Báo cáo khoa học: Relaxin-3⁄ insulin-like peptide 7, a neuropeptide involved in the stress response and food intake Masaki Tanaka pptx

MINIREVIEW Relaxin-3 ⁄ insulin-like peptide 7, a neuropeptide involved in the stress response and food intake Masaki Tanaka Department of Basic Geriatrics, Kyoto Prefectural University of Medicine, Japan Introduction Relaxin-3 ⁄ insulin-like peptide-7 (INSL7) has recently been identified as a new member of the insulin ⁄ relaxin family using human genomic databases [1]. The 142 amino acid human precursor polypeptide sequence is well conserved among humans, pigs, rats and mice [2]. Structurally, this precursor polypeptide consists of sig- nal peptides, and a B-chain, C-peptide and A-chain, and contains the RXXXRXXI motif in the B chain (B12–B19 in human) for binding to the relaxin recep- tor [3]. Similar to insulin, a mature two-chain peptide is produced after removal of the C-peptide and the for- mation of three disulfide bonds between respective cys- teine residues of the A-chain and B-chain [4]. An evolutionary study showed that relaxin-3 orthologs are present in fugu fish and zebrafish, but not in any inver- tebrate or prokaryote, and that these orthologs show high homology between different species in the mature peptide region. When compared with other insu- lin ⁄ relaxin superfamily members, relaxin-3 is con- strained by strong purifying selection, suggesting that this protein is an ancestral form and has a highly-con- served function [5]. In the present minireview, the expression of relaxin-3 in the brain, and particularly its functions, including the stress response and food intake, are described. Expression of relaxin-3 in the brain Relaxin-3 neurons in the brain Examination of relaxin-3 mRNA expression by north- ern blotting and reverse transcriptase-PCR revealed Keywords food intake; gene expression; hypothalamus; nucleus incertus; RXFP3; stress Correspondence M. Tanaka, Department of Basic Geriatrics, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602-8566, Japan Fax: +81 75 251 5797 Tel: +81 75 251 5797 E-mail: mtanaka@koto.kpu-m.ac.jp (Received 13 June 2010, revised 26 August 2010, accepted 18 October 2010) doi:10.1111/j.1742-4658.2010.07931.x Relaxin-3, also known as insulin-like peptide-7, is a newly-identified peptide of the insulin superfamily. All members of this superfamily have a similar structure, which consists of two subunits (A-chain and B-chain) linked by disulfide bonds. Relaxin-3 is so named because it has a motif that can interact with the relaxin receptor. By contrast to other relaxins, relaxin-3 is mainly expressed in the brain and testis. In rodent brain, ana- tomical studies have revealed its predominant expression in neurons of the nucleus incertus of the dorsal pons, and a few other regions of the brain- stem. On the other hand, relaxin-3-expressing nerve fibers and the relaxin-3 receptors, RXFP3 and RXFP1, are widely distributed in the forebrain, with the hypothalamus being one of the most densely-innervated regions. Therefore, relaxin-3 is considered to exert various actions through its ligand-receptor system. This minireview describes the expression of relaxin- 3 in the brain, as well as its functions in the hypothalamus, including the stress response and food intake. Abbreviations ARC, arcuate nucleus; CRF, corticotropin-releasing factor; CRFR1, CRF type 1 receptor; GnRH, gonadotropin-releasing hormone; HPA, hypothalamo-pituitary-adrenal; HPG, hypothalamo-pituitary-gonadal; INSL, insulin-like peptide; KO, knockout; LH, lateral hypothalamic area; NI, nucleus incertus; NPY, neuropeptide Y; PKA, protein kinase A; PVN, paraventricular hypothalamic nucleus; SON, supraoptic nucleus. 4990 FEBS Journal 277 (2010) 4990–4997 ª 2010 The Author Journal compilation ª 2010 FEBS that relaxin-3 is abundant in the brain, but not in female reproductive tissue such as the ovary and uterus [1,6]. By contrast, the expression of two other known relaxin genes (i.e. those encoding human relaxin-1 and -2) was detected in the ovarian corpus luteum during pregnancy, and in the deciduas trophoblast [7–9]. Thus, the physiological function of relaxin-3 is consid- ered to be different from that of other relaxin proteins involved in the growth and remodeling of reproductive and other tissues during pregnancy [10]. In the mouse and rat brain, relaxin-3 expression was reported to be localized to the central gray matter of the median dorsal pons near the fourth ventricle, termed the nucleus incertus (NI) [1,6,11]. We previ- ously reported details of relaxin-3 expression at the cel- lular level using immunocytochemistry and in situ hybridization [12]. In addition to the primary site of expression (i.e. the NI), where, in the rat, approxi- mately 2000 relaxin-3-positive neurons are found (Fig. 1A), a smaller number of these neurons are scattered in the pontine raphe nucleus, the periaqu- eductal gray matter, and the area dorsal to the substantia nigra in the midbrain reticular formation. By immunostaining using monoclonal antibody against the N-terminus of the human relaxin-3 A-chain [2], relaxin-3-immunoreactive fibers were observed to project densely to the septum, hippocampus, lateral hypothalamic area (LH) and intergeniculate leaflet of the thalamus (Fig. 1B). Ultrastructural examination revealed that relaxin-3 was localized to the dense-core vesicles in the perikarya, and it was also observed in the synaptic terminals of axons [12]. The NI comprises a distinct cell group in the caudoventral region of the pontine periventricular gray matter, adjacent to the ventromedial border of the caudal dorsal tegmental nucleus [13]. Studies involving neuronal tracing with anterograde and retrograde tracers have shown that the NI, together with the median raphe and interpe- duncular nuclei, may form a midline behavior control network, and many targets of the NI, such as the med- ial septum, hippocampus, hypothalamus, mammillary complex and amygdala, are involved in arousal mecha- nisms, including the synchronization and desynchroni- zation of the theta rhythm [14,15]. Recently, Ma et al. [16] reported that relaxin-3 neurons in the NI can help modulate spatial memory and the underlying hippo- campal theta activity. Using immunocytochemistry studies, relaxin-3-positive neurons in the NI have been shown to be GABAergic and to co-express corticotro- pin-releasing factor (CRF) type 1 receptors (CRFR1) [12,17]. Relaxin-3 receptor The cognate receptor for relaxin-3 is RXFP3, formally known as GPCR135 or SALPR [6,18]. Although it can also bind and activate RXFP1 and RXFP4, relaxin-3 binds RXPF3 with higher affinity (0.31 nm) than RXFP1 (2.0 nm) or RXFP4 (1.1 nm) [6,19]. RXFP3 mRNA is abundant in the olfactory bulb, paraventric- ular nucleus (PVN) and supraoptic nucleus (SON) in the hypothalamus amygdaloid–hippocampal area, as well as the bed nucleus stria terminalis, paraventricular thalamus, superior colliculus and interpeduncular nucleus in the brainstem. The distribution of RXFP3 approximately overlaps with the autoradiography pattern, showing selective RXFP3 binding of the chi- meric peptide, relaxin-3 B-chain ⁄ INSL5 A-chain [20]. In the brain, there is generally a close correlation between relaxin-3-positive nerve terminals and RXFP3 expression; however, the density of expression of ligand and receptor is not always equal. For example, the olfactory bulb exhibits abundant RXFP3 expres- DTg A B NIc 4V IP Hippocampus PAG RSC DB Hypothalamus LS NI DR NId MS mlf Fig. 1. (A) Relaxin-3 immunoreactivity in the NI. Relaxin-3 is expressed in neurons of both the pars compacta (NIc) and pars dissi- pata (NId) of the NI. DTg, dorsal tegmental nucleus; 4V, fourth ventri- cle; mlf, medial longitudinal fasciculus. Scale bars = 100 lm. (B) A schematic representation of the major projection of relaxin-3 in the forebrain. DB, diagonal band; DR, dorsal raphe nucleus; IP, interpe- duncular nucleus; LS, lateral septal nucleus; MS, medial septal nucleus; PAG, periaqueductal gray matter; RSC, retrosplenial cortex. M. Tanaka Relaxin-3 expression and function in the hypothalamus FEBS Journal 277 (2010) 4990–4997 ª 2010 The Author Journal compilation ª 2010 FEBS 4991 sion, whereas it has relatively low levels of relaxin-3- immunoreactive fibers. In the hypothalamus, relaxin-3 fibers densely innervate the lateral hypothalamic area, although RXFP3 is strongly expressed in the PVN and SON [12,17,21]. The structure and function of the relaxin family peptide receptors, including RXFP3 and RXFP4, were recently reviewed by Kong et al. [22]. Relaxin-3 expression in development and in other species During the development of the rat, relaxin-3 mRNA expression appears at embryonic day 18 near the fourth ventricle. Relaxin-3 peptide can be detected after birth by immunocytochemistry [23]. This develop- mental expression pattern is comparable with that of relaxin, the rodent equivalent of human relaxin-2, whose mRNA is not detectable in the rat brain at embryonic day 15, although it is detectable at postna- tal day 1 [24]. As well as rodents, the distribution of relaxin-3 in the brain has recently been reported for fish, monkeys and humans. In the zebrafish, the relaxin-3 gene is expressed in two neuron clusters in the brainstem: one is a midbrain cell cluster of the periaqueductal gray matter and the other is in a pos- terior region that could be homologous to the mam- malian NI [25]. Two groups have described the distribution of relaxin-3 in the primate brain. In the brain of Macaca fascicularis, relaxin-3-positive cell bodies were found to be distributed within a ventrome- dial region of the central gray matter of the pons and medulla, which appears to correspond to the NI in lower species [26]. In the rhesus macaque and humans, relaxin-3 immunostaining was predominantly observed in the ventral and dorsal tegmental nuclei of the brain- stem [27]. Thus, from fish to primates, this peptide is expressed in the dorsal tegmentum of the brain stem, corresponding to the NI in rodents. Regulation of relaxin-3 gene expression Concerning the regulation of relaxin-3 gene expression, relaxin-3 mRNA expression in the NI is enhanced by restraint stress or forced swim stress (Fig. 2A) [12,28]. This swim stress-induced increase in relaxin-3 tran- script levels is blunted by the systemic administration of CRFR1 antagonist [28]. Relaxin-3 transcript levels are also increased after treatment with p-chlorophenyl- alanine, a potent inhibitor of serotonin synthesis, indi- cating that serotonin negatively regulates relaxin-3 gene expression [23]. From these results, the expression of relaxin-3 may be observed to be dynamically altered under different physiological conditions. We found that relaxin-3 is expressed in a mouse neuroblastoma cell line, Neuro2a, and investigated the intracellular signaling that leads to activation of relaxin 3 gene transcription in vitro [29]. Using a clone stably-trans- fected with a relaxin-3 promoter-enhanced green fluo- rescent protein gene, we observed that the increase in intracellular cAMP induced by dibutyryl cAMP and forskolin treatment increased relaxin-3 promoter activ- ity. These increases were inhibited by pretreatment with the protein kinase A (PKA) inhibitors, H89 and KT5720. Moreover, the relaxin-3 promoter activity was enhanced by CRF treatment after the expression CRFR1 CRF cAMP G s PKA Relaxin-3 gene P Transcription factor AT P PKA Plasma membrane P Promoter Stress Nucleus 0 100 200 300 400 A B Cont PSL Stress * Cont Stress AC Fig. 2. (A) Relaxin-3 mRNA expression in the NI after 6 h of restrained stress. The upper panel shows a representative image of in situ hybridization using the [ 35 S]-labeled probe. The graph below indicates the calculated signal intensity of relaxin-3 mRNA. Data are shown as the mean ± SD of photostimulated luminescence (PSL) [12]. (B) A schematic representation of the intracellular signaling that regulates relaxin-3 gene expression. Downstream of CRFR1, the cAMP-PKA pathway is involved in the activation of relaxin-3 gene transcription. Relaxin-3 expression and function in the hypothalamus M. Tanaka 4992 FEBS Journal 277 (2010) 4990–4997 ª 2010 The Author Journal compilation ª 2010 FEBS of CRFR1 receptor in the cells. These results suggest that relaxin-3 transcription in vivo is activated via the cAMP-PKA pathway, which is downstream of CRFR1 [29] (Fig. 2B). The function of relaxin-3 in the brain Because relaxin-3-producing cells showed a relatively limited distribution, predominantly in neurons of the NI, the function of this peptide has been assessed based upon anatomical studies of the NI at the neuronal level [14,15]. The NI is composed of two subdivisions, the pars compacta and pars dissipata, and relaxin-3- positive neurons are found in both regions (Fig. 1A). With reference to the distribution of relaxin-3-positive nerve fibers and RXFP3 and RXFP1 expression, several functions of relaxin-3 in the brain have been demonstrated, including those related to neuroendo- crine processes, stress response, water intake and spatial memory [12,16,28,30–35]. Particularly, this peptide also regulates food intake, as well as other hypothalamic peptides described in this minireview series [36,37]. Stress response The NI is a region showing abundant expression of CRFR1, and strong c-Fos induction was observed in the NI in response to an intracerebroventricular injec- tion of CRF [38,39]. It is well known that CRF is expressed in parvocellular neurons of the PVN and, during the stress response, CRF activates the hypotha- lamic-pituitary-adrenal (HPA) axis, acting at CRFR1 on anterior pituitary corticotropes to stimulate the release of adrenocorticotropic hormone. There are also extrahypothalamic CRF-expressing neurons distributed through the brain in areas such as the neocortex and limbic regions, including the central amygdala and hippocampus [40,41]. The regulation of CRF expression may be involved in setting the ‘tone’ of stress-related behavior, including anxiety, as well as learning and memory [42,43]. CRF exerts its actions via two major receptors: CRFR1 and CRFR2. Both receptors belong to the class B subtype of G protein-coupled receptors, although they have a different distribution, suggesting that the two receptors have different functions. CRFR1 is considered to be involved in the acute phase of the stress response, whereas CRFR2 contributes to the maintenance and recovery phase that involves a gradual reduction of HPA axis activation [43,44]. In the rat NI, almost all relaxin-3-positive neurons coexpress CRFR1 and respond to CRF intracerebro- ventricular administration. Moreover, application of a water-restraint stress for 2–4 h induces c-Fos expres- sion and leads to an increase in relaxin-3 mRNA levels in the NI [12]. On the other hand, relaxin-3-positive neurons project fibers to the hypothalamus, and RXFP3 is intensely expressed in the PVN where hypo- thalamic CRF neurons exist. These results suggest that relaxin-3-expressing neurons respond immediately to stress and modulate the HPA axis. Recently, Banerjee et al. [28] reported that exposure of rats to a repeated forced swim for 10 min each time leads to a marked increase in relaxin-3 mRNA levels in the NI at 30–60 min after the second swim. Systemic treatment with the CRFR1 antagonist alarmin 30 min before the second swim blunted the stress-induced effect on relaxin-3 transcripts in the NI [28]. This supports the idea that relaxin-3-expressing neurons in the NI (and therefore relaxin-3) play a role in the central stress regulating system by mutual interaction with CRF- expressing neurons. Food intake Relaxin-3 was first reported to stimulate food intake when administered into the third ventricle or PVN of male Wistar rats. Administration of human relaxin-3, but not human relaxin-2, either intracerebroventricu- larly (180 pmol) or intra-PVN (18 pmol) increased 1-h food intake both in the early light and early dark phase (Fig. 3) [31]. The doses of relaxin-3 required to elicit a significant feeding response are in the picomo- lar range and are similar to the effective doses of other orexigenic peptides such as ghrelin (30 pmol; intra- PVN) and neuropeptide Y (NPY) (78 pmol; intra- PVN) [45,46]. Although RXFP3 and RXFP1 are expressed in the PVN, relaxin (specifically, human relaxin-2) binds RXFP1 but not RXFP3, suggesting that this feeding-promoting action of relaxin-3 is exerted through RXFP3 because the actions of relaxin have not been reported to include hyperphagia, but do include hemodynamic effects such as increasing arterial blood pressure and vasopressin release [47], or dipso- genesis [48]. In reverse, relaxin-3 was recently reported to facilitate water intake as well as relaxin, suggesting that RXFP1 was involved in this action [35]. Concern- ing the chronic administration of relaxin-3, intracere- broventricular injection for 14 days (600 pmolÆday )1 ) using osmotic minipumps led to a significant increase in food consumption and weight compared to vehicle infusion. There was no difference in locomotor activity between two groups either in the light phase or dark phase, suggesting that this effect of relaxin-3 is not a result of increased locomotor or arousal activity [34]. Chronic intra-PVN administration of human relaxin-3 (180 pmol twice a day for 7 days) also increased the M. Tanaka Relaxin-3 expression and function in the hypothalamus FEBS Journal 277 (2010) 4990–4997 ª 2010 The Author Journal compilation ª 2010 FEBS 4993 cumulative food intake in ad libitum-fed rats [32]. After such chronic administration, the plasma concentration of leptin and insulin was significantly increased [32]. In addition to the PVN, relaxin-3-administration into the SON or arcuate nucleus (ARC), but not into the LH, stimulated 1-h food intake [32]. The ARC and LH are well known as feeding centers where orexigenic peptides such as NPY, melanin-concentrating hor- mone and orexin are distributed. Although relaxin- 3-immunoreactive fibers are densely distributed, the RXFP3 level is relatively low in the ARC and LH. An electrophysiological study of neurons in these hypothalamic nuclei may help to resolve this disparity and clarify the hyperphagic mechanisms. Recently, relaxin-3 gene knockout (KO) mice of mixed background (129S5:B6) were examined in two studies. One group reported that KO mice are smaller and leaner than congenic controls [21], although the results obtained by the second group indicated that there was no genotypic difference in body weight or motor coordination [49]. Further studies using relaxin- 3 KO mice backcrossed to C57 ⁄ B6 should help to clar- ify the role of relaxin-3 in regulating body weight and metabolism. Actions of relaxin-3 at the hypothalamo-pituitary- gonadal (HPG) axis Recently, a role of relaxin-3 in regulation of the HPG axis was reported in that intracerebroventricular (5 nmol) and intra-PVN (540–1620 pmol) administra- tion of relaxin-3 in adult male rats significantly increased plasma luteinizing hormone levels. This effect was inhibited by pretreatment with a peripheral gona- dotropin-releasing hormone (GnRH) antagonist. By contrast, the central administration of human relaxin-2 was not found to influence the plasma luteinizing hor- mone concentration. Using hypothalamic explants and GT1-7 cells that express RXFP1 and RXFP3, relaxin- 3 was shown to dose-dependently stimulate GnRH release. GnRH neuronal cell bodies are found in sev- eral forebrain regions, including the medial septum, diagonal band, preoptic area and LH, where relaxin-3- positive fibers and RXFP3 are moderately-to-densely distributed [12,17,50]. These results suggest that relaxin-3 regulates the HPG axis via hypothalamic GnRH neurons. Thus, relaxin-3 is seen to belong to the group of neuropeptides that regulate energy homeostasis and reproduction (i.e. modulate both appetite and the HPG axis). This group includes NPY, orexin and galanin-like peptides [51–54]. Conclusions In this minireview, relaxin-3, which is the latest mem- ber of the insulin ⁄ relaxin family, is described in terms of its gene transcript and peptide expression in the brain, as well as its functional aspects that have thus far been reported. Although relaxin-3-expressing neu- rons show a confined distribution in the brainstem, being particularly dense in the NI of the dorsal tegmen- tal pons, their fibers and receptors (i.e. RXFP3 and RXFP1) are widely distributed in the forebrain. One of the target areas of relaxin-3 is the hypothalamus. Relaxin-3 is considered to have various actions medi- Fig. 3. Effect of intracerebroventricular administration of relaxin-3 in satiated male Wistar rats. (A) Effect of human relaxin-3 (H3) (18– 180 pmol) on 1-h food intake. *P < 0.05 versus vehicle in the early light phase. (B) Effect of H3 (18–180 pmol) on cumulative food intake over 4 h in the early light phase. & P < 0.05 at 18 pmol versus vehicle; *P < 0.05 at 54 pmol versus vehicle; # P < 0.05 at 180 pmol versus vehicle. Reproduced with permission [31]; ª 2005, The Endocrine Society). Relaxin-3 expression and function in the hypothalamus M. 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Tanaka Relaxin-3 expression and function in the hypothalamus FEBS Journal 277 (2010) 4990–4997 ª 2010 The Author Journal compilation ª 2010 FEBS 4997 . MINIREVIEW Relaxin-3 ⁄ insulin-like peptide 7, a neuropeptide involved in the stress response and food intake Masaki Tanaka Department of Basic Geriatrics, Kyoto Prefectural University. Relaxin-3 is so named because it has a motif that can interact with the relaxin receptor. By contrast to other relaxins, relaxin-3 is mainly expressed in the brain and testis. In rodent brain, ana- tomical. 1A) , a smaller number of these neurons are scattered in the pontine raphe nucleus, the periaqu- eductal gray matter, and the area dorsal to the substantia nigra in the midbrain reticular formation. By