Regulation of stress response on the hypothalamic-pituitary-gonadal axis via gonadotropin-inhibitory hormone
You Lee Son, Takayoshi Ubuka, Kazuyoshi Tsutsui
Frontiers in Neuroendocrinology, 64, 2022年01月
研究論文（学術雑誌）, 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.

Regulation of stress response on the hypothalamic-pituitary-gonadal axis via gonadotropin-inhibitory hormone.
You Lee Son, Takayoshi Ubuka, Kazuyoshi Tsutsui
Frontiers in neuroendocrinology, 64, 100953, 100953, 2022年01月, ［国際誌］
英語, 研究論文（学術雑誌）, 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.
Simon Miller, You Lee Son, Yoshiki Aikawa, Eri Makino, Yoshiko Nagai, Ashutosh Srivastava, Tsuyoshi Oshima, Akiko Sugiyama, Aya Hara, Kazuhiro Abe, Kunio Hirata, Shinya Oishi, Shinya Hagihara, Ayato Sato, Florence Tama, Kenichiro Itami, Steve A Kay, Megumi Hatori, Tsuyoshi Hirota
Nature chemical biology, 16, 6, 676, 685, 2020年03月30日, ［査読有り］, ［筆頭著者］, ［国際誌］
英語, 研究論文（学術雑誌）, 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
You Lee Son, Takayoshi Ubuka, Kazuyoshi Tsutsui
Frontiers in Endocrinology, 10, 110, 110, Frontiers Media SA, 2019年02月25日, ［査読有り］, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, 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
Kazuyoshi Tsutsui, Tomohiro Osugi, You Lee Son, Takayoshi Ubuka
General and Comparative Endocrinology, 264, 48, 57, Elsevier BV, 2018年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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
Kazuyoshi Tsutsui, You Lee Son, Mika Kiyohara, Ichiro Miyata
Endocrinology, 159, 1, 62, 68, The Endocrine Society, 2018年01月01日, ［査読有り］
研究論文（学術雑誌）,
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
Mika Kiyohara, You Lee Son, Kazuyoshi Tsutsui
Scientific Reports, 7, 1, Springer Science and Business Media LLC, 2017年12月, ［査読有り］, ［筆頭著者, 責任著者］
研究論文（学術雑誌）

Inhibitory action of gonadotropin-inhibitory hormone on the signaling pathways induced by kisspeptin and vasoactive intestinal polypeptide in GnRH neuronal cell line, GT1-7
You Lee Son, Takayoshi Ubuka, Tomoko Soga, Kazutoshi Yamamoto, George E. Bentley, Kazuyoshi Tsutsui
FASEB JOURNAL, 30, 6, 2198, 2210, FEDERATION AMER SOC EXP BIOL, 2016年06月, ［査読有り］
英語, 研究論文（学術雑誌）, 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
Takayoshi Ubuka, You Lee Son, Kazuyoshi Tsutsui
General and Comparative Endocrinology, 227, 27, 50, Elsevier BV, 2016年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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
Tomohiro Osugi, You Lee Son, Takayoshi Ubuka, Honoo Satake, Kazuyoshi Tsutsui
General and Comparative Endocrinology, 227, 94, 100, Elsevier BV, 2016年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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
Kazuyoshi Tsutsui, Takayoshi Ubuka, You Lee Son, George E. Bentley, Lance J. Kriegsfeld
Frontiers in Endocrinology, 6, 179, 179, Frontiers Media SA, 2015年11月23日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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
Yasuko Tobari, You Lee Son, Takayoshi Ubuka, Yoshihisa Hasegawa, Kazuyoshi Tsutsui
JOURNAL OF NEUROSCIENCE, 34, 29, 9803, 9811, SOC NEUROSCIENCE, 2014年07月, ［査読有り］
英語, 研究論文（学術雑誌）, 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 alpha 2A-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
Tomohiro Osugi, Tomoki Okamura, You Lee Son, Makoto Ohkubo, Takayoshi Ubuka, Yasuhisa Henmi, Kazuyoshi Tsutsui
PLoS ONE, 9, 7, e100962, e100962, Public Library of Science (PLoS), 2014年07月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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
You Lee Son, Takayoshi Ubuka, Misato Narihiro, Yujiro Fukuda, Itaru Hasunuma, Kazutoshi Yamamoto, Denise D. Belsham, Kazuyoshi Tsutsui
ENDOCRINOLOGY, 155, 5, 1817, 1826, ENDOCRINE SOC, 2014年05月, ［査読有り］
英語, 研究論文（学術雑誌）, 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
Takayoshi Ubuka, You Lee Son, Yasuko Tobari, Misato Narihiro, George E. Bentley, Lance J. Kriegsfeld, Kazuyoshi Tsutsui
Frontiers in Endocrinology, 5, 8, 8, Frontiers Media SA, 2014年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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 (LβT2 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
Tomohiro Osugi, Naohito Ohtaki, Yuya Sunakawa, You Lee Son, Makoto Ohkubo, Masayuki Iigo, Masafumi Amano, Kazuyoshi Tsutsui
Endocrinology, 154, 11, 4270, 4280, The Endocrine Society, 2013年11月01日, ［査読有り］
研究論文（学術雑誌）, 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
Takayoshi Ubuka, You Lee Son, George E. Bentley, Robert P. Millar, Kazuyoshi Tsutsui
General and Comparative Endocrinology, 190, 10, 17, Elsevier BV, 2013年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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 LβT2 cells, a mouse gonadotrope cell line. In this cell line, mouse GnIH inhibits GnRH-induced gonadotropin subunit, LHβ, FSHβ, 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 Inhibits GnRH-Induced Gonadotropin Subunit Gene Transcriptions by Inhibiting AC/cAMP/PKA-Dependent ERK Pathway in LβT2 Cells
You Lee Son, Takayoshi Ubuka, Robert P. Millar, Haruhiko Kanasaki, Kazuyoshi Tsutsui
Endocrinology, 153, 5, 2332, 2343, The Endocrine Society, 2012年05月01日, ［査読有り］
研究論文（学術雑誌）, 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, LβT2. First, we demonstrated the expression of GnIH receptor mRNA in LβT2 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 LβT2 cells.

総説：生殖を制御する新規脳ホルモンGnIHの起源と分子進化 比較内分泌学
筒井和義, 大杉知裕, 戸張靖子, 孫 ユリ, 産賀崇由
比較内分泌学, 38, 145, 76, 83, 日本比較内分泌学会, 2012年
日本語

Gonadotropin-inhibitory hormone action in the brain and pituitary
Takayoshi Ubuka, You Lee Son, Yasuko Tobari, Kazuyoshi Tsutsui
Frontiers in Endocrinology, 3, 148, 148, Frontiers Media SA, 2012年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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 (LβT2 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.

General and specific determinants of the selective interactions between SRC-1 NR box-2 and target nuclear receptors
You Lee Son, Min-Jung Park, Young Chul Lee
Molecular Biology Reports, 39, 1, 177, 184, Springer Science and Business Media LLC, 2012年01月, ［査読有り］
研究論文（学術雑誌）

Molecular determinants of the interactions between SRC-1 and LXR/RXR heterodimers
You Lee Son, Young Chul Lee
FEBS Letters, 584, 18, 3862, 3866, Wiley, 2010年09月24日, ［査読有り］
研究論文（学術雑誌）

Molecular Determinants Required for Selective Interactions between the Thyroid Hormone Receptor Homodimer and the Nuclear Receptor Corepressor N-CoR
Ji Young Kim, You Lee Son, Jeong-Sun Kim, Young Chul Lee
Journal of Molecular Biology, 396, 3, 747, 760, Elsevier BV, 2010年02月, ［査読有り］
研究論文（学術雑誌）

Molecular determinants of the interactions between LXR/RXR heterodimers and TRAP220
You Lee Son, Young Chul Lee
Biochemical and Biophysical Research Communications, 384, 3, 389, 393, Elsevier BV, 2009年07月, ［査読有り］
研究論文（学術雑誌）

Involvement of SMRT Corepressor in Transcriptional Repression by the Vitamin D Receptor
Ji Young Kim, You Lee Son, Young Chul Lee
MOLECULAR ENDOCRINOLOGY, 23, 2, 251, 264, ENDOCRINE SOC, 2009年02月, ［査読有り］
英語, 研究論文（学術雑誌）, To repress the expression of target genes, the unliganded nuclear receptor generally recruits the silencing mediator of retinoid and thyroid hormone receptor (SMRT)/nuclear receptor corepressor via its direct association with the conserved motif within bipartite nuclear receptor-interaction domains (IDs) of the corepressor. Here, we investigated the involvement of the SMRT corepressor in transcriptional repression by the unliganded vitamin D receptor (VDR). Using small interference RNA against SMRT in human embryonic kidney 293 cells, we demonstrated that SMRT is involved in the repression of the VDR-target genes, osteocalcin and vitamin D(3) 24-hydroxylase in vivo. Consistent with this, VDR and SMRT are recruited to the vitamin D response element of the endogenous osteocalcin promoter in the absence of 1 alpha, 25-(OH)(2)D(3) in chromatin immunoprecipitation assays. To address the involvement of the VDR-specific interaction of SMRT in this repression, we identified the molecular determinants of the interaction between VDR and SMRT. Interestingly, VDR specifically interacts with ID1 of the SMRT/nuclear receptor corepressor and that ID1 is required for their stable interaction. We also identified specific residues in the SMRT-ID1 that are required for VDR binding, using the one-plus two-hybrid system, a novel genetic selection method for specific missense mutations that disrupt protein-protein interactions. These mutational studies revealed that VDR interaction requires a wide range of the residues within and outside the extended helix motif of SMRT-ID1. Notably, SMRT mutants defective in the VDR interaction were also defective in the repression of endogenous VDR-target genes, indicating that the SMRT corepressor is directly involved in the VDR-mediated repression in vivo via an ID1-specific interaction with the VDR. (Molecular Endocrinology 23: 251-264, 2009)

A role of helix 12 of the vitamin D receptor in SMRT corepressor interaction
Ji Young Kim, You Lee Son, Young Chul Lee
Biochemical and Biophysical Research Communications, Elsevier BV, 2009年01月06日, ［査読有り］
研究論文（学術雑誌）

RXR heterodimerization allosterically activates LXR binding to the second NR box of activating signal co-integrator-2
You Lee Son, Ok Gu Park, Gwang Sik Kim, Jae Woon Lee, Young Chul Lee
BIOCHEMICAL JOURNAL, 410, 319, 330, PORTLAND PRESS LTD, 2008年03月, ［査読有り］
英語, 研究論文（学術雑誌）, ASC-2 (activating signal co-integrator-2) is a transcriptional co-activator that mediates the transactivation of NRs (nuclear receptors) via direct interactions with these receptors. ASC-2 contains two separate NR-interaction domains harbouring a core signature motif, LXXLL (where X is any amino acid), named the NR box. Although the first NR box (NR box-1) of ASC-2 interacts with many different NRs, the second NR box (NR box-2) specifically interacts with only LXR (liver X receptor), whose transactivation in vivo requires heterodimerization with RXR (retinoid X receptor). Interestingly, RXR has been shown to enhance the LXR transactivation, even in the absence of LXR ligand via a unique mechanism of allosteric regulation. In the present study we demonstrate that LXR binding to an ASC-2 fragment containing NR box-2 (Co4aN) is enhanced by RXR and even further by liganded RXR. We also identified specific residues in Co4aN involved in its interaction with LXR that were also required for the ASC-2-mediated transactivation of LXR in mammalian cells. Using these mutants, we found that the Co4aN-LXR interaction surface is not altered by the presence of RXR and RXR ligand and that the Ser(1490) residue is the critical determinant for the LXR-specific interaction of Co4aN. Notably the NR box-2, but not the NR box-1, is essential for ASC-2-mediated transactivation of LXR in vivo and for the interaction between LXR-RXR and ASC-2 in vitro. These results indicate that RXR does not interact directly with NR box-1 of ASC-2, but functions as an allosteric activator of LXR binding to NR box-2 of ASC-2.

性腺刺激ホルモン放出抑制ホルモン(GnIH)は甲状腺ホルモンによる生殖制御機構の新たな介在因子である
清原 美佳, 孫 ユリ, 宮田 市郎, 筒井 和義, 日本内分泌学会雑誌, 93, 1, 257, 257, 2017年04月
(一社)日本内分泌学会, 日本語

甲状腺ホルモンは性腺刺激ホルモン放出抑制ホルモン(GnIH)の発現を抑制する
清原 美佳, 孫 ユリ, 宮田 市郎, 筒井 和義, 日本内分泌学会雑誌, 92, 1, 259, 259, 2016年04月
(一社)日本内分泌学会, 日本語

雌の存在が雄ウズラの生殖内分泌システムを変化させる
戸張靖子, SON You Lee, 産賀崇由, 長谷川喜久, 筒井和義, Annual Meeting of Japanese Avian Endocrinology, 38th, 2014年

生殖生理と生殖行動を変化させる社会的刺激の脳内機構
戸張靖子, SON You Lee, 産賀崇由, 長谷川喜久, 筒井和義, 日本動物学会大会予稿集, 84th, 2013年

生殖生理と生殖行動を変化させる社会的刺激受容の神経内分泌機構
戸張靖子, SON You Lee, 産賀崇由, 長谷川喜久, 筒井和義, Annual Meeting of Japanese Avian Endocrinology, 37th, 2013年

褐色脂肪特異的に発現する細胞表面分子Eva1の機能解析
科学研究費助成事業 基盤研究(C)
2021年04月01日 - 2024年03月31日
孫 ユリ
体内の余分なエネルギーを蓄積する白色脂肪に対して、褐色脂肪は熱を産生しエネルギー消費を亢進させ肥満を防止する。老化と肥満に伴い褐色脂肪の機能は低下することから、脂肪細胞の褐色化誘導メカニズムは老化や肥満から生じる代謝異常症に対する新たな予防・治療の標的として注目されている。様々な褐色脂肪細胞のマーカー遺伝子が報告されているものの、その機能が明らかになっているのはごく一部しかない。本研究で注目している1回膜貫通型の細胞膜タンパク質であるEpithelial V-like Antigen 1（Eva1、別名；Myelin protein zero-like protein 2、Mpzl2）は、マウスの古典的褐色脂肪細胞の特徴的遺伝子として知られており、ヒトにおいても頚部と副腎周囲の褐色脂肪での発現が確認されているが、脂肪細胞におけるEva1の役割についてはまだ不明である。Eva1は細胞膜タンパク質として同種間の直接的な細胞接着（homophilic interaction）を仲介し、細胞の形態維持や細胞間の相互作用に関わっており、神経膠芽腫幹細胞においては増殖・幹細胞性維持を担っているとの報告があることから、肥満や加齢による脂肪細胞の形態変化および、脂肪幹細胞/前駆細胞の分化能にEva1が関与している可能性が考えられる。そこで本研究では、肥満や老化などによる褐色脂肪細胞の機能低下がEva1発現に及ぼす影響を明らかにし、褐色脂肪細胞におけるEva1発現の制御機構とEva1作用の分子機構の解明に取り組んでいる。
日本学術振興会, 基盤研究(C), 北海道大学, 21K06240

網膜光受容体メラノプシンの遺伝子破壊が代謝異常を招く分子メカニズム
科学研究費助成事業 基盤研究(C)
2018年04月01日 - 2021年03月31日
孫 ユリ
生体の恒常性は約24時間周期で自律振動する概日時計の働きによって維持されており、概日時計は光の入力と日々決まった時間帯に食事をすることで制御される。概日時計の中枢は視床下部の視交叉上核（suprachiasmatic nucleus；SCN）に存在し、外界の光に同調する。哺乳類の光受容感覚器は目の網膜であり、網膜神経節細胞（retinal ganglion cell；RGC）には青色光受容体であるメラノプシン（別名Opn4）が発現している。このメラノプシン発現網膜神経節細胞（melanopsin-expressing RGC；mRGC）はそれ自体が光受容能を保つとともに、桿体・錐体からの投射も受け、これらの光情報を統合して脳に伝達し、視覚以外の光応答である非視覚応答を担っている。mRGCはSCNをはじめ様々な脳部位に投射しており、mRGCの機能障害が起きると外界の光周期に同調できなくなり、体内リズムの乱れによる睡眠や代謝など様々な生体機能の異常を招くことが考えられる。本研究の目的は「非視覚光応答」と「摂食」といった外界からの入力情報がどのような生体内相互作用により概日時計及び代謝を制御しているのかを明らかにすることである。
本年度には、メラノプシン遺伝子破壊マウスの個体レベルにおける様々な代謝状態を中心的に調べた。具体的には、メラノプシン遺伝子破壊マウスに高脂肪食の自由摂食による肥満を誘導し、体重変化、摂食パターン、活動量及びリズム、ブドウ糖負荷試験 (GTT) とインスリン負荷試験 (ITT) による生体内糖代謝検査を行い、野生型の高脂肪食餌誘発性肥満マウスと比較することで、メラノプシンが担う非視覚光応答の破壊が代謝異常に及ぼす影響を調べた。また、CTによる体脂肪調査やH&E染色による脂肪組織の形態的変化を確認した。
日本学術振興会, 基盤研究(C), 慶應義塾大学, 18K06316

甲状腺ホルモンによる生殖制御におけるＧｎＩＨシステムの作用機構　　　　　　　　　　　　　　　
科学研究費助成事業 特別研究員奨励費
2015年04月24日 - 2017年03月31日
筒井 和義, SON YOU LEE
Thyroid disorders lead to abnormal pubertal development. Although interactions between the thyroid and reproductive endocrine systems are suggested, the detailed mechanisms of how thyroid hormone influences pubertal onset remain unclear. The hypothalamic peptide, gonadotropin-inhibitory hormone (GnIH) was shown to be decreased in the early prepubertal stage, suggesting the possible role of GnIH on pubertal onset. Therefore, we investigated the involvement of GnIH in thyroid status-induced pubertal disorders.


To do this, we induced hypothyroidism and hyperthyroidism in juvenile female mice, then analyzed the change in the regulatory factors of the reproductive axis. Hypothyroidism showed delayed pubertal onset with increased GnIH expression, however hypothyroidism-induced delayed puberty was prevented by GnIH-knockout, suggesting that GnIH may mediate the effect of hypothyroidism on pubertal delay. Reversely, hyperthyroidism had no effect on pubertal onset, however GnIH expression was significantly decreased, indicating the negative correlation between thyroid status and GnIH expression. Further, we found that hypothalamic GnIH neurons express thyroid hormone receptors (TRs), which may convey thyroid hormone signals directly to GnIH neurons. Although TRs did not directly bind to GnIH promoter region, thyroid status actively altered the chromatin modifications in GnIH promoter region.


Our findings indicate a novel function of GnIH to mediate the cross-talk between the hypothalamus-pituitary-thyroid and hypothalamus-pituitary-gonadal axes in proper pubertal development.
日本学術振興会, 特別研究員奨励費, 早稲田大学, 15F15909

時計遺伝子とメラトニンによるGnIH遺伝子の発現制御機構
科学研究費助成事業 特別研究員奨励費
2012年 - 2013年
筒井 和義, SON YouLee
従来の研究により、時計遺伝子やメラトニンが動物の生理状態や行動のリズムを制御することは分かっているが、その分子機構は不明である。本研究は、動物の生殖を制御する重要な新規脳ホルモンであるGnIH (gonadotropin-inhibitory hormlone)に着目して「時計遺伝子とメラトニンによるGnIH遺伝子の発現制御機構」を目的として研究を実施した。
1)時計遺伝子によるGnIH発現の制御機構
1-1)時計遺伝子の一つであるCry1/2の欠損マウス(Cry-nullマウス)でのGnIHの発現が正常マウスと比べて昼の時間帯に著しく増加することを明らかにした。1-2) GnIHニューロンには様々な時計遺伝子が発現していることをラット視床下部由来の細胞株(GnIHニューロン細胞株、rHypoE-23)を用い確認した。1-3) GnIH遺伝子上流に複数存在する時計遺伝子の作用部位であるE-box配列の機能をプロモーターアッセイにより調べた結果、時計遺伝子によるGnIH発現変動は直接E-boxを介した制御機構ではないことが分かった。1-4)体内時計の働きを制御する視交叉上核(SCN)から分泌される血管作動性腸管ペプチド(VIP)の受容体がGnIHニューロンに発現していることを明らかにし、VIPはGnIHニューロン細胞内でcAMP系のシグナルカスケードを活性化させることを明らかにした。
2)メラトニンによるGnIH発現の制御機構
2-1)メラトニン合成能のあるC3Hマウスではメラトニン合成能のないC57BLマウスに比較してGnIHの発現が著しく高いことを明らかにした。2-2) rHypoE-23細胞株にはメラトニン受容体が存在することを明らかにした。2-3)メラトニン受容体のシグナルカスケードの解析するためにrHypoE-23細胞株を用いレポーターアッセイ(CRE, sRE, NFAT-RE)を行った。
日本学術振興会, 特別研究員奨励費, 早稲田大学, 12F02082