Ghrelin: a multifunctional hormone in non-mammalian vertebrates

In mammals, ghrelin is a non-amidated peptide hormone, existing in both acylated and non-acylated forms, produced mainly from the X/A or ghrelin cells present in the mucosal layer of the stomach. Ghrelin is a natural ligand of the growth hormone (GH) secretagogue-receptor (GHS-R), and functions primarily as a GH-releasing hormone and an orexigen, as well as having several other biological actions. Among non-mammalian vertebrates, amino acid sequence of ghrelin has been reported in two species of cartilaginous fish, seven species of teleosts, two species of amphibians, one species of reptile and six species of birds. The structure and functions of ghrelin are highly conserved among vertebrates. This review presents a concise overview of ghrelin biology in non-mammalian vertebrates.     

Ghrelin: a multifunctional hormone in non-mammalian vertebrates.

In mammals, ghrelin is a non-amidated peptide hormone, existing in both acylated and non-acylated forms, produced mainly from the X/A or ghrelin cells present in the mucosal layer of the stomach. Ghrelin is a natural ligand of the growth hormone (GH) secretagogue-receptor (GHS-R), and functions primarily as a GH-releasing hormone and an orexigen, as well as having several other biological actions. Among non-mammalian vertebrates, amino acid sequence of ghrelin has been reported in two species of cartilaginous fish, seven species of teleosts, two species of amphibians, one species of reptile and six species of birds. The structure and functions of ghrelin are highly conserved among vertebrates. This review presents a concise overview of ghrelin biology in non-mammalian vertebrates.     

Evolution of interleukin-1beta

All jawed vertebrates possess a complex immune system, which is capable of anticipatory and innate immune responses. Jawless vertebrates posses an equally complex immune system but with no evidence of an anticipatory immune response. From these findings it has been speculated that the initiation and regulation of the immune system within vertebrates will be equally complex, although very little has been done to look at the evolution of cytokine genes, despite well-known biological activities within vertebrates. In recent years, cytokines, which have been well characterised within mammals, have begun to be cloned and sequenced within non-mammalian vertebrates, with the number of cytokine sequences available from primitive vertebrates growing rapidly. The identification of cytokines, which are mammalian homologues, will give a better insight into where immune system communicators arose and may also reveal molecules, which are unique to certain organisms. Work has focussed on interleukin-1 (IL-1), a major mediator of inflammation which initiates and/or increases a wide variety of non-structural, function associated genes that are characteristically expressed during inflammation. Other than mammalian IL-1β sequences there are now full cDNA sequences and genomic organisations available from bird, amphibian, bony fish and cartilaginous fish, with many of these genes having been obtained using an homology cloning approach. This review considers how the IL-1β gene has changed through vertebrate evolution and whether its role and regulation are conserved within selected non-mammalian vertebrates.     

Vertebrate sex determination: a new player in the field

Sex-determining genes have been identified in flies, worms and mammals but not, until recently, in non-mammalian vertebrates. Now, a gene has been isolated from the Y chromosome of the teleost fish medaka that is functionally comparable to the mammalian testis-determining gene, Sry.     

GPR50 is the mammalian ortholog of Mel1c: Evidence of rapid evolution in mammals

Background  

The melatonin receptor subfamily contains three members Mel1a, Mel1b and Mel1c, found in all vertebrates except for Mel1c which is found only in fish, Xenopus species and the chicken. Another receptor, the melatonin related receptor known as GPR50, found exclusively in mammals and later identified as a member of the melatonin receptor subfamily because of its identity to the three melatonin receptors despite its absence of affinity for melatonin. The aim of this study was to describe the evolutionary relationships between GPR50 and the three other members of the melatonin receptor subfamily.     

Differential ovarian expression of KiSS-1 and GPR-54 during the estrous cycle and photoperiod induced recrudescence in Siberian hamsters (Phodopus sungorus)

Kisspeptins, coded by the KiSS-1 gene, regulate aspects of the reproductive axis by stimulating GnRH release via the G protein coupled receptor, GPR54. Recent reports show that KiSS/GPR54 may be key mediators in photoperiod-controlled reproduction in seasonal breeders, and that KiSS-1/GPR54 are expressed in the hypothalamus, ovaries, placenta, and pancreas. This study examined the expression of KiSS-1/GPR54 mRNA and protein in ovaries of Siberian hamsters (Phodopus sungorus). Ovaries from cycling hamsters were collected during proestrus (P), estrus (E), diestrus I (DI), and diestrus II (DII). To examine KiSS-1/GPR54 during stimulated recrudescence, additional hamsters were maintained either in long day (LD 16L:8D, control) or short day (SD 8L:16D) for 14 weeks and then transferred to LD for 0-8 weeks. Staining of KiSS-1/GPR54 protein was detected by immunohistochemistry in steroidogenic cells of pre-antral and antral follicles, and corpora lutea. Immunostaining peaked in P and E, but decreased in the diestrus stages (P < 0.05). In recrudescing ovaries, KiSS-1/GPR54 immunostaining was low after 14 weeks of SD exposure (post-transfer [PT] week 0), and increased during the early weeks of recrudescence. Expression of KiSS-1/GPR54 mRNA was low with short day exposure, but increased during recrudescence and was higher at PT week 8 as compared to PT weeks 0 and 2 (P < 0.05). The elevated KiSS-1/GPR54 expression during P and E suggests a potential role in ovulation in Siberian hamsters. Transient increases in KiSS-1/GPR54 expression following LD stimulation are also suggestive of possible involvement in ovulation and/or restoration of ovarian function.     

Medaka dmY/dmrt1Y is not the universal primary sex-determining gene in fish

An outstanding candidate for a primary male-determining gene equivalent to Sry of mammals has been recently described from a non-mammalian vertebrate, the medaka fish (Oryzias latipes). However, the universality of dmY/dmrt1Y as the master sex-determining gene in fish is questionable. Phylogenetic analysis shows that dmY/dmrt1Y is an evolutionarily young Y chromosome-specific duplicate of a gene involved in testis development in vertebrates, and that this duplicate cannot be the primary sex-determining gene in most other fish species. Study of alternative fish models will probably uncover new genetic strategies controlling sexual dimorphism in vertebrates.     

Physical association of GPR54 C-terminal with protein phosphatase 2A

KiSS1 was discovered as a metastasis suppressor gene and subsequently found to encode kisspeptins (KP), ligands for a G protein coupled receptor (GPCR), GPR54. This ligand-receptor pair was later shown to play a critical role in the neuro-endocrine regulation of puberty. The C-terminal cytoplasmic (C-ter) domain of GPR54 contains a segment rich in proline and arginine residues that corresponds to the primary structure of four overlapping SH3 binding motifs. Yeast two hybrid experiments identified the catalytic subunit of protein phosphatase 2A (PP2A-C) as an interacting protein. Pull-down experiments with GST fusion proteins containing the GPR54 C-ter confirmed binding to PP2A-C in cell lysates and these complexes contained phosphatase activity. The proline arginine rich segment is necessary for these interactions. The GPR54 C-ter bound directly to purified recombinant PP2A-C, indicating the GPR54 C-ter may form complexes involving the catalytic subunit of PP2A that regulate phosphorylation of critical signaling intermediates.     

Structual comparison of dermatopontin amino acid sequences

Dermatopontin is a tyrosine-rich acidic extracellular matrix protein of 22 kD with possible functions in cellmatrix interactions and matrix assembly. Database of GenBank+EMBL+DDBJ sequences from Nucleotide, Gene, and Expressed Sequence Tag (EST) Divisions was searched with a keyword “dermatopontin” or mouse dermatopontin amino acid sequence. In addition to five mammals previously described, five mammalian, two bird, one fish dermatopontin genes were detected in vertebrates. Additionally, a goat EST was also shown as goat dermatopontin missing 5′-end of the coding region. Moreover, a mRNA sequence of rhesus monkey dermatopontin was identified, but the deduced amino acid sequence was terminated abruptly due to a nonsense codon. For three 6-residue repeat regions (D-R-E/Q-W-X-F/Y) that may function as part of a glycosaminoglycan binding site, the first repeat sequence is D-R-Q-W-N-Y in all mammals while Glutamine is substituted for Leucine in birds. The second and the third repeats are conserved in all vertebrates. The N-Y-D sequence, the consensus in many amine oxidases, is conserved in mammals except rodents. Asparagine is substituted for Threonine in birds. The tetrapeptide R-G-A-T sequence possibly recognizing the integrin family is conserved in mammals and birds, but Alanine was substituted for Glutamine in zebrafish resulting in loss of activity. In conclusion, functionally significant amino acid sequences in vertebrate dermatopontins are conserved in mammals, while they are not necessarily identified in birds and fish. The original function of vertebrate dermatopontins may be glycosaminoglycan binding and functions as a ligand for integrin and an amine oxidase may be gained in the process of evolution.     

The neurobiology and evolution of cannabinoid signalling

The plant Cannabis sativa has been used by humans for thousands of years because of its psychoactivity. The major psychoactive ingredient of cannabis is Delta(9)-tetrahydrocannabinol, which exerts effects in the brain by binding to a G-protein-coupled receptor known as the CB1 cannabinoid receptor. The discovery of this receptor indicated that endogenous cannabinoids may occur in the brain, which act as physiological ligands for CB1. Two putative endocannabinoid ligands, arachidonylethanolamide ('anandamide') and 2-arachidonylglycerol, have been identified, giving rise to the concept of a cannabinoid signalling system. Little is known about how or where these compounds are synthesized in the brain and how this relates to CB1 expression. However, detailed neuroanatomical and electrophysiological analysis of mammalian nervous systems has revealed that the CB1 receptor is targeted to the presynaptic terminals of neurons where it acts to inhibit release of 'classical' neurotransmitters. Moreover, an enzyme that inactivates endocannabinoids, fatty acid amide hydrolase, appears to be preferentially targeted to the somatodendritic compartment of neurons that are postsynaptic to CB1-expressing axon terminals. Based on these findings, we present here a model of cannabinoid signalling in which anandamide is synthesized by postsynaptic cells and acts as a retrograde messenger molecule to modulate neurotransmitter release from presynaptic terminals. Using this model as a framework, we discuss the role of cannabinoid signalling in different regions of the nervous system in relation to the characteristic physiological actions of cannabinoids in mammals, which include effects on movement, memory, pain and smooth muscle contractility. The discovery of the cannabinoid signalling system in mammals has prompted investigation of the occurrence of this pathway in non-mammalian animals. Here we review the evidence for the existence of cannabinoid receptors in non-mammalian vertebrates and invertebrates and discuss the evolution of the cannabinoid signalling system. Genes encoding orthologues of the mammalian CB1 receptor have been identified in a fish, an amphibian and a bird, indicating that CB1 receptors may occur throughout the vertebrates. Pharmacological actions of cannabinoids and specific binding sites for cannabinoids have been reported in several invertebrate species, but the molecular basis for these effects is not known. Importantly, however, the genomes of the protostomian invertebrates Drosophila melanogaster and Caenorhabditis elegans do not contain CB1 orthologues, indicating that CB1-like cannabinoid receptors may have evolved after the divergence of deuterostomes (e.g. vertebrates and echinoderms) and protostomes. Phylogenetic analysis of the relationship of vertebrate CB1 receptors with other G-protein-coupled receptors reveals that the paralogues that appear to share the most recent common evolutionary origin with CB1 are lysophospholipid receptors, melanocortin receptors and adenosine receptors. Interestingly, as with CB1, each of these receptor types does not appear to have Drosophila orthologues, indicating that this group of receptors may not occur in protostomian invertebrates. We conclude that the cannabinoid signalling system may be quite restricted in its phylogenetic distribution, probably occurring only in the deuterostomian clade of the animal kingdom and possibly only in vertebrates.     

Flt3 mutation activates p21WAF1/CIP1 gene expression through the action of STAT5

Metastin, a post-translationally modified variant of KiSS1, was recently identified as an endogenous peptide agonist for a novel G-protein coupled receptor, hOT7T175 (AXOR12, GPR54). In this study, we analyzed the role of KiSS1 and hOT7T175 in both pancreatic cancer tissues and pancreatic cancer cell lines. Furthermore, we synthesized novel short variant forms of metastin and tested the inhibitory effect of those variants on in vitro cell functions that are relevant to metastasis. Pancreatic cancer tissues showed significantly lower expression of KiSS1 mRNA than normal tissues (p=0.018), while cancer tissues showed significantly higher expression of hOT7T175 mRNA than normal pancreatic tissues (p=0.027). In human pancreatic cancer cell lines, KiSS1 mRNA was highly expressed in 2 out of 6 pancreatic cancer cell lines, while hOT7T175 mRNA was expressed in all cell lines at various degrees. PANC-1 cells showed the highest expression of hOT7T175. Exogenous metastin did not suppress cell proliferation but significantly reduced the in vitro migration of PANC-1 cells (p<0.01). Metastin induced activation of ERK1 in PANC-1 and AsPC-1 cells. Finally, we synthesized 3 novel short variant forms of metastin, FM053a2TFA, FM059a2TFA, and FM052a4TFA. These metastin variants significantly suppressed the migration of PANC-1 cells and activated ERK1. These data suggest that the metastin receptor, hOT7T175, is one of the promising targets for suppression of metastasis, and that small metastin variants could be an anti-metastatic agent to pancreatic cancer.     

The kisspeptin/gonadotropin-releasing hormone pathway and molecular signaling of puberty in fish

The mechanisms underlying the initiation of puberty in fish are poorly understood, and whether the Kiss1 receptor (Kiss1r; previously designated G protein-coupled receptor 54; GPR54) and its ligands, kisspeptins, play a significant role, as has been established in mammals, is not yet known. We determined (via real-time PCR) temporal patterns of expression in the brain of kiss1r, gnrh2, and gnrh3 and a suite of related genes in the hypothalamo-pituitary-gonadal (HPG) axis and analyzed them against the timing of gonadal germ cell development in male and female fathead minnow (Pimephales promelas). Full- or partial-length cDNAs for kiss1r (736 bp), gnrh2 (698 bp), and gnrh3 (804 bp) cloned from fathead minnow were found to be expressed only in the brain, testis, and ovary of adult fish. Localization of kiss1r, gnrh2, and gnrh3 within the brain provided evidence for their physiological roles and a likely hypophysiotropic role for GnRH3 in this species (which, like other cyprinids, does not appear to express gnrh1). In both sexes, kiss1r expression in the brain increased at the onset of puberty and reached maximal expression in males when spermatagonia type B appeared in the testis and in females when cortical alveolus-stage oocytes first appeared in the ovary, the timings of which differed for the two sexes. However, kiss1r expression was considerably lower during more advanced stages of spermatogenesis and oogenesis. The expression of kiss1r closely aligned with that of the gnrh genes (gnrh3 in particular), suggesting the Kiss1r/kisspeptin system in fish has a similar role in puberty to that occurring in mammals, and this hypothesis was supported by the induction of gnrh3 (2.25-fold) and kiss1r (1.5-fold) in early-mid pubertal fish injected with mammalian kisspeptin-10 (2 nmol/g wet weight). An intriguing finding, and contrasting that in mammals, was an elevated expression of esr1, ar, and cyp19a2 (genes involved in sex steroid signaling) in the brain at the onset of puberty, and in females slightly in advance of the elevation in the expression of kiss1r.