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

Yasuhiro Kishi,Masato Ota,Ryunosuke Ohkawa,Sumihare Noji,Masayuki Tanaka,Yutaka Yatomi,Hiroyuki Arai,Junken Aoki,Sachiko Iseki,Shinichi Okudaira

doi:10.1074/jbc.m605142200

Abstract

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.

Highlights

ATX was shown to have lysophospholipase D activity, which converts lysophosphatidylcholine to a bioactive lysophospholipid, lysophosphatidic acid (LPA) [5, 6]
LPA and sphingosine 1-phosphate (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]
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

Summary

MATERIALS AND METHODS

ATX Knock-out Mice—ATX knock-out mice (atxϪ/Ϫ) in the genetic background 129/SvEvBrd were produced by and obtained from Lexicon Genetics (The Woodlands, TX). B, genotyping of atxϩ/ϩ, atxϩ/Ϫ, and atxϪ/Ϫ 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). 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. Western blotting of ATX was performed as described using ATX-specific monoclonal antibody [15]. Primers used to detect ATX, LPA receptors, glyceraldehyde-3-phosphate dehydrogenase, and ␤-actin were described previously [17]. Allantois Culture—Allantoides were dissected from embryos Quantitative RT-PCR was performed as described [9] using at E8.5 and cultured in Dulbecco’s modified Eagle’s medium ABI PRISM7000 sequence detection system Each sample was normalized to the number of ␤-actin or sel formation, the allantois explants were cultured for 24 h glyceraldehyde-3-phosphate dehydrogenase transcripts

RESULTS

Embryo resorption

DISCUSSION