scholarly journals Estradiol Stimulates Progesterone Synthesis in Hypothalamic Astrocyte Cultures

Endocrinology ◽  
2007 ◽  
Vol 148 (2) ◽  
pp. 782-789 ◽  
Author(s):  
Paul E. Micevych ◽  
Victor Chaban ◽  
Julie Ogi ◽  
Phoebe Dewing ◽  
John K. H. Lu ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
De Novo ◽  
Regulatory Protein ◽  
Estradiol Treatment ◽  
Lh Surge ◽  
Ici 182,780 ◽  
Chain Cleavage ◽  
Intracellular Release ◽  
Progesterone Synthesis

The brain synthesizes steroids de novo, especially progesterone. Recently estradiol has been shown to stimulate progesterone synthesis in the hypothalamus and enriched astrocyte cultures derived from neonatal cortex. Estradiol-induced hypothalamic progesterone has been implicated in the control of the LH surge. The present studies were undertaken to determine whether hypothalamic astrocytes derived from female neonatal or female postpubertal rats increased production of progesterone in response to an estradiol challenge. Estradiol induced progesterone synthesis in postpubertal astrocytes but not neonatal astrocytes. This estradiol action was blocked by the estrogen receptor antagonist ICI 182,780. Previously we had demonstrated that estradiol stimulates a rapid increase in free cytosolic Ca2+ ([Ca2+]i) spikes in neonatal cortical astrocytes acting through a membrane estrogen receptor. We now report that estradiol also rapidly increased [Ca2+]i spikes in hypothalamic astrocytes. The membrane-impermeable estradiol-BSA construct also induced [Ca2+]i spikes. Both estradiol-BSA and estradiol were blocked by ICI 182,780. Depleting intracellular Ca2+ stores prevented the estradiol-induced increased [Ca2+]i spikes, whereas removing extracellular Ca2+ did not prevent estradiol-induced [Ca2+]i spikes. Together these results indicate that estradiol acts through a membrane-associated receptor to release intracellular stores of Ca2+. Thapsigargin, used to mimicked the intracellular release of Ca2+ by estradiol, increased progesterone synthesis, suggesting that estradiol-induced progesterone synthesis involves increases in [Ca2+]i. Estradiol treatment did not change levels of steroid acute regulatory protein, P450 side chain cleavage, 3β-hydroxysteroid dehydrogenase, and sterol carrier protein-2 mRNAs as measured by quantitative RT-PCR, suggesting that in vitro, estradiol regulation of progesterone synthesis in astrocytes does not depend on transcription of new steroidogenic proteins. The present results are consistent with our hypothesis that estrogen-positive feedback regulating the LH surge involves stimulating local progesterone synthesis by hypothalamic astrocytes.

scholarly journals Elacestrant (RAD1901) exhibits anti-tumor activity in multiple ER+ breast cancer models resistant to CDK4/6 inhibitors

2019 ◽  
Vol 21 (1) ◽  
Author(s):  
Hitisha K. Patel ◽  
Nianjun Tao ◽  
Kyung-Min Lee ◽  
Mariela Huerta ◽  
Heike Arlt ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Estrogen Receptor ◽  
De Novo ◽  
Single Agent ◽  
Metastatic Breast ◽  
Cancer Resistance ◽  
Treatment Paradigm ◽  
Anti Tumor Activity

Abstract Background Addition of CDK4/6 inhibitors (CDK4/6i) to endocrine therapy significantly increased progression-free survival, leading to their approval and incorporation into the metastatic breast cancer treatment paradigm. With these inhibitors being routinely used for patients with advanced estrogen receptor-positive (ER+) breast cancer, resistance to these agents and its impact on subsequent therapy needs to be understood. Considering the central role of ER in driving the growth of ER+ breast cancers, and thus endocrine agents being a mainstay in the treatment paradigm, the effects of prior CDK4/6i exposure on ER signaling and the relevance of ER-targeted therapy are important to investigate. The objective of this study was to evaluate the anti-tumor activity of elacestrant, a novel oral selective estrogen receptor degrader (SERD), in preclinical models of CDK4/6i resistance. Methods Elacestrant was evaluated as a single agent, and in combination with alpelisib or everolimus, in multiple in vitro models and patient-derived xenografts that represent acquired and “de novo” CDK4/6i resistance. Results Elacestrant demonstrated growth inhibition in cells resistant to all three approved CDK4/6i (palbociclib, abemaciclib, ribociclib) in both ESR1 wild-type and mutant backgrounds. Furthermore, we demonstrated that elacestrant, as a single agent and in combination, inhibited growth of patient-derived xenografts that have been derived from a patient previously treated with a CDK4/6i or exhibit de novo resistance to CDK4/6i. While the resistant lines demonstrate distinct alterations in cell cycle modulators, this did not affect elacestrant’s anti-tumor activity. In fact, we observe that elacestrant downregulates several key cell cycle players and halts cell cycle progression in vitro and in vivo. Conclusions We demonstrate that breast cancer tumor cells continue to rely on ER signaling to drive tumor growth despite exposure to CDK4/6i inhibitors. Importantly, elacestrant can inhibit this ER-dependent growth despite previously reported mechanisms of CDK4/6i resistance observed such as Rb loss, CDK6 overexpression, upregulated cyclinE1 and E2F1, among others. These data provide a scientific rationale for the evaluation of elacestrant in a post-CDK4/6i patient population. Additionally, elacestrant may also serve as an endocrine backbone for rational combinations to combat resistance.

scholarly journals Interaction of Vault Particles with Estrogen Receptor in the MCF-7 Breast Cancer Cell

1998 ◽  
Vol 141 (6) ◽  
pp. 1301-1310 ◽  
Author(s):  
Ciro Abbondanza ◽  
Valentina Rossi ◽  
Annarita Roscigno ◽  
Luigi Gallo ◽  
Angela Belsito ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Glucocorticoid Receptors ◽  
Sodium Molybdate ◽  
Nuclear Extract ◽  
Supernatant Fraction ◽  
Cdna Clones ◽  
Major Vault Protein ◽  
Estradiol Treatment ◽  
Mcf 7

A 104-kD protein was coimmunoprecipitated with the estrogen receptor from the flowtrough of a phosphocellulose chromatography of MCF-7 cell nuclear extract. mAbs to this protein identified several cDNA clones coding for the human 104-kD major vault protein. Vaults are large ribonucleoprotein particles of unknown function present in all eukaryotic cells. They have a complex morphology, including several small molecules of RNA, but a single protein species, the major vault protein, accounts for >70% of their mass. Their shape is reminiscent of the nucleopore central plug, but no proteins of known function have been described to interact with them. Western blot analysis of vaults purified on sucrose gradient showed the presence of estrogen receptor co-migrating with the vault peak. The AER317 antibody to estrogen receptor coimmunoprecipitated the major vault protein and the vault RNA also in the 20,000 g supernatant fraction. Reconstitution experiments of estrogen receptor fragments with the major vault protein mapped the site of the interaction between amino acids 241 and 280 of human estrogen receptor, where the nuclear localization signal sequences are located. Estradiol treatment of cells increased the amount of major vault protein present in the nuclear extract and coimmunoprecipitated with estrogen receptor, whereas the anti-estrogen ICI182,780 had no effect. The hormone-dependent interaction of vaults with estrogen receptor was reproducible in vitro and was prevented by sodium molybdate. Antibodies to progesterone and glucocorticoid receptors were able to coimmunoprecipitate the major vault protein. The association of nuclear receptors with vaults could be related to their intracellular traffic.

scholarly journals Chemerin, a Novel Regulator of Follicular Steroidogenesis and Its Potential Involvement in Polycystic Ovarian Syndrome

Endocrinology ◽  
2012 ◽  
Vol 153 (11) ◽  
pp. 5600-5611 ◽  
Author(s):  
Qi Wang ◽  
Ji Young Kim ◽  
Kai Xue ◽  
Jia-yin Liu ◽  
Arthur Leader ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Polycystic Ovarian Syndrome ◽  
Regulatory Protein ◽  
Hydroxysteroid Dehydrogenase ◽  
Negative Regulator ◽  
Estradiol Secretion ◽  
Chain Cleavage ◽  
Steroidogenic Acute Regulatory ◽  
Ovarian Syndrome

Abstract Polycystic ovarian syndrome (PCOS) is a heterogeneous syndrome associated with follicle growth arrest, minimal granulosa cell proliferation, dysregulated sex hormone profile, hyperthecosis, and insulin resistance. Using a 5α-dihydrotestosterone (DHT)-induced rat model that recapitulates the reproductive and metabolic phenotypes of human PCOS, we have examined the steroidogenic capability of granulosa cells from DHT-treated rats. Gene expression of several key steroidogenic enzymes including p450 side-chain cleavage enzyme (p450scc), aromatase, steroidogenic acute regulatory protein, hydroxysteroid dehydrogenase-17β, and hydroxysteroid dehydrogenase-3β were markedly lower in DHT-treated rats than the controls, although the responsiveness of their granulosa cells to FSH was higher. Expression of the adipokine chemerin and its receptor, chemokine receptor-like 1, was evident in control and DHT-treated rats, with significantly higher ovarian mRNA abundances and protein contents of chemerin and its receptor. Recombinant chemerin decreases basal estradiol secretion in granulosa cells from DHT-treated rats. When the inhibitory role of chemerin on steroidogenesis was further examined in vitro, chemerin suppressed FSH-induced progesterone and estradiol secretion in cultured preantral follicles and granulosa cells. Chemerin also inhibits FSH-induced aromatase and p450scc expression in granulosa cells. Overexpression of nuclear receptors NR5a1 and NR5a2 promotes p450scc and aromatase expression, respectively, which is suppressed by chemerin. These findings suggest that chemerin is a novel negative regulator of FSH-induced follicular steroidogenesis and may contribute to the pathogenesis of PCOS.

scholarly journals Neurosteroids and Female Reproduction: Estrogen Increases 3β-HSD mRNA and Activity in Rat Hypothalamus

Endocrinology ◽  
2005 ◽  
Vol 146 (10) ◽  
pp. 4386-4390 ◽  
Author(s):  
K. K. Soma ◽  
K. Sinchak ◽  
A. Lakhter ◽  
B. A. Schlinger ◽  
P. E. Micevych
Keyword(s):  
De Novo ◽  
Regulatory Protein ◽  
Feedback Mechanism ◽  
Estrogen Treatment ◽  
Ovariectomized Rats ◽  
Mrna Levels ◽  
Female Reproduction ◽  
Steroid Synthesis ◽  
Lh Surge ◽  
Adrenalectomized Rats

A central event in mammalian reproduction is the LH surge that induces ovulation and corpus luteum formation. Typically, the LH surge is initiated in ovariectomized rats by sequential treatment with estrogen and progesterone (PROG). The traditional explanation for this paradigm is that estrogen induces PROG receptors (PR) that are activated by exogenous PROG. Recent evidence suggests that whereas exogenous estrogen is necessary, exogenous PROG is not. In ovariectomized-adrenalectomized rats, estrogen treatment increases hypothalamic PROG levels before an LH surge. This estrogen-induced LH surge was blocked by an inhibitor of 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD), the proximal enzyme for PROG synthesis. These data indicate that estrogen induces de novo synthesis of PROG from cholesterol in the hypothalamus, which initiates the LH surge. The mechanism(s) by which estrogen up-regulates neuro-PROG is unknown. We investigated whether estrogen increases 1) mRNA levels for several proteins involved in PROG synthesis and/or 2) activity of 3β-HSD in the hypothalamus. In ovariectomized-adrenalectomized rats, estrogen treatment increased 3β-HSD mRNA in the hypothalamus, as measured by relative quantitative RT-PCR. The mRNAs for other proteins involved in steroid synthesis (sterol carrier protein 2, steroidogenic acute regulatory protein, and P450 side chain cleavage) were detectable in hypothalamus but not affected by estrogen. In a biochemical assay, estrogen treatment also increased 3β-HSD activity. These data support the hypothesis that PROG is a neurosteroid, produced locally in the hypothalamus from cholesterol, which functions in the estrogen positive-feedback mechanism driving the LH surge.

scholarly journals Prohibitin regulates the FSH signaling pathway in rat granulosa cell differentiation

2016 ◽  
Vol 56 (4) ◽  
pp. 325-336 ◽  
Author(s):  
Indrajit Chowdhury ◽  
Kelwyn Thomas ◽  
Anthony Zeleznik ◽  
Winston E Thompson
Keyword(s):  
Signaling Pathway ◽  
Morphological Changes ◽  
Regulatory Protein ◽  
Experimental Studies ◽  
Mitochondrial Fraction ◽  
Chain Cleavage ◽  
Cleavage Enzyme ◽  
Steroidogenic Acute Regulatory ◽  
Follicle Maturation

Published results from our laboratory identified prohibitin (PHB), a gene product expressed in granulosa cells (GCs) that progressively increases during follicle maturation. Our current in vitro studies demonstrate that follicle-stimulating hormone (FSH) stimulates Phb expression in rat primary GCs. The FSH-dependent expression of PHB was primarily localized within mitochondria, and positively correlates with the morphological changes in GCs organelles, and synthesis and secretions of estradiol (E2) and progesterone (P4). In order to confirm that PHB plays a regulatory role in rat GC differentiation, endogenous PHB-knockdown studies were carried out in undifferentiated GCs using adenoviral (Ad)-mediated RNA interference methodology. Knockdown of PHB in GCs resulted in the suppression of the key steroidogenic enzymes including steroidogenic acute regulatory protein (StAR), p450 cholesterol side-chain cleavage enzyme (p450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), and aromatase (Cyp19a1); and decreased E2 and P4 synthesis and secretions in the presence of FSH stimulation. Furthermore, these experimental studies also provided direct evidence that PHB within the mitochondrial fraction in GCs is phosphorylated at residues Y249, T258, and Y259 in response to FSH stimulation. The observed levels of phosphorylation of PHB at Y249, T258, and Y259 were significantly low in GCs in the absence of FSH stimulation. In addition, during GC differentiation FSH-induced expression of phospho-PHB (pPHB) requires the activation of MEK1-ERK1/2 signaling pathway. Taken together, these studies provide new evidence supporting FSH-dependent PHB/pPHB upregulation in GCs is required to sustain the differentiated state of GCs.

scholarly journals The RNA-mediated estrogen receptor α interactome of hormone-dependent human breast cancer cell nuclei

2019 ◽  
Vol 6 (1) ◽  
Author(s):  
Giovanni Nassa ◽  
Giorgio Giurato ◽  
Annamaria Salvati ◽  
Valerio Gigantino ◽  
Giovanni Pecoraro ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Estrogen Receptor ◽  
Estrogen Receptor Alpha ◽  
Molecular Mechanisms ◽  
Affinity Purification ◽  
Regulatory Protein ◽  
Estrogen Signaling ◽  
Target Cells ◽  
Cell Nuclei

Abstract Estrogen Receptor alpha (ERα) is a ligand-inducible transcription factor that mediates estrogen signaling in hormone-responsive cells, where it controls key cellular functions by assembling in gene-regulatory multiprotein complexes. For this reason, interaction proteomics has been shown to represent a useful tool to investigate the molecular mechanisms underlying ERα action in target cells. RNAs have emerged as bridging molecules, involved in both assembly and activity of transcription regulatory protein complexes. By applying Tandem Affinity Purification (TAP) coupled to mass spectrometry (MS) before and after RNase digestion in vitro, we generated a dataset of nuclear ERα molecular partners whose association with the receptor involves RNAs. These data provide a useful resource to elucidate the combined role of nuclear RNAs and the proteins identified here in ERα signaling to the genome in breast cancer and other cell types.

scholarly journals Estradiol Up-regulates AUF1p45 Binding to Stabilizing Regions within the 3′-Untranslated Region of Estrogen Receptor α mRNA

2007 ◽  
Vol 283 (3) ◽  
pp. 1764-1772 ◽  
Author(s):  
Nancy H. Ing ◽  
Dana A. Massuto ◽  
Laurie A. Jaeger
Keyword(s):  
Estrogen Receptor ◽  
Untranslated Region ◽  
Binding Proteins ◽  
Rna Binding ◽  
Regulation Of Gene Expression ◽  
Estrogen Receptor Α ◽  
Protein Isoforms ◽  
Dependent Manner ◽  
Estradiol Treatment

Estradiol up-regulates expression of the estrogen receptor α gene in the uterus by stabilizing estrogen receptor α mRNA. Previously, we defined two discrete minimal estradiol-modulated stability sequences (MEMSS) within the extensive 3′-untranslated region of estrogen receptor α mRNA with an in vitro stability assay using cytosolic extracts from sheep uterus. We report here that excess MEMSS RNA inhibited the enhanced stability of estrogen receptor α mRNA in extracts from estradiol-treated ewes compared with those from control ewes. Several estradiol-induced MEMSS-binding proteins were characterized by UV cross-linking in uterine extracts from ewes in a time course study (0, 8, 16, and 24 h after estradiol injection). The pattern of binding proteins changed at 16 h post-injection, concurrent with enhanced estrogen receptor α mRNA stability and the highest rate of accumulation of estrogen receptor α mRNA. The predominant MEMSS-binding protein induced by estradiol treatment was identified as AUF1 (A + U-rich RNA-binding factor 1) protein isoform p45 (a product of the heterogeneous nuclear ribonucleoprotein D gene). Immunoblot analysis indicated that only two of four AUF1 protein isoforms were present in the uterine cytosolic extracts and that estradiol treatment strongly increased the ratio of AUF1 isoforms p45 to p37. Nonphosphorylated recombinant AUF1p45 protected estrogen receptor α mRNA in vitro in a dose-dependent manner. These studies describe estrogenic induction of AUF1p45 binding to the estrogen receptor α mRNA as a molecular mechanism for post-transcriptional up-regulation of gene expression.

EFFECT OF AMINOGLUTETHIMIDE PHOSPHATE ON IN VITRO OESTRADIOL STIMULATED PROGESTERONE SYNTHESIS BY RABBIT CORPORA LUTEA

1978 ◽  
Vol 87 (1) ◽  
pp. 164-172 ◽  
Author(s):  
Gene B. Fuller ◽  
Barry M. Markaverich ◽  
William C. Hobson
Keyword(s):  
Cholesterol Synthesis ◽  
Side Chain ◽  
Corpora Lutea ◽  
Cholesterol Esters ◽  
Chain Cleavage ◽  
Progesterone Synthesis ◽  
Luteal Tissue ◽  
Time Periods ◽  
Sites Of Action

ABSTRACT Slices of rabbit corpora lutea were incubated for 3 and 8 h with oestradiol-17β in the presence or absence of aminoglutethimide phosphate (AGP)2) in order to identify a possible site(s) of action of oestrogen on progesterone synthesis. Oestradiol was unable to increase progesterone biosynthesis above controls in luteal tissue exposed to AGP during either incubation period. AGP alone significantly reduced progesterone concentrations and synthesis from [14C]acetate at both time periods while oestradiol alone increased progesterone mass at 8 h. Changes in 20α-OH progesterone typically paralleled those of progesterone at 3 h but no effect of the inhibitor was seen at 8 h. Except for an increase in 14C-incorporation into cholesterol esters in the AGP-treated groups at 3 h the concentration or synthesis of sterols from labelled acetate did not respond to treatment. These results suggested that cholesterol side chain cleavage and cholesterol synthesis are not major in vitro sites of action of oestradiol in the rabbit corpus luteum.

scholarly journals Hypoxic gene expression in chronic hepatitis B infected patients is not observed in state-of-art in vitro and mouse infection models

2020 ◽  
Author(s):  
PJ Liu ◽  
JM Harris ◽  
E Marchi ◽  
V D’Arienzo ◽  
T Michler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Hepatitis ◽  
Hepatitis B ◽  
Chronic Hepatitis B ◽  
De Novo ◽  
Regulatory Protein ◽  
Hbv Infection ◽  
Infection Models

ABSTRACTHepatitis B virus (HBV) is the leading cause of hepatocellular carcinoma (HCC) worldwide. The prolyl hydroxylase domain (PHD)-hypoxia inducible factor (HIF) pathway is a key mammalian oxygen sensing pathway and is frequently perturbed by pathological states including infection and inflammation. We discovered a significant upregulation of hypoxia regulated gene transcripts in patients with chronic hepatitis B (CHB) in the absence of liver cirrhosis. We used state-of-the-art in vitro and in vivo HBV infection models to evaluate a role for HBV infection and the viral regulatory protein HBx to drive HIF-signalling. HBx had no significant impact on HIF expression or associated transcriptional activity under normoxic or hypoxic conditions. Furthermore, we found no evidence of hypoxia gene expression in HBV de novo infection, HBV infected human liver chimeric mice or transgenic mice with integrated HBV genome. Collectively, our data show clear evidence of hypoxia gene induction in CHB that is not recapitulated in existing models for acute HBV infection, suggesting a role for inflammatory mediators in promoting hypoxia gene expression.

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