Induction of Angiogenesis by a Fragment of Human Tyrosyl-tRNA Synthetase*

The first step of protein synthesis is catalyzed by aminoacyl-tRNA synthetases. In addition, certain mammalian tRNA synthetases link protein synthesis to cytokine signaling pathways. In particular, human tyrosyl-tRNA synthetase (TyrRS) can be split by proteolysis into two fragments having distinct cytokine activities. One of the TyrRS fragments (mini TyrRS) contains features identical to those in CXC chemokines (like interleukin-8) that also act as angiogenic factors. Here mini TyrRS (but not full-length TyrRS) is shown to stimulate chemotaxis of endothelial cells in vitroand stimulate angiogenesis in each of two in vivo animal models. The angiogenic activity of mini TyrRS can be opposed by anti-angiogenic chemokines like IP-10. Thus, a biological fragment of human tyrosyl-tRNA synthetase links protein synthesis to regulation of angiogenesis.

Previous work on human tyrosyl-tRNA synthetase (TyrRS) 1 has shown that isolated domains of TyrRS possess cytokine activities (1). Initial studies were stimulated by the finding that human TyrRS contains a C-terminal domain with 49% sequence identity to the angiostatic cytokine human endothelial monocyte-activating polypeptide II (2). Surprisingly two separate signaling domains are embedded in human tyrosyl-tRNA synthetase, and both are released upon leukocyte elastase digestion (1,3). The isolated C-terminal domain (C-domain) had similar functional activities to its homologue endothelial monocyte-activating polypeptide II, such as inducing migration of mononuclear phagocytes and stimulating the production of tumor necrosis factor-␣ and tissue factor. In addition, the C-domain induced migration in polymorphonuclear leukocytes (PMNs) and stimulated them to release myeloperoxidase (1). The N-terminal domain (mini TyrRS), containing the Rossmann nucleotide-binding fold common to the 10 class I tRNA synthetases, induced directed migration of PMN cells with a bell-shaped concentration dependence like that of the CXC chemokine interleukin-8 . In contrast, full-length TyrRS was inactive in assays for these cytokine activities (1,3).
The similarity in PMN cell responses to mini TyrRS and IL-8 suggested a functional correlation between mini TyrRS and IL-8 activity. All CXC chemokines, such as IL-8, that function as PMN chemoattractants have a conserved Glu-Leu-Arg (ELR) motif near the N terminus. The ELR motif is critical for PMN receptor binding and PMN activation (4,5). Human mini TyrRS also has an ELR motif that is conserved among mammalian TyrRSs (3). The ELR motif occurs within the catalytic domain that contains a Rossmann nucleotide-binding fold. The close orthologue of mini TyrRS in bacteria and the yeast Saccharomyces cerevisiae lacked the cell signaling activity, thus supporting the idea that the cytokine activity was specific to the mammalian system. Mutagenesis experiments clarified that the ELR motif in human mini TyrRS is required for PMN receptor binding and for the cytokine activities of mini TyrRS just as it is for CXC chemokines (1). All CXC chemokines containing the ELR motif, such as IL-8, act as angiogenic factors (6 -10). Thus, the possibility was raised that human mini TyrRS can act as an angiogenic factor.

EXPERIMENTAL PROCEDURES
Protein Production and Biochemical Analysis-Recombinant human full-length and mini TyrRS were expressed and purified from Escherichia coli as described previously (1). Protein concentration was determined by the Bradford assay using the Bio-Rad Protein Assay reagent (Bio-Rad) with bovine serum albumin (Sigma) as a standard.
Human Umbilical Vein Endothelial Cell (HUVEC) Migration Assay-HUVECs were obtained from Clonetics (Walkersville, MD) and maintained in EGM ® -2 BulletKit ® medium (Clonetics) in an atmosphere of 5% CO 2 in air at 37°C according to the instructions of the supplier. Human vascular endothelial growth factor-165 (VEGF 165 ) (BIOSOURCE, Camarillo, CA) and human IP-10 (R&D Systems, Minneapolis, MN) were used in several experiments.
Angiogenesis Assays-Two different assays for angiogenesis were used to examine TyrRSs for activities in vivo (12)(13)(14)(15). The chicken chorioallantoic membrane (CAM) assay was performed as described previously (16,17) with 10-day-old chick embryos obtained from Mcintyre Poultry (Lakeside, CA). Cortisone acetate-treated 5-mm filter disks soaked with 20-l samples of VEGF 165 (1 pmol), human mini TyrRS (6 pmol), or full-length TyrRS (6 pmol) in phosphate-buffered saline (PBS) were placed onto the CAMs. Inhibitors and control samples (PBS alone, IP-10 (120 pmol), or mini tryptophanyl-tRNA synthetase (TrpRS) (60 * This work was supported by National Institutes of Health Grants GM23562 and CA92577 and by a fellowship from the National Foundation for Cancer Research. 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. § Supported by Japan Society for the Promotion of Science postdoctoral fellowships for research abroad (1997)(1998)(1999) pmol)) were added topically to the filter disks for 3 consecutive days. After 72 h, the CAM tissue associated with the filter disc was harvested and photographed at ϫ10 magnification on an Olympus Model SZH10 stereomicroscope. Angiogenesis was quantified by analyzing the number of blood vessel branches within the area of each disc.

RESULTS AND DISCUSSION
HUVEC Chemotaxis in Response to Mini TyrRS-In earlier experiments, we found that mini TyrRS stimulated PMN migration and that an ELR motif within human mini TyrRS was essential for the cytokine-like activity (1,3). In CXC chemokines, an ELR motif occurs in several members of the class, such as IL-8, Gro-␣, -␤, and -␥, and NAP-2, and confers proangiogenic activity to the cytokines (4,5,18,19). For example, IL-8 induces in vitro endothelial cell chemotaxis and in vivo corneal neovascularization (8). In contrast, CXC chemokines that lack the ELR motif, such as IP-10 and MIG, have potent anti-angiogenic activity (8, 20 -22).
To test our hypothesis that mini TyrRS (containing a natural ELR sequence) is an angiogenic factor as well as a PMN cell chemoattractant, we first evaluated if mini TyrRS induced endothelial cell migration using HUVECs. As shown in Fig. 1, human mini TyrRS stimulated HUVEC migration. In contrast, migration was not observed with full-length TyrRS. As a positive control, the potent angiogenic factor VEGF 165 stimulated HUVEC chemotaxis. That the ELR motif in mini TyrRS was required for activity was supported by an experiment showing that the R93Q mutant protein did not induce HUVEC chemotaxis (Fig. 1). The ability of human mini TyrRS to induce directed migration of endothelial cells in vitro encouraged the notion that mini TyrRS may induce angiogenesis in vivo.
Angiogenic Activity of Mini TyrRS on Chorioallantoic Membranes-We carried out in vivo angiogenesis assays on CAMs to establish if mini TyrRS had angiogenic activity in vivo. Strikingly human mini TyrRS induced angiogenesis in the CAMs as did VEGF 165 (Fig. 2). Full-length TyrRS had no activity in the CAM assay. Significantly the R93Q mutant of mini TyrRS did not stimulate angiogenesis in the CAMs just as it did not induce HUVEC migration. Thus, the ELR motif is important for angiogenic activity of mini TyrRS. In this respect, the activity of mini TyrRS parallels that of CXC chemokines (8).
Because mini TyrRS induced CAM angiogenesis in an ELRdependent manner, we evaluated whether mini TyrRS-stimulated angiogenesis could be inhibited by the anti-angiogenic CXC chemokine IP-10. This chemokine antagonizes the angiogenic activity of IL-8 in vivo (8). Treatment of the CAM with mini TyrRS followed by daily applications of IP-10 inhibited angiogenesis in this assay (Fig. 2). These data further support a connection between the activity of mini TyrRS and CXC chemokines.
Angiogenic Activity of Mini TyrRS in Mouse-To evaluate these findings in a mammalian system, the activity and potency of mini TyrRS was examined in a mouse matrigel plug assay (15). Mini TyrRS or other test agents were combined with matrigel, a growth factor-depleted gel formed from basement membranes, and injected subcutaneously into the mouse. Agents stimulating angiogenesis lead to the appearance of blood vessels within the matrigel plug, which are then visualized with a fluorescently labeled endothelial binding lectin, GSL-B4. The response to mini TyrRS was dose-dependent with angiogenesis occurring at doses of 60 nM or higher (Fig. 3). For comparison, the extent of blood vessel development within the matrigel plug containing VEGF 165 (20 nM, positive control) or PBS (negative control) was also measured.
Aware that angiogenic and angiostatic factors may work together to regulate angiogenesis (23)(24)(25), we investigated the effect of administration of mini TrpRS on the angiogenic activity of mini TyrRS. Mini TrpRS is an alternative splice variant of TrpRS that is induced by interferon-␥ (26,27), which, in turn, is known to stimulate production of angiostatic factors such as IP-10 and MIG (28,29). Indeed mini TrpRS-like fragments are potent inhibitors of angiogenesis (11,30). Angiogenesis in the mouse matrigel assay was inhibited by mini TrpRS, paralleling the finding of IP-10 inhibition of mini TyrRS-stimulated angiogenesis seen in the CAM assay (Fig. 3).
Conclusion-Several functional similarities between human mini TyrRS and IL-8 suggest a common mechanism of action. The similarities include chemoattraction of PMN cells, dependence on the presence of the ELR motif for activity, and binding to the CXCR1 receptor on PMN cells (1,3). Like IL-8, human mini TyrRS-induced angiogenesis in vivo in the CAM and the mouse matrigel assays.
To date two CXC receptors have been identified in human cell lines, CXCR1 and CXCR2, that bind to the ELR-containing chemokines. CXCR1 is selective for IL-8 (31), whereas the CXCR2 receptor binds to all ELR-containing chemokines (31)(32)(33). Angiogenesis in the mouse is thought to be mediated through CXCR2 because CXCR2Ϫ/Ϫ knockout mice do not have an angiogenic response to MIP-2 (the murine homologue of Gro-␤ and -␥) and a dramatically reduced response to human IL-8 in a mouse corneal micropocket assay (34). Additionally there is no known rodent homologue of CXCR1, reinforcing the probability that CXCR2 is responsible for angiogenic signaling in response to CXC chemokines. (The activity of the classical non-CXC angiogenic stimulants basic fibroblast growth factor and VEGF is normal in the CXCR2Ϫ/Ϫ mice (34).) However, earlier studies of mini TyrRS showed that mini TyrRS selectively bound to transfected rat basophilic leukemia cells expressing the human CXCR1 receptor (1). If CXCR1 is indeed absent in the mouse, then mini TyrRS must use a signaling mechanism distinct from CXCR1 to stimulate angiogenesis in the murine model studied here. As human and mouse TyrRS share 96% amino acid identity (GenBank TM accession numbers NM_003680 (human) and BC013552 (mouse)), it is likely that both human and mouse mini TyrRS signal through a common mechanism that is yet to be established.
Among class I tRNA synthetases, TrpRS and TyrRS are among the most closely related. Mini TrpRS lacks an N-terminal-appended domain that is peculiar to native TrpRS in higher eukaryotes. Mini TrpRS was shown previously to be anti-angiogenic in vitro and in three distinct assays in chick embryos and the mouse (11). Full-length TrpRS had no activity whatsoever. Particularly potent activity against angiogenesis with a mini TrpRS-like fragment was observed during retinal development in the mouse embryo (30). Interestingly angiogenesis stimulated by either mini TyrRS or VEGF 165 is inhibited by mini TrpRS in CAM and mouse matrigel models, raising the possibility that mini TyrRS and VEGF 165 stimulate a common downstream signaling event (Fig. 2). Thus, naturally occurring fragments of two proteins involved in translation, TyrRS and TrpRS, have opposing activity on endothelial cell migration and angiogenesis in the CAM and mouse matrigel assays. The opposing activities of two tRNA synthetases suggest tight regulation of the balance between pro-and anti-angiogenic stimuli.