Regulation of stress response on the hypothalamic-pituitary-gonadal axis via gonadotropin-inhibitory hormone.

Under stressful condition, reproductive function is impaired due to the activation of various components of the hypothalamic-pituitaryadrenal (HPA) axis, which can suppress the activity of the hypothalamic-pituitary-gonadal (HPG) axis at multiple levels. A hypothalamic neuropeptide, gonadotropin-inhibitory hormone (GnIH) is a key negative regulator of reproduction that governs the HPG axis. Converging lines of evidence have suggested that different stress types and their duration, such as physical or psychological, and acute or chronic, can modulate the GnIH system. To clarify the sensitivity and reactivity of the GnIH system in response to stress, we summarize and critically review the available studies that investigated the effects of various stressors, such as restraint, nutritional/metabolic and social stress, on GnIH expression and/or its neuronal activity leading to altered HPG action. In this review, we focus on GnIH as the potential novel mediator responsible for stress-induced reproductive dysfunction.

Isoform-selective regulation of mammalian cryptochromes.

CRY1 and CRY2 are essential components of the circadian clock controlling daily physiological rhythms. Accumulating evidences indicate distinct roles of these highly homologous proteins, in addition to redundant functions. Therefore, the development of isoform-selective compounds represents an effective approach towards understanding the similarities and differences of CRY1 and CRY2 by controlling each isoform individually. We conducted phenotypic screenings of circadian clock modulators, and identified KL101 and TH301 that selectively stabilize CRY1 and CRY2, respectively. Crystal structures of CRY-compound complexes revealed conservation of compound-binding sites between CRY1 and CRY2. We further discovered a unique mechanism underlying compound selectivity in which the disordered C-terminal region outside the pocket was required for the differential effects of KL101 and TH301 against CRY isoforms. By using these compounds, we found a new role of CRY1 and CRY2 as enhancers of brown adipocyte differentiation, providing the basis of CRY-mediated regulation of energy expenditure.

Molecular Mechanisms of Gonadotropin-Inhibitory Hormone (GnIH) Actions in Target Cells and Regulation of GnIH Expression.

Since gonadotropin-inhibitory hormone (GnIH) was discovered in 2000 as the first hypothalamic neuropeptide that actively inhibits gonadotropin release, researches conducted for the last 18 years have demonstrated that GnIH acts as a pronounced negative regulator of reproduction. Inhibitory effect of GnIH on reproduction is mainly accomplished at hypothalamic-pituitary levels; gonadotropin-releasing hormone (GnRH) neurons and gonadotropes are major targets of GnIH action based on the morphological interaction with GnIH neuronal fibers and the distribution of GnIH receptor. Here, we review molecular studies mainly focusing on the signal transduction pathway of GnIH in target cells, GnRH neurons, and gonadotropes. The use of well-defined cellular model systems allows the mechanistic study of signaling pathway occurring in target cells by demonstrating the direct cause-and-effect relationship. The insights gained through studying molecular mechanism of GnIH action contribute to deeper understanding of the mechanism of how GnIH communicates with other neuronal signaling systems to control our reproductive function. Reproductive axis closely interacts with other endocrine systems, thus GnIH expression levels would be changed by adrenal and thyroid status. We also briefly review molecular studies investigating the regulatory mechanisms of GnIH expression to understand the role of GnIH as a mediator between adrenal, thyroid and gonadal axes.

Review: Structure, function and evolution of GnIH.

Neuropeptides that possess the Arg-Phe-NH2 motif at their C-termini (i.e., RFamide peptides) have been characterized in the nervous system of both invertebrates and vertebrates. In vertebrates, RFamide peptides make a family and consist of the groups of gonadotropin-inhibitory hormone (GnIH), neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP), kisspeptin (kiss1 and kiss2), and pyroglutamylated RFamide peptide/26RFamide peptide (QRFP/26RFa). It now appears that these vertebrate RFamide peptides exert important neuroendocrine, behavioral, sensory, and autonomic functions. In 2000, GnIH was discovered as a novel hypothalamic RFamide peptide inhibiting gonadotropin release in quail. Subsequent studies have demonstrated that GnIH acts on the brain and pituitary to modulate reproductive physiology and behavior across vertebrates. To clarify the origin and evolution of GnIH, the existence of GnIH was investigated in agnathans, the most ancient lineage of vertebrates, and basal chordates, such as tunicates and cephalochordates (represented by amphioxus). This review first summarizes the structure and function of GnIH and other RFamide peptides, in particular NPFF having a similar C-terminal structure of GnIH, in vertebrates. Then, this review describes the evolutionary origin of GnIH based on the studies in agnathans and basal chordates.

Discovery of GnIH and Its Role in Hypothyroidism-Induced Delayed Puberty.

It is known that hypothyroidism delays puberty in mammals. Interaction between the hypothalamo-pituitary-thyroid (HPT) and hypothalamo-pituitary-gonadal (HPG) axes may be important processes in delayed puberty. Gonadotropin-inhibitory hormone (GnIH) is a newly discovered hypothalamic neuropeptide that inhibits gonadotropin synthesis and release in quail. It now appears that GnIH is conserved across various mammals and primates, including humans, and inhibits reproduction. We have further demonstrated that GnIH is involved in pubertal delay induced by thyroid dysfunction in female mice. Hypothyroidism delays pubertal onset with the increase in hypothalamic GnIH expression and the decrease in circulating gonadotropin and estradiol levels. Thyroid status regulates GnIH expression by epigenetic modification of the GnIH promoter region. Furthermore, knockout of GnIH gene abolishes the effect of hypothyroidism on delayed pubertal onset. Accordingly, it is considered that GnIH is a mediator of pubertal disorder induced by thyroid dysfunction. This is a novel function of GnIH that interacts between the HPT-HPG axes in pubertal onset delay. This mini-review summarizes the structure, expression, and function of GnIH and highlights the action of GnIH in pubertal disorder induced by thyroid dysfunction.

Involvement of gonadotropin-inhibitory hormone in pubertal disorders induced by thyroid status.

Thyroid disorders cause abnormal puberty, indicating interactions between the hypothalamus-pituitary-thyroid (HPT) and hypothalamus-pituitary-gonadal (HPG) axes, which are important in pubertal development. The hypothalamic gonadotropin-inhibitory hormone (GnIH) was shown to be decreased in the early prepubertal stage, suggesting the role of GnIH on pubertal onset. Here, we investigated whether thyroid dysfunction affects pubertal onset in female mice via GnIH regulation. Hypothyroidism showed delayed pubertal onset with increased GnIH expression and reduced pituitary-gonadal activity. Remarkably, knockout of GnIH prevented the effect of hypothyroidism to delay the pubertal onset, resulting in indistinguishable pubertal timing in GnIH-knockout female mice between control and hypothyroidism-induced group, indicating that increased GnIH expression induced by hypothyroidism may lead to delayed puberty. In contrast, hyperthyroidism led to a decrease in GnIH expression, however pubertal onset was normal, implying further reduction of the inhibitory GnIH had little effect on the phenotypical change. Critically, thyroid hormone suppressed GnIH expression in hypothalamic explants and GnIH neurons expressed thyroid hormone receptors to convey the thyroid status. Moreover, the thyroid status highly regulated the chromatin modifications of GnIH promoter, H3acetylation and H3K9tri-methylation. These findings indicate a novel function of GnIH to mediate HPT-HPG interactions that contribute to proper pubertal development.

Inhibitory action of gonadotropin-inhibitory hormone on the signaling pathways induced by kisspeptin and vasoactive intestinal polypeptide in GnRH neuronal cell line, GT1-7.

Gonadotropin-inhibitory hormone (GnIH) acts as a negative regulator of reproduction by acting on gonadotropes and gonadotropin-releasing hormone (GnRH) neurons. Despite its functional significance, the molecular mechanism of GnIH action in the target cells has not been fully elucidated. To expand our previous study on GnIH actions in gonadotropes, we investigated the potential signal transduction pathway that conveys the inhibitory action of GnIH in GnRH neurons by using the GnRH neuronal cell line, GT1-7. We examined whether GnIH inhibits the action of kisspeptin and vasoactive intestinal polypeptide (VIP), positive regulators of GnRH neurons. Although GnIH significantly suppressed the stimulatory effect of kisspeptin on GnRH release in hypothalamic culture, GnIH had no inhibitory effect on kisspeptin stimulation of serum response element and nuclear factor of activated T-cell response element activities and ERK phosphorylation, indicating that GnIH may not directly inhibit kisspeptin signaling in GnRH neurons. On the contrary, GnIH effectively eliminated the stimulatory effect of VIP on p38 and ERK phosphorylation, c-Fos mRNA expression, and GnRH release. The use of pharmacological modulators strongly demonstrated the specific inhibitory action of GnIH on the adenylate cyclase/cAMP/protein kinase A pathway, suggesting a common inhibitory mechanism of GnIH action in GnRH neurons and gonadotropes.-Son, Y. L., Ubuka, T., Soga, T., Yamamoto, K., Bentley, G. E., Tsutsui, K. Inhibitory action of gonadotropin-inhibitory hormone on the signaling pathways induced by kisspeptin and vasoactive intestinal polypeptide in GnRH neuronal cell line, GT1-7.

Molecular, cellular, morphological, physiological and behavioral aspects of gonadotropin-inhibitory hormone.

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that was isolated from the brains of Japanese quail in 2000, which inhibited luteinizing hormone release from the anterior pituitary gland. Here, we summarize the following fifteen years of researches that investigated on the mechanism of GnIH actions at molecular, cellular, morphological, physiological, and behavioral levels. The unique molecular structure of GnIH peptide is in its LPXRFamide (X=L or Q) motif at its C-terminal. The primary receptor for GnIH is GPR147. The cell signaling pathway triggered by GnIH is initiated by inhibiting adenylate cyclase and decreasing cAMP production in the target cell. GnIH neurons regulate not only gonadotropin synthesis and release in the pituitary, but also regulate various neurons in the brain, such as GnRH1, GnRH2, dopamine, POMC, NPY, orexin, MCH, CRH, oxytocin, and kisspeptin neurons. GnIH and GPR147 are also expressed in gonads and they may regulate steroidogenesis and germ cell maturation in an autocrine/paracrine manner. GnIH regulates reproductive development and activity. In female mammals, GnIH may regulate estrous or menstrual cycle. GnIH is also involved in the regulation of seasonal reproduction, but GnIH may finely tune reproductive activities in the breeding seasons. It is involved in stress responses not only in the brain but also in gonads. GnIH may inhibit male socio-sexual behavior by stimulating the activity of cytochrome P450 aromatase in the brain and stimulates feeding behavior by modulating the activities of hypothalamic and central amygdala neurons.

RFamide peptides in agnathans and basal chordates.

Since a peptide with a C-terminal Arg-Phe-NH2 (RFamide peptide) was first identified in the ganglia of the venus clam in 1977, RFamide peptides have been found in the nervous system of both invertebrates and vertebrates. In vertebrates, the RFamide peptide family includes gonadotropin-inhibitory hormone (GnIH), neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP), pyroglutamylated RFamide peptide/26RFamide peptide (QRFP/26RFa), and kisspeptins (kiss1 and kiss2). They are involved in important functions such as the release of hormones, regulation of sexual or social behavior, pain transmission, reproduction, and feeding. In contrast to tetrapods and jawed fish, the information available on RFamide peptides in agnathans and basal chordates is limited, thus preventing further insights into the evolution of RFamide peptides in vertebrates. In this review, we focus on the previous research and recent advances in the studies on RFamide peptides in agnathans and basal chordates. In agnathans, the genes encoding GnIH, NPFF, and PrRP precursors and the mature peptides have been identified in lamprey (Petromyzon marinus) and hagfish (Paramyxine atami). Putative kiss1 and kiss2 genes have also been found in the genome database of lamprey. In basal chordates, namely, in amphioxus (Branchiostoma japonicum), a common ancestral form of GnIH and NPFF genes and their mature peptides, as well as the ortholog of the QRFP gene have been identified. The studies revealed that the number of orthologs of vertebrate RFamide peptides present in agnathans and basal chordates is greater than expected, suggesting that the vertebrate RFamide peptides might have emerged and expanded at an early stage of chordate evolution.

Contribution of GnIH Research to the Progress of Reproductive Neuroendocrinology.

Since the discovery of gonadotropin-releasing hormone (GnRH) in mammals at the beginning of the 1970s, it was generally accepted that GnRH is the only hypothalamic neuropeptide regulating gonadotropin release in mammals and other vertebrates. In 2000, however, gonadotropin-inhibitory hormone (GnIH), a novel hypothalamic neuropeptide that actively inhibits gonadotropin release, was discovered in quail. Numerous studies over the past decade and a half have demonstrated that GnIH serves as a key player regulating reproduction across vertebrates, acting on the brain and pituitary to modulate reproductive physiology and behavior. In the latter case, recent evidence indicates that GnIH can regulate reproductive behavior through changes in neurosteroid, such as neuroestrogen, biosynthesis in the brain. This review summarizes the discovery of GnIH, and the contributions to GnIH research focused on its mode of action, regulation of biosynthesis, and how these findings advance our understanding of reproductive neuroendocrinology.

A new pathway mediating social effects on the endocrine system: female presence acting via norepinephrine release stimulates gonadotropin-inhibitory hormone in the paraventricular nucleus and suppresses luteinizing hormone in quail.

Rapid effects of social interactions on transient changes in hormonal levels are known in a wide variety of vertebrate taxa, ranging from fish to humans. Although these responses are mediated by the brain, neurochemical pathways that translate social signals into reproductive physiological changes are unclear. In this study, we analyzed how a female presence modifies synthesis and/or release of various neurochemicals, such as monoamines and neuropeptides, in the brain and downstream reproductive hormones in sexually active male Japanese quail. By viewing a female bird, sexually active males rapidly increased norepinephrine (NE) release in the paraventricular nucleus (PVN) of the hypothalamus, in which gonadotropin-inhibitory hormone (GnIH) neuronal cell bodies exist, increased GnIH precursor mRNA expression in the PVN, and decreased luteinizing hormone (LH) concentration in the plasma. GnIH is a hypothalamic neuropeptide that inhibits gonadotropin secretion from the pituitary. It was further shown that GnIH can rapidly suppress LH release after intravenous administration in this study. Centrally administered NE decreased plasma LH concentration in vivo. It was also shown that NE stimulated the release of GnIH from diencephalic tissue blocks in vitro. Fluorescence double-label immunohistochemistry indicated that GnIH neurons received noradrenergic innervations, and immunohistochemistry combined with in situ hybridization have further shown that GnIH neurons expressed α2A-adrenergic receptor mRNA. These results indicate that a female presence increases NE release in the PVN and stimulates GnIH release, resulting in the suppression of LH release in sexually active male quail.

Evolutionary origin of GnIH and NPFF in chordates: insights from novel amphioxus RFamide peptides.

Gonadotropin-inhibitory hormone (GnIH) is a newly identified hypothalamic neuropeptide that inhibits pituitary hormone secretion in vertebrates. GnIH has an LPXRFamide (X = L or Q) motif at the C-terminal in representative species of gnathostomes. On the other hand, neuropeptide FF (NPFF), a neuropeptide characterized as a pain-modulatory neuropeptide, in vertebrates has a PQRFamide motif similar to the C-terminal of GnIH, suggesting that GnIH and NPFF have diverged from a common ancestor. Because GnIH and NPFF belong to the RFamide peptide family in vertebrates, protochordate RFamide peptides may provide important insights into the evolutionary origin of GnIH and NPFF. In this study, we identified a novel gene encoding RFamide peptides and two genes of their putative receptors in the amphioxus Branchiostoma japonicum. Molecular phylogenetic analysis and synteny analysis indicated that these genes are closely related to the genes of GnIH and NPFF and their receptors of vertebrates. We further identified mature RFamide peptides and their receptors in protochordates. The identified amphioxus RFamide peptides inhibited forskolin induced cAMP signaling in the COS-7 cells with one of the identified amphioxus RFamide peptide receptors expressed. These results indicate that the identified protochordate RFamide peptide gene is a common ancestral form of GnIH and NPFF genes, suggesting that the origin of GnIH and NPFF may date back to the time of the emergence of early chordates. GnIH gene and NPFF gene may have diverged by whole-genome duplication in the course of vertebrate evolution.

Molecular basis for the activation of gonadotropin-inhibitory hormone gene transcription by corticosterone.

The inhibitory effect of stress on reproductive function is potentially mediated by high concentrations of circulating glucocorticoids (GCs) acting via the GC receptor (GR). Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that inhibits gonadotropin secretion. GnIH may mediate stress-induced reproductive dysfunction. However, it is not yet known whether GC-bound GR is directly involved in GnIH transcription. Here, we demonstrated the localization of GR mRNA in GnIH neurons in the paraventricular nucleus of quail, suggesting that GC can directly regulate GnIH transcription. We next showed that 24 hours of treatment with corticosterone (CORT) increase GnIH mRNA expression in the quail diencephalon. We further investigated the mechanism of activation of GnIH transcription by CORT using a GnIH-expressing neuronal cell line, rHypoE-23, derived from rat hypothalamus. We found the expression of GR mRNA in rHypoE-23 cells and increased GnIH mRNA expression by 24 hours of CORT treatment. We finally characterized the promoter activity of rat GnIH gene stimulated by CORT. Through DNA deletion analysis, we identified a CORT-responsive region at 2000-1501 bp upstream of GnIH precursor coding region. This region included 2 GC response elements (GREs) at -1665 and -1530 bp. Mutation of -1530 GRE abolished CORT responsiveness. We also found CORT-stimulated GR recruitment at the GnIH promoter region containing the -1530 GRE. These results provide a putative molecular basis for transcriptional activation of GnIH under stress by demonstrating that CORT directly induces GnIH transcription by recruitment of GR to its promoter.

Central and direct regulation of testicular activity by gonadotropin-inhibitory hormone and its receptor.

Gonadotropin-inhibitory hormone (GnIH) was first identified in Japanese quail to be an inhibitor of gonadotropin synthesis and release. GnIH peptides have since been identified in all vertebrates, and all share an LPXRFamide (X = L or Q) motif at their C-termini. The receptor for GnIH is the G protein-coupled receptor 147 (GPR147), which inhibits cAMP signaling. Cell bodies of GnIH neurons are located in the paraventricular nucleus (PVN) in birds and the dorsomedial hypothalamic area (DMH) in most mammals. GnIH neurons in the PVN or DMH project to the median eminence to control anterior pituitary function via GPR147 expressed in gonadotropes. Further, GnIH inhibits gonadotropin-releasing hormone (GnRH)-induced gonadotropin subunit gene transcription by inhibiting the adenylate cyclase/cAMP/PKA-dependent ERK pathway in an immortalized mouse gonadotrope cell line (LßT2 cells). GnIH neurons also project to GnRH neurons that express GPR147 in the preoptic area (POA) in birds and mammals. Accordingly, GnIH can inhibit gonadotropin synthesis and release by decreasing the activity of GnRH neurons as well as by directly inhibiting pituitary gonadotrope activity. GnIH and GPR147 can thus centrally suppress testosterone secretion and spermatogenesis by acting in the hypothalamic-pituitary-gonadal axis. GnIH and GPR147 are also expressed in the testis of birds and mammals, possibly acting in an autocrine/paracrine manner to suppress testosterone secretion and spermatogenesis. GnIH expression is also regulated by melatonin, stress, and social environment in birds and mammals. Accordingly, the GnIH-GPR147 system may play a role in transducing physical and social environmental information to regulate optimal testicular activity in birds and mammals. This review discusses central and direct inhibitory effects of GnIH and GPR147 on testosterone secretion and spermatogenesis in birds and mammals.

Molecular evolution of kiss2 genes and peptides in vertebrates.

The kiss1 peptide (kisspeptin), a product of the kiss1 gene, is one of the key neuropeptides regulating vertebrate reproduction. In 2009, we identified a paralogous gene of kiss1 in the brain of amphibians and named it kiss2. Currently, the presence of the kiss2 gene and the kiss2 peptide is still obscure in amniotes compared with that in other vertebrates. Therefore, we performed genome database analyses in primates and reptiles to investigate the molecular evolution of the kiss2 gene in vertebrates. Because the mature kiss2 peptide has been identified only in amphibians, we further performed immunoaffinity purification and mass spectrometry to identify the mature endogenous kiss2 peptide in the brains of salmon and turtle that possessed the kiss2 gene. Here we provide the first evidence for the presence of a kiss2-like gene in the genome database of primates including humans. Synthetic amidated human KISS2 peptide activated human GPR54 expressed in COS7 cells, but nonamidated KISS2 peptide was inactive. The endogenous amidated kiss2 peptide may not be produced in primates because of the lack of an amidation signal in the precursor polypeptide. The kiss2-like gene may be nonfunctional in crocodilians because of premature stop codons. We identified the mature amidated kiss2 peptide in turtles and fish and analyzed the localization of kiss2 peptide mRNA expression in fish. The present study suggests that the kiss2 gene may have mutated in primates and crocodilians and been lost in birds during the course of evolution. In contrast, the kiss2 gene and mature kiss2 peptide are present in turtles and fish.

Gonadotropin-inhibitory hormone (GnIH), GnIH receptor and cell signaling.

Gonadotropin-inhibitory hormone (GnIH) is an inhibitor of gonadotropin synthesis and release, which was originally identified in the hypothalamus of the Japanese quail (Coturnix japonica). The GnIH precursor polypeptide encodes one GnIH and two GnIH related peptides (GnIH-RP-1 and GnIH-RP-2) in birds that share the same C-terminal LPXRFamide (X=L or Q) motif. The receptor for GnIH is thought to be the G protein-coupled receptor 147 (GPR147) which has been shown to couple predominantly through the Gαi protein to inhibit cAMP production. The crude membrane fraction of COS-7 cells transfected with GPR147 cDNA specifically bound GnIH and GnIH-RPs in a concentration-dependent manner. Scatchard plot analysis of the binding showed that GPR147 possessed a single class of high-affinity binding sites. GnIH neurons project to the median eminence to control anterior pituitary function and GPR147 is expressed in the gonadotropes. GnIH neurons also project to gonadotropin-releasing hormone (GnRH)-I and GnRH-II neurons, and GnRH-I and GnRH-II neurons express GPR147. Thus, GnIH may inhibit gonadotropin synthesis and release by decreasing the activity of GnRH-I neurons as well as directly inhibiting the effects of GnRH on gonadotropes. GnIH may also partially inhibit reproductive behaviors by inhibiting GnRH-II neurons. GnIH and GPR147 are also expressed in the gonads, possibly acting in an autocrine/paracrine manner. The cell signaling process of GPR147 was extensively studied using LßT2 cells, a mouse gonadotrope cell line. In this cell line, mouse GnIH inhibits GnRH-induced gonadotropin subunit, LHß, FSHß, and common α, gene transcriptions by inhibiting adenylate cyclase/cAMP/PKA dependent ERK pathway. This review summarizes the functions of GnIH, GnIH receptor and its cell signaling processes in birds and discusses related findings in mammals.

Gonadotropin-inhibitory hormone action in the brain and pituitary.

Gonadotropin-inhibitory hormone (GnIH) was first identified in the Japanese quail as a hypothalamic neuropeptide inhibitor of gonadotropin secretion. Subsequent studies have shown that GnIH is present in the brains of birds including songbirds, and mammals including humans. The identified avian and mammalian GnIH peptides universally possess an LPXRFamide (X = L or Q) motif at their C-termini. Mammalian GnIH peptides are also designated as RFamide-related peptides from their structures. The receptor for GnIH is the G protein-coupled receptor 147 (GPR147), which is thought to be coupled to G(αi) protein. Cell bodies of GnIH neurons are located in the paraventricular nucleus (PVN) in birds and the dorsomedial hypothalamic area (DMH) in mammals. GnIH neurons in the PVN or DMH project to the median eminence to control anterior pituitary function. GPR147 is expressed in the gonadotropes and GnIH suppresses synthesis and release of gonadotropins. It was further shown in immortalized mouse gonadotrope cell line (LßT2 cells) that GnIH inhibits gonadotropin-releasing hormone (GnRH) induced gonadotropin subunit gene transcriptions by inhibiting adenylate cyclase/cAMP/PKA-dependent ERK pathway. GnIH neurons also project to GnRH neurons in the preoptic area, and GnRH neurons express GPR147 in birds and mammals. Accordingly, GnIH may inhibit gonadotropin synthesis and release by decreasing the activity of GnRH neurons as well as directly acting on the gonadotropes. GnIH also inhibits reproductive behavior possibly by acting within the brain. GnIH expression is regulated by a nocturnal hormone melatonin and stress in birds and mammals. Accordingly, GnIH may play a role in translating environmental information to inhibit reproductive physiology and behavior of birds and mammals. Finally, GnIH has therapeutic potential in the treatment of reproductive cycle and hormone-dependent diseases, such as precocious puberty, endometriosis, uterine fibroids, and prostatic and breast cancers.

Gonadotropin-inhibitory hormone inhibits GnRH-induced gonadotropin subunit gene transcriptions by inhibiting AC/cAMP/PKA-dependent ERK pathway in LßT2 cells.

A neuropeptide that directly inhibits gonadotropin secretion from the pituitary was discovered in quail and named gonadotropin-inhibitory hormone (GnIH). The presence and functional roles of GnIH orthologs, RF-amide-related peptides (RFRP), that possess a common C-terminal LPXRF-amide (X = L or Q) motif have also been demonstrated in mammals. GnIH orthologs inhibit gonadotropin synthesis and release by acting on pituitary gonadotropes and GnRH neurons in the hypothalamus via its receptor (GnIH receptor). It is becoming increasingly clear that GnIH is an important hypothalamic neuropeptide controlling reproduction, but the detailed signaling pathway mediating the inhibitory effect of GnIH on target cells is still unknown. In the present study, we investigated the pathway of GnIH cell signaling and its possible interaction with GnRH signaling using a mouse gonadotrope cell line, LßT2. First, we demonstrated the expression of GnIH receptor mRNA in LßT2 cells by RT-PCR. We then examined the inhibitory effects of mouse GnIH orthologs [mouse RFRP (mRFRP)] on GnRH-induced cell signaling events. We showed that mRFRP effectively inhibited GnRH-induced cAMP signaling by using a cAMP-sensitive reporter system and measuring cAMP levels, indicating that mRFRP function as an inhibitor of adenylate cyclase. We further showed that mRFRP inhibited GnRH-stimulated ERK phosphorylation, and this effect was mediated by the inhibition of the protein kinase A pathway. Finally, we demonstrated that mRFRP inhibited GnRH-stimulated gonadotropin subunit gene transcriptions and also LH release. Taken together, the results indicate that mRFRP function as GnIH to inhibit GnRH-induced gonadotropin subunit gene transcriptions by inhibiting adenylate cyclase/cAMP/protein kinase A-dependent ERK activation in LßT2 cells.

General and specific determinants of the selective interactions between SRC-1 NR box-2 and target nuclear receptors.

Nuclear receptors (NRs) associate with various coactivator proteins via direct interaction with a short LXXLL motif (also called NR box) that is present among coactivators. Here we identified the critical residues within or outside NR box-2 or -3 of SRC-1, which are required for the optimal interaction with LXR/RXR heterodimers using the yeast one- plus two-hybrid screening system. The critical residues of NR box-2 were broadly located from position -4 to +5 of the NR box (where +1 is the first L of LXXLL motif), whereas those of NR box-3 were located between -1 and +5. We assessed the functional and physical interactions between the isolated NR box-2 mutants and various NRs. Among the NR box-2 mutants, H-3Q, I-1T/V and H+2P mutants evidenced different interaction profiles depending on the target NRs, thereby indicating that these residues are the specific determinants required for the selective interaction between the SRC-1 NR box-2 and a given receptor.

Molecular determinants of the interactions between SRC-1 and LXR/RXR heterodimers.

Liver X receptor (LXR)/retinoid X receptor (RXR) heterodimers have been shown to perform critical functions in cholesterol and lipid metabolism. Here, we have conducted a comparative analysis of the contributions of LXR and RXR binding to steroid receptor coactivator-1 (SRC-1), which contains three copies of the NR box. We demonstrated that the coactivator-binding surface of LXR, but not that of RXR, is critically important for physical and functional interactions with SRC-1, thereby confirming that RXR functions as an allosteric activator of SRC-1-LXR interaction. Notably, we identified NR box-2 and -3 as the essential binding targets for the SRC-1-induced stimulation of LXR transactivity, and observed the competitive in vitro binding of NR box-2 and -3 to LXR.