Receptor Gpr1 Research Articles

Chemerin abundance in egg white and its expression with receptors in extra-embryonic annexes of Pekin ducks: implications for embryo development

Embryonic mortality is a significant problem in the commercial duck industry worldwide. Therefore, identification of new biomarkers for duck embryo development is necessary. In the chicken (order Galliformes), we previously showed that chemerin is a hormone locally produced by the reproductive tract in hens, particularly in the magnum area, leading to its accumulation in the egg white and within the embryo annexes during embryonic development. We therefore hypothesized that the chemerin concentration in egg white could be a biomarker of egg performance and reproductive parameters in Pekin ducks (order Anseriformes). Thus, we collected eggs from Pekin ducks over a 5-day period at three stages of the laying period (before the laying peak, after the laying peak, and at the end of the laying period) to measure the chemerin concentrations in egg white by enzyme-linked immunosorbent assay. The chemerin concentration in egg white decreased during the laying period and was not associated with reproductive parameters. We found negative correlations between the chemerin level in egg white and the albumen weight. Reverse-transcriptase quantitative polymerase chain reaction showed that chemerin and its three receptors CMKLR1, GPR1, and CCRL2 were expressed in the reproductive tract and within allantoic and amniotic annexes during embryo development. Chemerin concentrations strongly increased in amniotic fluid on embryonic day 16 (ED16) when the egg white was transferred into the amniotic sac. Finally, chemerin inhibition in egg white by in ovo injections of anti-chemerin antibodies (0.01, 0.1, and 1 µg) increased the embryo mortality rate. These data demonstrate the important role of the chemerin system during egg formation and embryo development in Pekin ducks, suggesting their potential use as biomarkers for determining the quality of poultry eggs and embryo development.

Expression of chemerin and its receptors in extra-embryonic annexes and role of chemerin and its GPR1 receptor in embryo development in layer and broiler hens

Intensive genetic selection of broiler breeders and layer hens resulted in differences in the mechanisms of growth and also in cell metabolism during embryogenesis. Previous research has shown that an adipokine named chemerin and one of these receptors, CMKLR1 were potentially involved in broiler embryo development. Here, our objectives were 1. to compare the expression of chemerin and its receptors CMKLR1, GPR1 and CCRL2 and chemerin concentration in extra-embryonic annexes (allantoic and amniotic membranes and fluids and plasma) in broiler and layer fertile eggs during the development (embryonic day (ED) 7, 14 and 18) by RT-qPCR and specific chicken ELISA and 2. to investigate the role of chemerin and one of its receptors GPR1 in embryo development after in ovo injections of neutralizing antibodies against chicken chemerin and GPR1. We found that chemerin expression in amniotic membranes was higher in layer than broiler eggs at ED7 and ED14 whereas the expression of the three receptors was higher in layer than broiler in the allantoic membranes at ED14 and ED18. Chemerin concentration was more important in layer than broiler at ED14 and ED18 in amniotic liquid and at all the studied stages in blood plasma. We also showed positive correlation between amniotic chemerin concentration and chemerin amniotic membrane expression, chemerin plasma concentration and embryo body weight in both breeds. Finally, in ovo injection of chicken chemerin and GPR1 neutralizing antibodies increased embryo mortality in both layer and broiler eggs. Taken together, even if chemerin concentration and chemerin system expression in embryonic membranes are mainly higher expressed in layer than in broiler, chemerin potentially through GPR1 could promote embryo development in both breeds.

Deciphering the regulatory mechanisms of the cAMP/protein kinase A pathway and their roles in the pathogenicity of Candida auris.

The emergence of multidrug-resistant fungal pathogens is a significant concern for global public health. Candida auris poses a considerable threat as a multidrug-resistant fungal pathogen. Our recent study revealed that the adenylyl cyclase Cyr1 and protein kinase A (PKA) pathways play distinct and redundant roles in drug resistance and pathogenicity of C. auris. However, the upstream and negative feedback regulatory mechanisms of C. auris are not yet fully understood. In this study, we discovered that the small GTPase Ras1, along with its nucleotide exchange factor Cdc25 and GTPase-activating protein Ira2, plays a major role in regulating cAMP/PKA-dependent traits, while G-protein-coupled receptor Gpr1 and heterotrimeric G-protein α subunit Gpa2 play a minor role. Pde2 plays a major role in negative feedback regulation of the cAMP/PKA pathway, while Pde1 plays a minor role. Hyperactivation of the Ras/cAMP/PKA pathway by deleting PDE2 or BCY1 renders C. auris cells thermosensitive and susceptible to nutrient deficiency, which leads to attenuated virulence. Our study demonstrates the distinct contributions of hyperactivation of the Ras/cAMP/PKA signaling pathway to C. auris pathogenesis and suggests potential therapeutic targets for C. auris-mediated candidiasis. IMPORTANCE Candida auris is a major concern as a multidrug-resistant fungal pathogen. While our previous studies highlighted the crucial roles of the cAMP/protein kinase A (PKA) pathway in regulating drug resistance, stress responses, morphogenesis, ploidy change, biofilm formation, and pathogenicity in this pathogen, their regulatory mechanism remains unclear. In our study, we provided evidence that the cAMP/PKA signaling pathway in C. auris is primarily governed by the small GTPase RAS rather than a G-protein-coupled receptor. Additionally, we discovered that the negative feedback regulation of cAMP, controlled by phosphodiesterases, is vital for C. auris virulence by promoting resistance to high temperatures and nutrient deficiencies. These findings underscore the diverse pathobiological significance of the Ras/cAMP/PKA signaling pathway in C. auris, shedding light on potential therapeutic targets and strategies for combating this multidrug-resistant fungal pathogen.

Atmospheric humidity regulates same-sex mating in Candida albicans through the trehalose and osmotic signaling pathways.

Sexual reproduction is prevalent in eukaryotic organisms and plays a critical role in the evolution of new traits and in the generation of genetic diversity. Environmental factors often have a direct impact on the occurrence and frequency of sexual reproduction in fungi. The regulatory effects of atmospheric relative humidity (RH) on sexual reproduction and pathogenesis in plant fungal pathogens and in soil fungi have been extensively investigated. However, the knowledge of how RH regulates the lifecycles of human fungal pathogens is limited. In this study, we report that low atmospheric RH promotes the development of mating projections and same-sex (homothallic) mating in the human fungal pathogen Candida albicans. Low RH causes water loss in C. albicans cells, which results in osmotic stress and the generation of intracellular reactive oxygen species (ROS) and trehalose. The water transporting aquaporin Aqy1, and the G-protein coupled receptor Gpr1 function as cell surface sensors of changes in atmospheric humidity. Perturbation of the trehalose metabolic pathway by inactivating trehalose synthase or trehalase promotes same-sex mating in C. albicans by increasing osmotic or ROS stresses, respectively. Intracellular trehalose and ROS signal the Hog1-osmotic and Hsf1-Hsp90 signaling pathways to regulate the mating response. We, therefore, propose that the cell surface sensors Aqy1 and Gpr1, intracellular trehalose and ROS, and the Hog1-osmotic and Hsf1-Hsp90 signaling pathways function coordinately to regulate sexual mating in response to low atmospheric RH conditions in C. albicans.

The influence of selection in wild pheasant (Phasianus colchicus) breeding on reproduction and the involvement of the chemerin system

Chemerin is a hormone produced mainly by adipose tissue and liver. We have recently shown that it is locally produced in the reproductive tract in hens, particularly at the magnum level, leading to its accumulation in the egg albumen. We have also determined that chemerin is necessary for egg fertilization, embryo development, and angiogenesis within the chorio-allantoic membrane in chicken species. We, therefore, hypothesize that chemerin, widely present in various gallinacean species, could be a marker of egg fertility in this animal order. To demonstrate this, we used a model close to the hen: the pheasant. By RT-qPCR, we have shown that chemerin and its three receptors CMKLR1, GPR1, and CCRL2 are expressed in the reproductive tract of females. In addition, chemerin is also produced predominantly in the magnum and accumulates in the egg albumen as determined by immunoblot. We then compared two lines of pheasants with different reproductive characteristics: the F11 and F22 breeds. F22 lays more eggs than F11, but have significantly lower fertility and hatchability rates. In addition, F22 exhibit a significantly lower amount of chemerin protein in their magnum (P < 0.01) and in the egg albumen (P < 0.0001) compared to F11. Finally, we observed a positive correlation between the chemerin amount in the albumen of F11 eggs and the hatching rate of the eggs (r=0.5; P=0.04) as well as a negative correlation between the chemerin quantity in the albumen of F22 eggs and the rate of unfertilized eggs (r=-0.37; P=0.04). Finally, chemerin system (ligand and receptors) is also expressed within embryo annexes (chorioallantoic and amniotic membranes) during incubation. These data demonstrate an interspecies conservation of chemerin production in the magnum, its accumulation in the egg albumen and its possible use as a marker for determining the quality of eggs in term of fertility and embryo development.

The Atypical Chemerin Receptor GPR1 Displays Different Modes of Interaction with β-Arrestins in Humans and Mice with Important Consequences on Subcellular Localization and Trafficking.

Atypical chemokine receptors (ACKRs) have emerged as a subfamily of chemokine receptors regulating the local bioavailability of their ligands through scavenging, concentration, or transport. The biological roles of ACKRs in human physiology and diseases are often studied by using transgenic mouse models. However, it is unknown whether mouse and human ACKRs share the same properties. In this study, we compared the properties of the human and mouse atypical chemerin receptor GPR1 and showed that they behave differently regarding their interaction with β-arrestins. Human hGPR1 interacts with β-arrestins as a result of chemerin stimulation, whereas its mouse orthologue mGPR1 displays a strong constitutive interaction with β-arrestins in basal conditions. The constitutive interaction of mGPR1 with β-arrestins is accompanied by a redistribution of the receptor from the plasma membrane to early and recycling endosomes. In addition, β-arrestins appear mandatory for the chemerin-induced internalization of mGPR1, whereas they are dispensable for the trafficking of hGPR1. However, mGPR1 scavenges chemerin and activates MAP kinases ERK1/2 similarly to hGPR1. Finally, we showed that the constitutive interaction of mGPR1 with β-arrestins required different structural constituents, including the receptor C-terminus and arginine 3.50 in the second intracellular loop. Altogether, our results show that sequence variations within cytosolic regions of GPR1 orthologues influence their ability to interact with β-arrestins, with important consequences on GPR1 subcellular distribution and trafficking.

Nutrient sensing and cAMP signaling in yeast: G-protein coupled receptor versus transceptor activation of PKA.

A major signal transduction pathway regulating cell growth and many associated physiological properties as a function of nutrient availability in the yeast Saccharomyces cerevisiae is the protein kinase A (PKA) pathway. Glucose activation of PKA is mediated by G-protein coupled receptor (GPCR) Gpr1, and secondary messenger cAMP. Other nutrients, including nitrogen, phosphate and sulfate, activate PKA in accordingly-starved cells through nutrient transceptors, but apparently without cAMP signaling. We have now used an optimized EPAC-based fluorescence resonance energy transfer (FRET) sensor to precisely monitor in vivo cAMP levels after nutrient addition. We show that GPCR-mediated glucose activation of PKA is correlated with a rapid transient increase in the cAMP level in vivo, whereas nutrient transceptor-mediated activation by nitrogen, phosphate or sulfate, is not associated with any significant increase in cAMP in vivo. We also demonstrate direct physical interaction between the Gap1 amino acid transceptor and the catalytic subunits of PKA, Tpk1, 2 and 3. In addition, we reveal a conserved consensus motif in the nutrient transceptors that is also present in Bcy1, the regulatory subunit of PKA. This suggests that nutrient transceptor activation of PKA may be mediated by direct release of bound PKA catalytic subunits, triggered by the conformational changes occurring during transport of the substrate by the transceptor. Our results support a model in which nutrient transceptors are evolutionary ancestors of GPCRs, employing a more primitive direct signaling mechanism compared to the indirect cAMP second-messenger signaling mechanism used by GPCRs for activation of PKA.

A Screened GPR1 Peptide Exerts Antitumor Effects on Triple-Negative Breast Cancer

The adipokine chemerin has been considered an important regulator of tumor immune surveillance. Chemerin recruits leukocytes through the receptor CMKLR1 to improve clinical outcomes of tumors and overall patient survival, but the role of GPR1 in tumors has not been widely investigated. Here, we found that GPR1 expression is elevated in breast cancer—especially triple-negative breast cancer (TNBC) tissues and cell lines. Herein, we screened a phage display peptide library to identify LRH7-G5, a peptide antagonist that blocks chemerin/GPR1 signaling. This peptide performed as an anticancer agent to suppress the proliferation of the TNBC cell lines MDA-MB-231 and HCC1937 but has little effect on T47D cells. LRH7-G5 treatment significantly blocked tumor growth in a TNBC cell-bearing orthotopic mouse model. Last, our results showed that this peptide’s antitumor role is mediated through the PI3K/AKT signaling pathway. In conclusion, these data collectively suggest that the chemerin receptor GPR1 is a novel target for controlling TNBC progression and establish peptide LRH7-G5 as a new therapeutic agent for suppressing TNBC tumor growth.

Expression of chemerin receptors CMKLR1, GPR1 and CCRL2 in the porcine pituitary during the oestrous cycle and early pregnancy and the effect of chemerin on MAPK/Erk1/2, Akt and AMPK signalling pathways

Studies on adipokines, substances that are produced in adipose tissue, indicate that they influence both metabolism and reproduction. Chemerin is a novel addition to the adipokine family. It is believed that chemerin receptors are expressed in different structures of the hypothalamic-pituitary-gonadal (HPG) axis, which are crucial for endocrine control of reproductive functions, including the pituitary. The aim of this study was to investigate the expression of chemerin receptors (CMKLR1, GPR1, CCRL2) genes and proteins in the porcine pituitary. The effect of chemerin on MAPK/Erk1/2, Akt and AMPK signalling pathways was also investigated. The anterior (AP) and posterior (PP) lobes of the pituitary were examined on days 2 to 3, 10 to 12, 14 to 16, and 17 to 19 of the oestrous cycle and on days 10 to 11, 12 to 13, 15 to 16, and 27 to 28 of pregnancy. This is the first study to demonstrate that CMKLR1, GPR1 and CCRL2 are expressed in the porcine AP and PP, which implies that this gland is sensitive to chemerin action. The expression of the studied chemerin receptors fluctuated during different phases of the cycle and early gestation, which could be related to changes in the endocrine status of female pigs. The study also revealed that CMKLR1 and CCRL2 proteins were present in gonadotrophs and thyrotrophs, whereas CCRL2 was also present in somatotrophs, during the cycle and early pregnancy. We observed that chemerin affected MAPK/Erk1/2, Akt and AMPK signalling pathways in the porcine AP. These results suggest that chemerin may participate in the regulation of reproductive functions at the level of the pituitary.

GPR1 Promotes Breast Cancer Tumorigenesis and Metastasis via PI3K/Akt Pathway

Background: Obesity has been shown to be highly correlated with breast cancer risk and therapy resistance. However, the underlying mechanism has yet been undetermined. The adipokine chemerin was shown to be associated with the prognosis of breast cancer and downregulated in patients. A survey of public whole genome expression datasets found that the receptor GPR1 is upregulated in breast cancers as well as in other human tumors. Moreover, high chemerin expression in tumors was correlated with adverse outcomes in breast cancer. Methods: GPR1 expression was ablated in human breast cancer cell line (MDA-MB-231) by shRNA. Tumor cell line proliferation, migration, colony formation, cell cycle and apoptosis was monitored in MDA-MB-231 cell line, and then the xenograft beared on nude mice was measured. Findings: Here, we report that disruption of GPR1, through shRNA in breast cancer cell MDA-MB-231, significantly inhibited tumor cell growth and invasion in vitro and tumor xenograft in vivo. This inhibition was involved in the suppression of phosphorylation of the PI3K/Akt signaling pathway. Interpretation: These results show that GPR1, as the receptor of adipokine chemerin, can promote the tumor growth and invasion through AKT/PI3K signaling, suggesting that obesity-derived chemerin may have a role in tumor genesis and invasion. Funding Statement: This work was partially supported by National Key Research and Development Program of China (2018YFC1003703), the National Major Basic Research Program of China (2013CB945503 ), NSFC grant (81830041, 81830041, 31671562), China Postdoctoral Science Foundation Funded Project (2018M640843 ), Guangdong grant (2018A030310058), and Shenzhen grant (JCYJ20170815090309586, JCYJ20170412140326739, JCYJ20170817143406822). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: All procedures related to animal were carried out according to the guidelines of the Committee on the Use of Live Animals for Teaching and Research, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences.

The Gpr1-regulated Sur7 family protein Sfp2 is required for hyphal growth and cell wall stability in the mycoparasite Trichoderma atroviride

Mycoparasites, e.g. fungi feeding on other fungi, are prominent within the genus Trichoderma and represent a promising alternative to chemical fungicides for plant disease control. We previously showed that the seven-transmembrane receptor Gpr1 regulates mycelial growth and asexual development and governs mycoparasitism-related processes in Trichoderma atroviride. We now describe the identification of genes being targeted by Gpr1 under mycoparasitic conditions. The identified gene set includes a candidate, sfp2, encoding a protein of the fungal-specific Sur7 superfamily, whose upregulation in T. atroviride upon interaction with a fungal prey is dependent on Gpr1. Sur7 family proteins are typical residents of membrane microdomains such as the membrane compartment of Can1 (MCC)/eisosome in yeast. We found that GFP-labeled Gpr1 and Sfp2 proteins show partly overlapping localization patterns in T. atroviride hyphae, which may point to shared functions and potential interaction during signal perception and endocytosis. Deletion of sfp2 caused heavily altered colony morphology, defects in polarized growth, cell wall integrity and endocytosis, and significantly reduced mycoparasitic activity, whereas sfp2 overexpression enhanced full overgrowth and killing of the prey. Transcriptional activation of a chitinase specific for hyphal growth and network formation and strong downregulation of chitin synthase-encoding genes were observed in Δsfp2. Taken together, these findings imply crucial functions of Sfp2 in hyphal morphogenesis of T. atroviride and its interaction with prey fungi.

Inhibition of chemerin/CMKLR1 axis in neuroblastoma cells reduces clonogenicity and cell viability in vitro and impairs tumor growth in vivo.

Pro-inflammatory cells, cytokines, and chemokines are essential in promoting a tumor supporting microenvironment. Chemerin is a chemotactic protein and a natural ligand for the receptors CMKLR1, GPR1, and CCRL2. The chemerin/CMKLR1 axis is involved in immunity and inflammation, and it has also been implicated in obesity and cancer.In neuroblastoma, a childhood tumor of the peripheral nervous system we identified correlations between high CMKLR1 and GPR1 expression and reduced overall survival probability. CMKLR1, GPR1, and chemerin RNA and protein were detected in neuroblastoma cell lines and neuroblastoma primary tumor tissue. Chemerin induced calcium mobilization, increased MMP-2 synthesis as well as MAP-kinase- and Akt-mediated signaling in neuroblastoma cells. Stimulation of neuroblastoma cells with serum, TNFα or IL-1β increased chemerin secretion. The small molecule CMKLR1 antagonist α-NETA reduced the clonogenicity and viability of neuroblastoma cell lines indicating the chemerin/CMKLR1 axis as a promoting factor in neuroblastoma tumorigenesis. Furthermore, nude mice carrying neuroblastoma SK-N-AS cells as xenografts showed impaired tumor growth when treated daily with α-NETA from day 1 after tumor cell injection.This study demonstrates the potential of the chemerin/CMKLR1 axis as a prognostic factor and possible therapeutic target in neuroblastoma.

Lactate signalling regulates fungal β-glucan masking and immune evasion.

As they proliferate, fungi expose antigens at their cell surface that are potent stimulators of the innate immune response, and yet the commensal fungus Candida albicans is able to colonize immuno competent individuals. We show that C. albicans may evade immune detection by presenting a moving immunological target. We report that the exposure of β-glucan, a key pathogen-associated molecular pattern (PAMP) located at the cell surface of C. albicans and other pathogenic Candida species, is modulated in response to changes in the carbon source. Exposure to lactate induces β-glucan masking in C. albicans via a signalling pathway that has recruited an evolutionarily conserved receptor (Gpr1) and transcriptional factor (Crz1) from other well-characterized pathways. In response to lactate, these regulators control the expression of cell-wall-related genes that contribute to β-glucan masking. This represents the first description of active PAMP masking by a Candida species, a process that reduces the visibility of the fungus to the immune system.

Abstract P624: Characterisation of the Chemerin Axis in the Human Vasculature

Objective: The peptide chemerin is an emerging novel vasoconstrictor which has been implicated in altering vascular function in animal models. CMKLR1 is a known chemerin receptor which functions predominately in immune response. Chemerin has also been proposed as the ligand for orphan receptor GPR1, which our group has recently confirmed. We tested the hypothesis that chemerin was vasoactive in human vessels and identified which receptor mediated this action. Method and Results: Immunohistochemistry revealed that chemerin was expressed in endothelial cells and receptors CMKLR1 and GPR1 were expressed in the smooth muscle cells of human vessels. Quantitative PCR analysis of human vessels supported this with CMKLR1 10 fold more abundant than GPR1. C-terminal fragment chemerin149-157 (C9) caused a concentration dependent contraction in isolated endothelium denuded human saphenous vein (hSV) with a pD2=7.4±0.3 (n=4), which was shifted to the right by antagonist CCX832 (100mM), pD2=6.1±0.2 (n=3). Full characterisation of the novel antagonist CCX832 with receptor-transfected cells confirmed it to be selective for the CMKLR1 receptor, and had no effect on GPR1. In β-arrestin recruitment assays, CCX832 was functionally active at the CMKLR1 receptor causing a rightward shift of the C9 response, logKD=-8.2±0.1 (n=3); however no effect was seen at the GPR1 receptor. Radiolabelled competition binding experiments showed that CCX832 competes for binding with [125I]-C9 at the CMKLR1 receptor, pIC50=9.0±0.1 (n=5), however it does not at the GPR1 receptor. In competition binding experiments on human saphenous vein homogenate, CCX832 competes for binding with [125I]-C9, pIC50=8.6±0.4 (n=6). This is in agreement with the in vitro pharmacology data, that chemerin’s vasoactivity is mediated by CMKLR1. Data is expressed as mean±SEM. Conclusion: These data support the emerging role of chemerin as an endogenous vasoconstrictor through its receptor CMKLR1. It identifies for the first time the components of the chemerin axis in the human vasculature, and confirms that chemerin causes vasoconstriction of human vessels. This study identifies a novel therapeutic drug target for hypertension in humans.

Abstract 3279: Expression of chemerin and chemerin receptors in neuroblastoma: implications in tumorigenesis

Abstract Chemerin is an adipokine and immunomodulating factor that promotes chemotaxis of immature dendritic cells, natural killer cells, macrophages and endothelial cells. Secreted as prochemerin with low activity, it can be C-terminally processed by different proteases expressed by a broad range of cell types and tissues. The resulting isoforms vary in receptor affinity and biological activity and are natural ligands for the G protein-coupled receptors (GPCRs) CMKLR1, GPR1 and CCLR2. To date, the activation of CMKLR1 (chemokine-like receptor 1) by chemerin and its role in metabolism and metabolic disorders as well as inflammation is best understood. Neuroblastoma (NB) is a malignancy of the sympathetic nervous system and the most common extracranial solid pediatric tumor. Several chemoattractant GPCRs have been suggested to promote tumor progression, angiogenesis and metastasis in NB. Although for some cancers a potential function has been suggested, the role of chemerin and its receptors in the NB tumor microenvironment remains unknown. In our study, the screening of microarray databases and analysis of neuroblastoma expression data showed a correlation between high CMKLR1, GPR1 and CCLR2 expression and a reduction in the overall survival probability. Expression of CMKLR1, GPR1, and chemerin was shown in nine neuroblastoma cell lines using RT-PCR, Western blot and immunocytochemistry. Furthermore, chemerin and CMKLR1 were also detected in neuroblastoma tumor tissue by immunohistochemistry. Stimulation of NB cell lines with active chemerin induced calcium mobilization and increased phosphorylation of MEK1/2 and ERK1/2 indicating an activation of the MAPK signaling pathway. Furthermore, chemerin stimulation led to increased NF-κB phosphorylation and translocation to the nucleus. The induction of NF-κB mediated signaling was observed by luciferase reporter assay. TNFα, IL-1β or serum stimulation increased chemerin protein expression and secretion in neuroblastoma cells. To assess the functional significance of chemerin and its receptors in neuroblastoma tumorigenesis, cell clones overexpressing or silenced for CMKLR1/ Chemerin/ GPR1 are used in NB animal models. Citation Format: Conny Tuemmler, Igor Snapkov, Ugo L. Moens, Per Kogner, John Inge Johnsen, Baldur Sveinbjørnsson. Expression of chemerin and chemerin receptors in neuroblastoma: implications in tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3279. doi:10.1158/1538-7445.AM2015-3279

Abstract 3990: Chemerin and chemerin receptors in neuroblastoma tumor microenvironment

Abstract Tumor promoting inflammatory cells as well as inflammatory mediators such as chemokines are important contributors to the tumor microenvironment as they can support tumor progression, angiogenesis and metastasis. Chemerin (also known as TIG-2 or RARRES2) is a chemoattractant factor for macrophages, immature DCs and NK-cells and a known adipokine involved in inflammation, metabolism and adipogenesis. Synthesized as a 163aa preproprotein, chemerin is N-terminally cleaved and secreted as inactive prochemerin. Following secretion, chemerin can be cleaved at the C-terminus by a variety of extracellular proteases resulting in several isoforms with varying length and biological activity. During inflammation initiation, maintenance and resolution the different chemerin isoforms may function pro- and/ or anti- inflammatory. Chemerin is a natural ligand for the G protein- coupled receptors CMKLR1 (Chem23) and GPR1. The role of chemerin and CMKLR1 in the tumor microenvironment has not been extensively studied. The aim of this work is to study the function of chemerin and its receptors in neuroblastoma (NB). Screening of mRNA expression arrays showed a correlation between high expression of CMKLR1 and GPR1 and a worsened prognosis in NB. Chemerin, CMKLR1 and GPR1 expression was detected in different neuroblastoma cell lines by RT-PCR, immunocytochemistry and Western Blot. Stimulation with chemerin resulted in rapid and transient ERK1/2 and Akt phosphorylation. TNF-α and IL-1β treatment increased chemerin and CMKLR1 protein levels. The functional significance of chemerin/ CMKLR1 in NB will be assessed by the use of NB animal models using cell clones silenced for chemerin/ CMKLR1. Citation Format: Conny Tuemmler, Igor Snapkov, Ugo L. Moens, Baldur Sveinbjørnsson. Chemerin and chemerin receptors in neuroblastoma tumor microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3990. doi:10.1158/1538-7445.AM2014-3990

The Yeast Sks1p Kinase Signaling Network Regulates Pseudohyphal Growth and Glucose Response

The yeast Saccharomyces cerevisiae undergoes a dramatic growth transition from its unicellular form to a filamentous state, marked by the formation of pseudohyphal filaments of elongated and connected cells. Yeast pseudohyphal growth is regulated by signaling pathways responsive to reductions in the availability of nitrogen and glucose, but the molecular link between pseudohyphal filamentation and glucose signaling is not fully understood. Here, we identify the glucose-responsive Sks1p kinase as a signaling protein required for pseudohyphal growth induced by nitrogen limitation and coupled nitrogen/glucose limitation. To identify the Sks1p signaling network, we applied mass spectrometry-based quantitative phosphoproteomics, profiling over 900 phosphosites for phosphorylation changes dependent upon Sks1p kinase activity. From this analysis, we report a set of novel phosphorylation sites and highlight Sks1p-dependent phosphorylation in Bud6p, Itr1p, Lrg1p, Npr3p, and Pda1p. In particular, we analyzed the Y309 and S313 phosphosites in the pyruvate dehydrogenase subunit Pda1p; these residues are required for pseudohyphal growth, and Y309A mutants exhibit phenotypes indicative of impaired aerobic respiration and decreased mitochondrial number. Epistasis studies place SKS1 downstream of the G-protein coupled receptor GPR1 and the G-protein RAS2 but upstream of or at the level of cAMP-dependent PKA. The pseudohyphal growth and glucose signaling transcription factors Flo8p, Mss11p, and Rgt1p are required to achieve wild-type SKS1 transcript levels. SKS1 is conserved, and deletion of the SKS1 ortholog SHA3 in the pathogenic fungus Candida albicans results in abnormal colony morphology. Collectively, these results identify Sks1p as an important regulator of filamentation and glucose signaling, with additional relevance towards understanding stress-responsive signaling in C. albicans.

The effects of Gpr1 and Gpa2 on the mating pathway in Saccharomyces cerevisiae

Nutrient sensing and mating are clearly linked in a number of different fungal species. The mechanisms by which these two processes interact appear to differ in different species. We have observed that glucose sensing and mating are closely linked in Saccharomyces cerevisiae, with disruptions in the glucose sensing pathway causing reduction in mating pathway activity. In this study we sought to determine the mechanisms by which this reduction occurs. In a series of gene replacement experiments we have shown that overexpressing the mating receptor in glucose receptor (Gpr1) deficient cells restores the mating pathway. Likewise, overexpression of the g protein associated with glucose sensing, Gpa2, is able to restore the mating pathway in Gpr1蜐 cells. These effects seem to be independent of the activity of the g‐protein. Dominant negative effects were seen with some modified forms of Gpr1. We propose a mechanism of interaction that involves the formation of a complex involving potentially Ste2, Gpr1, Gpa2, and Gpa1. This research was funded by the NSF, Project #0952519

The seven-transmembrane receptor Gpr1 governs processes relevant for the antagonistic interaction of Trichoderma atroviride with its host

Mycoparasitic Trichoderma species are applied as biocontrol agents in agriculture to guard plants against fungal diseases. During mycoparasitism, Trichoderma directly interacts with phytopathogenic fungi, preceded by a specific recognition of the host and resulting in its disarming and killing. In various fungal pathogens, including mycoparasites, signalling via heterotrimeric G proteins plays a major role in regulating pathogenicity-related functions. However, the corresponding receptors involved in the recognition of host-derived signals are largely unknown. Functional characterization of Trichoderma atroviride Gpr1 revealed a prominent role of this seven-transmembrane protein of the cAMP-receptor-like family of fungal G-protein-coupled receptors in the antagonistic interaction with the host fungus and governing of mycoparasitism-related processes. Silencing of gpr1 led to an avirulent phenotype accompanied by an inability to attach to host hyphae. Furthermore, gpr1-silenced transformants were unable to respond to the presence of living host fungi with the expression of chitinase- and protease-encoding genes. Addition of exogenous cAMP was able to restore host attachment in gpr1-silenced transformants but could not restore mycoparasitic overgrowth. A search for downstream targets of the signalling pathway(s) involving Gpr1 resulted in the isolation of genes encoding e.g. a member of the cyclin-like superfamily and a small secreted cysteine-rich protein. Although silencing of gpr1 caused defects similar to those of mutants lacking the Tga3 Gα protein, no direct interaction between Gpr1 and Tga3 was observed in a split-ubiquitin two-hybrid assay.

Cloning of porcine chemerin, ChemR23 and GPR1 and their involvement in regulation of lipogenesis

Chemerin is a novel adipokine which is abundant in adipose tissue to promote adipocyte differentiation and with significant relativity to BMI and insulin sensitivity. We report here the molecular characterization of porcine chemerin and its receptors ChemR23 and GPR1, as well as their transcriptional regulation during lipogenesis. Chemerin was mainly expressed in liver, intestine, kidney and adipose tissue, consistent with the expression pattern of GPR1, but not ChemR23, which was predominantly present in spleen and temperately in adipose tissue. We further investigated the lipogenesis-related transcriptional activation of <TEX>$PPAR{\gamma}$</TEX> and KLF15 on chemerin and its receptors. The data showed that KLF15, but not <TEX>$PPAR{\gamma}$</TEX>, can up-regulate the mRNA level of chemerin, ChemR23 and GPR1, which was consistent with the results of luciferase assay that confirmed the effect of KLF15 on ChemR23 promoter. Taken together, our data provide basic molecular information for the further investigation on the function of chemerin in lipogenesis.