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1.
FEBS Lett ; 586(20): 3681-91, 2012 Oct 19.
Article in English | MEDLINE | ID: mdl-22975311

ABSTRACT

The motility, angiogenesis and metastasis-stimulating factor Autotaxin (Atx), over expressed by human neuroblastomas (NB), is constitutively expressed by human Nmyc-amplified SK-N-BE and non-Nmyc-amplified SH-SY5Y NB cells. Here, we characterise a novel Atx transcriptional mechanism, utilised by both cell lines, that is restricted to the first 285bp of the Atx promoter and involves AP-1 and SP transcription factors, acting through a CRE/AP-1-like element at position -142 to -149 and a GAbox at position -227 to -235 relative to the Atx translational start site. This novel transcriptional mechanism can be inhibited by internally initiated SP-3 and the natural phenol curcumin.


Subject(s)
Curcumin/pharmacology , Neuroblastoma/pathology , Phosphoric Diester Hydrolases/genetics , Proto-Oncogene Proteins c-myc/metabolism , Sp Transcription Factors/metabolism , Transcription Factor AP-1/metabolism , Transcription, Genetic/drug effects , Cell Line, Tumor , Cyclic AMP/genetics , Gene Deletion , Genes, Reporter/genetics , Humans , Phosphoric Diester Hydrolases/deficiency , Response Elements/drug effects , Response Elements/genetics , Transcriptional Activation/drug effects
2.
Mol Cell Biol ; 30(10): 2401-10, 2010 May.
Article in English | MEDLINE | ID: mdl-20231358

ABSTRACT

We previously reported that vascular endothelial growth factor (VEGF)-dependent activation of phospholipase Cgamma1 (PLCgamma) regulated tube stability by competing with phosphoinositide 3-kinase (PI3K) for their common substrate. Here we describe an additional mechanism by which PLCgamma promoted regression of tubes and blood vessels. Namely, it increased the level of autotaxin (ATX), which is a secreted form of lysophospholipase D that produces lysophosphatidic acid (LPA). LPA promoted motility of endothelial cells, leading to disorganization/regression of tubes in vitro. Furthermore, mice that under- or overexpressed members of this intrinsic destabilization pathway showed either delayed or accelerated, respectively, regression of blood vessels. We conclude that endothelial cells can be instructed to engage a PLCgamma-dependent intrinsic destabilization pathway that results in the production of soluble regression factors such as ATX and LPA. These findings are likely to potentiate ongoing efforts to prevent, manage, and eradicate numerous angiogenesis-based diseases such as proliferative diabetic retinopathy and solid tumors.


Subject(s)
Endothelial Cells/drug effects , Endothelial Cells/metabolism , Lysophospholipids , Multienzyme Complexes/metabolism , Neovascularization, Physiologic/physiology , Phosphodiesterase I/metabolism , Phospholipase C gamma/metabolism , Pyrophosphatases/metabolism , Animals , Calcineurin/metabolism , Cattle , Cells, Cultured , Endothelial Cells/cytology , Enzyme Activation , Eye/blood supply , Humans , Lysophospholipids/metabolism , Lysophospholipids/pharmacology , Mice , Mice, Transgenic , Multienzyme Complexes/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphodiesterase I/genetics , Phospholipase C gamma/genetics , Phosphoric Diester Hydrolases , Pyrophosphatases/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Vascular Endothelial Growth Factor A/metabolism
3.
Mol Cancer Res ; 8(3): 309-21, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20197381

ABSTRACT

Important roles for vascular endothelial growth factor (VEGF) and autotaxin (ATX) have been established for embryonic vasculogenesis and cancer progression. We examined whether these two angiogenic factors cooperate in regulation of endothelial cell migratory responses. VEGF stimulated expression of ATX and LPA1, a receptor for the ATX enzymatic product lysophosphatidic acid (LPA), in human umbilical vein endothelial cells. Knockdown of ATX expression significantly decreased mRNA levels for the receptors LPA1, LPA2, S1P1, S1P2, S1P3, and VEGFR2 and abolished cell migration to lysophosphatidylcholine, LPA, recombinant ATX, and VEGF. Migration to sphingosylphosphorylcholine and sphinogosine-1-phosphate was also reduced in ATX knockdown cells, whereas migration to serum remained unchanged. Furthermore, ATX knockdown decreased Akt2 mRNA levels, whereas LPA treatment strongly stimulated Akt2 expression. We propose that VEGF stimulates LPA production by inducing ATX expression. VEGF also increases LPA1 signaling, which in turn increases Akt2 expression. Akt2 is strongly associated with cancer progression, cellular migration, and promotion of epithelial-mesenchymal transition. These data show a role for ATX in maintaining expression of receptors required for VEGF and lysophospholipids to accelerate angiogenesis. Because VEGF and ATX are upregulated in many cancers, the regulatory mechanism proposed in these studies could apply to cancer-related angiogenesis and cancer progression. These data further suggest that ATX could be a prognostic factor or a target for therapeutic intervention in several cancers.


Subject(s)
Cell Movement/physiology , Endothelial Cells/metabolism , Multienzyme Complexes/metabolism , Phosphodiesterase I/metabolism , Pyrophosphatases/metabolism , Receptors, Lysophosphatidic Acid/metabolism , Signal Transduction/physiology , Vascular Endothelial Growth Factor A/metabolism , Cell Movement/drug effects , Cells, Cultured , Endothelial Cells/cytology , Endothelial Cells/drug effects , Humans , Multienzyme Complexes/drug effects , Multienzyme Complexes/genetics , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/physiopathology , Phosphodiesterase I/drug effects , Phosphodiesterase I/genetics , Phosphoric Diester Hydrolases , Proto-Oncogene Proteins c-akt/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Pyrophosphatases/drug effects , Pyrophosphatases/genetics , RNA Interference/physiology , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Receptors, Lysophosphatidic Acid/drug effects , Receptors, Lysosphingolipid/drug effects , Receptors, Lysosphingolipid/metabolism , Receptors, Vascular Endothelial Growth Factor/drug effects , Receptors, Vascular Endothelial Growth Factor/metabolism , Signal Transduction/drug effects , Up-Regulation/drug effects , Up-Regulation/physiology , Vascular Endothelial Growth Factor A/pharmacology
4.
Lipids Health Dis ; 8: 4, 2009 Feb 17.
Article in English | MEDLINE | ID: mdl-19222837

ABSTRACT

BACKGROUND: The secreted enzyme autotaxin (ATX) stimulates tumor cell migration, tumorigenesis, angiogenesis, and metastasis. ATX hydrolyzes nucleotides, but its hydrolysis of lysophospholipids to produce lysophosphatidic acid (LPA) accounts for its biological activities. ATX has been identified only as a constitutively active enzyme, and regulation of its activity is largely unexplored. In spite of its presence in plasma along with abundant putative substrate LPC, the product LPA is found in plasma at unexpectedly low concentrations. It is plausible that the LPA-producing activity of ATX is regulated by its expression and by access to substrate(s). For this reason studying the interaction of enzyme with substrate is paramount to understanding the regulation of LPA production. RESULTS: In this study we determine ATX hydrolytic activities toward several artificial and natural substrates. Two novel point mutations near the enzyme active site (H226Q and H434Q) confer attenuated activity toward all substrates tested. The Vmax for LPC compounds depends upon chain length and saturation; but this order does not differ among wild type and mutants. However the mutant forms show disproportionately low activity toward two artificial substrates, pNpTMP and FS-3. The mutant forms did not significantly stimulate migration responses at concentrations that produced a maximum response for WT-ATX, but this defect could be rescued by inclusion of exogenous LPC. CONCLUSION: H226Q-ATX and H434Q-ATX are the first point mutations of ATX/NPP2 demonstrated to differentially impair substrate hydrolysis, with hydrolysis of artificial substrates being disproportionately lower than that of LPC. This implies that H226 and H434 are important for substrate interaction. Assays that rely on hydrolyses of artificial substrates (FS-3 and pNpTMP), or that rely on hydrolysis of cell-derived substrate, might fail to detect certain mutated forms of ATX that are nonetheless capable of producing LPA in the presence of sufficient exogenous substrate. H420Q-ATX could not be differentiated from WT-ATX, indicating that histidine at position 420 is not required for any of the activities of ATX tested in this study.


Subject(s)
Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Mutant Proteins/metabolism , Phosphodiesterase I/genetics , Phosphodiesterase I/metabolism , Point Mutation/genetics , Pyrophosphatases/genetics , Pyrophosphatases/metabolism , Amino Acid Substitution/drug effects , Cell Movement/drug effects , Fatty Acids/metabolism , Humans , Hydrolysis/drug effects , Immunoblotting , Kinetics , Lysophospholipids/pharmacology , Mutant Proteins/genetics , Phosphoric Diester Hydrolases , Substrate Specificity/drug effects
5.
Mol Cancer Res ; 6(3): 352-63, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18337445

ABSTRACT

Tumor cell migration, invasion, and angiogenesis are important determinants of tumor aggressiveness, and these traits have been associated with the motility stimulating protein autotaxin (ATX). This protein is a member of the ectonucleotide pyrophosphatase and phosphodiesterase family of enzymes, but unlike other members of this group, ATX possesses lysophospholipase D activity. This enzymatic activity hydrolyzes lysophosphatidylcholine to generate the potent tumor growth factor and motogen lysophosphatidic acid (LPA). In the current study, we show a link between ATX expression, LPA, and vascular endothelial growth factor (VEGF) signaling in ovarian cancer cell lines. Exogenous addition of VEGF-A to cultured cells induces ATX expression and secretion, resulting in increased extracellular LPA production. This elevated LPA, acting through LPA(4), modulates VEGF responsiveness by inducing VEGF receptor (VEGFR)-2 expression. Down-regulation of ATX secretion in SKOV3 cells using antisense morpholino oligomers significantly attenuates cell motility responses to VEGF, ATX, LPA, and lysophosphatidylcholine. These effects are accompanied by decreased LPA(4) and VEGFR2 expression as well as by increased release of soluble VEGFR1. Because LPA was previously shown to increase VEGF expression in ovarian cancer, our data suggest a positive feedback loop involving VEGF, ATX, and its product LPA that could affect tumor progression in ovarian cancer cells.


Subject(s)
Multienzyme Complexes/genetics , Ovarian Neoplasms/genetics , Phosphodiesterase I/genetics , Pyrophosphatases/genetics , Vascular Endothelial Growth Factor A/genetics , Antibodies/pharmacology , Cell Line, Tumor , Cell Movement , Cyclic AMP/physiology , DNA Primers , Female , Gene Deletion , Humans , Lysophospholipids/physiology , Ovarian Neoplasms/enzymology , Ovarian Neoplasms/pathology , Phosphoric Diester Hydrolases , Polymerase Chain Reaction , RNA, Messenger/genetics , Signal Transduction , Vascular Endothelial Growth Factor Receptor-1/immunology , Vascular Endothelial Growth Factor Receptor-2/immunology
6.
Cell Signal ; 19(6): 1328-38, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17307336

ABSTRACT

Lysophosphatidic acid (LPA) stimulates sphingosine-1-phosphate (S1P)-sensitive motility in NIH3T3 clone7 cells. S1P inhibits motility only when added to the bottom well of the Boyden chamber, suggesting that pseudopodia can respond to their microenvironment. In order to study and localize this effect, we utilized a Transwell insert system to isolate pseudopodia. LPA stimulates protrusion of pseudopodia that are enriched in RhoA compared to cell bodies. Removal of LPA results in slow retraction with loss of vinculin-rich adhesion complexes and prolonged activation of RhoA. However, RhoA, ROCK and mDia are not required for this process. In contrast, rapid retraction, induced by adding S1P to the bottom well, is associated with a quick spike of activated RhoA and coalescence of adhesion complexes that colocalize with the ends of stress fibers. S1P-induced retraction requires RhoA and ROCK but is only delayed by inhibition of mDia. These data indicate that pseudopodia sense and integrate signals initiated by localized bioactive lipids, affecting both cellular polarity and their own function in motility.


Subject(s)
Lysophospholipids/pharmacology , Pseudopodia/drug effects , Pseudopodia/enzymology , Sphingosine/analogs & derivatives , rhoA GTP-Binding Protein/metabolism , Actins/metabolism , Animals , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/metabolism , Cell Adhesion/drug effects , Cell Line , Cell Movement/drug effects , Cell Polarity/drug effects , Enzyme Activation/drug effects , Formins , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Microtubules/drug effects , Microtubules/metabolism , NIH 3T3 Cells , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Protein Transport/drug effects , Sphingosine/pharmacology , Vinculin/metabolism , rac GTP-Binding Proteins/metabolism , rho-Associated Kinases , rhoA GTP-Binding Protein/antagonists & inhibitors
7.
Cancer Lett ; 250(1): 53-62, 2007 May 18.
Article in English | MEDLINE | ID: mdl-17189669

ABSTRACT

The histone deacetylase inhibitor, trichostatin A (TSA), and the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (Aza-dC), induced epigenetic regulation of sphingosine-1-phosphate (S1P) receptors in human melanoma cells, switching S1P from motility inhibitor to stimulator. Quantitative PCR revealed increased expression of S1P(1) and S1P(3), associated with S1P-induced chemotaxis, and decreased expression of S1P(2), associated with motility inhibition. Expression of lysophosphatidic acid (LPA) receptors was less affected. The TSA effect was reversible suggesting no mutational change, and Aza-dC treatment resulted in demethylation of a putative S1P(1) promoter. S1P receptors, therefore, appear to be susceptible to epigenetic regulation, accompanied by altered cellular functionality.


Subject(s)
Azacitidine/analogs & derivatives , Cell Movement/drug effects , Epigenesis, Genetic , Hydroxamic Acids/pharmacology , Lysophospholipids/pharmacology , Melanoma/genetics , Receptors, Lysosphingolipid/genetics , Sphingosine/analogs & derivatives , Azacitidine/pharmacology , Cell Line, Tumor , DNA Methylation , Decitabine , Gene Expression Regulation , Humans , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Receptors, Lysophospholipid , Sphingosine/pharmacology
8.
Lipids Health Dis ; 4: 5, 2005 Feb 28.
Article in English | MEDLINE | ID: mdl-15737239

ABSTRACT

BACKGROUND: Autotaxin (ATX, NPP-2), originally purified as a potent tumor cell motility factor, is now known to be the long-sought plasma lysophospholipase D (LPLD). The integrity of the enzymatic active site, including three crucial histidine moieties, is required for motility stimulation, as well as LPLD and 5'nucleotide phosphodiesterase (PDE) activities. Except for relatively non-specific chelation agents, there are no known inhibitors of the ATX LPLD activity. RESULTS: We show that millimolar concentrations of L-histidine inhibit ATX-stimulated but not LPA-stimulated motility in two tumor cell lines, as well as inhibiting enzymatic activities. Inhibition is reversed by 20-fold lower concentrations of zinc salt. L-histidine has no significant effect on the Km of LPLD, but reduces the Vmax by greater than 50%, acting as a non-competitive inhibitor. Several histidine analogs also inhibit the LPLD activity of ATX; however, none has greater potency than L-histidine and all decrease cell viability or adhesion. CONCLUSION: L-histidine inhibition of LPLD is not a simple stoichiometric chelation of metal ions but is more likely a complex interaction with a variety of moieties, including the metal cation, at or near the active site. The inhibitory effect of L-histidine requires all three major functional groups of histidine: the alpha amino group, the alpha carboxyl group, and the metal-binding imidazole side chain. Because of LPA's involvement in pathological processes, regulation of its formation by ATX may give insight into possible novel therapeutic approaches.


Subject(s)
Cytokines/pharmacology , Histidine/pharmacology , Lysophospholipids/biosynthesis , Multienzyme Complexes/pharmacology , Neoplasms/metabolism , Phosphodiesterase I/pharmacology , Pyrophosphatases/pharmacology , Cations, Divalent/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Chelating Agents/pharmacology , Enzyme Activation/drug effects , Histidine/analogs & derivatives , Humans , Molecular Structure , Neoplasms/pathology , Phosphoric Diester Hydrolases/metabolism , Substrate Specificity , Zinc/chemistry , Zinc/pharmacology
9.
Cancer Res ; 63(17): 5446-53, 2003 Sep 01.
Article in English | MEDLINE | ID: mdl-14500380

ABSTRACT

Autotaxin (ATX) is an exoenzyme that potently induces tumor cell motility, and enhances experimental metastasis and angiogenesis. ATX was shown recently to be identical to serum lysophospholipase D activity, producing lysophosphatidic acid (LPA) from lyso-glycerophospholipids. LPA, itself a strong chemoattractant for tumor cells, may mediate the actions of ATX. We now extend the substrate specificity to sphingosylphosphorylcholine (SPC), which ATX hydrolyzes to sphingosine-1-phosphate (S1P). Under migration assay conditions, this novel reaction for the production of S1P has a substrate (SPC) K(m) = 0.23 +/- 0.07 mM. In our responder cell lines (NIH3T3 clone7 and A2058), S1P exerts maximal biological effects at concentrations of 10-100 nM and is mimicked in its biological effects by ATX plus SPC. These effects include inhibition of ATX- and LPA-stimulated motility, and elevation of activated Rho. In NIH3T3 clone7 cells stimulated with platelet-derived growth factor and treated with 10-25 nM S1P, motility is not inhibited and activation of Rho is unaffected, indicating that S1P possesses specificity in its effects. The exoenzyme ATX can potentially regulate diverse processes such as motility and angiogenesis via the S1P family of receptors. Because ATX hydrolyzes nucleotides, lyso-glycerophospholipids, and phosphosphingolipids into bioactive products, it possesses the ability, depending on the availability of substrates, to act as positive or negative regulator of receptor-mediated activity in the cellular microenvironment.


Subject(s)
Glucose-6-Phosphate Isomerase/pharmacology , Glycoproteins/pharmacology , Lysophospholipids , Multienzyme Complexes , Phosphorylcholine/analogs & derivatives , Phosphorylcholine/metabolism , Receptors, G-Protein-Coupled , Sphingosine/analogs & derivatives , Sphingosine/biosynthesis , Sphingosine/metabolism , 3T3 Cells , Animals , COS Cells , Catalysis , Cell Movement/physiology , Chlorocebus aethiops , Hydrolysis/drug effects , Mice , Phosphodiesterase I , Phosphoric Diester Hydrolases , Pyrophosphatases , Receptors, Cell Surface/biosynthesis , Receptors, Lysophospholipid , rho GTP-Binding Proteins/metabolism
10.
FEBS Lett ; 515(1-3): 137-40, 2002 Mar 27.
Article in English | MEDLINE | ID: mdl-11943209

ABSTRACT

Autotaxin (ATX), an exo-nucleotide pyrophosphatase and phosphodiesterase, stimulates tumor cell motility at sub-nanomolar levels and augments invasiveness and angiogenesis. We investigated the role of G protein-coupled phosphoinositide 3-kinase gamma (PI3Kgamma) in ATX-mediated tumor cell motility stimulation. Pretreatment of human melanoma cell line A2058 with wortmannin or LY294002 inhibited ATX-induced motility. ATX increased the PI3K activity in p110gamma, but not p85, immunoprecipitates. This effect was abrogated by PI3K inhibitors or inhibited by pertussis toxin. Furthermore, stimulation of tumor cell motility by ATX was inhibited by catalytically inactive form of PI3Kgamma, strongly indicating the crucial role of PI3Kgamma for ATX-mediated motility in human melanoma cells


Subject(s)
Cell Movement/drug effects , GTP-Binding Proteins/metabolism , Glucose-6-Phosphate Isomerase/pharmacology , Glycoproteins/pharmacology , Isoenzymes/metabolism , Melanoma/metabolism , Multienzyme Complexes , Phosphatidylinositol 3-Kinases/metabolism , Androstadienes/pharmacology , Animals , COS Cells , Chromones/pharmacology , Class Ib Phosphatidylinositol 3-Kinase , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/pharmacology , Melanoma/drug therapy , Morpholines/pharmacology , Pertussis Toxin , Phosphatidylinositol 3-Kinases/pharmacology , Phosphodiesterase I , Phosphoinositide-3 Kinase Inhibitors , Phosphoric Diester Hydrolases , Protein Subunits , Pyrophosphatases , Tumor Cells, Cultured , Virulence Factors, Bordetella/pharmacology , Wortmannin
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