LPP3 localizes LPA6 signalling to non-contact site in endothelial cells

Lysophosphatidic acid (LPA) is an emerging angiogenic factor, as knockdown of its producing enzyme, autotaxin and receptors caused severe developmental vascular defects in both mice and fish. In addition, overexpression of autotaxin in mice caused 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), and showed that LPP3 was localized in specific cell-cell contact sites of endothelial cells and suppresses LPA signalling via LPA6 receptor. In HEK293 cells, overexpression of LPP3 dramatically suppressed activation of LPA6. In human umbilical vein endothelial cells (HUVECs), LPA induced actin stress fiber formation via LPA6, which was prominently up-regulated by LPP3 knockdown. LPP3 was localized to cell-cell contact sites and was missing in non-contact sites to which LPA-induced actin stress fiber formation mediated by LPA6 was restricted. Interestingly, the expression of LPP3 in HUVECs was dramatically increased after forskolin-treatment, in which Notch signalling was involved. These results indicate that LPP3 regulates and localizes LPA signalling in endothelial cells thereby stabilizing vessels via Notch signalling for proper vasculature. Jo ur na l o f C el l S ci en ce A cc ep te d m an us cr ip t Introduction LPA regulates a wide variety of cellular processes in vertebrates including the migration, adhesion, proliferation, differentiation and cell death, and thereby influences multiple in vivo events ranging from organogenesis to development of cancer (Ishii et al., 2004; Lin et al., 2010). LPA is mainly produced by autotaxin (ATX), an extracellular enzyme that converts lysophospholipids to LPA, and exerts its action through at least six G protein-coupled receptors (LPA1-6) specific to LPA (Chun et al., 2010). A major question in this field is how extracellularly-produced LPA is controlled. Lipid phosphate phosphatases (LPPs) are key factors controlling LPA metabolism. LPPs are integral membrane enzymes that dephosphorylate lipid phosphates such as sphingosine-1-phosphate (S1P) and LPA (Brindley and Pilquil, 2009; Pyne et al., 2005). In vertebrates, at least three LPP genes (LPP1-3) have been identified (Kai et al., 1997; Roberts et al., 1998). LPA under the control of these LPA-related genes was recently shown to be a key regulator of the development of embryonic vasculature in both mice and zebrafish (Escalante-Alcalde et al., 2003; Panchatcharam et al., 2014; Tanaka et al., 2006; Yukiura et al., 2011). ATX knockout mice died around E9.5 due to the defects of blood vessel formation in the yolk sac, allantois and embryos (Tanaka et al., 2006). Similar vascular defects were observed when ATX expression was suppressed in zebrafish embryos (Yukiura et al., 2011). In the zebrafish embryos, ATX knockdown caused retarded elongation (or sprouting) of intersegmental vessels (ISVs). Knockdown of multiple LPA receptors (LPA1, LPA4 and LPA6) resulted in similar defects in ISV formation (Yukiura et al., Jo ur na l o f C el l S ci en ce A cc ep te d m an us cr ip t 2011). LPP3 knockout mice failed to form a chorio-allantoic placenta and yolk sac vasculature around E9.5 (Escalante-Alcalde et al., 2003). This phenotype was also reproduced in endothelial cell-specific LPP3 knockout mice (Panchatcharam et al., 2014). Very recently, we established transgenic mice overexpressing ATX and found that overexpression of ATX in embryo caused similar vascular defects, as well as vascular defects in retina when ATX was overexpressed conditionally in neonate. Because endothelial cells but not mural cells are involved in vascular formation in the embryonic stages, it is assumed that the level of LPA, balanced by ATX and LPP3, affects endothelial cell functions via LPA receptors in a highly coordinated fashion, thereby regulating blood vessel formation. To understand regulation of LPA signalling, we examined the roles of LPA and LPP3 using human umbilical vein endothelial cells