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Abstract In the tumor microenvironment, macrophages play crucial roles resulting in tumor suppression and progression, depending on M1 and M2 macrophages, respectively. In particular, macrophage-derived exosomes modulate the gene expression of cancer cells by delivering miRNAs which downregulate specific genes. The communication between macrophages and cancer cells is especially important in immunogenic tumors such as melanoma, where the cancer pogression is significantly influenced by the surrounding immune cells. In this study, we identified that M1 macrophages secrete exosomal miR-29c-3p in the co-culture system with melanoma cells. Simultaneously, ENPP2, the target of miR-29c-3p, decreased in the melanoma cells which are co-cultured with M1 macrophages. Additionally, we observed that the reduction of ENPP2 alleviates melanoma cell migration and invasion, due to the changes of cholesterol metabolism and ECM remodeling. Based on these findings, we demonstrated that M1 macrophages suppress aggressiveness of melanoma cells via exosomal miR-29c-3p-mediated knock-down of ENPP2 in cancer cells. Graphical Abstract

In the tumor microenvironment, macrophages play crucial roles resulting in tumor suppression and progression, depending on M1 and M2 macrophages, respectively. In particular, macrophage-derived exosomes modulate the gene expression of cancer cells by delivering miRNAs which downregulate specific genes. The communication between macrophages and cancer cells is especially important in immunogenic tumors such as melanoma, where the cancer pogression is significantly influenced by the surrounding immune cells. In this study, we identified that M1 macrophages secrete exosomal miR-29c-3p in the co-culture system with melanoma cells. Simultaneously, ENPP2, the target of miR-29c-3p, decreased in the melanoma cells which are co-cultured with M1 macrophages. Additionally, we observed that the reduction of ENPP2 alleviates melanoma cell migration and invasion, due to the changes of cholesterol metabolism and ECM remodeling. Based on these findings, we demonstrated that M1 macrophages suppress aggressiveness of melanoma cells via exosomal miR-29c-3p-mediated knock-down of ENPP2 in cancer cells. [ABSTRACT FROM AUTHOR]

Human periodontal ligament stem cells (hPDLSCs) differentiate into periodontal ligament (PDL) fibroblasts, osteoblasts, and cementoblasts. To identify inducers of PDL fibroblastic differentiation, monoclonal antibody series were developed a series of against membrane/extracellular matrix (ECM) molecules through decoy immunization. The anti-PDL13 antibody targets ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), renowned for regulating skeletal and soft tissue mineralization. ENPP1 accumulates in the periodontal ligament region of tooth roots, and specifically localizes to the cell boundaries and elongated processes of the fibroblastic cells. As ENPP1 expression increases during fibroblastic differentiation, mineralization induced by tissue-nonspecific alkaline phosphatase (TNAP), a pyrophosphate-degrading enzyme, is completely inhibited. This is consistent with ENPP1 and TNAP acting in opposition, and TGF-ß1-induced ENPP1 expression creates an essential environment for PDL fibroblast differentiation. Representative fibroblastic differentiation markers decrease with endogenous ENPP1 inhibition by siRNA and antibody blocking. ENPP2 generates lipid signaling molecules. In contrast to ENPP1, ENPP2 disappears in TGF-ß1-induced PDL fibroblasts. Ectopic expression of ENPP2 hinders TGF-ß1-induced PDL fibroblastic differentiation. Suppression of ENPP1 and ENPP2 leads to severe defects in undifferentiated and differentiated cells, demonstrating that these two factors play opposing roles in soft and hard tissue differentiation but can complement each other for cell survival. In conclusion, increased ENPP1 is crucial for TGF-ß1-induced PDL differentiation, while ENPP2 and TNAP can inhibit ENPP1. ENPP1 and ENPP2 exhibit complementary functions in the cell survival. [ABSTRACT FROM AUTHOR]

Autotaxin (ATX) is a member of the ectonucleotide pyrophosphate/phosphodiesterase (ENPP) family; it is encoded by the ENPP2 gene. ATX is a secreted glycoprotein and catalyzes the hydrolysis of lysophosphatidylcholine to lysophosphatidic acid (LPA). LPA is responsible for the transduction of various signal pathways through the interaction with at least six G protein-coupled receptors, LPA Receptors 1 to 6 (LPAR1–6). The ATX–LPA axis is involved in various physiological and pathological processes, such as angiogenesis, embryonic development, inflammation, fibrosis, and obesity. However, significant research also reported its connection to carcinogenesis, immune escape, metastasis, tumor microenvironment, cancer stem cells, and therapeutic resistance. Moreover, several studies suggested ATX and LPA as relevant biomarkers and/or therapeutic targets. In this review of the literature, we aimed to deepen knowledge about the role of the ATX–LPA axis as a promoter of cancer development, progression and invasion, and therapeutic resistance. Finally, we explored its potential application as a prognostic/predictive biomarker and therapeutic target for tumor treatment. [ABSTRACT FROM AUTHOR]

Autotaxin (ATX) converts lysophosphatidylcholine and sphingosyl-phosphorylcholine into lysophosphatidic acid and sphingosine 1-phosphate, respectively. Despite the pivotal function of ATX in lipid metabolism, mechanisms by which ATX regulates immune and inflammatory disorders remain elusive. Here, using myeloid cell lineage-restricted Atx knockout mice, we show that Atx deficiency disrupts membrane microdomains and lipid rafts, resulting in the inhibition of Toll-like receptor 4 (TLR4) complex formation and the suppression of adaptor recruitment, thereby inhibiting TLR4-mediated responses in macrophages. Accordingly, TLR4-induced innate immune functions, including phagocytosis and iNOS expression, are attenuated in Atx-deficient macrophages. Consequently, Atx-/- mice exhibit a higher bacterial prevalence in the intestinal mucosa compared to controls. When combined with global Il10-/- mice, which show spontaneous colitis due to the translocation of luminal commensal microbes into the mucosa, myeloid cell lineage-restricted Atx knockout accelerates colitis development compared to control littermates. Collectively, our data reveal that Atx deficiency compromises innate immune responses, thereby promoting microbe-associated gut inflammation.

Lysophosphatidic acid is a growth factor-like bioactive phospholipid recognising LPA receptors and mediating signalling pathways that regulate embryonic development, wound healing, carcinogenesis, and fibrosis, via effects on cell migration, proliferation and differentiation. Extracellular LPA is generated from lysophospholipids by the secreted hydrolase—ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2; also, AUTOTAXIN/ATX) and metabolised by different membrane-bound phospholipid phosphatases (PLPPs). Here, we use public bulk and single-cell RNA sequencing datasets to explore the expression of Lpar1–6, Enpp2, and Plpp genes under skeletal muscle homeostasis and regeneration conditions. We show that the skeletal muscle system dynamically expresses the Enpp2-Lpar-Plpp gene axis, with Lpar1 being the highest expressed member among LPARs. Lpar1 was expressed by mesenchymal fibro-adipogenic progenitors and tenocytes, whereas FAPs mainly expressed Enpp2. Clustering of FAPs identified populations representing distinct cell states with robust Lpar1 and Enpp2 transcriptome signatures in homeostatic cells expressing higher levels of markers Dpp4 and Hsd11b1. However, tissue injury induced transient repression of Lpar genes and Enpp2. The role of LPA in modulating the fate and differentiation of tissue-resident FAPs has not yet been explored. Ex vivo, LPAR1/3 and ENPP2 inhibition significantly decreased the cell-cycle activity of FAPs and impaired fibro-adipogenic differentiation, implicating LPA signalling in the modulation of the proliferative and differentiative fate of FAPs. Together, our results demonstrate the importance of the ENPP2-LPAR-PLPP axis in different muscle cell types and FAP lineage populations in homeostasis and injury, paving the way for further research on the role of this signalling pathway in skeletal muscle homeostasis and regeneration, and that of other organs and tissues, in vivo.

Ferroptosis and immune infiltration play an important role in the pathogenesis of intervertebral disc degeneration (IDD). However, there is still a lack of comprehensive analysis on the interaction between ferroptosis-related genes (FRGs) and immune microenvironment in IDD patients. Therefore, this study aims to explore the correlation between FRGs characteristics and immune infiltration in the progression of IDD. The expression profiles (GSE56081 and GSE70362) and FRGs were downloaded from the comprehensive gene expression omnibus (GEO) and FerrDb database, respectively, and the differences were analyzed using R. The intersection of IDD related differential genes (DEGs) and FRGs was taken as differentially expressed FRGs (DE-FRGs) and GO and KEGG enrichment analysis was conducted. Then, we used least absolute shrinkage and selection operator (LASSO) regression algorithm and support vector machine (SVM) algorithm to screen feature genes and draw ROC curve judge the diagnostic value of key DE-FRGs. Then CIBERSORT algorithm is used to evaluate the infiltration of immune cells and analyze the correlation between key DE-FRGs and immune infiltration. Based on the analysis results, we conducted single gene GSEA analysis on key DE-FRGs. RT-PCR and immunohistochemistry further verified the clinical value of the results of biochemical analysis and screening. Seven key DE-FRGs were screened, including the upregulated genes NOX4 and PIR, and the downregulated genes TIMM9, ATF3, ENPP2, FADS2 and TFAP2A. Single gene GSEA analysis further elucidates the role of DE-FRGs in IDD associated with ferroptosis. Correlation analysis showed that seven key DE-FRGs were closely related to immune infiltration in the development of IDD. Finally, RT-PCR and immunohistochemical staining showed that NOX4, ENPP2, FADS2 and TFAP2A were statistically significant differences. In this study, we explored the connection between ferroptosis related characteristics and immune infiltration in IDD, and confirmed that NOX4, ENPP2, FADS2, and TFAP2A may become biomarkers and potential therapeutic targets for IDD.

Simple Summary: Autotaxin (ATX) has been linked with the pathogenesis of several cancers and especially with breast cancer (BC). BC is one of the most common cancers among women and although significant steps have been made regarding its early detection and treatment options, challenges still remain. This review aims to summarize the current knowledge of the role and regulation of ATX in BC and to shed light on its potential for clinical applications. 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. [ABSTRACT FROM AUTHOR]

Autotaxin (ATX), encoded by the ctonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) gene, is a key enzyme in lysophosphatidic acid (LPA) synthesis. We have recently described ENPP2 methylation profiles in health and multiple malignancies and demonstrated correlation to its aberrant expression. Here we focus on breast cancer (BrCa), analyzing in silico publicly available BrCa methylome datasets, to identify differentially methylated CpGs (DMCs) and correlate them with expression. Numerous DMCs were identified between BrCa and healthy breast tissues in the gene body and promoter-associated regions (PA). PA DMCs were upregulated in BrCa tissues in relation to normal, in metastatic BrCa in relation to primary, and in stage I BrCa in relation to normal, and this was correlated to decreased mRNA expression. The first exon DMC was also investigated in circulating cell free DNA (ccfDNA) isolated by BrCa patients; methylation was increased in BrCa in relation to ccfDNA from healthy individuals, confirming in silico results. It also differed between patient groups and was correlated to the presence of multiple metastatic sites. Our data indicate that promoter methylation of ENPP2 arrests its transcription in BrCa and introduce first exon methylation as a putative biomarker for diagnosis and monitoring which can be assessed in liquid biopsy. [ABSTRACT FROM AUTHOR]

• LPA signalling inhibition reverses CAIA-induced pain-like behavior. • Autotaxin expression is locally upregulated in DRG neurons. • Satellite glia cells express LPA1 in humans and mice. • LPA stimulates satellite glia cells in vivo and in vitro. Inflammatory and neuropathic-like components underlie rheumatoid arthritis (RA)-associated pain, and lysophosphatidic acid (LPA) is linked to both joint inflammation in RA patients and to neuropathic pain. Thus, we investigated a role for LPA signalling using the collagen antibody-induced arthritis (CAIA) model. Pain-like behavior during the inflammatory phase and the late, neuropathic-like phase of CAIA was reversed by a neutralizing antibody generated against LPA and by an LPA 1/3 receptor inhibitor, but joint inflammation was not affected. Autotaxin, an LPA synthesizing enzyme was upregulated in dorsal root ganglia (DRG) neurons during both CAIA phases, but not in joints or spinal cord. Late-phase pronociceptive neurochemical changes in the DRG were blocked in Lpar1 receptor deficient mice and reversed by LPA neutralization. In vitro and in vivo studies indicated that LPA regulates pain-like behavior via the LPA 1 receptor on satellite glia cells (SGCs), which is expressed by both human and mouse SGCs in the DRG. Furthermore, CAIA-induced SGC activity is reversed by phospholipid neutralization and blocked in Lpar1 deficient mice. Our findings suggest that the regulation of CAIA-induced pain-like behavior by LPA signalling is a peripheral event, associated with the DRGs and involving increased pronociceptive activity of SGCs, which in turn act on sensory neurons. [ABSTRACT FROM AUTHOR]

Human periodontal ligament stem cells (hPDLSCs) differentiate into periodontal ligament (PDL) fibroblasts, osteoblasts, and cementoblasts. To identify inducers of PDL fibroblastic differentiation, monoclonal antibody series were developed a series of against membrane/extracellular matrix (ECM) molecules through decoy immunization. The anti-PDL13 antibody targets ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), renowned for regulating skeletal and soft tissue mineralization. ENPP1 accumulates in the periodontal ligament region of tooth roots, and specifically localizes to the cell boundaries and elongated processes of the fibroblastic cells. As ENPP1 expression increases during fibroblastic differentiation, mineralization induced by tissue-nonspecific alkaline phosphatase (TNAP), a pyrophosphate-degrading enzyme, is completely inhibited. This is consistent with ENPP1 and TNAP acting in opposition, and TGF-β1-induced ENPP1 expression creates an essential environment for PDL fibroblast differentiation. Representative fibroblastic differentiation markers decrease with endogenous ENPP1 inhibition by siRNA and antibody blocking. ENPP2 generates lipid signaling molecules. In contrast to ENPP1, ENPP2 disappears in TGF-β1-induced PDL fibroblasts. Ectopic expression of ENPP2 hinders TGF-β1-induced PDL fibroblastic differentiation. Suppression of ENPP1 and ENPP2 leads to severe defects in undifferentiated and differentiated cells, demonstrating that these two factors play opposing roles in soft and hard tissue differentiation but can complement each other for cell survival. In conclusion, increased ENPP1 is crucial for TGF-β1-induced PDL differentiation, while ENPP2 and TNAP can inhibit ENPP1. ENPP1 and ENPP2 exhibit complementary functions in the cell survival., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ju, Ko and Jang.)

Anne-Li Lind,1 David Just,2 Maria Mikus,2 Claudia Fredolini,2 Marina Ioannou,2 Björn Gerdle,3 Bijar Ghafouri,3 Emmanuel Bäckryd,3 Lars Tanum,4 Torsten Gordh,1 Anna Månberg2 1Department of Surgical Sciences, Uppsala University, Uppsala, Sweden; 2Division of Affinity Proteomics, SciLifeLab, Deptartment of Protein Science, KTH Royal Institute of Technology, Stockholm, Sweden; 3Pain and Rehabilitation Center, and Department of Medical and Health Sciences, Linköping University, Linköping, Sweden; 4Department of R&D in Mental Health, Akershus University Hospital, Lørenskog, NorwayCorrespondence: Anna MånbergDivision of Affinity Proteomics, SciLifeLab, Department of Protein Science, KTH - Royal Institute of Technology, Tomtebodvägen 23A, Solna, Stockholm, SwedenTel +46 8 790 9803Email anna.manberg@scilifelab.seObjective: Neuropathic pain and fibromyalgia are two common and poorly understood chronic pain conditions that lack satisfactory treatments, cause substantial suffering and societal costs. Today, there are no biological markers on which to base chronic pain diagnoses, treatment choices or to understand the pathophysiology of pain for the individual patient. This study aimed to investigate cerebrospinal fluid (CSF) protein profiles potentially associated with fibromyalgia and neuropathic pain.Methods: CSF samples were collected from 25 patients with neuropathic pain (two independent sets, n=14 patients for discovery, and n=11 for verification), 40 patients with fibromyalgia and 134 controls without neurological disease from two different populations. CSF protein profiling of 55 proteins was performed using antibody suspension bead array technology.Results: We found increased levels of apolipoprotein C1 (APOC1) in CSF of neuropathic pain patients compared to controls and there was a trend for increased levels also in fibromyalgia patients. In addition, levels of ectonucleotide pyrophosphatase family member 2 (ENPP2, also referred to as autotaxin) were increased in the CSF of fibromyalgia patients compared to all other groups including patients with neuropathic pain.Conclusion: The increased levels of APOC1 and ENPP2 found in neuropathic pain and fibromyalgia patients may shed light on the underlying mechanisms of these conditions. Further investigation is required to elucidate their role in maintaining pain and other main symptoms of these disorders.Keywords: cerebrospinal fluid, neuropathic pain, fibromyalgia, antibody suspension bead arrays, APOC1, ENPP2

Background and Aim: Uncontrolled proliferation is the most prominent biological feature of tumors. In order to rapidly proliferate, tumor cells regulate their metabolic behavior by controlling the expression of metabolism-related genes (MRGs) to maximize the utilization of available nutrients. In this study, we aimed to construct prognosis models for colorectal adenocarcinoma (COAD) and rectum adenocarcinoma (READ) using MRGs to predict the prognoses of patients. Methods: We first acquired the gene expression profiles of COAD and READ from the TCGA database, and then utilized univariate Cox analysis, Lasso regression, and multivariable Cox analysis to identify the MRGs for risk models. Results: Eight genes (CPT1C, PLCB2, PLA2G2D, GAMT, ENPP2, PIP4K2B, GPX3, and GSR) in the colon cancer risk model and six genes (TDO2, PKLR, GAMT, EARS2, ACO1, and WAS) in the rectal cancer risk model were identified successfully. Multivariate Cox analysis indicated that these two models could accurately and independently predict overall survival (OS) for patients with COAD or READ. Furthermore, functional enrichment analysis was used to identify the metabolism pathway of MRGs in the risk models and analyzed these genes comprehensively. Then, we verified the prognosis model in independent COAD cohorts (GSE17538) and detected the correlations of the protein expression levels of GSR and ENPP2 with prognosis for COAD or READ. Conclusion: In this study, 14 MRGs were identified as potential prognostic biomarkers and therapeutic targets for colorectal cancer. [ABSTRACT FROM AUTHOR]

Background/Aims: Signaling of Gs protein-coupled receptors (GsPCRs) is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA) and Epac, and an efflux of cAMP, the function of which is still unclear. Methods: Activation of adenylyl cyclase by GsPCR agonists or cholera toxin was monitored by measurement of the intracellular cAMP concentration by ELISA, anti-phospho-PKA substrate motif phosphorylation by immunoblotting, and an Epac-FRET assay in the presence and absence of adenosine receptor antagonists or ecto-nucleotide phosphodiesterase/pyrophosphatase2 (eNPP2) inhibitors. The production of AMP from cAMP by recombinant eNPP2 was measured by HPLC. Extracellular adenosine was determined by LC-MS/MS, extracellular ATP by luciferase and LC-MS/MS. The expression of eNPP isoenzymes 1-3 was examined by RT-PCR. The expression of multidrug resistance protein 4 was suppressed by siRNA. Results: Here we show that the activation of GsPCRs and the GsPCRs-independent activation of Gs proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A2A or A2B adenosine receptors). In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A2A adenosine receptors by an autocrine signaling pathway involving cAMP efflux through multidrug resistance protein 4 and hydrolysis of released cAMP to AMP by eNPP2. In contrast, in PC3 cells cholera toxin- and GsPCR-induced stimulation of adenylyl cyclase resulted in the activation of A2B adenosine receptors. Conclusion: Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors.

Enpp2 is an enzyme that catalyzes the conversion of lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), which exhibits a wide variety of biological functions. Here, we examined the biological effects of Enpp2 on dendritic cells (DCs), which are specialized antigen-presenting cells (APCs) characterized by their ability to migrate into secondary lymphoid organs and activate naïve T-cells. DCs were generated from bone marrow progenitors obtained from C57BL/6 mice. Enpp2 levels in DCs were regulated using small interfering (si)RNA or recombinant Enpp2. Expression of Enpp2 in LPS-stimulated mature (m)DCs was high, however, knocking down Enpp2 inhibited mDC function. In addition, the migratory capacity of mDCs increased after treatment with rmEnpp2; this phenomenon was mediated via the RhoA-mediated signaling pathway. Enpp2-treated mDCs showed a markedly increased capacity to migrate to lymph nodes in vivo. These findings strongly suggest that Enpp2 is necessary for mDC migration capacity, thereby increasing our understanding of DC biology. We postulate that regulating Enpp2 improves DC migration to lymph nodes, thus improving the effectiveness of cancer vaccines based on DC.

Overcoming and preventing cancer therapy resistance is the most pressing challenge in modern breast cancer management. Consequently, most modern breast cancer research is aimed at understanding and blocking these therapy resistance mechanisms. One increasingly promising therapeutic target is the autotaxin (ATX)-lysophosphatidate (LPA)-lipid phosphate phosphatase (LPP) axis. Extracellular LPA, produced from albumin-bound lysophosphatidylcholine by ATX and degraded by the ecto-activity of the LPPs, is a potent cell-signaling mediator of tumor growth, invasion, angiogenesis, immune evasion, and resistance to cancer treatment modalities. LPA signaling in the post-natal organism has central roles in physiological wound healing, but these mechanisms are subverted to fuel pathogenesis in diseases that arise from chronic inflammatory processes, including cancer. Over the last 10 years, our understanding of the role of LPA signaling in the breast tumor microenvironment has begun to mature. Tumor-promoting inflammation in breast cancer leads to increased ATX production within the tumor microenvironment. This results in increased local concentrations of LPA that are maintained in part by decreased overall cancer cell LPP expression that would otherwise more rapidly break it down. LPA signaling through six G-protein-coupled LPA receptors expressed by cancer cells can then activate virtually every known tumorigenic pathway. Consequently, to target therapy resistance and tumor growth mediated by LPA signaling, multiple inhibitors against the LPA signaling axis are entering clinical trials. In this review, we summarize recent developments in LPA breast cancer biology, and illustrate how these novel therapeutics against the LPA signaling pathway may be excellent adjuncts to extend the efficacy of evolving breast cancer treatments., Competing Interests: The authors report no proprietary or commercial interest in any product mentioned or concept discussed in this article. D.N.B. is a member of the Scientific and Clinical Advisory Boards of iOnctura and receives funding for a postdoctoral fellow from iOnctura to work on an ATX inhibitor, but he has no commercial interests in the company. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results., (Copyright 2024, Benesch et al.)

Aims Calcific aortic valve stenosis (CAVS) is characterized by a fibrocalcific process. Studies have shown an association between CAVS and the activation of platelets. It is believed that shear stress associated with CAVS promotes the activation of platelets. However, whether platelets actively participate to the mineralization of the aortic valve (AV) and the progression of CAVS is presently unknown. To identify the role of platelets into the pathobiology of CAVS. Methods and results Explanted control non-mineralized and mineralized AVs were examined by scanning electron microscope (SEM) for the presence of activated platelets. In-depth functional assays were carried out with isolated human valve interstitial cells (VICs) and platelets as well as in LDLR−/− apoB100/100 IGFII (IGFII) mice. Scanning electron microscope and immunogold markings for glycoprotein IIb/IIIa (GPIIb/IIIa) revealed the presence of platelet aggregates with fibrin in endothelium-denuded areas of CAVS. In isolated VICs, collagen-activated platelets induced an osteogenic programme. Platelet-derived adenosine diphosphate induced the release of autotaxin (ATX) by VICs. The binding of ATX to GPIIb/IIIa of platelets generated lysophosphatidic acid (LysoPA) with pro-osteogenic properties. In IGFII mice with CAVS, platelet aggregates were found at the surface of AVs. Administration of activated platelets to IGFII mice accelerated the development of CAVS by 2.1-fold, whereas a treatment with Ki16425, an antagonist of LysoPA receptors, prevented platelet-induced mineralization of the AV and the progression of CAVS. Conclusions These findings suggest a novel role for platelets in the progression of CAVS. View large Download slide View large Download slide [ABSTRACT FROM AUTHOR]

Abstract The lipid mediator lysophosphatidic acid (LPA) in biological fluids is primarily produced by cleavage of lysophospholipids by the lysophospholipase D enzyme Autotaxin (ATX). LPA has been identified and abundantly detected in the culture medium of various cancer cell types, tumor effusates, and ascites fluid of cancer patients. Our current understanding of the physiological role of LPA established its role in fundamental biological responses that include cell proliferation, metabolism, neuronal differentiation, angiogenesis, cell migration, hematopoiesis, inflammation, immunity, wound healing, regulation of cell excitability, and the promotion of cell survival by protecting against apoptotic death. These essential biological responses elicited by LPA are seemingly hijacked by cancer cells in many ways; transcriptional upregulation of ATX leading to increased LPA levels, enhanced expression of multiple LPA GPCR subtypes, and the downregulation of its metabolic breakdown. Recent studies have shown that overexpression of ATX and LPA GPCR can lead to malignant transformation, enhanced proliferation of cancer stem cells, increased invasion and metastasis, reprogramming of the tumor microenvironment and the metastatic niche, and development of resistance to chemo-, immuno-, and radiation-therapy of cancer. The fundamental role of LPA in cancer progression and the therapeutic inhibition of the ATX-LPA axis, although highly appealing, remains unexploited as drug development to these targets has not reached into the clinic yet. The purpose of this brief review is to highlight some unique signaling mechanisms engaged by the ATX-LPA axis and emphasize the therapeutic potential that lies in blocking the molecular targets of the LPA system. [ABSTRACT FROM AUTHOR]

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 24 h and 48 h 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 24 h and 48 h 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 48 h 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 10 μM 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. [ABSTRACT FROM AUTHOR]

Ferroptosis is an iron- and oxidative-dependent form of regulated cell death and may play important roles in maintaining myocardium homeostasis and pathology of cardiovascular diseases. Currently, the regulatory roles of lipid signals in regulating cardiomyocytes ferroptosis has not been explored. In this study, we show that ENPP2, as a lipid kinase involved in lipid metabolism, protects against erastin-induced ferroptosis in cardiomyocytes. The classical ferroptosis inducer erastin remarkably inhibits the growth which could be rescued by the small molecule Fer-1 in H9c2 cells. Adenovirus mediated ENPP2 overexpression modestly promotes migration and proliferation and significantly inhibits erastin-induced ferroptosis of H9c2 cells. ENPP2 overexpression leads to increase the LPA level in supernatant of H9c2 cells. H9c2 cells express the LPAR1, LPAR3, LPAR4 and LPAR5 receptors. The supernatant of ENPP2 transduced cardiomyocytes could protects the cells from erastin-induced ferroptosis of H9c2 cells. Furthermore, we observed that ENPP2 overexpression regulates ferroptosis-associated gene GPX4, ACSL4 and NRF2 expression and modulates MAPK and AKT signal in H9c2 cells. Collectively, these findings demonstrated that ENPP2/LPA protects cardiomyocytes from erastin-induced ferroptosis through modulating GPX4, ACSL4 and NRF2 expression and enhancing AKT survival signal. [ABSTRACT FROM AUTHOR]

Background/Aims: Signaling of Gs protein-coupled receptors (GsPCRs) is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA) and Epac, and an efflux of cAMP, the function of which is still unclear. Methods: Activation of adenylyl cyclase by GsPCR agonists or cholera toxin was monitored by measurement of the intracellular cAMP concentration by ELISA, anti-phospho-PKA substrate motif phosphorylation by immunoblotting, and an Epac-FRET assay in the presence and absence of adenosine receptor antagonists or ecto-nucleotide phosphodiesterase/pyrophosphatase2 (eNPP2) inhibitors. The production of AMP from cAMP by recombinant eNPP2 was measured by HPLC. Extracellular adenosine was determined by LC-MS/MS, extracellular ATP by luciferase and LC-MS/MS. The expression of eNPP isoenzymes 1-3 was examined by RT-PCR. The expression of multidrug resistance protein 4 was suppressed by siRNA. Results: Here we show that the activation of GsPCRs and the GsPCRs-independent activation of Gs proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A2A or A2B adenosine receptors). In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A2A adenosine receptors by an autocrine signaling pathway involving cAMP efflux through multidrug resistance protein 4 and hydrolysis of released cAMP to AMP by eNPP2. In contrast, in PC3 cells cholera toxin- and GsPCR-induced stimulation of adenylyl cyclase resulted in the activation of A2B adenosine receptors. Conclusion: Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors. [ABSTRACT FROM AUTHOR]

Inhibition of Autotaxin (ATX) is a potential treatment strategy for several diseases, including tumors with elevated ATX-lysophosphatidic acid (LPA) signaling. Combining structure-based virtual screening together with hen egg-white Autotaxin (ewATX) activity assays enabled the discovery of novel small-molecule ATX inhibitors with a 2,4-dihydropyrano[2,3- c ]pyrazole scaffold. These compounds are suggested to bind to the lipophilic pocket, leaving the active site unrestrained. Our most potent compound, ( S )-6-amino-4-(3,4-dichlorophenyl)-3-(4-[(4-fluorobenzyl)oxy]phenyl)-2,4-dihydropyrano[2,3- c ]pyrazole-5-carbonitrile [( S )- 25 ], inhibited human ATX (hATX) with an IC 50 -value of 134 nM. It also blocked ATX-evoked but not LPA-mediated A2058 melanoma cell migration. Noteworthy, molecular modeling correctly predicted the biologically active enantiomer of 2,4-dihydropyrano[2,3- c ]pyrazoles, as verified by compound crystallization and activity assays. Our study established the ewATX activity assay as a valid and affordable tool in ATX inhibitor discovery. Overall, our study offers novel insights and approaches into design of novel ATX inhibitors targeting the hydrophobic pocket instead of the active site. [ABSTRACT FROM AUTHOR]

Somatic cell nuclear transfer (SCNT) is a useful tool for animal cloning, but the efficiency of producing viable offspring by SCNT is very low. To improve this efficiency in the production of cloned pigs, it is critical to understand the interactions between uterine function and cloned embryos during implantation. Lysophosphatidic acid (LPA) is a lipid mediator that plays an important role in the establishment of pregnancy in pigs; however, LPA production in the uterine endometrium of pigs carrying SCNT-cloned conceptuses has not been determined. Therefore, we investigated expression of ENPP2, an LPA-generating enzyme, in the uterine endometrium of gilts with conceptuses derived from SCNT during the implantation period. Uterine endometrial tissue and uterine flushing were obtained from gilts carrying SCNT-derived conceptuses and from gilts carrying conceptuses resulting from natural mating on d 12 of pregnancy. Our results demonstrated no difference in the level of ENPP2 mRNA expression in the uterine endometrium between gilts carrying SCNT-derived conceptuses and gilts carrying naturally-conceived conceptuses, but secretion of ENPP2 protein into the uterine lumen did decrease significantly in pigs with SCNT-derived conceptuses. These results indicate that expression and secretion of ENPP2, which are critical for appropriate LPA production and successful pregnancy, are dysregulated in the uterine endometrium of pigs carrying SCNT-derived conceptuses.

• Genetic variants in ferroptosis-related genes (rs2305128 in ENPP2 and rs1868088 in EPAS1) may increase HDM-induced allergic rhinitis (AR) risk. • A significant increased risk of HDM-induced AR was correlated with an increasing number of risk genotypes. • The joint effect of the two variants was more pronounced among AR patients with moderate-to-severe symptoms. Ferroptosis-related genes disrupt iron homeostasis and enhance lipid peroxidation to initiate respiratory system diseases. However, the association between genetic variants in the ferroptosis-related genes with house dust mite (HDM)-induced allergic rhinitis (AR) susceptibility remains unclear. A case-control study, involving 222 cases and 237 healthy controls from a Chinese population, was conducted to evaluate the relationship between single nucleotide polymorphisms (SNPs) in ferroptosis-related genes and HDM-induced AR risk. A gene-based analysis was performed by multi-marker analysis of genomic annotation (MAGMA) to identify candidate associated ferroptosis-related genes. A logistic regression model and joint analysis were used to assess the effect of SNPs on HDM-induced AR susceptibility. Two independent SNPs (rs2305128 in ENPP2 and rs1868088 in EPAS1) were significantly associated with HDM-induced AR risk (OR = 1.82, 95% CI = 1.19–2.79, P = 5.98 × 10−3, P FDR = 4.88 × 10−2; OR = 2.14, 95% CI = 1.23–3.72, P = 6.95 × 10−3, P FDR = 4.87 × 10−2, respectively). Moreover, combined analysis of these two SNPs revealed that an increased risk of HDM-induced AR was positively associated with an increasing number of risk genotypes (P trend = 8.48 × 10−5). The stratification analysis showed that the cumulative effect of two SNPs on HDM-induced AR risk was more pronounced among patients presenting more serious symptoms and harboring one or two risk genotypes. These findings suggest that the genetic variants in ferroptosis-related genes ENPP2 and EPAS1 may increase HDM-induced AR risk and serve as potential predictors of HDM-induced AR susceptibility. [ABSTRACT FROM AUTHOR]

Autotaxin (ATX; ENPP2) produces the lipid mediator lysophosphatidic acid (LPA) that signals through disparate EDG (LPA 1–3) and P2Y (LPA 4–6) G protein-coupled receptors. ATX/LPA promotes several (patho)physiological processes, including in pulmonary fibrosis, thus serving as an attractive drug target. However, it remains unclear if clinical outcome depends on how different types of ATX inhibitors modulate the ATX/LPA signaling axis. Here, we show that the ATX "tunnel" is crucial for conferring key aspects of ATX/LPA signaling and dictates cellular responses independent of ATX catalytic activity, with a preference for activation of P2Y LPA receptors. The efficacy of the ATX/LPA signaling responses are abrogated more efficiently by tunnel-binding inhibitors, such as ziritaxestat (GLPG1690), compared with inhibitors that exclusively target the active site, as shown in primary lung fibroblasts and a murine model of radiation-induced pulmonary fibrosis. Our results uncover a receptor-selective signaling mechanism for ATX, implying clinical benefit for tunnel-targeting ATX inhibitors. [Display omitted] • ATX is a dual-function protein acting as an LPA-producing enzyme and LPA chaperone • Structural integrity of the ATX tunnel is essential to mediate signaling functions • ATX-bound LPA signals preferentially via P2Y-family LPA receptors • Occupancy of the ATX tunnel is crucial for ziritaxestat to exert inhibition in vivo Salgado-Polo et al. report that occupancy of the tunnel of the LPA-producing enzyme ATX regulates signaling outcome, through P2Y-type LPA receptors. Inhibiting this chaperone function of ATX by tunnel-binding inhibitors, such as ziritaxestat, correlates with drug efficacy in a mouse lung fibrosis model. [ABSTRACT FROM AUTHOR]

Lysophosphatidic acid is a growth factor-like bioactive phospholipid recognising LPA receptors and mediating signalling pathways that regulate embryonic development, wound healing, carcinogenesis, and fibrosis, via effects on cell migration, proliferation and differentiation. Extracellular LPA is generated from lysophospholipids by the secreted hydrolase-ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2; also, AUTOTAXIN/ATX) and metabolised by different membrane-bound phospholipid phosphatases (PLPPs). Here, we use public bulk and single-cell RNA sequencing datasets to explore the expression of Lpar 1-6 , Enpp2 , and Plpp genes under skeletal muscle homeostasis and regeneration conditions. We show that the skeletal muscle system dynamically expresses the Enpp2 - Lpar - Plpp gene axis, with Lpar1 being the highest expressed member among LPARs. Lpar1 was expressed by mesenchymal fibro-adipogenic progenitors and tenocytes, whereas FAPs mainly expressed Enpp2 . Clustering of FAPs identified populations representing distinct cell states with robust Lpar1 and Enpp2 transcriptome signatures in homeostatic cells expressing higher levels of markers Dpp4 and Hsd11b1 . However, tissue injury induced transient repression of Lpar genes and Enpp2 , LPAR1/3 and ENPP2 inhibition significantly decreased the cell-cycle activity of FAPs and impaired fibro-adipogenic differentiation, implicating LPA signalling in the modulation of the proliferative and differentiative fate of FAPs. Together, our results demonstrate the importance of the ENPP2-LPAR-PLPP axis in different muscle cell types and FAP lineage populations in homeostasis and injury, paving the way for further research on the role of this signalling pathway in skeletal muscle homeostasis and regeneration, and that of other organs and tissues, Ex vivo , LPAR1/3 and ENPP2 inhibition significantly decreased the cell-cycle activity of FAPs and impaired fibro-adipogenic differentiation, implicating LPA signalling in the modulation of the proliferative and differentiative fate of FAPs. Together, our results demonstrate the importance of the ENPP2-LPAR-PLPP axis in different muscle cell types and FAP lineage populations in homeostasis and injury, paving the way for further research on the role of this signalling pathway in skeletal muscle homeostasis and regeneration, and that of other organs and tissues, in vivo ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Contreras and Harvey.)

Ferroptosis and immune infiltration play an important role in the pathogenesis of intervertebral disc degeneration (IDD). However, there is still a lack of comprehensive analysis on the interaction between ferroptosis-related genes (FRGs) and immune microenvironment in IDD patients. Therefore, this study aims to explore the correlation between FRGs characteristics and immune infiltration in the progression of IDD. The expression profiles (GSE56081 and GSE70362) and FRGs were downloaded from the comprehensive gene expression omnibus (GEO) and FerrDb database, respectively, and the differences were analyzed using R. The intersection of IDD related differential genes (DEGs) and FRGs was taken as differentially expressed FRGs (DE-FRGs) and GO and KEGG enrichment analysis was conducted. Then, we used least absolute shrinkage and selection operator (LASSO) regression algorithm and support vector machine (SVM) algorithm to screen feature genes and draw ROC curve judge the diagnostic value of key DE-FRGs. Then CIBERSORT algorithm is used to evaluate the infiltration of immune cells and analyze the correlation between key DE-FRGs and immune infiltration. Based on the analysis results, we conducted single gene GSEA analysis on key DE-FRGs. RT-PCR and immunohistochemistry further verified the clinical value of the results of biochemical analysis and screening. Seven key DE-FRGs were screened, including the upregulated genes NOX4 and PIR, and the downregulated genes TIMM9, ATF3, ENPP2, FADS2 and TFAP2A. Single gene GSEA analysis further elucidates the role of DE-FRGs in IDD associated with ferroptosis. Correlation analysis showed that seven key DE-FRGs were closely related to immune infiltration in the development of IDD. Finally, RT-PCR and immunohistochemical staining showed that NOX4, ENPP2, FADS2 and TFAP2A were statistically significant differences. In this study, we explored the connection between ferroptosis related characteristics and immune infiltration in IDD, and confirmed that NOX4, ENPP2, FADS2, and TFAP2A may become biomarkers and potential therapeutic targets for IDD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhang, Cui, Wang, Cheng, Zhai, Jiao, Wang, Cui and Yu.)

Autotaxin, also known as ecto-nucleotide pyrophosphatase/phosphodiesterase family member 2, is a secreted glycoprotein that plays multiple roles in human physiology and cancer pathology. This protein, by converting lysophosphatidylcholine into lysophosphatidic acid, initiates a complex signalling cascade with significant biological implications. The article outlines the autotaxin gene and protein structure, expression regulation and physiological functions, but focuses mainly on the role of autotaxin in cancer development and progression. Autotaxin and lysophosphatidic acid signalling influence several aspects of cancer, including cell proliferation, migration, metastasis, therapy resistance, and interactions with the immune system. The potential of autotaxin as a diagnostic biomarker and promising drug target is also examined.

Background—Mendelian randomization studies have highlighted that lipoprotein(a) [Lp(a)] was associated with calcific aortic valve disease (CAVD). Lp(a) transports oxidized phospholipids with a high content in lysophosphatidylcholine. Autotaxin (ATX) transforms lysophosphatidylcholine into lysophosphatidic acid. We hypothesized that ATXlysophosphatidic acid could promote inflammation/mineralization of the aortic valve. Methods and Results—We have documented the expression of ATX in control and mineralized aortic valves. By using different approaches, we have also investigated the role of ATX-lysophosphatidic acid in the mineralization of isolated valve interstitial cells and in a mouse model of CAVD. Enzyme-specific ATX activity was elevated by 60% in mineralized aortic valves in comparison with control valves. Immunohistochemistry studies showed a high level of ATX in mineralized aortic valves, which colocalized with oxidized phospholipids and apolipoprotein(a). We detected a high level of ATX activity in the Lp(a) fraction in circulation. Interaction between ATX and Lp(a) was confirmed by in situ proximity ligation assay. Moreover, we documented that valve interstitial cells also expressed ATX in CAVD. We showed that ATX-lysophosphatidic acid promotes the mineralization of the aortic valve through a nuclear factor κB/interleukin 6/bone morphogenetic protein pathway. In LDLR-/-/ApoB100/100/IGFII mice, ATX is overexpressed and lysophosphatidic acid promotes a strong deposition of hydroxyapatite of calcium in aortic valve leaflets and accelerates the development of CAVD. Conclusions—ATX is transported in the aortic valve by Lp(a) and is also secreted by valve interstitial cells. ATXlysophosphatidic acid promotes inflammation and mineralization of the aortic valve and thus could represent a novel therapeutic target in CAVD. [ABSTRACT FROM AUTHOR]

Genomic amplification of the c-Jun proto-oncogene has been identified in ~30% of dedifferentiated liposarcomas (DDLPS), but the functional contribution of c-Jun to the progression of DDLPS remains poorly understood. In previous work we showed that knock-down of c-Jun by RNA interference impaired the in vitro proliferation and in vivo growth of a DDLPS cell line (LP6) with genomic amplification of the c-Jun locus. Here, we used gene expression analysis and functional studies in a broad panel of cell lines to further define the role of c-Jun in DDLPS and other soft tissue sarcomas. We show that c-Jun knock-down impairs transition through the G1 phase of the cell cycle in multiple DDLPS cell lines. We also found that high levels of c-Jun expression are both necessary and sufficient to promote DDLPS cell migration and invasion in vitro. Our data suggest that high levels of c-Jun enhance motility in part by driving the expression of ENPP2/Autotaxin. c-Jun over-expression has minimal effects on in vitro proliferation but substantially enhances the in vivo growth of weakly tumourigenic DDLPS cell lines. Finally, we provide evidence that c-Jun genomic amplification and over-expression may have similar functional consequences in other types of soft tissue sarcoma. Our data suggest a model in which relatively low levels of c-Jun are sufficient for in vitro proliferation, but high levels of c-Jun enhance invasiveness and capacity for in vivo tumour growth. These observations provide an explanation for the selective advantage provided by c-Jun genomic amplification in vivo and suggest that sarcomas with elevated c-Jun levels are likely to have a particularly high malignant potential. Data from exon array and RNA-Seq experiments have been deposited in the GEO database (Accession No. GSE57531). [ABSTRACT FROM AUTHOR]

Massive infiltrated and enriched decidual macrophages (dMφ) have been widely regarded as important regulators of maternal-fetal immune tolerance and trophoblast invasion, contributing to normal pregnancy. However, the characteristics of metabolic profile and the underlying mechanism of dMφ residence remain largely unknown. Here, we observe that dMφ display an active glycerophospholipid metabolism. The activation of ENPP2-lysophosphatidic acid (LPA) facilitates the adhesion and retention, and M2 differentiation of dMφ during normal pregnancy. Mechanistically, this process is mediated through activation of the LPA receptors (LPAR1 and PPARG/PPARγ)-DDIT4-macroautophagy/autophagy axis, and further upregulation of multiple adhesion factors (e.g., cadherins and selectins) in a CLDN7 (claudin 7)-dependent manner. Additionally, poor trophoblast invasion and placenta development, and a high ratio of embryo loss are observed in Enpp2 ± , lpar1 and spontaneous abortion mouse models, which should be dependent on the activation of DDIT4-autophagy-CLDN7-adhesion molecules axis. This observation reveals that inactivation of ENPP2-LPA metabolism and insufficient autophagy of dMφ result in resident obstacle of dMφ and further increase the risk of spontaneous abortion, and provides potential therapeutic strategies to prevent spontaneous abortion. -/- ACTB: actin beta; ADGRE1/F4/80: adhesion G protein-coupled receptor E1; Atg5: autophagy related 5; ATG13: autophagy related 13; BECN1: beclin 1; CDH1/E-cadherin: cadherin 1; CDH5/VE-cadherin: cadherin 5; CFSE: carboxyfluorescein succinimidyl ester; CLDN7: claudin 7; CSF1/M-CSF: colony stimulating factor 1; CSF2/GM-CSF: colony stimulating factor 2; Ctrl: control; CXCL10/IP-10: chemokine (C-X-C) ligand 10; DDIT4: DNA damage inducible transcript 4; dMφ: decidual macrophage; DSC: decidual stromal cells; ENPP2/ATX: ectonucleotide pyrophosphatase/phosphodiesterase 2; Enpp2 ± heterozygous knockout mouse; ENPP2i/PF-8380: ENPP2 inhibitor; EPCAM: epithelial cell adhesion molecule; ESC: endometrial stromal cells; FGF2/b-FGF: fibroblast growth factor 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GPCPD1: glycerophosphocholine phosphodiesterase 1; HE: heterozygote; HIF1A: hypoxia inducible factor 1 subunit alpha; HNF4A: hepatocyte nuclear factor 4 alpha; HO: homozygote; ICAM2: intercellular adhesion molecule 2; IL: interleukin; ITGAV/CD51: integrin subunit alpha V; ITGAM/CD11b: integrin subunit alpha M; ITGAX/CD11b: integrin subunit alpha X; ITGB3/CD61: integrin subunit beta 3; KLRB1/NK1.1: killer cell lectin like receptor B1; KRT7/cytokeratin 7: keratin 7; LPA: lysophosphatidic acid; LPAR: lysophosphatidic acid receptor; Abbreviations: ACTB: actin beta; ADGRE1/F4/80: adhesion G protein-coupled receptor E1; Atg5: autophagy related 5; ATG13: autophagy related 13; BECN1: beclin 1; CDH1/E-cadherin: cadherin 1; CDH5/VE-cadherin: cadherin 5; CFSE: carboxyfluorescein succinimidyl ester; CLDN7: claudin 7; CSF1/M-CSF: colony stimulating factor 1; CSF2/GM-CSF: colony stimulating factor 2; Ctrl: control; CXCL10/IP-10: chemokine (C-X-C) ligand 10; DDIT4: DNA damage inducible transcript 4; dMφ: decidual macrophage; DSC: decidual stromal cells; ENPP2/ATX: ectonucleotide pyrophosphatase/phosphodiesterase 2; Enpp2 ± : Enpp2 heterozygous knockout mouse; ENPP2i/PF-8380: ENPP2 inhibitor; EPCAM: epithelial cell adhesion molecule; ESC: endometrial stromal cells; FGF2/b-FGF: fibroblast growth factor 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GPCPD1: glycerophosphocholine phosphodiesterase 1; HE: heterozygote; HIF1A: hypoxia inducible factor 1 subunit alpha; HNF4A: hepatocyte nuclear factor 4 alpha; HO: homozygote; ICAM2: intercellular adhesion molecule 2; IL: interleukin; ITGAV/CD51: integrin subunit alpha V; ITGAM/CD11b: integrin subunit alpha M; ITGAX/CD11b: integrin subunit alpha X; ITGB3/CD61: integrin subunit beta 3; KLRB1/NK1.1: killer cell lectin like receptor B1; KRT7/cytokeratin 7: keratin 7; LPA: lysophosphatidic acid; LPAR: lysophosphatidic acid receptor; lpar1 -/- : lpar1 homozygous knockout mouse; LPAR1i/AM966: LPAR1 inhibitor; LY6C: lymphocyte antigen 6 complex, locus C1; LYPLA1: lysophospholipase 1; LYPLA2: lysophospholipase 2; Lyz2 : lysozyme 2; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MARVELD2: MARVEL domain containing 2; 3-MA: 3-methyladenine; MBOAT2: membrane bound O-acyltransferase domain containing 2; MGLL: monoglyceride lipase; MRC1/CD206: mannose receptor C-type 1; MTOR: mechanistic target of rapamycin kinase; NP: normal pregnancy; PDGF: platelet derived growth factor; PLA1A: phospholipase A1 member A; PLA2G4A: phospholipase A2 group IVA; PLPP1: phospholipid phosphatase 1; pMo: peripheral blood monocytes; p-MTOR: phosphorylated MTOR; PPAR: peroxisome proliferator activated receptor; PPARG/PPARγ: peroxisome proliferator activated receptor gamma; PPARGi/GW9662: PPARG inhibitor; PTPRC/CD45: protein tyrosine phosphatase receptor type, C; Rapa: rapamycin; RHEB: Ras homolog, mTORC1 binding; SA: spontaneous abortion; SELE: selectin E; SELL: selectin L; si CLDN7: CLDN7 -silenced; STAT: signal transducer and activator of transcription; SQSTM1: sequestosome 1; TJP1: tight junction protein 1; VCAM1: vascular cell adhesion molecule 1; WT: wild type.

Prior studies with crosses of the FVB/ NJ ( FVB; seizure-induced cell death-susceptible) mouse and the C57 BL/6J ( B6; seizure-induced cell death-resistant) mouse revealed the presence of a quantitative trait locus ( QTL) on chromosome 15 that influenced susceptibility to kainic acid-induced cell death ( Sicd2). In an earlier study, we confirmed that the Sicd2 interval harbors gene(s) conferring strong protection against seizure-induced cell death through the creation of the FVB.B6- Sicd2 congenic strain, and created three interval-specific congenic lines ( ISCLs) that encompass Sicd2 on chromosome 15 to fine-map this locus. To further localise this Sicd2 QTL, an additional congenic line carrying overlapping intervals of the B6 segment was created ( ISCL-4), and compared with the previously created ISCL-1- ISCL-3 and assessed for seizure-induced cell death phenotype. Whereas all of the ISCLs showed reduced cell death associated with the B6 phenotype, ISCL-4, showed the most extensive reduction in seizure-induced cell death throughout all hippocampal subfields. In order to characterise the susceptibility loci on Sicd2 by use of this ISCL and identify compelling candidate genes, we undertook an integrative genomic strategy of comparing exon transcript abundance in the hippocampus of this newly developed chromosome 15 subcongenic line ( ISCL-4) and FVB-like littermates. We identified 10 putative candidate genes that are alternatively spliced between the strains and may govern strain-dependent differences in susceptibility to seizure-induced excitotoxic cell death. These results illustrate the importance of identifying transcriptomics variants in expression studies, and implicate novel candidate genes conferring susceptibility to seizure-induced cell death. [ABSTRACT FROM AUTHOR]

Somatic cell nuclear transfer (SCNT) is a useful tool for animal cloning, but the efficiency of producing viable offspring by SCNT is very low. To improve this efficiency in the production of cloned pigs, it is critical to understand the interactions between uterine function and cloned embryos during implantation. Lysophosphatidic acid (LPA) is a lipid mediator that plays an important role in the establishment of pregnancy in pigs; however, LPA production in the uterine endometrium of pigs carrying SCNT-cloned conceptuses has not been determined. Therefore, we investigated expression of ENPP2, an LPA-generating enzyme, in the uterine endometrium of gilts with conceptuses derived from SCNT during the implantation period. Uterine endometrial tissue and uterine flushing were obtained from gilts carrying SCNT-derived conceptuses and from gilts carrying conceptuses resulting from natural mating on d 12 of pregnancy. Our results demonstrated no difference in the level of ENPP2 mRNA expression in the uterine endometrium between gilts carrying SCNT-derived conceptuses and gilts carrying naturally-conceived conceptuses, but secretion of ENPP2 protein into the uterine lumen did decrease significantly in pigs with SCNT-derived conceptuses. These results indicate that expression and secretion of ENPP2, which are critical for appropriate LPA production and successful pregnancy, are dysregulated in the uterine endometrium of pigs carrying SCNT-derived conceptuses. [ABSTRACT FROM AUTHOR]

Background: Epidemiologic studies and animal models suggest that in utero arsenic exposure affects fetal health, with a negative association between maternal arsenic ingestion and infant birth weight often observed. However, the molecular mechanisms for this association remain elusive. In the present study, we aimed to increase our understanding of the impact of low-dose arsenic exposure on fetal health by identifying possible arsenic-associated fetal tissue biomarkers in a cohort of pregnant women exposed to arsenic at low levels.Methods: Arsenic concentrations were determined from the urine samples of a cohort of 133 pregnant women from New Hampshire. Placental tissue samples collected from enrollees were homogenized and profiled for gene expression across a panel of candidate genes, including known arsenic regulated targets and genes involved in arsenic transport, metabolism, or disease susceptibility. Multivariable adjusted linear regression models were used to examine the relationship of candidate gene expression with arsenic exposure or with birth weight of the baby.Results: Placental expression of the arsenic transporter AQP9 was positively associated with maternal urinary arsenic levels during pregnancy (coefficient estimate: 0.25; 95% confidence interval: 0.05 - 0.45). Placental expression of AQP9 related to expression of the phospholipase ENPP2 which was positively associated with infant birth weight (coefficient estimate: 0.28; 95% CI: 0.09 - 0.47). A structural equation model indicated that these genes may mediate arsenic's effect on infant birth weight (coefficient estimate: -0.009; 95% confidence interval: -0.032 - -0.001; 10,000 replications for bootstrapping).Conclusions: We identified the expression of AQP9 as a potential fetal biomarker for arsenic exposure. Further, we identified a positive association between the placental expression of phospholipase ENPP2 and infant birth weight. These findings suggest a path by which arsenic may affect birth outcomes. [ABSTRACT FROM AUTHOR]

Abstract: We analyzed global gene expression profiles of IL-4 induced alternatively activated as well as IFNγ+TNFα stimulated classically activated human monocyte derived macrophages and identified novel IL-4 regulated alternative activation marker genes including MS4A4A, SLA, CD180, and ENPP2. Transcription factor prediction analysis of IL-4 regulated genes suggested that the regulated genes are involved in a complex regulation of lipid metabolism, defense against cell metabolism derived reactive oxygen species, and basal expression of inflammation linked genes. Both an in silico transcription activation prediction as well as experimental data suggested the presence of alternative macrophage activation specific endogenous PPARγ ligand producing mechanisms. We found the induction of three enzymes whose activity can potentially generate endogenous PPARγ ligands in an IL-4 dependent manner. These are MAOA, ENPP2, and ALOX15 producing 5-methoxy-indole acetate, lysophosphatidic acid (LPA) and 13-hydroxyoctadienoic acid (13-HODE), and/or 15-hydroxyeicosatetraenoic acid (15-HETE), respectively. Our data suggest that global gene expression profiling, combined with computational transcription activity prediction, can lead to identification of transcriptional networks that underpin cellular subtype specification. [Copyright &y& Elsevier]

Ectonucleotide pyrophosphatase/phosphodiestrase 2 (Enpp2) isolated from the supernatant of human melanoma cells is a lysophospholipase D that transforms lysophosphatidylcholine into lysophospatidic acid. Although multiple analyses have investigated the function of Enpp2 in the hypothalamus, its role in the uterus during the estrous cycle is not well understood. In the present study, rat uterine Enpp2 was analyzed by RT-PCR, Western blotting, and immunohistochemistry. Quantitative PCR analysis demonstrated that uterine Enpp2 mRNA was decreased during estrus compared to proestrus and diestrus. To determine whether uterine Enpp2 expression is affected by sex steroid hormones, immature rats were treated with 17β-estradiol (E2), progesterone, or both on postnatal days 14 to 16. Interestingly, the expression of Enpp2 mRNA and protein were down-regulated by E2 in the uterus during estrus but not during proestrus or diestrus, suggesting that Enpp2 may play a role in uterine function during estrus. Enpp2 is primarily localized in the stromal cells of the endometrium during proestrus and estrus. During diestrus, Enpp2 was highly expressed in the epithelial cells of the endometrium. Taken together, these results suggest that uterine Enpp2 may be regulated by E2 and plays a role in reproductive functions during female rat development. [ABSTRACT FROM AUTHOR]

Abstract: Autotaxin, encoded by the Enpp2 gene, generates lysophosphatidic acid (LPA) extracellularly, eliciting various cellular responses through specific LPA receptors. Previous studies have revealed that Enpp2 −/− mice die at E9.5 owing to angiogenic defects in the yolk sac. Moreover, Enpp2 −/− embryos show growth retardation, allantois malformation, no axial turning, and head cavity formation. We have also demonstrated that lysosome biogenesis is impaired in yolk sac visceral endoderm cells of Enpp2 −/− embryos as a result of the downregulation of the Rho-ROCK (Rho-associated coiled-coil containing protein kinase)-LIM kinase pathway. In this study, we examine what signaling defect(s) is responsible for head cavity formation and yolk sac angiogenic defects. By using a whole embryo culture system, we show that 10μM Ki16425, an antagonist for the LPA receptors, induces head cavity formation and yolk sac angiogenic defects in wild-type embryos. Moreover, 1μM Ki16425 induces both phenotypes in Enpp2 heterozygous embryos at significantly higher incidence than in wild-type embryos, suggesting an interaction between autotaxin and LPA receptor signaling. Furthermore, we show that inhibition of the Rho-ROCK pathway induces head cavity formation, whereas multiple pathways are involved in yolk sac angiogenic defects. These results reveal the signal transduction defects that underlie the abnormalities in Enpp2 −/− embryos. [ABSTRACT FROM AUTHOR]

Autotaxin (ATX or ENPP2) is a secreted glycosylated mammalian enzyme that exhibits lysophospholipase D activity, hydrolyzing lysophosphatidylcholine to the signalling lipid lysophosphatidic acid. ATX is an ∼100 kDa multi-domain protein encompassing two N-terminal somatomedin B-like domains, a central catalytic phosphodiesterase domain and a C-terminal nuclease-like domain. Protocols for the efficient expression of ATX from stably transfected mammalian HEK293 cells in amounts sufficient for crystallographic studies are reported. Purification resulted in protein that crystallized readily, but various attempts to grow crystals suitable in size for routine crystallographic structure determination were not successful. However, the available micrometre-thick plates diffracted X-rays beyond 2.0 Å resolution and allowed the collection of complete diffraction data to about 2.6 Å resolution. The problems encountered and the current advantages and limitations of diffraction data collection from thin crystal plates are discussed. [ABSTRACT FROM AUTHOR]

Introduction: Hepatitis C is the most significant risk factor for development of hepatocellular carcinoma. Inflammation, fibrosis, and liver cell proliferation may contribute to cancer development either through malignant hepatocyte transformation or extracellular matrix remodeling within the tumor microenvironment. The study objective was to investigate differences in gene expression between patients with Hepatitis C (+/- cancer) and normal that might explain the increased cancer risk.Methods: Liver tissue was collected from three patient groups: 1) healthy patients, 2) Hepatitis C patients without cancer, 3) patients with Hepatitis C and hepatocellular carcinoma. Microarray analysis was performed on samples from each group. Western blot and real-time polymerase chain reaction (PCR) analyses corroborated the microarray data. A p value of 0.05 was set as significant.Results: Microarray analysis showed overexpression of autotaxin in patients with cancer versus hepatitis patients or normal patients. Rho GTPase binding proteins (Cdc42s) associated with lysophosphatidic acid signaling were also overexpressed in cancer patients. Real-time polymerase chain reaction showed overexpression of several factors associated with autotaxin in patients with Hepatitis C (+/- cancer) versus normal patients.Conclusions: Patients with Hepatitis C and hepatocellular carcinoma show differential expression of various components of the autotaxin pathway versus normal patients. This merits further investigation in the context of early diagnosis. [ABSTRACT FROM AUTHOR]

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.