Adipocyte-Cancer Cell Interactions in the Bone Microenvironment.

When compared to adipocytes in other anatomical sites, the interaction of bone marrow resident adipocytes with the other cells in their microenvironment is less well understood. Bone marrow adipocytes originate from a resident, self-renewing population of multipotent bone marrow stromal cells which can also give rise to other lineages such as osteoblasts. The differentiation fate of these mesenchymal progenitors can be influenced to favour adipogenesis by several factors, including the administration of thiazolidinediones and increased age. Experimental data suggests that increases in bone marrow adipose tissue volume may make bone both more attractive to metastasis and conducive to cancer cell growth. Bone marrow adipocytes are known to secrete a variety of lipids, cytokines and bioactive signaling molecules known as adipokines, which have been implicated as mediators of the interaction between adipocytes and cancer cells. Recent studies have provided new insight into the impact of bone marrow adipose tissue volume expansion in regard to supporting and exacerbating the effects of bone metastasis from solid tumors, focusing on prostate, breast and lung cancer and blood cancers, focusing on multiple myeloma. In this mini-review, recent research developments pertaining to the role of factors which increase bone marrow adipose tissue volume, as well as the role of adipocyte secreted factors, in the progression of bone metastatic prostate and breast cancer are assessed. In particular, recent findings regarding the complex cross-talk between adipocytes and metastatic cells of both lung and prostate cancer are highlighted.

Hepatocyte expressed chemerin-156 does not protect from experimental non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is a rapidly growing liver disease. The chemoattractant chemerin is abundant in hepatocytes, and hepatocyte expressed prochemerin protected from NASH. Prochemerin is inactive and different active isoforms have been described. Here, the effect of hepatocyte expressed muChem-156, a highly active murine chemerin isoform, was studied in the methionine-choline deficient dietary model of NASH. Mice overexpressing muChem-156 had higher hepatic chemerin protein. Serum chemerin levels and the capability of serum to activate the chemerin receptors was unchanged showing that the liver did not release active chemerin. Notably, activation of the chemerin receptors by hepatic vein blood did not increase in parallel to total chemerin protein in patients with liver cirrhosis. In experimental NASH, muChem-156 had no effect on liver lipids. Accordingly, overexpression of active chemerin in hepatocytes or treatment of hepatocytes with recombinant chemerin did not affect cellular triglyceride and cholesterol levels. Importantly, overexpression of muChem-156 in the murine liver did not change the hepatic expression of inflammatory and profibrotic genes. The downstream targets of chemerin such as p38 kinase were neither activated in the liver of muChem-156 producing mice nor in HepG2, Huh7 and Hepa1-6 cells overexpressing this isoform. Recombinant chemerin had no effect on global gene expression of primary human hepatocytes and hepatic stellate cells within 24 h of incubation. Phosphorylation of p38 kinase was, however, increased upon short-time incubation of HepG2 cells with chemerin. These findings show that muChem-156 overexpression in hepatocytes does not protect from liver steatosis and inflammation.

Mechanism of action and therapeutic route for a muscular dystrophy caused by a genetic defect in lipid metabolism.

CHKB encodes one of two mammalian choline kinase enzymes that catalyze the first step in the synthesis of the membrane phospholipid phosphatidylcholine. In humans and mice, inactivation of the CHKB gene (Chkb in mice) causes a recessive rostral-to-caudal muscular dystrophy. Using Chkb knockout mice, we reveal that at no stage of the disease is phosphatidylcholine level significantly altered. We observe that in affected muscle a temporal change in lipid metabolism occurs with an initial inability to utilize fatty acids for energy via mitochondrial ß-oxidation resulting in shunting of fatty acids into triacyglycerol as the disease progresses. There is a decrease in peroxisome proliferator-activated receptors and target gene expression specific to Chkb-/- affected muscle. Treatment of Chkb-/- myocytes with peroxisome proliferator-activated receptor agonists enables fatty acids to be used for ß-oxidation and prevents triacyglyerol accumulation, while simultaneously increasing expression of the compensatory choline kinase alpha (Chka) isoform, preventing muscle cell injury.

Chemerin Overexpression in the Liver Protects against Inflammation in Experimental Non-Alcoholic Steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is marked by macrophage infiltration and inflammation. Chemerin is a chemoattractant protein and is abundant in hepatocytes. The aim of this study was to gain insight into the role of hepatocyte-produced prochemerin in NASH. Therefore, mice were infected with adeno-associated virus 8 to direct hepatic overexpression of prochemerin in a methionine-choline deficient dietary model of NASH. At the end of the study, hepatic and serum chemerin were higher in the chemerin-expressing mice. These animals had less hepatic oxidative stress, F4/80 and CC-chemokine ligand 2 (CCL2) protein, and mRNA levels of inflammatory genes than the respective control animals. In order to identify the underlying mechanisms, prochemerin was expressed in hepatocytes and the hepatic stellate cells, LX-2. Here, chemerin had no effect on cell viability, production of inflammatory, or pro-fibrotic factors. Notably, cultivation of human peripheral blood mononuclear cells (PBMCs) in the supernatant of Huh7 cells overexpressing chemerin reduced CCL2, interleukin-6, and osteopontin levels in cell media. CCL2 was also low in RAW264.7 cells exposed to Hepa1-6 cell produced chemerin. In summary, the current study showed that prochemerin overexpression had little effect on hepatocytes and hepatic stellate cells. Of note, hepatocyte-produced chemerin deactivated PBMCs and protected against inflammation in experimental NASH.

Chemerin-156 is the Active Isoform in Human Hepatic Stellate Cells.

The chemokine chemerin exists as C-terminally processed isoforms whose biological functions are mostly unknown. A highly active human chemerin variant (huChem-157) was protective in experimental hepatocellular carcinoma (HCC) models. Hepatic stellate cells (HSCs) are central mediators of hepatic fibrogenesis and carcinogenesis and express the chemerin receptors chemokine-like receptor 1 (CMKLR1) and G protein-coupled receptor 1 (GPR1). Here we aimed to analyse the effect of chemerin isoforms on the viability, proliferation and secretome of the human HSC cell line LX-2. Therefore, huChem-157, 156 and 155 were over-expressed in LX-2 cells, which have low endogenous chemerin levels. HuChem-157 produced in LX-2 cells activated CMKLR1 and GPR1, and huChem-156 modestly induced GPR1 signaling. HuChem-155 is an inactive chemerin variant. Chemerin isoforms had no effect on cell viability and proliferation. Cellular expression of the fibrotic proteins galectin-3 and alpha-smooth muscle actin was not regulated by any chemerin isoform. HuChem-156 increased IL-6, IL-8 and galectin-3 in cell media. HuChem-157 was ineffective, and accordingly, did not enhance levels of these proteins in media of primary human hepatic stellate cells when added exogenously. These analyses provide evidence that huChem-156 is the biologic active chemerin variant in hepatic stellate cells and acts as a pro-inflammatory factor.

Chemerin Isoform-Specific Effects on Hepatocyte Migration and Immune Cell Inflammation.

Murine chemerin is C-terminally processed to the bioactive isoforms, muChem-156 and muChem-155, among which the longer variant protects from hepatocellular carcinoma (HCC). However, the role of muChem-155 is mostly unknown. Here, we aimed to compare the effects of these isoforms on the proliferation, migration and the secretome of the human hepatocyte cell lines HepG2 and Huh7 and the murine Hepa1-6 cell line. Therefore, huChem-157 and -156 were overexpressed in the human cells, and the respective murine variants, muChem-156 and -155, in the murine hepatocytes. Both chemerin isoforms produced by HepG2 and Hepa1-6 cells activated the chemerin receptors chemokine-like receptor 1 (CMKLR1) and G protein-coupled receptor 1 (GPR1). HuChem-157 was the active isoform in the Huh7 cell culture medium. The potencies of muChem-155 and muChem-156 to activate human GPR1 and mouse CMKLR1 were equivalent. Human CMKLR1 was most responsive to muChem-156. Chemerin variants showed no effect on cell viability and proliferation. Activation of the mitogen-activated protein kinases Erk1/2 and p38, and protein levels of the epithelial-mesenchymal transition marker, E-cadherin, were not regulated by the chemerin variants. Migration was reduced in HepG2 and Hepa1-6 cells by the longer isoform. Protective effects of chemerin in HCC include the modulation of cytokines but huChem-156 and huChem-157 overexpression did not change IL-8, CCL20 or osteopontin in the hepatocytes. The conditioned medium of the transfected hepatocytes failed to alter these soluble factors in the cell culture medium of peripheral blood mononuclear cells (PBMCs). Interestingly, the cell culture medium of Huh7 cells producing the inactive variant huChem-155 reduced CCL2 and IL-8 in PBMCs. To sum up, huChem-157 and muChem-156 inhibited hepatocyte migration and may protect from HCC metastasis. HuChem-155 was the only human isoform exerting anti-inflammatory effects on immune cells.

At the Crossroads of the Adipocyte and Osteoclast Differentiation Programs: Future Therapeutic Perspectives.

: The coordinated development and function of bone-forming (osteoblasts) and bone-resorbing (osteoclasts) cells is critical for the maintenance of skeletal integrity and calcium homeostasis. An enhanced adipogenic versus osteogenic potential of bone marrow mesenchymal stem cells (MSCs) has been linked to bone loss associated with diseases such as diabetes mellitus, as well as aging and postmenopause. In addition to an inherent decrease in bone formation due to reduced osteoblast numbers, recent experimental evidence indicates that an increase in bone marrow adipocytes contributes to a disproportionate increase in osteoclast formation. Therefore, a potential strategy for therapeutic intervention in chronic bone loss disorders such as osteoporosis is to interfere with the pro-osteoclastogenic influence of marrow adipocytes. However, application of this approach is limited by the extremely complex regulatory processes in the osteoclastogenic program. For example, key regulators of osteoclastogenesis such as the receptor activator of nuclear factor-kappaB ligand (RANKL) and the soluble decoy receptor osteoprotegerin (OPG) are not only secreted by both osteoblasts and adipocytes, but are also regulated through several cytokines produced by these cell types. In this context, biologically active signaling molecules secreted from bone marrow adipocytes, such as chemerin, adiponectin, leptin, visfatin and resistin, can have a profound influence on the osteoclast differentiation program of hematopoietic stem cells (HSCs), and thus, hold therapeutic potential under disease conditions. In addition to these paracrine signals, adipogenic transcription factors including CCAAT/enhancer binding protein alpha (C/EBPα), C/EBP beta (C/EBPß) and peroxisome proliferator-associated receptor gamma (PPARγ) are also expressed by osteoclastogenic cells. However, in contrast to MSCs, activation of these adipogenic transcription factors in HSCs promotes the differentiation of osteoclast precursors into mature osteoclasts. Herein, we discuss the molecular mechanisms that link adipogenic signaling molecules and transcription factors to the osteoclast differentiation program and highlight therapeutic strategies targeting these mechanisms for promoting bone homeostasis.

More Than an Adipokine: The Complex Roles of Chemerin Signaling in Cancer.

Chemerin is widely recognized as an adipokine, with diverse biological roles in cellular differentiation and metabolism, as well as a leukocyte chemoattractant. Research investigating the role of chemerin in the obesity-cancer relationship has provided evidence both for pro- and anti-cancer effects. The tumor-promoting effects of chemerin primarily involve direct effects on migration, invasion, and metastasis as well as growth and proliferation of cancer cells. Chemerin can also promote tumor growth via the recruitment of tumor-supporting mesenchymal stromal cells and stimulation of angiogenesis pathways in endothelial cells. In contrast, the majority of evidence supports that the tumor-suppressing effects of chemerin are immune-mediated and result in a shift from immunosuppressive to immunogenic cell populations within the tumor microenvironment. Systemic chemerin and chemerin produced within the tumor microenvironment may contribute to these effects via signaling through CMKLR1 (chemerin1), GPR1 (chemerin2), and CCLR2 on target cells. As such, inhibition or activation of chemerin signaling could be beneficial as a therapeutic approach depending on the type of cancer. Additional studies are required to determine if obesity influences cancer initiation or progression through increased adipose tissue production of chemerin and/or altered chemerin processing that leads to changes in chemerin signaling in the tumor microenvironment.

Positive allosteric modulation of the type 1 cannabinoid receptor reduces the signs and symptoms of Huntington's disease in the R6/2 mouse model.

Huntington's disease (HD) is an inherited progressive neurodegenerative disease characterized by motor, cognitive, and behavioural changes. One of the earliest changes to occur in HD is a reduction in cannabinoid 1 receptor (CB1) levels in the striatum, which is strongly correlated with HD pathogenesis. CB1 positive allosteric modulators (PAM) enhance receptor affinity for, and efficacy of activation by, orthosteric ligands, including the endocannabinoids anandamide and 2-arachidonoylglycerol. The goal of this study was to determine whether the recently characterized CB1 allosteric modulators GAT211 (racemic), GAT228 (R-enantiomer), and GAT229 (S-enantiomer), affected the signs and symptoms of HD. GAT211, GAT228, and GAT229 were evaluated in normal and HD cell models, and in a transgenic mouse model of HD (7-week-old male R6/2 mice, 10 mg/kg/d, 21 d, i.p.). GAT229 was a CB1 PAM that improved cell viability in HD cells and improved motor coordination, delayed symptom onset, and normalized gene expression in R6/2 HD mice. GAT228 was an allosteric agonist that did not enhance endocannabinoid signaling or change symptom progression in R6/2 mice. GAT211 displayed intermediate effects between its enantiomers. The compounds used here are not drugs, but probe compounds used to determine the potential utility of CB1 PAMs in HD. Changes in gene expression, and not protein, were quantified in R6/2 HD mice because HD pathogenesis is associated with dysregulation of mRNA levels. The data presented here provide the first proof of principle for the use of CB1 PAMs to treat the signs and symptoms of HD.

Overexpression of Hepatocyte Chemerin-156 Lowers Tumor Burden in a Murine Model of Diethylnitrosamine-Induced Hepatocellular Carcinoma.

The tumor inhibitory potential of the highly active chemerin-156 isoform was described in orthotopic models of hepatocellular carcinoma (HCC). The majority of HCC arises in the fibrotic liver, which was not reproduced in these studies. Here, a potential therapeutic activity of chemerin-156 was evaluated in diethylnitrosamine (DEN)-induced liver cancer, which mimics fibrosis-associated HCC. Mice were infected with adeno-associated virus (AAV) six months after DEN injection to overexpress chemerin-156 in the liver, and animals injected with non-recombinant-AAV served as controls. Three months later, the animals were killed. Both groups were comparable with regard to liver steatosis and fibrosis. Of note, the number of very small tumors was reduced by chemerin-156. Anyhow, the expression of inflammatory and profibrotic genes was similar in larger tumors of control and chemerin-156-AAV-infected animals. Although genes with a role in lipid metabolism, like 3-hydroxy-3-methylglutaryl-coenzym-A--reductase, were overexpressed in tumors of animals with high chemerin-156, total hepatic cholesterol, diacylglycerol and triglyceride levels, and distribution of individual lipid species were normal. Chemerin-156-AAV-infected mice had elevated hepatic and systemic chemerin. Ex vivo activation of the chemerin receptor chemokine-like receptor 1 increased in parallel with serum chemerin, illustrating the biological activity of the recombinant protein. In the tumors, chemerin-155 was the most abundant variant. Chemerin-156 was not detected in tumors of the controls and was hardly found in chemerin-156-AAV infected animals. In conclusion, the present study showed that chemerin-156 overexpression caused a decline in the number of small lesions but did not prevent the growth of pre-existing neoplasms.

The impact of chemerin or chemokine-like receptor 1 loss on the mouse gut microbiome.

Chemerin is an adipocyte derived signalling molecule (adipokine) that serves as a ligand activator of Chemokine-like receptor 1(CMKLR1). Chemerin/CMKLR1 signalling is well established to regulate fundamental processes in metabolism and inflammation. The composition and function of gut microbiota has also been shown to impact the development of metabolic and inflammatory diseases such as obesity, diabetes and inflammatory bowel disease. In this study, we assessed the microbiome composition of fecal samples isolated from wildtype, chemerin, or CMKLR1 knockout mice using Illumina-based sequencing. Moreover, the knockout mice and respective wildtype mice used in this study were housed at different universities allowing us to compare facility-dependent effects on microbiome composition. While there was no difference in alpha diversity within samples when compared by either facility or genotype, we observed a dramatic difference in the presence and abundance of numerous taxa between facilities. There were minor differences in bacterial abundance between wildtype and chemerin knockout mice, but significantly more differences in taxa abundance between wildtype and CMKLR1 knockout mice. Specifically, CMKLR1 knockout mice exhibited decreased abundance of Akkermansia and Prevotella, which correlated with body weight in CMKLR1 knockout, but not wildtype mice. This is the first study to investigate a linkage between chemerin/CMKLR1 signaling and microbiome composition. The results of our study suggest that chemerin/CMKLR1 signaling influences metabolic processes through effects on the gut microbiome. Furthermore, the dramatic difference in microbiome composition between facilities might contribute to discrepancies in the metabolic phenotype of CMKLR1 knockout mice reported by independent groups. Considered altogether, these findings establish a foundation for future studies to investigate the relationship between chemerin signaling and the gut microbiome on the development and progression of metabolic and inflammatory disease.

Bone Marrow Adipose Tissue and Skeletal Health.

PURPOSE OF REVIEW: To summarize and discuss recent progress and novel signaling mechanisms relevant to bone marrow adipocyte formation and its physiological/pathophysiological implications for bone remodeling. RECENT FINDINGS: Skeletal remodeling is a coordinated process entailing removal of old bone and formation of new bone. Several bone loss disorders such as osteoporosis are commonly associated with increased bone marrow adipose tissue. Experimental and clinical evidence supports that a reduction in osteoblastogenesis from mesenchymal stem cells at the expense of adipogenesis, as well as the deleterious effects of adipocyte-derived signaling, contributes to the etiology of osteoporosis as well as bone loss associated with aging, diabetes mellitus, post-menopause, and chronic drug therapy. However, this view is challenged by findings indicating that, in some contexts, bone marrow adipose tissue may have a beneficial impact on skeletal health. Further research is needed to better define the role of marrow adipocytes in bone physiology/pathophysiology and to determine the therapeutic potential of manipulating mesenchymal stem cell differentiation.

The chemerin knockout rat reveals chemerin dependence in female, but not male, experimental hypertension.

Measures of the adipokine chemerin are elevated in multiple cardiovascular diseases, including hypertension, but little mechanistic work has been done to implicate chemerin as being causative in such diseases. The chemerin knockout (KO) rat was created to test the hypothesis that removal of chemerin would reduce pressure in the normal and hypertensive state. Western analyses confirmed loss of chemerin in the plasma and tissues of the KO vs. wild-type (WT) rats. Chemerin concentration in plasma and tissues was lower in WT females than in WT males, as determined by Western analysis. Conscious male and female KO rats had modest differences in baseline measures vs. the WT that included systolic, diastolic, mean arterial and pulse pressures, and heart rate, all measured telemetrically. The mineralocorticoid deoxycorticosterone acetate (DOCA) and salt water, combined with uninephrectomy as a hypertensive stimulus, elevated mean and systolic blood pressures of the male KO higher than the male WT. By contrast, all pressures in the female KO were lower than their WT throughout DOCA-salt treatment. These results revealed an unexpected sex difference in chemerin expression and the ability of chemerin to modify blood pressure in response to a hypertensive challenge.-Watts, S. W., Darios, E. S., Mullick, A. E., Garver, H., Saunders, T. L., Hughes, E. D., Filipiak, W. E., Zeidler, M. G., McMullen, N., Sinal, C. J., Kumar, R. K., Ferland, D. J., Fink, G. D. The chemerin knockout rat reveals chemerin dependence in female, but not male, experimental hypertension.

Chemerin in a Mouse Model of Non-alcoholic Steatohepatitis and Hepatocarcinogenesis.

BACKGROUND/AIM: Non-alcoholic steatohepatitis (NASH) is a risk factor for hepatocellular carcinoma (HCC). The adipokine chemerin protects from HCC and is reduced in human HCC. In this study, chemerin expression was analyzed in a murine model of NASH-HCC. MATERIALS AND METHODS: Serum and hepatic chemerin, and ex vivo chemerin receptor activation were monitored in NASH and NASH-HCC in mice fed a low-methionine diet deficient in choline after initiation of tumors by injection of diethylnitrosamine. RESULTS: In non-tumorous liver tissues, the extent of hepatic steatosis, and the levels of proteins regulating hepatic lipids and liver fibrosis were similar in NASH and NASH-associated HCC. Systemic and hepatic chemerin, and chemerin receptor activation were not changed in HCC. Liver tumors only developed in diethylnitrosamine-injected mice and their number was increased in NASH. Chemerin protein was induced in liver in NASH, but was unchanged in HCC tissues. CONCLUSION: Hepatic and serum chemerin and ex vivo analyzed chemerin receptor activation do not differ in murine NASH-associated HCC when compared to NASH. Hepatic tumors still develop despite high endogenous levels of serum and liver chemerin protein.

Ex vivo analysis of serum chemerin activity in murine models of obesity.

OBJECTIVES: Chemerin is an adipokine with established roles in inflammation, adipogenesis and the regulation of glucose and lipid homeostasis. Extracellular proteolytic processing of chemerin generates a spectrum of isoforms that differ significantly with respect to the ability to activate the cognate receptors chemokine-like receptor 1 (CMKLR1) and G-protein-coupled receptor 1 (GPR1). Increased total serum chemerin has been widely reported in obese humans as well as in preclinical rodent models of adiposity. However, very little information is available regarding the correspondence, if any, of changes in total serum chemerin protein with chemerin bioactivity. METHODS: Total serum chemerin and ex vivo CMKLR1 and GPR1 activation was compared using two widely used murine obesity models: high fat diet feeding (HFD) and leptin deficiency (ob/ob). RESULTS: Total serum chemerin levels and ex vivo CMKLR1 and GPR1 activation were significantly induced in HFD. The bioactivity ratio (bioactive chemerin/total chemerin) was also increased when measured with CMKLR1, but not GPR1. In contrast, while ob/ob mice exhibited increased total serum chemerin protein, ex vivo receptor activation was observed with GPR1, but not CMKLR1. There was no change in bioactivity ratio for either receptor. Of note, GPR1 but not CMKLR1 bioactivity positively correlated with adipose tissue inflammation. CONCLUSIONS: While increased total serum chemerin is a consistent finding among rodent obesity models, this may not accurately reflect changes in chemerin bioactivity which is the major determinant of biological effects.

The Hippo component YAP localizes in the nucleus of human papilloma virus positive oropharyngeal squamous cell carcinoma.

BACKGROUND: HPV infection causes cervical cancer, mediated in part by the degradation of Scribble via the HPV E6 oncoprotein. Recently, Scribble has been shown to be an important regulator of the Hippo signaling cascade. Deregulation of the Hippo pathway induces an abnormal cellular transformation, epithelial to mesenchymal transition, which promotes oncogenic progression. Given the recent rise in oropharyngeal HPV squamous cell carcinoma we sought to determine if Hippo signaling components are implicated in oropharyngeal squamous cell carcinoma. METHODS: Molecular and cellular techniques including immunoprecipiations, Western blotting and immunocytochemistry were used to identify the key Hippo pathway effector Yes-Associated Protein (YAP)1. Oropharyngeal tissue was collected from CO2 laser resections, and probed with YAP1 antibody in tumor and pre-malignant regions of HPV positive OPSCC tissue. RESULTS: This study reveals that the Scribble binding protein Nitric Oxide Synthase 1 Adaptor Protein (NOS1AP) forms a complex with YAP. Further, the NOS1APa and NOS1APc isoforms show differential association with activated and non-activated YAP, and impact cellular proliferation. Consistent with deregulated Hippo signaling in OPSCC HPV tumors, we see a delocalization of Scribble and increased nuclear accumulation of YAP1 in an HPV-positive OPSCC. CONCLUSION: Our preliminary data indicates that NOS1AP isoforms differentially associate with YAP1, which, together with our previous findings, predicts that loss of YAP1 enhances cellular transformation. Moreover, YAP1 is highly accumulated in the nucleus of HPV-positive OPSCC, implying that Hippo signaling and possibly NOS1AP expression are de-regulated in OPSCC. Further studies will help determine if NOS1AP isoforms, Scribble and Hippo components will be useful biomarkers in OPSCC tumor biology.

Chemokine-Like Receptor 1 Is a Novel Wnt Target Gene that Regulates Mesenchymal Stem Cell Differentiation.

Bone remodeling is a dynamic process requiring the coordinated action of formative (osteoblast) and resorptive (osteoclast) cell populations. An imbalance of the development and function of these cell types underlies several chronic bone loss disorders such as osteoporosis. Increased bone marrow adipocyte numbers commonly occur with bone loss disorders and numerous studies have documented an inverse relationship between bone marrow fat and bone formation. Osteoblasts and adipocytes derive in a competitive fashion from a common mesenchymal stem cell (MSC) precursor. Generally, factors that promote MSC adipogenesis inhibit osteoblastogenesis and thereby, reduce bone formation. Previously we established that the secreted protein chemerin regulates adipogenic and osteoblastogenic differentiation of MSCs by signaling through chemokine-like receptor 1 (CMKLR1). However, the fundamental mechanisms by which chemerin/CMKLR1 influences lineage determination remain largely uncharacterized. Herein, we provide experimental evidence that chemerin/CMKLR1 regulates canonical Wnt signaling in MSCs by influencing the expression, subcellular location, and transcriptional activity of the central Wnt transducer, ß-catenin. Moreover, we provide evidence that CMKLR1 is a novel Wnt responsive gene that functions in a negative feedback loop to limit osteoblastogenic Wnt signaling. Mechanistically, this entails Notch-dependent changes in the expression and function of key adipogenic and osteoblastogenic transcription factors, cell cycle proteins and chromatin remodeling enzymes. Consistent with this, MSCs from CMKLR1 knockout (-/-) mice exhibited similar dependency on Notch signaling to maintain osteoblastogenic differentiation. Taken together, our findings support a fundamental biological function for chemerin/CMKLR1 to balance osteoblastogenic and adipogenic signaling and thereby contribute to the maintenance of pluripotency in MSCs. Stem Cells 2017;35:711-724.

CMKLR1 activation ex vivo does not increase proportionally to serum total chemerin in obese humans.

Prochemerin is the inactive precursor of the adipokine chemerin. Proteolytic processing is obligatory for the conversion of prochemerin into active chemerin and subsequent regulation of cellular processes via the chemokine-like receptor 1 (CMKLR1). Elevated plasma or serum chemerin concentrations and differential processing of prochemerin have been reported in obese humans. The impact of these changes on CMKLR1 signalling in humans is unknown. The objective of this pilot study was to develop a cellular bioassay to measure CMKLR1 activation by chemerin present in human serum and to characterise how obesity modifies serum activation of CMKLR1 under fasted and fed conditions. Blood samples were collected from control (N = 4, BMI 20-25) and obese (N = 4, BMI >30) female subjects after an overnight fast (n = 2) and at regular intervals (n = 7) following consumption of breakfast over a period of 6 h. A cellular CMKLR1-luminescent reporter assay and a pan-chemerin ELISA were used to determine CMKLR1 activation and total chemerin concentrations, respectively. Serum total chemerin concentration (averaged across all samples) was higher in obese vs control subjects (17.9 ± 1.8 vs 10.9 ± 0.5 nM, P < 0.05), but serum activation of CMKLR1 was similar in both groups. The CMKLR1 activation/total chemerin ratio was lower in obese vs control subjects (0.33 ± 0.04 vs 0.58 ± 0.05, P < 0.05). After breakfast, serum total chemerin or CMKLR1 activation did not differ from baseline values. In conclusion, the unexpected observation that obese serum activation of CMKLR1 did not match increased total chemerin concentrations suggests impaired processing to and/or enhanced degradation of active chemerin in serum of obese humans.

Adipocyte-secreted chemerin is processed to a variety of isoforms and influences MMP3 and chemokine secretion through an NFkB-dependent mechanism.

Obesity is associated with white adipose tissue (WAT) remodelling characterized by changes in cellular composition, size, and adipokine secretion. Levels of the adipokine chemerin are positively associated with obesity; however, the biological function of chemerin in WAT is poorly understood. We identified factors involved in WAT remodelling, including matrix metalloproteinase (Mmp)3 and chemokines (Ccl2, 3, 5, 7), as novel targets of chemerin signalling in mature adipocytes. Inhibition of chemerin signalling increased MMP activity and the recruitment of macrophages towards adipocyte-conditioned media. These effects were mediated through increases in NFkB signalling, suggesting that chemerin exerts an anti-inflammatory influence. We also demonstrate that multiple chemerin isoforms are present in adipocyte-conditioned media and that adipocyte-secreted chemerin, but not synthetic chemerin, recapitulates the activity of endogenous chemerin. Considered altogether, this suggests that endogenously secreted chemerin plays an autocrine/paracrine role in WAT, identifying chemerin as a therapeutic target to modulate adipose remodelling.

Effects of alpha-lipoic acid on chemerin secretion in 3T3-L1 and human adipocytes.

Chemerin is a novel adipokine associated with obesity and insulin resistance. α-Lipoic acid (α-LA) has shown beneficial properties on diabetes and obesity. The aim of this study was to examine the effects of α-LA on chemerin production in adipocytes in absence or presence of TNF-α, insulin and AICAR. The potential signaling pathways involved in α-LA effects on chemerin were also analyzed. α-LA actions on chemerin were tested in differentiated 3T3-L1 adipocytes and in some cases in human subcutaneous and omental adipocytes. Chemerin mRNA levels were measured by RT-PCR and the amount of chemerin secreted to culture media was determined by ELISA. α-LA induced a concentration-dependent inhibition on both chemerin secretion and mRNA levels in 3T3-L1 adipocytes. The AMPK activator AICAR and the PI3K inhibitor LY294002 dramatically abrogated both chemerin secretion and gene expression, and further potentiated the inhibitory effect of α-LA on chemerin secretion. Insulin was able to partially reverse the inhibitory action of α-LA on chemerin secretion. α-LA also reduced basal chemerin secretion in both subcutaneous and omental adipocytes from overweight/obese subjects. Moreover, α-LA was able to abolish the stimulatory effects of the pro-inflammatory cytokine TNF-α on chemerin secretion. Our data demonstrated the ability of α-LA to inhibit chemerin production, an adipokine associated to obesity and metabolic syndrome, suggesting that the reduction of chemerin could contribute to the antiobesity/antidiabetic properties described for α-LA.