Expression Of ENPP2 Research Articles

Regulation of TGF-β1-induced fibroblast differentiation of human periodontal ligament stem cells through the mutually antagonistic action of ectonucleotide pyrophosphatase/phosphodiesterase 1 and 2.

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.

Role of enterocyte Enpp2 and autotaxin in regulating lipopolysaccharide levels, systemic inflammation, and atherosclerosis

Conversion of lysophosphatidylcholine to lysophosphatidic acid (LPA) by autotaxin, a secreted phospholipase D, is a major pathway for producing LPA. We previously reported that feeding Ldlr−/− mice standard mouse chow supplemented with unsaturated LPA or lysophosphatidylcholine qualitatively mimicked the dyslipidemia and atherosclerosis induced by feeding a Western diet (WD). Here, we report that adding unsaturated LPA to standard mouse chow also increased the content of reactive oxygen species and oxidized phospholipids (OxPLs) in jejunum mucus. To determine the role of intestinal autotaxin, enterocyte-specific Ldlr−/−/Enpp2 KO (intestinal KO) mice were generated. In control mice, the WD increased enterocyte Enpp2 expression and raised autotaxin levels. Ex vivo, addition of OxPL to jejunum from Ldlr−/− mice on a chow diet induced expression of Enpp2. In control mice, the WD raised OxPL levels in jejunum mucus and decreased gene expression in enterocytes for a number of peptides and proteins that affect antimicrobial activity. On the WD, the control mice developed elevated levels of lipopolysaccharide in jejunum mucus and plasma, with increased dyslipidemia and increased atherosclerosis. All these changes were reduced in the intestinal KO mice. We conclude that the WD increases the formation of intestinal OxPL, which i) induce enterocyte Enpp2 and autotaxin resulting in higher enterocyte LPA levels; that ii) contribute to the formation of reactive oxygen species that help to maintain the high OxPL levels; iii) decrease intestinal antimicrobial activity; and iv) raise plasma lipopolysaccharide levels that promote systemic inflammation and enhance atherosclerosis.

Investigation of Lipid Metabolism Dysregulation in Chronic Lymphocytic Leukemia and the Role of ENPP2 in Tumorigenesis of Chronic Lymphocytic Leukemia

Introduction Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western world, and although the advent of novel targeted drugs has greatly improved the prognosis of CLL patients, CLL remains incurable at present. Improving the risk stratification of CLL patients and finding new CLL therapeutic targets are urgent issues to be addressed. Methods We performed untargeted metabolomic analysis using LC-MS on sera from CLL patients and healthy controls (HC). Statistical analysis was used to screen for differential metabolites (DMs). Differentially expressed genes (DEGs) were also screened for enrichment pathway enrichment relying on transcriptome data analysis through the GEO database. Finally, DMs and DEGs were analyzed together to explore CLL metabolic alteration pathways. In this study, we targeted the gene ENPP2. Peripheral blood samples from 82 CLL patients were collected at the Department of Hematology in Shandong Provincial Hospital. Expressions levels of ENPP2 in CLL patients were determined by quantitative RT-PCR. In addition, cell viability and apoptosis were assessed by cell counting kit-8 and annexin V-PE/7AAD staining. Results A total of 52 differential metabolites were annotated, 40 of them were up-regulated and 12 were down-regulated. There were significant differences in sphingolipids (SP), glycerolipids (GL), glycerophospholipids (GP), and fatty acids (FA) levels in CLL patients compared to health controls. Fifty-two differential metabolites between samples from CLL patients and HC were analyzed by pathway analysis using metaboanalyst v5.0. KEGG enrichment showed that differential metabolites in CLL patients and HC were mainly annotated and enriched in human diseases, metabolism, and organismal systems (Figure 1A). Furthermore, we obtained differential expression information from the GEO database for CLL patients with HC. A total of 539 DEGs were screened, of which 279 were up-regulated and 260 were down-regulated. A total of 257 KEGG pathways were enriched. In the current work, a joint analysis of the two histological data was performed to validate each other. A total of 20 KEGG pathways were repeatedly obtained from metabolomics and transcriptomics data-based pathway analysis, including pathways in cancer, insulin resistance, long-term depression, regulation of lipolysis in adipocytes metabolic pathways, glycerophospholipid metabolism, glycerolipid metabolism, sphingolipid metabolism, arachidonic acid metabolism, ether lipid metabolism, inositol phosphate metabolism, choline metabolism in cancer, phospholipase D signaling pathway. A PPI network was constructed for the identified significantly different genes, from which we also screened for the gene ENPP2, a secreted lysophospholipase D, that we desired to investigate further (Figure 1B). We validated the difference in ENPP2 expression between CLL patients and healthy donors in the GEO database and its guiding effect on CLL prognosis. The results showed that elevated expression of ENPP2 in CLL patients and relatively high expression in CLL patients implied a worse prognosis. In addition, to validate the role of ENPP2 in CLL, we did the further investigation in CLL patients in our own center. Aberrantly increased expression of ENPP2 were detected in CLL primary cells and cell line (MEC1) at mRNA compared with normal B cells. To investigate the biological processes of ENPP2 involving in CLL progression, functional assays were performed. The survival rate of CLL cells treated with ENPP2 inhibitor PF-8380 was significantly decreased, while apoptosis was increased. In addition, we performed a combination of ibrutinib with PF8380, which showed a further decrease in cell viability and showed better anti-CLL effects. Conclusion Taken together, the present work provides evidence that the identified metabolic biomarkers can be used for CLL diagnosis and screening. The combined analysis of metabolomics and transcriptomics data provides a comprehensive understanding of the metabolic pathways in CLL. Besides, this study was the first investigation on the role of ENPP2 in CLL. ENPP2 expression was increased in CLL cells, and the ENPP2 inhibitor PF-8380 inhibited cell proliferation and induced apoptosis, with therapeutic potential. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Autotaxin is a potential link between genetic risk factors and immunological disturbances of plasmacytoid dendritic cells in systematic lupus erythematosus.

The importance of autotaxin, an enzyme that catalyzes lysophospholipid production, has recently been recognized in various diseases, including cancer and autoimmune diseases. Herein, we examined the role of autotaxin in systemic lupus erythematosus (SLE), utilizing data from ImmuNexUT, a comprehensive database consisting of transcriptome data and expression quantitative trait locus (eQTL) data of immune cells from patients with immune-mediated disorders. Serum autotaxin concentrations in patients with SLE and healthy controls (HCs) were compared. The transcriptome data of patients with SLE and age- and sex-matched HCs were obtained from ImmuNexUT. The expression of ENPP2, the gene encoding autotaxin, was examined in peripheral blood immune cells. Next, weighted gene correlation network analysis (WGCNA) was performed to identify genes with expression patterns similar to ENPP2. The ImmuNexUT eQTL database and public epigenomic databases were used to infer the relationship between autotaxin and pathogenesis of SLE. Autotaxin levels were elevated in the serum of patients with SLE compared to HCs. Furthermore, the expression of ENPP2 was higher in plasmacytoid dendritic cells (pDCs) than in other immune cell subsets, and its expression was elevated in pDCs of patients with SLE compared to HCs. In WGCNA, ENPP2 belonged to a module that correlated with disease activity. This module was enriched in interferon-associated genes and included genes whose expression was influenced by single-nucleotide polymorphisms associated with SLE, suggesting that it is a key module connecting genetic risk factors of SLE with disease pathogenesis. Analysis utilizing the ImmuNexUT eQTL database and public epigenomic databases suggested that the increased expression of ENPP2 in pDCs from patients with SLE may be caused by increased expression of interferon-associated genes and increased binding of STAT3 complexes to the regulatory region of ENPP2. Autotaxin may play a critical role in connecting genetic risk factors of SLE to disease pathogenesis in pDCs.

Autotaxin signaling facilitates β cell dedifferentiation and dysfunction induced by Sirtuin 3 deficiency

Objectiveβ cell dedifferentiation may underlie the reversible reduction in pancreatic β cell mass and function in type 2 diabetes (T2D). We previously reported that β cell-specific Sirt3 knockout (Sirt3f/f;Cre/+) mice developed impaired glucose tolerance and glucose-stimulated insulin secretion after feeding with high fat diet (HFD). RNA sequencing showed that Sirt3-deficient islets had enhanced expression of Enpp2 (Autotaxin, or ATX), a secreted lysophospholipase which produces lysophosphatidic acid (LPA). Here, we hypothesized that activation of the ATX/LPA pathway contributed to pancreatic β cell dedifferentiation in Sirt3-deficient β cells. MethodsWe applied LPA, or lysophosphatidylcoline (LPC), the substrate of ATX for producing LPA, to MIN6 cell line and mouse islets with altered Sirt3 expression to investigate the effect of LPA on β cell dedifferentiation and its underlying mechanisms. To examine the pathological effects of ATX/LPA pathway, we injected the β cell selective adeno-associated virus (AAV-Atx-shRNA) or negative control AAV-scramble in Sirt3f/f and Sirt3f/f;Cre/+ mice followed by 6-week of HFD feeding. ResultsIn Sirt3f/f;Cre/+ mouse islets and Sirt3 knockdown MIN6 cells, ATX upregulation led to increased LPC with increased production of LPA. The latter not only induced reversible dedifferentiation in MIN6 cells and mouse islets, but also reduced glucose-stimulated insulin secretion from islets. In MIN6 cells, LPA induced phosphorylation of JNK/p38 MAPK which was accompanied by β cell dedifferentiation. The latter was suppressed by inhibitors of LPA receptor, JNK, and p38 MAPK. Importantly, inhibiting ATX in vivo improved insulin secretion and reduced β cell dedifferentiation in HFD-fed Sirt3f/f;Cre/+ mice. ConclusionsSirt3 prevents β cell dedifferentiation by inhibiting ATX expression and upregulation of LPA. These findings support a long-range signaling effect of Sirt3 which modulates the ATX-LPA pathway to reverse β cell dysfunction associated with glucolipotoxicity.

Enpp2 Expression by Dendritic Cells Is a Key Regulator in Migration.

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.

Enhancer-mediated enrichment of interacting JMJD3-DDX21 to ENPP2 locus prevents R-loop formation and promotes transcription.

ENPP2, which encodes for the enzyme autotaxin (ATX), is overexpressed during chronic inflammatory diseases and various cancers. However, the molecular mechanism involved in the ENPP2 transcription remains elusive. Here, in HEK 293T cells, we demonstrated that lipopolysaccharide (LPS) increased the transcription process at ENPP2 locus through a NF-кB pathway and a reduction of H3K27me3 level, a histone repressive mark, by the demethylase UTX. Simultaneously, the H3K27me3 demethylase JMJD3/KDM6B was recruited to the transcription start site (TSS), within the gene body and controlled the expression of ENPP2 in a non-enzymatic manner. Mass spectrometry data revealed a novel interaction for JMJD3 with DDX21, a RNA helicase that unwinds R-loops created by nascent transcript and DNA template. Upon LPS treatment, JMJD3 is necessary for DDX21 recruitment at ENPP2 locus allowing the resolution of aberrant R-loops. CRISPR-Cas9-mediated deletion of a distant-acting enhancer decreased the expression of ENPP2 and lowered the recruitment of JMJD3–DDX21 complex at TSS and its progression through the gene body. Taken together, these findings revealed that enhancer-mediated enrichment of novel JMJD3–DDX21 interaction at ENPP2 locus is necessary for nascent transcript synthesis via the resolution of aberrant R-loops formation in response to inflammatory stimulus.

RNA Expression Profile Using Targeted NGS As a Potential Predictor of Early Molecular Response and Relapse in Core-Binding Factor Acute Myeloid Leukemia

Background: Core-binding factor (CBF) acute myeloid leukemias (AML) are classified as favorable prognosis subgroup but outcomes do not approach that of acute promyelocytic leukemia. We and others have shown that that 3-log reduction in fusion transcript by quantitative PCR (qPCR) at the end of induction portends for better outcome. In this study, we focused on finding gene expressions by RNA sequencing from samples at first diagnosis that could predict early molecular remission. In addition, RNA seq allowed us to identify biological difference distinguishing between the two cytogenetic subgroups [Inversion (Inv) 16 vs t (8;21)] that potentially can be used therapeutic approaches and to detect low frequency mutations.Methods: Samples collected at diagnosis from forty-five patients with CBF-AML, treated with fludarabine, cytarabine and granulocyte colony stimulating factor (FLAG) based regimen were analyzed. RNA sequencing was performed by hybridization based targeted panel that detects fusion, expression, and mutations in the 1385 genes (Illumina, San Diego, CA). Each sample was deeply sequenced (>55M reads) using Illumina paired-end 75 bp sequencing and duplicate reads were removed before fragment per kilobase of transcript per million mapped reads (FPKMs) were calculated for each gene. The expression data was mean centered around expression of ABL1. Cytogenetic subgroups were classified by a Bayes multi-variate logistic regression model. The models were evaluated by the Leave One Out Cross-validation technique.Results: The median age of patients was 36 years (range, 22-78); 26 patients (58%) had Inv16 and 19 (42%) had t(8;21). All patients achieved remission and 7 patients relapsed; 4 had t(8;21) and 3 had Inv16. The expression of RUNX1T1, NR6A1, VEGFA, PLCG1, and CD58 mRNA was significantly (P<0.00001) higher and MYH11, IRF8, SPECC1, LPAR1, and TNFRSF11A were significantly (P<0.00001) lower in t(8;21) than in Inv16. High expressions of GSN (p = 0.0005), TACC3 (p = 0.0012) were significantly associated with 3-log reduction in qPCR post-induction in patients with Inv16, as compared to CCDC6, SDHA, NPM1 (all p < 0.005) in patients with t(8;21). In addition high expressions of CD44, FUS, MKL1, TCF7L2 and ELK4 were associated with t(8;21) with a relatively lower degree of significance (all p<.015). Expression of ENPP2, SOCS3, and CIC correlated negatively (P<0.001) with relapse. In multivariate analysis, expression of CIC, ENPP2, and USP6 genes were strong predictor of relapse for all patients (F-score=92.6%), while the expression of USP6 and FASLG genes were predictor of relapse in Inv16 patients (F-score= 97.9) and the expression of CSBPD and PRICKLE1 were predictor of relapse in t(8;21) with F-score=100%.Our analysis also identified several mutations not commonly described in CBF AML; RICTOR (n=5), ACACA (n=4), FIP1L1, KDM5A, MSH6 (all n=3), KMT2D, ATM, PRKCA (all n= 2). Mutations in 43 other genes were seen in only one patient each. Presence of KIT (n = 16, 36%) or FLT3 (n = 10, 22%) mutations did not predict for relapse and this was consistent our prior analysis.Conclusion: Our data suggests that baseline targeted RNA NGS profiling, not only will detect the Inv16 and t(8;21) fusion RNA, but might help to predict early molecular response and relapse in patients with CBF-AML. Pathway based analysis to understand underlying biology of cytogenetic subgroups and molecular response prediction is ongoing. Our next steps will include accessing publicly available databases for further validation of data. DisclosuresCortes:novartis: Research Funding. DiNardo:Karyopharm: Honoraria; Agios: Consultancy; Abbvie: Honoraria; Bayer: Honoraria; Medimmune: Honoraria; Celgene: Honoraria. Kadia:Novartis: Consultancy; Jazz: Consultancy, Research Funding; Celgene: Research Funding; Celgene: Research Funding; Abbvie: Consultancy; Amgen: Consultancy, Research Funding; Novartis: Consultancy; BMS: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Abbvie: Consultancy; Amgen: Consultancy, Research Funding; Takeda: Consultancy; Pfizer: Consultancy, Research Funding; BMS: Research Funding; Takeda: Consultancy.

Proteomics Based Identification of Autotaxin As An Anti-Hepatitis B Virus Factor and a Promoter of Hepatoma Cell Invasion and Migration

Background/Aims: Hepatitis B virus (HBV) infection is a major cause of cirrhosis and hepatocellular carcinoma. Therefore, we aimed to obtain further information on HBV pathogenesis, and to search for novel putative molecules for anti-HBV therapy. Methods: We utilized Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) to identify the secretory proteins that are differentially expressed in the HBV DNA-transfected HepG2.2.15 cell line and its parental HepG2 cell line. Immunohistochemistry (IHC) was employed to assess the clinical relevance of the observations. Small interfering (si)RNA-based silencing transfection methods were carried out to study the function of ENPP2. Results: Totally, 133 unique proteins were identified as differentially expressed in HepG2.2.15 cell line compared with HepG2 cell line. Ectonucleotide pyrophosphatase/phosphodiesterase family member 2 precursor (ENPP2) is one of the most significantly up-regulated secretory proteins associated with HBV replication. This differential expression of ENPP2 was further validated by real-time quantitative RT-PCR, Western Blot and immunohistochemical analysis. To study the function of ENPP2, we knockdown ENPP2 expression in HepG2.2.15 cell line by RNA interference. ENPP2 silencing increased HBV replication approximately 2.3-fold by enhancing, via the type I IFN signaling pathway, HBV cccDNA (covalently closed circular DNA) translation into viral RNA. Moreover, attenuation of ENPP2 expression inhibited both the invasion and migration ability of hepatoma cells in vitro via interacting with the molecules in the tumor microenvironment. Conclusion: Our study demonstrates that ENPP2 may be a novel anti-HBV target and indicate that suppression of its expression may inhibit the invasion and migration ability of hepatoma cells.

453 The role of purinergic signaling in development of irritant dermatitis of acrodermatitis enteropathica

Purinergic signaling is crucial to maintain homeostasis of inflammation. We previously elucidated that disruption of the homeostasis of skin inflammation by Zn deficiency-mediated ablation of Langerhans cells (LCs) that expressed CD39 (E-NTPDase1) was causative for development of dermatitis in acrodermatitis enteropathica. ATP was hydrolyzed into AMP by E-NTPDases (Entpd), E-NPPs (Enpp), and tissue non-specific alkaline phosphatases (TNAP). It has been well known that LCs express Entpd1 (CD39) and keratinocytes (KCs) do not express it in both mice and humans. However, the expression of other ATP-hydrolyzing molecules such as Entpd 2-3, Enpp 1-3, and TNAP in LCs and KCs is not addressed so far. The purpose of this study is 1. to determine the expression of these ATP-hydrolyzing molecules in LCs and KCs of both mice and humans by qPCR, and 2. to examine the alteration of expression of these ATP-hydrolyzing molecules under Zn deficient condition by qPCR. Normal murine KCs expressed Entpd2, Enpp1, and low levels of Enpp2 and TNAP, whereas normal human epidermal KCs (NHEKs) expressed Entpd2, Entpd3, and Enpp1. Normal murine LCs expressed Entpd1 (CD39), Entpd2, and low level of Enpp1, whereas normal human LCs expressed Entpd1 (CD39), and low levels of Enpp2 and Enpp3. KCs from Zn-deficient diet mice showed increased expression of Entpd2 and Enpp1, and loss of expression of Enpp2 and TNAP. Finally, we examined the impact of LCs in ATP hydrolysis. ATP hydrolysis activity was determined by measuring released phosphate. ATP hydrolysis activity was impaired up to 82% in LC-depleted murine epidermis compared with LC-existing murine epidermis. These data suggested that the expression of ATP-hydrolyzing molecules in LCs and KCs were different between mice and humans. Additionally, Zn deficiency altered the expression of ATP-hydrolyzing molecules in KCs. Lastly, Entpd1 (CD39)- and Entpd2-expressing LCs assumed about 80% of ATP hydrolysis, whereas Entpd2- and Enpp1-expressing KCs assumed the remaining 20% ATP hydrolysis in murine epidermis.

ENPP2 contributes to adipose tissue expansion and insulin resistance in diet-induced obesity.

Body weight is tightly regulated by food intake and energy dissipation, and obesity is related to decreased energy expenditure (EE). Herein, we show that nucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2, autotaxin) is an adipose-derived, secreted enzyme that controls adipose expansion, brown adipose tissue (BAT) function, and EE. In mice, Enpp2 was highly expressed in visceral white adipose tissue and BAT and is downregulated in hypertrophied adipocytes/adipose tissue. Enpp2(+/-) mice and adipocyte-specific Enpp2 knockout mice fed a high-fat diet showed smaller body weight gains and less insulin resistance than control mice fed the same diet. BAT was functionally more active and EE was increased in Enpp2-deficient mice. In humans, ENPP2 expression in subcutaneous fat and ENPP2 levels in serum were reduced in obese subjects. Taken together, our results establish ENPP2 as an adipose-derived, secreted enzyme that regulates adipose obesity and systemic metabolism. They also suggest ENPP2 could be a useful therapeutic target for the treatment of metabolic disease.

Analysis of ENPP2 in the Uterine Endometrium of Pigs Carrying Somatic Cell Nuclear Transfer Cloned Embryos.

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.

Analysis of the Lysophosphatidic Acid-Generating Enzyme ENPP2 in the Uterus During Pregnancy in Pigs1

Lysophosphatidic acid (LPA), a simple phospholipid, plays a critical role in the establishment of pregnancy in pigs. LPA production is mediated by the action of ENPP2, a secreted lysophospholipase D (lysoPLD) that converts lysophosphatidylcholine to LPA. However, the mechanism that regulates LPA production by ENPP2 in the porcine uterus is not well understood. In this study, we evaluated ENPP2 expression during the estrous cycle and pregnancy in the uterine endometrium and in early stage conceptuses. We also evaluated lysoPLD activity in the uterine lumen. ENPP2 transcripts and proteins were detected in the uterine endometrium at all stages of the estrous cycle and pregnancy, with higher levels on Day (D) 12 and D15 of the estrous cycle and pregnancy. ENPP2 expression was localized mainly in luminal and glandular epithelial cells in the endometrium and was also detected in conceptuses on D12 of pregnancy. Secreted ENPP2 protein was detected in fluid flushing samples from the uterine lumen on D12 of the estrous cycle and pregnancy, with higher levels on D12 of pregnancy. LysoPLD activity was detected in uterine flushings on D12 of the estrous cycle and pregnancy, with higher levels on D12 of pregnancy. This study showed that uterine endometrium and conceptuses produce ENPP2 and secreted it into the uterine lumen where it has lysoPLD activity. These results suggest that ENPP2 may play an important role in the establishment of pregnancy in pigs by regulating LPA production at the maternal-conceptus interface.

Expression and regulation of Enpp2 in rat uterus during the estrous cycle

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.

The Expression of ENPP2 in Rat Uterus During the Estrous Cycle.

The Expression of ENPP2 in Rat Uterus During the Estrous Cycle.

High Expression of ENPP2/Autotaxin in Patients with FLT3-ITD Positive Acute Myeloid Leukemia.

The Fms-like tyrosine kinase 3 (FLT-3) receptor tyrosine kinase acts as an important player in controlling the growth and differentiation of haematopoietic progenitor cells. It was shown that activating mutations of FLT-3 are involved in the pathogenesis of acute myeloid leukemia (AML). The most frequent aberration is an internal tandem duplication (ITD) of the juxtamembrane domain coding sequence, which is present in up to 27% of the patients with AML and has been associated with poorer prognosis. To further understand the factors involved, we performed microarray analysis to identify genes specifically upregulated in cases with FLT3-ITD mutations. One gene we identified was ENPP2/Autotaxin (ENPP2/ATX). The ENPP2/ATX protein acts as a lysophospholipase D through generating lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). These two bioactive small lipids have crucial functions in cell migration and proliferation and act via G-protein coupled receptors. ENPP2/ATX has been associated with tumor cell migration in several solid tumors, expression has not been reported in myeloid cells before. The aim of the current study was to investigate the expression pattern of ENPP2/ATX in normal haematopoiesis and leukemia as well as its role in the pathogenesis of AML. Real-Time PCR analysis of 215 primary AML samples from all major cytogenetic risk groups confirmed a significant upregulation in FLT3-ITD patients. High expression was also found in cases with complex karyotype and −5 and −7, but not in cases with good risk cytogenetics. To explore the function of ENPP2/ATX in the haematopoietic system, we stably overexpressed two different splice variants of ENPP2/ATX in several leukemic cell lines. The expression was detected at both, mRNA and protein level by RT-PCR and Western blot analyses, respectively. The lysophospholipase D activity of the protein could be measured by a functional assay using an artificial substrate for ENPP2/ATX. Transwell migration assays showed an increased migration capacity of cells with high expression of ENPP2, which indicates that the overexpressed protein is functional. Based on our results, we suggest that ENPP2/ATX is involved in leukemic cell migration. Our findings point to a close link between ENPP2/ATX and FLT3 in the pathogenesis of AML. Current work focuses on its role in cell survival and prognosis.