VE-cadherin Links tRNA Synthetase Cytokine to Anti-angiogenic Function

Abstract

A natural fragment of an enzyme that catalyzes the first step of protein synthesis-human tryptophanyltRNA synthetase (T2-TrpRS) has potent anti-angiogenic activity. A cellular receptor through which T2-TrpRS exerts its anti-angiogenic activity has not previously been identified. Here T2-TrpRS was shown to bind at intercellular junctions of endothelial cells (ECs). Using genetic knock-outs, binding was established to depend on VE-cadherin, a calcium-dependent adhesion molecule, which is selectively expressed in ECs, concentrated at adherens junctions, and is essential for normal vascular development. In contrast, T2-TrpRS binding to EC junctions was not dependent on platelet endothelial cell adhesion molecule type-1, another adhesion molecule found at EC junctions. Pull-down assays confirmed direct complex formation between T2-TrpRS and VEcadherin. Binding of T2-TrpRS inhibited VEGF-induced ERK activation and EC migration. Thus, a VE-cadherindependent pathway is proposed to link T2-TrpRS to inhibition of new blood vessel formation.During their long evolution, aminoacyl-tRNA synthetases, enzymes that catalyze the first step of protein synthesisacquired additional functions, including regulation of transcription and translation, RNA splicing, and cytokine activities in inflammatory and angiogenic signaling pathways (1, 2). Recently, fragments of the closely related human tyrosyl-tRNA and tryptophanyl-tRNA synthetase (TrpRS) 1 were discovered to regulate angiogenesis (2-6). In mammalian cells, TrpRS is activated for anti-angiogenic signaling by proteolysis or alternative splicing to give two natural isoforms-mini-TrpRS and T2-TrpRS (6). Expression of mini-TrpRS is strongly induced (along with other angiostatic factors such as IP-10 and MIG) by the anti-proliferative cytokine interferon-␥ (7-9). Mini-TrpRS and T2-TrpRS inhibit development of new vessels without affecting pre-established vasculature (4, 6, 10). Regions within mini-TrpRS that contribute to angiostatic activity have been proposed (11,12). The anti-angiogenic activity of T2-TrpRS and mini-TrpRS was demonstrated in several cell-based assays in vitro and also in vivo in the chick embryo and in the neonatal and adult mouse (4, 6). More recently, in vitro studies on endothelial cells (ECs) have shown that the inhibitory activity of mini-TrpRS and T2-TrpRS abrogates cellular responses involved in angiogenesis, such as motility and migration of ECs (4, 6) and activation of extracellular signal-regulated kinase (ERK1/2) and Akt (13). T2-TrpRS inhibited several additional EC responses to the mechanical force of shear stress including endothelial nitric-oxide synthase activation, shear stress-induced gene expression, and cell alignment (13). While the protein binds specifically to the ends of newly formed vessels, the connection of this binding to the inhibition of angiogenic signal transduction pathways remains unknown. To further elucidate the mechanism of T2-TrpRS, we sought to identify the EC target responsible for the potent anti-angiogenic activity. EXPER...

Small interfering RNA knockdown of mini-TyrRS and mini-TrpRS effects angiogenesis in human umbilical vein endothelial cells in hypoxic culture

“…*p \ 0.05, compared with the mock transfection group and negative control group. There were no statistic difference between the mock transfection group and the negative control group (Lee et al 2004;Tzima et al 2005). The same phenomenon is also present between full-length TyrRS and mini-TyrRS.…”

Antioxidant or neurotrophic factor treatment preserves function in a mouse model of neovascularization-associated oxidative stress

“…Intravitreal injection of Macugen, an RNA aptamer that blocks VEGF 165 , reduced intra- and subretinal NV confirming that VEGF upregulation in the deep retina is critical for abnormal vessel formation in the Vldlr -/-mouse ( Figure 3C). In order to further evaluate effects of antiangiogenic therapy on subretinal vessels, we used a combination of angiostatic agents previously shown to synergistically inhibit retinal angiogenesis (28): Macugen, an integrin α v β 3 /α v β 5 antagonist, and T2-TrpRS, a fragment of tryptophan transfer RNA synthetase with angiostatic activity (29)(30)(31). Using combination therapy, we observed substantial reductions in intraretinal vascular sprouts and total area of subretinal NV (70% and 60%, respectively; Figure 3, D and E).…”

An alternative conformation of human TrpRS suggests a role of zinc in activating non-enzymatic function

“…7,8,9 Our previous study has demonstrated that T2-TpRS binds the two tryptophan residues (Trp2 and Trp4) through its tryptophan and AMP binding pockets in the active site, respectively, and thereby inhibits the VE-cadherin mediated endothelial cell-cell junctions and disrupts the formation of new blood vessels. 8 Notably, human TrpRS manifests its anti-angiogenic activity only when the WHEP domain is removed.…”