Kyeong-Hoon Jeong

Impaired diurnal adrenal rhythmicity restored by constant infusion of corticotropin-releasing hormone in corticotropin-releasing hormone-deficient mice.

The normal pattern of daily glucocorticoid production in mammals requires circadian modulation of hypothalamicpituitary-adrenal axis activity. To assess both the factors responsible for imparting this diurnal profile and its physiologic importance, we have exploited corticotropin-releasing hormone (CRH)-deficient mice generated by homologous recombination in embryonic stem cells. CRH-deficient mice have lost normal circadian variations in plasma ACTH and glucocorticoid while maintaining normal circadian locomotor activity. Constant peripheral infusion of CRH produced marked diurnal excursions of plasma glucocorticoid, indicating that CRH acts in part as a permissive factor for other circadian modulators of adrenocortical activity. The presence of atrophic adrenals in CRH-deficient mice without an overt deficit in basal plasma ACTH concentration suggests that the diurnal increase in ACTH is essential to maintain normal adrenal function.

Identification and Characterization of the Gonadotropin-releasing Hormone Response Elements in the Mouse Gonadotropin-releasing Hormone Receptor Gene

The response of the pituitary gonadotrope to gonadotropin-releasing hormone (GnRH) correlates directly with the concentration of GnRH receptors (GnRHR) on the cell surface, which is mediated in part at the level of GnRHR gene expression. Several hormones have been implicated in this regulation, most notably GnRH itself. Despite these observations and the central role that GnRH is known to play in reproductive development and function, the molecular mechanism(s) by which GnRH regulates transcription of the GnRHR gene has not been well elucidated. Previous studies in this laboratory have identified and partially characterized the promoter region of the mouse GnRHR gene and demonstrated that the regulatory elements for tissue-specific expression as well as for GnRH regulation are present within the 1.2-kilobase 5′-flanking sequence. By using deletion and mutational analysis as well as functional transfection studies in the murine gonadotrope-derived αT3-1 cell line, we have localized GnRH responsiveness of the mouse GnRHR gene to two DNA sequences at −276/−269 (designated Sequence UnderlyingResponsiveness to GnRH-2 (SURG-2), which contains the consensus sequence for the activating protein-1-binding site) and −292/−285 (a novel element designated SURG-1), and demonstrated that this response is mediated via protein kinase C. By using the electrophoretic mobility shift assay, we further demonstrate that a member(s) of the Fos/Jun heterodimer superfamily is responsible in part for the DNA-protein complexes formed on SURG-2, using αT3-1 nuclear extracts. These data define a bipartite GnRH response element in the mouse GnRHR 5′-flanking sequence and suggest that the activating protein-1 complex plays a central role in conferring GnRH responsiveness to the murine GnRHR gene.

Impaired Basal and Restraint-Induced Epinephrine Secretion in Corticotropin-Releasing Hormone- Deficient Mice1

CRH is thought to play a role in responses of the adrenocortical and adrenomedullary systems during stress. To investigate the role of CRH in stress-induced secretions of corticosterone and epinephrine, we subjected wild-type (WT) and CRH-deficient (knockout, KO) mice to restraint, and analyzed plasma corticosterone, plasma catecholamines, and adrenal phenylethanolamine N-methyltransferase (PNMT) gene expression and activity before and during 3 h of restraint. Plasma corticosterone increased over 40-fold in WT mice, but minimally in CRH KO mice. Adrenal corticosterone content tended to increase in CRH KO mice, although to levels 5-fold lower than that in WT mice. CRH KO mice had significantly lower plasma epinephrine and higher norepinephrine than WT mice at baseline, and delayed epinephrine secretion during restraint. Adrenal PNMT messenger RNA content in CRH KO mice tended to be lower than that in WT mice, though the degree of induction was similar in both genotypes. PNMT enzyme activity was significant...

Direct Binding of AP-1 (Fos/Jun) Proteins to a SMAD Binding Element Facilitates Both Gonadotropin-releasing Hormone (GnRH)- and Activin-mediated Transcriptional Activation of the Mouse GnRH Receptor Gene

The response of pituitary gonadotropes to gonadotropin-releasing hormone (GnRH) correlates directly with the concentration of GnRH receptors (GnRHR) on the cell surface, which is mediated in part at the level of gene expression. Several factors are known to affect expression of the mouse GnRHR (mGnRHR) gene, including GnRH and activin. We have previously shown that activin augments GnRH-mediated transcriptional activation of mGnRHR gene, and that region −387/−308 appears to be necessary to mediate this effect. This region contains two overlapping cis-regulatory elements of interest: GnRHR activating sequence (GRAS) and a putative SMAD-binding element (SBE). This study investigates the role of these elements and their cognate transcription factors in transactivation of the mGnRHR gene. Transfection studies confirm the presence of GnRH- and activin-response elements within −387/−308 of mGnRHR gene promoter. Competition electrophoretic mobility shift assay experiments using −335/−312 as probe and αT3–1 nuclear extract or SMAD, Jun, and Fos proteins demonstrate direct binding of AP-1 (Fos/Jun) protein complexes to −327/−322 and SMAD proteins to −329/−328. Further transfection studies using mutant constructs of these cis-regulatory elements confirm that both are functionally important. These data define a novel cis-regulatory element comprised of an overlapping SBE and newly characterized non-consensus AP-1 binding sequence that integrates the stimulatory transcriptional effects of both GnRH and activin on the mGnRHR gene.

Activin A Augments GnRH-Mediated Transcriptional Activation of the Mouse GnRH Receptor Gene

The response of pituitary gonadotropes to GnRH correlates directly with the concentration of GnRH receptors (GnRHRs) on the cell surface, which is mediated in part at the level of GnRHR gene expression. We have previously localized GnRH responsiveness in the mouse GnRHR (mGnRHR) gene promoter to two elements: activating protein-1 and sequence underlying responsiveness to GnRH-1. This study was designed to investigate potential synergy between GnRH and activin A in transcriptional activation of the mGnRHR gene. In functional transfection studies using αT3-1 cells, GnRH agonist stimulation of the mGnRHR gene promoter (−765/+62) resulted in a 10.9-fold increase in activity, which was further increased 2-fold (to 21.3-fold) following activin pretreatment. Activin pretreatment alone had no effect. Deletion of region −387/−308 or mutation of a putative SMAD-binding element at −331/−324 (5′-GTCTAG[T]C-3′) abrogated the augmented response to GnRH in the presence of activin but not the response to GnRH alone. Over...

The physiology of corticotropin-releasing hormone deficiency in mice.

A review of the generation and characterization of corticotropin-releasing hormone (CRH)-deficient mice is presented. The studies summarized demonstrate the central role of CRH in the pituitary-adrenal axis response to stress, circadian stimulation, and glucocorticoid withdrawal. Additionally, pro-inflammatory actions of CRH at sites of local inflammation are given further support. In contrast, behavioral effects during stress that had been ascribed to CRH action are not altered in CRH-deficient mice. The normal behavioral response to stress in CRH-deficient mice strongly suggests the importance of other, possibly as yet undiscovered, CRH-like molecules.

Molecular Mechanisms of Gonadotropin-Releasing Hormone Receptor Gene Regulation

GnRH plays a critical role in regulating mammalian reproductive development and function. At the level of the anterior pituitary, GnRH binds to the GnRH recetor (GnRHR) on the cell surface of pituitary gonadotropes. Here, it activates intracellular signal transduction pathways to effect both the snthesis and intermittent release of the gonadotropins LH and FSH. These hormones then enter the systemic circulation to regualte gonadal function, including steroid hormone synthesis and gametogenesis. The response of pituitary gonadotropes to GnRH correlates directly with the concentration of GnRHR on the cell surface, which is mediated, at least in part, at the level of gene expression. A number of endocrine, paracrine, and autocrine factors are known to regulate GnRHR gene expression. This article reviews in detail the role of the GnRHR in the hypothalamic-pituitary-gonadal axis and the factors mediating expression of this gene. A better understanding of the molecular mechanisms that regulate transcription of the GnRHR gene will further our knowledge about the role of this receptor in mammalian reproductive physiology in health and disease.

Essential Role of the Homeodomain for Pituitary Homeobox 1 Activation of Mouse Gonadotropin-Releasing Hormone Receptor Gene Expression through Interactions with c-Jun and DNA

ABSTRACT The gonadotropin-releasing hormone receptor (GnRHR) is expressed primarily in the gonadotropes of the anterior pituitary. Pituitary homeobox 1 (Pitx-1) has been shown to activate pituitary-specific gene expression by direct DNA binding and/or protein-protein interaction with other transcription factors. We hypothesized that Pitx-1 might also dictate tissue-specific expression of the mouse GnRHR (mGnRHR) gene in a similar manner. Pitx-1 activated the mGnRHR gene promoter, and transactivation was localized to sequences between −308 and −264. Pitx-1 bound to this region only with low affinity. This region includes an activating protein 1 (AP-1) site, which was previously shown to be important for mGnRHR gene expression. Further characterization indicated that an intact AP-1 site was required for full Pitx-1 responsiveness. Furthermore, Pitx-1 and AP-1 were synergistic in the activation of the mGnRHR gene promoter. A Pitx-1 homeodomain (HD) point mutation, which eliminated DNA binding ability, caused only a partial reduction of transactivation, whereas deletion of the HD completely prevented transactivation. Pitx-1 interacted directly with c-Jun, and the HD was sufficient for this interaction. While the point mutation in the Pitx-1 HD did not affect interaction with c-Jun, deletion of the HD eliminated the interaction. Taken together, our studies indicate that Pitx-1 can direct transactivation of the mGnRHR gene, in part by DNA binding and in part by an action of Pitx-1 as a cofactor for AP-1, augmenting AP-1 activity through a novel protein-protein interaction between c-Jun and the HD of Pitx-1.

CHAPTER 31 – Gonadotropin-Releasing Hormone Regulation of Gonadotropin Biosynthesis and Secretion

This chapter focuses on gonadotropin-releasing hormone (GnRH) regulation of gonadotropin biosynthesis and secretion. GnRH is secreted from the hypothalamus in a pulsatile fashion, and pulsatile GnRH stimulates luteinizing hormone (LH) and follicle-stimulating hormone (FSH) biosynthesis and secretion. Agonist occupancy of plasma membrane GnRH receptors is required to stimulate LH release. GnRH regulates gonadotropin release by means of the rapid increase in [Ca 2+ ] i after its binding to the GnRH receptor (GnRHR). Through G protein–coupled signal transduction, GnRH binding rapidly activates phospholipase C (PLC), and PLC produces 1,4,5-trisphosphate (IP 3 ) and 1,2-diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate (PIP 2 ), which in turn rapidly mobilizes transient intracellular Ca 2+ to trigger a burst initiation of exocytosis, causing rapid LH secretion within 10 seconds and lasting for 100 seconds. Besides stimulating the acute release of gonadotropins, GnRH regulates long-term maintenance of pituitary responsiveness to the hormone. The pulse pattern of GnRH administration to the pituitary is a critical determinant of gonadotropin release over periods of time.

Calcium‐sensing receptor stimulates secretion of an interferon‐γ‐induced monokine (CXCL10) and monocyte chemoattractant protein‐3 in immortalized GnRH neurons

Biology of GnRH neurons is critically dependent on extracellular Ca2+ (Ca2+o). We evaluated differences in gene expression patterns with low and high Ca2+o in an immortalized GnRH neuron line, GT1‐7 cells. Mouse global oligonucleotide microarray was used to evaluate transcriptional differences among the genes regulated by elevated Ca2+o. Our result identified two interferon‐γ (IFNγ)‐inducible chemokines, CXCL9 and CXCL10, and a beta chemokine, monocyte chemoattractant protein‐3 (MCP‐3/CCL7), being up‐regulated in GT1‐7 cells treated with high Ca2+o (3.0 mM) compared with low Ca2+o (0.5 mM). Up‐regulation of these mRNAs by elevated Ca2+o was confirmed by quantitative PCR. Elevated Ca2+o stimulated secretion of CXCL10 and MCP‐3 but not CXCL9 in GT1‐7 cells, and this effect was mediated by an extracellular calcium‐sensing receptor (CaR) as the dominant negative CaR attenuated secretion of CXCL10 and MCP‐3. CXCL10 and MCP‐3 were localized in mouse GnRH neurons in the preoptic hypothalamus. Suppression of K+ channels (BK channels) with 25 nM charybdotoxin inhibited high‐Ca2+o‐stimulated CXCL10 release. Accordingly, CaR activation by a specific CaR agonist, NPS‐467, resulted in the activation of a Ca2+‐activated K+ channel in these cells. CaR‐mediated MCP‐3 secretion involves the PI3 kinase pathway in GT1‐7 cells. MCP‐3 stimulated chemotaxis of astrocytes treated with transforming growth factor‐β (TGFβ). With TGFβ‐treated astrocytes, we next observed that conditioned medium from GT1‐7 cells treated with high Ca2+ promoted chemotaxis of astrocytes, and this effect was attenuated by a neutralizing antibody to MCP‐3. These results implicate CaR as an important regulator of GnRH neuron function in vivo by stimulating secretion of heretofore unsuspected cytokines, i.e., CXCL10 and MCP‐3.