Kegg Pathway: GnRH signaling pathway

Endocrinology. 2009 Nov 3;
Chen Q, Weiner RI, Blackman BE

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.

Endocrinology. 2009 Oct 28;
Constantin S, Jasoni CL, Wadas B, Herbison AE

Multiple factors regulate the activity of the GnRH neurons responsible for controlling fertility. Foremost among neuronal inputs to GnRH neurons are those using the amino acids glutamate and gamma-aminobutyric acid (GABA). The present study used a GnRH-Pericam transgenic mouse line, enabling live cell imaging of intracellular calcium concentrations ([Ca(2+)]i) to evaluate the effects of glutamate and GABA signaling on [Ca(2+)]i in peripubertal and adult mouse GnRH neurons. Activation of GABAA, N-methyl-D-aspartate, or alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionate acid (AMPA) receptors was found to evoke an increase in [Ca(2+)]i, in subpopulations of GnRH neurons. Approximately 70% of GnRH neurons responded to GABA, regardless of postnatal age or sex. Many fewer ( approximately 20%) GnRH neurons responded to N-methyl-D-aspartate, and this was not influenced by postnatal age or sex. In contrast, about 65% of adult male and female GnRH neurons responded to AMPA compared with about 14% of male and female peripubertal mice (P < 0.05). The mechanisms underlying the ability of GABA and AMPA to increase [Ca(2+)]i in adult GnRH neurons were evaluated pharmacologically. Both GABA and AMPA were found to evoke [Ca(2+)]i increases through a calcium-induced calcium release mechanism involving internal calcium stores and inositol-1,4,5-trisphosphate receptors. For GABA, the initial increase in [Ca(2+)]i originated from GABAA receptor-mediated activation of L-type voltage-gated calcium channels, whereas for AMPA this appeared to involve direct calcium entry through the AMPA receptor. These observations show that all of the principal amino acid receptors are able to control [Ca(2+)]i in GnRH neurons but that they do so in a postnatal age- and intracellular pathway-specific manner.

J Biol Chem. 2009 Oct 26;
Armstrong SP, Caunt CJ, Fowkes RC, Tsaneva-Atanasova K, McArdle CA

Gonadotropin-releasing hormone (GnRH) acts via 7 transmembrane region receptors on gonadotropes to stimulate synthesis and secretion of the luteinizing hormone (LH) and follicle-stimulating hormone (FSH). It is secreted in pulses and its effects depend on pulse frequency but decoding mechanisms are unknown. Here we have used NFAT2-EFP (nuclear factor of activated T-cells 2-emerald fluorescent protein) to monitor GnRH signaling. Increasing [Ca(2+)](i) causes calmodulin/calcineurin-dependent nuclear NFAT translocation, a response involving proteins (calmodulins and NFATs) that decode frequency in other systems. Using live cell imaging, pulsatile GnRH caused dose- and frequency-dependent increases in nuclear NFAT2-EFP and at low frequency, translocation simply tracked GnRH exposure (albeit with slower kinetics). At high frequency (30 min intervals), failure to return to basal conditions before repeat stimulation caused integrative tracking, illustrating how the relative dynamics of up- and down-stream signals can increase efficiency of GnRH action. Mathematical modelling predicted desensitization of GnRH effects on [Ca(2+)](i) and that desensitization would increase with dose, frequency and receptor number but no such desensitization was seen in HeLa and/or LbetaT2 cells, possibly because pulsatile GnRH did not reduce receptor expression (measured by immunofluorescence). GnRH also caused dose- and frequency-dependent activation of alphaGSU, LHbeta and FSHbeta luciferase reporters, effects that were blocked by calcineurin inhibition. Pulsatile GnRH also activated an NFAT-responsive luciferase reporter but this response was directly related to cumulative pulse duration. This, together with the lack of desensitization of translocation responses suggests that NFAT may mediate GnRH action but is not a genuine decoder of GnRH pulse frequency.

J Neuroendocrinol. 2009 Oct 14;
Chan YM, Broder-Fingert S, Wong KM, Seminara SB

Abstract The kisspeptin/Gpr54 signaling pathway plays a critical role in reproduction by stimulating the secretion of GnRH, yet mice carrying mutations in Kiss1 (which encodes kisspeptin) or Gpr54 exhibit partial sexual maturation. For instance, a proportion of female Kiss1(-/-) and Gpr54(-/-) mice exhibit vaginal oestrus, and some male Kiss1(-/-) and Gpr54(-/-) mice exhibit spermatogenesis. To characterise this partial sexual maturation, we examined the vaginal cytology of female Kiss1(-/-) and Gpr54(-/-) mice over time. Nearly all mutant mice eventually enter oestrus, then spontaneously transition from oestrus to dioestrus and back to oestrus again. These transitions are not associated with ovulation, and the frequency of these transitions increases with age. The oestrus exhibited by female Kiss1(-/-) and Gpr54(-/-) mice was disrupted by administration of the competitive GnRH antagonist acyline, which also resulted in lower uterine weights and, in Kiss1(-/-) mice, lower serum FSH and LH concentrations. Similarly, male Kiss1(-/-) and Gpr54(-/-) mice treated with acyline had smaller testicular sizes and absence of mature sperm. In addition to examining intact Kiss1(-/-) and Gpr54(-/-) mice, we also assessed the effects of acyline on gonadotrophin concentrations in gonadectomised mice. Gonadectomy resulted in a significant increase in serum FSH concentrations in male Gpr54(-/-) and Kiss1(-/-) mice. Acyline administration to gonadectomised Kiss1(-/-) and Gpr54(-/-) male mice lowered serum FSH and LH concentrations significantly. In contrast to males, gonadectomy did not result in significant gonadotrophin changes in female Kiss1(-/-) and Gpr54(-/-) mice, but acyline administration was followed by a decrease in LH concentrations. These results demonstrate that, while kisspeptin signaling is critical for the high levels of GnRH activity required for normal sexual maturation and for ovulation, Kiss1(-/-) and Gpr54(-/-) mice retain some degree of GnRH activity. This GnRH activity is sufficient to produce significant effects on vaginal cytology and uterine weights in female mice and on spermatogenesis and testicular weights in male mice.

Endocrinology. 2009 Oct 16;
Cunningham RL, Giuffrida A, Roberts JL

Aged men have a greater incidence of Parkinson's disease (PD) than women. PD is a neurodegenerative condition associated with the loss of dopamine neurons in the nigrostriatal pathway. This study examined the neurotoxic effects of androgens in a dopaminergic cell line (N27 cells) and the downstream signaling pathways activated by androgens. Treatment of N27 cells with testosterone- and dihydrotestosterone-induced mitochondrial dysfunction, protein kinase C (PKC)-delta cleavage, and apoptosis in dopaminergic neuronal cells. Inhibition of caspase-3 prevented the cleavage of PKCdelta from the full-length element to the catalytic fragment and apoptosis in N27 cells, suggesting that androgen-induced apoptosis is mediated by caspase-3-dependent activation of PKCdelta. Androgen-induced apoptosis may be specific to dopamine neurons as evidenced by a lack of testosterone-induced apoptosis in GnRH neurons. These results support a neurotoxic consequence of testosterone on dopaminergic neurons and may provide insight into the gender bias found in PD.

J Neurosci. 2009 Sep 23; 29(38): 11859-66
Navarro VM, Gottsch ML, Chavkin C, Okamura H, Clifton DK, Steiner RA

Kisspeptin is encoded by the Kiss1 gene, and kisspeptin signaling plays a critical role in reproduction. In rodents, kisspeptin neurons in the arcuate nucleus (Arc) provide tonic drive to gonadotropin-releasing hormone (GnRH) neurons, which in turn supports basal luteinizing hormone (LH) secretion. Our objectives were to determine whether preprodynorphin (Dyn) and neurokinin B (NKB) are coexpressed in Kiss1 neurons in the mouse and to evaluate its physiological significance. Using in situ hybridization, we found that Kiss1 neurons in the Arc of female mice not only express the Dyn and NKB genes but also the NKB receptor gene (NK3) and the Dyn receptor [the kappa opioid receptor (KOR)] gene. We also found that expression of the Dyn, NKB, KOR, and NK3 in the Arc are inhibited by estradiol, as has been established for Kiss1, and confirmed that Dyn and NKB inhibit LH secretion. Moreover, using Dyn and KOR knock-out mice, we found that long-term disruption of Dyn/KOR signaling compromises the rise of LH after ovariectomy. We propose a model whereby NKB and dynorphin act autosynaptically on kisspeptin neurons in the Arc to synchronize and shape the pulsatile secretion of kisspeptin and drive the release of GnRH from fibers in the median eminence.

Endocr Rev. 2009 Oct; 30(6): 713-43
Oakley AE, Clifton DK, Steiner RA

Kisspeptin (a product of the Kiss1 gene) and its receptor (GPR54 or Kiss1r) have emerged as key players in the regulation of reproduction. Mutations in humans or genetically targeted deletions in mice of either Kiss1 or Kiss1r cause profound hypogonadotropic hypogonadism. Neurons that express Kiss1/kisspeptin are found in discrete nuclei in the hypothalamus, as well as other brain regions in many vertebrates, and their distribution, regulation, and function varies widely across species. Kisspeptin neurons directly innervate and stimulate GnRH neurons, which are the final common pathway through which the brain regulates reproduction. Kisspeptin neurons are sexually differentiated with respect to cell number and transcriptional activity in certain brain nuclei, and some kisspeptin neurons express other cotransmitters, including dynorphin and neurokinin B (whose physiological significance is unknown). Kisspeptin neurons express the estrogen receptor and the androgen receptor, and these cells are direct targets for the action of gonadal steroids in both male and female animals. Kisspeptin signaling in the brain has been implicated in mediating the negative feedback action of sex steroids on gonadotropin secretion, generating the preovulatory GnRH/LH surge, triggering and guiding the tempo of sexual maturation at puberty, controlling seasonal reproduction, and restraining reproductive activity during lactation. Kisspeptin signaling may also serve diverse functions outside of the classical realm of reproductive neuroendocrinology, including the regulation of metastasis in certain cancers, vascular dynamics, placental physiology, and perhaps even higher-order brain function.

J Clin Endocrinol Metab. 2009 Oct; 94(10): 3633-9
Guran T, Tolhurst G, Bereket A, Rocha N, Porter K, Turan S, Gribble FM, Kotan LD, Akcay T, Atay Z, Canan H, Serin A, O'Rahilly S, Reimann F, Semple RK, Topaloglu AK

CONTEXT: The neurokinin B (NKB) receptor, encoded by TACR3, is widely expressed within the central nervous system, including hypothalamic nuclei involved in regulating GnRH release. We have recently reported two mutations in transmembrane segments of the receptor and a missense mutation in NKB in patients with normosmic isolated hypogonadotropic hypogonadism (nIHH). PATIENTS AND METHODS: We sequenced the TACR3 gene in a family in which three siblings had nIHH. The novel mutant receptor thus identified was studied in a heterologous expression system using calcium flux as the functional readout. RESULTS: All affected siblings were homozygous for the His148Leu mutation, in the first extracellular loop of the NKB receptor. The His148Leu mutant receptor exhibited profoundly impaired signaling in response to NKB (EC(50) = 3 +/- 0.1 nm and >5 microm for wild-type and His148Leu, respectively). The location of the mutation in an extracellular part of the receptor led us also to test whether senktide, a synthetic NKB analog, may retain ability to stimulate the mutant receptor. However, the signaling activity of the His148Leu receptor in response to senktide was also severely impaired (EC(50) = 1 +/- 1 nm for wild-type and no significant response of His148Leu to 10 microm). CONCLUSIONS: Homozygosity for the TACR3 His148Leu mutation leads to failure of sexual maturation in humans, whereas signaling by the mutant receptor in vitro in response to either NKB or senktide is severely impaired. These observations further strengthen the link between NKB, the NKB receptor, and regulation of human reproductive function.

Endocrinology. 2009 Nov; 150(11): 5016-26
Roa J, Garcia-Galiano D, Varela L, Sánchez-Garrido MA, Pineda R, Castellano JM, Ruiz-Pino F, Romero M, Aguilar E, López M, Gaytan F, Diéguez C, Pinilla L, Tena-Sempere M

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that operates as sensor of cellular energy status and effector for its coupling to cell growth and proliferation. At the hypothalamic arcuate nucleus, mTOR signaling has been recently proposed as transducer for leptin effects on energy homeostasis and food intake. However, whether central mTOR also participates in metabolic regulation of fertility remains unexplored. We provide herein evidence for the involvement of mTOR in the control of puberty onset and LH secretion, likely via modulation of hypothalamic expression of Kiss1. Acute activation of mTOR by l-leucine stimulated LH secretion in pubertal female rats, whereas chronic l-leucine infusion partially rescued the state of hypogonadotropism induced by food restriction. Conversely, blockade of central mTOR signaling by rapamycin caused inhibition of the gonadotropic axis at puberty, with significantly delayed vaginal opening, decreased LH and estradiol levels, and ovarian and uterine atrophy. Inactivation of mTOR also blunted the positive effects of leptin on puberty onset in food-restricted females. Yet the GnRH/LH system retained their ability to respond to ovariectomy and kisspeptin-10 after sustained blockade of mTOR, ruling out the possibility of unspecific disruption of GnRH function by rapamycin. Finally, mTOR inactivation evoked a significant decrease of Kiss1 expression at the hypothalamus, with dramatic suppression of Kiss1 mRNA levels at the arcuate nucleus. Altogether our results unveil the role of central mTOR signaling in the control of puberty onset and gonadotropin secretion, a phenomenon that involves the regulation of Kiss1 and may contribute to the functional coupling between energy balance and gonadal activation and function.

J Neurosci. 2009 Aug 5; 29(31): 9809-18
Chen P, Moenter SM

Gonadotropin-releasing hormone (GnRH) neurons are the central regulators of fertility. GnRH stimulates or inhibits GnRH neuronal activity depending on dose. The mechanisms for these actions remain unknown. We hypothesized GnRH acts in part by altering fast synaptic transmission to GnRH neurons. GABAergic and glutamatergic postsynaptic currents (PSCs), both of which can excite these neurons, were recorded from GnRH neurons in brain slices from adult intact and orchidectomized (ORX) males. ORX enhanced the frequency of GABA transmission to GnRH neurons, but had no effect on glutamatergic transmission. Effects of ORX on GABAergic transmission were reversed by estradiol replacement, suggesting GABA is a mediator of steroid feedback in males. GABAergic neurons express type-1 GnRH receptor (GnRHR-1). Low GnRH (20 nm) reduced GABAergic PSC frequency in GnRH neurons from both ORX and intact mice. High GnRH (2 microm) had no effect on either GABAergic or glutamatergic transmission to GnRH neurons. To investigate mechanisms mediating low-dose GnRH suppression of GABAergic transmission, GABAergic PSCs were recorded after arresting G(alphai) activity with pertussis toxin (PTX). PTX abolished the suppressive effect of low GnRH. Moreover, PTX uncovered a stimulatory effect of high GnRH on GABAergic transmission. These data suggest low-dose GnRH suppresses GnRH firing rate in part by decreasing GABAergic transmission to the GnRH neurons, independent of gonadal hormone milieu. Low-dose GnRH appears to exert the suppressive effect by activating GnRHR-I coupled to G(alphai). The concentration-dependent effects of GnRH may be mediated in part by changes in affinity of GnRH to GnRHR-I coupled to different G(alpha) proteins.

Cancer Res. 2009 Aug 15; 69(16): 6473-81
Fister S, Günthert AR, Aicher B, Paulini KW, Emons G, Gründker C

Recently, we could show that gonadotropin-releasing hormone (GnRH)-II antagonists induce apoptosis in human endometrial, ovarian, and breast cancer cells in vitro and in vivo. In the present study, we have ascertained receptor binding and effects of GnRH-II antagonists on mitogenic signal transduction and on activation of proapoptotic protein Bax. The GnRH-II antagonists tested showed EC50 values for GnRH-I receptor binding in the range of 1 to 2 nmol/L. The GnRH-II agonist [D-Lys6]GnRH-II showed an EC50 value for GnRH-I receptor binding of approximately 1,000 nmol/L. Agonistic activity on GnRH-I receptor function with an EC50 of 13 nmol/L has been determined for [D-Lys6]GnRH-II. Antagonistic activities with EC50 values in the range of 1 nmol/L were determined for the GnRH-II antagonists. Treatment of human endometrial, ovarian, and breast cancer cells with GnRH-II antagonists resulted in time-dependent activation of stress-induced mitogen-activated protein kinases p38 and c-Jun NH2-terminal kinase. In addition, treatment with GnRH-II antagonists induced time-dependent activation of proapoptotic protein Bax. GnRH-II antagonists are not involved in activation of protein kinase B/Akt or extracellular signal-regulated kinase 1/2. The GnRH-II antagonists tested had similar binding affinities to the GnRH-I receptor comparable with that of GnRH-I antagonist Cetrorelix. Referring to the cyclic AMP response element reporter gene activation assay, the GnRH-II agonist [D-Lys6]GnRH-II has to be classified as an agonist at the GnRH-I receptor, whereas the GnRH-II antagonists tested are clear antagonists at the GnRH-I receptor. GnRH-II antagonists induce apoptotic cell death in human endometrial, ovarian, and breast cancer cells via activation of stress-induced mitogen-activated protein kinases p38 and c-Jun NH2-terminal kinase followed by activation of proapoptotic protein Bax.

Gen Comp Endocrinol. 2010 Jan 15; 165(2): 262-268
Palevitch O, Abraham E, Borodovsky N, Levkowitz G, Zohar Y, Gothilf Y

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons control pituitary gonadotropin secretion and gametogenesis. In the course of development, these neurons migrate from the olfactory placode to the hypothalamus. The precise molecular mechanism of this neuronal migration is unclear. Here, we investigated whether the chemokine receptor, Cxcr4b, and its cognate ligand, Cxcl12a, are required for proper migration of GnRH3 neurons in zebrafish. Deviated GnRH3 axonal projections and neuronal migration were detected in larvae that carry a homozygote cxcr4b mutation. Similarly, knockdown of Cxcr4b or Cxcl12a led to the appearance of abnormal GnRH3 axonal projections and cell migration, including absence of the characteristic lateral crossing of GnRH3 axons at the anterior commissure and optic chiasm. Double-labeling analysis has shown that cxcr4b and cxcl12a are expressed along the GnRH3 migration pathway (i.e. olfactory placode, terminal nerve and the optic chiasm). The results of this study suggest that the Cxcl12a-Cxcr4b ligand-receptor pair are involved in the migration of GnRH3 neurons in zebrafish, and are therefore crucial for the development of this system.

Am J Physiol Cell Physiol. 2009 Sep; 297(3): C591-600
Finch AR, Caunt CJ, Armstrong SP, McArdle CA

Gonadotropin-releasing hormone (GnRH) acts via seven transmembrane receptors to stimulate gonadotropin secretion. Sustained stimulation desensitizes GnRH receptor (GnRHR)-mediated gonadotropin secretion, and this underlies agonist use in hormone-dependent cancers. Since type I mammalian GnRHR do not desensitize, agonist-induced internalization and downregulation may underlie desensitization of GnRH-stimulated gonadotropin secretion; however, research focus has recently shifted to anterograde trafficking, with the finding that human (h)GnRHR are mostly intracellular. Moreover, there is little direct evidence for agonist-induced trafficking of hGnRHR, and whether or not type I mammalian GnRHR show agonist-induced internalization is controversial. Here we use automated imaging to monitor expression and internalization of hemagglutinin (HA)-tagged hGnRHRs, mouse (m) GnRHR, Xenopus (X) GnRHRs, and chimeric receptors (hGnRHR with added XGnRHR COOH tails, h.XGnRHR) expressed by adenoviral transduction in HeLa cells. We find that agonists stimulate downregulation and/or internalization of mGnRHR and XGnRHR, that GnRH stimulates trafficking of hGnRHR and can stimulate internalization or downregulation of hGnRHR when steps are taken to increase cell surface expression (addition of the XGnRHR COOH tail or pretreatment with pharmacological chaperone). Agonist effects on internalization (of h.XGnRHR) and downregulation (of hGnRHR and h.XGnRHR) were not mimicked by a peptide antagonist and were prevented by a mutation that prevents GnRHR signaling, demonstrating dependence on receptor signaling as well as agonist occupancy. Thus agonist-induced internalization and downregulation of type I mammalian GnRHR occurs in HeLa cells, and we suggest that the high throughput imaging systems described here will facilitate study of the molecular mechanisms involved.

Zhong Xi Yi Jie He Xue Bao. 2009 Jun; 7(6): 563-8
Guan F, Ma SL, Chen BY

OBJECTIVE: To compare the varieties and contents of the main nerval information molecules in perfusate from hypothalamic medial preoptic area (MPOA) of the rats in different sexual cycles and the ovariectomized rats treated by electro-acupuncture, so as to observe the similarities and differences of hypothalamic neuroendocrine signal transduction pathway under the physiological and pathological status, and to explore the mechanisms of neuroendocrine signal transduction of electro-acupuncture therapeutic effect in perimenopausal syndrome. METHODS: The stereo localization technique and push-and-pull perfusion of the rat brain nucleus were adopted for collecting the hypothalamic MPOA perfusate of the female rats with normal sexual cycle, and also for collecting the MPOA perfusate of ovariectomized rats after electro-acupuncture treatment as acupuncture perfusate (AP). After being respectively microinjected into MPOA of the ovariectomized rats, the influence of the different perfusates on vagina cytology and serum estradiol (E2) level was observed. The contents of gonadotropin-releasing hormone (GnRH), dopamine (DA), gamma-aminobutyric acid (GABA), glutamate (Glu), aspartate (Asp) and beta-endorphin (beta-EP) in the perfusate of each group were detected by radioimmunoassay or high performance liquid chromatography, and then the varieties and contents of these substances in the perfusate of each group were compared and analyzed. RESULTS: The contents of neural active substances including DA, GABA, Glu, and beta-EP in the perfusate from the rats' MPOA during different stages of sexual cycle showed some regular changes. After the perfusate was microinjected respectively into the MPOA of the ovariectomized rats, the changes of animal vaginal exfoliated cells and serum E2 level showed the similar four-stage cycle characteristics as normal rats; the changes of vaginal exfoliated cells and serum E2 level of the ovariectomized rats without electro-acupuncture treatment showed the acupuncture-like effects following the microinjection of AP in the MPOA. CONCLUSION: The information molecules in the perfusate from MPOA of rats in normal sexual cycle or electro-acupuncture-treated rats include classical neurotransmitters, amino acid neurotransmitters, and neuropeptides. Acupuncture may modulate the release and synthesis of these information molecules to normalize the subnormal function of MPOA in perimenopausal period. Thus, AP may be a neuroendocrine signal carrier of acupuncture effects for acupuncture signal transduction from one individual to another. This may provide a new thinking and method for studying acupuncture signal transduction of the acupuncture therapeutic effects on perimenopausal syndrome.

Mol Cell Endocrinol. 2009 Nov 13; 311(1-2): 126-34
Novaira HJ, Ng Y, Wolfe A, Radovick S

Kisspeptins, and their G-protein coupled receptor 54 (GPR54), are key components in the regulation of gonadotropin-releasing hormone (GnRH) secretion in humans and other mammals. Several studies demonstrate that the central or systemic administration of kisspeptin increases GnRH and gonadotropin secretion in both prepubertal and adult animals; however, the cellular targets and intracellular mechanisms of action in the central reproductive axis are unclear. In this study, we documented the presence of GPR54 in two GnRH secreting neuronal cell lines (GT1-7 and GN11). Kisspeptin treatment increases GnRH secretion and GnRH mRNA levels in a dose and time dependent manner. 10(-9)M kisspeptin maximally stimulated GnRH secretion by 2-fold and GnRH mRNA levels up to 4-fold after 4h of treatment in both cell lines. Negative regulation by 17beta-estradiol of GnRH secretion and GnRH mRNA was antagonized by kisspeptin. Co-treatment with kisspeptin and 17beta-estradiol increased GnRH secretion by 2-fold and GnRH mRNA by 4-fold over estradiol alone in both cell lines. Intracellular signaling pathway studies showed that an ERK1/2 MAPK inhibitor (PD98059) and a PI3K inhibitor, LY29402, attenuated the effects of kisspeptin on GnRH mRNA modulation. Furthermore, Western blot analysis showed that phosphorylation of both MAPK and Akt substrates increased with kisspeptin treatment. This work demonstrates that the kisspeptin-GPR54 system plays a significant role stimulating GnRH secretion and positive regulation of GnRH mRNA levels in GnRH neurons in culture, and also, demonstrates the activation of MAPK and Akt signaling pathways by kisspeptin in GT1-7 and GN11 cell lines.

Clin Breast Cancer. 2009 Jun; 9 Suppl 1: S6-S17
Cleator SJ, Ahamed E, Coombes RC, Palmieri C

In up to 75% of breast cancers, estrogen receptor (ER) signaling is a key promoter of tumor proliferation, and inhibition of this pathway has clear therapeutic efficacy. The principal clinical means of inhibiting ER signaling comprise selective ER modulators, such as tamoxifen, that act as partial receptor agonists; measures to reduce the circulating level of estrogen, including ovarian ablation, gonadotropin-releasing hormone analogues, and aromatase inhibition; and antagonism and downregulation of ER by the antiestrogen fulvestrant. Each of these therapies is effective in a proportion of ER-positive breast cancers, but de novo and acquired resistance remain significant problems. Emerging knowledge of the biology of ER signaling will provide insights into the mechanisms of resistance and help guide development of therapeutic strategies to maximize response. This review summarizes the contemporary treatment of early-stage and advanced ER-positive breast cancer in premenopausal and postmenopausal women, with an emphasis on recently published or presented data. Mechanisms of resistance to endocrine interventions and trials exploring strategies to overcome them will also be discussed.

Mol Cell Endocrinol. 2009 Sep 24; 308(1-2): 17-25
Kelly MJ, Rønnekleiv OK

It is well known that many of the actions of 17beta-estradiol (E2) in the central nervous system (CNS) are mediated via intracellular receptor/transcription factors that interact with steroid response elements on target genes. However, there is compelling evidence for membrane-associated steroid receptors for E2 in hypothalamic and other brain neurons. Indeed, we are just beginning to understand how E2 signals via membrane receptors, and how these signals impact not only membrane excitability but also gene transcription in neurons. We know that E2 can rapidly alter neuronal activity within seconds, indicating that some cellular effects can occur via membrane-delimited events. In addition, E2 can affect second messenger systems including calcium mobilization and a plethora of kinases to alter cell signaling. This review will concentrate on rapid membrane-initiated and intracellular signaling by E2 in the hypothalamus and hippocampus, the nature of receptors involved and how they contribute to CNS functions.

Endocrinology. 2009 Sep; 150(9): 4203-12
Acosta-Martínez M, Luo J, Elias C, Wolfe A, Levine JE

GnRH neurosecretion is subject to regulation by insulin, IGF-I, leptin, and other neuroendocrine modulators whose effects may be conveyed by activation of phosphoinositide 3-kinase (PI3K)-mediated pathways. It is not known, however, whether any of these regulatory actions are exerted directly, via activation of PI3K in GnRH neurons, or whether they are primarily conveyed via effects on afferent circuitries governing GnRH neurosecretion. To investigate the role of PI3K signaling in GnRH neurons, we used conditional gene targeting to ablate expression of the major PI3K regulatory subunit, p85alpha, in GnRH neurons. Combined in situ hybridization and immunohistochemistry confirmed reduction of p85alpha mRNA expression in GnRH neurons of GnRH-p85alpha knockout (KO) animals. Females of both genotypes exhibited estrous cyclicity and had comparable serum LH, estradiol-17beta, and FSH levels. In male GnRH-p85alphaKO mice, serum LH, testosterone, and sperm counts were significantly reduced compared with wild type. To investigate the role of the other major regulatory subunit, p85beta, on the direct control of GnRH neuronal function, we generated mice with a GnRH-neuron-specific p85alpha deletion on a global betaKO background. No additional reproductive effects in male or female mice were found, suggesting that p85beta does not substitute p85 activity toward PI3K function in GnRH neurons. Our results suggest that p85alpha, and thus PI3K activity, participates in the control of GnRH neuronal activity in male mice. The sex-specific phenotype in these mice raises the possibility that PI3K activation during early development may establish sex differences in GnRH neuronal function.

Mol Cell Endocrinol. 2009 Aug 13; 307(1-2): 125-32
Mutiara S, Kanasaki H, Oride A, Purwana IN, Shimasaki S, Yamamoto H, Miyazaki K

The purpose of the present study was to examine the signal transduction pathways involved in follistatin gene expression induced by GnRH in the LbetaT2 cell line. The LHbeta-subunit was predominantly increased by high frequency GnRH pulses (30 min interval); whereas low frequency pulses (120 min) increased FSHbeta. In a static culture, follistatin expression was significantly increased at 12 h (2.35 +/- 0.80-fold) after the addition of GnRH. Following pulsatile stimulation, follistatin mRNA was increased by high frequency GnRH pulses, but not by low frequency pulses. In a static culture, GnRH maximally activated extracellular signal-regulated kinase (ERK) 10 min (3.2 +/- 0.55-fold) after treatment. In addition, intracellular cAMP accumulated up to 2.1 +/- 0.76-fold. Follistatin promoter activity was significantly increased following transfection with either a constitutively active cAMP dependent protein kinase (PKA) or a constitutively active MEK kinase (MEKK). The induction of follistatin gene expression by GnRH was completely inhibited by H89, a protein kinase A inhibitor, and U0126, a MEK inhibitor. Follistatin gene expression was also activated by both PACAP and CPT-cAMP under static culture conditions. Maximal ERK activation levels were nearly identical regardless of GnRH pulse frequency; however, high frequency GnRH pulses elevated both the intracellular cAMP level as well as cAMP-response element (Cre) promoter activity. These results suggest that both the PKA and ERK pathways are necessary for the induction of the follistatin promoter. Furthermore, the intracellular cAMP level, but not ERK activity, determined whether follistatin was induced following high frequency GnRH pulses.

Curr Opin Endocrinol Diabetes Obes. 2009 Aug; 16(4): 321-7
Ciccone NA, Kaiser UB

PURPOSE OF REVIEW: To discuss recent progress in our understanding of pituitary gonadotroph development and gonadotropin gene regulation, with an emphasis on differential luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion and subunit synthesis, and the implications this may have on female reproductive health. RECENT FINDINGS: In the mature gonadotroph, there is an emerging concept that differential synthesis of gonadotropin beta-subunit genes, essential for cyclic reproductive function, is associated with modification of activation and/or stability of important regulatory proteins and transcription factors. Recent studies suggest that cellular events, which affect histone modification, play an essential role in both gonadotroph development and the ontogeny of gonadotropin subunit gene expression. Such dynamic events are under the orchestration of the hypothalamic neuropeptide gonadotropin-releasing hormone (GnRH), potentially through the ability of GnRH to activate several distinct signaling cascades within the gonadotroph. SUMMARY: Greater insight into the cellular events that are key to gonadotroph physiology will contribute to our understanding of abnormal gonadotropin secretion in disorders such as hypothalamic amenorrhea and polycystic ovarian syndrome (PCOS), and provide a context for the design of novel therapeutic approaches.