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Published as doi: 10.1096/fj.07-9973com.
 (The FASEB Journal. 2008;22:1597-1605.)
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The novel fragment of tyrosyl tRNA synthetase, mini-TyrRS, is secreted to induce an angiogenic response in endothelial cells

Y. Greenberg*, M. King*, W. B. Kiosses{dagger}, K. Ewalt{dagger}, X. Yang{dagger}, P. Schimmel{dagger}, J. S. Reader{ddagger},§ and E. Tzima*,§,1

* Department of Cell and Molecular Physiology,

{dagger} Department of Molecular Biology, The Scripps Research Institute,

{ddagger} Department of Medicine, and

§ Carolina Cardiovascular Biology Center, University of North Carolina, Chapel Hill, Chapel Hill, North Carolina

1Correspondence: Department of Cell and Molecular Physiology, University of North Carolina, Chapel Hill, 103 Mason Farm Rd., Chapel Hill, NC 27599-7545. E-mail: etzima{at}med.unc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aminoacyl tRNA synthetases—enzymes that catalyze the first step of protein synthesis—in mammalian cells are now known to have expanded functions, including activities in signal transduction pathways, such as those for angiogenesis and inflammation. The native synthetases themselves are procytokines, having no signal transduction activities. After alternative splicing or natural proteolysis, specific fragments that are potent cytokines and that interact with specific receptors on cell surfaces are released. In this manner, a natural fragment of human tyrosyl tRNA synthetase (TyrRS), mini-TyrRS, has been shown to act as a proangiogenic cytokine. The mechanistic basis for the action of mini-TyrRS in angiogenesis has yet to be established. Here, we show that mini-TyrRS is exported from endothelial cells when they are treated with tumor necrosis factor-{alpha}. Mini-TyrRS binds to vascular endothelial cells and activates an array of angiogenic signal transduction pathways. Mini-TyrRS-induced angiogenesis requires the activation of vascular endothelial growth factor receptor-2 (VEGFR2/Flk-1/KDR). Mini-TyrRS stimulates VEGFR2 phosphorylation in a VEGF-independent manner, suggesting VEGFR2 transactivation. Transactivation of VEGFR2 and downstream angiogenesis require an intact Glu-Leu-Arg (ELR) motif in mini-TyrRS, which is important for its cytokine activity. These studies therefore suggest a mechanism by which mini-TyrRS induces angiogenesis in endothelial cells and provide further insight into the role of mini-TyrRS as a link between translation and angiogenesis.—Greenberg, Y., King, M., Kiosses, W. B., Ewalt, K., Yang, X., Schimmel, P., Reader, J. S., and Tzima, E. The novel fragment of tyrosyl tRNA synthetase, mini-TyrRS, is secreted to induce an angiogenic response in endothelial cells.


Key Words: angiogenesis • vascular endothelial growth factor receptor 2 • noncanonical


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
AMINOACYL-TRNA SYNTHETASES (AARSS) are key components for protein synthesis because they link specific amino acids to their cognate tRNAs. Once considered a class of "housekeeping" enzymes, AARSs are now known to be multifunctional: they participate in a variety of functions, including transcription, translation, splicing, inflammation, angiogenesis, and apoptosis (reviewed in refs. 1 2 3 ). In mammals, the synthetases developed sophisticated activities that vary depending on the type of AARS and its cellular location. In this manner, YRS, WRS, and KRS are secreted to trigger signaling pathways; EPRS and QRS express their noncanonical activities in the cytoplasm, whereas KRS and MRS exert nuclear functions. The canonical and noncanonical activities of AARSs are further refined by their association with nonenzymatic factors known as AARS-interacting multifunctional proteins, in particular p43, p38, and p18. The importance of the noncanonical functions of AARSs in human disease has recently been highlighted by the identification of an active dominant mutation of glycyl-tRNA synthetase that causes neuropathy in a Charcot-Marie-Tooth 2D mouse model (4) , whereas YRS might have an additional axon terminal-specific function playing a role in Charcot-Marie-Tooth neuropathies (5 6 7) .

Human tyrosyl-tRNA synthetase is inactive in cell signaling. However, it can be split (by natural proteolysis) and activated for cell signaling into two fragments, each with their own cytokine activities (8) . One fragment, which is endothelial monocyte-activating polypeptide II-like, has robust leukocyte and monocyte chemotaxis activity and stimulates production of tissue factor, myeloperoxidase, and tumor necrosis factor-{alpha} (TNF-{alpha}) (8 , 9) . The other fragment, which contains the aminoacylation catalytic domain, and is designated as mini-TyrRS was discovered to also have cytokine activity (8 , 9) . Mini-TyrRS specifically stimulated migration of polymorphonuclear (PMN) cells with a bell-shaped dose dependence (as seen with CXC chemokines; refs. 10 , 11 ), whereas full-length, unsplit TyrRS was inactive. The surprising cytokine activity of mini-TyrRS was unique to the mammalian enzyme and mimicked that of interleukin (IL) -8 (8 , 9) . CXC chemokines such as IL-8 that are active as PMN chemoattractants have an Glu-Leu-Arg (ELR) motif (10 , 12) . This motif is essential for binding to PMN receptors and for PMN activity. The ELR motif in mini-TyrRS is also important for its cytokine activity (refs. 9 , 13 and unpublished results).

In addition, CXC chemokines containing the ELR motif are active as proangiogenic factors (14 , 15) . Indeed, mini-TyrRS also activated angiogenesis as assayed in vivo in the angiogenesis assay with chick chorioallantoic membranes (CAMs) and in a murine matrigel activity of angiogenesis (16) . In the matrigel assay, a dose-response with mini-TyrRS was seen, with angiogenesis occurring at 60 nM or higher (17) . In the CAM assay, the proangiogenic activity of mini-TyrRS was opposed by the angiostatic agent IP10 and by the potent angiostatic activity of mini-TrpRS (17) . TrpRS is structurally related to TyrRS but embeds an antiangiogenic activity (16 , 18) . The structural and evolutionary relationship between TrpRS and TyrRS suggests that the cell-signaling activities in these two AARSs might have developed together (19) . Alternative mRNA splicing or protein proteolysis is thought to be involved in the removal of an N-terminal domain to produce active antiangiogenic factors (mini- and T2-TrpRS), which inhibit mini-TyrRS and vascular endothelial growth factor (VEGF) -induced blood vessel development and endothelial cell (EC) signaling (16 , 20 , 21) . Cell binding is dependent on the vascular EC-specific cadherin, VE-cadherin (22) , which is required for angiogenesis (23) .

Here, we show for the first time that mini-TyrRS is secreted by intact ECs in response to TNF-{alpha} treatment. The secreted mini-TyrRS binds to ECs and activates an array of angiogenic signal transduction pathways through transactivation of vascular endothelial growth factor receptor-2 (VEGFR2).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
Bovine aortic endothelial cells (BAECs) were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) with 10% FBS (Invitrogen), 10 µg/ml penicillin, and 0.25 µg/ml streptomycin (Invitrogen). Human umbilical vein endothelial cells (HUVECs) were obtained from Cambrex Corp. (East Rutherford, NJ, USA and were maintained in EC medium (EGM; Cambrex) supplemented to 10% FBS and cultivated on gelatin-coated surfaces in an atmosphere of 5% CO2 in air at 37°C according to the supplier’s instructions. Fluorescently labeled mini-TyrRS was prepared by modification with AlexaFluor 488 (Alexa488-mini-TyrRS; 5 mol dye/mol protein) according to the manufacturer’s instructions (Molecular Probes Inc., Eugene, OR, USA). Protein concentration was determined by the Bradford assay using the Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA) with BSA (Sigma-Aldrich Corp., St. Louis, MO, USA) as a standard. Mitogen-activated protein kinase kinase (MEK) inhibitor U0126 (Promega Corp., Madison, WI, USA) and VEGFR tyrosine kinase inhibitor (Calbiochem, San Diego, CA, USA) were used at 50 µM and 400 nM, respectively.

Mini-TyrRS binding to ECs
Cell binding was performed by incubating Alexa488-mini-TyrRS at 30 µg/ml for 15 min. Cells were fixed for 30 min in 2% formaldehyde, rinsed twice with PBS, and mounted in immunofluorescence mounting medium (ICN Biotech, Irvine, CA, USA). Images of fixed cells were acquired using a Leica SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany). In some experiments, cell binding was performed by incubating His-mini-TyrRS at 30 µg/ml for 15 min, lysing cells, and assessing bound mini-TyrRS by immunoblotting with anti-His antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and IRDye 680-labeled goat anti-rabbit secondary antibody.

TyrRS secretion
BAECs were cultured to confluence, washed twice with PBS (Invitrogen) and further cultured in medium containing 2% FBS, with or without 50 ng/ml TNF-{alpha} (BioSource International, Camarillo, CA, USA), VEGF (Upstate Biotechnology, Lake Placid, NY, USA), or IL-8 (PeproTech Inc., Rocky Hill, NJ, USA) for 16 h. Media were collected, and cells were lysed in sample buffer containing β-mercaptoethanol. To investigate whether TyrRS was secreted from BAECs, culture supernatants were concentrated using Centricon-10 (Millipore Co., Bedford, MA, USA) and analyzed by Western blot analysis. Further, concentrated culture supernatants were precleared using protein A/G Sepharose beads (Santa Cruz Biotechnology, Inc.). Cleared supernatants were then incubated with protein A/G Sepharose previously conjugated with anti-TyrRS antibody (raised in rabbit) for 3 h at 4°C with continuous mixing. Samples were washed thoroughly in lysis buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, and 0.1% SDS] supplemented with 1 mM aprotinin, 1 µg/ml leupeptin, 1 mM PMSF, 1 mM Na3VO4, 10 mM NaF, 1 mM sodium pyrophosphate, and 1 mM β-glycerophosphate), and complexes were eluted using SDS-PAGE sample buffer. Samples were loaded on SDS-PAGE and Western blotting was performed using primary antibodies and HRP-conjugated anti-mouse or anti-rabbit antibodies (Jackson Immunochemical Laboratories, Bar Harbor, ME, USA). Immunoreactive proteins were visualized by enhanced chemiluminescence (GE Healthcare, Little Chalfont, Buckinghamshire, UK). In some experiments, IRDye 680 or IRDye 800 antibodies (Rockland, Gilbertsville, PA, USA) were used as secondaries in conjunction with the Odyssey Imaging System (Li-COR, Lincoln, NE, USA).

Proliferation assays
For proliferation assays, the Promega nonradioactive kit was used according to the manufacturer’s instructions. Briefly, BAECs or HUVECs were split into 96-well plates at 32,000 cells/well. Cells were allowed to grow overnight and were serum-starved for 16 h, and 50 ng/ml of mini-TyrRS or VEGF was added for 48 h. Then 15 ml of dye solution was added, and the incubation was continued for 4 h at 37 C; 100 µl of stop/solubilization solution was added, and the incubation was continued overnight. Absorbance was taken at 570 nm.

Migration assays
Migration was measured by a transwell filter assay using 8-µm pore transwells (Costar, Cambridge, MA, USA) coated with 10 mg/ml fibronectin. BAECs that had been serum-starved were placed in the upper chamber (3.5x104 cells) and starvation media supplemented with 50 ng/ml mini-TyrRS or VEGF was placed in the lower chamber. As a control, media without an added chemotactic factor was placed in the lower chamber. After 16 h of incubation, filters were washed in PBS, fixed in 4% paraformaldehyde, and stained with 0.5% crystal violet. After adherent cells were removed from the upper side of the filter with a cotton swab, cells that had migrated and adhered to the underside of the filter were counted with an inverted microscope.

Wound healing assays were performed in six-well plates using confluent BAECs. After serum starvation overnight, a wound was made using a pipette tip, 50 ng/ml of mini-TyrRS or VEGF was added overnight, and migration was monitored.

Phosphorylation assays
BAECs were cultured to confluency and serum-starved overnight in media containing 0.5% FBS. Mini-TyrRS was added at the indicated concentrations and for the indicated times. BAECs were lysed in 0.5% Nonidet P-40, 20 mM Tris (pH 7.6), 250 mM NaCl supplemented with 1 mM aprotinin, 1 µg/ml leupeptin, 1 mM PMSF, 1 mM Na3VO4, 10 mM NaF, 1 mM sodium pyrophosphate, and 1 mM β-glycerophosphate and centrifuged at 13,000 rpm for 10 min, and the supernatants were mixed in SDS sample buffer. Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and incubated with anti-phospho-extracellular signal-regulated kinase (ERK) 1/2 and total ERK1/2 (Cell Signaling Technology Inc., Danvers, MA, USA), phospho-Akt and total Akt (Cell Signaling Technology Inc.), phospho-Tyr-418Src (BioSource International) and total Src (Upstate Biotechnology[b]), phospho-Ser-1179 endothelial nitric oxide synthase (eNOS) and total eNOS (Cell Signaling Technology Inc.), and phospho-Tyr-VEGFR2 (BioSource International) and total VEGFR2 (Santa Cruz Biotechnology Inc.).

Permeability assay
Transwells (3.0 µm pore; Costar) were used and coated with collagen (Chemicon International Inc., Temecula, CA, USA); 200 µl of 1–2 x 106 cells/ml was added to each transwell together with 500 µl of medium. Cells were allowed to grow to confluent monolayers (~72 h) and serum-starved (in 0.5% FBS) overnight. Mini-TyrRS or VEGF was added to the top compartment for the indicated times. Fluorescein isothiocyanate (FITC) -dextran (3000D; Sigma-Aldrich Corp.) was added for 5 min, and the reaction was stopped by removing the transwells from the wells. The plate solution was mixed thoroughly, and 100 µl was transferred to 96-well pates and read using a fluorescent plate reader with 485 and 530 nm filter settings.

In vitro angiogenesis
In vitro angiogenesis was assayed using a kit from Chemicon International Inc.. Briefly, HUVECs (7500/well) were added on top of the extracellular matrix in a 96-well plate in medium containing 1% FBS. In some wells, 50 ng/ml of mini-TyrRS or VEGF was added, and tube formation was monitored with time. Approximately 7 h later, images were obtained. In some experiments, we used chamber slides that were coated with Matrigel (BD Biosciences, San Jose, CA, USA). After addition of miniTyrRS or VEGF (7 h later), cells were fixed 20 min in PBS containing 2% formaldehyde, permeabilized with 0.2% Triton X-100, and blocked with PBS containing 10% goat serum and 1% BSA for 1 h at room temperature. Antibody incubations were performed as described previously (24) using tetramethylrhodamine B isothiocyanate (TRITC) -phalloidin and 4',6'-diamidino-2-phenylidole (DAPI), and slides were mounted in Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA, USA). Images were obtained using a Nikon Eclipse E800 microscope and a Hamamatsu ORCA-ER digital camera.

Matrix metalloproteinase (MMP) assays
Confluent BAECs were serum-starved overnight (in 0.5% FBS) and treated with VEGF at 50 ng/ml or mini-TyrRS at 50 ng/ml for 24 h. Culture supernatants were concentrated using Centricon-10 (Millipore) and mixed with Tris-glycine SDS sample buffer. Activities of MMPs secreted were detected by using zymography on gelatin-containing polyacrylamide gels (Invitrogen).

Statistical analysis
Each experimental group was analyzed using single-factor analysis of variance. P values were obtained by performing two-tailed Student’s t tests using Microsoft Excel. Statistical significance was defined as P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TNF-{alpha}-induced secretion of mini-TyrRS
To see whether endogenous mini-TyrRS or full-length TyrRS can be secreted in a signal-dependent manner, we treated ECs with cytokines and growth factors, such as TNF-{alpha}, VEGF, or IL-8. After treatment of cells with TNF-{alpha}, VEGF, or IL-8 for 12 h, the media were collected without disrupting cell membranes. We verified that there was no apoptosis during treatment with cytokines (not shown). In BAECs, secretion of mini-TyrRS was observed by TNF-{alpha} but not with VEGF or IL-8. No actin or lactate dehydrogenase was leaked to the media, suggesting that no cell lysis occurred (Fig. 1 A). Importantly, intracellular levels of TyrRS did not change. To confirm that the secreted protein was mini-TyrRS, anti-TyrRS antibodies were used to immunoprecipitate culture supernatants from TNF-{alpha}-stimulated cultures. A single band at ~43 kDa, corresponding to mini-TyrRS, was observed (Fig. 1B ). Thus, secretion of mini-TyrRS appears to be a signal-dependent process and is stimulated in response to TNF- {alpha}.


Figure 1
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  		Figure 1. Mini-TyrRS is exported from ECs. A) BAECs were treated with 10 ng/ml TNF-{alpha}, 50 ng/ml IL-8, or 50 ng/ml VEGF, and the expression and secretion of mini-TyrRS were determined by Western blotting of the whole cell lysates and culture media. No lactate dehydrogenase (LDH) was present in the medium, suggesting no cell lysis. B) The culture supernatants from TNF-{alpha}-stimulated cultures were further subjected to immunoprecipitation using anti-TyrRS antibody. The immunoprecipitate was subjected to electrophoresis and a band at ~43 kDa, corresponding to mini-TyrRS, was observed in Western blots using anti-TyrRS antibody (lane 2). Nonspecific IgG was used as a control.

Mini-TyrRS binding to ECs
Because mini-TyrRS has angiogenic activity in vivo, we hypothesized that the secreted mini-TyrRS may act on ECs. We investigated the binding of mini-TyrRS to ECs. The binding of Alexa488-labeled mini-TyrRS was observed by immunofluorescence (Fig. 2 A), although binding was not observed at lower concentrations, probably owing to a threshold effect. The cell binding of mini-TyrRS was blocked by a titration with an anti-TyrRS antibody or unlabeled mini-TyrRS, indicating that the fluorescent signal is attributable to the specific cell binding of mini-TyrRS (not shown). Surface binding was also verified by subsequent extraction with Triton X-100 (not shown). The cell binding of mini-TyrRS was also verified by Western blotting of the protein extracts. Total proteins were extracted from ECs after treatment with His-mini-TyrRS and subjected to Western blotting with anti-His antibody. The amount of exogenously added mini-TyrRS increased in a dose-dependent manner (Fig. 2B ).


Figure 2
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  		Figure 2. Binding of mini-TyrRS to ECs. A) BAECs were incubated for 10 min with Alexa488-mini-TyrRS and visualized using a confocal microscope. Binding of mini-TyrRS is seen homogeneously on the surface of BAECs. B) His tagged mini-TyrRS visualized by Western blotting with anti-His antibody.

Mini-TyrRS induces proliferation and migration of ECs
Although vascular ECs rarely divide, angiogenic factors induce proliferation of ECs (25) . To better understand the function of mini-TyrRS we checked its effect on the proliferation of ECs. As shown in Fig. 3 , mini-TyrRS increased the proliferation of ECs similar to VEGF. The activity of mini-TyrRS was also tested in two migration assays. First, we used a wound migration assay: cultivated ECs were scraped with a pipette tip and then allowed to migrate in the presence or absence of mini-TyrRS. As shown in Fig. 4 , migration of ECs was enhanced at 50 ng/ml mini-TyrRS compared with untreated cells. Second, the chemotactic activity of mini-TyrRS on ECs was tested using the transwell migration system. Different amounts of mini-TyrRS were added to the lower chamber, and the cells migrating from the upper to the lower chamber were counted. The migrated cells were stained with hematoxylin, and the cell counting was performed in high-power fields. The cell migration was increased to ~2.5-fold, similar to VEGF (Fig. 4) .


Figure 3
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  		Figure 3. Mini-TyrRS induces EC proliferation. The effect of mini-TyrRS on BAECs and HUVECs was monitored by the cellular conversion of a tetrazolium salt into a formazan product detected using a plate reader. VEGF was included as a positive control. The proliferation of the untreated cells was taken as 1. The values represent the average of three independent experiments.


Figure 4
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  		Figure 4. Mini-TyrRS induces activation of MMP2 and EC migration. A) BAECs on culture dishes were scraped with a pipette tip and allowed to migrate in the media containing 0 or 50 ng/ml mini-TyrRS. The lines stand for the boundary of the wounds introduced by the pipette tip. B) The effect of mini-TyrRS on EC migration was also assayed using a Transwell chamber with a gelatin-coated polycarbonate membrane. BAECs were suspended in the upper chamber, and the indicated concentrations of mini-TyrRS were filled in the lower chamber. VEGF (50 ng/ml) was used as a positive control. The cells migrating to the lower chambers were stained with hematoxylin and counted. C) BAECs were treated with the indicated concentrations of mini-TyrRS, and the activity of MMP2 was determined by zymography. The data are shown as fold increase compared with untreated control cells and are the averages of the three independent experiments.

Mini-TyrRS activates MMP2
Proteinases of the plasminogen activator and MMPs degrade matrix molecules and thus liberate growth factors sequestered within the extracellular matrix (26) . Breakdown of matrix by MMPs also permits the migration of ECs. In particular, the gelatinases MMP2 and MMP9, which are capable of degrading native collagen type IV, allow vascular cell migration and invasion (27 , 28) . Because we used gelatin-coated membrane for the transwell cell migration assay (Fig. 4B ), we tested whether these two proteinases are involved in the mini-TyrRS-induced cell migration. The activities of these two enzymes were determined by their ability to digest gelatin in the gel matrix (zymography). As shown in Fig. 4C , mini-TyrRS induced MMP2 activation as determined by zymography.

Mini-TyrRS activates signaling mediators ERK, Akt, and Src
To address whether mini-TyrRS affects the activities of major signaling molecules, we assayed activation of ERK, Src, and Akt by phosphorylation after treatment of ECs with mini-TyrRS for 10 min. All three signaling mediators showed activation by addition of mini-TyrRS to BAECs, although they showed different sensitivities to the concentration of mini-TyrRS (Fig. 5 ). Interestingly, mini-TyrRS showed dose-dependent bell-shaped activity in the activation of ERK, Src, and Akt. To determine the functional significance of ERK for mini-TyrRS signaling, we pretreated the cells with U0126 that inhibits MEK, the upstream kinase for ERK (29) . The U0126 compound blocked the mini-TyrRS-induced migration (Fig. 6 ). Thus, ERK seems to mediate the mini-TyrRS-dependent cell migration.


Figure 5
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  		Figure 5. Activation of intracellular signaling pathways by mini-TyrRS. Phosphorylation of the intracellular-signaling proteins ERK, Akt, and Src was analyzed in BAECs stimulated with different concentration of mini-TyrRS. Activation of ERK, Akt and Src is shown as fold over control (untreated) cells. The values stand for the average of three independent experiments.


Figure 6
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  		Figure 6. ERK mediates mini-TyrRS-dependent BAEC migration. BAECs were pretreated with 50 µM U0126 (MEK inhibitor), and 50 ng/ml mini-TyrRS was subsequently added. The effect of ERK inhibition on mini-TyrRS-dependent migration was determined using a transwell assay. Cells migrated through the membrane were detected by hematoxylin staining.

Mini-TyrRS induces a transient increase in permeability
Considering that vascular permeability is a key component of the angiogenic process in vivo and that activation of eNOS mediates permeability, we assessed whether treatment with mini-TyrRS activates eNOS in BAECs. Several studies have emphasized the importance of phosphorylation in the regulation of eNOS activity (30) ; phosphorylation of Ser-1179 leads to increased enzyme activity and increased nitric oxide (NO) production. As shown in Fig. 7 , mini-TyrRS induced phosphorylation of eNOS on Ser-1179, suggesting activation of this enzyme. Importantly, NO can induce angiogenesis in vitro (31 , 32) and in vivo (33) ; NO also modulates angiogenesis in response to tissue ischemia (34) . To determine the effect of mini-TyrRS on permeability, confluent monolayers of ECs were grown on transwell filter inserts and treated with mini-TyrRS (50 ng/ml) or VEGF (50 ng/ml) for various times. Permeability of the monolayer for fluorescently labeled dextran (FITC-coupled dextran) was determined by measuring the fluorescence intensity of the medium in the lower compartment. Our data show that both mini-TyrRS and VEGF induce a transient increase in dextran permeability, suggesting that mini-TyrRS exerts its proangiogenic effects through modulation of endothelial permeability.


Figure 7
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  		Figure 7. Mini-TyrRS induces activation of eNOS and a transient increase in EC permeability. The effect of mini-TyrRS on the activity of eNOS was investigated in BAECs using phosphoSer1179 eNOS-specific antibodies and antibody against total eNOS. Endothelial monolayer permeability was measured by using the transwell system with a fluorescence-labeled dextran tracer. BAECs were treated with 50 ng/ml VEGF or 50 ng/ml mini-TyrRS for 30 or 60 min. The values stand for the average of three independent experiments.

Mini-TyrRS induces EC tube formation
An important step in angiogenesis is the organization of ECs into networks of cords that subsequently acquire a lumen (25) . The effect of mini-TyrRS on tube formation was tested on Matrigel. HUVECs were cultivated on Matrigel containing different amounts of mini-TyrRS. Untreated cells failed to assemble into tubes, whereas cells treated with either mini-TyrRS or VEGF formed long tubes with several branch points (Fig. 8 ).


Figure 8
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  		Figure 8. Mini-TyrRS induces tube formation in ECs. Tube formation of HUVECs was observed by immunofluorescence microscopy using TRITC-phalloidin to visualize actin filaments and DAPI to visualize nuclei.

Mini-TyrRS transactivates VEGFR2
Because mini-TyrRS induced EC tube formation similar to VEGF, we investigated the relationship between VEGF and mini-TyrRS in this process. The effects of VEGF on angiogenesis are mediated by VEGFR2; thus, we examined whether VEGFR2 functions downstream of mini-TyrRS using an inhibitor of VEGFR tyrosine kinase activity. We found that the VEGFR inhibitor blocked mini-TyrRS-induced EC tube formation in vitro (Fig. 9 B). These data suggest that VEGFR2 is critical in mini-TyrRS-induced lumen formation.


Figure 9
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  		Figure 9. Mini-TyrRS transactivates VEGFR2. A) Confluent BAECs were treated with mini-TyrRS at various concentrations, for 10 min or with 50 ng/ml mini-TyrRS for various time points, followed by immunoblot analysis of cell lysates. B) ECs were preincubated with 600 nM VEGFR inhibitor for 15 min and were then allowed to form tubes in Matrigel. Where indicated, 50 ng/ml of E91N mini-TyrRS was included instead of wild-type (WT) mini-TyrRS. C) Confluent BAECs were treated with mini-TyrRS or mutant mini-TyrRS at 50 ng/ml for 10 min, followed by immunoblot analysis of cell lysates. P, phospho.

To confirm activation of the VEGFR2 receptor, we investigated whether mini-TyrRS induces phosphorylation of this receptor. We assayed phosphorylation of Y1054 and Y1214 and found that mini-TyrRS stimulates phosphorylation/activation of VEGFR2 on Y1054 in a dose-dependent manner, with maximal phosphorylation at concentrations of 5 ng/ml (Fig. 9A ). These results suggested two possibilities for mini-TyrRS-induced VEGFR2 activation: 1) mini-TyrRS stimulates the production of VEGF, which then activates VEGFR2, or 2) mini-TyrRS stimulates the activation of VEGFR2 by transactivation mechanisms. To test the first possibility, we assayed production of VEGF in response to mini-TyrRS and found no differences in the amount of VEGF protein either in cell lysates or cell media in response to mini-TyrRS (not shown). We therefore focused our efforts on the second possibility. To determine the time course of phosphorylation of VEGFR2 after treatment with mini-TyrRS, we exposed ECs to mini-TyrRS and detected phosphorylation of the receptor after various incubation times. VEGFR2 was phosphorylated in a time-dependent manner and its phosphorylation was transient (Fig. 9A ).

It has shown previously that the ELR motif in mini-TyrRS is important for its cytokine activity (9) . The ELR motif resides within the catalytic domain that consists of a Rossmann nucleotide-binding fold. To investigate the significance of this motif in VEGFR2 transactivation, we used three mini-TyrRS mutants: E91N, L92Y, and R93Q. The mutant mini-TyrRS did not induce phosphorylation of VEGFR2, suggesting that the ELR motif in mini-TyrRS has an important role in transactivation of VEGFR2 (Fig. 9C ). Importantly, the mini-TyrRS mutants failed to induce EC tube formation, in contrast to the effects of wild-type mini-TyrRS (Fig. 9B ), further suggesting that the ELR region of mini-TyrRS is required for downstream signaling and the induction of angiogenesis.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Among the human AARSs, TyrRS, TrpRS, and LysRS are secreted and function as extracellular signaling molecules. This secretion does not seem to utilize the classic endoplasmic reticulum/Golgi export pathway because AARSs lack the typical signal peptides for secretion (20) .

TrpRS is structurally related to TyrRS but embeds an antiangiogenic activity (16 , 18) . The structural and evolutionary relationship between TrpRS and TyrRS suggests that the cell-signaling activities in these two AARSs might have developed together (21) . Alternatively, mRNA splicing or protein proteolysis is thought to be involved in the removal of an N-terminal domain to produce active antiangiogenic factors (mini- and T2-TrpRS), which inhibit mini-TyrRS and VEGF-induced blood vessel development and EC signaling (16 , 20 , 21) . Cell binding is dependent on the vascular EC-specific cadherin, VE-cadherin (22) , which is required for angiogenesis (23) .

LysRS exhibits a diversity of functions. Human LysRS is involved in the packaging of HIV virion via the interaction of its N-terminal motif with the C-terminal capsid region of the Gag protein of HIV (35) . Human LysRS is also secreted from various cell lines in response to TNF-{alpha} and stimulates macrophages and peripheral blood mononuclear cells to enhance migration and TNF-{alpha} production (2) . Thus, LysRS and TNF-{alpha} seem to form a positive feedback loop to amplify the secretion of both factors. Several other AARSs, such as HisRS, AsnRS, and SerRS, also stimulate immune cells through their interactions with cell surface chemokine receptors (36) .

We have now shown that mini-TyrRS is secreted from ECs in response to TNF-{alpha} treatment. Human TyrRS is secreted and processed into two protein fragments. The resulting N-terminal fragment, mini-TyrRS, becomes a proangiogenic factor, and the carboxyl-terminal protein becomes an immune cell stimulant for migration and production of TNF-{alpha}, tissue factor, and myeloperoxidase (8 , 17) . In this study, we investigate the mechanism of induction of angiogenesis by mini-TyrRS.

Angiogenesis is a multistep process in which quiescent blood vessels give rise to new blood vessels (37) . After ECs are exposed to an angiogenic factor, the endothelium is destabilized, leading to a decrease in EC adhesion and an increase in vascular permeability. Simultaneously, MMPs are produced and activated, which degrade the basal lamina in discrete regions of the blood vessel. The ECs are then able to proliferate and migrate into surrounding connective tissue, forming a "sprout," or cord of ECs, which subsequently develops a lumen; sprouts from adjacent arterioles and venules fuse to form a network of blood vessels. The nascent vessels then recruit peri-ECs, smooth muscle-like cells that stabilize the endothelium by promoting basal lamina deposition and intercellular adhesions (38 , 39) . In the present study, we have shown that mini-TyrRS stimulates proliferation and migration of primary human and bovine ECs. Second, mini-TyrRS induces phosphorylation of the protein kinases Akt, Src, and ERK, which are known to transduce cell growth signals in ECs. Third, mini-TyrRS induces a transient increase in EC permeability, possibly through activation of eNOS. Fourth, mini-TyrRS induces activation of MMP2 and promotes formation of endothelial tubes. Intriguingly, mini-TyrRS was found to transactivate VEGFR2, and this transactivation is required for downstream tube formation. Importantly, just as the ELR motif is required for its angiogenic activity (8 , 17) , we found that it is also essential for transactivation of VEGFR2, suggesting that activation of this receptor supports downstream signaling. Other molecules have been described to transactivate VEGFR2 (40 41 42 43 44) ; in addition, transactivation of VEGFR2 has been reported in response to the mechanical force of shear stress (45 , 46) . We are currently investigating the physiological significance of the mini-TyrRS-induced VEGFR2 transactivation in the angiogenic cascade.

New agents are needed for therapeutic angiogenesis in cardiovascular disease. Induction of angiogenesis by the novel biological mini-TyrRS is not only an outstanding conundrum in basic science, but is also of great interest from the perspective of developing efficient and safe therapies for angiogenesis. The expanded functions of tRNA synthetases are probably related to a persistent need for new functions in the development of complex organisms and to meet the demands of a constant array of new selective pressures (2) .


   ACKNOWLEDGMENTS
 
The technical help of Ms. Lindsey Buckingham is gratefully acknowledged. We thank members of the Jim Faber laboratory for helpful discussions. This work was funded by a gift from aTyr Pharma, Inc. (San Diego, CA, USA)

Received for publication October 1, 2007. Accepted for publication November 29, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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