Ectonucleotide Pyrophosphatase/phosphodiesterase 2 Research Articles

High levels of autotaxin and lysophosphatidic acid predict poor outcome in treatment of resectable gastric carcinoma

BackgroundAlthough early-stage gastric cancer is a candidate for curative surgical resection, the absence of specific early symptoms results in a late diagnosis and consequently most patients present advanced or metastatic disease. Identifying noveland tumor-specific biomarkers is needed to increase early detection and match patients to the appropriate treatment. The present study focused on the possible prognostic role of Ectonucleotide Pyrophosphatase/Phosphodiesterase 2 (ENPP2)/Autotaxin (ATX) and lysophosphatidic acid (LPA) in Gastro-Esophageal Adenocarcinoma (GEA). High levels of ATX/LPA are associated with several malignancies including gastrointestinal tumors. MethodsUsing a bioinformatics analysis, the incidence of ENPP2 mutations together with its expression in the tumor tissues and the correlation between the presence of mutations and the survival rate were examined in databases of GEA patients. Furthermore, circulating levels of ATX and LPA were studied retrospectively and longitudinally both in patients receiving frontal surgery and in patients receiving preoperative chemotherapy. ResultsOverall findings suggested that although ENPP2 mutations occur at low incidence, their presence was associated with a particular poor Overall Survival (OS). Furthermore, removal of the tumour by surgery resulted in a decrease in serum ATX and LPA levels within five days, regardless of any previous chemotherapy. Basal circulating ATX were associated with the aggressive diffuse GEA and could be considered of negative prognostic value, mainly in combination models with circulating Carcino-Embryonic Antigen (CEA). ConclusionsBased on these observations, clinical trials with ATX-targeted drugs and standard chemotherapy regimens may benefit from selecting GEA patients based on their levels of ATX, LPA and CEA.

Keloid nomogram prediction model based on weighted gene co-expression network analysis and machine learning

Keloids are benign skin tumors resulting from the excessive proliferation of connective tissue in wound skin. Precise prediction of keloid risk in trauma patients and timely early diagnosis are of paramount importance for in-depth keloid management and control of its progression. This study analyzed four keloid datasets in the high-throughput gene expression omnibus (GEO) database, identified diagnostic markers for keloids, and established a nomogram prediction model. Initially, 37 core protein-encoding genes were selected through weighted gene co-expression network analysis (WGCNA), differential expression analysis, and the centrality algorithm of the protein-protein interaction network. Subsequently, two machine learning algorithms including the least absolute shrinkage and selection operator (LASSO) and the support vector machine-recursive feature elimination (SVM-RFE) were used to further screen out four diagnostic markers with the highest predictive power for keloids, which included hepatocyte growth factor (HGF), syndecan-4 (SDC4), ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), and Rho family guanosine triphophatase 3 (RND3). Potential biological pathways involved were explored through gene set enrichment analysis (GSEA) of single-gene. Finally, univariate and multivariate logistic regression analyses of diagnostic markers were performed, and a nomogram prediction model was constructed. Internal and external validations revealed that the calibration curve of this model closely approximates the ideal curve, the decision curve is superior to other strategies, and the area under the receiver operating characteristic curve is higher than the control model (with optimal cutoff value of 0.588). This indicates that the model possesses high calibration, clinical benefit rate, and predictive power, and is promising to provide effective early means for clinical diagnosis.

High expression of ENPP2 is an independent predictor of poor prognosis in liver cancer

Ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) has been identified as a potential biomarker in lung and prostate cancers; however, its expression and clinical relevance in hepatocellular carcinoma (HCC) remain incompletely understood. This study comprehensively assessed ENPP2 expression in pan-cancer using bioinformatics. We analyzed the expression of ENPP2 mRNA in primary liver cancer and adjacent tissues of patients with HCC using data from the TCGA database. Cox regression and Kaplan–Meier methods were used to identify clinicopathological factors associated with survival, and the diagnostic value of ENPP2 expression was evaluated using receiver operating characteristic curve analysis. We also validated our findings by performing real-time PCR on clinical liver cancer samples. Furthermore, we conducted gene set enrichment analysis using the Cancer Genome Atlas dataset to gain additional insights into the biological significance of ENPP2 in HCC. High ENPP2 expression in HCC patients is associated with gender and clinical stage, and is a significant prognostic factor for worse outcomes. ENPP2 expression is an independent risk factor for progression-free and disease-specific survival in both cohorts, suggesting its potential as an HCC biomarker. ENPP2’s diagnostic value in HCC patients was confirmed by the area under the receiver operating characteristic curve, which was 0.806. real-time PCR analysis validated the higher expression of ENPP2 in clinical liver cancer tissues. Gene set enrichment analysis identified pathways enriched in HCC patients with high ENPP2 expression, including those related to the cell cycle, MTOR and T cell receptor signaling, and phosphatidylinositol signaling systems. We have demonstrated that ENPP2 is highly expressed in HCC and is a potential independent molecular marker for the diagnosis and prognosis of HCC.

The Role of Autotaxin and LPA Signaling in Embryonic Development, Pathophysiology and Cancer

Autotaxin (ATX) or Ectonucleotide Pyrophosphatase/Phosphodiesterase 2 (ENPP2) is a secreted enzyme with lysophospholipase D activity, with its primary function being the extracellular hydrolysis of lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), a bioactive lipid [...].

Autotaxin in Breast Cancer: Role, Epigenetic Regulation and Clinical Implications.

Autotaxin (ATX), the protein product of Ectonucleotide Pyrophosphatase Phosphodiesterase 2 (ENPP2), is a secreted lysophospholipase D (lysoPLD) responsible for the extracellular production of lysophosphatidic acid (LPA). ATX-LPA pathway signaling participates in several normal biological functions, but it has also been connected to cancer progression, metastasis and inflammatory processes. Significant research has established a role in breast cancer and it has been suggested as a therapeutic target and/or a clinically relevant biomarker. Recently, ENPP2 methylation was described, revealing a potential for clinical exploitation in liquid biopsy. The current review aims to gather the latest findings about aberrant signaling through ATX-LPA in breast cancer and discusses the role of ENPP2 expression and epigenetic modification, giving insights with translational value.

Endothelial ENPP2 (Ectonucleotide Pyrophosphatase/Phosphodiesterase 2) Increases Atherosclerosis in Female and Male Mice.

Maladapted endothelial cells (ECs) secrete ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase 2; autotaxin)-a lysophospholipase D that generates lysophosphatidic acids (LPAs). ENPP2 derived from the arterial wall promotes atherogenic monocyte adhesion induced by generating LPAs, such as arachidonoyl-LPA (LPA20:4), from oxidized lipoproteins. Here, we aimed to determine the role of endothelial ENPP2 in the production of LPAs and atherosclerosis. We quantified atherosclerosis in mice harboring loxP-flanked Enpp2 alleles crossed with Apoe-/- mice expressing tamoxifen-inducible Cre recombinase under the control of the EC-specific bone marrow X kinase promoter after 12 weeks of high-fat diet feeding. A tamoxifen-induced EC-specific Enpp2 knockout decreased atherosclerosis, accumulation of lesional macrophages, monocyte adhesion, and expression of endothelial CXCL (C-X-C motif chemokine ligand) 1 in male and female Apoe-/- mice. In vitro, ENPP2 mediated the mildly oxidized LDL (low-density lipoprotein)-induced expression of CXCL1 in aortic ECs by generating LPA20:4, palmitoyl-LPA (LPA16:0), and oleoyl-LPA (LPA18:1). ENPP2 and its activity were detected on the endothelial surface by confocal imaging. The expression of endothelial Enpp2 established a strong correlation between the plasma levels of LPA16:0, stearoyl-LPA (LPA18:0), and LPA18:1 and plaque size and a strong negative correlation between the LPA levels and ENPP2 activity in the plasma. Moreover, endothelial Enpp2 knockout increased the weight of high-fat diet-fed male Apoe-/- mice. We demonstrated that the expression of ENPP2 in ECs promotes atherosclerosis and endothelial inflammation in a sex-independent manner. This might be due to the generation of LPA20:4, LPA16:0, and LPA18:1 from mildly oxidized lipoproteins on the endothelial surface.

Aberrant ENPP2 expression promotes tumor progression in multiple myeloma

Ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) has been recently linked to tumor development. However, its role in modulating multiple myeloma (MM) disease progression remains unclear. Here, we demonstrated that CD138+ cells isolated from MM patients presented with higher expression of ENPP2 compared with CD138- cells. Treatment of MM cells with IL-6 resulted in ENPP2 upregulation. ENPP2 overexpression promoted proliferation, inhibited apoptosis, increased lysophosphatidic acid (LPA) generation, and upregulated osteoclastogenesis mediator expression in MM cells. In contrast, ENPP2 inhibition induced apoptosis, suppressed proliferation and survival, decreased LPA generation and downregulated osteoclastogenesis mediator expression. In an MM xenograft mouse model, ENPP2 knockdown significantly reduced MM tumor burden by inhibiting cell proliferation and inducing apoptosis. Furthermore, ENPP2 knockdown decreased the levels of LPA, osteoclastogenesis mediators in sera of mice with MM. Our findings revealed the tumor-promoting role of ENPP2 in MM, thus providing new molecular evidence for targeting the ENPP2-LPA axis in MM therapy.

Hepatitis B Virus Promotes Hepatocellular Carcinoma Progression Synergistically With Hepatic Stellate Cells via Facilitating the Expression and Secretion of ENPP2.

Background: Hepatitis B virus (HBV) infection is a major risk factor causing hepatocellular carcinoma (HCC) development, but the molecular mechanisms are not fully elucidated. It has been reported that virus infection induces ectonucleotide pyrophosphatase-phosphodiesterase 2 (ENPP2) expression, the latter participates in tumor progression. Therefore, the aim of the present study was to investigate whether HBV induced HCC malignancy via ENPP2. Methods: HCC patient clinical data were collected and prognosis was analyzed. Transient transfection and stable ectopic expression of the HBV genome were established in hepatoma cell lines. Immunohistochemical staining, RT-qPCR, western blot, and ELISA assays were used to detect the expression and secretion of ENPP2. Finally, CCK-8, colony formation, and migration assays as well as a subcutaneous xenograft mouse model were used to investigate the influence of HBV infection, ENPP2 expression, and activated hepatic stellate cells (aHSCs) on HCC progression in vitro and in vivo. Results: The data from cancer databases indicated that the level of ENPP2 was significant higher in HCC compared within normal liver tissues. Clinical relevance analysis using 158 HCC patients displayed that ENPP2 expression was positively correlated with poor overall survival and disease-free survival. Statistical analysis revealed that compared to HBV-negative HCC tissues, HBV-positive tissues expressed a higher level of ENPP2. In vitro, HBV upregulated ENPP2 expression and secretion in hepatoma cells and promoted hepatoma cell proliferation, colony formation, and migration via enhancement of ENPP2; downregulation of ENPP2 expression or inhibition of its function suppressed HCC progression. In addition, aHSCs strengthened hepatoma cell proliferation, migration in vitro, and promoted tumorigenesis synergistically with HBV in vivo; a loss-function assay further verified that ENPP2 is essential for HBV/aHSC-induced HCC progression. Conclusion: HBV enhanced the expression and secretion of ENPP2 in hepatoma cells, combined with aHSCs to promote HCC progression via ENPP2.

ENPP2 Methylation in Health and Cancer.

Autotaxin (ATX) encoded by Ectonucleotide Pyrophosphatase/Phosphodiesterase 2 (ENPP2) is a key enzyme in Lysophosphatidic Acid (LPA) synthesis implicated in cancer. Although its aberrant expression has been reported, ENPP2 methylation profiles in health and malignancy are not described. We examined in silico the methylation of ENPP2 analyzing publicly available methylome datasets, to identify Differentially Methylated CpGs (DMCs) which were then correlated with expression at gene and isoform levels. Significance indication was set to be FDR corrected p-value < 0.05. Healthy tissues presented methylation in all gene body CGs and lower levels in Promoter Associated (PA) regions, whereas in the majority of the tumors examined (HCC, melanoma, CRC, LC and PC) the methylation pattern was reversed. DMCs identified in the promoter were located in sites recognized by multiple transcription factors, suggesting involvement in gene expression. Alterations in methylation were correlated to an aggressive phenotype in cancer cell lines. In prostate and lung adenocarcinomas, increased methylation of PA CGs was correlated to decreased ENPP2 mRNA expression and to poor prognosis parameters. Collectively, our results corroborate that methylation is an active level of ATX expression regulation in cancer. Our study provides an extended description of the methylation status of ENPP2 in health and cancer and points out specific DMCs of value as prognostic biomarkers.

Plasmacytoid dendritic cell activation is dependent on coordinated expression of distinct amino acid transporters

Human plasmacytoid dendritic cells (pDCs) are interleukin-3 (IL-3)-dependent cells implicated in autoimmunity, but the role of IL-3 in pDC biology is poorly understood. We found that IL-3-induced Janus kinase 2-dependent expression of SLC7A5 and SLC3A2, which comprise the large neutral amino acid transporter, was required for mammalian target of rapamycin complex 1 (mTORC1) nutrient sensor activation in response to toll-like receptor agonists. mTORC1 facilitated increased anabolic activity resulting in type I interferon, tumor necrosis factor, and chemokine production and the expression of the cystine transporter SLC7A11. Loss of function of these amino acid transporters synergistically blocked cytokine production by pDCs. Comparison of invitro-activated pDCs with those from lupus nephritis lesions identified not only SLC7A5, SLC3A2, and SLC7A11 but also ectonucleotide pyrophosphatase-phosphodiesterase 2 (ENPP2) as components of a shared transcriptional signature, and ENPP2 inhibition also blocked cytokine production. Our data identify additional therapeutic targets for autoimmune diseases in which pDCs are implicated.

Transcriptional activation of ENPP2 by FoxO4 protects cardiomyocytes from doxorubicin‑induced toxicity.

It has been shown that ferroptosis is involved in doxorubicin (DOX)-induced cardiotoxicity and that ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) can protect cardiomyocytes from ferroptosis. Thus, the present study aimed to investigate whether ENPP2 could protect cardiomyocytes from DOX-induced injury by inhibiting ferroptosis. H9c2 cardiomyocytes were exposed to various concentrations (0.625, 1.25, 2.5, 5 or 10 µM) of DOX for different time periods. Cell viability and ENPP2 expression were determined. ENPP2-overexpressing H9c2 cells were treated with DOX and subsequently cell viability, oxidative stress, autophagy and ferroptosis were measured using the corresponding assays (MTT assay, commercial kits and western blot analysis). Dual-luciferase reporter and chromatin immunoprecipitation assays, as well as bioinformatics analysis, were applied to detect the interaction between ENPP2 and FoxO4. Following FoxO4 overexpression in H9c2 cells, the aforementioned cellular processes were assessed. The results indicated that ENPP2 expression was downregulated following treatment of the cells with DOX. DOX also led to the decreased cell viability, reduced autophagy and elevated ferroptosis in H9c2 cells, which were notably reversed by ENPP2 overexpression. In addition, FoxO4 bound to the ENPP2 promoter, resulting in inhibition of its expression. Following FoxO4 overexpression in H9c2 cells, further experiments conducted using commercial kits and western blot analysis revealed that FoxO4 overexpression partially inhibited the effects of ENPP2 overexpression on DOX-induced oxidative stress, autophagy and ferroptosis in H9c2 cells. In conclusion, the data indicated that ENPP2 was transcriptionally regulated by FoxO4 to protect cardiomyocytes from DOX-induced toxicity by inhibiting ferroptosis. Therefore, specific treatment approaches targeting the FoxO4/ENPP2 axis and ferroptosis may provide potential therapies for alleviating DOX-induced cardiotoxicity.

Hepatitis B Virus Infection Promotes Hepatocellular Carcinoma Progression Through LPC/ENPP2/LPA Axis

Background: Hepatitis B virus (HBV) infection is considered as an independent risk factor for poor prognosis in hepatocellular carcinoma (HCC) patients, but the molecular mechanisms have not been fully elucidated. Ectonucleotide pyrophosphatase-phosphodiesterase 2 (ENPP2) hydrolyzes lysophosphatidylcholine (LPC) to produce lysophosphatidic acid (LPA) and participates in virus replication and tumor progress. This study aimed to elucidate whether HBV infection inducing HCC progression via LPC/ENPP2/LPA axis. Methods: The clinical data for HCC patients were collected and prognosis was analyzed. The in vitro and in vivo experiments were used to investigate the influences of HBV, ENPP2 and activated hepatic stellate cell (HSC) on HCC progression. Findings: We found that ENPP2 expression was positively related to HBV infection and poor prognosis. HBV-induced HCC progression via ENPP2 was enhanced by LPC which was secreted by activated HSC. HCC patients with high ENPP2 combined with cirrhosis had worse prognosis than non-cirrhotic patients. In cirrhotic HCC patients, high ENPP2 expression cohort also had worse prognosis than low expression cohort. Interpretation: In HBV positive cirrhotic HCC patients, HBV enhanced host cell secretion of ENPP2, combined with LPC secreted by fibroblast to promote tumor progression via LPC/ENPP2/LPA. These findings may provide insights into novel approaches to HBV associated HCC therapy. Funding Statement: This study was supported by the National Natural Science Foundation of China (81801999, 81772597, 81672412, 81972255), the Guangdong Science and Technology Foundation (2017A030311002), the Science Foundation of Jiangxi (20181BAB214002), the Education Department Science and Technology Foundation of Jiangxi (GJJ170936) and grant from Guangdong Science and Technology Department (2017B030314026). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The clinicopathologic information of HCC patient (n = 774) and tumor tissues (n = 158) were obtained from Sun Yat-sen Memorial Hospital after approval by the hospital Ethical Committee. Animal studies were carried out in the South China University of Technology and approved by the Laboratory Animal Welfare and Ethics Committee of the South China University of Technology. All animal experiments conformed to the approved guidelines of Animal Care and Use Committee of South China University of Technology.

Potential Role of H-Ferritin in Mitigating Valvular Mineralization.

Objective- Calcific aortic valve disease is a prominent finding in elderly and in patients with chronic kidney disease. We investigated the potential role of iron metabolism in the pathogenesis of calcific aortic valve disease. Approach and Results- Cultured valvular interstitial cells of stenotic aortic valve with calcification from patients undergoing valve replacement exhibited significant susceptibility to mineralization/osteoblastic transdifferentiation in response to phosphate. This process was abrogated by iron via induction of H-ferritin as reflected by lowering ALP and osteocalcin secretion and preventing extracellular calcium deposition. Cellular phosphate uptake and accumulation of lysosomal phosphate were decreased. Accordingly, expression of phosphate transporters Pit1 and Pit2 were repressed. Translocation of ferritin into lysosomes occurred with high phosphate-binding capacity. Importantly, ferritin reduced nuclear accumulation of RUNX2 (Runt-related transcription factor 2), and as a reciprocal effect, it enhanced nuclear localization of transcription factor Sox9 (SRY [sex-determining region Y]-box 9). Pyrophosphate generation was also increased via upregulation of ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase-2). 3H-1, 2-dithiole-3-thione mimicked these beneficial effects in valvular interstitial cell via induction of H-ferritin. Ferroxidase activity of H-ferritin was essential for this function, as ceruloplasmin exhibited similar inhibitory functions. Histological analysis of stenotic aortic valve revealed high expression of H-ferritin without iron accumulation and its relative dominance over ALP in noncalcified regions. Increased expression of H-ferritin accompanied by elevation of TNF-α (tumor necrosis factor-α) and IL-1β (interleukin-1β) levels, inducers of H-ferritin, corroborates the essential role of ferritin/ferroxidase via attenuating inflammation in calcific aortic valve disease. Conclusions- Our results indicate that H-ferritin is a stratagem in mitigating valvular mineralization/osteoblastic differentiation. Utilization of 3H-1, 2-dithiole-3-thione to induce ferritin expression may prove a novel therapeutic potential in valvular mineralization.

Autotaxin in Pathophysiology and Pulmonary Fibrosis.

Lysophospholipid signaling is emerging as a druggable regulator of pathophysiological responses, and especially fibrosis, exemplified by the relative ongoing clinical trials in idiopathic pulmonary fibrosis (IPF) patients. In this review, we focus on ectonucleotide pyrophosphatase-phosphodiesterase 2 (ENPP2), or as more widely known Autotaxin (ATX), a secreted lysophospholipase D (lysoPLD) largely responsible for extracellular lysophosphatidic acid (LPA) production. In turn, LPA is a bioactive phospholipid autacoid, forming locally upon increased ATX levels and acting also locally through its receptors, likely guided by ATX's structural conformation and cell surface associations. Increased ATX activity levels have been detected in many inflammatory and fibroproliferative conditions, while genetic and pharmacologic studies have confirmed a pleiotropic participation of ATX/LPA in different processes and disorders. In pulmonary fibrosis, ATX levels rise in the broncheoalveolar fluid (BALF) and stimulate LPA production. LPA engagement of its receptors activate multiple G-protein mediated signal transduction pathways leading to different responses from pulmonary cells including the production of pro-inflammatory signals from stressed epithelial cells, the modulation of endothelial physiology, the activation of TGF signaling and the stimulation of fibroblast accumulation. Genetic or pharmacologic targeting of the ATX/LPA axis attenuated disease development in animal models, thus providing the proof of principle for therapeutic interventions.

Oxidative stress stimulates invasive potential in rat C6 and human U-87 MG glioblastoma cells via activation and cross-talk between PKM2, ENPP2 and APE1 enzymes

Maintaining genomic integrity is essential for cell survival and viability. Reactive oxygen species (ROS) overproduction results in oxidative stress leading to the genomic instability via generation of small base lesions in DNA and these unrepaired DNA damages lead to various cellular consequences including cancer. Recent data support the concept "oxidative stress is an indispensable participant in fostering proliferation, survival, and migration" in various cancer cell types including glioblastoma cells. In this study we demonstrate that treatment of non-cytotoxic doses of oxidants such as amyloid beta [Aβ(25-35)] peptide, glucose oxidase (GO), and hydrogen peroxide (H2O2) for 24h and 48h time points found to increase the expression level and activity of a multifunctional enzyme Apurinic/apyrimidinic endonuclease (APE1), a key enzyme of base excision repair (BER) pathway which takes care of base damages; and also resulted in modulation in the expression levels of downstream BER-pathway enzymes viz. PARP-1, XRCC1, DNA polβ, and ligase IIIα was observed upon oxidative stress in C6 and U-87 MG cells. Oxidants treatment to the C6 and U-87 MG cells also resulted in an elevation in the intracellular expression of glycolytic pathway enzyme Pyruvate kinase M2 (PKM2) and the metastasis inducer protein Ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) as analyzed using Western blotting and Immunofluorescence microscopic studies. Our study also reports that oxidative stress induced for 24h and 48h in C6 and U-87 MG cells resulted in extracellular secretion of APE1 and ENPP2 as analyzed using Western blotting in conditioned media. However, the biological significance of extracellular secreted APE1 remains elusive. Oxidative stress also elevated the ENPP2's LysoPLD activity in conditioned media of C6 and U-87 MG cells. Our results also demonstrate that oxidative stress affects the expression level and localization of APE1, PKM2, and ENPP2 in C6 and U-87 MG cells. As evidenced by the colocalization pattern at 24 h and 48h time points, it can be attributed that oxidative stress mediates crosstalk between APE1, PKM2, and ENPP2. In addition, when C6 and U-87 MG cells were treated with lysophosphatidic acid (LPA), a bioactive lipid that negatively regulates ENPP2's LysoPLD activity at 10μM concentration, demonstrated strong migratory potential in C6 and U-87 MG cells, and also induced migration upon oxidative stress. Altogether, the findings demonstrate the potential of C6 and U-87 MG cells to utilize three proteins viz. APE1, PKM2, and ENPP2 towards migration and survival of gliomas. Thus the knowledge on oxidative stress induced APE1's interaction with PKM2 and ENPP2 opens a new channel for the therapeutic target(s) for gliomas.

Selective export of autotaxin from the endoplasmic reticulum

Autotaxin (ATX) or ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) is a secretory glycoprotein and functions as the key enzyme for lysophosphatidic acid generation. The mechanism of ATX protein trafficking is largely unknown. Here, we demonstrated that p23, a member of the p24 protein family, was the protein-sorting receptor required for endoplasmic reticulum (ER) export of ATX. A di-phenylalanine (Phe-838/Phe-839) motif in the human ATX C-terminal region was identified as a transport signal essential for the ATX-p23 interaction. Knockdown of individual Sec24 isoforms by siRNA revealed that ER export of ATX was impaired only if Sec24C was down-regulated. These results suggest that ATX is selectively exported from the ER through a p23, Sec24C-dependent pathway. In addition, it was found that AKT signaling played a role in ATX secretion regulation to facilitate ATX ER export by enhancing the nuclear factor of activated T cell-mediated p23 expression. Furthermore, the di-hydrophobic amino acid motifs (FY) also existed in the C-terminal regions of human ENPP1 and ENPP3. Such a p23, Sec24C-dependent selective ER export mechanism is conserved among these ENPP family members.

Genistein and menaquinone-4 treatment-induced alterations in the expression of mRNAs and their products are beneficial to osteoblastic MC3T3-E1 cell functions.

The aim of the present study was to determine the molecular basis of the beneficial effects of genistein and/or menaquinone‑4 (MK‑4) on bone quality. Initially, 1µM genistein was applied to MC3T3‑E1 cells for 24h and the upregulated mRNAs that were detected by microarray were selected for further examination by reverse transcription‑quantitative‑polymerase chain reaction. Among them, alterations were observed in the level of GATA‑binding protein 6 (GATA6), Notch gene homolog 2 (NOTCH2), Wnt family member 5A (WNT5A), bone γ‑carboxyglutamate protein (BGLAP), chondroadherin (CHAD), dipeptidyl peptidase 4 (DPP4), ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), alkaline phosphatase (ALP) 3 and ATPase phospholipid‑transporting 11A (ATP11A) in response to treatment with 0.1µM 17‑β‑estradiol, 1µM genistein, and/or 1µM MK‑4. GATA6, NOTCH2 and WNT5A are considered to be associated with osteoclast, but not osteoblast, function; however, increases in osteoblastic mRNAs, including BGLAP and CHAD, were observed in each of the treatment groups at 48h. Immunocytochemical analysis confirmed an increase in CHAD and DPP4 proteins following the administration of genistein + MK‑4. Furthermore, genistein + MK‑4 led to alterations in cell morphology to spindle or oval shapes, and increased the intensity of ALP staining. Although the level of ALP mRNA was not consistently altered in response to the treatments, a marked increase in ALP activity was observed following 96h treatment with genistein + MK‑4. Therefore, the simultaneous intake of genistein and MK‑4 appears to be beneficial for the maintenance of bone quality.

A rhodium(III)-based inhibitor of autotaxin with antiproliferative activity

BackgroundCancer of the skin is by far the most common of all cancers. Melanoma accounts for only about 1% of skin cancers but causes a large majority of skin cancer deaths. Autotaxin (ATX), also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), regulates physiological and pathological functions of lysophosphatidic acid (LPA), and is thus an important therapeutic target. MethodsWe synthesized ten metal-based complexes and a novel cyclometalated rhodium(III) complex 1 was identified as an ATX enzymatic inhibitor using multiple methods, including ATX enzymatic assay, thermal shift assay, western immunoblotting and so on. ResultsProtein thermal shift assays showed that 1 increased the melting temperature (Tm) of ATX by 3.5°C. 1 also reduced ATX-LPA mediated downstream survival signal pathway proteins such as ERK and AKT, and inhibited the activation of the transcription factor nuclear factor κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3). 1 also exhibited strong anti-proliferative activity against A2058 melanoma cells (IC50=0.58μM). Structure-activity relationship indicated that both the rhodium(III) center and the auxiliary ligands of complex 1 are important for bioactivity. Conclusions1 represents a promising scaffold for the development of small-molecule ATX inhibitors for anti-tumor applications. To our knowledge, complex 1 is the first metal-based ATX inhibitor reported to date. General significanceRhodium complexes will have the increased attention in therapeutic and bioanalytical applications.

Abstract 576: The Role of Adipose Derived Autotaxin on Inflammation Associated with Cardiovascular Disease

Obesity is an established risk factor for cardiovascular disease (CVD) and stroke, and the cardiovascular complications of obesity are a leading cause of potentially preventable death. Recent evidence suggests that increased cardiovascular mortality in patients with obesity may not be fully explained by associated risk factors such as dyslipidemia, hyperglycemia, insulin resistance and hypertension. In addition to contributing to traditional CVD risk factors, obesity is also characterized by a chronic sub-acute inflammatory state termed “metainflammation” involving increased circulating inflammatory cytokines. Autotaxin (ATX), encoded by the ecto-nucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) gene, is a potent cell motility-stimulating factor that is secreted by adipose tissue. ATX hydrolyzes lysophosphatidylcholine (LPC), a lipid that is abundant in the circulation, to lysophosphatidic acid (LPA). The main objective of this study was to explore whether adipose ATX contributes to systemic inflammatory responses in obesity. We established two animal models with reduced ATX expression: MX-1 Cre-mediated deletion of the ENPP2 gene to generate a global ATX loss (Mx1) and Adipoq-Cre mediated loss of ATX expression in adipocytes (AdipoQ). Mice were fed a high fat diet for up to 20 weeks. No differences in body weight between the ENPP2 loss and littler mate controls were found. RNA and proteins were extracted, qPCR and western blots were performed for ATX and inflammatory cytokines; MCP, TNF and IFN. Results demonstrate that mice lacking global or adipose-derived ATX display approx 2 fold lower adipose inflammation. Furthermore, LPS was used to provoke a systemic response in mice that had been on a HFD for 10 weeks. Six hours after LPS injection, RNA was extracted and qPCR performed for IL6, IL1beta and IFN gamma. In response to a LPS challenge, AdipoQ mice showed a 2 fold reduction in inflammatory response in adipose tissue. The data generated from these experiments will provide important insight into potential mechanisms of how ATX and adipose tissue influence obesity induced inflammation and results from this and future projects may identify potential therapeutic targets to prevent and treat obesity induced inflammation and CVD.

Novel CSF Biomarker of Metabolic Dysfunction Predicts AD‐like Associations across the Alzheimer's Spectrum

Obesity and insulin resistance(IR) are associated with brain atrophy and cognitive decline, particularly in Alzheimer's disease (AD). IR is correlated with temporal and frontal amyloid deposition and brain atrophy in late middle‐aged participants at risk for AD, as well as less glucose metabolism in cognitively normal elders and AD participants. Because insulin‐degrading enzyme complicates the measurement of insulin levels in cerebrospinal fluid (CSF), an alternative biomarker for metabolic dysregulation in CSF should be used to draw conclusions between central hyper insulinemia and associations with brain atrophy and hypometabolism. Ecto‐nucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), also known as autotaxin, is studied in tumor cell mobility and is produced by beige adipose tissue. ENPP2 regulates energy metabolism, and is higher in AD prefrontal cortex (PFC). Additionally, ENPP2 stimulates the formation of lysophosphatidic acid, which promotes the generation of tissue fibrosis in vivo and in vitro. We studied Alzheimer's Disease Neuroimaging Initiative(ADNI) participants who were cognitively normal (CN; n=86) or had Mild Cognitive impairment (MCI; n=135) or AD (n=66). Statistical analyses were conducted using SPSS software. Multinomial regression analyses tested if higher ENPP2 was associated with higher relative risk ratios for MCI or AD diagnosis, relative to the CN reference group. Mixed model analyses were used to regress ENPP2 against metabolic, MRI, FDG‐PET and cognitive outcomes. Then on‐parametric Spearman's statistic was used to correlate ENPP2 and CSF biomarker values. T1‐weighted MRI images were preprocessed using FreeSurfer 4.3, and cortical thickness was examined in8 bilateral regions of interest. CSF ENPP2 levels strongly corresponded with CSF insulin‐like growth factor binding protein 2, an established biomarker of glucoregulatory function, and systemic fasting glucose. ENPP2 levels were higher in MCI and AD, and each point increase in log‐based ENPP2 values corresponded to a 3.5 to 5 times increase in the odds of having some degree of clinically relevant memory impairment. Higher ENPP2 predicted thinner PFC cortical thickness in most regions of interest in AD such as medialorbitofrontal cortex, and pars orbital is in MCI. In addition, less bilateral prefrontal cortex glucose metabolism was seen in AD. ENPP2 was also positively correlated with levels of total tau, p‐Tau181, total tau/Aβ1‐42, as well as pTau‐181/Aβ1‐42 to a marginal degree. This pattern suggests that CSF ENPP2 predicts AD neuropathology in a manner similar to dysmetabolism. Future studies should examine the potential benefit of decreasing ENPP2 levels in individuals with MCI or AD.Support or Funding InformationThis study was funded by Iowa State University and NIH K99 AG047282. Neither funding source had any involvement in the report. Data collection and sharing for this project were funded by the ADNI (National Institutes of Health Grant U01‐AG‐024904) and Department of Defense ADNI (award number W81XWH‐12‐2‐0012). ADNI is funded by the National Institute on Aging, the National Institute of Biomedical Imaging and Bioengineering, and through generous contributions from the Alzheimer's Association and the Alzheimer's Drug Discovery Foundation. The Canadian Institutes of Health Research is providing funds to support ADNI clinical sites in Canada. Private‐sector contributions are facilitated by the Foundation for the National Institutes of Health (www.fnih.org). The grantee organization is the Northern California Institute for Research and Education, and the study is coordinated by the Alzheimer's Disease Cooperative Study at the University of California, San Diego. ADNI data are disseminated by the Laboratory for Neuro Imaging at the University of Southern California. The data used in the preparation of this article were obtained from the ADNI database (adni.loni.usc.edu). As such, the investigators within the ADNI contributed to the design and implementation of ADNI and/or provided data but did not participate in analysis or writing of this report.