Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M605142200 on July 6, 2006

J. Biol. Chem., Vol. 281, Issue 35, 25822-25830, September 1, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/35/25822    most recent
M605142200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tanaka, M.
Right arrow Articles by Arai, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tanaka, M.
Right arrow Articles by Arai, H.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Autotaxin Stabilizes Blood Vessels and Is Required for Embryonic Vasculature by Producing Lysophosphatidic Acid*

Masayuki Tanaka{ddagger}, Shinichi Okudaira{ddagger}, Yasuhiro Kishi{ddagger}, Ryunosuke Ohkawa§, Sachiko Iseki, Masato Ota, Sumihare Noji||, Yutaka Yatomi§, Junken Aoki{ddagger}**1, and Hiroyuki Arai{ddagger}

From the {ddagger}Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, §Department of Clinical Laboratory, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Department of Developmental Biology, Graduate School of Dentistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, ||Department of Biological Science and Technology, Faculty of Engineering, University of Tokushima, 2-1 Minamijosanjima-cho, Tokushima 770-8506, and **Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Corporation, 4-1-8 Honcho, Kawaguchi City, Saitama 332-0012, Japan

Received for publication, May 30, 2006 , and in revised form, July 5, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Autotaxin (ATX) is a cancer-associated motogen that has multiple biological activities in vitro through the production of bioactive small lipids, lysophosphatidic acid (LPA). ATX and LPA are abundantly present in circulating blood. However, their roles in circulation remain to be solved. To uncover the physiological role of ATX we analyzed ATX knock-out mice. In ATX-null embryos, early blood vessels appeared to form properly, but they failed to develop into mature vessels. As a result ATX-null mice are lethal around embryonic day 10.5. The phenotype is much more severe than those of LPA receptor knock-out mice reported so far. In cultured allantois explants, neither ATX nor LPA was angiogenic. However, both of them helped to maintain preformed vessels by preventing disassembly of the vessels that was not antagonized by Ki16425, an LPA receptor antagonist. In serum from heterozygous mice both lysophospholipase D activity and LPA level were about half of those from wild-type mice, showing that ATX is responsible for the bulk of LPA production in serum. The present study revealed a previously unassigned role of ATX in stabilizing vessels through novel LPA signaling pathways.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Autotaxin (ATX)2 is a motogen-like phosphodiesterase originally isolated from conditioned medium of human melanoma cells (1). Enforced expression of ATX in Ras-transformed NIH3T3 cells greatly enhances their invasive, tumorigenic, and metastatic potentials (2). In addition, enhanced expression of ATX has been demonstrated in various malignant tumor tissues (3). Thus, ATX is implicated in tumorigenic and metastatic potentials of cancer cells. ATX is also expressed in various tissues and is present at high concentration in various biological fluids including plasma, serum, and seminal plasma (4), implying specific roles of ATX in circulation.

Recently, ATX was shown to have lysophospholipase D (lysoPLD) activity, which converts lysophosphatidylcholine to a bioactive lysophospholipid, lysophosphatidic acid (LPA) (5, 6). ATX also converts sphingosylphosphorylcholine into another bioactive lysophospholipid, sphingosine 1-phosphate (S1P) in vitro (7). Because LPA and S1P are regulators of cell motility and proliferation in various cell systems, they might be the effectors of the motogenic actions of ATX. LPA and S1P have been shown to have diverse roles in many biological processes that are mediated by G protein-coupled receptors (GPCRs) specific to LPA or S1P; there are five GPCRs for LPA (LPA1-5) and five for S1P (S1P1-5) with a number of putative GPCRs (8). Thus, ATX may exert its functions through these receptors. Indeed, ATX stimulates cell motility of tumor cells through one of the LPA receptors, LPA1 (9), and ATX positively or negatively modulates cell motility depending on S1P receptor subtypes (7, 10). To uncover the physiological role of ATX and to identify the endogenous product of ATX, we investigated ATX knock-out mice. In this study we show that ATX produces LPA, but not S1P, in circulating blood and that it contributes to blood vessel stability through novel LPA signaling pathways.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
ATX Knock-out Mice—ATX knock-out mice (atx-/-) in the genetic background 129/SvEvBrd were produced by and obtained from Lexicon Genetics (The Woodlands, TX). The ATX gene was targeted with an ATX gene-targeting vector, which was designed to replace the initiation codon and first 45 amino acids encoded by exons 1 and 2 with a lacZ-neo cassette in the vector pKOS (Fig. 1A). The ATX knock-out mice were backcrossed with C57BL/6j mice at least six times before being used. Mice were genotyped by both Southern blotting and PCR of genomic DNA. The PCR primers for detection of wild-type alleles are 5'-ccgaatctctccgatcactac-3' and 5'-tccaacattaaagcgataacc-3', and those for detection of mutant alleles are 5'-gcagcgcatcgccttctatc-3' and 5'-tccaacattaaagcgataacc-3'. Flk-1-laxZ knock-in mice (11) were kindly donated by Dr. Janet Rossant (The Hospital for Sick Children, Toronto, Canada). All mice used in this study were bred and maintained at the Animal Care Facility in the Graduate School of Pharmaceutical Sciences, the University of Tokyo, under specific pathogen-free conditions in accordance with institutional guidelines.


Figure 1
View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 1.
atx knock-out mice. A, targeting strategy. Top, partial restriction map of the atx locus. Middle, construct of atx gene targeting vector. Bottom, the expected mutant locus. The DNA fragment used as a probe for Southern blotting is also shown on the top figure. B, genotyping of atx+/+, atx+/-, and atx-/- E8.5 embryos by PCR using genomic DNA and two sets of primers (fw and rev for detection of wild-type allele and Neo and rev for detection of mutant allele, see in panel A). C, quantitative real-time RT-PCR analysis of atx+/+, atx+/-, and atx-/- embryos at E8.5. The level of ATX mRNA was measured using quantitative real-time RT-PCR and is expressed as a relative value to glyceraldehyde-3-phosphate dehydrogenase mRNA.

 
Recombinant ATX—Two recombinant mouse ATX proteins (wild-type ATX and catalytically inactive T209A mutant ATX) with a His tag at the N terminus were expressed and purified using a baculovirus system and nickel column chromatography (HisTrap HP; GE Healthcare), respectively.

Preparation of ATX-depleted Serum—To establish anti-mouse ATX monoclonal antibody, the recombinant mouse ATX protein (50 µg) was used to immunize rats (WKY/Izm strain) with Freund's complete adjuvant into the hind footpads. Cells from the enlarged medial iliac and inguinal lymph nodes were fused with mouse myeloma (PAI) cells. The antibody-secreting hybridoma cells were selected by screening with an enzyme-linked immunosorbent assay and immunoprecipitation. One clone (5E5 (rat IgG1)) was found to have activity to immunoprecipitate ATX in mouse serum. 5E5 reacted with mouse, human, and bovine ATX but does not react with rat ATX. The monoclonal antibody was purified from culture supernatant of hybridoma cells and was coupled to Sepharose 4B beads (2 mg/ml) (GE Healthcare). To deplete ATX from mouse serum, mouse serum (1 ml) was incubated with the 5E5-Sepahrose 4B (40 µl)for 2 h at 4 °C,andthe resulting supernatant was used for measuring lysoPLD activity and LPA production. ATX bound to the 5E5-Sepahrose 4B was eluted with 100 mM glycine, pH 2.5. This gave rise to a single 100-kDa band on sodium dodecyl sulfate polyacrylamide gel electrophoresis, showing that 5E5 is specific to ATX.

Immunohistochemistry, Whole-mount Immunostaining, and Whole-mount LacZ Staining—Tissue sections (5 µm) were dehydrated, embedded in paraffin, incubated in 3% (v/v) H2O2 for 20 min, incubated overnight at 4 °C with first antibodies (EPOS anti-a-SMA/HRP; DAKO), stained with diaminobenzidine according to the manufacturer's protocol, and counter-stained with hematoxylin. For whole-mount embryo immuno-staining, embryos were fixed in 4% paraformaldehyde, dehydrated, incubated with 5% hydrogen peroxide in methanol, rehydrated through a methanol series to phosphate-buffered saline, incubated in phosphate-buffered saline containing 4% bovine serum albumin and 0.1% Triton X-100, incubated with rat anti-PECAM monoclonal antibody (BD Biosciences), incubated with peroxidase-conjugated goat anti-rat IgG (American Qualex), and stained with diaminobenzidine as a peroxidase substrate. LacZ staining was performed as described (11).


Figure 2
View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 2.
lysoPLD activity and ATX protein in plasma and amniotic fluids. A, lysophospholipase D activity of plasma and amniotic fluids isolated from atx+/+ and atx+/- adult mice or atx+/+ and atx+/- embryos at E11.5, E12.5, and E13.5. Note that lysoPLD activities in amniotic fluids are comparable with those in plasma. B, Western blot of ATX in plasma isolated from atx+/+ and atx+/- adult mice. Plasma samples from four different individual mice in each genotype were loaded in each lane. C, densitometric analysis of Western blots in panel B. Results represent the percentage of ATX protein expression in atx+/- mouse plasma relative to atx+/+. D, Western blot analysis of ATX in amniotic fluids isolated from atx+/+, atx+/-, and atx-/- embryos at E8.5-E13.5. Amniotic fluids from different individual embryos were loaded in each lane. E, Western blot analysis of ATX in amniotic fluids and whole lysates isolated from embryos and yolk sacs at E9.5. Samples from two different individual embryos were loaded in each lane (10 µg/lane). Note that ATX expression was detected in amniotic fluids but not in whole lysates of embryos and yolk sacs. Statistical significance was analyzed using Student's t test. Statistically significant deferences (p < 0.05) are indicated by asterisks in panels A and C. For Western blot analysis the same gels were stained with Coomassie Brilliant Blue and used as loading controls in panels B, D, and E (data not shown).

 


Figure 3
View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 3.
ATX is responsible for bulk LPA production in plasma. A, LPA levels in plasma from atx+/+ and atx+/- mice determined by a coloriometric method. Plasma LPA levels were reduced by about one half in atx+/- mice. B, lysoPLD activity of ATX-depleted mouse serum. lysoPLD activity was determined by liberation of choline from lysophosphatidylcholine (LPC) using 14:0-LPC as a substrate. C, production of LPA in ATX-depleted mouse serum during incubation at 37 °C. Open circles represent ATX-depleted serum, and closed circles represent control serum. D, S1P level in plasma from atx+/+ and atx+/- mice. Plasma S1P levels were not significantly different. Statistical significance was analyzed using Student's t test. Statistically significant differences (p < 0.05) are indicated by asterisks in panels A and B.

 
Allantois Culture—Allantoides were dissected from embryos at E8.5 and cultured in Dulbecco's modified Eagle's medium containing glutamine and antibiotics (18). To perform the vessel formation, the allantois explants were cultured for 24 h (37 °C; 5% CO2) in the presence or absence of the factors tested. In some cases, to examine the effect of factors on stabilization of preformed vessels the allantoides were first cultured for 24 h in the presence of amniotic fluids (isolated from E12.5 embryos) to allow formation of stable vascular networks and then cultured for an additional 18 h in the presence or absence of the factors tested. Cells were fixed with 4% paraformaldehyde and then immunostained with anti-PECAM monoclonal antibody.

Determination of LPA and S1P Concentration, lysoPLD Activity, and Western Blotting—LPA and S1P concentrations in plasma and serum were determined as described previously (12-14). lysoPLD activity was determined as described using 14:0 lysophosphatidylcholine as substrate (5). Western blotting of ATX was performed as described using ATX-specific monoclonal antibody (15).

In Situ Hybridization—Expression of ATX and LPA1 was detected in paraffin-embedded or frozen sections by in situ hybridization using digoxigenin-labeled RNA probes for ATX and LPA1 as previously described (16). Specimens were treated with proteinase K at 2 µg/ml in phosphate-buffered saline for 5 min and then refixed in 4% paraformaldehyde/phosphate-buffered saline for 20 min, followed by acetylation. Finally, digoxigenin was immunodetected with 5-bromo-4-chloro-3-indolyl phosphate (Roche Applied Science).

Quantitative RT-PCR—From embryos and yolk sacs at E8.5-E10.5, total RNA was isolated using ISOGEN (Nippongene, Toyama, Japan) and reverse transcribed using the SuperScript first-strand synthesis system for RT-PCR (Invitrogen). Primers used to detect ATX, LPA receptors, glyceraldehyde-3-phosphate dehydrogenase, and beta-actin were described previously (17). Quantitative RT-PCR was performed as described (9) using ABI PRISM7000 sequence detection system (Applied Biosystems). Each sample was normalized to the number of beta-actin or glyceraldehyde-3-phosphate dehydrogenase transcripts.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Vascular Defects in ATX-null Embryos—To understand the physiological role of ATX, we investigated atx knock-out mice. The ATX gene was targeted with an ATX gene-targeting vector that was designed to replace the initiation codon and first 45 amino acids encoded by exons 1 and 2 with a lacZ-neo cassette in the vector pKOS (Fig. 1A). The generation of mutant mice was confirmed by Southern blotting (not shown) and polymerase chain reaction (PCR) analyses of tail genomic DNA (Fig. 1B). RT-PCR analysis of embryos carrying the knock-out allele confirmed that ATX mRNA was not produced from the disrupted locus (Fig. 1C). The plasma lysoPLD activity and plasma ATX protein level in atx+/- mice were about half the values found in atx+/+ mice (Fig. 2, A and B), showing that ATX is responsible for most of the lysoPLD activity in plasma. The plasma LPA concentrations in atx+/- mice were about half of those in atx+/+ mice (Fig. 3A). In addition, in ATX-depleted mouse serum, which is prepared by mixing the mouse serum with Sepharose 4B beads coupled with anti-ATX monoclonal antibody (5E5), both lysoPLD activity and LPA production during incubation at 37 °C were hardly detected (Fig. 3, B and C). These results clearly indicate that ATX is a major enzyme that produces LPA in serum. By contrast, the S1P levels in plasma (Fig. 3D) and serum (not shown) from atx+/+ mice were unchanged. Despite the reduced LPA level, heterozygous mice appeared phenotypically normal. Viability and fecundity were similar to those in wild-type mice.


Figure 4
View larger version (65K):
[in this window]
[in a new window]
  		FIGURE 4.
Morphologies of atx+/+ and atx-/- embryo proper at E8. 5, E9.5, and E10.5. At E8.5 (A-C) about half of the atx-/- embryos appear to be normal compared with atx+/+ embryos (A and C), whereas the other half of embryos showed abnormal morphologies in head regions (B, arrow). At E9.5 (D-F) and E10.5 (G and H) atx-/- embryos are easily distinguishable from atx+/+ or atx+/- littermates. atx-/- embryos exhibit several defects such as pericardial effusion (E, arrow) and open and kinky nural tube (F, arrowhead). Scale bars, 100 µm in panels A-F and 200 µm in panels G and H.

 
Intercrossing of heterozygous animals produced no homozygous pups, indicating that ATX mutations are recessive embryonic lethal. To determine when homozygous embryos were dying, embryos were isolated at various stages of gestation. Homozygous embryos survived at least to embryonic day 10.5 (E10.5) (Table 1). From E9.5, homozygous embryos were clearly abnormal, and at E11.5 almost all of them were dead in utero. Several defects were evident in the homozygous embryo proper. These included growth retardation, open and kinky neural tubes, pericardial effusion, reduced number of somites, incomplete turning, pallor, and fragility (Fig. 4). The hearts beat until E10.5, but contractions were weak and irregular. In addition, yolk sacs were apparently abnormal in homozygous (atx-/-) embryos. The yolk sac of atx-/- embryos completely lacked large vitelline vessels at E9.5 and E10.5, whereas wild-type (atx+/+) yolk sacs had well developed vitelline vessels (Fig. 5A). Histological analysis also showed a clear difference in morphology between atx+/+ and atx-/- tissues. Individual vessels were clearly visible in atx+/+ tissue, with numerous regularly spaced attachments between the visceral endoderm and mesoderm layers (Fig. 5A). Visceral endoderm and mesoderm were more widely separated in atx-/- tissue, with occasional attachments forming numerous large spaces that were sparsely populated with blood cells (Fig. 5A). Despite the vascular defects, embryonic erythrocytes were present (Fig. 5A, arrows), indicating that blood cells differentiated. To specifically visualize the endothelial vasculature in the developing yolk sac and embryo, we utilized flk-1 heterozygous mice, in which expression of lacZ is regulated by the promoter of the flk-1 gene and is restricted to endothelial cells (11). We generated atx+/+flk+/- and atx-/-flk+/- embryos with flk-1 promoter-regulated lacZ expression. To do this we first crossed atx+/- mice with flk+/- (flk.LacZ) mice and generated atx+/-flk+/-. Then atx+/+flk+/- and atx-/-flk+/- embryos were generated by crossing atx+/-flk+/- mice. At E8.5, atx-/-flk+/- yolk sacs had slightly rough but almost comparable tubular structures to atx+/+flk+/- yolk sacs (Fig. 5B). In contrast, at E9.5, atx-/-flk+/- yolk sacs were found to have severe vascular defects, i.e. they had no well developed fine blood vessels (Fig. 5B). Consistent with this observation, both normal and abnormal vessels were visible in atx-/-flk+/- yolk sacs at E9.0 (Fig. 5B). Embryos at E8.5 atx-/- had vascular defects in the head (arrows) and cardiac (sinus venosus) regions but not in the dorsal aortae (Fig. 5B). Staining with antibodies against PECAM, another endothelial cell-specific marker, confirmed the vascular defects of the yolk sac in homozygous embryos (not shown). Incomplete vascular remodeling was also observed in the embryo proper. Whole-mount anti-PECAM staining revealed well developed thin and fine tubule-like structures in atx+/+ embryos (Fig. 5C). In sharp contrast, the structures were thick and irregular-shaped in atx-/- embryos (Fig. 5C). At E9.5, the atx+/+ placenta had both embryonic and maternal blood vessels (distinguishable by the size of their erythrocytes), whereas the atx-/- placenta had only maternal blood vessels (Fig. 5D). At E9.5, chorioallantoic attachment and fusion were observed both in atx+/+ and atx-/- embryos (Fig. 5E). However, it was evident that atx-/- allantois lacked the numerous vessel lumen visible in the atx+/+ allantois (Fig. 5E). Immunostaining of the yolk sac and embryo proper at E9.5 with anti-{alpha}-smooth muscle actin, a specific marker of vascular smooth muscle cells, showed that smooth muscle cells were present and surrounded the endothelial cells in both the yolk sac (Fig. 5F) and embryo proper (not shown). This indicates that mural cell investment is not the cause of vascular defects in atx-/- embryos. These analyses suggest that initial blood vessel formation occurs properly in atx-/- embryos but the newly formed blood vessels fail to develop into mature vessels in the absence of ATX.


View this table:
[in this window]
[in a new window]
  		TABLE 1
Genotype distributions of offspring of atx+/- intercrosses at different stages of development

 


Figure 5
View larger version (84K):
[in this window]
[in a new window]
  		FIGURE 5.
Defects in vascular remodeling in atx-/- embryos. A, defects in the yolk sac vasculature. Yolk sac from atx+/+ and atx-/- embryos at E9.5 and E10.5. Sections are stained with hematoxylin and eosin E. Note the normal blood vessels (arrowheads) in atx+/+ and their absence in atx-/- yolk sac. Arrows indicate that erythrocytes are present in atx-/- yolk sacs. e, erythrocytes. B, whole-mount LacZ staining of atx+/+ flk+/- and atx-/-flk+/- embryos (E8.5) and yolk sacs (E8.5, E9.0, and E9.5). At E9.0 both normal and abnormal (arrowhead) vessels are visible in atx-/- yolk sacs, whereas only normal vessels are visible in atx+/+ yolk sacs. DA, dorsal aorta; H, heart; VA, vitelline artery; SV, sinus venosus. C, whole-mount PECAM immunostaining of atx+/+ and atx-/- embryos at E9.5. Magnifications of head regions are shown at right. D, sections of atx+/+ and atx-/- placenta at E9.5 were stained with haematoxylin and eosin. Arrowheads, maternal blood vessels; arrows, embryonic blood vessels. E, histological analysis of allantoic development and vascularization. In atx+/+ embryos, the allantois forms a large, erythrocyte-filled vessel connected to placenta at E9.5. atx-/- allantois is sparse and lacks the numerous vessel lumen visible in the atx+/+ allantois. F, expression of {alpha}-smooth muscle actin-positive cells in atx+/+ and atx-/- yolk sacs at E9.5. e, erythrocytes. Scales are indicated in each picture.

 


Figure 6
View larger version (63K):
[in this window]
[in a new window]
  		FIGURE 6.
Expression of ATX and LPA receptors in embryos. A, quantitative RT-PCR analysis of ATX in mouse embryos and yolk sacs at E8.5-E10.5. B, in situ hybridization of ATX in yolk sac at E8.5. C, quantitative RT-PCR analysis of four LPA receptors in yolk sacs at E8.5-E10.5. Note among the four LPA receptors LPA1 is predominantly expressed in yolk sacs. D, in situ hybridization of LPA1 in yolk sac at E8.5.

 
RT-PCR experiments detected modest expression of ATX mRNA in both the yolk sac and embryo proper until E10.5 (Fig. 6A). In situ hybridization experiments detected weak expression in endodermal cells that surround the yolk sac at E8.5 (Fig. 6B). By contrast, significant ATX protein expression was observed in amniotic fluids in all embryonic stages tested, and its level was nearly equivalent to the plasma level as judged by both Western blotting and lysoPLD activity (Fig. 2, A and C). As was observed in plasma, the lysoPLD activity and ATX protein in amniotic fluids from atx+/- embryos at E11.5, 12.5, and 13.5 were almost half the value found in atx+/+ embryos (Fig. 2, A and C). Western blot analysis also showed that ATX expression was not detectable in either yolk sac or embryo proper at E9.5, although ATX protein was highly expressed in amniotic fluids (Fig. 2, D and E). These analyses indicate that most of the ATX proteins produced in embryos are released and concentrated in amniotic fluids.


Figure 7
View larger version (97K):
[in this window]
[in a new window]
  		FIGURE 7.
ATX and LPA stabilize preformed vessels and prevent endothelial disassembly in allantois explants. A, anti-PECAM-labeled fluorescent microscopy images of E8.5 allantois cultured for 24 h in amniotic fluids isolated from E12.5 embryos or Dulbecco's modified Eagle's medium containing recombinant ATX (1 µg/ml), LPA (1 µM), and fetal calf serum (10%). Neither ATX nor LPA shows angiogenic activity. The amniotic fluids promote blood vessel formation of allantoides isolated from atx-/- embryos. B, allantoides were cultured for 24 h in the presence of amniotic fluids to form blood vessels. Then, the vessels were cultured for 18 h in Dulbecco's modified Eagle's medium in the presence or absence of recombinant ATX (1 µg/ml), catalytically inactive mutant ATX (T209A mutant, 1 µg/ml), LPA (1 µM), S1P (1 µM), or amniotic fluids. ATX and LPA prevent endothelial disassembly and stabilize vessels in allantois explants. Experiments were performed at least three times in triplicate, and the representative results were shown.

 
ATX and LPA Prevent Disassembly of Vessels in Allatois Culture System—To determine the role of ATX and to assess its enzymatic activity in vascular remodeling, we utilized the allantois explant culture system (18). As reported by Argraves et al. (18), when E8.5 allantois explants were cultured in the presence of 10% fetal calf serum, a highly branched network of PECAM-positive vessels was visible after 24 h of culture (Fig. 7A). The amniotic fluids isolated from E12.5 wild-type embryos were found to have a similar but an even more pronounced effect on vessel formation than fetal calf serum (Fig. 7A). Allantois explants from atx-/- embryos also showed a highly branched network of PECAM-positive vessels when they were cultured in the presence of amniotic fluids isolated from E12.5 wild-type embryos (Fig. 7A) or fetal calf serum (not shown). Adding LPA or recombinant ATX to this system had little effect on vessel formation (Fig. 7A), showing that ATX and LPA themselves are not angiogenic. In the presence of amniotic fluids, the vessels that formed within the first 24 h of culture kept their tubular structure during the next 18 h of culture (Fig. 7B). However, in the absence of amniotic fluid, the preformed nascent vessels were unstable and disassembled (Fig. 7B). The recombinant ATX, but not catalytically inactive ATX, prevented the disassembly (Fig. 7B). LPA also prevented the disassembly of preformed vessels, whereas S1P had no effect (Fig. 7B). These results indicate that ATX in the amniotic fluids has a role in stabilizing vessels.

To know the molecular mechanisms of how LPA stabilizes blood vessels, we examined the expression of LPA receptors. RT-PCR and in situ hybridization experiments showed that among the four LPA receptors LPA1 is predominantly expressed in the yolk sac and embryo proper at E8.5, E9.5, and E10.5 (Fig. 6C), where it is expressed by cells in the endodermal layer other than endothelial cells (Fig. 6D). Expression levels of LPA2, LPA3, and LPA4 were revealed to be low. Thus, we tested the effect of an LPA receptor antagonist, Ki16425, on the vessel-stabilizing effect of ATX and LPA. The Ki values of Ki16425 are 0.25 µM for LPA1, 5.6 µM for LPA2, and 0.36 µM for LPA3 (19). Ki16425 did not show antagonistic activity toward LPA4 (19). Adding Ki16425 at 5 µM in the allantois culture system did not affect the vessel-stabilizing effects of ATX and LPA (Fig. 8). Thus, it is unlikely that LPA1, LPA2, and LPA3 are involved in the vessel-stabilizing effect of ATX and LPA.


Figure 8
View larger version (104K):
[in this window]
[in a new window]
  		FIGURE 8.
An LPA receptor antagonist, Ki16425, does not affect ATX- or LPA-induced prevention of endothelial disassembly in allantois explants. Allantoides were cultured for 24 h in the presence of amniotic fluids to form blood vessels. Then, the vessels were cultured for 18 h in Dulbecco's modified Eagle's medium containing recombinant ATX (1 µg/ml) or LPA (1 µM) in the presence or absence of Ki16425 (5 µM). Vessels were stained with anti-PECAM antibody.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
ATX was originally identified as a cell motility-stimulating factor for cancer cells (1). Later, ATX was found to have enzymatic activity to produce a bioactive phospholipid, LPA (5, 6, 20). LPA has been defined as a growth factor-like lipid with numerous biological activities, including stimulation of cell motility (3). In this study to elucidate the physiological role of ATX we analyzed ATX knock-out mice. To our surprise ATX knock-out mice were embryonic lethal, showing that ATX is indispensable for embryonic development. Blood vessel formation was severely affected in ATX-null embryos, suggesting novel roles of ATX and LPA in the formation of blood vessels in the embryonic stage. In addition, the present study confirmed that ATX is responsible for bulk LPA production in blood, but not for S1P production. The phenotype of ATX-null embryos was quite different from those of LPA receptors reported so far (21-23). These results raise a possibility that ATX and LPA contribute to embryonic blood vessel formation through as yet unidentified LPA receptors.

Nam et al. (24) reported that injection of Matrigel mixed with purified ATX into athymic nude mice resulted in new blood vessel formation within the plug and that ATX stimulated human umbilical vein endothelial cells grown on Matrigel to form tubules. From these results they suggested that ATX was an angiogenic factor. Our present data suggest that ATX itself is not angiogenic but it stabilizes preformed vessels through an unknown mechanism. Because Matrigel contains several factors that promote vessel formation, ATX may contribute to the vessel formation possibly in cooperation with such factors.

The formation of vasculature by vasculogenesis and angiogenesis is essential not only for embryonic development but also for the unrestrained growth of tumors (25). ATX stimulates both cell proliferation and cell motility of cancer cells through LPA production (5). In addition, overexpression of ATX is frequently associated with malignant tumors such as small cell lung cancer (26), renal cell cancer (27), hepatocellular carcinoma (28, 29), breast cancer (30, 31), Hodgkin lymphoma (32), thyroid cancer (33), and glioblastoma (17). Thus, ATX has been implicated in the progression of malignant tumors. In this study we showed that ATX has an additional role in blood vessel formation, possibly by stabilizing preformed blood vessels. The present study raises the possibility that ATX, in addition to stimulating proliferation and motility of tumor cells (5), contributes to the progression of tumors by stabilizing blood vessels in the vicinity of tumors.

ATX is capable of producing S1P because ATX also catalyzes a reaction to convert sphingosylphosphocholine to S1P (7). We showed in this study that ATX is a major producing enzyme for LPA, but not for S1P, in blood. It is accepted in recent reports that S1P is produced intracellularly from sphingosine through phosphorylation reaction mediated by two isozymes of sphingosine kinase (sphK) (sphingosine kinase 1 and sphingosine kinase 2) (34, 35). Interestingly, vascular defects were also observed in sphingosine kinase-null and S1P receptor-null embryos. Both sphk1-/-,sphk2-/- double mutant embryos (36) and s1p1-/-,s1p2-/-, s1p3-/- triple mutant embryos (37) died around E12.5, showing that the phenotypes are milder than that of atx-/- embryos. Thus, despite their similar structures, LPA and S1P have distinct synthetic pathways, targets, and functions.

Among the four LPA receptors reported so far, LPA1 is predominantly expressed in embryos around E9.5, whereas LPA2, LPA3, and LPA4 are weakly expressed (Fig. 6, C and D). We showed that Ki16425, an antagonist for LPA1, LPA2, and LPA3, had little effect on vessel-stabilizing activity of ATX and LPA (Fig. 8). Thus, it is unlikely that LPA1, LPA2, and LPA3 are involved in this process. Consistent with this, phenotypes of LPA receptor knock-out mice are quite different from that of ATX knock-out mice. LPA1, LPA2, and LPA3 single and LPA1 and LPA2 double knock-out mice are reported to be viable (21-23). LPA also exerts its role through other targets such as GPR23/LPA4 (38). Very recently a novel G protein-coupled receptor, GPR92/LPA5, was identified as a fifth cellular receptor for LPA (39, 40), which showed the highest expression in gut. LPA4 and LPA5 knock-out mice have not been reported so far. It is possible that ATX exerts its role in blood vessel stabilization through these newly identified LPA receptors via signaling pathways independent of classical LPA receptors (LPA1, LPA2, and LPA3).


   FOOTNOTES
 
* This research was supported by grants from National Institute of Biomedical Innovation, Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Corporation, the 21st Century Center of Excellence Program, and the Ministry of Education, Science, Sports, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 81-3-5841-4723; Fax: 81-3-3818-3173; E-mail: jaoki{at}mol.f.u-tokyo.ac.jp.

2 The abbreviations used are: ATX, autotaxin; LPA, lysophosphatidic acid; S1P, sphingosine 1-phosphate; PECAM, platelet-endothelial cell adhesion molecule; E, embryonic day; lysoPLD, lysophospholipase D; RT-PCR, reverse transcription PCR. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Wouter H. Moolenaar and Masayuki Masu for sharing unpublished results.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Stracke, M. L., Krutzsch, H. C., Unsworth, E. J., Arestad, A., Cioce, V., Schiffmann, E., and Liotta, L. A. (1992) J. Biol. Chem. 267, 2524-2529[Abstract/Free Full Text]
  2. Nam, S. W., Clair, T., Campo, C. K., Lee, H. Y., Liotta, L. A., and Stracke, M. L. (2000) Oncogene 19, 241-247[CrossRef][Medline] [Order article via Infotrieve]
  3. Mills, G. B., and Moolenaar, W. H. (2003) Nat. Rev. Cancer 3, 582-591[CrossRef][Medline] [Order article via Infotrieve]
  4. Aoki, J. (2004) Semin. Cell Dev. Biol. 15, 477-489[CrossRef][Medline] [Order article via Infotrieve]
  5. Umezu-Goto, M., Kishi, Y., Taira, A., Hama, K., Dohmae, N., Takio, K., Yamori, T., Mills, G. B., Inoue, K., Aoki, J., and Arai, H. (2002) J. Cell Biol. 158, 227-233[Abstract/Free Full Text]
  6. Tokumura, A., Majima, E., Kariya, Y., Tominaga, K., Kogure, K., Yasuda, K., and Fukuzawa, K. (2002) J. Biol. Chem. 277, 39436-39442[Abstract/Free Full Text]
  7. Clair, T., Aoki, J., Koh, E., Bandle, R. W., Nam, S. W., Ptaszynska, M. M., Mills, G. B., Schiffmann, E., Liotta, L. A., and Stracke, M. L. (2003) Cancer Res. 63, 5446-5453[Abstract/Free Full Text]
  8. Ishii, I., Fukushima, N., Ye, X., and Chun, J. (2004) Annu. Rev. Biochem. 73, 321-354[CrossRef][Medline] [Order article via Infotrieve]
  9. Hama, K., Aoki, J., Fukaya, M., Kishi, Y., Sakai, T., Suzuki, R., Ohta, H., Yamori, T., Watanabe, M., Chun, J., and Arai, H. (2004) J. Biol. Chem. 279, 17634-17639[Abstract/Free Full Text]
  10. Takuwa, Y. (2002) Biochim. Biophys. Acta 1582, 112-120[Medline] [Order article via Infotrieve]
  11. Shalaby, F., Rossant, J., Yamaguchi, T. P., Gertsenstein, M., Wu, X. F., Breitman, M. L., and Schuh, A. C. (1995) Nature 376, 62-66[CrossRef][Medline] [Order article via Infotrieve]
  12. Kishimoto, T., Matsuoka, T., Imamura, S., and Mizuno, K. (2003) Clin. Chim. Acta 333, 59-67[CrossRef][Medline] [Order article via Infotrieve]
  13. Yatomi, Y., Ruan, F., Ohta, J., Welch, R. J., Hakomori, S., and Igarashi, Y. (1995) Anal. Biochem. 230, 315-320[CrossRef][Medline] [Order article via Infotrieve]
  14. Ruwisch, L., Schafer, K. M., and Kleuser, B. (2001) Naunyn-Schmiedebergs Arch. Pharmacol. 363, 358-363[CrossRef][Medline] [Order article via Infotrieve]
  15. Tanaka, M., Kishi, Y., Takanezawa, Y., Kakehi, Y., Aoki, J., and Arai, H. (2004) FEBS Lett. 571, 197-204[CrossRef][Medline] [Order article via Infotrieve]
  16. Iseki, S., Wilkie, A. O., Heath, J. K., Ishimaru, T., Eto, K., and Morriss, K. G. (1997) Development 124, 3375-3384[Abstract]
  17. Kishi, Y., Okudaira, S., Tanaka, M., Hama, K., Shida, D., Kitayama, J., Yamori, T., Aoki, J., Fujimaki, T., and Arai, H. (2006) J. Biol. Chem. 281, 17492-17500[Abstract/Free Full Text]
  18. Argraves, W. S., Larue, A. C., Fleming, P. A., and Drake, C. J. (2002) Dev. Dyn. 225, 298-304[CrossRef][Medline] [Order article via Infotrieve]
  19. Ohta, H., Sato, K., Murata, N., Damirin, A., Malchinkhuu, E., Kon, J., Kimura, T., Tobo, M., Yamazaki, Y., Watanabe, T., Yagi, M., Sato, M., Suzuki, R., Murooka, H., Sakai, T., Nishitoba, T., Im, D. S., Nochi, H., Tamoto, K., Tomura, H., and Okajima, F. (2003) Mol. Pharmacol. 64, 994-1005[Abstract/Free Full Text]
  20. Aoki, J., Taira, A., Takanezawa, Y., Kishi, Y., Hama, K., Kishimoto, T., Mizuno, K., Saku, K., Taguchi, R., and Arai, H. (2002) J. Biol. Chem. 277, 48737-48744[Abstract/Free Full Text]
  21. Contos, J. J., Fukushima, N., Weiner, J. A., Kaushal, D., and Chun, J. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 13384-13389[Abstract/Free Full Text]
  22. Contos, J. J., Ishii, I., Fukushima, N., Kingsbury, M. A., Ye, X., Kawamura, S., Brown, J. H., and Chun, J. (2002) Mol. Cell. Biol. 22, 6921-6929[Abstract/Free Full Text]
  23. Ye, X., Hama, K., Contos, J. J., Anliker, B., Inoue, A., Skinner, M. K., Suzuki, H., Amano, T., Kennedy, G., Arai, H., Aoki, J., and Chun, J. (2005) Nature 435, 104-108[CrossRef][Medline] [Order article via Infotrieve]
  24. Nam, S. W., Clair, T., Kim, Y. S., McMarlin, A., Schiffmann, E., Liotta, L. A., and Stracke, M. L. (2001) Cancer Res. 61, 6938-6944[Abstract/Free Full Text]
  25. Folkman, J. (1995) Nat. Med. 1, 27-31[CrossRef][Medline] [Order article via Infotrieve]
  26. Yang, Y., Mou, L. J., Liu, N., and Tsao, M. S. (1999) Am. J. Respir. Cell Mol. Biol. 21, 216-222[Abstract/Free Full Text]
  27. Stassar, M. J., Devitt, G., Brosius, M., Rinnab, L., Prang, J., Schradin, T., Simon, J., Petersen, S., Kopp, S. A., and Zoller, M. (2001) Br. J. Cancer 85, 1372-1382[CrossRef][Medline] [Order article via Infotrieve]
  28. Zhao, Z., Xu, S., and Zhang, G. (1999) Zhonghua Gan Zang Bing Za Zhi 7, 140-141[Medline] [Order article via Infotrieve]
  29. Zhang, G., Zhao, Z., Xu, S., Ni, L., and Wang, X. (1999) Chin. Med. J. 112, 330-332[Medline] [Order article via Infotrieve]
  30. Euer, N., Schwirzke, M., Evtimova, V., Burtscher, H., Jarsch, M., Tarin, D., and Weidle, U. H. (2002) Anticancer Res. 22, 733-740[Medline] [Order article via Infotrieve]
  31. Yang, S. Y., Lee, J., Park, C. G., Kim, S., Hong, S., Chung, H. C., Min, S. K., Han, J. W., Lee, H. W., and Lee, H. Y. (2002) Clin. Exp. Metastasis 19, 603-608[CrossRef][Medline] [Order article via Infotrieve]
  32. Baumforth, K. R., Flavell, J. R., Reynolds, G. M., Davies, G., Pettit, T. R., Wei, W., Morgan, S., Stankovic, T., Kishi, Y., Arai, H., Nowakova, M., Pratt, G., Aoki, J., Wakelam, M. J., Young, L. S., and Murray, P. G. (2005) Blood 106, 2138-2146[Abstract/Free Full Text]
  33. Kehlen, A., Englert, N., Seifert, A., Klonisch, T., Dralle, H., Langner, J., and Hoang-Vu, C. (2004) Int. J. Cancer 109, 833-838[CrossRef][Medline] [Order article via Infotrieve]
  34. Allende, M. L., Sasaki, T., Kawai, H., Olivera, A., Mi, Y., van Echten-Deckert, G., Hajdu, R., Rosenbach, M., Keohane, C. A., Mandala, S., Spiegel, S., and Proia, R. L. (2004) J. Biol. Chem. 279, 52487-52492[Abstract/Free Full Text]
  35. Kharel, Y., Lee, S., Snyder, A. H., Sheasley, O. S., Morris, M. A., Setiady, Y., Zhu, R., Zigler, M. A., Burcin, T. L., Ley, K., Tung, K. S., Engelhard, V. H., Macdonald, T. L., Pearson, W. S., and Lynch, K. R. (2005) J. Biol. Chem. 280, 36865-36872[Abstract/Free Full Text]
  36. Mizugishi, K., Yamashita, T., Olivera, A., Miller, G. F., Spiegel, S., and Proia, R. L. (2005) Mol. Cell. Biol. 25, 11113-11121[Abstract/Free Full Text]
  37. Kono, M., Mi, Y., Liu, Y., Sasaki, T., Allende, M. L., Wu, Y. P., Yamashita, T., and Proia, R. L. (2004) J. Biol. Chem. 279, 29367-29373[Abstract/Free Full Text]
  38. Noguchi, K., Ishii, S., and Shimizu, T. (2003) J. Biol. Chem. 278, 25600-25606[Abstract/Free Full Text]
  39. Lee, C. W., Rivera, R., Gardell, S., Dubin, A. E., and Chun, J. (June 14, 2006) J. Biol. Chem. doi: 10.1074/jbc.M603670200[Abstract/Free Full Text]
  40. Kotarsky, K., Boketoft, A., Bristulf, J., Nilsson, N. E., Norberg, A., Hansson, S., Sillard, R., Owman, C., Leeb-Lundberg, F. L., and Olde, B. (2006) J. Pharmacol. Exp. Ther. 318, 619-628[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Cancer Res.Home page
H. Zhang, X. Xu, J. Gajewiak, R. Tsukahara, Y. Fujiwara, J. Liu, J. I. Fells, D. Perygin, A. L. Parrill, G. Tigyi, et al.
Dual Activity Lysophosphatidic Acid Receptor Pan-Antagonist/Autotaxin Inhibitor Reduces Breast Cancer Cell Migration In vitro and Causes Tumor Regression In vivo
Cancer Res., July 1, 2009; 69(13): 5441 - 5449.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Yanagida, K. Masago, H. Nakanishi, Y. Kihara, F. Hamano, Y. Tajima, R. Taguchi, T. Shimizu, and S. Ishii
Identification and Characterization of a Novel Lysophosphatidic Acid Receptor, p2y5/LPA6
J. Biol. Chem., June 26, 2009; 284(26): 17731 - 17741.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Jansen, M. Andries, R. Derua, E. Waelkens, and M. Bollen
Domain Interplay Mediated by an Essential Disulfide Linkage Is Critical for the Activity and Secretion of the Metastasis-promoting Enzyme Autotaxin
J. Biol. Chem., May 22, 2009; 284(21): 14296 - 14302.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
A. Tokumura, T. Kume, S. Taira, K. Yasuda, and H. Kanzaki
Altered activity of lysophospholipase D, which produces bioactive lysophosphatidic acid and choline, in serum from women with pathological pregnancy
Mol. Hum. Reprod., May 1, 2009; 15(5): 301 - 310.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Z. Pamuklar, L. Federico, S. Liu, M. Umezu-Goto, A. Dong, M. Panchatcharam, Z. Fulerson, E. Berdyshev, V. Natarajan, X. Fang, et al.
Autotaxin/Lysopholipase D and Lysophosphatidic Acid Regulate Murine Hemostasis and Thrombosis
J. Biol. Chem., March 13, 2009; 284(11): 7385 - 7394.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
A. Ambesi and P. J. McKeown-Longo
Anastellin, the Angiostatic Fibronectin Peptide, Is a Selective Inhibitor of Lysophospholipid Signaling
Mol. Cancer Res., February 1, 2009; 7(2): 255 - 265.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
E. Liszewska, P. Reinaud, E. Billon-Denis, O. Dubois, P. Robin, and G. Charpigny
Lysophosphatidic Acid Signaling during Embryo Development in Sheep: Involvement in Prostaglandin Synthesis
Endocrinology, January 1, 2009; 150(1): 422 - 434.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
G. Ferry, N. Moulharat, J.-P. Pradere, P. Desos, A. Try, A. Genton, A. Giganti, M. Beucher-Gaudin, M. Lonchampt, M. Bertrand, et al.
S32826, A Nanomolar Inhibitor of Autotaxin: Discovery, Synthesis and Applications as a Pharmacological Tool
J. Pharmacol. Exp. Ther., December 1, 2008; 327(3): 809 - 819.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
Z. Lee, C.-T. Cheng, H. Zhang, M. A. Subler, J. Wu, A. Mukherjee, J. J. Windle, C.-K. Chen, and X. Fang
Role of LPA4/p2y9/GPR23 in Negative Regulation of Cell Motility
Mol. Biol. Cell, December 1, 2008; 19(12): 5435 - 5445.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
T. Nakasaki, T. Tanaka, S. Okudaira, M. Hirosawa, E. Umemoto, K. Otani, S. Jin, Z. Bai, H. Hayasaka, Y. Fukui, et al.
Involvement of the Lysophosphatidic Acid-Generating Enzyme Autotaxin in Lymphocyte-Endothelial Cell Interactions
Am. J. Pathol., November 1, 2008; 173(5): 1566 - 1576.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
L. P. Saunders, A. Ouellette, R. Bandle, W. C. Chang, H. Zhou, R. N. Misra, E. M. De La Cruz, and D. T. Braddock
Identification of small-molecule inhibitors of autotaxin that inhibit melanoma cell migration and invasion
Mol. Cancer Ther., October 1, 2008; 7(10): 3352 - 3362.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
M. Panchatcharam, S. Miriyala, F. Yang, M. Rojas, C. End, C. Vallant, A. Dong, K. Lynch, J. Chun, A. J. Morris, et al.
Lysophosphatidic Acid Receptors 1 and 2 Play Roles in Regulation of Vascular Injury Responses but Not Blood Pressure
Circ. Res., September 12, 2008; 103(6): 662 - 670.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
X. Ye
Lysophospholipid signaling in the function and pathology of the reproductive system
Hum. Reprod. Update, September 1, 2008; 14(5): 519 - 536.
[Abstract] [Full Text] [PDF]

Home page
 Ann Clin BiochemHome page
S. Hosogaya, Y. Yatomi, K. Nakamura, R. Ohkawa, S. Okubo, H. Yokota, M. Ohta, H. Yamazaki, T. Koike, and Y. Ozaki
Measurement of plasma lysophosphatidic acid concentration in healthy subjects: strong correlation with lysophospholipase D activity
Ann Clin Biochem, July 1, 2008; 45(4): 364 - 368.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Giganti, M. Rodriguez, B. Fould, N. Moulharat, F. Coge, P. Chomarat, J.-P. Galizzi, P. Valet, J.-S. Saulnier-Blache, J. A. Boutin, et al.
Murine and Human Autotaxin {alpha}, {beta}, and {gamma} Isoforms: GENE ORGANIZATION, TISSUE DISTRIBUTION, AND BIOCHEMICAL CHARACTERIZATION
J. Biol. Chem., March 21, 2008; 283(12): 7776 - 7789.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
M. M. Ptaszynska, M. L. Pendrak, R. W. Bandle, M. L. Stracke, and D. D. Roberts
Positive Feedback between Vascular Endothelial Growth Factor-A and Autotaxin in Ovarian Cancer Cells
Mol. Cancer Res., March 1, 2008; 6(3): 352 - 363.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. C. Kienesberger, A. Lass, K. Preiss-Landl, H. Wolinski, S. D. Kohlwein, R. Zimmermann, and R. Zechner
Identification of an Insulin-regulated Lysophospholipase with Homology to Neuropathy Target Esterase
J. Biol. Chem., February 29, 2008; 283(9): 5908 - 5917.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Jansen, N. Callewaert, I. Dewerte, M. Andries, H. Ceulemans, and M. Bollen
An Essential Oligomannosidic Glycan Chain in the Catalytic Domain of Autotaxin, a Secreted Lysophospholipase-D
J. Biol. Chem., April 13, 2007; 282(15): 11084 - 11091.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/35/25822    most recent
M605142200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tanaka, M.
Right arrow Articles by Arai, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tanaka, M.
Right arrow Articles by Arai, H.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement