RESUMO
The gonadotropin-releasing hormone (GnRH) is the master regulator of fertility and kisspeptin (KP) is a potent trigger of GnRH secretion from GnRH neurons. KP signals via KISS1R, a Gαq/11-coupled receptor, and mice bearing a global deletion of Kiss1r (Kiss1r(-/-)) or a GnRH neuron-specific deletion of Kiss1r (Kiss1r(d/d)) display hypogonadotropic hypogonadism and infertility. KISS1R also signals via ß-arrestin, and in mice lacking ß-arrestin-1 or -2, KP-triggered GnRH secretion is significantly diminished. Based on these findings, we hypothesized that ablation of Gαq/11 in GnRH neurons would diminish but not completely block KP-triggered GnRH secretion and that Gαq/11-independent GnRH secretion would be sufficient to maintain fertility. To test this, Gnaq (encodes Gαq) was selectively inactivated in the GnRH neurons of global Gna11 (encodes Gα11)-null mice by crossing Gnrh-Cre and Gnaq(fl/fl);Gna11(-/-) mice. Experimental Gnaq(fl/fl);Gna11(-/-);Gnrh-Cre (Gnaq(d/d)) and control Gnaq(fl/fl);Gna11(-/-) (Gnaq(fl/fl)) littermate mice were generated and subjected to reproductive profiling. This process revealed that testicular development and spermatogenesis, preputial separation, and anogenital distance in males and day of vaginal opening and of first estrus in females were significantly less affected in Gnaq(d/d) mice than in previously characterized Kiss1r(-/-) or Kiss1r(d/d) mice. Additionally, Gnaq(d/d) males were subfertile, and although Gnaq(d/d) females did not ovulate spontaneously, they responded efficiently to a single dose of gonadotropins. Finally, KP stimulation triggered a significant increase in gonadotropins and testosterone levels in Gnaq(d/d) mice. We therefore conclude that the milder reproductive phenotypes and maintained responsiveness to KP and gonadotropins reflect Gαq/11-independent GnRH secretion and activation of the neuroendocrine-reproductive axis in Gnaq(d/d) mice. SIGNIFICANCE STATEMENT: The gonadotropin-releasing hormone (GnRH) is the master regulator of fertility. Over the last decade, several studies have established that the KISS1 receptor, KISS1R, is a potent trigger of GnRH secretion and inactivation of KISS1R on the GnRH neuron results in infertility. While KISS1R is best understood as a Gαq/11-coupled receptor, we previously demonstrated that it could couple to and signal via non-Gαq/11-coupled pathways. The present study confirms these findings and, more importantly, while it establishes Gαq/11-coupled signaling as a major conduit of GnRH secretion, it also uncovers a significant role for non-Gαq/11-coupled signaling in potentiating reproductive development and function. This study further suggests that by augmenting signaling via these pathways, GnRH secretion can be enhanced to treat some forms of infertility.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/deficiência , Hormônio Liberador de Gonadotropina/fisiologia , Hipogonadismo/fisiopatologia , Infertilidade Feminina/fisiopatologia , Infertilidade Masculina/fisiopatologia , Animais , Blastocisto/patologia , Desenvolvimento Embrionário , Feminino , Subunidades alfa de Proteínas de Ligação ao GTP/fisiologia , Perfilação da Expressão Gênica , Genitália Feminina/patologia , Genitália Feminina/fisiopatologia , Genitália Masculina/patologia , Genitália Masculina/fisiopatologia , Hormônios Esteroides Gonadais/metabolismo , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Gonadotropinas Hipofisárias/metabolismo , Gonadotropinas Hipofisárias/farmacologia , Hipogonadismo/genética , Hipogonadismo/patologia , Sistema Hipotálamo-Hipofisário/fisiopatologia , Hipotálamo/patologia , Infertilidade Feminina/embriologia , Infertilidade Feminina/genética , Infertilidade Masculina/embriologia , Infertilidade Masculina/genética , Kisspeptinas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Oligopeptídeos/farmacologia , Ovariectomia , Ovulação/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Peptídeos/farmacologia , Fenótipo , Receptores Acoplados a Proteínas G , Receptores de Kisspeptina-1 , EspermatogêneseRESUMO
There is a critical need for techniques that directly monitor protein synthesis within cells isolated from normal and diseased tissue. Fibrotic disease, for which there is no drug treatment, is characterized by the overexpression of collagens. Here, we use a bioinformatics approach to identify a pair of glycine and proline isoacceptor tRNAs as being specific for the decoding of collagen mRNAs, leading to development of a FRET-based approach, dicodon monitoring of protein synthesis (DiCoMPS), that directly monitors the synthesis of collagen. DiCoMPS aimed at detecting collagen synthesis will be helpful in identifying novel anti-fibrotic compounds in cells derived from patients with fibrosis of any etiology, and, suitably adapted, should be widely applicable in monitoring the synthesis of other proteins in cells.
Assuntos
Colágeno/biossíntese , Fibroblastos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Microscopia Confocal , RNA de Transferência de Glicina/metabolismo , RNA de Transferência de Prolina/metabolismo , Animais , Carbocianinas/metabolismo , Células Cultivadas , Fibroblastos/patologia , Fibrose , Corantes Fluorescentes/metabolismo , Humanos , Cinética , Camundongos , Camundongos Knockout , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/genética , RNA de Transferência de Glicina/genética , RNA de Transferência de Prolina/genética , TransfecçãoRESUMO
During the first trimester of human pregnancy, cytotrophoblasts proliferate within the tips of the chorionic villi to form cell columns that anchor the placenta to the uterus. This migration coincides with a widespread change in the adhesion molecule repertoire of these trophoblasts. Kisspeptin and its receptor, KISS1R, are best known as potent triggers of gonadotropin-releasing hormone secretion. The kisspeptin/KISS1R signaling system is also highly expressed in the human placenta, where it was demonstrated to inhibit extra-villous trophoblast (EVT) migration and invasion in vitro. Here we show that kisspeptin, in a dose- and time-dependent manner, induces increased adhesion of human EVTs to type-I collagen, a major component of the human placenta. This increased adhesion was both rapid and transient, suggesting that it likely occurred through the activation of KISS1R secondary effectors such as PKC and ERK, which underwent rapid and transient kisspeptin-dependent activation in EVTs. We then showed that inhibition of both PKC and ERK1/2 attenuated the kisspeptin-dependent increase in EVT adhesion, suggesting that these molecules are key positive regulators of trophoblast adhesion. We therefore propose that kisspeptin/KISS1R signaling potentiates EVT adhesion to type-I collagen via "inside-out signaling." Furthermore, kisspeptin treatment increased mouse blastocyst adhesion to collagen I, suggesting that kisspeptin signaling is a key regulator of trophoblast function during implantation as well as early placentation.
Assuntos
Adesão Celular/fisiologia , Colágeno Tipo I/metabolismo , Kisspeptinas/metabolismo , Placenta/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Trofoblastos/fisiologia , Análise de Variância , Animais , Western Blotting , Primers do DNA/genética , Implantação do Embrião/fisiologia , Feminino , Imunofluorescência , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Microscopia Confocal , Gravidez , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Receptores de Kisspeptina-1 , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Group I metabotropic glutamate receptors (mGluRs) are coupled via phospholipase Cbeta to the hydrolysis of phosphoinositides and function to modulate neuronal excitability and synaptic transmission at glutamatergic synapses. The desensitization of Group I mGluR signaling is thought to be mediated primarily via second messenger-dependent protein kinases and G protein-coupled receptor kinases. We show here that both mGluR1 and mGluR5 interact with the calcineurin inhibitor protein (CAIN). CAIN is co-immunoprecipitated in a complex with Group I mGluRs from both HEK 293 cells and mouse cortical brain lysates. Purified CAIN and its C-terminal domain specifically interact with glutathione S-transferase fusion proteins corresponding to the second intracellular loop and the distal C-terminal tail domains of mGluR1. The interaction of CAIN with mGluR1 could also be blocked using a Tat-tagged peptide corresponding to the mGluR1 second intracellular loop domain. Overexpression of full-length CAIN attenuates the agonist-stimulated endocytosis of both mGluR1a and mGluR5a in HEK 293 cells, but expression of the CAIN C-terminal domain does not alter mGluR5a internalization. In contrast, overexpression of either full-length CAIN or the CAIN C-terminal domain impairs agonist-stimulated inositol phosphate formation in HEK 293 cells expressing mGluR1a. This CAIN-mediated antagonism of mGluR1a signaling appears to involve the disruption of receptor-Galpha(q/11) complexes. Taken together, these observations suggest that the association of CAIN with intracellular domains involved in mGluR/G protein coupling provides an additional mechanism by which Group I mGluR endocytosis and signaling are regulated.
Assuntos
Calcineurina/fisiologia , Receptores de Glutamato Metabotrópico/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Calcineurina/química , Cálcio/metabolismo , Linhagem Celular , Endocitose , Humanos , Fosfatos de Inositol/química , Microscopia de Fluorescência/métodos , Modelos Biológicos , Fosforilação , Plasmídeos/metabolismo , Ligação Proteica , Proteína Quinase C/metabolismo , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
Placental expression of gonadotropin-releasing hormone (GnRH)-I and II, as well as their cognate receptor, coincides with a period of extensive remodeling of the maternal-fetal interface, near the end of the first trimester of pregnancy. To further define the role of GnRH in human placentation, we performed a microarray screen of HTR-8/SVneo trophoblasts to identify GnRH-regulated genes and their roles in placentation. This screen revealed that GnRH regulates the expression of four angiogenic chemokines: CXCL2, CXCL3, CXCL6, and CXCL8. The microarray data were subsequently confirmed by an extensive Q-PCR time-course analysis. CXCL8, a representative chemokine, was selected for further analysis and shown to be strongly expressed by trophoblasts at the maternal-fetal interface of the human placenta, as well as to accumulate in a GnRH-dependent manner in trophoblast-conditioned media in culture. Trophoblasts were subsequently shown to recruit lymphocytes (Jurkat T cells and primary peripheral blood T and uterine natural killer cells) in chemotaxis assays and this was shown to be GnRH dependent. Furthermore, this recruitment was shown to occur via the release of CXCR1/CXCR2 interacting chemokines, such as the CXCLs investigated in this study. This novel regulation of chemokines by GnRH signaling demonstrates the role of GnRH in regulating the recruitment of lymphocytes to the decidua and the possibility of a direct effect on spiral artery remodeling via the release of proangiogenic chemokines and secondary effects via release of angiogenic factors by recruited lymphocytes.
Assuntos
Proteínas Angiogênicas/metabolismo , Quimiocinas/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Placentação , Trofoblastos/imunologia , Proteínas Angiogênicas/genética , Busserrelina/farmacologia , Linhagem Celular Transformada , Quimiocina CXCL2/metabolismo , Quimiocina CXCL6/metabolismo , Quimiocinas/genética , Quimiocinas CXC/metabolismo , Quimiotaxia de Leucócito , Meios de Cultivo Condicionados/metabolismo , Feminino , Imunofluorescência , Perfilação da Expressão Gênica/métodos , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Antagonistas de Hormônios/farmacologia , Humanos , Interleucina-8/metabolismo , Células Jurkat , Células Matadoras Naturais/imunologia , Neovascularização Fisiológica , Análise de Sequência com Séries de Oligonucleotídeos , Oligopeptídeos/farmacologia , Placentação/efeitos dos fármacos , Gravidez , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/metabolismo , Receptores LHRH/genética , Receptores LHRH/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Linfócitos T/imunologia , Fatores de Tempo , Trofoblastos/efeitos dos fármacos , Trofoblastos/metabolismoRESUMO
Glutamate exerts its effects through binding and activation of two classes of specific receptors: ionotropic (iGluRs) and metabotropic (mGluRs). Group I mGluR includes mGluR1 and mGluR5 subtypes, group II includes mGluR2 and mGluR3 subtypes and group III includes the subtypes mGluR 4, 6, 7 and 8. Glutamate and its receptors are found in all key hypothalamic areas critically involved in reproduction and neuroendocrine function. To date, considerable data support an important role for iGluRs in the control of neuroendocrine function; however, the role of mGluRs as regulators of hypothalamic-pituitary function has not been clearly elucidated. mGluRs could be exerting a fine tune on the release of hypothalamic factors that regulate hormone release such as Substance P, GABA, alpha-MSH and CRH. Group II mGluR exert a direct inhibitory effect on anterior pituitary prolactin and GH secretion. Moreover, some group II mGluR agonists, like LY 354,740 and LY 379,268, can modulate PRL secretion from the anterior pituitary through their actions as dopamine receptor agonists. Evidence suggests a role for group III mGluR subtypes in stress-related behavioral disorders. Several reports indicate that selective ligands for mGluR subtypes have potential for the treatment of a wide variety of neurological and psychiatric disorders, including depression, anxiety disorders, schizophrenia, epilepsy and Alzheimer's disease among others. Since converging lines of evidence suggest a role for mGluRs subtypes in neuroendocrine regulation of hormone secretion, mGluRs neuroendocrine actions must be taken in consideration to insure proper treatment of these diseases. Moreover, discovery of selective agonists provides an opportunity to investigate the physiological role of mGluR subtypes and to directly test the neuroendocrine actions of mGluRs. Finally, mGluRs selective agonists may have an impact in the treatment of conditions involving chronic stress, such as depression and anxiety disorders, since they regulate neuroendocrine stress circuits involving the HPA axis and stress-sensitive hormones such as oxytocin and prolactin. This review aims to provide a survey of our current understanding of the effects of mGluR activation on neuroendocrine function.
Assuntos
Sistemas Neurossecretores/fisiologia , Receptores de Glutamato Metabotrópico/fisiologia , Animais , HumanosRESUMO
Purpose: The ubiquitously expressed transmembrane glycoprotein CD47 delivers an anti-phagocytic (do not eat) signal by binding signal-regulatory protein α (SIRPα) on macrophages. CD47 is overexpressed in cancer cells and its expression is associated with poor clinical outcomes. TTI-621 (SIRPαFc) is a fully human recombinant fusion protein that blocks the CD47-SIRPα axis by binding to human CD47 and enhancing phagocytosis of malignant cells. Blockade of this inhibitory axis using TTI-621 has emerged as a promising therapeutic strategy to promote tumor cell eradication.Experimental Design: The ability of TTI-621 to promote macrophage-mediated phagocytosis of human tumor cells was assessed using both confocal microscopy and flow cytometry. In vivo antitumor efficacy was evaluated in xenograft and syngeneic models and the role of the Fc region in antitumor activity was evaluated using SIRPαFc constructs with different Fc tails.Results: TTI-621 enhanced macrophage-mediated phagocytosis of both hematologic and solid tumor cells, while sparing normal cells. In vivo, TTI-621 effectively controlled the growth of aggressive AML and B lymphoma xenografts and was efficacious in a syngeneic B lymphoma model. The IgG1 Fc tail of TTI-621 plays a critical role in its antitumor activity, presumably by engaging activating Fcγ receptors on macrophages. Finally, TTI-621 exhibits minimal binding to human erythrocytes, thereby differentiating it from CD47 blocking antibodies.Conclusions: These data indicate that TTI-621 is active across a broad range of human tumors. These results further establish CD47 as a critical regulator of innate immune surveillance and form the basis for clinical development of TTI-621 in multiple oncology indications. Clin Cancer Res; 23(4); 1068-79. ©2016 AACR.
Assuntos
Antígenos de Diferenciação/genética , Antígeno CD47/genética , Imunoglobulina G/imunologia , Neoplasias/tratamento farmacológico , Receptores Imunológicos/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Animais , Anticorpos Bloqueadores/administração & dosagem , Anticorpos Bloqueadores/imunologia , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Antígeno CD47/antagonistas & inibidores , Linhagem Celular Tumoral , Humanos , Camundongos , Neoplasias/imunologia , Neoplasias/patologia , Fagocitose/efeitos dos fármacos , Ligação Proteica , Mapas de Interação de Proteínas/efeitos dos fármacos , Receptores Imunológicos/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
GPCR internalization is a critical regulatory step in determining receptor activity. While internalization terminates G protein-coupled signaling, it might be required for G protein-independent signaling. A large number of clinical therapies are based on preventing or promoting GPCR internalization. Thus, for any given GPCR, it is important to characterize its internalization and understand the factors that regulate such internalization. Here we describe different experimental protocols to evaluate the internalization of any GPCR transiently expressed in HEK 293 cells. The protocols describe the use of immunofluorescence and imaging techniques as well as flow cytometry. The techniques described use the FLAG-tagged kisspeptin receptor (KISS1R) as an example but are equally applicable to any other GPCR.
Assuntos
Proteínas de Bactérias/metabolismo , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Proteínas de Fluorescência Verde/metabolismo , Luciferases/metabolismo , Proteínas Luminescentes/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Bactérias/genética , Recuperação de Fluorescência Após Fotodegradação , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Luciferases/genética , Substâncias Luminescentes/química , Proteínas Luminescentes/genética , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Transporte Proteico , Receptor A2A de Adenosina/genética , Receptores de Dopamina D2/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Kisspeptina-1 , TransfecçãoRESUMO
Indoleamine 2,3-dioxygenase-1 (IDO) is an immune regulatory enzyme expressed by most human tumors. IDO levels in tumor cells correlate with increased metastasis and poor patient outcome and IDO is linked to tumor cell resistance to immunotherapy, radiation therapy, and chemotherapy. Knowledge of tumor cell-autonomous effects of IDO, independent of its well-known role in regulating and suppressing anti-tumor immune responses, is limited. Clonal populations of A549 human lung adenocarcinoma cells stably transfected with anti-IDO shRNA or scrambled control shRNA were used to study IDO effects on drug sensitivity and resistance. IFNγ was used to induce IDO in those cells. We show, for the first time, that IDO mediates human tumor cell resistance to the candidate anticancer drugs FK866 (an NAD+ inhibitor), methoxyamine (MX, a base excision repair [BER] inhibitor) and approved anticancer drugs pemetrexed (a folate anti-metabolite) and gemcitabine (a nucleoside analogue), and combined treatment with pemetrexed and MX, in the absence of immune cells. Concurrent knockdown of IDO and thymidylate synthase (TS, a key rate-limiting enzyme in DNA synthesis and repair) sensitizes human lung cancer cells to pemetrexed and 5FUdR to a greater degree than knockdown of either target alone. We conclude that BER in IDO-expressing A549 cells plays a major role in mediating resistance to a range of approved and candidate anticancer drugs. IDO inhibitors are undergoing clinical trials primarily to improve antitumor immune responses. We show that targeting IDO alone or in combination with TS is a potentially valuable therapeutic strategy for cancer treatment, independent of immune activity and in combination with conventional chemotherapy.
Assuntos
Acrilamidas/farmacologia , Desoxicitidina/análogos & derivados , Regulação para Baixo/efeitos dos fármacos , Hidroxilaminas/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Pemetrexede/farmacologia , Piperidinas/farmacologia , Animais , Proteína BRCA2/metabolismo , Linhagem Celular Tumoral , Células Clonais , Reparo do DNA/efeitos dos fármacos , Desoxicitidina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Floxuridina/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Camundongos SCID , NAD/metabolismo , RNA Interferente Pequeno/metabolismo , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
Since D-aspartate stimulates prolactin and LH release, our objective was to determine whether D-aspartate modifies the release of hypothalamic and posterior pituitary factors involved in the control of their secretion and whether its effects on these tissues are exerted through NMDA receptors and mediated by nitric oxide. In the hypothalamus, D-aspartate stimulated luteinizing hormone-releasing hormone (LHRH), alpha-melanocyte-stimulating hormone (alpha-MSH) and GABA release and inhibited dopamine release through interaction with NMDA receptors. It increased nitric oxide synthase (NOS) activity, and its effects on LHRH and hypothalamic GABA release were blunted when NOS was inhibited. In the posterior pituitary gland, D-aspartate inhibited GABA release but had no effect on dopamine or alpha-MSH release. We report that D-aspartate differentially affects the release of hypothalamic and posterior pituitary factors involved in the regulation of pituitary hormone secretion.
Assuntos
Ácido D-Aspártico/metabolismo , Dopamina/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo/metabolismo , Vias Neurais/metabolismo , Neuro-Hipófise/metabolismo , alfa-MSH/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Ácido D-Aspártico/farmacologia , Inibidores Enzimáticos/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Hipotálamo/efeitos dos fármacos , Masculino , N-Metilaspartato/farmacologia , Vias Neurais/efeitos dos fármacos , Neurônios Nitrérgicos/efeitos dos fármacos , Neurônios Nitrérgicos/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/efeitos dos fármacos , Óxido Nítrico Sintase/metabolismo , Neuro-Hipófise/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
The aim of the present study was to investigate the effect of metabotropic glutamate receptor (mGluR) activation on gamma-aminobutyric acid (GABA) and alpha-melanocyte stimulating hormone (alpha-MSH) release from hypothalamic fragments and posterior pituitaries. The actions of a number of subtype-selective mGluR agonists were monitored. A group I mGluR agonist, (S)-3-hydroxyphenylglycine (3-HPG; 0.5 mM), decreased K+-induced hypothalamic GABA release. (RS)-1-Aminoindan-1,5-dicarboxylic acid (AIDA), a specific group I mGluR antagonist (0.2 mM), blocked the effect of 3-HPG. (2S, 1'S, 2'S)-2-(Carboxycyclopropyl) glycine (L-CCG-I) and L-serine-O-phosphate (L-SOP; 0.01-1 mM), agonists of group II and III mGluRs, respectively, did not modify hypothalamic evoked GABA release. Group I mGluR activation decreased, whereas group III increased and group II induced no changes in GABA release from the posterior pituitary. 3-HPG (1 mM) and L-CCG-I (0.1 mM) decreased, whereas L-SOP (0.01-0.1 mM) did not change alpha-MSH release from hypothalamic fragments. No agonists of the three mGluR groups modified alpha-MSH release from the posterior pituitary. These results indicate that activation of mGluRs differentially affects GABA and alpha-MSH release from the hypothalamus and the posterior pituitary.
Assuntos
Glicina/análogos & derivados , Hipotálamo/metabolismo , Neuro-Hipófise/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , alfa-MSH/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Cicloleucina/análogos & derivados , Cicloleucina/farmacologia , Inibidores Enzimáticos/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Glicina/farmacologia , Indanos/farmacologia , Masculino , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos WistarRESUMO
The hypothalamic kisspeptin signaling system is a major positive regulator of the reproductive neuroendocrine axis, and loss of Kiss1 in the mouse results in infertility, a condition generally attributed to its hypogonadotropic hypogonadism. We demonstrate that in Kiss1(-/-) female mice, acute replacement of gonadotropins and estradiol restores ovulation, mating, and fertilization; however, these mice are still unable to achieve pregnancy because embryos fail to implant. Progesterone treatment did not overcome this defect. Kiss1(+/-) embryos transferred to a wild-type female mouse can successfully implant, demonstrating the defect is due to maternal factors. Kisspeptin and its receptor are expressed in the mouse uterus, and we suggest that it is the absence of uterine kisspeptin signaling that underlies the implantation failure. This absence, however, does not prevent the closure of the uterine implantation chamber, proper alignment of the embryo, and the ability of the uterus to undergo decidualization. Instead, the loss of Kiss1 expression specifically disrupts embryo attachment to the uterus. We observed that on the day of implantation, leukemia inhibitory factor (Lif), a cytokine that is absolutely required for implantation in mice, is weakly expressed in Kiss1(-/-) uterine glands and that the administration of exogenous Lif to hormone-primed Kiss1(-/-) female mice is sufficient to partially rescue implantation. Taken together, our study reveals that uterine kisspeptin signaling regulates glandular Lif levels, thereby identifying a novel and critical role for kisspeptin in regulating embryo implantation in the mouse. This study provides compelling reasons to explore this role in other species, particularly livestock and humans.
Assuntos
Implantação do Embrião , Kisspeptinas/fisiologia , Fator Inibidor de Leucemia/fisiologia , Prenhez/fisiologia , Útero/fisiologia , Animais , Estradiol/fisiologia , Feminino , Gonadotropinas/fisiologia , Kisspeptinas/deficiência , Kisspeptinas/genética , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Gravidez , Progesterona/metabolismo , SuperovulaçãoRESUMO
Hypothalamic GnRH is the master regulator of the neuroendocrine reproductive axis, and its secretion is regulated by many factors. Among these is kisspeptin (Kp), a potent trigger of GnRH secretion. Kp signals via the Kp receptor (KISS1R), a Gαq/11-coupled 7-transmembrane-spanning receptor. Until this study, it was understood that KISS1R mediates GnRH secretion via the Gαq/11-coupled pathway in an ERK1/2-dependent manner. We recently demonstrated that KISS1R also signals independently of Gαq/11 via ß-arrestin and that this pathway also mediates ERK1/2 activation. Because GnRH secretion is ERK1/2-dependent, we hypothesized that KISS1R regulates GnRH secretion via both the Gαq/11- and ß-arrestin-coupled pathways. To test this hypothesis, we measured LH secretion, a surrogate marker of GnRH secretion, in mice lacking either ß-arrestin-1 or ß-arrestin-2. Results revealed that Kp-dependent LH secretion was significantly diminished relative to wild-type mice (P < .001), thus supporting that ß-arrestin mediates Kp-induced GnRH secretion. Based on this, we hypothesized that Gαq/11-uncoupled KISS1R mutants, like L148S, will display Gαq/11-independent signaling. To test this hypothesis, L148S was expressed in HEK 293 cells. and results confirmed that, although strongly uncoupled from Gαq/11, L148S retained the ability to trigger significant Kp-dependent ERK1/2 phosphorylation (P < .05). Furthermore, using mouse embryonic fibroblasts lacking ß-arrestin-1 and -2, we demonstrated that L148S-mediated ERK1/2 phosphorylation is ß-arrestin-dependent. Overall, we conclude that KISS1R signals via Gαq/11 and ß-arrestin to regulate GnRH secretion. This novel and important finding could explain why patients bearing some types of Gαq/11-uncoupled KISS1R mutants display partial gonadotropic deficiency and even a reversal of the condition, idiopathic hypogonadotropic hypogonadism.
Assuntos
Arrestinas/fisiologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Hormônio Luteinizante/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Animais , Arrestinas/genética , Busserrelina/farmacologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Kisspeptina-1 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , beta-Arrestina 1 , beta-Arrestina 2 , beta-ArrestinasRESUMO
Kisspeptins (KPs), peptide products of the KISS1 metastasis-suppressor gene, are endogenous ligands for a G protein-coupled receptor (KISS1R). KISS1 acts as a metastasis suppressor in numerous human cancers. However, recent studies have demonstrated that an increase in KISS1 and KISS1R expression in patient breast tumors correlates with higher tumor grade and metastatic potential. We have shown that KP-10 stimulates invasion of estrogen receptor α (ERα)-negative MDA-MB-231 breast cancer cells via transactivation of the epidermal growth factor receptor (EGFR). Here, we report that either KP-10 treatment of ERα-negative nonmalignant mammary epithelial MCF10A cells or expression of KISS1R in MCF10A cells induced a mesenchymal phenotype and stimulated invasiveness. Similarly, exogenous expression of KISS1R in ERα-negative SKBR3 breast cancer cells was sufficient to trigger invasion and induced extravasation in vivo. In contrast, KP-10 failed to transactivate EGFR or stimulate invasiveness in the ERα-positive MCF7 and T47D breast cancer cells. This suggested that ERα negatively regulates KISS1R-dependent breast cancer cell migration, invasion, and EGFR transactivation. In support of this, we found that these KP-10-induced effects were ablated upon exogenous expression of ERα in the MDA-MB-231 cells, by down-regulating KISS1R expression. Lastly, we have identified IQGAP1, an actin cytoskeletal binding protein as a novel binding partner of KISS1R, and have shown that KISS1R regulates EGFR transactivation in breast cancer cells in an IQGAP1-dependent manner. Overall, our data strongly suggest that the ERα status of mammary cells dictates whether KISS1R may be a novel clinical target for treating breast cancer metastasis.
Assuntos
Movimento Celular , Células Epiteliais/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Embrião de Galinha , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Receptor alfa de Estrogênio/genética , Feminino , Humanos , Fosfatos de Inositol/metabolismo , Kisspeptinas/farmacologia , Células MCF-7 , Glândulas Mamárias Humanas , Microscopia de Fluorescência , Invasividade Neoplásica , Interferência de RNA , Receptores Acoplados a Proteínas G/genética , Receptores de Kisspeptina-1 , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismoRESUMO
The kisspeptin receptor (KISS1R) is a Gα(q/11)-coupled seven-transmembrane receptor activated by a group of peptides referred to as kisspeptins (Kps). The Kp/KISS1R signaling system is a powerful regulator of GnRH secretion, and inactivating mutations in this system are associated with hypogonadotropic hypogonadism. A recent study revealed that Kp triggers prolonged signaling; not from the inability of the receptor to undergo rapid desensitization, but instead from the maintenance of a dynamic and active pool of KISS1R at the cell surface. To investigate this further, we hypothesized that if a dynamic pool of receptor is maintained at the cell surface for a protracted period, chronic Kp-10 treatment would trigger the sustained activation of Gα(q/11) as evidenced through the prolonged activation of phospholipase C, protein kinase C, and prolonged mobilization of intracellular Ca(2+). Through single-cell analyses, we tested our hypothesis in human embryonic kidney (HEK) 293 cells and found that was indeed the case. We subsequently determined that prolonged KISS1R signaling was not a phenomenon specific to HEK 293 cells but is likely a conserved property of KISS1R-expressing cells because evidence of sustained KISS1R signaling was also observed in the GT1-7 GnRH neuronal and Chinese hamster ovary cell lines. While exploring the regulation of prolonged KISS1R signaling, we identified a critical role for extracellular Ca(2+). We found that although free intracellular Ca(2+), primarily derived from intracellular stores, was sufficient to trigger the acute activation of a major KISS1R secondary effector, protein kinase C, it was insufficient to sustain chronic KISS1R signaling; instead extracellular Ca(2+) was absolutely required for this.
Assuntos
Cálcio/metabolismo , Kisspeptinas/metabolismo , Receptores Acoplados a Proteínas G/biossíntese , Animais , Células CHO , Cálcio/química , Cricetinae , Células HEK293 , Humanos , Microscopia Confocal/métodos , Modelos Biológicos , Neurônios/metabolismo , Peptídeos/química , Receptores de Kisspeptina-1 , Transdução de SinaisRESUMO
Kisspeptins (Kp), peptide products of the Kisspeptin-1 (KISS1) gene are endogenous ligands for a G protein-coupled receptor 54 (GPR54). Previous findings have shown that KISS1 acts as a metastasis suppressor in numerous cancers in humans. However, recent studies have demonstrated that an increase in KISS1 and GPR54 expression in human breast tumors correlates with higher tumor grade and metastatic potential. At present, whether or not Kp signaling promotes breast cancer cell invasiveness, required for metastasis and the underlying mechanisms, is unknown. We have found that kisspeptin-10 (Kp-10), the most potent Kp, stimulates the invasion of human breast cancer MDA-MB-231 and Hs578T cells using Matrigel-coated Transwell chamber assays and induces the formation of invasive stellate structures in three-dimensional invasion assays. Furthermore, Kp-10 stimulated an increase in matrix metalloprotease (MMP)-9 activity. We also found that Kp-10 induced the transactivation of epidermal growth factor receptor (EGFR). Knockdown of the GPCR scaffolding protein, ß-arrestin 2, inhibited Kp-10-induced EGFR transactivation as well as Kp-10 induced invasion of breast cancer cells via modulation of MMP-9 secretion and activity. Finally, we found that the two receptors associate with each other under basal conditions, and FRET analysis revealed that GPR54 interacts directly with EGFR. The stability of the receptor complex formation was increased upon treatment of cells by Kp-10. Taken together, our findings suggest a novel mechanism by which Kp signaling via GPR54 stimulates breast cancer cell invasiveness.
Assuntos
Neoplasias da Mama/metabolismo , Receptores ErbB/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Kisspeptinas/farmacologia , Metaloproteinase 9 da Matriz/metabolismo , Receptores de Kisspeptina-1RESUMO
The kisspeptin/GPR54 signaling system positively regulates GnRH secretion, thereby acting as an important regulator of the hypothalamic-pituitary-gonadal axis. It also negatively regulates tumor metastases and placental trophoblast invasion. GPR54 is a G(q/11)-coupled GPCR and activation by kisspeptin stimulates PIP(2) hydrolysis and inositol phosphate (IP) formation, Ca(2+) mobilization, arachidonic acid release, and ERK1/2 and p38 MAP kinase phosphorylation. Recently, we reported that GPR54 displays constitutive activity and internalization in the heterologous human embryonic kidney 293 cell system. Given the physiological and clinical importance of GPR54 as well as other GPCRs, we present assays for measuring constitutive receptor internalization and activity. Specifically, we describe the use of immunofluorescence coupled to confocal imaging, flow cytometry and indirect receptor radiolabeling to measure constitutive receptor internalization, and IP turnover in intact cells to measure constitutive activity. While we use the FLAG-tagged GPR54 molecule as an example to describe these assays, the assays can be applied to a wide range of GPCRs.
Assuntos
Bioensaio/métodos , Proteínas Supressoras de Tumor/metabolismo , Linhagem Celular , Citometria de Fluxo , Imunofluorescência , Humanos , Fosfatos de Inositol , KisspeptinasRESUMO
G protein-coupled receptor 54 (GPR54) is a G(q/11)-coupled 7 transmembrane-spanning receptor (7TMR). Activation of GPR54 by kisspeptin (Kp) stimulates PIP(2) hydrolysis, Ca(2+) mobilization and ERK1/2 MAPK phosphorylation. Kp and GPR54 are established regulators of the hypothalamic-pituitary-gonadal (HPG) axis and loss-of-function mutations in GPR54 are associated with an absence of puberty and hypogonadotropic hypogonadism, thus defining an important role of the Kp/GPR54 signaling system in reproductive function. Given the tremendous physiological and clinical importance of the Kp/GPR54 signaling system, we explored the contributions of the GPR54-coupled G(q/11) and ß-arrestin pathways on the activation of a major downstream signaling molecule, ERK, using G(q/11) and ß-arrestin knockout mouse embryonic fibroblasts. Our study revealed that GPR54 employs the G(q/11) and ß-arrestin-2 pathways in a co-dependent and temporally overlapping manner to positively regulate ERK activity and pERK nuclear localization. We also show that while ß-arrestin-2 potentiates GPR54 signaling to ERK, ß-arrestin-1 inhibits it. Our data also revealed that diminished ß-arrestin-1 and -2 expression in the GT1-7 GnRH hypothalamic neuronal cell line triggered distinct patterns of gene expression following Kp-10 treatment. Thus, ß-arrestin-1 and -2 also regulate distinct downstream responses in gene expression. Finally, we showed that GPR54, when uncoupled from the G(q/11) pathway, as is the case for several naturally occurring GPR54 mutants associated with hypogonadotropic hypogonadism, continues to regulate gene expression in a G protein-independent manner. These new and exciting findings add significantly to our mechanistic understanding of how this important receptor signals intracellularly in response to kisspeptin stimulation.
Assuntos
Arrestinas/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Hipotálamo/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Arrestinas/genética , Linhagem Celular , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Hipotálamo/enzimologia , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Kisspeptina-1 , beta-Arrestina 1 , beta-Arrestina 2 , beta-ArrestinasRESUMO
Group I metabotropic glutamate receptors (mGluRs) are coupled via Galphaq/11 to the activation of phospholipase Cbeta, which hydrolyzes membrane phospholipids to form inositol 1,4,5 trisphosphate and diacylglycerol. This results in the release of Ca2+ from intracellular stores and the activation of protein kinase C. The activation of Group I mGluRs also results in ERK1/2 phosphorylation. We show here, that the proline-rich tyrosine kinase 2 (Pyk2) interacts with both mGluR1 and mGluR5 and is precipitated with both receptors from rat brain. Pyk2 also interacts with GST-fusion proteins corresponding to the second intracellular loop and the distal carboxyl-terminal tail domains of mGluR1a. Pyk2 colocalizes with mGluR1a at the plasma membrane in human embryonic kidney (HEK293) cells and with endogenous mGluR5 in cortical neurons. Pyk2 overexpression in HEK293 results in attenuated basal and agonist-stimulated inositol phosphate formation in mGluR1 expressing cells and involves a mechanism whereby Pyk2 displaces Galphaq/11 from the receptor. The activation of endogenous mGluR1 in primary mouse cortical neuron stimulates ERK1/2 phosphorylation. Treatments that prevent Pyk2 phosphorylation in cortical neurons, and the overexpression of Pyk2 dominant-negative and catalytically inactive Pyk2 mutants in HEK293 cells, prevent ERK1/2 phosphorylation. The Pyk2 mediated activation of ERK1/2 phosphorylation is also Src-, calmodulin- and protein kinase C-dependent. Our data reveal that Pyk2 couples the activation mGluRs to the mitogen-activated protein kinase pathway even though it attenuates mGluR1-dependent G protein signaling.
Assuntos
Quinase 2 de Adesão Focal/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transdução de Sinais/fisiologia , Animais , Ativação Enzimática , Inibidores Enzimáticos/metabolismo , Agonistas de Aminoácidos Excitatórios/metabolismo , Quinase 2 de Adesão Focal/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Imidazóis/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Neurônios/metabolismo , Fosforilação , Conformação Proteica , Ácido Quisquálico/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Receptor de Glutamato Metabotrópico 5 , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tirfostinas/metabolismoRESUMO
The mammalian type I gonadotropin releasing hormone receptor (GnRH-R) is a structurally unique G protein-coupled receptor (GPCR) that lacks cytoplasmic tail sequences and displays inefficient plasma membrane expression (PME). Compared to its murine counterparts, the primate type I receptor is inefficiently folded and retained in the endoplasmic reticulum (ER) leading to a further reduction in PME. The decrease in PME and concomitant increase in intracellular localization of the mammalian GnRH-RI led us to characterize the spatial distribution of the human and mouse GnRH receptors in two human cell lines, HEK 293 and HTR-8/SVneo. In both human cell lines we found the receptors were expressed in the cytoplasm and were associated with the ER and nuclear membrane. A molecular analysis of the receptor protein sequence led us to identify a putative monopartite nuclear localization sequence (NLS) in the first intracellular loop of GnRH-RI. Surprisingly, however, neither the deletion of the NLS nor the addition of the Xenopus GnRH-R cytoplasmic tail sequences to the human receptor altered its spatial distribution. Finally, we demonstrate that GnRH treatment of nuclei isolated from HEK 293 cells expressing exogenous GnRH-RI triggers a significant increase in the acetylation and phosphorylation of histone H3, thereby revealing that the nuclear-localized receptor is functional. Based on our findings, we conclude that the mammalian GnRH-RI is an intracellular GPCR that is expressed on the nuclear membrane. This major and novel discovery causes us to reassess the signaling potential of this physiologically and clinically important receptor.