RESUMO
Toxic effects of nanoparticles on female reproductive health have been documented but the underlying mechanisms still need to be clarified. Here, we investigated the effect of carbon black nanoparticles (CB NPs) on the pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which are key regulators of gonadal gametogenesis and steroidogenesis. To that purpose, we subjected adult female mice to a weekly non-surgical intratracheal administration of CB NPs at an occupationally relevant dose over 4 weeks. We also analyzed the effects of CB NPs in vitro, using both primary cultures of pituitary cells and the LßT2 gonadotrope cell line. We report here that exposure to CB NPs does not disrupt estrous cyclicity but increases both circulating FSH levels and pituitary FSH ß-subunit gene (Fshb) expression in female mice without altering circulating LH levels. Similarly, treatment of anterior pituitary or gonadotrope LßT2 cells with increasing concentrations of CB NPs dose-dependently up-regulates FSH but not LH gene expression or release. Moreover, CB NPs enhance the stimulatory effect of GnRH on Fshb expression in LßT2 cells without interfering with LH regulation. We provide evidence that CB NPs are internalized by LßT2 cells and rapidly activate the cAMP/PKA pathway. We further show that pharmacological inhibition of PKA significantly attenuates the stimulatory effect of CB NPs on Fshb expression. Altogether, our study demonstrates that exposure to CB NPs alters FSH but not LH expression and may thus lead to gonadotropin imbalance.
RESUMO
Reproductive function is under the control of the neurohormone GnRH, which activates a G-protein-coupled receptor (GnRHR) expressed in pituitary gonadotrope cells. GnRHR activates a complex signaling network to regulate synthesis and secretion of the two gonadotropin hormones, luteinizing hormone and follicle-stimulating hormone, both regulating gametogenesis and steroidogenesis in gonads. Recently, in an attempt to identify the mechanisms underlying GnRHR signaling plasticity, we identified the first interacting partner of GnRHR, the proto-oncogene SET. We showed that SET binds to intracellular domains of GnRHR to enhance its coupling to cAMP pathway in αT3-1 gonadotrope cells. Here, we demonstrate that SET protein is rapidly regulated by GnRH, which increases SET phosphorylation state and decreases dose-dependently SET protein level. Our results highlight a post-translational regulation of SET protein involving the proteasome pathway. We determined that SET phosphorylation upon GnRH stimulation is mediated by PKC and that PKC mediates GnRH-induced SET down-regulation. Phosphorylation on serine 9 targets SET for degradation into the proteasome. Furthermore, a non-phosphorylatable SET mutant on serine 9 is resistant to GnRH-induced down-regulation. Altogether, these data suggest that GnRH-induced SET phosphorylation on serine 9 mediates SET protein down-regulation through the proteasome pathway. Noteworthy, SET down-regulation was also observed in response to pulsatile GnRH stimulation in LßT2 gonadotrope cells as well as in vivo in prepubertal female mice supporting its physiological relevance. In conclusion, this study highlights a regulation of SET protein by the neurohormone GnRH and identifies some of the mechanisms involved.
Assuntos
Gonadotrofos/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/farmacologia , Proteínas Oncogênicas/genética , Animais , Animais Recém-Nascidos , Células Cultivadas , Proteínas de Ligação a DNA , Regulação da Expressão Gênica/efeitos dos fármacos , Gonadotrofos/metabolismo , Chaperonas de Histonas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Oncogênicas/metabolismo , Fosforilação , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteína Quinase C/metabolismo , Proto-Oncogene MasRESUMO
Secretion of 17-ß-estradiol (E2) by human granulosa cells can be disrupted by various environmental toxicants. In the current study, we investigated whether carbon black nanoparticles (CB NPs) affect the steroidogenic activity of cultured human granulosa cells. The human granulosa cell line KGN and granulosa cells from patients undergoing in vitro fertilization were treated with increasing concentrations of CB NPs (1 to 100 µg/mL) together or not with follicle-stimulating hormone (FSH). We observed that CB NPs are internalized in KGN cells without affecting cell viability. CB NPs could be localized in the cytoplasm, within mitochondria and in association with the outer face of the endoplasmic reticulum membrane. In both cell types, CB NPs reduced in a dose-dependent manner the activity of aromatase enzyme, as reflected by a decrease in E2 secretion. A significant decrease was observed in response to CB NPs concentrations from 25 and 50 µg/mL in KGN cell line and primary cultures, respectively. Furthermore, CB NPs decreased aromatase protein levels in both cells and reduced aromatase transcript levels in KGN cells. CB NPs rapidly activated extracellular signal-regulated kinase 1 and 2 in KGN cells and pharmacological inhibition of this signaling pathway using PD 98059 significantly attenuated the inhibitory effects of CB NPs on CYP19A1 gene expression and aromatase activity. CB NPs also inhibited the stimulatory effect of FSH on aromatase expression and activity. Altogether, our study on cultured ovarian granulosa cells reveals that CB NPs decrease estrogens production and highlights possible detrimental effect of these common NPs on female reproductive health.
Assuntos
Inibidores da Aromatase/farmacologia , Estradiol/metabolismo , Células da Granulosa/enzimologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Nanopartículas/administração & dosagem , Fuligem/farmacologia , Aromatase/genética , Aromatase/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Disruptores Endócrinos/farmacologia , Estradiol/biossíntese , Antagonistas de Estrogênios , Feminino , Fertilização in vitro , Hormônio Foliculoestimulante/administração & dosagem , Células da Granulosa/química , Células da Granulosa/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Nanopartículas/análise , Fuligem/administração & dosagem , Fuligem/análiseRESUMO
Glucocorticoid hormones (GC) are the main stress mediators associated with reproductive disorders. GC exert their effects through activation of the glucocorticoid receptor (GR) principally acting as a transcription factor. Beside well-established GR-mediated genomic actions, several lines of evidence suggest a role for rapid membrane-initiated GC signaling in gonadotrope cells triggered by a membrane-associated GR. Herein, we demonstrate the existence of a specific membrane-initiated GC signaling in LßT2 gonadotrope cells involving two related phosphoproteins: Ca2+/Calmodulin-dependent protein kinase II (CaMKII) and synapsin-I. Within 5 min, LßT2 cells treated with stress range of 10-7 M Corticosterone or a membrane impermeable-GC, BSA-conjugated corticosterone, exhibited a 2-fold increase in levels of phospho-CaMKII and phospho-synapsin-I. Biochemical approaches revealed that this rapid signaling is promoted by a palmitoylated GR. Importantly, GC significantly alter GnRH-induced CaMKII phosphorylation, consistent with a novel cross-talk between the GnRH receptor and GC. This negative effect of GC on GnRH signaling was further observed on LH release by mouse pituitary explants. Altogether, our work provides new findings in GC field by bringing novel understanding on how GR integrates plasma membrane, allowing GC membrane-initiated signaling that differs in presence of GnRH to disrupt GnRH-dependent signaling and LH secretion.
Assuntos
Genoma , Glucocorticoides/metabolismo , Gonadotrofos/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Lipoilação , Receptores de Glucocorticoides/metabolismo , Transdução de Sinais , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Dexametasona , Células HEK293 , Humanos , Hormônio Luteinizante/metabolismo , Masculino , Camundongos , Fosforilação , Sinapsinas/metabolismoRESUMO
Reproductive function is highly dependent on nutritional input. We recently provided evidence that the unsaturated ω6 fatty acid (FA), linoleic acid (linoleic), interferes with transcription and secretion of the gonadotropin LH, highlighting the existence of a lipid sensing in pituitary gonadotropes. Here, we show, using a combination of in vivo and in vitro models, that linoleic differentially regulates Lhb and Fshb expression. Central exposure of rats to linoleic over 7 days was associated with increase of Lhb but not Fshb transcript levels. Consistently, exposure of rat pituitary cells or LßT2 cells to linoleic increased Lhb, whereas it dramatically decreased Fshb transcript levels without affecting its stability. This effect was also induced by ω9 and ω3-polyunsaturated FA but not by saturated palmitic acid. Analysis of the underlying mechanisms in LßT2 cells using small interfering RNA revealed that early growth response protein 1 mediates linoleic stimulation of Lhb expression. Furthermore, we demonstrated that linoleic counteracts activin and bone morphogenetic protein-2 stimulation of Fshb expression. Using Western blotting and Smad-responsive reporter gene assays, linoleic was shown to decrease basal Smad2/3 phosphorylation levels as well as activin- and bone morphogenetic protein-2-dependent activation of Smad, uncovering a new FA-sensitive signaling cascade. Finally, the protein phosphatase magnesium-dependent 1A was shown to mediate linoleic inhibition of basal Smad phosphorylation and Fshb expression, identifying protein phosphatase magnesium-dependent 1A as a new target of FA in gonadotropes. Altogether, this study provides a novel mechanism by which FAs target gene expression and underlines the relevant role of pituitary gonadotropes in mediating the effects of nutritional FA on reproductive function.
Assuntos
Ácidos Graxos Insaturados/farmacologia , Subunidade beta do Hormônio Folículoestimulante/genética , Gonadotrofos/metabolismo , Ácido Linoleico/farmacologia , Proteínas Smad/metabolismo , Animais , Células Cultivadas , Subunidade beta do Hormônio Folículoestimulante/metabolismo , Expressão Gênica/efeitos dos fármacos , Gonadotrofos/efeitos dos fármacos , Masculino , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/fisiologia , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
In mammals, the receptor of the neuropeptide gonadotropin-releasing hormone (GnRHR) is unique among the G protein-coupled receptor (GPCR) family because it lacks the carboxyl-terminal tail involved in GPCR desensitization. Therefore, mechanisms involved in the regulation of GnRHR signaling are currently poorly known. Here, using immunoprecipitation and GST pull-down experiments, we demonstrated that SET interacts with GnRHR and targets the first and third intracellular loops. We delineated, by site-directed mutagenesis, SET binding sites to the basic amino acids (66)KRKK(69) and (246)RK(247), located next to sequences required for receptor signaling. The impact of SET on GnRHR signaling was assessed by decreasing endogenous expression of SET with siRNA in gonadotrope cells. Using cAMP and calcium biosensors in gonadotrope living cells, we showed that SET knockdown specifically decreases GnRHR-mediated mobilization of intracellular cAMP, whereas it increases its intracellular calcium signaling. This suggests that SET influences signal transfer between GnRHR and G proteins to enhance GnRHR signaling to cAMP. Accordingly, complexing endogenous SET by introduction of the first intracellular loop of GnRHR in αT3-1 cells significantly reduced GnRHR activation of the cAMP pathway. Furthermore, decreasing SET expression prevented cAMP-mediated GnRH stimulation of Gnrhr promoter activity, highlighting a role of SET in gonadotropin-releasing hormone regulation of gene expression. In conclusion, we identified SET as the first direct interacting partner of mammalian GnRHR and showed that SET contributes to a switch of GnRHR signaling toward the cAMP pathway.
Assuntos
Cálcio/metabolismo , AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Gonadotrofos/metabolismo , Receptores LHRH/química , Animais , Sinalização do Cálcio , Proteínas de Ligação ao GTP/metabolismo , Humanos , Cinética , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Ratos , Receptores LHRH/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
The GnRH receptor (GnRHR) plays a central role in the development and maintenance of reproductive function in mammals. Following stimulation by GnRH originating from the hypothalamus, GnRHR triggers multiple signaling events that ultimately stimulate the synthesis and the periodic release of the gonadotropins, luteinizing-stimulating hormone (LH) and follicle-stimulating hormones (FSH) which, in turn, regulate gonadal functions including steroidogenesis and gametogenesis. The concentration of GnRHR at the cell surface is essential for the amplitude and the specificity of gonadotrope responsiveness. The number of GnRHR is submitted to strong regulatory control during pituitary development, estrous cycle, pregnancy, lactation, or after gonadectomy. These modulations take place, at least in part, at the transcriptional level. To analyze this facet of the reproductive function, the 5' regulatory sequences of the gene encoding the GnRHR have been isolated and characterized through in vitro and in vivo approaches. This review summarizes results obtained with the mouse, rat, human, and ovine promoters either by transient transfection assays or by means of transgenic mice.
RESUMO
The gonadotropin-releasing hormone (GnRH) pulsatile pattern is critical for appropriate regulation of gonadotrope activity but only little is known about the signaling mechanisms by which gonadotrope cells decode such pulsatile pattern. Here, we review recent lines of evidence showing that the GnRH receptor (GnRH-R) activates the cyclic AMP (cAMP) pathway in gonadotrope cells, thus ending a long-lasting controversy. Interestingly, coupling of GnRH-R to the cAMP pathway as well as induction of nitric oxide synthase 1 (NOS1) or follistatin through this signaling pathway take place preferentially under high GnRH pulsatility. The preovulatory surge of GnRH in vivo is indeed associated with an important increase of pituitary cAMP and NOS1 expression levels, both being markedly inhibited by treatment with a GnRH antagonist. Altogether, this suggests that due to its atypical structure and desensitization properties, the GnRH-R may continue to signal through the cAMP pathway under conditions inducing desensitization for most other receptors. Such a mechanism may contribute to decode high GnRH pulsatile pattern and enable gonadotrope cell plasticity during the estrus cycle.
RESUMO
The proto-oncogene and inhibitor of protein phosphatase 2A (PP2A), SET, interacts with the third intracellular loop of the M3 muscarinic receptor (M3-MR), and SET knockdown with small interfering RNA (siRNA) in Chinese hamster ovary (CHO) cells augments M3-MR signaling. However, the mechanism of this action of SET on receptor signaling has not been defined, and we initiated studies to address this question. Knockdown of SET by siRNA in CHO cells stably expressing the M3-MR did not alter agonist-induced receptor phosphorylation or receptor internalization. Instead, it increased the extent of receptor dephosphorylation after agonist removal by â¼60%. In competition binding assays, SET knockdown increased high-affinity binding of agonist in intact cells and membrane preparations. Glutathione transferase pull-down assays and site-directed mutagenesis revealed a SET binding site adjacent to and perhaps overlapping the G protein-binding site within the third intracellular loop of the receptor. Mutation of this region in the M3-MR altered receptor coupling to G protein. These data indicate that SET decreases M3-MR dephosphorylation and regulates receptor engagement with G protein, both of which may contribute to the inhibitory action of SET on M3-MR signaling.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Chaperonas de Histonas/metabolismo , Receptor Muscarínico M3/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Células CHO , Cricetinae , Proteínas de Ligação a DNA , Proteínas de Ligação ao GTP/genética , Chaperonas de Histonas/genética , Humanos , Mutagênese Sítio-Dirigida/métodos , Mutação , Fosforilação , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptor Muscarínico M3/genética , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
The activity of pituitary gonadotrope cells, crucial for reproductive function, is regulated by numerous factors including signals related to nutritional status. In this work, we demonstrated, for the first time, that in vivo central exposure of rats to lipids intracarotid infusion of a heparinized triglyceride emulsion selectively increases the expression of pituitary LH subunit genes without any alteration of pituitary GnRH receptor and hypothalamic GnRH or Kiss-1 transcript levels. Furthermore, we showed that unsaturated fatty acids (UFA), oleate and linoleate, increase LH release in a dose-dependent manner as well as LHß mRNA levels in both immortalized LßT2 gonadotrope cell line and rat primary cell cultures. In contrast, the saturated palmitate was ineffective. ACTH or TSH secretion was unaffected by UFA treatment. We demonstrated in LßT2 cells that linoleate effect is mediated neither by activation of membrane fatty acid (FA) receptors GPR40 or GPR120 although we characterized these receptors in LßT2 cells, nor through nuclear peroxisome proliferator-activated receptors. Furthermore, linoleate ß-oxidation is not required for its action on LH secretion. In contrast, pharmacological inhibition of protein kinase C (PKC) or ERK pathways significantly prevented linoleate-stimulated LH release. Accordingly, linoleate was shown to activate novel PKC isoforms, PKCε and -θ, as well as ERK1/2 in LßT2 cells. Lastly, unsaturated, but not saturated, FA inhibited GnRH-induced LH secretion in LßT2 cells as well as in pituitary cell cultures. Altogether, these results suggest that the pituitary is a relevant site of FA action and that UFA may influence reproduction by directly interfering with basal and GnRH-dependent gonadotrope activity.
Assuntos
Ácidos Graxos Insaturados/farmacologia , Gonadotrofos/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/farmacologia , Isoenzimas/fisiologia , Hormônio Luteinizante/metabolismo , Proteína Quinase C-épsilon/fisiologia , Proteína Quinase C/fisiologia , Animais , Células Cultivadas , Gonadotrofos/metabolismo , Masculino , Proteína Quinase C-theta , RatosRESUMO
Previous in vivo studies have established that pituitary nitric oxide synthase type 1 (NOS1) is regulated by gonadotropin-releasing hormone (GnRH). The aim of our study was to elucidate the mechanisms of NOS1 regulation by GnRH in rat pituitary cells. Using a perifused cell system, we demonstrated that NOS1 induction was sensitive to GnRH pulse frequency and was maximally induced under continuous GnRH stimulation. In primary cultures of rat pituitary cells, sustained stimulation with the GnRH agonist triptorelin (GnRHa) increased NOS1 protein levels, whereas NOS2 and NOS3 levels were unaffected. NOS1 up-regulation occurred in gonadotroph cells only, in a time-dependent and concentration-dependent manner (maximum increase, 2.5-fold; half-maximal concentration, 0.17 nM). GnRHa effect was mimicked by cAMP pathway activators and, most importantly, was blocked by disruption of the protein kinase A (PKA) pathway using pharmacological inhibitors such as Rp-cAMP or drug phosphatase technology-protein kinase inhibitor (DPT-PKI), a cell-permeant PKI peptide. In contrast, modulation of the PKC pathway and inhibition of the MAPK cascade were ineffective. Overall, these experiments demonstrated that GnRH-induced up-regulation of pituitary NOS1 is mediated notably by the cAMP/PKA pathway. Last, in vivo administration of a GnRH antagonist markedly inhibited the pituitary cAMP rise at proestrus in addition to suppressing NOS1 increase. Altogether, our data suggest that the cAMP/PKA signaling pathway is preferentially recruited under sustained GnRH stimulation in vivo during proestrus, allowing the expression of a specific set of PKA-regulated proteins, including NOS1, in gonadotroph cells.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Óxido Nítrico Sintase/metabolismo , Hipófise/enzimologia , Proestro/metabolismo , Animais , Técnicas de Cultura de Células , AMP Cíclico/análise , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Feminino , Hormônio Liberador de Gonadotropina/farmacologia , Óxido Nítrico Sintase/análise , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo II/análise , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/análise , Óxido Nítrico Sintase Tipo III/metabolismo , Proestro/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
AGS3, a receptor-independent activator of G-protein signaling, is involved in unexpected functional diversity for G-protein signaling systems. AGS3 has seven tetratricopeptide (TPR) motifs upstream of four G-protein regulatory (GPR) motifs that serve as docking sites for Gialpha-GDP. The positioning of AGS3 within the cell and the intramolecular dynamics between different domains of the proteins are likely key determinants of their ability to influence G-protein signaling. We report that AGS3 enters into the aggresome pathway and that distribution of the protein is regulated by the AGS3 binding partners Gialpha and mammalian Inscuteable (mInsc). Gialpha rescues AGS3 from the aggresome, whereas mInsc augments the aggresome-like distribution of AGS3. The distribution of AGS3 to the aggresome is dependent upon the TPR domain, and it is accelerated by disruption of the TPR organizational structure or introduction of a nonsynonymous single-nucleotide polymorphism. These data present AGS3, G-proteins, and mInsc as candidate proteins involved in regulating cellular stress associated with protein-processing pathologies.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Estruturas Citoplasmáticas/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Sequências Repetitivas de Aminoácidos , Substituição de Aminoácidos/efeitos dos fármacos , Aminoácidos , Animais , Proteínas de Transporte/genética , Linhagem Celular , Estruturas Citoplasmáticas/efeitos dos fármacos , Inibidores de Dissociação do Nucleotídeo Guanina , Humanos , Leupeptinas/farmacologia , Proteínas Mutantes/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Inibidores de Proteassoma , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Ratos , Deleção de Sequência/efeitos dos fármacos , Relação Estrutura-Atividade , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismoRESUMO
Brain control of the reproductive system is mediated through hypothalamic gonadotropin-releasing hormone (GnRH) which activates specific receptors (GnRHR) present at the surface of the pituitary gonadotropes to trigger secretion of the two gonadotropins LH and FSH. A unique feature of this system is the high dependence on the secretion mode of GnRH, which is basically pulsatile but undergoes considerable fluctuations in pulse frequency pattern in response to endogenous or external factors. How the physiological fluctuations of GnRH secretion that orchestrate normal reproduction are decoded by the gonadotrope cell machinery to ultimately control gonadotropin release and/or subunit gene transcription has been the subject of intensive studies during the past decades. Surprisingly, the mammalian GnRHR is unique among G protein-coupled receptor family as it lacks the carboxy-terminal tail usually involved in classical endocytotic process. Accordingly, it does not desensitize properly and internalizes very poorly. Both this atypical intrinsic property and post-receptor events may thus contribute to decode the GnRH signal. This includes the participation of a network of signaling pathways that differently respond to GnRH together with a growing amount of genes differentially sensitive to pulse frequency. Among these are two pairs of genes, the transcription factors EGR-1 and NAB, and the regulatory factors activin and follistatin, that function as intracellular autoregulatory feedback loops controlling respectively LHbeta and FSHbeta gene expression and hence, LH and FSH synthesis. Pituitary gonadotropes thus represent a unique model of cells functionally adapted to respond to a considerably fluctuating neuroendocrine stimulation, from short individual pulses to sustained GnRH as observed at the proestrus of ovarian cycle. Altogether, the data emphasize the adaptative reciprocal complementarity of hypothalamic GnRH neurones and pituitary gonadotropes to function as an original unit.
Assuntos
Hormônio Luteinizante , Receptores LHRH , Animais , Hormônio Foliculoestimulante , Regulação da Expressão Gênica , Hormônio Liberador de Gonadotropina/metabolismo , Hipófise/metabolismoRESUMO
Gonadotrope cells are primarily regulated by GnRH but are also targets of the pituitary adenylyl cyclase-activating polypeptide (PACAP). Although it has been reported that reciprocal interactions between both neuropeptides contribute to regulation of gonadotrope function, the underlying mechanisms remain poorly understood. In this study, we reevaluated PACAP coupling to the cAMP pathway in LbetaT2 gonadotrope cells and analyzed GnRH effect on PACAP signaling. We established that PACAP38 markedly increases intracellular cAMP levels (EC50 of 4.7 +/- 1.3 nm) through the PACAP type 1 receptor (PAC1-R), as evidenced by pharmacological and RT-PCR studies. Interestingly, although GnRH couples to cAMP pathway in LbetaT2 cells, the effects of both neuropeptides were not synergistic. Instead, the GnRH agonist (GnRHa) triptorelin rapidly and strongly inhibited (70% inhibition as early as 5 min) PACAP38-induced cAMP production. Inhibition was calcium independent, mimicked by the phorbol ester phorbol 12-myristate 13-acetate, and blocked by the protein kinase C (PKC) inhibitor bisindoylmaleimide, indicating that GnRHa inhibitory action relies on PKC. Selective down-regulation of both conventional and novel PKC prevented a GnRHa effect, whereas pharmacological inhibition of conventional PKC only was ineffective, strongly suggesting the involvement of novel PKC isoforms. GnRHa did not inhibit forskolin- or cholera toxin-stimulated cAMP accumulation, suggesting that PAC1-R is the predominant target of GnRH. Accordingly, we demonstrated for the first time that GnRH increases PAC1-R phosphorylation through PKC, providing a potential molecular mechanism which may account for GnRH inhibitory effect.
Assuntos
AMP Cíclico/metabolismo , Gonadotrofos/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Proteína Quinase C/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Animais , Western Blotting , Cálcio/metabolismo , Linhagem Celular , Gonadotrofos/citologia , Gonadotrofos/metabolismo , Hormônio Liberador de Gonadotropina/agonistas , Camundongos , Fosforilação/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Ligação Proteica/efeitos dos fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pamoato de Triptorrelina/farmacologiaRESUMO
GnRH regulates the reproductive system by stimulating synthesis and release of gonadotropins. GnRH acts through a receptor coupled to multiple intracellular events including a rapid phosphoinositide turnover. Although the cAMP pathway is essential for gonadotrope function, the ability of GnRH to induce cAMP, as well as the coupling mechanisms involved, remain controversial. In this study, we established that GnRH increases intracellular cAMP levels in a concentration-dependent manner in LbetaT2 gonadotrope cells (maximal increase, 2.5-fold; EC(50), 0.30 nm), and this was further evidenced by GnRH activation of a cAMP-sensitive reporter gene. The GnRH effect was Ca(2+) independent, mimicked by the phorbol ester phorbol 12-myristate 13-acetate, and blocked by the protein kinase C (PKC) inhibitor bisindolylmaleimide, indicating that the GnRH effect was mediated by PKC. Pharmacological inhibition of conventional PKC isoforms with Gö6976 did not prevent GnRH-induced cAMP production, whereas down-regulation of novel PKCdelta, -epsilon, and -theta by a long-term treatment with GnRH markedly reduced it. Expression of dominant-negative (DN) mutants of PKCdelta or -epsilon but not PKCtheta impaired GnRH activation of a cAMP-sensitive promoter, demonstrating that PKCdelta and -epsilon are the two endogenous isoforms mediating GnRH activation of the adenylyl cyclase (AC) pathway in LbetaT2 cells. Accordingly, we identified by RT-PCR and immunocytochemical analysis, two PKC-sensitive AC isoforms, i.e. AC5 and AC7 as potential targets for GnRH. Lastly, we showed that only sustained stimulation of GnRH receptor significantly increased cAMP, suggesting that in vivo, the cAMP signaling pathway may be selectively recruited under intense GnRH release such as the preovulatory GnRH surge.
Assuntos
AMP Cíclico/metabolismo , Gonadotrofos , Hormônio Liberador de Gonadotropina/fisiologia , Proteína Quinase C-delta/fisiologia , Proteína Quinase C-épsilon/fisiologia , Adenilil Ciclases/metabolismo , Animais , Cálcio/fisiologia , Linhagem Celular , Gonadotrofos/efeitos dos fármacos , Gonadotrofos/metabolismo , Hormônio Liberador de Gonadotropina/agonistas , Isoenzimas/fisiologia , Camundongos , Proteína Quinase C-delta/metabolismo , Proteína Quinase C-épsilon/metabolismo , Receptores LHRH/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
G protein-coupled receptors mediate cell responses to extracellular stimuli and likely function in the context of a larger signal transduction complex. Utilizing the third intracellular loop of a G protein-coupled receptor in glutathione S-transferase pulldown assays from rat brain lysates coupled with high sensitivity detection methods and subsequent functional studies, we report the identification of SET as a regulator of muscarinic receptor signaling. SET is a putative oncogene reported to inhibit protein phosphatase 2A and regulate gene transcription. SET binds the carboxyl region of the M3-muscarinic receptor i3 loop, and endogenous SET co-immunoprecipitates with intact M3 muscarinic receptor expressed in cells. Small interfering RNA knockdown of endogenous SET in Chinese hamster ovary cells stably expressing the M3 muscarinic receptor augmented receptor-mediated mobilization of intracellular calcium by approximately 35% with no change in agonist EC(50), indicating that interaction of SET with the M3 muscarinic receptor reduces its signaling capacity. SET knockdown had no effect on the mobilization of intracellular calcium by the P2-purinergic receptor, ionomycin, or a direct activator of phospholipase C, indicating a specific regulation of M3 muscarinic receptor signaling. These data provide expanded functionality for SET and a previously unrecognized mechanism for regulation of GPCR signaling capacity.
Assuntos
Proteínas Cromossômicas não Histona/fisiologia , Receptor Muscarínico M2/fisiologia , Receptor Muscarínico M3/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Animais , Células CHO , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cricetinae , Proteínas de Ligação a DNA , Chaperonas de Histonas , Humanos , Líquido Intracelular/metabolismo , Líquido Intracelular/fisiologia , Camundongos , Dados de Sequência Molecular , Ligação Proteica/genética , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/genética , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , RNA Interferente Pequeno/genética , Ratos , Receptor Muscarínico M2/biossíntese , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M3/antagonistas & inibidores , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Accessory proteins involved in signal processing through heterotrimeric G proteins are generally defined as proteins distinct from G protein-coupled receptor (GPCR), G protein, or classical effectors that regulate the strength/efficiency/specificity of signal transfer upon receptor activation or position these entities in the right microenvironment, contributing to the formation of a functional signal transduction complex. A flurry of recent studies have implicated an additional class of accessory proteins for this system that provide signal input to heterotrimeric G proteins in the absence of a cell surface receptor, serve as alternative binding partners for G protein subunits, provide unexpected modes of G protein regulation, and have introduced additional functional roles for G proteins. This group of accessory proteins includes the recently discovered Activators of G protein Signaling (AGS) proteins identified in a functional screen for receptor-independent activators of G protein signaling as well as several proteins identified in protein interaction screens and genetic screens in model organisms. These accessory proteins may influence GDP dissociation and nucleotide exchange at the G(alpha) subunit, alter subunit interactions within heterotrimeric G(alphabetagamma) independent of nucleotide exchange, or form complexes with G(alpha) or G(betagamma) independent of the typical G(alphabetagamma) heterotrimer. AGS and related accessory proteins reveal unexpected diversity in G protein subunits as signal transducers within the cell.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Transdução de Sinais/fisiologia , Animais , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Nucleotídeos/metabolismoRESUMO
We previously reported that G protein-coupled receptor kinase (GRK) may contribute to beta-adrenergic receptor (beta-AR) uncoupling occurring just before parturition in rat uterine muscle (myometrium). To identify the GRK involved, we set up in this study a primary cell culture retaining the morphological and functional characteristics of myometrial tissue as well as the in vivo pattern of GRK expression (GRK2, GRK5, and GRK6). In this model, homologous beta-AR desensitization was assessed by an approximately 60% decrease in cAMP production to a subsequent challenge with the beta-agonist, isoproterenol. Desensitization was reduced by 36% with a GRK inhibitor, heparin, and by 31% with a protein kinase A in-hibitor, H89. Using antibodies known to specifically inhibit either GRK2/3 or GRK4-6 families, we demonstrated that only the GRK4-6 family mediated beta-AR desensitization. To discriminate between endogenous GRK5 and GRK6, we attempted to inhibit their action by introducing, into myometrial cells, kinase-dead dominant-negative mutants ((K215R)GRK5 and (K215R)GRK6). Expression of (K215R)GRK6 increased by approximately 70% the cAMP response to isoproterenol without effect on forskolin stimulation. Conversely, expression of (K215R)GRK5 or (K220R)GRK2 had no effect on beta-adrenergic signaling. These results strongly suggest that endogenous GRK6 mediate homologous beta-AR desensitization in myometrial cells.