Kisspepeptin-GPR54 signaling in the neuroendocrine reproductive axis

Molecular and Cellular Endocrinology 254–255 (2006) 91–96

Kisspepeptin-GPR54 signaling in the neuroendocrine reproductive axis M.L. Gottsch a , D.K. Clifton b , R.A. Steiner a,b,c,∗ a b

Department of Physiology & Biophysics, University of Washington, Seattle, WA 98195-7290, USA Department of Obstetrics & Gynecology, University of Washington, Seattle, WA 98195-7290, USA c Department of Biology, University of Washington, Seattle, WA 98195-7290, USA

Abstract Kisspeptins, which are products of the Kiss1 gene, and their receptor, GPR54, have emerged as key players in the regulation of gonadotropinreleasing hormone (GnRH) secretion. Mutations or targeted deletions of GPR54 produce isolated hypogonadotropic hypogonadism in humans and mice, indicating that signaling through this receptor is a prerequisite for sexual maturation. Centrally administered kisspeptins stimulate GnRH and gonadotropin secretion in prepubertal and adult animals. Kisspeptin-expressing neurons are direct targets for the negative and positive feedback actions of sex steroids, which differentially regulate the expression of KiSS-1 mRNA in various regions of the forebrain. This review highlights what is currently known about kisspeptin-GPR54 signaling in the regulation of the neuroendocrine reproductive axis. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Kisspeptins; Gonadotropin-releasing hormone; Neuroendocrine reproductive axis

1. The discovery of kisspeptins and GPR54 and their role in puberty Biologists studying cancer were the first to characterize the G protein-coupled receptor GPR54 and its ligand (Lee et al., 1999). Lee et al. discovered that the ligand for GPR54 was a product of the metastasis suppressor gene, Kiss1—the letters “ss” indicate a “suppressor sequence” and “Ki” was added as a prefix to reflect the fact that the molecule was discovered in Hershey, Pennsylvania, home of the Hershey chocolate “Kiss” (Lee et al., 1996; Lee and Welch, 1997). Because products of the Kiss1 gene were found to suppress metastasis of melanoma and breast cancer cells, the 54 amino acid protein product of Kiss1 was named “metastin” (aka kisspeptin-54) (Lee et al., 1996; Lee and Welch, 1997). It was later shown that kisspeptin-54, as well as shorter fragments (collectively called kisspeptins), all bind Abbreviations: AR, androgen receptor; E, estradiol; ER, estrogen receptor; FSH, follicle stimulating hormone; GABA, ␥-aminobutyric acid; GnRH, gonadotropin releasing hormone; GPR54, G-protein coupled receptor-54; GPR54 KO, GPR54 knockout; HPG, hypothalamic–pituitary–gonadal axis; LH, luteinizing hormone; NPY, neuropeptide Y; P, progesterone; PR, progesterone receptor; T, testosterone; UK, United Kingdom ∗ Corresponding author at: Department of Physiology & Biophysics, Health Sciences Building (G-424), School of Medicine, University of Washington, Box # 357290, 1959 NE Pacific Street, Seattle, WA 98195-7290, USA. Tel.: +1 206 543 8712; fax: +1 206 685 0619. E-mail address: [email protected] (R.A. Steiner). 0303-7207/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2006.04.030

and activate GPR54 (Muir et al., 2001; Clements et al., 2001; Kotani et al., 2001; Hori et al., 2001; Stafford et al., 2002). The role of kisspeptins and GPR54 in cancer continues to be investigated; however, their emerging role in regulation of the hypothalamic–pituitary–gonadal (HPG) axis will be the focus of this review. The onset of puberty is triggered by activation of neurons in the forebrain that produce GnRH. The amplified secretion of GnRH evokes the release of LH and FSH, which then awaken the gonads [reviewed in (Ebling and Cronin, 2000; Plant and Barker-Gibb, 2004; Shahab et al., 2005)]. Although, this cascade has been well characterized for many mammalian species, the molecular and cellular events in the forebrain that actually initiate this process remain shrouded in mystery. There are numerous suspects that have been implicated as “triggers”, including molecules that are intrinsic to the brain, such as GABA and NPY, and peripherally derived hormonal cues, such as leptin (Terasawa et al., 1999; Richter and Terasawa, 2001; Urbanski, 2001; Cheung et al., 1997; Cunningham et al., 1999). Despite the drive to describe the mechanisms of pubertal onset, genetic approaches to reveal a “smoking gun” for the initiation of puberty have only added ambiguity (Chehab et al., 1996, 1997, 2002; Chehab, 2000) that is until it was discovered that GPR54 is an indispensable conduit for signaling the onset of puberty in the human and mouse. Three groups announced the essential role of GPR54 in puberty almost simultaneously. In an effort to decipher the

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physiological significance of GPR54, investigators at Paradigm Therapeutics in the UK produced genetically targeted deletions in the GPR54 gene and found a remarkable phenotype—failure of puberty in mice (Seminara et al., 2003) (reviewed in Colledge, 2004). While scientists at Paradigm Therapeutics were busy characterizing the GPR54 KO mouse, de Roux et al. (2003) and Seminara et al. (2003), were both concurrently pursuing analysis of a gene in patients of consanguineous families, who had been diagnosed with idiopathic hypogonadotropic hypogonadism. Both de Roux et al. and Seminara et al. reported that a number of their patients had mutations in GPR54. These patients appeared to be healthy except they failed to progress through puberty and had hypogonadotropic hypogonadism, like the GPR54 KO mice. Soon thereafter, Funes et al. (2003) presented a description of their own GPR54 KO mice, which had the same phenotype. The most remarkable common denominator was the finding that disruption of GPR54 signaling produced a profound – yet isolated – phenotype, which was virtually identical in the human and mouse. Thus, signaling through GPR54 appears to be a molecular corridor that must be open to permit the onset of puberty. Developmental changes in the expression of KiSS-1 and GPR54 may play a role in timing the onset of puberty, and could conceivably be the proximate triggering event for pubertal maturation. Navarro et al. (2004a) have reported that both KiSS-1 mRNA and GPR54 mRNA increase as a function of sexual development in the rat. Shahab et al. (2005) have described a similar trend in the monkey, observing that KiSS-1 mRNA increases across puberty—at least in intact females and agonadal males. This suggests there may be a developmental increase in the expression of KiSS-1 that occurs independently of sex steroids, which normally increases with the onset of puberty in the intact animal. The situation with GPR54 is somewhat different, in that the expression of GPR54 mRNA was shown to increase with puberty in the intact female but not in the agonadal male (neither intact males nor agonadal females were studied in this report) (Shahab et al., 2005), suggesting that either males are different from females in this regard or that sex steroids induce the expression of GPR54 (reflected by the developmental increase in GPR54 mRNA in the intact female). It will be important to revisit the question of what happens to the expression of KiSS-1 and GPR54 over puberty with techniques that allow careful anatomical dissection of precisely which, where, and how certain populations of kisspeptin/GPR54-expressing cells are regulated as a function of puberty, and to compare the rodent and primate. 2. Activational effects of Kisspeptins on GnRH neurons Additional studies have revealed that kisspeptin-GPR54 signaling may serve a regulatory function in the neuroendocrine reproductive axis—beyond acting as a simple “gate” for the onset of puberty (Seminara et al., 2003; de Roux et al., 2003; Funes et al., 2003; Semple et al., 2005). First, it was discovered that centrally administered kisspeptins stimulate LH secretion via a GnRH-dependent mechanism (in both adult and prepubertal animals) (Gottsch et al., 2004; Thompson et

al., 2004; Irwig et al., 2004; Navarro et al., 2005); second, it was observed that kisspeptins delivered into the brain can induce ovulation and advance the onset of puberty in rodents (Navarro et al., 2004b; Kinoshita et al., 2005; Matsui et al., 2004). Third, KiSS-1 mRNA was also discovered to be expressed in brain regions that are known to be important for the regulation of GnRH secretion, including the arcuate nucleus (Arc), the periventricular nucleus (PeN), and the anteroventral periventricular nucleus (AVPV) (Gottsch et al., 2004). These observations suggest that kisspeptins and GPR54 play a pivotal function in the regulation of gonadotropin secretion in the adult, as well as the developing animal. Remarkably, kisspeptins stimulate the hypothalamic– pituitary axis and induce LH secretion, regardless of the route of administration, either centrally (into the cerebral ventricles) or peripherally (intraperitoneally or systemically). Moreover, kisspeptins have this effect at exceedingly low doses (Gottsch et al., 2004; Thompson et al., 2004; Navarro et al., 2004b)—doses substantially lower than any other previously studied secretagogues for LH (Sahu et al., 1987; Horvath et al., 2001; Hohmann et al., 2000; Ohtaki et al., 2001; Ebling et al., 1993; Krasnow et al., 2003). The potency of kisspeptins maybe explained by their having a direct action on GnRH neurons, most of which express GPR54 and show rapid induction of Fos following exposure to kisspeptin (Irwig et al., 2004; Parhar et al., 2004; Messager et al., 2005). The relative uniformity in the expression of GPR54 among GnRH neurons and the induction of Fos in those neurons following exposure to kisspeptin is astonishing—particularly considering the phenotypic heterogeneity of GnRH neurons (Todman et al., 2005). The expression of GPR54 by GnRH neurons and the ability of kisspeptins to activate these cells would appear to be an important common denominator among most – if not all – GnRH neurons. The fact that kisspeptins do not stimulate LH secretion in GPR54 KO mice—despite the fact that GnRH neurons in these mice are phenotypically normal otherwise (Messager et al., 2005), would suggest that GPR54 is the only receptor for kisspeptins on GnRH neurons and that their primary physiological function is to support GnRH secretion. Although it would appear that kisspeptin-GPR54 signaling is a prerequisite for GnRH (and gonadotropin) secretion, we know relatively little about either the cellular physiology of kisspeptin neurons or the intracellular signaling mechanism by which kisspeptins act on GnRH neurons. We do know that GPR54 signaling occurs via a Gq-coupled G protein and that its activation can result in an increase in intracellular calcium (Kotani et al., 2001), PI turnover, and ERK activation through MAP kinase (Kotani et al., 2001; Stafford et al., 2002; Becker et al., 2005; Masui et al., 2004; Ringel et al., 2002), but we do not know precisely how these pathways stimulate GnRH secretion. Although it is evident that kisspeptins activate GnRH neurons, we cannot be certain that kisspeptin-producing cells make direct synaptic contact with GnRH neurons (despite the unequivocal expression of GPR54 by GnRH neurons). Likewise, even if it is proven that some kisspeptin neurons do make synaptic contact with GnRH neurons, it will be necessary to establish, by rigorous tract-tracing techniques, precisely which populations of kisspeptin neurons do so. It is also conceivable that there

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is an as yet uncharacterized role(s) of GPR54 and kisspeptins in the brain, which is evidenced by the fact that there are other populations of cells in the forebrain, besides GnRH neurons, that also express GPR54 and Fos, following activation by kisspeptins (Irwig et al., 2004). Revealing the phenotype of these “other” kisspeptin target cells could reveal more about the nature of GPR54 and kisspeptin regulation of puberty and reproductive function in the adult animal. 3. Regulation of KiSS-1 and GPR54 mRNAs by sex steroids Emerging evidence suggests that kisspeptin-GPR54 signaling serves as an afferent stimulatory input to GnRH neurons. However, the Kiss1 and GPR54 genes may be targets for regulation by sex steroids as part of the circuitry regulating the HPG axis. Navarro et al. (2004a) and Irwig et al. (2004) have provided evidence that KiSS-1- and GPR54-expressing neurons are targets for regulation by sex steroids. These teams have suggested that sex steroids estrogen (E) and testosterone (T) feed back to inhibit the expression of KiSS-1 mRNA in the hypothalamus. Studies of KiSS-1 mRNA by RT-PCR and in situ hybridization (ISH) have indicated that the dominant influence of these sex steroids on the expression of KiSS-1 mRNA in the medial basal hypothalamus (specifically, in the Arc) is inhibition, perhaps reflecting the hypothalamic circuit that orchestrates the negative feedback regulation of GnRH and gonadotropin secretion. The report by Navarro et al. (2004a) also indicates that GPR54 mRNA is regulated in a similar fashion as KiSS-1 mRNA. Together, these observations suggest that kisspeptin-GPR54 signaling provides tonic stimulatory input to GnRH neurons, which is governed by the negative feedback effects of sex steroids acting on kisspeptin neurons. Thus, in the case of these kisspeptin neurons in the Arc, rising titers of sex steroids inhibit the expression (and secretion) of kisspeptins, dampening their stimulatory drive to GnRH neurons and diminishing the output of GnRH. Likewise, with falling titers of sex steroids, the kisspeptin neurons in the Arc are disinhibited, amplifying their expression of kisspeptins, and increasing their stimulatory drive to GnRH neurons—a classical negative feedback circuit. Confidence in the simple negative feedback model has been recently eroded by the discovery that sex steroids induce the expression KiSS-1 mRNA in some areas of the brain, while inhibiting KiSS-1 expression in other areas (Smith et al., 2005a). These observations demonstrate that sex steroids produce a differential regulation of KiSS-1 mRNA among the various nuclear groups in the forebrain. Smith et al., have shown that in the adult male mouse, T inhibits the expression of KiSS-1 mRNA in the Arc but stimulates KiSS-1 expression in the AVPV. The complex effects of T are mediated by the androgen receptor (AR) as well as the estrogen receptor (ER, after aromatization of T to E). In the Arc, the AR, which shows ∼ 65% colocalization with KiSS-1 mRNA, and ER␣, which shows ∼ 88% colocalization with KiSS-1 mRNA, both appear to contribute to the steroid-dependent inhibition in the expression of KiSS-1 mRNA. However, in the AVPV, ER␣ appears to mediate the T-dependent induction of KiSS-1 mRNA–again, following aromatization of T

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to E (Smith et al., 2005a). The differential expression of AR and ER between these two nuclei may explain the opposite effects of T on the regulation of KiSS-1 mRNA in these two regions; however, this remains to be carefully dissected at the molecular level. In the female mouse, the expression of KiSS-1 mRNA is also differentially regulated between the Arc and AVPV, with E inhibiting KiSS-1 expression in the Arc and inducing its expression in the AVPV (Smith et al., 2005b). In the female, nearly all KiSS-1 cells are ER␣ positive, whereas only ∼25–30% of KiSS-1 cells are ER␤ positive (Smith et al., 2005b). In ER␣ KO mice, the E-dependent regulation of KiSS-1 mRNA is lost, but in the ER␤ KO mice, E-dependent regulation remains intact, indicating that the regulation of KiSS-1 mRNA by E is mediated primarily by ER␣. The molecular basis for the differential effects of E on KiSS-1 expression in the Arc and AVPV is unknown, but it is plausible that another receptor, possibly the progesterone receptor (PR), contributes to the phenomenon. Preliminary data from our lab suggests that most KiSS-1 neurons colocalize with or are in very close proximity to PR positive neurons (unpublished observations); however, the role, if any, of progesterone in KiSS-1 mRNA regulation has yet to be elucidated. Another factor that could contribute to the differential regulation of E in the female Arc and AVPV is dopamine. Tyrosine hydroxylase (the rate-limiting enzyme in dopamine synthesis) and KiSS-1 mRNA colocalize in the AVPV but not the Arc (Lee et al., 2005). Thus, it seems possible that dopamine plays some role in the induction of KiSS-1 expression in the AVPV by E. The differential regulation of KiSS-1 mRNA among nuclei in the forebrain has some fascinating implications for the physiological role of KiSS-1 in the HPG axis. The Arc is a nodal point for mediating the negative feedback regulation of GnRH and gonadotropin secretion, whereas the AVPV has been implicated in the positive feedback regulation of the LH surge in the female (Simerly et al., 1990; Shughrue et al., 1997; Mitra et al., 2003; Canteras et al., 1994; Simonian et al., 1999; Soper and Weick, 1980; Simerly, 1998, 2002; Gu and Simerly, 1997; Le et al., 1999). The AVPV is unlike other sexually dimorphic nuclei in that it is one of the few that is larger in the female than the male (Simerly, 1998). Furthermore, neurons from the AVPV are thought to synapse onto GnRH neurons (Simerly, 1998; Gu and Simerly, 1997). Within the AVPV there is an abundance of ER␣, -␤ and PR, which are poised to respond to their ligands and augment the LH surge (Simerly et al., 1996). In the female, it could be that the E-dependent induction of KiSS-1 mRNA in the AVPV plays a role in mediating the preovulatory GnRH/LH surge—the so-called “positive feedback” phenomenon that drives ovulation. Indeed, the expression of KiSS-1 mRNA is sexually differentiated in the AVPV, with much greater expression in the female than the male (Fig. 1) (Smith et al., 2005a,b), suggesting that kisspeptin neurons in the AVPV are involved in sexually differentiated processes, such as the generation of the GnRH/LH surge (Kinoshita et al., 2005) or the regulation of sexual behavior. Unlike the AVPV, KiSS-1 mRNA levels in the Arc are not different between males and females. This observation is consistent with the hypothesis that kisspeptin neurons in the Arc play the same roles in both sexes, such as the negative feedback regulation of gonadotropin secretion by gonadal steroids.

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4. Beyond reproductive neuroendocrinology

Fig. 1. The expression of KiSS-1 mRNA in the AVPV and Arc of male and female mice. Asterisk (* ) indicates significant difference between males and females (p < 0.005). Bars represent means (n = 6 (male); 8 (female)) and tick marks represent the standard errors. (Figure adapted from Smith et al., 2005a,b).

Even though KiSS-1 expression in the AVPV of the male is lower than that of the female, T still positively regulates it. However, the physiological significance of this phenomenon is not immediately apparent. It is possible that the phenomenon of Tdependent induction of KiSS-1 in the AVPV in the male reflects only vestigial expression that remains following its “erasure” during the critical period of sexual differentiation (Navarro et al., 2004a). If this were true, it may offer an explanation of why E cannot elicit a GnRH/LH surge in the male rodent. Alternatively, perhaps the T-dependent induction of KiSS-1 mRNA in the AVPV in the male is linked to another aspect of reproduction, such as sexual behavior. A model for the role of kisspeptin neurons in the HPG axis is shown in Fig. 2.

It is clear that kisspeptpin-GPR54 signaling is a critical component for normal reproductive function. Yet, there remain many unanswered questions in this new field of ‘kisspeptinology.’ GPR54 mRNA is expressed in other regions of the body, besides the brain (Lee et al., 1999; Muir et al., 2001; Clements et al., 2001; Kotani et al., 2001; Ohtaki et al., 2001); yet, we have no clue about its physiological relevance in these areas. Furthermore, most G protein-coupled receptors have several isoforms; yet, so far, no other GPR54 isotypes have been identified. The placenta is a major source of kisspeptins (Horikoshi et al., 2003; Terao et al., 2004; Bilban et al., 2004), but the physiological relevance of kisspeptins in pregnancy is unknown. It has been postulated that kisspeptins act as growth factors and may control invasion and implantation of the trophoblast (Terao et al., 2004; Bilban et al., 2004). Whether kisspeptins produced by the placenta can act on the hypothalamus or other sites in the body where GPR54 is expressed remains unexplored. It is conceivable that the exponential rise in kisspeptins that occurs in the maternal plasma with the progression of pregnancy could play a role fetal development or parturition—but again, there has been no systematic investigation of these ideas. 5. Looking ahead Finally, what lies ahead for investigations of kisspeptinGPR54 signaling? First, the anti-metastatic properties of kisspeptin will continue to be investigated. New developments in this arena could provide insight concerning the molecular signals that cause cancers in situ to become metastatic and thereby render clues about strategies for its treatment. Second, learn-

Fig. 2. A schematic representation of our current understanding of GPR54 and KiSS-1 signaling in the forebrain of the mouse. Kisspeptin stimulates GnRH secretion by a direct effect on GnRH neurons, most of which express the kisspeptin receptor, GPR54. Neurons that express KiSS-1 mRNA reside in the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (arcuate). In the arcuate, estradiol (E) and testosterone (T) inhibit the expression of KiSS-1 mRNA, whereas, in the AVPV, these same sex steroid hormones induce KiSS-1 mRNA expression.

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ing more about the role of kisspeptins that are produced by the placenta may help us to understand the biology of implantation and placentation and could suggest new approaches to treat certain disorders of pregnancy, such as preeclampia and placenta previa. Finally, continuing to investigate the role of KiSS-1 and GPR54 in reproductive neuroendocrinology may help to solve some old, intractable mysteries–What initiates puberty? How does E stimulate the preovulatory GnRH/LH surge? How is the GnRH surge mechanism coupled to the circadian oscillator in the suprachiasmatic nucleus? What is the basis for sexual differentiation of the GnRH surge mechanism? Answering these questions should keep us busy for some time! Acknowledgments We are grateful to Jeremy Smith, Heather Dungan, Sonya Jakawich and Kathy Lee for their critical review of this manuscript. This work was supported by grants from the National Institutes of Health [R01 HD27142, SCCPRR (U54) HD12629, R01 DK61517] and the National Science Foundation (IBN 0110686). References Becker, J.A., Mirjolet, J.F., Bernard, J., Burgeon, E., Simons, M.J., Vassart, G., Parmentier, M., Libert, F., 2005. Activation of GPR54 promotes cell cycle arrest and apoptosis of human tumor cells through a specific transcriptional program not shared by other Gq-coupled receptors. Biochem. Biophys. Res. Commun. 326, 677–686. Bilban, M., Ghaffari-Tabrizi, N., Hintermann, E., Bauer, S., Molzer, S., Zoratti, C., Malli, R., Sharabi, A., Hiden, U., Graier, W., Knofler, M., Andreae, F., Wagner, O., Quaranta, V., Desoye, G., 2004. Kisspeptin10, a KiSS-1/metastin-derived decapeptide, is a physiological invasion inhibitor of primary human trophoblasts. J. Cell Sci. 117, 1319–1328. Canteras, N.S., Simerly, R.B., Swanson, L.W., 1994. Organization of projections from the ventromedial nucleus of the hypothalamus: a Phaseolus vulgaris-leucoagglutinin study in the rat. J. Comp. Neurol. 348, 41–79. Chehab, F.F., 2000. Leptin as a regulator of adipose mass and reproduction. Trends Pharmacol. Sci. 21, 309–314. Chehab, F.F., Lim, M.E., Lu, R., 1996. Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nat. Genet. 12, 318–320. Chehab, F.F., Mounzih, K., Lu, R., Lim, M.E., 1997. Early onset of reproductive function in normal female mice treated with leptin. Science 275, 88–90. Chehab, F.F., Qiu, J., Mounzih, K., Ewart-Toland, A., Ogus, S., 2002. Leptin and reproduction. Nutr. Rev. 60, S39–S46 (discussion S68-84, 85-7). Cheung, C.C., Thornton, J.E., Kuijper, J.L., Weigle, D.S., Clifton, D.K., Steiner, R.A., 1997. Leptin is a metabolic gate for the onset of puberty in the female rat. Endocrinology 138, 855–858. Clements, M.K., McDonald, T.P., Wang, R., Xie, G., O’Dowd, B.F., George, S.R., Austin, C.P., Liu, Q., 2001. FMRFamide-related neuropeptides are agonists of the orphan G-protein-coupled receptor GPR54. Biochem. Biophys. Res. Commun. 284, 1189–1193. Colledge, W.H., 2004. GPR54 and puberty. Trends Endocrinol. Metab. 15, 448–453. Cunningham, M.J., Clifton, D.K., Steiner, R.A., 1999. Leptin’s actions on the reproductive axis: perspectives and mechanisms. Biol. Reprod. 60, 216–222. de Roux, N., Genin, E., Carel, J.C., Matsuda, F., Chaussain, J.L., Milgrom, E., 2003. Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proc. Natl. Acad. Sci. U.S.A. 100, 10972–10976.

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