Lipid Phosphate Phosphatase-3 Research Articles

Abstract 401: Functional Analysis of Coronary Artery Disease-associated PLPP3 Gene Variants

Heritable variants of the PLPP3 gene are associated with human coronary artery disease risk. Expression of the PLPP3 gene product, lipid phosphate phosphatase 3 (LPP3) is strongly increased in blood and vascular cells by pro-atherogenic, pro-inflammatory stimuli including oxidized LDL. PLPP3 is essential for vascular development in mice but animals with inducible post-natal or tissue specific inactivation of the gene exhibit accelerated development of aortic atherosclerosis. These observations suggest the hypothesis that PLPP3 normally functions to suppress the development and progression of atherosclerosis. The coronary artery disease-associated PPAP2B haplotype block is in the final intron of the gene. Recently published analysis of chromatin remodeling in oxidized LDL stimulated human monocytes identifies dynamic changes in these coronary artery disease associated loci. We show that several of these loci correspond to potential or identified regulatory genomic elements. Functional analysis of these elements identifies multiple cFos/AP1 and cEBP regulated transcriptional enhancer sequences that exhibit allelic variation that could impact on their function as regulators of PLPP3 gene expression. Some of these enhancer sequences bind their transcription factor targets in an allele specific manner and are functional activators of transcription from the PLPP3 promoter. Because haplotypes containing the coronary artery disease associated enchancer sequence variants are also associated with attenuated increases in PPAP2B expression in oxidized LDL treated human monocytes our observations support the hypothesis that heritable cardiovascular disease risk is associated with attenuation of cis acting enhancer sequences that normally promote LPP3 expression. These observations also raise the possibility that these intronic enhancers have longer distance or trans-acting effects on other genes that also contribute to coronary artery disease risk

Endothelial lipid phosphate phosphatase-3 deficiency that disrupts the endothelial barrier function is a modifier of cardiovascular development.

AimsLipid phosphate phosphatase-3 (LPP3) is expressed at high levels in endothelial cells (ECs). Although LPP3 is known to hydrolyse the phosphate group from lysolipids such as spingosine-1-phosphate and its structural homologues, the function of Lpp3 in ECs is not completely understood. In this study, we investigated how tyrosine-protein kinase receptor (TEK or Tie2) promoter–dependent deletion of Lpp3 alters EC activities.Methods and resultsLpp3fl/fl mice were crossed with the tg.Tie2Cre transgenic line. Vasculogenesis occurred normally in embryos with Tie2Cre-mediated deletion of Lpp3 (called Lpp3ECKO), but embryonic lethality occurred in two waves, the first wave between E8.5 and E10.5, while the second between E11.5 and E13.5. Lethality in Lpp3ECKO embryos after E11.5 was accompanied by vascular leakage and haemorrhage, which likely resulted in insufficient cardiovascular development. Analyses of haematoxylin- and eosin-stained heart sections from E11.5 Lpp3ECKO embryos showed insufficient heart growth associated with decreased trabeculation, reduced growth of the compact wall, and absence of cardiac cushions. Staining followed by microscopic analyses of Lpp3ECKO embryos revealed the presence of apoptotic ECs. Furthermore, Lpp3-deficient ECs showed decreased gene expression and protein levels of Cyclin-D1, VE-cadherin, Fibronectin, Klf2, and Klf4. To determine the underlying mechanisms of vascular leakage and barrier disruption, we performed knockdown and rescue experiments in cultured ECs. LPP3 knockdown decreased transendothelial electrical resistance and increased permeability. Re-expression of β-catenin cDNA in LPP3-knockdown ECs partially restored the effect of the LPP3 loss, whereas re-expression of p120ctn cDNA did not.ConclusionThese findings demonstrate the essential roles of LPP3 in the maturation of EC barrier integrity and normal cardiovascular development.

Abstract 342: Lack of Lipid Phosphate Phosphatase 3 Promotes Cardiac Dysfunction and Early Mortality in Mice

Lysophosphatidic acid (LPA) is a naturally occurring glycerophospholipid and has been reported to increase heart rate and left ventricular pressure in the heart in vivo . The primary route of circulating LPA production involves hydrolysis of lysophosphatidylcholine by the secreted enzyme autotaxin (ATX). Lipid phosphate phosphatase-3 (LPP3) is a plasma membrane enzyme that regulates the availability of LPA by dephosphorylation. We made the novel discovery that tissue-specific deficiency of Ppap2b (gene that encodes LPP3) leads to embryonic lethality (endothelial LPP3), exaggerates vascular inflammation, increases heart rate, enhances endothelial permeability, and promotes the development of the neointima after vascular injury. Based on our earlier reports, we have generated mice that specifically lack LPP3 in cardiomyocytes. These mice showed early mortality ~8 months due to cardiac dysfunction. Whereas lack of LPP1 or LPP2 (global knockouts) didn’t had any obvious phenotypic effect. Lack of LPP3 accounts for less than 10 percent activity in cardiomyocytes purified from the Myh6-Ppap2b Δ , which augments our previous finding that the other two LPP isoforms have a lesser role in the cardiovascular system. Blood pressure was similar in Ppap2b fl/fl (96 ± 9 mmHg; n = 19) and Myh6-Ppap2b Δ mice (92 ± 7 mmHg; n = 19), although heart rates were significantly higher in Myh6-Ppap2b Δ 3-month old mice (642 ± 21 bpm, compared to Ppap2b fl/fl with 600± 17 bpm; P<0.001). Knockdown of LPP3 enhanced cardiomyocyte hypertrophy induced by LPA based on analysis of sarcomere organization, cell surface area, levels of fetal genes ANP and BNP, and ANF release from nuclei, which are hallmarks of cardiomyocyte hypertrophy, indicating that LPP3 negatively regulates cardiac dysfunction caused by LPA. We observed an increase in ATX levels accompanied by a decrease in LPP3 expression following infarction in the myocardium of Ppap2b fl/fl mice. Infarction induced expression of IL-6 and KC, were 3 ± 0.5-fold and 2 ± 0.6-fold higher, respectively, in Myh6-Ppap2b Δ mice. Analysis of plasma by cytokine antibody array confirmed the elevation in IL-6 and KC, whereas G-CSF and sICAM-1 appeared lower than in Ppap2b fl/fl .

Abstract 158: PPAP2B Expression Regulates the Development of Atherosclerosis

Coronary artery disease (CAD) is the leading cause of death in both men and women worldwide. The bioactive lipid lysophosphatidic acid (LPA) accumulates in human atheroma compared to levels in healthy control tissue. LPA can be degraded by the membrane protein lipid phosphate phosphatase 3 (LPP3), encoded by the gene PPAP2B. Noncoding polymorphisms in PPAP2B associate with CAD risk, and gene expression analysis indicates that individuals possessing the risk allele exhibit lower levels of PPAP2B mRNA in leukocytes and vascular cells. We hypothesize that decreased LPP3, as a result of low PPAP2B expression, accelerates experimental atherosclerosis. Murine models of tissue specific LPP3 knockdown were generated using the Cre-Lox system driven by either the MX-1 promoter (MX-1Δ) or the smooth muscle cell specific SM22 promoter (SM22Δ) in mice on the atherogenic LDLr -/- background. Following 12 weeks on Western diet, knockdown of LPP3 in both the MX-1Δ and the SM22Δ mice resulted in significantly more atherosclerosis by en face analysis compared to LPP3-fl/fl littermate controls. LPA content determined using LC/MS/MS tended to be higher in the proximal aortas of MX-1Δ and SM22Δ knockdown mice compared to controls. Significantly higher macrophage gene expression was observed in the MX-1Δ mice while IL-6 levels were significantly higher in the SM22Δ mice compared to controls. Finally, targeting LPP3 expression in the MX-1Δ, but not the SM22Δ, mice resulted in significantly higher circulating levels of LPA compared to controls. These results are consistent with accelerated atherosclerosis in LPP3 knockdown mice and suggest a protective role for LPP3 in CAD. Increased vascular LPA content, as a consequence of decreased LPP3 expression, may promote the infiltration of monocyte/macrophages into lesions as well as exacerbate inflammation to accelerate the development of CAD. Our findings provide mechanistic insight into the genome wide association studies that linked genetic variation in PPAP2B with risk of CAD and focus attention on the LPA/ LPP3 signaling nexus as a novel therapeutic strategy to prevent atherosclerosis.

Abstract 656: Lipid Phosphate Phosphatase-3 Mediated Inflammation and Angiogenesis

A recent meta-analysis of genome-wide association studies (GWAS) of coronary artery disease (CAD) involving over 86,000 individuals identified PPAP2B (gene that encodes lipid phosphate phosphatase-3; LPP3) as 1 of 13 new loci that associate with CAD. The PPAP2B loci independently predicted CAD (P = 3.81 X 10-19) and lacked association with traditional risk factors. The lipid phosphate phosphatase 3 is a cell surface integral membrane protein that regulates the bio availability of major bioactive lysophospholipids, like lysophosphatidic acid (LPA). Our group also made the surprising discovery that tissue-specific deficiency of LPP3 exaggerates vascular inflammation and promotes the development of neointima after vascular injury. Here we present evidence that enothelial cell LPP3 serves as an intrinsic negative regulator of vascular inflammation, promotes endothelial barrier function. Our results suggest that loss of LPP3 by siRNA mediated knockdown enhanced proliferation with elevated pErk signaling whereas migration was inhibited in human microvascular endothelial cells (HMEC). The endothelial cells that were silenced for LPP3 were further subjected LPA receptor knockdown and we found that although LPA-induced Erk phosphorylation was similar between LPA 1 and LPA 4 silencing, LPA 4 silencing resulted in enhancement of Akt phosphorylation at Thr-308 in response to LPA. Dual inhibition of LPA 1 and LPA 4 abolished LPA mediated migration in LPP3 silenced HMEC suggesting that the signaling response is mediated through the LPA 1 receptor. Rac activation in response to LPA was barely detectable in LPP3 silenced HMEC, which correlated with its weak migratory response. In contrast, a prominent increase in Rac-GTP levels was observed upon LPA 4 silencing under these conditions. Activation of HMEC with LPA increased phosphorylation of MSK downstream of p38, and CREB phosphorylation was inhibited upon LPA 4 receptor blockade in LPP3 silenced endothelial cells.

LPP3 localizes LPA6 signalling to non-contact sites in endothelial cells.

ABSTRACTLysophosphatidic acid (LPA) is emerging as an angiogenic factor, because knockdown of the enzyme that produces it (autotaxin, also known as ENPP2) and its receptors cause severe developmental vascular defects in both mice and fish. In addition, overexpression of autotaxin in mice causes similar vascular defects, indicating that the extracellular amount of LPA must be tightly regulated. Here, we focused on an LPA-degrading enzyme, lipid phosphate phosphatase 3 (LPP3, also known as PPAP2B), and showed that LPP3 was localized in specific cell–cell contact sites of endothelial cells and suppresses LPA signalling through the LPA6 receptor (also known as LPAR6). In HEK293 cells, overexpression of LPP3 dramatically suppressed activation of LPA6. In human umbilical vein endothelial cells (HUVECs), LPA induced actin stress fibre formation through LPA6, which was substantially upregulated by LPP3 knockdown. LPP3 was localized to cell–cell contact sites and was missing in non-contact sites to which LPA-induced actin stress fibre formation mediated by LPA6 was restricted. Interestingly, the expression of LPP3 in HUVECs was dramatically increased after forskolin treatment in a process involving Notch signalling. These results indicate that LPP3 regulates and localizes LPA signalling in endothelial cells, thereby stabilizing vessels through Notch signalling for proper vasculature.

Abstract 43: Mice With Cardiac-Specific Inactivation Of Ppap2b (Lipid Phosphate Phosphatase 3) Validates Genome-Wide Association Studies.

A recent meta-analysis of Genome-Wide Association Studies (GWAS) of coronary artery disease (CAD) involving over 86,000 individuals identified PPAP2B (encodes lipid phosphate phosphatase-3; LPP3) as a new loci (SNP rs17114036; P= 3.81 X 10 −19 ) that independently predicts CAD. LPP3 is an integral membrane enzyme that dephosphorylates lysophosphatidic acid, sphingosine-1-phosphate and related bioactive lipids. Strikingly, we found that targeted inactivation of LPP3 in endothelial cells results in early embryonic lethality, in part to due to vascular patterning defects. In addition, we found that constitutive inactivation of LPP3 in the myocardium results in cardiac dysfunction, indicating that dysregulation of LPP3-dependent cardiomyocyte cell function. Heart rate was significantly higher in conditional Ppap2b Δ mice (P<0.001), which may indicate a role for LPP3 in regulating heart rate and/or function. Further, we found that myocardial LPP3 levels are altered following myocardial infarction in mice and these results are in accord with our myocardial infarct data from patient that had down-regulated myocardial LPP3 levels. The major rs17114036A allele was associated with a 1.17 odds ratio for CAD. This SNP is located in the final intron of the six exon PPAP2B gene. At least seven SNPs are in robust linkage disequilbrium (r2>0.9) with rs17114036. The sequence surrounding the SNP rs17114036 matches a U1 spliceosome recognition sequence, and the variant base is located at the final position of a sequence with high homology to a U1 spliceosome 5’ spice site recognition motif. Binding of the U1 spliceosome may alter mRNA stability or processing, perhaps by masking cryptic splice sites, thus enabling efficient splicing or promoting polyadenylation of the mature message. To address the polymorphism, we transfected MDA MB 453 cells, which are homozygous for the “T’ allele of rs17114036 SNP, with a short synthetic 2’-O-methyl phosphorothioate RNA. We found a dramatic decrease in both LPP3 protein expression and LPP3 mRNA levels. These results imply that the region containing the rs17114036 SNP may be important for proper processing and/or stability of the LPP3 transcript thereby functionally validating the GWAS study.

Mice With Targeted Inactivation of Ppap2b in Endothelial and Hematopoietic Cells Display Enhanced Vascular Inflammation and Permeability

Lipid phosphate phosphatase 3 (LPP3), encoded by the PPAP2B gene, is an integral membrane enzyme that dephosphorylates, and thereby terminates, the G-protein-coupled receptor-mediated signaling actions of lysophosphatidic acid (LPA) and sphingosine-1-phosphate. LPP3 is essential for normal vascular development in mice, and a common PPAP2B polymorphism is associated with increased risk of coronary artery disease in humans. Herein, we investigate the function of endothelial LPP3 to understand its role in the development and human disease. We developed mouse models with selective LPP3 deficiency in endothelial and hematopoietic cells. Tyrosine kinase Tek promoter-mediated inactivation of Ppap2b resulted in embryonic lethality because of vascular defects. LPP3 deficiency in adult mice, achieved using a tamoxifen-inducible Cre transgene under the control of the Tyrosine kinase Tek promoter, enhanced local and systemic inflammatory responses. Endothelial, but not hematopoietic, cell LPP3 deficiency led to significant increases in vascular permeability at baseline and enhanced sensitivity to inflammation-induced vascular leak. Endothelial barrier function was restored by pharmacological or genetic inhibition of either LPA production by the circulating lysophospholipase D autotaxin or of G-protein-coupled receptor-dependent LPA signaling. Our results identify a role for the autotaxin/LPA-signaling nexus as a mediator of endothelial permeability in inflammation and demonstrate that LPP3 limits these effects. These findings have implications for therapeutic targets to maintain vascular barrier function in inflammatory states.

Lipid Phosphate Phosphatase 3 Negatively Regulates Smooth Muscle Cell Phenotypic Modulation to Limit Intimal Hyperplasia

The lipid phosphate phosphatase 3 (LPP3) degrades bioactive lysophospholipids, including lysophosphatidic acid and sphingosine-1-phosphate, and thereby terminates their signaling effects. Although emerging evidence links lysophosphatidic acid to atherosclerosis and vascular injury responses, little is known about the role of vascular LPP3. The goal of this study was to determine the role of LPP3 in the development of vascular neointima formation and smooth muscle cells (SMC) responses. We report that LPP3 is expressed in vascular SMC after experimental arterial injury. Using gain- and loss-of-function approaches, we establish that a major function of LPP3 in isolated SMC cells is to attenuate proliferation (extracellular signal-regulated kinases) activity, Rho activation, and migration in response to serum and lysophosphatidic acid. These effects are at least partially a consequence of LPP3-catalyzed lysophosphatidic acid hydrolysis. Mice with selective inactivation of LPP3 in SMC display an exaggerated neointimal response to injury. Our observations suggest that LPP3 serves as an intrinsic negative regulator of SMC phenotypic modulation and inflammation after vascular injury, in part, by regulating lysophospholipid signaling. These findings may provide a mechanistic link to explain the association between a PPAP2B polymorphism and coronary artery disease risk.

Abstract 382: Mice with Endothelial-Targeted Inactivation of PPAP2B (Lipid Phosphate Phosphatase 3) Display Enhanced Vascular Inflammation and Permeability

Meta-analysis of data from a series of genome-wide association studies (GWAS) revealed a striking association between the PPAP2B loci and coronary artery disease. The PPAP2B gene encodes for the lipid phosphate phosphatase 3 (LPP3) integral membrane enzyme, that dephosphorylates lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P) and related bioactive lipids. While emerging evidence suggests a role for both LPA and S1P in experimental models of atherosclerosis, essentially nothing is understood about the pathophysiologic role of LPP3, largely due to the fact that genetic inactivation results embryonic lethality. We report that constitutive inactivation of LPP3 in vascular endothelial cells accomplished by breeding mice with floxed Ppap2b to mice expressing Cre recombinase under control of the Tie1 promoter results in embryonic lethality, indicating that dysregulation of LPP3-dependent vascular endothelial cell function likely underlies the developmental phenotype observed in Ppap2b -null embryos. Using an estrogen inducible Cre transgene under control of the Tie1 promoter, we have inactivated Ppap2b expression in endothelial cells in adult mice and confirmed the lack of vascular endothelial LPP3 by immunologic and biochemical approaches. The absence of vascular endothelial LPP3 results in a 2.2±0.5 fold increase in basal vascular permeability, as assessed by Evans blue dye extravasation in lung tissue. The permeability difference is exaggerated following an inflammatory challenge (LPS; 2 mg/kg), and LPS-induced expression of plasma inflammatory markers IL6 and KC were 3.3±0.5 and 1.9±0.6 fold higher in mice lacking endothelial LPP3. LPS-enhanced permeability in the absence of LPP3 was attenuated by administration of an LPA receptor antagonist. Mice lacking LPA receptors 1 and 2 (LPA1, LPA2) or LPA receptor 4 (LPA4) were protected from LPS-mediated permeability. These results demonstrate a fundamental role for LPP3, possibility via modulation of local LPA levels and signaling through LPA1, LPA2, and LPA4 receptors, in maintaining the integrity of the vascular endothelium.

The role of lipid phosphate phosphatase 1 in lymphocyte egress from lymphoid organs (173.39)

Abstract The signaling lipid sphingosine 1-phosphate (S1P) regulates vascular permeability, lymphocyte trafficking, and inflammation. The concentration of S1P is high in blood and lymph compared to lymphoid organs, and this difference guides lymphocyte exit from lymphoid organs into circulation. However, little is known about how S1P distribution is controlled in vivo, and it is not clear how a ubiquitously made lipid functions as a signal that requires precise spatial and temporal control. Lipid phosphate phosphatase 3 (LPP3) is essential to maintain low S1P in the thymus, and we asked whether its homolog LPP1 cooperates with LPP3 to regulate lymphoid S1P. Measurements of Lpp1 messenger RNA indicate that, like Lpp3, Lpp1 is highly expressed by thymic endothelial and epithelial cells, and lymph node (LN) lymphatic endothelial, blood endothelial, and fibroblastic reticular cells. Using mice deficient in LPP1, we have found that LPP1 is not required to maintain low S1P in thymus or LN or to enable efficient lymphocyte egress. Mice doubly deficient in LPP1 and LPP3 preliminarily have a small accumulation of mature thymocytes and minor reduction in the number of T cells in lymph compared to mice deficient in LPP3 alone, suggestive of an egress defect. Further work will test the hypothesis that LPP1 and LPP3 work together to maintain low lymphoid organ S1P and efficient lymphocyte circulation.

Abstract 531: Tie-2/Cre--Mediated Conditional Deletion of Lipid Phosphate Phosphatase-3 Reveals Its Role in Endothelial Cell Apoptosis

Rationale: A recent genome-wide association study (GWAS) has linked a frequently occurring variation in the LPP3 (also known as PPAP2b) loci to increased risk of coronary heart disease (CAD). However, the in vivo function of LPP3 in vascular endothelial cell is incompletely understood. Goal: To address the endothelial cell (EC) specific function of Lpp3 in mice. Results: Tie-2/Cre mediated Lpp3 deletion did not affect normal vasculogenesis in early embryonic development, in contrast, in late embryonic stages it led to impaired angiogenesis associated with hemorrhage, edema and late embryonic lethal phenotype. Immunohistochemical staining followed by microscopic analyses of mutant embryos revealed reduced fibronectin and VE-cadherin expression throughout different vascular bed, and increased apoptosis in CD31+ vascular structures. Transmission electron microscopy (TEM) showed the presence of apoptotic endothelial cells and disruption of adherens junctions in mutant embryos. LPP3-knockdown in vitro showed an increase in p53 and p21 protein levels, with concomitant decrease in cell proliferation. LPP3-knockdown also decreased transendothelial electrical resistance (TER), interestingly re-expression of ß-catenin cDNA into LPP3-depleted endothelial cells partially restored the effect of loss of LPP3. Conclusion: These results suggest the ability of LPP3 to regulate survival and apoptotic activities of endothelial cells during patho/physiological angiogenesis.

Abstract 17554: Mice with Endothelial-Targeted Inactivation of Ppap2b (Lipid Phosphate Phosphatase 3) Display Enhanced Vascular Inflammation

Meta-analysis of data from a series of genome-wide association studies (GWAS) revealed a striking association between the PPAP2B locus and coronary artery disease. The PPAP2B gene encodes for lipid phosphate phosphatase 3 (LPP3), an integral membrane enzyme that dephosphorylates lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P) and related bioactive lipids. While emerging evidence suggests a role for both LPA and S1P in experimental models of atherosclerosis, essentially nothing is understood about the pathophysiologic role of LPP3, largely due to the fact that genetic deletion results embryonic lethality in mice. We report that constitutive inactivation of LPP3 in vascular endothelial cells accomplished by breeding mice with floxed Ppap2b to mice expressing Cre recombinase under control of the Tie1 promoter results in embryonic lethality and a similar defect in vasculogenesis as is observed in mice with global inactivation of Ppap2b , indicating that dysregulation of LPP3-dependent vascular endothelial cell function likely underlies the developmental phenotype observed in Ppap2b -null embryos. Using an estrogen inducible Cre transgene under control of the Tie1 promoter, we have inactivated Ppap2b expression in endothelial cells in adult mice and confirmed the lack of vascular endothelial LPP3 by immunologic and biochemical approaches. The absence of vascular endothelial LPP3 results in a 2.2 ± 0.5 fold increase in basal vascular permeability, as assessed by Evans blue dye extravasation in lung tissue. The permeability difference is exaggerated following an inflammatory challenge (LPS; 2 mg/kg), and LPS-induced expression of plasma inflammatory markers IL6 and KC are 3.3 ± 0.5 and 1.9 ± 0.6 fold higher in mice lacking endothelial LPP3. In addition, loss of endothelial LPP3 impairs angiogenesis in matrigel implanted in mice. These results demonstrate a fundamental role for LPP3 in maintaining the integrity of the vascular endothelium. Importantly, our findings suggest that polymorphisms associated with a reduction in LPP3 expression could promote vascular inflammation and thereby heighten risk of coronary artery disease and myocardial infarction.

Lipid phosphate phosphatase 3 enables efficient thymic egress

The signaling lipid sphingosine-1-phosphate (S1P) stabilizes the vasculature, directs lymphocyte egress from lymphoid organs, and shapes inflammatory responses. However, little is known about how S1P distribution is controlled in vivo, and it is not clear how a ubiquitously made lipid functions as a signal that requires precise spatial and temporal control. We have found that lipid phosphate phosphatase 3 (LPP3) enables efficient export of mature T cells from the thymus into circulation, and several lines of evidence suggest that LPP3 promotes exit by destroying thymic S1P. Although five additional S1P-degrading enzymes are expressed in the thymus, they cannot compensate for the loss of LPP3. Moreover, conditional deletion of LPP3 in either epithelial cells or endothelial cells is sufficient to inhibit egress. These results suggest that S1P generation and destruction are tightly regulated and that LPP3 is essential to establish the balance.

Lipid phosphate phosphatase-3 regulates tumor growth via β-catenin and Cyclin-D1 signaling

BackgroundThe acquisition of proliferative and invasive phenotypes is considered a hallmark of neoplastic transformation; however, the underlying mechanisms are less well known. Lipid phosphate phosphatase-3 (LPP3) not only catalyzes the dephosphorylation of the bioactive lipid sphingosine-1-phosphate (S1P) to generate sphingosine but also may regulate embryonic development and angiogenesis via the Wnt pathway. The goal of this study was to determine the role of LPP3 in tumor cells.ResultsWe observed increased expression of LPP3 in glioblastoma primary tumors and in U87 and U118 glioblastoma cell lines. We demonstrate that LPP3-knockdown inhibited both U87 and U118 glioblastoma cell proliferation in culture and tumor growth in xenograft assays. Biochemical experiments provided evidence that LPP3-knockdown reduced β-catenin, CYCLIN-D1, and CD133 expression, with a concomitant increase in phosphorylated β-catenin. In a converse experiment, the forced expression of LPP3 in human colon tumor (SW480) cells potentiated tumor growth via increased β-catenin stability and CYCLIN-D1 synthesis. In contrast, elevated expression of LPP3 had no tumorigenic effects on primary cells.ConclusionsThese results demonstrate for the first time an unexpected role of LPP3 in regulating glioblastoma progression by amplifying β-catenin and CYCLIN-D1 activities.

Lipid phosphate phosphatase 3 maintains low thymic sphingosine-1-phosphate and enables efficient thymic egress (149.8)

Abstract The signaling lipid sphingosine-1-phosphate (S1P) stabilizes the vasculature, directs lymphocyte circulation, and shapes inflammatory responses. However, little is known about how S1P distribution is controlled in vivo, and it is not clear how a ubiquitously made lipid functions as a signal that requires precise spatial and temporal control. Here we have found that lipid phosphate phosphatase 3 (LPP3) maintains low thymic S1P and efficient egress of mature T cells from the thymus into circulation. Although five additional S1P-degrading enzymes are expressed in the thymus, they cannot compensate for the loss of LPP3. Moreover, conditional deletion of LPP3 in either epithelial cells or endothelial cells is sufficient to increase thymic S1P. These results suggest that S1P generation and destruction are tightly regulated, and LPP3 is essential to establish the balance.

Expression of LPP3 in Bergmann glia is required for proper cerebellar sphingosine-1-phosphate metabolism/signaling and development.

Bioactive lipids serve as intracellular and extracellular mediators in cell signaling in normal and pathological conditions. Here we describe that an important regulator of some of these lipids, the lipid phosphate phosphatase-3 (LPP3), is abundantly expressed in specific plasma membrane domains of Bergmann glia (BG), a specialized type of astrocyte with key roles in cerebellum development and physiology. Mice selectively lacking expression of LPP3/Ppap2b in the nervous system are viable and fertile but exhibit defects in postnatal cerebellum development and modifications in the cytoarchitecture and arrangement of BG with a mild non-progressive motor coordination defect. Lipid and gene profiling studies in combination with pharmacological treatments suggest that most of these effects are associated with alterations in sphingosine-1-phosphate (S1P) metabolism and signaling. Altogether our data indicate that LPP3 participates in several aspects of neuron-glia communication required for proper cerebellum development.

Lipid Phosphate Phosphatase 3 Stabilization of β-Catenin Induces Endothelial Cell Migration and Formation of Branching Point Structures

Endothelial cell (EC) migration, cell-cell adhesion, and the formation of branching point structures are considered hallmarks of angiogenesis; however, the underlying mechanisms of these processes are not well understood. Lipid phosphate phosphatase 3 (LPP3) is a recently described p120-catenin-associated integrin ligand localized in adherens junctions (AJs) of ECs. Here, we tested the hypothesis that LPP3 stimulates beta-catenin/lymphoid enhancer binding factor 1 (beta-catenin/LEF-1) to induce EC migration and formation of branching point structures. In subconfluent ECs, LPP3 induced expression of fibronectin via beta-catenin/LEF-1 signaling in a phosphatase and tensin homologue (PTEN)-dependent manner. In confluent ECs, depletion of p120-catenin restored LPP3-mediated beta-catenin/LEF-1 signaling. Depletion of LPP3 resulted in destabilization of beta-catenin, which in turn reduced fibronectin synthesis and deposition, which resulted in inhibition of EC migration. Accordingly, reexpression of beta-catenin but not p120-catenin in LPP3-depleted ECs restored de novo synthesis of fibronectin, which mediated EC migration and formation of branching point structures. In confluent ECs, however, a fraction of p120-catenin associated and colocalized with LPP3 at the plasma membrane, via the C-terminal cytoplasmic domain, thereby limiting the ability of LPP3 to stimulate beta-catenin/LEF-1 signaling. Thus, our study identified a key role for LPP3 in orchestrating PTEN-mediated beta-catenin/LEF-1 signaling in EC migration, cell-cell adhesion, and formation of branching point structures.

Inducible Capillary Formation in Lymphatic Endothelial Cells by Blocking Lipid Phosphate Phosphatase-3 Activity

Lymphangiogenesis plays critical roles under normal and/or pathological conditions; however, the molecular contributors to this event were unknown until recently. In the present study, we first employed gene chip analysis and confirmed that lipid phosphate phosphatase-3 (LPP3) expression was increased until capillary formation in the conditionally immortalized rat lymphatic endothelial cell line. Signaling responses occur when several lipids induce acute biological functions; further, lipid phosphate phosphatases (LPPs) control their functions via dephosphorylation; however, there is no report on the association between LPP3 and lymphangiogenesis. siRNA-targeted LPP3 significantly increased capillary formation of human lymphatic endothelial cells; in contrast, it decreased cell adhesion to the basement membrane matrix. Furthermore, the inducible effect of the LPP inhibitor on capillary formation was observed. For the first time, we report that LPP3 abolishes accelerated abnormal lymphangiogenesis. Blocking LPP3 activities may aid in the development of novel therapy for lymph vessel defects.