Regulation of the Hypothalamic GnRH Neuron

Child Health And Human Development

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Abstract

The pulsatile secretory activity of the network of GnRH-producing neurons in the hypothalamus, and consequently of the GnRH-regulated pituitary gonadotroph cells, is essential for maintenance of the gonadotropin secretory profiles that ensure normal reproductive function. Based on the capacity of cultured fetal hypothalamic cells and immortalized GnRH neurons (GT1-7 cells) for prolonged pulsatile release of GnRH, we have analyzed the cellular and biochemical mechanisms of episodic neurosecretion in vitro. An analysis of the cellular and biochemical mechanisms of episodic neurosecretion in fetal hypothalamic cells and GT1-7 neuronal cells revealed that pulsatile GnRH secretion is calcium-dependent, and is regulated by cyclic AMP and cell excitability. It is also dependent on autocrine agonist activation of the endogenous GnRH receptor (GnRH-R) that we have identified in GnRH neurons, and is abolished by GnRH antagonists. The agonist-activated GnRH-R couples primarily to Gq, and also to Gs and Gi proteins according to the prevailing GnRH concentration. These and other findings indicate that an agonist-induced concentration-dependent switch in coupling of the GnRH-R between specific G proteins regulates Gq/11-InsP3/Ca2+ signaling, as well as Gs/cAMP-induced stimulatory and Gi/cAMP-dependent inhibitory reponses. Our in vitro studies suggest that this autocrine inhibitory mechanism can serve as a timer to regulate the frequency of Ca2+- and cAMP-dependent episodes of GnRH release. In related studies, we have found that hypothalamic GnRH neurons and their immortalized counterparts (GT1-7 cells) express not only nuclear but also cell membrane receptors for the two estrogen receptor subtypes (ERalpha and ERbeta). The membrane-associated ERs expressed in GnRH neurons undergo high-affinity interactions with adenylyl cyclase inhibitory G proteins, modulate intracellular cAMP signaling, and regulate the GnRH secretory profile. The sensitivity of this interaction to picomolar estradiol concentrations suggests that this process represents a physiological negative feedback action of estrogen on the GnRH neuron. G protein-coupled receptors (GPCRs) mediate cellular responses to a diverse array of extracellular messenger molecules. Recent studies have shown that GPCR-mediated signaling pathways include transactivation of receptor tyrosine kinases (RTKs), including receptors for epidermal growth factor (EGFR), platelet-derived growth factor, neurotrophins and fibroblast growth factor. Cross-communication between GPCRs and RTKs is complex process, and the sets of signaling molecules utilized during this process are primarily determined by cell context and the types of receptors activated. The differential involvement of RTKs and downstream signaling pathways activated in response to GPCR stimulation elicits a variety of cellular effects during development, proliferation, differentiation, survival, repair and synaptic transmission in the central nervous system. GnRH utilizes multiple signaling pathways to activate extracellularly regulated MAP kinases, such as ERK1/2, in normal and immortalized pituitary gonadotrophs and GnRH neurons (GT1-7 cells), and in transfected cells expressing the GnRH receptor. In GT1-7 cells, which express receptors for GnRH and epidermal growth factor (EGF), these agonists and phorbol myristate acetate (PMA) stimulate ERK1/2 phosphorylation. The actions of GnRH and PMA, but not that of EGF, were dependent on activation of protein kinase C (PKC) and on transactivation and phosphorylation of the EGF receptor. Both GnRH and EGF increased tyrosine phosphorylation of the EGF receptor. GnRH and PMA, but not EGF, caused rapid phosphorylation of the proline-rich tyrosine kinase, Pyk2, at Tyr 402. This was dependent on Ca2+ and activation of PKC. GnRH stimulation caused translocation of PKCalpha and epsilon to the cell membrane and enhanced the association of Src with the activated PKC isoforms, Pyk2, and the EGF receptor. Inhibition of Src kinase, and dominant negative Pyk2, attenuated ERK1/2 activation by GnRH and PMA, but not by EGF. These findings indicate that Src and Pyk2 act upstream of the EGF-receptor to mediate its transactivation, which is essential for GnRH-induced ERK1/2 phosphorylation in hypothalamic GnRH neurons. In related studies, the mechanism of agonist-induced activation of Pyk2 and its relationship with ERK1/2 phosphorylation was analyzed in non-neuronal HEK293 cells stably expressing the GnRH receptor. In this system, GnRH stimulation caused rapid and sustained phosphorylation of Pyk2 and ERK1/2 that was accompanied by their nuclear translocation. Pyk2 was also localized as expected on cell membranes and focal adhesions. However, dominant regulatory Pyk2 had no effect on GnRH-induced ERK1/2 phosphorylation and c-fos expression. The actions of GnRH on ERK1/2 and Pyk2 were mimicked by activation of PKC and were abolished by its inhibition. GnRH caused translocation of protein kinase C (PKC) alpha and delta isoforms to the cell membrane, as well as phosphorylation of Raf at Ser 338, a major site in the activation of MEK/ERK1/2. Stimulation of HEK293 cells by EGF caused marked ERK1/2 phosphorylation that was mediated by activation of the EGFR receptor. However, GnRH-induced ERK activation was completely independent of EGF-R activation. These results indicate that activation of PKC is responsible for GnRH-induced phosphorylation of both ERK1/2 and Pyk2 in HEK cells, and that Pyk2 activation does not contribute to GnRH signaling. Moreover, GnRH-induced phosphorylation of ERK1/2 and expression of c-fos in HEK cells is independent of Src and EGF-R transactivation, and is mediated through the PKC/Raf/MEK cascade. The agonist-induced translocation of Pyk2 to the nucleus suggests that it may have exert actions therein as well as at the periphery of the cell. The duration as well as the magnitude of MAP kinase activation has been proposed to regulate gene expression and other specific intracellular responses in individual cell types. GnRH-induced activation of ERK1/2 by GnRH is transient in immortalized GT1-7 neurons, but is sustained in alphaT3-1 pituitary gonadotropes and GnRHR-transfected HEK293 cells. Each of these cell types also expresses the EGF receptor (EGFR) and responds to EGF stimulation with marked and transient ERK1/2 phosphorylation. However, GnRH-induced ERK1/2 phosphorylation due to EGFR transactivation was confined to GT1-7 cells. In these cells, neither EGF nor GnRH receptor activation caused translocation of phospho-ERK1/2 into the nucleus. In contrast, agonist stimulation of GnRHRs expressed in HEK293 cells caused sustained phosphorylation and nuclear translocation of ERK1/2 by a PKC-dependent but EGF-R-independent pathway. GnRH-induced activation of ERK1/2 was dependent on Src kinase in GT1-7, but not in HEK293 cells. However, in GT1-7 cells, both EGF and GnRH markedly stimulated phosphorylation of the ERK1/2-dependent protein, p90RSK-1 (RSK-1) at Ser360/Thr364, and caused its translocation into the nucleus. Whereas activation of the Gq/PKC pathway in HEK293 cells causes sustained phosphorylation and translocation of ERK1/2 to the nucleus, transactivation of the EGFR by GnRH in GT1-7 cells elicits transient ERK1/2 phosphorylation without nuclear accumulation. These findings indicate that the duration of ERK1/2 activation depends on the signaling pathways utilized by GnRH in specific target cells, and that transactivation of the tightly regulated EGF receptor can account for the transient ERK1/2 responses that are elicited by stimulation of certain GPCRs.