Phoenixin-14 as a novel direct regulator of porcine luteal cell functions†.

Phoenixin is a neuropeptide with a well-established role in the central regulation of reproductive processes; however, knowledge regarding its role in the ovary is limited. One of the main active phoenixin isoforms is phoenixin-14, which acts through G protein-coupled receptor 173. Our research hypothesis was that phoenixin-14 is expressed in porcine corpus luteum and exerts luteotropic action by affecting the endocrine function of luteal cells through G protein-coupled receptor 173 and protein kinase signaling. Luteal cells were cultured to investigate the effect of phoenixin-14 (1-1000 nM) on endocrine function. We showed that phoenixin-14 and G protein-coupled receptor 173 are produced locally in porcine corpus luteum and their levels change during the estrous cycle. We detected phoenixin-14 immunostaining in the cytoplasm and G protein-coupled receptor 173 in the cell membrane. Plasma phoenixin levels were highest during the early luteal phase. Interestingly, insulin, luteinizing hormone, progesterone, and prostaglandins decreased phoenixin-14 levels in luteal cells. Phoenixin-14 increased progesterone, estradiol, and prostaglandin E2 secretion, but decreased prostaglandin F2α, upregulated the expression of steroidogenic enzymes, and downregulated receptors for luteinizing hormone and prostaglandin. Also, phoenixin-14 increased the expression of G protein-coupled receptor 173 and the phosphorylation of extracellular signal-regulated kinase 1/2, protein kinase B, inhibited the phosphorylation of protein kinase A, and had mixed effect on AMP-activated protein kinase alpha and protein kinase C. G protein-coupled receptor 173 and extracellular signal-regulated kinase 1/2 mediated the effect of phoenixin-14 on endocrine function of luteal cells. Our results suggest that phoenixin is produced by porcine luteal cells and can be a new regulator of their function.

Characterizing the SREB G protein-coupled receptor family in fish: Brain gene expression and genomic differences in upstream transcription factor binding sites.

The SREB (Super-conserved Receptors Expressed in Brain) family of orphan G protein-coupled receptors is highly conserved in vertebrates and consists of three members: SREB1 (orphan designation GPR27), SREB2 (GPR85), and SREB3 (GPR173). SREBs are associated with processes ranging from neuronal plasticity to reproductive control. Relatively little is known about similarities across the entire family, or how mammalian gene expression patterns compare to non-mammalian vertebrates. In fish, this system may be particularly complex, as some species have gained a fourth member (SREB3B) while others have lost genes. To better understand the system, the present study aimed to: 1) use qPCR to characterize sreb and related gene expression patterns in the brains of three fish species with different systems, and 2) identify possible differences in transcriptional regulation among the receptors, using upstream transcription factor binding sites across 70 ray-finned fish genomes. Overall, regional patterns of sreb expression were abundant in forebrain-related areas. However, some species-specific patterns were detected, such as abundant expression of receptors in zebrafish (Danio rerio) hypothalamic-containing sections, and divergence between sreb3a and sreb3b in pufferfish (Dichotomyctere nigroviridis). In addition, a gene possibly related to the system (dkk3a) was spatially correlated with the receptors in all three species. Genomic regions upstream of sreb2 and sreb3b, but largely not sreb1 or sreb3a, contained many highly conserved transcription factor binding sites. These results provide novel information about expression differences and transcriptional regulation across fish that may inform future research to better understand these receptors.

Expression and localization of the neuropeptide phoenixin-14 and its receptor GRP173 in the canine reproductive organs and periovarian adipose tissue.

Phoenixin-14 (PNX-14) is a regulatory neuropeptide encoded by the SMIM20 gene, which has been implicated in the reproductive cycle by modulating the hypothalamic-pituitary-gonadal (HPG) axis. Recently, we showed that PNX-14 is downregulated in bitches with cystic endometrial hyperplasia and pyometra. The objective of this study was to determine the expression of Smim20, PNX-14, and its putative receptor GRP173 in the canine ovary (both healthy and those with ovarian cysts), periovarian adipose tissue (PAT) and in the endometrium during the oestrous cycle. The expression was analysed by RT-qPCR and Western blot. In tissue sections, peptides were localised by immunofluorescent assays, and blood plasma concentrations of PNX-14 were detected by EIA. The results demonstrated increased levels of PNX in bitches in the anestrus groups compared to diestrus animals. The expression of GPR173 increased in PAT during the diestrus phase and endometrial tissue in late diestrus bitches. In the ovary, strong signals of PNX-14 and GPR173 were detected in the luteal and follicular cells. Furthermore, bitches with cystic ovaries were characterised by elevated circulating PNX levels and a significantly higher expression of PNX and GPR173 in gonadal tissues, when compared with healthy animals. Moreover, a positive correlation between PNX and progesterone in the blood of healthy bitches was noted, which changed to a negative correlation in females affected by cystic ovaries. These studies expand the knowledge regarding the expression and localization of the PNX/GRP173 system in canine reproductive organs during physiological and pathological conditions.

A Novel CCK Receptor GPR173 Mediates Potentiation of GABAergic Inhibition.

Cholecystokinin (CCK) enables excitatory circuit long-term potentiation (LTP). Here, we investigated its involvement in the enhancement of inhibitory synapses. Activation of GABA neurons suppressed neuronal responses in the neocortex to a forthcoming auditory stimulus in mice of both sexes. High-frequency laser stimulation (HFLS) of GABAergic neurons potentiated this suppression. HFLS of CCK interneurons could induce the LTP of their inhibition toward pyramidal neurons. This potentiation was abolished in CCK knock-out mice but intact in mice with both CCK1R and 2R knockout of both sexes. Next, we combined bioinformatics analysis, multiple unbiased cell-based assays, and histology examinations to identify a novel CCK receptor, GPR173. We propose GPR173 as CCK3R, which mediates the relationship between cortical CCK interneuron signaling and inhibitory LTP in the mice of either sex. Thus, GPR173 might represent a promising therapeutic target for brain disorders related to excitation and inhibition imbalance in the cortex.SIGNIFICANCE STATEMENT CCK, the most abundant and widely distributed neuropeptide in the CNS, colocalizes with many neurotransmitters and modulators. GABA is one of the important inhibitory neurotransmitters, and much evidence shows that CCK may be involved in modulating GABA signaling in many brain areas. However, the role of CCK-GABA neurons in the cortical microcircuits is still unclear. We identified a novel CCK receptor, GPR173, localized in the CCK-GABA synapses and mediated the enhancement of the GABA inhibition effect, which might represent a promising therapeutic target for brain disorders related to excitation and inhibition imbalance in the cortex.

The role of phoenixin in the proliferation and migration of ectopic epithelial cells in vitro.

AIM: Endometriosis is one of the most common gynecologic diseases in women of reproductive age. The pathophysiology of endometriosis is still not fully understood. Phoenixin (PNX-14) is a newly discovered neuropeptide that regulates the hypothalamo-pituitary-gonadal (HPG) axis and reproductive functions. Recently, we reported that PNX-14, its precursor protein and receptor were expressed in human endometrium. Moreover, PNX-14 serum levels in endometriosis were reduced. This study aimed to evaluate the in vitro biological functions of physiological PNX-14 concentrations on the ectopic endometrium Z12 cells. METHODS: The proliferation and migration of Z12 cells were assessed using the xCELLigence® RTCA DP system following 72 h of stimulation with 0.05 and 0.2 nM of PNX-14. GPR173 and small integral membrane protein 20 (SMIM20) gene expression was evaluated using quantitative polymerase chain reaction (qPCR) and the protein levels of GPR173 were analyzed using Western blot analysis. RESULTS: PNX-14 at the concentration observed in the serum of patients with endometriosis (0.05 nM) reduced GPR173 and increased SMIM20 expression, while protein levels of GPR173 remained unchanged. Cell proliferation was increased by the 0.02 nM PNX-14- the concentration found in healthy subjects. The 0.2 nM of PNX-14 decreased SMIM20 expression with no change to GPR173 expression and reduced ectopic epithelial cell proliferation during the first 5 h after stimulation. However, at 72 h, the proliferation increased. CONCLUSIONS: This study shows that PNX-14 at endometriosis specific concentration desensitized ectopic epithelium to PNX-14, and increased the expression of SMIM20 to restore the physiological levels of PNX-14.

Regulation and physiological functions of phoenixin.

Phoenixin is a newly discovered neuropeptide generated from small integral membrane protein 20. Phoenixin is a ligand for the G protein-coupled receptor 173 (GPR173) and has been detected in central and peripheral tissues of human, rats, mice, bovine, and zebrafish. It was initially involved in regulating reproductive function by stimulating the luteinizing hormone release from pituitary cells by increasing the level of gonadotropin-releasing hormone. Recently, many functions of phoenixin have been generalized, including regulation of food intake, memory, Alzheimer's disease, anxiety, inflammation, neuronal and microglial activity, energy metabolism and body fluid balance, cardiovascular function, and endocrine activity. In addition, the interaction between phoenixin and nesfatin-1 have been revealed. The present article summarized the latest research progress on physiological function of phoenixin, suggesting that it is a potential target for novel drug development and clinical application.

Superconserved receptors expressed in the brain: Expression, function, motifs and evolution of an orphan receptor family.

GPR27, GPR85 and GPR173 constitute a small family of G protein-coupled receptors (GPCR) that share the distinctive characteristics of being highly conserved throughout vertebrate evolution and predominantly expressed in the brain. Accordingly, they have been coined as "Superconserved Receptors Expressed in the Brain" (SREB), although their expression profile is more complex than what was originally thought. SREBs have no known validated endogenous ligands and are thus labeled as "orphan" receptors. The investigation of this particular category of uncharacterized receptors holds great promise both in terms of physiology and drug development. In the largest GPCR family, the Rhodopsin-like or Class A, around 100 receptors are considered orphans. Because GPCRs are the most successful source of drug targets, the discovery of a novel function or ligand most likely will lead to significant breakthroughs for the discovery of innovative therapies. The high level of conservation is one of the characteristic features of the SREBs. We propose herein a detailed analysis of the putative evolutionary origin of this family. We highlight the properties that distinguish SREBs from other rhodopsin-like GPCRs. We present the current evidence for these receptors downstream signaling pathways and functions. We discuss the pharmacological challenge for the identification of natural or synthetic ligands of orphan receptors like SREBs. The different SREB-related scientific questions are presented with a highlight on what should be addressed in the near future, including the confirmation of published evidence and their validation as drug targets. In particular, we discuss in which pathological conditions these receptors may be of great relevance to solve unmet medical needs.

Super-conserved receptors expressed in the brain: biology and medicinal chemistry efforts.

The super-conserved receptors expressed in the brain (SREB) constitute a family of orphan G protein-coupled receptors that include GPR27 (SREB1), GPR85 (SREB2) and GPR173 (SREB3). Their sequences are highly conserved in vertebrates, and they are almost exclusively expressed in the central nervous system. This family of receptors has attracted much attention due to their putative physiological functions and their potential as novel drug targets. The SREB family has been postulated to play important roles in a wide range of different diseases, including pancreatic ß-cell insulin secretion and regulation, schizophrenia, autism and atherosclerosis. This review intends to provide a comprehensive overview of the SREB family and its recent advances in biology and medicinal chemistry.

In recent years, the super-conserved receptors expressed in the brain called GPR27, GPR85 and GPR173 have attracted much interest in the field of medicinal science. They have one important feature in common: they are all almost entirely found in the brain. Researchers have investigated their functions in the body in various animal models, as well as their utility in future drug development. GPR27 has been found to be involved in insulin and blood sugar processes in the body and therefore may be important for diabetes treatment. GPR85 is thought to be linked to brain diseases such as schizophrenia and autism. GPR173 is linked to many different illnesses, including atherosclerosis (the buildup of fats, cholesterol and other substances in arteries) and Type 2 diabetes.

Phoenixin-20 ameliorates brain infarction by promoting microglia M2 polarization in an ischemic stroke model.

Ischemic stroke is one of the most common causes of death worldwide. The transformation of microglia from the classic M1 to the alternative M2 state has been shown to have both deleterious and immunosuppressive roles in neuroinflammation. Microglial polarization toward the M2 phase is currently proposed to be a beneficial phenotype in brain ischemic injury. Phoenixin-20 is a newly identified pleiotropic neuropeptide expressed abundantly in different brain regions. In this study, we found that administration of Phoenixin-20 in ischemic stroke middle cerebral artery occlusion (MCAO) mice significantly reduced the brain infarction area but improved the neurological deficit score. Gene expression analysis showed Phoenixin-20 treatment inhibited pro-inflammatory M1 phase microglial markers: a cluster of differentiation molecule 11b (CD11b), cluster of differentiation molecule 86 (CD86), inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and increased anti-inflammatory M2 phase markers (found in Inflammatory Zone 1 (FIZZ1), Arginase 1 (Arg-1), Chitinase 3-like 3 (YM1), and interleukin-10 (IL-10)) in the infarcted brain. We further investigated the molecular mechanism of Phoenixin-20 in cultured microglia. We found that treatment with it induced signature genes expression in microglial M2 state, including Fizz1, Arg-1, YM1, and IL-10, indicating the promotion of microglial polarization toward the M2 state. Furthermore, we found that treatment with the M2 phase cytokine interleukin 4 (IL-4) induced the expression of microglial G Protein-Coupled Receptor (GPR173), which is the receptor of Phoenixin-20. Silencing of the microglial signal transducer and activator of transcription 6 (STAT6) partially blocked the effect of IL-4 on GPR173, suggesting that STAT6 is the upstream regulator of GPR173. Finally, we showed that the silencing of GPR173 completely abolished the effect of Phoenixin-20 in microglia, indicating the dependency of its regulatory role on GPR173. Collectively, our study demonstrates that Phoenixin-20 has a protective role in the acute stroke model. Our cell-based study demonstrates Phoenixin-20 promotes microglia toward M2 transformation, which could be the mechanism of its neuroprotection.

Canine cystic endometrial hyperplasia and pyometra may downregulate neuropeptide phoenixin and GPR173 receptor expression.

The most common uterine diseases affecting bitches are cystic endometrial hyperplasia (CEH) and pyometra. The neuropeptide phoenixin (PNX) and its receptor (GPR173) are potential key factors involved in the proliferative and inflammatory regulation of the reproductive system in females. This study aimed to evaluate the expression of PNX and GPR173 by qPCR, western blot and immunofluorescence assays in the endometrium of bitches suffering from CEH or pyometra compared to clinically healthy females. Additionally, PNX and progesterone (P4) plasma concentrations were analysed. The results showed a significantly lower expression levels of PNX and GPR173 (mRNA and protein production) in bitches with the CEH or pyometra groups compared to healthy animals. Immunofluorescence staining examination also confirmed a lower concentration of PNX and GPR173 signals in bitches with pathological uteri. Moreover, a lower concentration of PNX blood levels in bitches suffering from pyometra was observed. The PNX concentration was negatively correlated with P4 but only in healthy bitches. These results illustrate that the development of canine uterine disorders may cause a lower expression of PNX and its receptor GPR173.

The Regulation of Phoenixin: A Fascinating Multidimensional Peptide.

The phoenixin (PNX) peptide is linked to the control of reproduction, food intake, stress, and inflammation. However, little is known about what regulates its gene and protein expression, information that is critical to understand the physiological role of PNX. In this review, we summarize what is known about the transcriptional control of Pnx and its receptor Gpr173. A main function of PNX is as a positive regulator of the hypothalamic-pituitary-gonadal axis, but there is a lack of research on its control by reproductive hormones and peptides. PNX is also associated with food intake, and its expression is linked to feeding status, fatty acids, and glucose. It is influenced by environmental and hormonal-induced stress. The regulation of Pnx in most contexts remains an enigma, in part due to conflicting and negative results. An extensive analysis of the response of the Pnx gene to factors related to reproduction, metabolism, stress, and inflammation is required. Analysis of the Pnx promoter and epigenetic regulation must be considered to understand how this level of control contributes to its pleiotropic effects. PNX is now linked to a broad range of functions, but more research on its gene regulation is required to understand its place in overall physiology and therapeutic potential.

Phoenixin as a New Target in the Development of Strategies for Endometriosis Diagnosis and Treatment.

Small integral membrane protein 20/phoenixin (SMIM20/PNX) and its receptor GPR173 (G Protein-Coupled Receptor 173) play a role in the regulation of the hypothalamic-pituitary-gonadal axis (HPG). The aim of the study was to determine PNX, FSH, LH, and 17ß-estradiol association in women with endometriosis, and the expression of SMIM20/PNX signaling via GPR173. Serum PNX, FSH, LH, and 17ß-estradiol concentrations were measured by enzyme and electrochemiluminescence immunoassay. SMIM20/PNX and GPR173 expression in the eutopic and ectopic endometrium was assessed by qPCR and immunohistochemistry. Reduced PNX level, increased LH/FSH ratio and elevated 17ß-estradiol concentration were found in patients with endometriosis. No differences in SMIM20 expression were observed between the studied endometria. GPR173 expression was lower in ectopic than in eutopic endometria. SMIM20 expression was mainly restricted to stroma. GPR173 was detected in some eutopic and ectopic stromal cells and in eutopic glandular epithelial cells. Discriminant analysis indicates the diagnostic relevance of PNX and LH/FSH ratio in patients with endometriosis. In women with endometriosis, reduced PNX levels and GPR173 expression may be responsible for HPG axis dysregulation. These new insights may contribute to a better understanding of the pathophysiology of endometriosis and provide the basis for a new strategy for diagnosis and treatment of endometriosis.

Phoenixin-20 ameliorates gestational diabetes mellitus (GDM) symptoms and placental insults in an experimental mouse model.

BACKGROUND AND PURPOSE: Gestational diabetes mellitus (GDM) is a complication commonly observed in pregnancy, closely associated with increased oxidative stress, inflammatory response, and endoplasmic reticulum (ER) stress. Phoenixin-20 (PNX-20) is a newly reproductive hormone from the hypothalamus that has displayed pleiotropic effects. The promising inhibitory effects of PNX-20 on inflammation have recently been widely reported. The present study aims to investigate the protective effect of PNX-20 on GDM induced placental insults. METHODS: A GDM model was established on C57BLKsJ db/+ mice. The expression level of GPR173 was evaluated using RT-PCR and western blotting analysis. The serum level of glucose, insulin, lipid profiles, and oxidative stress indicators were detected with commercial kits. Fetal analysis was performed to evaluate the reproductive ability. ELISA was used to detect the production of inflammatory factors. The expressions of p-eIF-2α, ATF4, and GRP78 were evaluated with western blotting assay. RESULTS: Firstly, we found that GPR173 is expressed in the placenta tissue. Secondly, the elevated blood glucose level and lipid level, declined serum insulin level, fetus alive ratio, fetal and placenta weight, and shorten crown-rump length, were observed in the placenta tissue of GDM mice, which were reversed by treatment with PNX-20. Thirdly, the excessively released inflammatory factors and activated oxidative stress in GDM mice were alleviated by the administration of PNX-20. Lastly, the activated eIF-2α/ATF4 ER stress signaling pathway in GDM mice was dramatically suppressed by PNX-20. CONCLUSION: Our data revealed a protective property of PNX-20 against placental insults resulted from GDM.

Role of the Novel Peptide Phoenixin in Stress Response and Possible Interactions with Nesfatin-1.

The novel peptide phoenixin was shown to be involved in several physiological processes ranging from reproduction to food intake. Interest in this protein has steadily increased over the last few years and its known implications have become much broader, playing a role in glucose homeostasis, anxiety, nociception, and pruritus. Phoenixin is expressed in a multitude of organs such as the small intestine, pancreas, and in the hypothalamus, as well as several other brain nuclei influencing numerous physiological functions. Its highly conserved amino-acid sequence amongst species leads to the assumption, that phoenixin might be involved in essential physiological functions. Its co-expression and opposing functionality to the extensively studied peptide nesfatin-1 has given rise to the idea of a possible counterbalancing role. Several recent publications focused on phoenixin's role in stress reactions, namely restraint stress and lipopolysaccharide-induced inflammation response, in which also nesfatin-1 is known to be altered. This review provides an overview on the phoenixins and nesfatin-1 properties and putative effects, and especially highlights the recent developments on their role and interaction in the response to response.

Structure-activity relationships of agonists for the orphan G protein-coupled receptor GPR27.

GPR27 belongs, with GPR85 and GPR173, to a small subfamily of three receptors called "Super-Conserved Receptors Expressed in the Brain" (SREB). It has been postulated to participate in key physiological processes such as neuronal plasticity, energy metabolism, and pancreatic ß-cell insulin secretion and regulation. Recently, we reported the first selective GPR27 agonist, 2,4-dichloro-N-(4-(N-phenylsulfamoyl)phenyl)benzamide (I, pEC50 6.34, Emax 100%). Here, we describe the synthesis and structure-activity relationships of a series of new derivatives and analogs of I. All products were evaluated for their ability to activate GPR27 in an arrestin recruitment assay. As a result, agonists were identified with a broad range of efficacies including partial and full agonists, showing higher efficacies than the lead compound I. The most potent agonist was 4-chloro-2,5-difluoro-N-(4-(N-phenylsulfamoyl)phenyl)benzamide (7y, pEC50 6.85, Emax 37%), and the agonists with higher efficacies were 4-chloro-2-methyl-N-(4-(N-phenylsulfamoyl)phenyl)benzamide (7p, pEC50 6.04, Emax 123%), and 2-bromo-4-chloro-N-(4-(N-phenylsulfamoyl)phenyl)benzamide (7r, pEC50 5.99, Emax 123%). Docking studies predicted the putative binding site and interactions of agonist 7p with GPR27. Selected potent agonists were found to be soluble and devoid of cellular toxicity within the range of their pharmacological activity. Therefore, they represent important new tools to further characterize the (patho)physiological roles of GPR27.

Phoenixin-20 Prevents ox-LDL-Induced Attachment of Monocytes to Human Aortic Endothelial Cells (HAECs): A Protective Implication in Atherosclerosis.

The exact cause of atherosclerosis is not known, and therefore, the current treatment options are limited. The activation of endothelial cells by oxidized low-density lipoprotein (ox-LDL) plays a key role in the initiation and progression of atherosclerosis. Phoenixin-20 is one of the newly identified neuropeptides with pleiotropic effects in the regulation of reproduction and other biological functions. G-protein receptor-coupled 173 (GPR173) is the putative receptor of Phoenixin-20. In the present study, we show that endothelial GPR173 is repressed upon ox-LDL stimulation in human aortic endothelial cells (HAECs). We further elaborate on the hypothesis that GPR173 could be involved in the pathogenesis of atherosclerosis through a series of experiments. Our results indicate that ox-LDL remarkably triggers the increase of ROS, NOX-4, pro-inflammatory cytokines IL-1ß, IL-8, and MCP-1 expression, as well as adhesion molecules ICAM-1 and VCAM-1 release. However, the agonism of GPR173 using Phoenixin-20 significantly ameliorates all of these harmful effects from ox-LDL by suppressing the NF-κB pathway. Furthermore, we show that agonism of GPR173 by Phoenixin-20 prevents the attachment of monocytes THP-1 to endothelial cells, which is an important therapeutic approach to preventing atherogenesis. In conclusion, our study demonstrates that GPR173 agonism by Phoenixin-20 plays a protective role against ox-LDL-induced endothelial dysfunction, implying that Phoenixin-20 may have therapeutic implications in atherosclerosis.

phoenixin(smim20), a gene coding for a novel reproductive ligand, is expressed in the brain of adult zebrafish.

Gonadotropin-releasing hormone (GnRH) is a highly conserved neuroendocrine decapeptide that is essential for the onset of puberty and the maintenance of the reproductive state. In addition to its role as hypothalamic releasing hormone, GnRH has multiple functions including modulator of neural activity within the nervous system and of resulting behaviors. These multiple functions are reflected by the existence of multiple isoforms. Despite its importance as a critical hypothalamic releasing hormone, the gnrh1 gene has been lost in zebrafish, and its reproductive function is not compensated for by other GnRH isoforms (GnRH2 and GnRH3), suggesting that, surprisingly, zebrafish do not use any of the GnRH peptides to control reproduction and fertility. Previously we proposed that Phoenixin/SMIM20, a novel peptide identified in mammals and the ligand for the orphan GPR173, is a potential candidate to control the initiation of sexual development and fertility in the zebrafish. Here we confirm the sequence of the zebrafish phoenixin/smim20 gene and by RT-PCR show that it is expressed early in development through adulthood. Subsequently we show that phoenixin/smim20 is expressed in the adult brain including the regions of the hypothalamus important in the control of fertility and reproduction.

Phoenixin: More than Reproductive Peptide.

Phoenixin (PNX) neuropeptide is a cleaved product of the Smim20 protein. Its most common isoforms are the 14- and 20-amino acid peptides. The biological functions of PNX are mediated via the activation of the GPR173 receptor. PNX plays an important role in the central nervous system (CNS) and in the female reproductive system where it potentiates LH secretion and controls the estrus cycle. Moreover, it stimulates oocyte maturation and increases the number of ovulated oocytes. Nevertheless, PNX not only regulates the reproduction system but also exerts anxiolytic, anti-inflammatory, and cell-protective effects. Furthermore, it is involved in behavior, food intake, sensory perception, memory, and energy metabolism. Outside the CNS, PNX exerts its effects on the heart, ovaries, adipose tissue, and pancreatic islets. This review presents all the currently available studies demonstrating the pleiotropic effects of PNX.

GPR173 agonist phoenixin 20 promotes osteoblastic differentiation of MC3T3-E1 cells.

Osteogenic differentiation is critical to bone homeostasis, and its imbalance plays a key role in the progression of osteoporosis. Osteoblast cells are responsible for synthesizing new bone tissue, and understanding how to control osteoblastic differentiation is vital to the treatment of osteoporosis. Herein, we show that GPR173 signaling is involved in the regulation of osteoblastic differentiation in MC3T3-E1 cells. Our data reveals that GPR173 is abundantly expressed in MC3T3-E1 cells, and its expression is inducible upon the introduction of osteogenic media. The activation of GPR173 by its selective agonist phoenixin 20 induces the expression of several osteoblast signature genes including collagen type 1 alpha 1 (Col-I), osteocalcin (OCN), alkaline phosphatase (ALP) as well as increased matrix mineralization and ALP activity, suggesting that the activation of GPR173 promotes osteoblastic differentiation. Moreover, we show that the effect of phoenixin 20 is mediated by its induction on the key regulator runt-Related Transcription Factor 2 (Runx2). Mechanistically, we display that the action of phoenixin 20 requires the activation of MAPK kinase p38, and deactivation of p38 by its inhibitor SB203580 weakens the phoenixin 20-mediated induction of RUNX-2, ALP, and matrix mineralization. Silencing of GPR173 attenuates phoenixin 20-mediated osteoblastic differentiation, indicating its dependence on the receptor. Collectively, our study reveals a new role of GPR173 and its agonist phoenixin 20 in osteoblastic differentiation.

Phoenixin-20 suppresses lipopolysaccharide-induced inflammation in dental pulp cells.

Pulpal infection is one of the most common causes of dental emergency admission. Tooth pain due to infection caused by gram-negative bacteria is the main manifestation of this sort of dental problem. The GPR173 signaling pathway is a highly conserved G-protein-coupled receptor that mediates neurological and reproductive function. In this study, we found that GPR173 was fairly expressed in isolated human dental pulp cells, and its expression was reduced in response to pro-inflammatory lipopolysaccharide (LPS) treatment. The activation of GPR173 by its ligand Phoenixin-20 reduced LPS-induced cytotoxicity, as revealed by a reduction in the release of LDH. Additionally, Phoenixin-20 suppressed LPS-induced release of pro-inflammatory cytokines and inflammatory mediators, including IL-6, MCP-1, VCAM-1, and ICAM-1, as well as MMP-2 and MMP-9. Mechanistically, we showed the suppressive action of Phoenixin-20 on LPS-induced activation of TLR-4 and Myd88 as well as the activation of the NF-κB pathway. Collectively, our study demonstrates that the GPR173 signaling pathway is an important mediator of LPS-induced inflammation, and the activation of GPR173 by its natural ligand Phoenixin-20 exhibits robust anti-inflammatory effects in dental pulp cells, suggesting that GPR173 is an interesting target molecule in the development of pulp cell-based therapies.