ATX-LPA 1 axis contributes to proliferation of chondrocytes by regulating fibronectin assembly leading to proper cartilage formation

The lipid mediator lysophosphatidic acid (LPA) signals via six distinct G protein-coupled receptors to mediate both unique and overlapping biological effects, including cell migration, proliferation and survival. LPA is produced extracellularly by autotaxin (ATX), a secreted lysophospholipase D, from lysophosphatidylcholine. ATX-LPA receptor signaling is essential for normal development and implicated in various (patho)physiological processes, but underlying mechanisms remain incompletely understood. Through gene targeting approaches in zebrafish and mice, we show here that loss of ATX-LPA(1) signaling leads to disorganization of chondrocytes, causing severe defects in cartilage formation. Mechanistically, ATX-LPA(1) signaling acts by promoting S-phase entry and cell proliferation of chondrocytes both in vitro and in vivo, at least in part through β1-integrin translocation leading to fibronectin assembly and further extracellular matrix deposition; this in turn promotes chondrocyte-matrix adhesion and cell proliferation. Thus, the ATX-LPA(1) axis is a key regulator of cartilage formation

Autotaxin Overexpression Causes Embryonic Lethality and Vascular Defects

<div><p>Autotaxin (ATX) is a secretory protein, which converts lysophospholipids to lysophosphatidic acid (LPA), and is essential for embryonic vascular formation. ATX is abundantly detected in various biological fluids and its level is elevated in some pathophysiological conditions. However, the roles of elevated ATX levels remain to be elucidated. In this study, we generated conditional transgenic (Tg) mice overexpressing ATX and examined the effects of excess LPA signalling. We found that ATX overexpression in the embryonic period caused severe vascular defects and was lethal around E9.5. ATX was conditionally overexpressed in the neonatal period using the Cre/loxP system, which resulted in a marked increase in the plasma LPA level. This resulted in retinal vascular defects including abnormal vascular plexus and increased vascular regression. Our findings indicate that the ATX level must be carefully regulated to ensure coordinated vascular formation</p></div

Overexpression of ATX delays retinal vascularization and decreases vessel branching.

<p>(A and B) Vascular defects in retina from ATX cTg mice at P6. Retina vasculature was visualized by staining the vessels with isolectin B4. Scale bar, 500 μm. (C) Magnification view of vascular plexus in retina from ATX cTg mice at P6. Scale bar, 100 μm. (D) The vascular defects were evaluated by determining the branching points quantitatively. Error bars indicate <i>s</i>.<i>d</i>. (control; n = 12, ATX cTg; n = 7). P-values were estimated by student’s <i>t</i>-test, ***P < 0.001. Data were pooled from three independent experiments.</p

Overexpression of ATX causes abnormal vessel morphology and vessel regression.

<p>(A and B) Magnification view of angiogenic front in retina from ATX cTg mice at P6. Control and ATX cTg retinas had similar filopodia protrusion. Scale bar, 50 μm. TM, tamoxifen. (C and D) ATX cTg retinas displayed vessel regression at vascular plexus. Control (wild type) and ATX cTg retinas labeled for CD31 (green) and collagen IV (red). Arrows highlight empty collagen IV sleeves, indicating vessel regression. Scale bar, 100 μm. (E and F) Vessel regression was evaluated quantitatively. Error bars indicate <i>s</i>.<i>d</i>. (control; n = 7, ATX cTg; n = 5). P-values were estimated by student’s <i>t</i>-test, ***P < 0.001. Data were pooled from three independent experiments.</p

Transient ATX overexpression decreases vessel branching but does not delay retinal vascularization.

<p>(A) Schematic of the experimental strategy to assess initial defects in retinal vasculature in ATX cTg mice. (B) LysoPLD activity of ATX cTg mice plasma. Error bars indicate <i>s</i>.<i>d</i>. (control; n = 9, ATX cTg; n = 4). (C and D) Vascular defects in retina from ATX cTg mice at P6. Retina vasculature was visualized by staining the vessels with isolectin B4. Scale bar, 500 μm. (E) Magnification view of vascular plexus in retina from ATX cTg mice at P6. Scale bar, 100 μm. (F) The vascular defects were evaluated by determining the branching points quantitatively. Error bars indicate <i>s</i>.<i>d</i>. (control; n = 9, ATX cTg; n = 4). P-values were estimated by student’s <i>t</i>-test, **P < 0.01. Data in (B) and (F) were pooled from three independent experiments.</p

Overexpression of ATX in embryos led to lethality with severe defects.

<p>(A) Schematic diagram of the construction of ATX Tg mice. The ATX transgene was inserted at the downstream of the neo^r/pA cassette. This fragment, in which the ATX transgene is silent, was introduced into mice, and the transgene-positive offspring were then mated with CAG-Cre Tg mice. At this stage, the LNL (for loxP-neo^r/pA-loxP) cassette was excised by Cre recombinase, and the ATX transgene was activated under control of the CAG promoter in the transgene-positive embryos. (B) PCR genotyping of ATX Tg mice. After mating of LNL-ATX Tg mice with CAG-Cre Tg mice, PCR genotyping was performed. Fragments of 1.6 and 0.2 kb were amplified for LNL-ATX Tg and CAG-ATX Tg (ATX Tg) mice, respectively, whereas these products were not detected in WT littermates. (C) A picture of embryos and placentas at E11.5. (D) Defects in the yolk sac vasculature. Yolk sac from control (wild type) and ATX Tg embryos at E10.5. (E-G) Morphologies of control (wild type) (left) and ATX Tg (right) embryo proper at E9.5 and E10.5. At E10.5 (E) and E9.5 (F and G), ATX Tg embryos are easily distinguishable from control littermates (wild type). ATX Tg embryos exhibit several defects such as growth retardation (E), open and kinky neural tube (F, arrow) and abnormal allantois (G, arrow). Scale bars, 200 μm in panels D and E and 100 μm in panels F and G. (H) Quantitative RT-PCR analysis of ATX mRNA in mouse embryos at E8.5. (wild type; n = 3, CAG-Cre; n = 3, LNL-ATX Tg; n = 2, ATX Tg; n = 3,).</p

ATX expression is increased in ATX conditional transgenic mice.

<p>(A) Schematic of the experimental strategy to assess formation of the retinal vasculature in ATX conditional Tg (ATX cTg) mice. (B) LysoPLD activity of ATX cTg mice plasma. LysoPLD activity was determined by liberation of choline from lysophosphatidylcholine (LPC) using 14:0-LPC as a substrate. Error bars indicate <i>s</i>.<i>d</i>. (control; n = 12, ATX cTg; n = 7). P-values were estimated by student’s <i>t</i>-test, ***P < 0.001. (C) Relative abundance of five major LPA species (16:0, 18:1, 18:2, 20:4 and 22:6-LPA) in mice plasma. Lipids in plasma were extracted with methanol and analyzed by LC-MS/MS. Error bars indicate <i>s</i>.<i>d</i>. (control; n = 12, ATX cTg; n = 7). P-values were estimated by one-way ANOVA with Bonferroni’s posttest analyses, *P < 0.05, ***P < 0.001. (D) Western blot analysis of ATX in plasma isolated from CreER and ATX cTg mice. Data in (B) and (C) were pooled from three independent experiments.</p

Genotype distribution of offspring from LNL-ATX Tg females crossed with CAG-Cre males.

<p>Genotype distribution of offspring from LNL-ATX Tg females crossed with CAG-Cre males.</p