Estrogens are used extensively to treat hot flashes in menopausal women. Some of the beneficial effects of estrogens in hormone therapy on the brain might be due to nongenomic effects in neurons such as the rapid stimulation of calcium oscillations. Most studies have examined the nongenomic effects of estrogen receptors (ER) in primary neurons or brain slices from the rodent brain. However, these cells can not be maintained continuously in culture because neurons are post-mitotic. Neurons derived from embryonic stem cells could be a potential continuous, cell-based model to study nongenomic actions of estrogens in neurons if they are responsive to estrogens after differentiation. In this study ER-subtype specific estrogens were used to examine the role of ERalpha and ERbeta on calcium oscillations in neurons derived from human (hES) and mouse embryonic stem cells. Unlike the undifferentiated hES cells the differentiated cells expressed neuronal markers, ERbeta, but not ERalpha. The non-selective ER agonist 17beta-estradiol (E(2)) rapidly increased [Ca2+]i oscillations and synchronizations within a few minutes. No change in calcium oscillations was observed with the selective ERalpha agonist 4,4',4''-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT). In contrast, the selective ERbeta agonists, 2,3-bis(4-Hydroxyphenyl)-propionitrile (DPN), MF101, and 2-(3-fluoro-4-hydroxyphenyl)-7-vinyl-1,3 benzoxazol-5-ol (ERB-041; WAY-202041) stimulated calcium oscillations similar to E(2). The ERbeta agonists also increased calcium oscillations and phosphorylated PKC, AKT and ERK1/2 in neurons derived from mouse ES cells, which was inhibited by nifedipine demonstrating that ERbeta activates L-type voltage gated calcium channels to regulate neuronal activity. Our results demonstrate that ERbeta signaling regulates nongenomic pathways in neurons derived from ES cells, and suggest that these cells might be useful to study the nongenomic mechanisms of estrogenic compounds.

The frequency of intrinsic pulsatile GnRH secretion from endogenous GnRH neurons and GT1 GnRH cell lines is stimulated by increased intracellular cAMP levels. The downstream molecules comprising the cAMP signaling pathway are organized in microdomains by a family of scaffolding proteins, A-kinase anchoring proteins (AKAPs). These molecules tether protein kinase A, cAMP-specific phosphodiesterases, phosphatases to known substrates. In neurons AKAP150 organizes many of the signaling molecules known to regulate the excitability and intrinsic pulsatile activity of GnRH neurons. AKAP150 was expressed in both the GT1-1 and GT1-7 cells. We determined the role of AKAP150 in coordinating GT1-1 cell excitability and intrinsic GnRH pulsatile secretion by lowering AKAP150 levels with a small interfering RNA (siRNA) adenovirus construct to AKAP150 (Ad-AKAP150-siRNA). Infection with Ad-AKAP150-siRNA specifically decreased AKAP150 mRNA levels by 74% and protein levels by 53% relative to uninfected cells or cells infected with a luciferase control adenovirus siRNA vector. In GT1 cells, spontaneous Ca(2+) oscillations, an index of neuron excitability, are stimulated by increased levels of intracellular cAMP and lowered by decreased levels. The frequency of spontaneous Ca(2+) oscillations in Ad-AKAP150-siRNA-treated GT1-1 cells decreased by 47.2% relative to controls. A dramatic decrease in the number of spontaneous GnRH pulses was also observed after infection with Ad-AKAP150-siRNA. The interpulse interval increased to 143 +/- 20.25 min in Ad-AKAP150-siRNA infected cells from 32.2 +/- 7.3 min in luciferase control adenovirus siRNA vector-infected cells. These data demonstrate an important role of AKAP150 in coordinating signaling events regulating the frequency of intrinsic pulsatile GnRH secretion.

We have previously shown that the 16-kDa N-terminal fr gment of human prolactin (16K hPRL) has antiangiogenic properties, including the ability to induce apoptosis in vascular endothelial cells. Here, we examined whether the NF -κB signaling pathway was involved in mediating the apoptotic action of 16K hPRL in bovin e adrenal cortex capillary endothelial (BACE) cells. In a dose-dependent manner, treatmen t with 16K hPRL induced I κB-α degradation permitting translocation of NFκB to the nucleus and reporter gene activation. Inhibition of NF-κB activation by overexpression of a non-degradable IκB-α mutant or treatment with NFκB inhibitors blocked 16K hPRL-induced apoptosis. Tr eatment with 16K hPRL activated the initiator caspases 8 and 9 and t he effector caspase 3, all of which were essential for stimulation of DNA fragmentation. Thi s activation of the caspases cascade by 16K hPRL was also NFκB-dependent. These findings support the conclusion that NF-κB signaling plays a central role in 16K hPRL-induced apoptosis in vascular endothelial cells.

Other Types| November 19 2008 Remembrances of Claude Kordon Subject Area: Endocrinology , Neurology and Neuroscience Richard Weiner; Richard Weiner aDepartment of Obstetrics and Gynecology, Reproductive Sciences, UCSF, San Francisco, Calif., USA; Search for other works by this author on: This Site PubMed Google Scholar Jacques Epelbaum; Jacques Epelbaum bINSERM, Paris, and Search for other works by this author on: This Site PubMed Google Scholar Alain Enjalbert Alain Enjalbert cICNE UMR 6544, CNRS, Institut Jean Roche, Faculté de Médecine, Université de la Méditerranée, Marseille, France Search for other works by this author on: This Site PubMed Google Scholar Neuroendocrinology (2008) 88 (4): 243–245. https://doi.org/10.1159/000174144 Article history Published Online: November 19 2008 Content Tools Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Tools Icon Tools Get Permissions Cite Icon Cite Search Site Citation Richard Weiner, Jacques Epelbaum, Alain Enjalbert; Remembrances of Claude Kordon. Neuroendocrinology 1 November 2008; 88 (4): 243–245. https://doi.org/10.1159/000174144 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsNeuroendocrinology Search Advanced Search Article PDF first page preview Close Modal 2008Copyright / Drug Dosage / DisclaimerCopyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements. You do not currently have access to this content.

Cultures of endogenous GnRH neurons and the GT1 GnRH neuronal cell line release GnRH in pulses (intrinsic pulsatile release) with an interpulse frequency similar to that seen in castrated animals. In both GT1 cells and transgenic rats, lowering cAMP levels by expression of a phosphodiesterase decreased the frequency of intrinsic GnRH pulsatility. We asked whether the cyclic nucleotide-gated cation (CNG) channels expressed in GT1 cells participated in cAMP modulation of intrinsic GnRH pulsatility. Because expression of the CNGA2 subunit is essential for formation of functional CNG channels, we developed an adenovirus (Ad) vector expressing a short interference RNA (siRNA) to the CNGA2 subunit (Ad-CNG-siRNA) or as an infection control, to the coding region of luciferase (Ad-Luc-siRNA). Infection with the Ad-CNG-siRNA of COS cells transfected with a CNGA2 expression vector significantly inhibited CNGA2 protein levels by 74% by Western blot. Infection of GT1-1 cells with Ad-CNG-siRNA resulted in a 68% decrease in the levels of CNGA2 mRNA, a 44% decrease in protein levels, and a clear decrease in immunostaining with an antibody to CNGA2. Infection of GT1-1 cells with Ad-CNG-siRNA decreased spontaneous Ca2+ oscillations compared with Ad-Luc-siRNA-infected or uninfected cells by 71%. Furthermore infection with Ad-CNG-siRNA resulted in a 2-fold increase in the interpulse interval in GnRH secretion (49.4+/-9.1 min) compared with uninfected cells (25.9+/-2.5 min) or Ad-Luc-siRNA (29.3+/-2.8 min)-infected cells. These data provide the first direct evidence that the CNG channel is a downstream signaling molecule in the regulation of the frequency of intrinsic GnRH pulsatility by cAMP.

In some species such as flies, worms, frogs and fish, the key to forming and maintaining early germ cell populations is the assembly of germ plasm, microscopically distinct egg cytoplasm that is rich in RNAs, RNA-binding proteins and ribosomes. Cells which inherit germ plasm are destined for the germ cell lineage. In contrast, in mammals, germ cells are formed and maintained later in development as a result of inductive signaling from one embryonic cell type to another. Research advances, using complementary approaches, including identification of key signaling factors that act during the initial stages of germ cell development, differentiation of germ cells in vitro from mouse and human embryonic stem cells and the demonstration that homologs of germ plasm components are conserved in mammals, have shed light on key elements in the early development of mammalian germ cells. Here, we use FRET (Fluorescence Resonance Energy Transfer) to demonstrate that living mammalian germ cells possess specific RNA/protein complexes that contain germ plasm homologs, beginning in the earliest stages of development examined. Moreover, we demonstrate that, although both human and mouse germ cells and embryonic stem cells express the same proteins, germ cell-specific protein/protein interactions distinguish germ cells from precursor embryonic stem cells in vitro; interactions also determine sub-cellular localization of complex components. Finally, we suggest that assembly of similar protein complexes may be central to differentiation of diverse cell lineages and provide useful diagnostic tools for isolation of specific cell types from the assorted types differentiated from embryonic stem cells.

Increasing evidence suggests that fibroblast growth factors (FGFs) are neurotrophic in GnRH neurons. However, the extent to which FGFs are involved in establishing a functional GnRH system in the whole organism has not been investigated. In this study, transgenic mice with the expression of a dominant-negative FGF receptor mutant (FGFRm) targeted to GnRH neurons were generated to examine the consequence of disrupted FGF signaling on the formation of the GnRH system. To first test the effectiveness of this strategy, GT1 cells, a GnRH neuronal cell line, were stably transfected with FGFRm. The transfected cells showed attenuated neurite outgrowth, diminished FGF-2 responsiveness in a cell survival assay, and blunted activation of the signaling pathway in response to FGF-2. Transgenic mice expressing FGFRm in a GnRH neuron-specific manner exhibited a 30% reduction in GnRH neuron number, but the anatomical distribution of GnRH neurons was unaltered. Although these mice were initially fertile, they displayed several reproductive defects, including delayed puberty, reduced litter size, and early reproductive senescence. Overall, our results are the first to show, at the level of the organism, that FGFs are one of the important components involved in the formation and maintenance of the GnRH system.

Expression of a cAMP-specific phosphodiesterase in GnRH neurons in the GPR-4 transgenic rat resulted in decreased LH levels and pulse frequency and diminished fertility. We have characterized changes in fertility, adiposity, and reproductive and metabolic hormones with age. Although LH levels were decreased in 3-, 6-, and 9-month-old GPR-4 females relative to wild-type (WT) controls, GPR-4 females did not become anovulatory until 6 months of age. No differences were observed in FSH, estradiol, or androstenedione levels in 3-, 6-, or 9-month-old GPR-4 and WT females. At 9 months of age, GPR-4 females had significantly increased abdominal and sc fat depot weights that were associated with increased leptin and insulin levels not observed in WT females. We tested the hypothesis that metabolic changes observed at 9 months of age were the result of dysregulation of the mechanisms controlling energy balance. Two-month-old female GPR-4 rats placed on a high-energy diet gained weight at a rate significantly greater than WT females and, after 24 d, developed the same metabolic phenotype observed in 9-month-old GRP-4 females (increased abdominal and sc fat associated with elevated leptin and insulin concentrations). Overeating did not correlate with changes in estradiol or androstenedione levels. We conclude that decreased GnRH neuronal activity is closely associated with decreased reproductive function and dysregulation of food intake.

We report studies on the mechanism of action of bisphenol A (BPA) on the differentiation of oligodendrocyte precursor cells (OPCs). Our results show that: (1) BPA inhibits the differentiation of OPCs induced by exposure to thyroid hormone (T3). (2) The effect is mediated through various mechanisms via the thyroid hormone receptor (TRβ1) which is considered to be responsible for OPC differentiation. (3) The action of BPA on OPC differentiation does not involve the FcRγ-Fyn-myelin basic protein (MBP) cascade as an inducer of OPC differentiation nor does it suppress CREB phosphorylation, which is considered to be induced by the T3-TR complex. (4) The presence of MBP isoforms (21.5, 18.5, 17.0 and 14.0 kDa) was detected in OPCs, and the expression of exon 2-containing isoforms (i.e. 17.0 and 21.5 kDa) was upregulated upon treatment with T3. In contrast, expression of MBP was inhibited by BPA.

PURPOSE To examine the ability and mechanism of the 16 kDa N-terminal fragment of human prolactin (16K hPRL) in the inhibition of abnormal retinal neovascularization. METHODS The 16K hPRL-encoding sequence was inserted into an adenoviral vector (16K-Ad). Western blot analysis verified the expression of 16K hPRL and inhibition of proliferation, confirming functional activity of the 16K hPRL in virus-infected adult bovine aortic endothelial (ABAE) cells. 16K hPRL inhibited retinal neovascularization in a mouse model of oxygen-induced retinopathy. The ability of recombinant 16K hPRL expressed in E. coli (r16K hPRL) was compared to that of endostatin in inducing apoptosis of cultured human retinal endothelial cells (HREC). RESULTS 16K was expressed in virus-infected ABAE cells and resulted in a dose-dependent inhibition of cell proliferation. Eyes injected with 16K-Ad showed a reduction in preretinal neovascularization of 82.3 +/- 9.3% (P < 0.00001) when compared to uninjected controls. r16K hPRL was 100 times more potent than endostatin in inducing apoptosis in HRECs. CONCLUSIONS Intravitreal administration of 16K hPRL inhibited neovascularization in the mouse model of oxygen-induced retinopathy. 16K hPRL stimulated apoptosis in HRECs and inhibited cell proliferation in ABAE cells. These results suggested a potential therapeutic role for 16K hPRL in the treatment of proliferative retinopathies.

Pharmacologically increasing cyclic adenosine monophosphate (cAMP) levels in GT1 gonadotropin-releasing hormone (GnRH) cell lines increased the secretion of GnRH. Dopamine (DA) increased the GnRH secretion in GT1 cells via a DA receptor positively coupled to adenylate cyclase. We then asked whether inhibition of the DA-induced increase in cAMP would block the stimulatory effect of DA on GnRH release. Expression of the cAMP-specific phosphodiesterase (PDE4D1) was used in a genetic approach to inhibit the DA-induced increase in cAMP levels. Cells were infected with an adenovirus vector (Ad) expressing PDE4D1 (PDE-Ad) or, for controls, with an empty Ad (Null-Ad). Infection with the PDE-Ad completely blocked the forskolin-induced stimulation of GnRH secretion and [Ca2+]i and decreased the majority of the release of cAMP into the culture medium. In contrast, although PDE-Ad infection blocked virtually all of the DA-induced increase in extracellular cAMP, the release of GnRH and the increase in [Ca2+]i were only delayed for approximately 15 min. GT1 cells express the D1 DA receptor which is positively coupled to adenylate cyclase but not the D5 DA receptor. These data suggest that the initial phase of the DA-induced secretion of GnRH is dependent on an increase in cAMP levels. However, it appears that an additional non-cAMP-regulated signaling pathway is involved in the stimulation of GnRH release via the D1 DA receptor.

We have previously shown that the 16-kDa N-terminal fragment of human prolactin (16K hPRL) has antiangiogenic properties, including the ability to induce apoptosis in vascular endothelial cells. Here, we examined whether the nuclear factor-kappaB (NF-kappaB) signaling pathway was involved in mediating the apoptotic action of 16K hPRL in bovine adrenal cortex capillary endothelial cells. In a dose-dependent manner, treatment with 16K hPRL induced inhibitor kappaB-alpha degradation permitting translocation of NF-kappaB to the nucleus and reporter gene activation. Inhibition of NF-kappaB activation by overexpression of a nondegradable inhibitor kappaB-alpha mutant or treatment with NF-kappaB inhibitors blocked 16K hPRL-induced apoptosis. Treatment with 16K hPRL activated the initiator caspases-8 and -9 and the effector caspase-3, all of which were essential for stimulation of DNA fragmentation. This activation of the caspase cascade by 16K hPRL was also NF-kappaB dependent. These findings support the conclusion that NF-kappaB signaling plays a central role in 16K hPRL-induced apoptosis in vascular endothelial cells.

Genetic targeting of the cAMP-specific phosphodiesterase 4D1 (PDE4D1) to gonadotropin-releasing hormone (GnRH) neurons in the GPR-4 transgenic rat resulted in decreased luteinizing hormone (LH) pulse frequency in castrated female and male rats. A similar decrease in the intrinsic GnRH pulse frequency was observed in GT1 GnRH cells expressing the PDE4D1 phosphodiesterase. We have extended these findings in ovariectomized (OVX) GPR-4 rats by asking what effect transgene expression had on pulsatile LH and follicle-stimulating hormone (FSH) secretion, plasma and pituitary levels of LH and FSH, and levels of the α-glycoprotein hormone subunit (α-GSU), LH-β and FSH-β subunit mRNAs. In OVX GPR-4 rats the LH pulse frequency but not pulse amplitude was decreased by 50% compared to wild-type littermate controls. Assaying the same samples for FSH, the FSH pulse frequency and amplitude were unchanged. The plasma and anterior pituitary levels of LH in the GPR-4 rats were significantly decreased by approximately 45%, while the plasma but not anterior pituitary level of FSH was significantly decreased by 25%. As measured by real-time RT-PCR, the mRNA levels for the α-GSU in the GPR-4 rats were significantly decreased by 41%, the LH-β subunit by 38% and the FSH-β subunit by 28%. We conclude that in the castrated female GPR-4 rats the decreased GnRH pulse frequency results in decreased levels of LH and FSH and in the α- and β-subunit mRNA levels.

Pulsatile GnRH secretion is an intrinsic property of GnRH neurons. Since increases in cAMP levels increase excitability and GnRH secretion in the GT1-1 GnRH cell line, we asked whether cAMP levels play a role in timing excitability and intrinsic pulsatile GnRH secretion. The expression of the cAMP-specific phosphodiesterase (PDE4D1) was used in a genetic approach to lower cAMP levels. Cells were infected with an adenovirus vector (Ad) expressing PDE4D1 (PDE-Ad), or for controls with an empty Ad (Null-Ad) or an Ad expressing green fluorescent protein (GFP-Ad). Infection with the PDE-Ad significantly inhibited forskolin-induced increases in cAMP production, GnRH secretion, and Ca2+ oscillations. Infection of GT1-1 cells with the PDE-Ad vs. GFP-Ad or Null-Ad controls significantly decreased spontaneous Ca2+ oscillations and inhibited the frequency of GnRH pulses. These data support the hypothesis that the level of cAMP in GT1 neurons is a component of the biological clock timing neuron excitability and pulsatile GnRH secretion. Genetically targeted expression of PDE4D1 represents a powerful approach to study the role of cAMP levels in specific populations of neurons in transgenic animals.