. Histomorphometric analyses revealed that ovaries collected from transgenic female mice possessed significantly fewer atretic small preantral follicles compared with wild-type sisters, resulting in a larger population of healthy maturing follicles per ovary. However, the number of oocytes ovulated in response to exogenous gonadotropin priming and the number of pups per litter were not significantly different among wild-type vs. transgenic female mice. Nonetheless, oocytes obtained from transgenic mice and cultured in vitro were found to be resistant to spontaneous and anticancer drug-induced apoptosis. We conclude that targeted expression of Bcl-2 only in oocytes can be achieved as a means to convey resistance of the female germ line to naturally occurring and chemotherapy-induced apoptosis.
Adenovirus-Mediated Delivery of a Dominant Negative Estrogen Receptor Gene Abrogates Estrogen-Stimulated Gene Expression and Breast Cancer Cell Proliferation
Authors: Lazennec Gwendal , Alcorn Joseph L. , Katzenellenbogen Benita S.
Published: January 2014

Dominant negative estrogen receptors are transcriptionally inactive, altered forms of the estrogen receptor (ER) that can dimerize with the ER and have the potential to inactivate the biological functions of this receptor. Here, we provide the first report that adenoviral delivery of a dominant negative ER to ER-positive breast cancer cells is able to effectively suppress estrogen-stimulated cell proliferation and the hormonal induction of endogenous genes. We constructed recombinant adenoviral vectors expressing a dominant negative ER (S554 fs, Ad-fs) or, for comparison, antisense ER (Ad-AS), or the sense wild-type ER (Ad-WT). Expression of the dominant negative ER or antisense ER, but not wild-type ER, blocked estradiol stimulation of the estrogen-responsive genes pS2 and c-myc. The dominant negative ER also fully abolished the estradiol-induced increase in proliferation of MCF-7 breast cancer cells, as did the antisense ER. The antiproliferative effects of the dominant negative and antisense ERs are explained by an increase in the number of cells in the G0/G1 stage of the cell cycle and decrease in the number of cells in G2/M as determined by flow cytometry, and also by a significant increase in the percentage of cells undergoing apoptosis. Our data strongly support the idea that targeting ER action using recombinant viral delivery of dominant negative ERs is an effective way to suppress ER-positive breast cancer cell proliferation and suggests the potential attractiveness of dominant negative gene therapy approaches targeted to the ER for the treatment of hormone-responsive breast cancer.
The Nuclear Receptor Corepressor N-CoR Regulates Differentiation: N-CoR Directly Interacts with MyoD
Authors: Bailey Peter , Downes Michael , Lau Patrick , Harris Jonathan , Chen Shen Liang , Hamamori Yasuo , Sartorelli Vittorio , Muscat George E. O.
Published: January 2014

Classical ligand-activated nuclear receptors (e.g. thyroid hormone receptor, retinoic acid receptor), orphan nuclear receptors (e.g. Rev-erbAα/β), Mad/Max bHLH (basic helix loop helix)-LZ proteins, and oncoproteins, PLZF and LAZ3/BCL6, bind DNA and silence transcription by recruiting a repressor complex that contains N-CoR (nuclear receptor corepressor)/SMRT (silencing mediator of retinoic acid and thyroid hormone receptor), Sin3A/B, and HDAc-1/-2 proteins. The function of the corepressor, N-CoR, in the process of cellular differentiation and coupled phenotypic acquisition, has not been investigated. We examined the functional role of N-CoR in myogenesis (muscle differentiation), an ideal paradigm for the analysis of the determinative events that govern the cell’s decision to divide or differentiate. We observed that the mRNA encoding N-CoR was suppressed as proliferating myoblasts exited the cell cycle, and formed morphologically and biochemically differentiated myotubes. Exogenous expression of N-CoR (but not RIP13) in myogenic cells ablated 1) myogenic differentiation, 2) the expression of the myoD gene family that encode the myogenic specific bHLH proteins, and 3) the crucial cell cycle regulator, p21Waf-1/Cip-1 mRNA. Furthermore, N-CoR expression efficiently inhibits the myoD-mediated myogenic conversion of pluripotential C3H10T1/2 cells. We demonstrate that MyoD-mediated transactivation and activity are repressed by N-CoR. The mechanism involves direct interactions between MyoD and N-CoR; moreover, the interaction was dependent on the amino-terminal repression domain (RD1) of N-CoR and the bHLH region of MyoD. Trichostatin A treatment significantly stimulated the activity of MyoD by approximately 10-fold and inhibited the ability of N-CoR to repress MyoD-mediated transactivation, consistent with the involvement of the corepressor and the recruitment of a histone deacteylase activity in the process. This work demonstrates that the corepressor N-CoR is a key regulator of MyoD activity and mammalian differentiation, and that N-CoR has a multifaceted role in myogenesis.
Functional Analysis of the Mouse ICER (Inducible cAMP Early Repressor) Promoter: Evidence for a Protein That Blocks Calcium Responsiveness of the CAREs (cAMP Autoregulatory Elements)
Authors: Krueger Darcy A. , Mao Dailing , Warner Elizabeth A. , Dowd Diane R.
Published: January 2014

Although Ca2+ and cAMP mediate their effects through distinct pathways, both signals converge upon the phosphorylation of the cAMP response element (CRE) binding protein, CREB, thereby activating transcription of CRE-regulated genes. In WEHI7.2 thymocytes, cAMP increases the expression of the inducible cAMP early repressor (ICER) gene through CRE-like elements, known as cAMP autoregulatory elements (CAREs). Because Ca2+- and cAMP-mediated transcription converge in WEHI7.2 thymocytes, we examined the effect of Ca2+ fluxes on the expression of the ICER gene in these cells. Despite the presence of multiple CAREs within its promoter, ICER gene transcription was not activated by Ca2+. Moreover, Ca2+ attenuated the stimulatory effect of cAMP on ICER expression. Transient expression of reporter constructs demonstrated that when these CAREs were placed in a different DNA promoter context, the elements became responsive to Ca2+. Detailed studies using chimeric promoter constructs to map the region responsible for blocking the transcriptional response to Ca2+ indicated that a small portion of the ICER promoter was necessary for the effect. Southwestern blot analysis identified a 83-kDa nuclear protein that bound specifically to that region. The relative binding activity of the factor to the ICER promoter and mutant promoter sequences correlated with an inhibition of Ca2+-activated gene expression in WEHI7.2 cells. These data suggest that the factor functions as a putative Ca2+-activated repressor of CREB/CRE-mediated transcription. Thus, depending on the surrounding context in which the CRE is located, CREs of individual genes can be regulated separately by Ca2+ and cAMP despite the convergence of these two signaling pathways.
Synergistic Activation of the Prolactin Promoter by Vitamin D Receptor and GHF-1: Role of the Coactivators, CREB-Binding Protein and Steroid Hormone Receptor Coactivator-1 (SRC-1)
Authors: Castillo Ana I. , Jimenez-Lara Ana M. , Tolon Rosa M. , Aranda Ana
Published: January 2014

PRL gene expression is dependent on the presence of the pituitary-specific transcription factor GHF-1/Pit-1, which is transcribed in a highly restricted manner in cells of the anterior pituitary. In pituitary GH3 cells, vitamin D increases the levels of PRL transcripts and stimulates the PRL promoter. We have analyzed the role of GHF-1 and of the vitamin D receptor (VDR) to confer vitamin D responsiveness to the PRL promoter. For this purpose we have used nonpituitary HeLa cells, which do not express GHF-1. We found that VDR activates the PRL promoter both in a ligand-dependent and -independent manner through a sequence located between positions− 45/−27 in the proximal 5′-flanking region. This sequence also confers VDR and vitamin D responsiveness to a heterologous promoter. In the context of the PRL gene, VDR requires the presence of GHF-1 to activate the promoter. Truncation of the last 12 C-terminal amino acids of VDR, which contain the ligand-dependent activation function (AF2), abolishes regulation by vitamin D, suggesting that binding of coactivators to this region mediates ligand-dependent stimulation of the PRL promoter by the receptor. Indeed, expression of the coactivators, steroid hormone receptor coactivator-1 (SRC-1) and CREB-binding protein (CBP), significantly enhances the stimulatory effect of vitamin D mediated by the wild-type VDR but not by the AF2 mutant receptor. Furthermore, CBP also increases the activation of the PRL promoter by GHF-1 and the ligand-independent activation by both wild-type and mutant VDR.
Progesterone Induces Focal Adhesion in Breast Cancer Cells MDA-MB-231 Transfected with Progesterone Receptor Complementary DNA
Authors: Lin Valerie C.-L. , Ng Eng Hen , Aw Swee Eng , Tan Michelle G.-K. , Ng Esther H.-L. , Bay Boon Huat
Published: January 2014

Since the effects of progesterone are mediated mainly via estrogen-dependent progesterone receptor (PR), the expression of the effects of progesterone may be masked or overridden by the influence of estrogen under conditions in which priming with estrogens is required. We have established a PR-positive but estrogen receptor-α (ER-α) negative breast cancer cell model by transfecting PR cDNA into ER-α- and PR-negative MDA-MB-231 cells in order that the functions of progesterone can be studied independently of estrogens. We have demonstrated using this model that progesterone markedly inhibited cell growth. We have also discovered that progesterone induced remarkable changes in cell morphology and specific adhesion structures. Progesterone-treated cells became considerably more flattened and well spread than vehicle-treated control cells. This was associated with a striking increase of stress fibers, both in number and diameter, and increased focal contacts as shown by the staining of focal adhesion proteins paxillin and talin. There were also distinct increases in tyrosine phosphorylation of focal adhesion protein paxillin and focal adhesion kinase in association with increased focal adhesion. The staining of tyrosine-phosphorylated proteins was concentrated at focal adhesions in progesterone-treated cells. More interestingly, monoclonal antibody (Ab) to β1 integrin was able to inhibit progesterone-induced cell spreading and formation of actin cytoskeleton. To our knowledge, this is the first report describing a direct effect of progesterone in inducing spreading and adhesion of breast cancer cells, and β1-integrin appeared to play an essential role in the effect. It is known that the initial step of tumor metastasis is the breakaway of tumor cells from primary tumor mass when they lose the ability to attach. Hence, progesterone-induced cell spreading and adhesion may have significant implications in tumor metastasis.