Chemerin Receptor Research Articles

The adipokine chemerin amplifies electrical field-stimulated contraction in the isolated rat superior mesenteric artery.

The adipokine chemerin causes arterial contraction and is implicated in blood pressure regulation, especially in obese subjects with elevated levels of circulating chemerin. Because chemerin is expressed in the perivascular adipose tissue (PVAT) that surrounds the sympathetic innervation of the blood vessel, we tested the hypothesis that chemerin (endogenous and exogenous) amplifies the sympathetic nervous system in mediating electrical field-stimulated (EFS) contraction. The superior mesenteric artery, with or without PVAT and with endothelium and sympathetic nerve intact, was mounted into isolated tissue baths and used for isometric contraction and stimulation. Immunohistochemistry validated a robust expression of chemerin in the PVAT surrounding the superior mesenteric artery. EFS (0.3-20 Hz) caused a frequency-dependent contraction in isolated arteries that was reduced by the chemerin receptor ChemR23 antagonist CCX832 alone (100 nM; with, but not without, PVAT), but not by the inactive congener CCX826 (100 nM). Exogenous chemerin-9 (1 μM)-amplified EFS-induced contraction in arteries (with and without PVAT) was blocked by CCX832 and the α-adrenergic receptor antagonist prazosin. CCX832 did not directly inhibit, nor did chemerin directly amplify, norepinephrine-induced contraction. Whole mount immunohistochemical experiments support colocalization of ChemR23 with the sympathetic nerve marker tyrosine hydroxylase in superior mesenteric PVAT and, to a lesser extent, in arteries and veins. These studies support the idea that exogenous chemerin modifies sympathetic nerve-mediated contraction through ChemR23 and that ChemR23 may be endogenously activated. This is significant because of the well-appreciated role of the sympathetic nervous system in blood pressure control.

Distinctive downmodulation of plasmacytoid dendritic cell functions by vitamin D3 analogue calcipotriol

BackgroundIn relation to Th17 cell actions, interferon (IFN)-α production by plasmacytoid dendritic cells (pDCs) are involved in the pathogenesis of psoriasis. Vitamin D3 analogues are widely used in the treatment of psoriasis, however, their actions on pDCs are not well understood. ObjectiveTo investigate the effects of Vitamin D3 analogue calcipotriol (CAL) on pDCs, focusing on the cytokine production and chemotactic activity. MethodsWe compared in mice the effects of CAL, cyclosporine A (CyA), and triamcinolone acetonide (TA) on the cytokine production by pDCs (IFN-α), conventional DCs (TNF-α), and γd T cells (IL-17A). pDCs isolated from mouse spleen cells were stimulated with CpG-ODN in the presence or absence of each drug for 48h. Purified splenic conventional DCs (cDCs) and lymph node γδ T cells were stimulated with CpG-ODN or with anti-CD3/CD28 antibody, respectively. IFN-α, TNF-α and IL-17A in the 48-h culture supernatants were quantified by ELISA. We also studied the ability of CAL to inhibit the chemotaxis of freshly isolated pDCs toward chemerin and VEGF-A, representative chemoattractants of pDCs, by a real-time monitoring method, EZ-Taxiscan. To assess the effect of CAL on pDC accumulation in vivo, we painted CAL ointment to the mouse skin inflamed by topical application of imiquimod cream (IMQ) for 4 consecutive days. In the skin samples, we enumerated 440c+ pDCs by immunohistochemistry and evaluated the mRNA expression of cytokines by real-time PCR. ResultsCAL significantly inhibited CpG-enhanced pDC IFN-α production at a comparable level to T cell IL-17A production, whereas its effect on cDC TNF-α production was minimal. Accordingly, CAL suppressed the CpG-augmented expression of TLR9 and MyD88. On the contrary, CyA strongly suppressed the production of TNF-α and IL-17A, but not IFN-α. TA inhibited the production of all the cytokines tested. The effect of CAL on the chemotactic activity of pDCs was also evaluated, demonstrating a significant downmodulation by exposure to the reagent. CAL depressed chemerin receptor CMKLR1 expression in pDCs. The in vivo mouse study showed that simultaneous application of CAL to the imiquimod-applied skin reduce both the recruitment of pDCs and the expression of IFN-α2 in the skin. ConclusionsOur findings suggest that CAL uniquely downmodulates the cytokine production and chemotactic activity of pDCs. The CAL suppression of the in vivo pDC accumulation to the skin suggests that these actions are therapeutically relevant.

Abstract 249: Increased Vascular CCRL2 Expression by Disturbed Blood Flow Promotes Atherosclerotic Plaque Formation in ApoE Deficient Mice

Atherosclerosis is a chronic inflammatory disease of the arterial wall elicited by accumulation of LDL and leucocytes in the subendothelium at predilection sites with disturbed laminar flow. Chemokines and their receptors appear to act as critical players in atherosclerosis as they not only direct atherogenic recruitment of leucocytes but also exert cell hemostatic functions by chemokine ligand-receptor axes and their specific or combined contributions. Atypical chemokine (C-C motif) receptor-like 2 (CCRL2) cooperates with its ligand chemerin and leukocyte-expressed chemerin receptor chemokine-like receptor 1 (CMKLR1) to regulate cell trafficking and inflammatory responses,but its role in atherosclerosis is not clear. To investigate whether CCRL2 contributes to the pathomechanism of atherogenesis, we generated CCRL2 -/- mice in hyperlipidemic atherosclerosis-prone ApoE -/- background and found that the atherosclerotic plaque area of the total aorta was significantly reduced compared with CCRL2 +/+ ApoE -/- mice on a high fat diet. The protective effect of CCRL2 deficiency was anatomically isolated primarily to the site of disturbed blood flow (D-flow) in the aortic arch but not in the descending aorta. Endothelial CCRL2 was upregulated in response to D-flow and either CCRL2 or CMKLR1 deletion reduced plaque formation. Further studies showed that CCRL2 co-localized with CMKLR1 and chemerin within the atherosclerotic aorta root. CCRL2 deficiency led to significantly less lipid deposition in aortic root, reduced CMKLR1 + leukocyte rolling on lesional vascular endothelium, diminished macrophage accumulation and foam cell formation, and polarized macrophage to an M2-like phenotype. These results demonstrate that D-flow induction of vascular CCRL2 is required for optimal formation of atherosclerotic plaques via coordinating the accumulation of CMKLR1 + monocytes/macrophages within the vascular wall, and thus identifies CCRL2 as a novel drug target to prevent or treat atherosclerosis. This work was supported by Natural Science Foundation of China (grant 81370373 to L.Z. and 31300781 to C.T.) Key Words: atherosclerosis, CCRL2, chemerin, macrophage

075 Ephrin-A loss in cutaneous squamous cell carcinoma progression and metastasis

075 Ephrin-A loss in cutaneous squamous cell carcinoma progression and metastasis

078 Dsg2 enhances spontaneous BCC formation in Ptc1+/−mice

078 Dsg2 enhances spontaneous BCC formation in Ptc1+/−mice

The role of chemerin and ChemR23 in stimulating the invasion of squamous oesophageal cancer cells.

Background:Stromal cells, including cancer-associated myofibroblasts (CAMs), are recognised to be determinants of cancer progression, but the mechanisms remain uncertain. The chemokine-like protein, chemerin, is upregulated in oesophageal squamous cancer (OSC) CAMs compared with adjacent tissue myofibroblasts (ATMs). In this study, we hypothesised that chemerin stimulates OSC cell invasion.Methods:Expression of the chemerin receptor, ChemR23, in OSC was examined by immunohistochemistry. The invasion of OSC cells was studied using Boyden chambers and organotypic assays, and the role of chemerin was explored using siRNA, immunoneutralisation and a ChemR23 receptor antagonist. Matrix metalloproteinases (MMPs) were detected by western blot, enzyme assays or immunohistochemistry.Results:Immunohistochemistry indicated expression of the putative chemerin receptor ChemR23 in OSC. It was also expressed in the OSC cell line, OE21. Chemerin stimulated OE21 cell migration and invasion in Boyden chambers. Conditioned medium (CM) from OSC CAMs also stimulated OE21 cell invasion and this was inhibited by chemerin immunoneutralisation, the ChemR23 antagonist CCX832, and by pretreatment of CAMs with chemerin siRNA. In organotypic cultures of OE21 cells on Matrigel seeded with either CAMs or ATMs, there was increased OE21 cell invasion by CAMs that was again inhibited by CCX832. Chemerin increased MMP-1, MMP-2 and MMP-3 abundance, and activity in OE21 cell media, and this was decreased by inhibiting protein kinase C and p44/42 MAPK kinase but not PI-3 kinase.Conclusions:The data indicate that OSC myofibroblasts release chemerin that stimulates OSC cell invasion. Treatments directed at inhibiting chemerin–ChemR23 interactions might be therapeutically useful in delaying progression in OSC.

Mesenchymal Stem Cells Exhibit Regulated Exocytosis in Response to Chemerin and IGF.

Mesenchymal stem cells (MSCs) play important roles in tissue repair and cancer progression. Our recent work suggests that some mesenchymal cells, notably myofibroblasts exhibit regulated exocytosis resembling that seen in neuroendocrine cells. We now report that MSCs also exhibit regulated exocytosis. Both a G-protein coupled receptor agonist, chemerin, and a receptor tyrosine kinase stimulant, IGF-II, evoked rapid increases in secretion of a marker protein, TGFβig-h3. The calcium ionophore, ionomycin, also rapidly increased secretion of TGFβig-h3 while inhibitors of translation (cycloheximide) or secretory protein transport (brefeldin A) had no effect, indicating secretion from preformed secretory vesicles. Inhibitors of the chemerin and IGF receptors specifically reduced the secretory response. Confocal microscopy of MSCs loaded with Fluo-4 revealed chemerin and IGF-II triggered intracellular Ca2+ oscillations requiring extracellular calcium. Immunocytochemistry showed co-localisation of TGFβig-h3 and MMP-2 to secretory vesicles, and transmission electron-microscopy showed dense-core secretory vesicles in proximity to the Golgi apparatus. Proteomic studies on the MSC secretome identified 64 proteins including TGFβig-h3 and MMP-2 that exhibited increased secretion in response to IGF-II treatment for 30min and western blot of selected proteins confirmed these data. Gene ontology analysis of proteins exhibiting regulated secretion indicated functions primarily associated with cell adhesion and in bioassays chemerin increased adhesion of MSCs and adhesion, proliferation and migration of myofibroblasts. Thus, MSCs exhibit regulated exocytosis that is compatible with an early role in tissue remodelling.

CMKLR1 and GPR1 mediate chemerin signaling through the RhoA/ROCK pathway

Chemerin is an adipose-derived hormone that regulates immunity and energy homesotasis. To date, all known chemerin functions have been attributed to activation of the G protein-coupled receptor chemokine-like receptor-1 (CMKLR1). Chemerin is also the only known ligand for a second receptor, G protein-coupled receptor-1 (GPR1), whose signaling and function remains unknown. This study investigated the in vitro signal transduction mechanisms of CMKLR1 and GPR1 using a panel of luciferase-reporters and pathway-specific inhibitors. Herein we report the novel finding that chemerin signals through a RhoA and rho-associated protein kinase (ROCK)-dependent pathway for activation of the transcriptional regulator serum-response factor (SRF). Despite similarities in RhoA/ROCK, Gαi/o, and MAPK signaling, we also demonstrate species-specific and receptor-dependent variations in GPR1 and CMKLR1 signaling and expression of the SRF target genes EGR1, FOS and VCL. Moreover, we demonstrate that signaling through p38, Gαi/o, RhoA, and ROCK is required for chemerin-mediated chemotaxis of L1.2 lymphocytes and AGS gastric adenocarcinoma cells. These results provide, to our knowledge, the first empirical evidence that GPR1 is a functional chemerin receptor and identify RhoA/SRF as a novel chemerin-signaling axis via both CMKLR1 and GPR1.

Chemerin is a novel regulator of lactogenesis in bovine mammary epithelial cells

Chemerin is a chemoattractant cytokine (chemokine) produced by adipocytes and hepatocytes; it regulates insulin sensitivity and adipocyte differentiation. The objective of this study was to investigate the effect of chemerin on the expression of genes related to lactogenesis and the regulators of chemerin signaling in a bovine mammary epithelial cell line (MAC-T). Two types of chemerin receptors, chemokine like-receptor 1 (CMKLR1) and chemokine (C–C motif) receptor-like 2 (CCRL2), were detected in cultured MAC-T cells, whereas chemerin was not detected. G protein-coupled receptor 1 (GPR1), another receptor of chemerin, was undetectable in MAC-T cells. Chemerin upregulated transcript expression of CMKLR1, CCRL2, and genes associated with fatty acid synthesis, glucose uptake, insulin signaling, and casein synthesis in MAC-T cells. Lactogenic hormones (insulin, growth hormone, and prolactin) downregulated the expression of CMKLR1 in MAC-T cells. Adiponectin suppressed CMKLR1 expression. TNF-α suppressed CMKLR1, but induced CCRL2 expression. These data suggest chemerin is a novel regulator of lactogenesis via its own receptor in bovine mammary epithelial cells.

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 MP07: Endogenous Chemerin Amplifies Nerve-Dependent Arterial Contraction

The adipokine chemerin causes contraction of isolated arteries and is implicated in blood pressure regulation, especially in the obese population that have elevated levels of circulating chemerin. Because chemerin is expressed in the perivascular adipose tissue (PVAT) that facilitates the sympathetic innervation of the blood vessel, we tested the hypothesis that chemerin (endogenous and exogenous) would amplify the effects of the sympathetic nervous system in mediating electrical field stimulated (EFS) contraction. The model was the superior mesenteric artery with PVAT, mounted into tissue baths for isometric contraction. Immunohistochemistry validated a robust expression of chemerin in the PVAT surrounding the superior mesenteric artery. EFS (0.3-20 Hz) caused a frequency-dependent, prazosin-sensitive contraction that was reduced (~40%) by the chemerin receptor ChemR23 antagonist CCX832 (100 nM; figure) but not by the inactive congener CCX826 (100 nM). Exogenous chemerin (1 μM) amplified EFS-induced contraction in a manner that was also blocked by CCX832. Chemerin did not directly modify contraction of the superior mesenteric artery (-PVAT) to cumulative concentrations of norepinephrine (1 nM - 10 μM), supporting that contractile amplification by chemerin was not at the level of smooth muscle. These studies raise the interesting possibilities that endogenous chemerin and/or ChemR23 modifies nerve-mediated contraction. This is significant because of the well appreciated role of the sympathetic nervous system in blood pressure control.

Adventitia and perivascular cells.

The past few years have seen significant advances in our understanding of the multiple and dynamic roles of the adventitia and its companion perivascular tissues for vessel wall homeostasis and disease. It is now becoming clear that signals originating from within the adventitia and from perivascular cells play essential roles in regulation of vascular development, physiology, artery wall remodeling, immune surveillance, and vascular disease.1–4 The adventitia is in contact with and completely surrounds the media and is the interface between the vessel wall and its neighboring tissues. It contains many different interacting cell types including fibroblasts, microvascular endothelium, nerves, resident macrophages, T cells, B cells, mast cells, and dendritic cells.5–9 The adventitia is also home to resident vascular progenitor cells whose formation and maintenance depend, in part, on sonic hedgehog signaling.1,10–12 Perivascular cells are in close contact with the adventitia, particularly for the aorta and coronary arteries. Perivascular tissue includes adipocytes, lymphatic vessels, perivascular nerves, and stromal cells exhibiting mesenchymal stem cell–like properties.3,13 The adventitia and periadventitial cells function in concert. They are linked by microvessels, nerves, and migratory cells to regulate vascular physiology, homeostasis, structural remodeling, and exert major influences on the progression or regression of vascular disease. Crosstalk between intima, media, and adventitia further links the adventitia-periadventitial unit to the rest of the vessel wall. Much of the work advancing our concepts of the adventitia and periadventitial tissues has been published in ATVB , and some of those key studies are discussed in this highlights article. In the following summary, areas that have attracted the most interest over the past 2 years in ATVB are reviewed including the multiple roles of perivascular adipose tissue (PVAT) on control of vascular physiology and remodeling, adventitial progenitor …

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

Chemerin Expression in the Peritoneal Fluid, Serum, and Ovarian Endometrioma of Women with Endometriosis.

Inflammation is an essential process in the pathogenesis of endometriosis. Serum and peritoneal fluid (PF) samples were collected from women with endometriosis (n = 31) and women without endometriosis (n = 48). Chemerin, tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) levels in serum and PF samples were determined with a specific enzyme-linked immunosorbent assay. Eutopic endometrial tissue from controls and ovarian endometriotic cysts were obtained during surgery. Expression of chemerin and chemerin receptors in ectopic and eutopic endometrial tissues was measured on real-time reverse transcriptase-polymerase chain reaction. Protein expression was examined with Western blot and densitometric analysis. Chemerin concentrations were higher in PF from women with endometriosis than that in that from controls. PF chemerin concentrations were significantly correlated with both TNF-α and IL-6 levels in PF. The mRNA and protein of chemerin and its receptor were significantly increased in the ovarian endometrioma tissue compared with eutopic endometrium of controls. These findings suggest that chemerin plays a role in endometriosis-related pelvic inflammation.

ChemR23, the receptor for chemerin and resolvin E1, is expressed and functional on M1 but not on M2 macrophages.

ChemR23 is a G protein-coupled receptor that is triggered by two ligands, the peptide chemerin and the eicosapentaenoic acid-derived lipid mediator resolvin E1 (RvE1). Chemerin acts as a chemoattractant for monocytes and macrophages, whereas RvE1 promotes resolution of inflammation-inducing macrophage phagocytosis of apoptotic neutrophils. Although ChemR23-mediated signaling plays a role in mononuclear cell migration to inflamed tissue, as well as in the resolution of inflammation, its regulation in different polarization states of macrophages is largely unknown. We analyzed the expression and function of ChemR23 in monocytes and differently activated human primary macrophages. Using 5' RACE, we identified three transcription start sites and several splice variants of ChemR23 in both monocytes and macrophages. Although the promoters P1 and P3 are used equally in unpolarized macrophages, stimulation with LPS or IFN-γ leads to increased transcription from P3 in inflammatory M1 macrophages. Such ChemR23-expressing M1 macrophages are chemotactic to chemerin, whereas M2 macrophages not expressing ChemR23 surface receptor are not. Repolarization of ChemR23-expressing M1 macrophages with 10 nM RvE1 increases IL-10 transcription and phagocytosis of microbial particles, leading to a resolution-type macrophage distinct from the M2 phenotype. These results show that ChemR23 is tightly regulated in response to inflammatory and anti-inflammatory stimuli. The restricted expression of ChemR23 in naive and M1 macrophages supports the role of ChemR23 in the attraction of macrophages to inflamed tissue by chemerin and in the initiation of resolution of inflammation through RvE1-mediated repolarization of human M1 macrophages toward resolution-type macrophages.

Evidence from studies in rodents and in isolated adipocytes that agonists of the chemerin receptor CMKLR1 may be beneficial in the treatment of type 2 diabetes.

The literature is unclear on whether the adipokine chemerin has pro- or anti-inflammatory properties or plays any role in the aetiology of type 2 diabetes or obesity. To address these questions, and in particular the potential of agonists or antagonists of the chemerin receptor CMKLR1 in the treatment of type 2 diabetes and obesity, we studied the metabolic phenotypes of both male and female, CMKLR1 knockout and heterozygote mice. We also investigated changes in plasma chemerin levels and chemerin gene mRNA content in adipose tissue in models of obesity and diabetes, and in response to fasting or administration of the insulin sensitizing drug rosiglitazone, which also has anti-inflammatory properties. The effects of murine chemerin and specific C-terminal peptides on glucose uptake in wild-type and CMKLR1 knockout adipocytes were investigated as a possible mechanism by which chemerin affects the blood glucose concentration. Both male and female CMKLR1 knockout and heterozygote mice displayed a mild tendency to obesity and impaired glucose homeostasis, but only when they were fed on a high-fat died, rather than a standard low-fat diet. Obesity and impaired glucose homeostasis did not occur concurrently, suggesting that obesity was not the sole cause of impaired glucose homeostasis. Picomolar concentrations of chemerin and its C15- and C19-terminal peptides stimulated glucose uptake in the presence of insulin by rat and mouse wild-type epididymal adipocytes, but not by murine CMKLR1 knockout adipocytes. The insulin concentration-response curve was shifted to the left in the presence of 40 pM chemerin or its C-15 terminal peptide. The plasma chemerin level was raised in diet-induced obesity and ob/ob but not db/db mice, and was reduced by fasting and, in ob/ob mice, by treatment with rosiglitazone. These findings suggest that an agonist of CMKLR1 is more likely than an antagonist to be of value in the treatment of type 2 diabetes and to have associated anti-obesity and anti-inflammatory activities. One mechanism by which an agonist of CMKLR1 might improve glucose homeostasis is by increasing insulin-stimulated glucose uptake by adipocytes.

Chemerin Induces Insulin Resistance in Rat Cardiomyocytes in Part through the ERK1/2 Signaling Pathway

Aims: Chemerin is a novel adipokine that is closely associated with cardiovascular diseases and glucose homeostasis. This study aimed to investigate the effects of chemerin on insulin resistance in rat cardiomyocytes. Methods: Rat cardiomyocytes were treated with high concentrations of glucose and tumor necrosis factor-alpha (TNF-E), and chemerin and chemokine-like receptor 1 (CMKLR1) were measured by Western blot analysis. Then, the cardiomyocytes were treated with chemerin and insulin. Glucose uptake was evaluated using a fluorescence microplate reader. Western blot analysis was used to evaluate the phosphorylation of Akt, insulin receptor substrate-1, p38 mitogen-activated protein kinase (MAPK), as well as extracellular signal-regulated kinase (ERK)1/2. Results: Chemerin and CMKLR1 were found to be expressed in rat cardiomyocytes. Pretreatment with chemerin caused decreases in glucose uptake and phosphorylation of Akt in insulin-stimulated cardiomyocytes. Furthermore, chemerin activated the phosphorylation of p38 MAPK and ERK1/2 in insulin-stimulated cardiomyocytes. Inhibition of ERK partially rescued chemerin-induced insulin resistance. Conclusion: Chemerin is a novel adipokine that induces insulin resistance in rat cardiomyocytes in part through the ERK1/2 pathway. i 2014 S. Karger AG, Basel

Chemerin expression in Chinese pregnant women with and without gestational diabetes mellitus

ObjectivesThe aim of this study was to determine the effect of obesity, gestational diabetes mellitus (GDM) on circulating chemerin concentrations and chemerin gene expression of adipotissue in pregnancy women. Material and methodsTotally 42 normal glucose tolerant (NGT) women and 48 women with GDM were included in this study. Their clinical features and biochemical parameters were analyzed. The NGT and GDM women were subgrouped by prepregnancy BMI as normal-weight group, overweight group, and obese group, respectively. Serum chemerin and tumor necrosis factor α (TNF-α) of these individuals were determined by ELISA methods, and subcutaneous adipose tissues’ mRNA expressions of chemerin and CMKLR1 (encoding the receptor of chemerin) were analyzed by real-time PCR. ResultsSerum chemerin in obese-NGT group and normal-weight-GDM group was significantly higher than that of normal-weight-NGT group. Chemerin and CMKLR1 mRNA expression of subcutaneous adipose tissue was lower in the obese-NGT group than normal-weight-NGT group. There was no significant difference of CMKLR1 mRNA expression between normal-weight-NGT and normal-weight-GDM group. Serum chemerin significantly and positively correlated with triglycerides (TG) and homeostasis model assessment of insulin resistance (HOMA-IR) assessed both by uni- and multivariate. ConclusionsGestational obesity, GDM may contribute to the elevating serum chemerin. Serum chemerin in pregnancy was associated with insulin resistance and triglycerides. Chemerin gene may play a role both in obese and GDM patients. CMKLR1 might exert its action only in obese individuals, not in GDM patients before delivery.

The chemerin receptor CMKLR1 is a functional receptor for amyloid-β peptide.

Amyloid-β peptides such as Aβ1-42 (Aβ42) play a pivotal role in the progression of Alzheimer's disease (AD). Aβ42 is neurotoxic and can activate microglial cells. These cells in turn migrate toward senile (neuritic) plaques and help to clear Aβ deposits through an endocytotic mechanism. It is of potential significance to characterize the Aβ42 receptors that mediate microglia chemotaxis and Aβ42 uptake. We found that the transcript of the chemerin receptor CMKLR1 was upregulated in the brain of AD patients and in mouse brain tissue following systemic LPS administration. CMKLR1 and Aβ42 colocalized in hippocampus and cortex of AβPP/PS1 transgenic mice. Moreover, Aβ42 bound specifically to CMKLR1 in stably transfected rat basophilic leukemia (RBL) cells (CMKLR1-RBL), suggesting that CMKLR1 is a receptor for Aβ42. Aβ42 induced migration of primary microglia, the mouse microglial cell line N9, and CMKLR1-RBL cells, but not untransfected RBL-2H3 cells. Mechanistic studies showed that Aβ42 induced CMKLR1-dependent cell migration through activation of the ERK1/2, PKA, and Akt pathways, but not Ca2+ mobilization. Aβ42 stimulation of CMKLR1-RBL cells and primary glial cells led to internalization of the Aβ42-CMKLR1 complex, suggesting a potential role for CMKLR1 in Aβ42 clearance. Taken together, these results indicate that Aβ42 activates CMKLR1, leading to glia cell migration and clearance of Aβ42. CMKLR1 is a new addition to the repertoire of cell surface molecules that are responsible for Aβ processing and clearance.

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