Domain 2 of uPAR regulates single-chain urokinase-mediated angiogenesis through β₁-integrin and VEGFR2

Abstract

How single-chain urokinase (ScuPA) mediates angiogenesis is incompletely understood. ScuPA (≥4 nM) induces phosphorylated (p)ERK1/2 (MAPK44 and MAPK42) and pAkt (Ser⁴⁷³) in umbilical vein and dermal microvascular endothelial cells. Activation of pERK1/2 by ScuPA is blocked by PD-98059 or U-0126, and pAkt (Ser⁴⁷³) activation is inhibited by wortmannin or LY-294002. ScuPA (32 nM) or protease-inhibited two-chain urokinase stimulates pERK1/2 to the same extent, indicating that signaling is not dependent on enzymatic activity. ScuPA induces pERK1/2, but not pAkt (Ser⁴⁷³), in SIN1^−/− cells, indicating that the two pathways are not identical. Peptides from domain 2 of the urokinase plasminogen activator receptor (uPAR) or domain 5 of high-molecular-weight kininogen compete with ScuPA for the induction of pERK1/2 and pAkt (Ser⁴⁷³). A peptide of the integrin-binding site on uPAR, a β₁-integrin peptide that binds uPAR, antibody 6S6 to β₁-integrin, tyrosine kinase inhibitors AG-1478 or PP3, and small interfering RNA knockdown of VEFG receptor 2, but not HER1–HER4, blocked ScuPA-induced pERK1/2 and pAkt (Ser⁴⁷³). ScuPA-induced endothelial cell proliferation was blocked by inhibitors of pERK1/2 and pAkt (Ser⁴⁷³), antibody 6S6, and uPAR or kininogen peptides. ScuPA initiated aortic sprouts and Matrigel plug angiogenesis in normal, but not uPAR-deficient, mouse aortae or mice, respectively, but these were blocked by PD-98059, LY-294002, AG-1478, or cleaved high-molecular-weight kininogen. In summary, this investigation indicates a novel, a nonproteolytic signaling pathway initiated by zymogen ScuPA and mediated by domain 2 of uPAR, β₁-integrins, and VEGF receptor 2 leading to angiogenesis. Kininogens or peptides from it downregulate this pathway.

single-chain urokinase (ScuPA) is a zymogen that is activated by plasma kallikrein or plasmin to form two-chain urokinase (TcuPA). ScuPA binding to the urokinase receptor [urokinase plasminogen activator receptor (uPAR)] allows for its activation on cell membranes, accelerating plasminogen activation. ScuPA also initiates intracellular signaling through interactions with uPARs and other lateral-binding partners including integrins, lipoprotein receptor protein, the HER (ErbB) family of receptors, and PDGF receptors, among others (22). ScuPA interacts with each of the three domains of uPAR (3, 34, 35), a glycophosphoinositol-linked protein that regulates cell adhesion, migration, proliferation, chemotaxis, invasion, oncogenesis, angiogenesis, and efferocytosis (22, 62). In turn, this receptor is known to interact with at least 33 lateral partners and 9 soluble ligands to form the uPAR interactome (22).

uPAR mediates angiogenesis through protease-dependent and -independent mechanisms (3, 6, 7, 9, 51, 55, 57). Prager et al. (57) proposed a protease-dependent pathway whereby VEGF, through its receptor [VEGF receptor 2 (VEGFR2; Flk-1/KDR)], induces the expression of matrix metalloproteinase (MMP)-2, leading to the activation of ScuPA bound to uPAR as an early step in the angiogenesis process. Urokinase or ScuPA itself, upon binding to uPAR, produces plasminogen activation to plasmin, which degrades the extracellular matrix, facilitating endothelial cell (EC) migration (55, 56, 63). This pathway leads to ERK1/2 phosphorylation that is inhibited by density-enhanced phosphatase-1 (9). Margheri et al. (51) reported that the angiogenic properties of endothelial colony-forming cells depend on caveolae integrity and full-length uPAR. TcuPA also enhances uPAR's interactions with vitronectin to promote angiogenesis (66). On a vitronectin matrix, uPAR-deficient cells have decreased angiogenic function of membrane protrusion and lamellipoda formation mediated by focal adhesion kinase, paxillin, and vinculin (2, 62).

Alternatively, soluble uPAR, through its Ser⁸⁸-Arg-Ser-Arg-Tyr⁹² chemotactic peptide sequence from the domain I–II linker region alone, stimulates vessel sprouting through a protease-independent mechanism (7). It has been recognized that diisopropylfluorophosphate (DFP)-treated urokinase and noncleavable uPAR influence cell proliferation mediated by integrins (38, 52). Although the growth factor domain of urokinase interacts with domain 1 of uPAR, domain 2 of uPAR also is a recognized binding site for ScuPA (4, 18, 23, 50). High-molecular-weight kininogen (HK), vitronectin, and factor XII, uPAR-soluble ligands, also bind to the same region on domain 2 as ScuPA (15, 28, 42, 43, 50).

ScuPA stimulates ERK1/2 and Akt phosphorylation in ECs and smooth muscle cells (25, 27, 31, 41). Phosphorylation of ERK1/2 by FGF-2 induces angiogenesis in ECs, and Akt mediates EC survival and cytoprotection (25, 31). These investigations along with those detailed above suggest that there is no single cohesive pathway for ScuPA-mediated angiogenesis through uPAR. A recent study (42) from our laboratory indicated that factor XII binds to domain 2 of uPAR to initiate a signaling pathway through phosphorylated (p)ERK1/2 and pAkt (Ser⁴⁷³), leading to cell proliferation and angiogenesis (42). Alternatively, intact or cleaved HK binds to domain 2 of uPAR to inhibit EC migration and invasion, reduce pERK1/2 and p-phosphatidylinositol 3-kinase (pPI3K)-Akt signaling, and block angiogenesis (11, 16, 40, 42, 46, 48, 70). These observations suggest that exposure and regulation of the activity of domain 2 of uPAR may have a gatekeeping function in signaling and angiogenesis.

In the present study, we examined the hypothesis that the zymogen ScuPA, like TcuPA and factor XII (42), stimulates a signaling pathway leading to cell proliferation and angiogenesis. This pathway is mediated in part through β-integrins and is downregulated by HK. Additionally, we show that VEGFR2 (Flk-1/KDR) participates upstream of pERK1/2 and pAkt (Ser⁴⁷³) after zymogen ScuPA stimulation. These investigations indicate a direct, nonproteolytic pathway where ScuPA, through VEGFR2, stimulates angiogenesis.

MATERIALS AND METHODS

Materials.

Single-chain HK, with a specific activity of 13 U/mg, and two-chain HK [plasma kallikrein-cleaved HKa (HKa)] were purchased from Enzyme Research Laboratories. TcuPA was purchased from Molecular Innovations, and ScuPA was prepared as previously described (4). With SDS-PAGE, ScuPA migrated as a single band of ∼46 kDa on reduced or nonreduced gradient gels. p-A-PMSF (APMSF; 3 mM)-treated TcuPA was prepared and dialyzed overnight before use. Prestained molecular weight standards, nitrocellulose, and polyacrylamide were purchased from Bio-Rad. ECL Western blot detection reagents were purchased from GE Healthcare. Wortmannin, 2′-amino-3′-methoxyflavone (PD-98059), 2-(4-morholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY-294002), 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG-1478), 4-amino-5(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), and 4-amino-7-phenylpyrazol[3,4-d]pyrimidine (PP3) were purchased from Calbiochem. 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U-0126) was purchased from Cell Signaling. VEGF (VEGF₁₆₅) was purchased from R&D Systems. FGF was obtained from BD Biosciences. uPAR knockout (KO; Plaur^−/−) mice were generously provided by Dr. Thomas Bugge (National Institutes of Health) (10). All animals studies were approved by Institutional Animal Care and Use Committee of Case Western Reserve University (Protocol Number 2011-0061).

Peptides and antibodies.

Peptides from domain 5 of HK were synthesized at the Carbohydrate Structure Facility of the University of Michigan (Ann Arbor, MI) and by the Polypeptide Group (Table 1) (33). Peptides comprising regions within domain 2 of uPAR (Table 1) (50), a scrambled peptide comprising uPAR Pro¹⁷⁶ to Thr¹⁹⁵ with the sequence H-NSFGCNHDFKGPTHNCLTNF-OH, a peptide from domain 2 of uPAR that binds integrins [H-¹³⁰IQEGEEGRPKDDR¹⁴²-OH (IQE13)], and a peptide from the β-propeller of the β₁-integrin α-subunit that binds uPAR [H-²²⁴NLDSPEGGF²³²-OH (NLD9)] were prepared by the Polypeptide Group (20, 67). Antibodies to ERK1/2 (catalog no. 9102) and p-p44/42 ERK1/2 (Thr²⁰²/Tyr²⁰⁴) (pERK1/2; catalog no. 9101) were purchased from Cell Signaling. Antibodies to Akt (catalog no. 9072) and pAkt (Ser⁴⁷³) (catalog no. 4058S) were also obtained from Cell Signaling. Monoclonal antibodies to β₁-integrin clones P4C10 and 6S6, α₅β₁-integrin clone HA5, and α₃-integrin clone ASC-1 were purchased from Chemicon. Monoclonal antibody to α₅-integrin clone P1D6 was purchased from Upstate. Another monoclonal antibody to β₁-integrin (AIIB2) was purchased from the Developmental Studies Hybridoma Bank. Rabbit antibodies to human EGF receptor (EGFR, ErbB1/HER1; catalog no. SC-03, used at 1:1,000) were purchased from Santa Cruz Biotechnology. Rabbit antibodies to human ErbB2 (HER2; catalog no. 4290, 1:1,000), ErbB3 (HER3; catalog no. 4754, 1:1,000), VEGFR2 (Flk-1/KDR; catalog no. 2479, 1:2,000), and pVEGFR2 (catalog no. 2478, 1:2,000) were purchased from Cell Signaling Technologies. Rabbit antibodies to human ErbB4 (HER4, catalog no. 2218-1, 1:500) were purchased from Epitomics. A peroxidase-conjugated AffiniPure donkey anti-rabbit IgG (H+L; catalog no. 711-035-152, 1:2,000–4,000) was obtained from Jackson ImmunoResearch Laboratories. Modeling of uPAR from the structure provided in Ref. 3 was performed with the Cn3D4.3 program available at the National Center for Biotechnology Inforation website under the “Structure” heading.

Table 1.

Peptides used to influence ERK1/2 and Akt phosphorylation

	Domain	Sequence*
uPAR
LRG20	2	H-144LRGCGYLPGCPGSNGFHNND163-OH
YLP20	2	H-149YLPGCPGSNGFHNNDTFHFL168-OH
PGS20	2	H-154PGSNGFHNNDTFHFLKCCNT173-OH
FHN20	2	H-159FHNNDTFHFLKCCNTTKCNE178-OH
TKC19	2	H-174TKCNEGPILELENLPQNGR192-OH
High-molecular-weight  kininogen
HKH20	5	479HKHGHGHGKHKNKGKKNGKH498
HVL24	5	471HVLDHGHKHKHGHGHGKHKNKGKK494
GGH18	5	469GGHVLDHGHKHKHGHGHG486
GKE19	5	402GKEQGHTRRHDWGHEKQRK420

The peptides for the urokinase plasminogen activator receptor (uPAR) are described in Ref. 50. The numbering of the amino acids shown represents the location of the amino acid in the mature protein sequence as indicated by the crystal structure of uPAR in Ref. 3 and available from the National Center for Biotechnology Information structure website. Note: the single letter code is used for amino acids. The peptides for high-molecular-weight kininogen are described in Ref. 33. The numbering of the amino acids shown represents the location of the amino acid in the mature protein sequence.

EC culture.

Human umbilical vein ECs (HUVEC) were obtained from AllCells, and dermal human microvascular ECs (HMEC) were obtained from Invitrogen. Trypsin-EDTA and trypsin neutralizing solutions were purchased from Lonza. Cells were cultured on gelatin in endothelial growth medium (EGM), unless otherwise stated, according to procedures from AllCells. As specified, HUVECs were cultured on 1 μg/ml collagen (BD Biosciences), fibrinogen (Sigma), fibronectin (Sigma), or vitronectin (Sigma). In preparation for signaling experiments, cells between the first and third passages were subcultured onto gelatin-coated, six-well plates in CS-C medium (Cell Systems) 24 h before the start of experiments (24). Cell viability was determined using trypan blue exclusion. Cell numbers were determined by counting on a hemocytometer. In certain experiments, 32 nM ScuPA, 32 nM TcuPA, or 32 nM APMSF-treated TcuPA were incubated with monolayers of HUVECs for 30 min in the presence of 0.8 mM l-pyroglutamyl-Gly-l-Arg-pNA HCl (S2444; Diapharma) in CS media. In other experiments, the same proteins in the absence or presence of 50 μM PD-98059 were incubated with serum-starved HUVEC monolayers for 7 min and then immunoblotted for ERK1/2 and total ERK. Mouse embryonic fibroblasts (MEFs) from SIN1^−/− mice were provided by Dr. Bing Su (Yale University) and were cultured in DMEM with 15% FBS (37).

ERK1/2 and Akt phosphorylation experiments.

HUVECs or HMECs were cultured in six-well microtiter plates for 24 h in serum-containing medium. Cultures were then washed in serum-free media (CS-C medium kit) without growth factors and starved for 2 h in the absence or presence of inhibitors. Unless stated otherwise, PD-98059 was added at 100 μM, U1026 at 50 μM, wortmannin at 30–50 nM, LY-294002 at 50 μM, and peptides at 300 μM. At the end of the incubation, cells were incubated with 4–200 nM ScuPA or vehicle for 5–7 min, and the reaction was stopped by a wash with cold PBS. In all cases, cells were scraped into 100 μl of 2× Laemmli sample buffer from Bio-Rad, which was reduced with 5% β-mercaptoethanol. Equal amounts of cell lysates were subjected to 10% SDS-PAGE and then electrotransferred to nitrocellulose membranes. Membranes then were incubated in blocking buffer that included 5% nonfat dried milk followed by an overnight incubation at 4°C with monoclonal antibodies to ERK and p44/42 ERK1/2 (Thr²⁰²/Tyr²⁰⁴) or Akt and pAkt (Ser⁴⁷³) diluted at 1:1,000. After being washed, membranes were incubated at room temperature with horseradish peroxidase-conjugated anti-rabbit IgG (1:2,000) for 1 h. The secondary antibody was detected with an ECL system from GE Healthcare. Blots were scanned using Scion image software, and the intensity of the inhibited samples presented as an arbitrary number was compared with uninhibited ScuPA-stimulated controls on the same immunoblot. For the graphic presentation of each experiment, the percent control value was determined by making the arbitrary number of the scan for the ScuPA-stimulated samples 100% and the lowest inhibited sample 0%, followed by analysis using the following formula: (sample number − number of lowest inhibited specimen)/(ScuPA number − number of lowest inhibited specimen) × 100.

Small interfering RNA knockdown experiments.

Small interfering (si)RNA to HER1–HER4 (ErbB1-ErbB4) or VEGFR2 (Flt-1/KDR) were obtained from Dharmacon (Thermo Scientific) and were used according to their recommendations. Each gene was targeted with a pool of four siRNA prepared by the manufacturer. EGFR (ErbB1 or HER1) was targeted with Smartpool M-003114-03-0010, ErbB2 (HER2) was targeted with Smartpool l-003126-00-0010, ErbB3 (HER3) was targeted with Smartpool L-003127-00-0010, ErbB4 (HER4) was targeted with Smartpool L-003128-00-0010, and VEGFR2 (Flt-1/KDR) was targeted with Smartpool M-003148-01-0010. The sequences of each siRNA are available from the manufacturer. In each experiment, ∼50 nM siRNA final concentration was mixed with OPTI-MEM in 200 μl. This material then was mixed with an equal volume of 10 μl Dharmafect transfection reagent in OPTI-MEM. After an incubation, mixture was added to washed cells (2 ml/well in a 6-well plate) and incubated overnight with HMECs. The next day, cells were washed and replaced in serum-free medium for starvation before a signaling experiment.

EC proliferation assays.

Cell proliferation was monitored by measuring the amount of soluble formazan produced by the cellular reduction of the MTS tetrazolium compound (Owen's reagent) using the Celltiter 96^R Aq_ueous One Solution Cell Proliferation Assay (Promega). Samples were read in a microtiter plate reader at 490 nm. HUVECs were cultured in 96-well microtiter plates on gelatin at 1 × 10⁴ cells/well for 24 h in 2% serum-containing medium. Cultures were then washed with serum-free medium without growth factors and serum starved for 2 h followed by stimulation with 64 nM ScuPA or vehicle for an additional 24 h. MTS/formazan was then measured by absorbance at 490 nm. In other experiments, peptides from domain 2 of uPAR or domain 5 of HK, antibodies to β₁-integrin, or inhibitors upstream to pERK1/2 or pAkt were added to the reaction mixture to determine if they would influence cell proliferation (Table 1).

EC 5-bromo-2′-deoxyuridine assay.

HUVECs were cultured in 96-well microtiter plates on gelatin at 1 × 10⁴ cells/well for 24 h in EGM from AllCells. Cells were then washed with serum-free medium without growth factor and serum starved for 2 h followed by treatment with 64 nM ScuPA or vehicle with or without upstream inhibitors to pERK1/2, pAkt, peptides to domain 2 of uPAR or domain 5 of HK, or antibodies to various integrins and in the presence of 5-bromo-2′-deoxyuridine (BrdU) labeling solution according to the manufacturer's specifications (Roche Applied Science). After 24 h, the culture medium was removed, and cells were washed twice with medium containing 10% serum. Cells were then fixed with precooled fixative (70% ethanol in 0.5 M HCl) and incubated for 30 min at −15 to −25°C. Cells were then washed three times with medium containing 10% serum and treated with nucleases for 30 min at 37°C in the absence of CO₂. After the nuclease solution had been removed, cells were washed three times with medium containing 10% serum and then treated with anti-BrdU-POD Fab fragments for 30 min at 37°C. Cells were then washed and treated with peroxidase substrate followed by an incubation at room temperature until positive samples showed a green color. Samples were read in a microplate reader at 450 nm.

Angiogenesis experiments.

For aortic sprouting experiments, thoracic aortae were isolated from 8- to 12-wk-old mice and transferred to a compartmentalized Felsen dish containing phenol-red free EGM (AllCells) as previously described (8). The surrounding fibroadipose tissue was dissected, and aortae were subjected to eight sequential washes in EGM. Aortae were sectioned into ∼1-mm segments. In some experiments, aortic segments were embedded into a 15-μl drop of rat tail collagen I (1.81 mg/ml, BD Biosciences) in 12-well dishes (3 rings/well). The final collagen formulation was composed of 1.81 mg/ml collagen I in sterile PBS containing 2.7 mM CaCl₂ and 23 mM NaOH. Collagen was then allowed to gel at 37°C under 5% CO₂ in a cell culture incubator for 30 min, after which 1 ml of CS-C complete medium was added to each well containing test reagents. Aortic ring sprouts were photographed on days 4–6. Sprouting was quantified by counting the number of sprout roots around the entire aortic section from at least three independent experiments unless otherwise stated (42). In other experiments, aortic segments were suspended in reduced growth factor Matrigel (BD Biosciences) containing EGM with VEGF (5 nM). Aortic ring sprouts were photographed on days 4 and 5 depending on the experiment. Sprout areas on day 5 were determined by morphometric analysis using MetaMorph by dividing the area of the sprouts by the aortic perimeter. Sprouting images were obtained using a Nikon TE200 microscope with a ×10/0.25 objective lens.

For Matrigel angiogenesis, growth factor-free Matrigel was injected subcutaneously into murine flanks as previously described (42). The Matrigel contained heparin (60 U/ml) in the absence or presence of FGF (6 nM) or ScuPA (32 nM). Plugs were added to the flanks of wild-type (WT) or uPAR KO mice (2 plugs/mouse). In other ScuPA-induced angiogenesis experiments on WT mice, LY-294002 (50 μM), HKa (128 nM), AG-1478 (50 nM), or PD-98059 (50 μM) was added to the plug. Plugs were harvested 9 days after injection, and vessel hemoglobin content was measured with Drabkin's assay (Ricca Chemical) of homogenized Matrigel pieces normalized by weight. Matrigel plugs also were flash frozen in OCT, and sections were cut at 4 μm for staining with 4′,6-diamidino-2-phenylindole and anti-CD31. Photographs of the Matrigel plugs were obtained using a Leica MZ 16FA microscope with a ×10 lens. Angiogenesis microscopic images were obtained using a Nikon TE200 microscope with a ×20/0.45 lens.

Statistical analysis.

Differences between inhibited samples and controls were determined using Student's t-test for nongrouped data. Significance was defined as P values of <0.05. For comparisons between three groups, one-way ANOVA was used with the Bonferonni/Dunn test to determine the statistical significance between groups. Unless otherwise stated, comparisons using the t-test are presented in the text.

RESULTS

ScuPA stimulates pERK1/2 and pAkt (Ser⁴⁷³) expression in ECs.

To begin our examination for a signaling pathway for ScuPA's induction of EC angiogenesis, we studied ScuPA-initiated ERK1/2 and Akt phosphorylation as previously described (25, 27, 31, 41, 62). ScuPA stimulated expression of pERK1/2 in cultured HUVECs grown on gelatin over a wide range of concentrations (4–200 nM). Based on this, concentrations of 16–32 nM were chosen for subsequent signaling experiments. Induction of pERK1/2 was blocked by MEK1 inhibitors U-0126 and PD-98059 (P < 0.001; Fig. 1, A and C). Wortmannin (50 nM, P < 0.003) but not LY-294002 (P = 0.13) also reduced ScuPA-induced pERK1/2 in HUVECs (Fig. 1, A and C). Wortmannin at this concentration had a toxic effect on HUVECs, resulting in the apparent pERK1/2 inhibition. ScuPA also stimulated pAkt (Ser⁴⁷³). Akt phosphorylation was >95% inhibited by either wortmannin or LY-294002 (P < 0.001; Figs 1, A and C). Neither U-0126 nor PD-98059 inhibited the induction of pAkt (Ser⁴⁷³) (Fig. 1, B and D). These investigations confirm that ScuPA stimulated both pERK1/2 and pAkt (Ser⁴⁷³).

Fig. 1.

Single-chain urokinase (ScuPA) stimulates phosphorylated (p)ERK1/2 and pAkt (Ser⁴⁷³) in human umbilical vein endothelial cells (HUVECs; left) and human microvascular endothelial cells (HMECs; right). A: immunoblots of HUVECs using antibodies to pERK1/2 and ERK1/2 (top) and antibodies to pAkt (Ser⁴⁷³) [pAkt (S473)] and total Akt (bottom). B: same experiments except HMECs were used. In both A and B, lane 1 (from the left) is untreated, serum-starved control cells, lane 2 is 16 nM ScuPA alone, and lanes 3–6 are treated 16 nM ScuPA in the presence of an inhibitor [for pERK1/2/ERK 1/2 experiments, lane 3 shows wortmannin (50 nM) treatment, lane 4 shows U-0126 (50 μM) treatment, lane 5 shows LY-294002 (50 μM) treatment, and lane 6 shows PD-98059 (50 μM) treatment; for pAkt (Ser⁴⁷³)/Akt experiments, lane 5 shows PD-98059 treatment and lane 6 shows LY-294002 treatment]. Lane 8 in all immunoblots shows serum-treated cells incubated with 10% serum. A and B show representative immunoblots from 3 or more experiments. C and D: bar graphs of means ± SD of scans of 3 or more immunoblots shown in A and B, respectively, for both pERK1/2 and pAkt (Ser⁴⁷³). *P ≤ 0.05 compared with ScuPA-stimulated HUVECs alone. NS, not significant.

Since HUVECs are not adult cells, we performed similar experiments with HMECs. Again, the induction of pERK1/2 was blocked by MEK1 inhibitors U-0126 and PD-98059 (P < 0.001; Fig. 1, B and D). HMECs were hardier cells than HUVECs because wortmannin (50 nM) did not reduce ScuPA-induced pERK1/2 (Fig. 1, B and D). ScuPA-stimulated pAkt (Ser⁴⁷³) was >95% inhibited by either wortmannin or LY-294002 (P < 0.001; Fig. 1, B and D). Neither U-0126 nor PD-98059 inhibited the induction of pAkt (Ser⁴⁷³) (Fig. 1, B and D). These combined experiments indicated that ScuPA-stimulated a similar signaling pathway in two different ECs. We also examined SIN1^−/− MEFs to confirm that ScuPA-stimulated pERK1/2 was independent of pAkt (Ser⁴⁷³). SIN1^−/− MEFs cannot phosphorylate Akt (Ser⁴⁷³) because SIN1 is necessary for mammalian target of rapamycin (mTOR) complex (mTORC)2 assembly for the phosphorylation of Akt (Ser⁴⁷³) (35). ScuPA-induced pERK1/2, but not pAkt (Ser⁴⁷³), in SIN1^−/− cells. These data indicate that the induction of pERK1/2 did not require pAkt (Ser⁴⁷³).

Finally, since ScuPA initiated this signaling cascade in HUVECs cultured on gelatin, we determined if these events were cell matrix dependent (Fig. 2, A and B). ScuPA stimulated pAkt (Ser⁴⁷³) expression to the same extent in HUVECs grown on collagen, fibrinogen, vitronectin, or fibronectin and pERK1/2 expression on collagen and fibrinogen as on gelatin (Fig. 2, A and B). On vitronectin and fibronectin, untreated control pERK1/2 samples had higher backgrounds, making the ScuPA-stimulated samples appear less magnified.

Fig. 2.

A and B: influence of cell matrix on the ability of ScuPA to induce pERK1/2 and pAkt (Ser⁴⁷³) in HUVECs. In these experiments, serum-starved HUVECs alone (control) or cells incubated with 16 nM ScuPA in the absence or presence of 50 μM U-0126 or 50 μM LY-294002 were incubated with HUVECs cultured on collagen or fibrinogen (A) or on vitronectin or fibrinogen (B). Shown is a single experiment using each matrix for culture. C: hydrolytic activity of 32 nM of ScuPA, two-chain urokinase (TcuPA), or p-A-PMSF (APMSF)-treated TcuPA (TcuPA + APMSF) on 0.8 mM l-pyroglutamyl- Gly-l-Arg-pNA HCl. “Cells” is the amidolytic activity of substrate alone on the cells for the incubation period. The green arrow indicates the time of incubation (∼7 min) of ScuPA in its various forms with HUVECs for the immunoblot in D. This graph is representative of 2 such experiments. D: pERK1/2 expression by serum-starved HUVECs alone (control) or after incubation with 32 nM ScuPA in the absence or presence of 100 μM PD-98059 and 32 nM TcuPA in the absence or presence of PD-98059 or 32 nM APMSF-treated TcuPA (TcuPA + APMSF) in the absence or presence of PD-98059. Shown is a single experiment.

Signaling by ScuPA versus tcuPA.

We next asked if signaling in ECs requires the conversion of ScuPA to its proteolytically active TcuPA counterpart. In contrast to TcuPA, the addition of 32 nM ScuPA or APMSF-treated TcuPA to HUVEC monolayers did not generate amidolytic activity in the 7-min incubation period used to induce pERK1/2 (Fig. 2C). However, the addition of 32 nM ScuPA, TcuPA, or APMSF-treated TcuPA induced comparable phosphorylation of ERK1/2 (Fig. 2D). These experiments indicated that neither conversion of ScuPA to TcuPA, which may expose novel epitopes, nor the presence of its catalytic activity is required for ScuPA to induce pERK1/2 in HUVECs.

ScuPA-induced pERK1/2 and pAkt are mediated by uPAR.

Human ScuPA does not activate murine cells in a single-cell proteolytic plaque assay, and the β-hairpin of the growth factor domain within the NH₂-terminal fragment of murine ScuPA does not interact with human uPAR (18, 23). The growth factor domain of ScuPA is known to interact with domain 1 of uPAR (18). Therefore, it was not clear how human ScuPA induced pERK1/2 in SIN1^−/− MEFs. Although murine and human ScuPA share only 60% overall sequence homology, there is 77% amino acid sequence homology in domain 2 of uPAR from Leu¹⁴⁴ to Thr¹⁷³, the region that expresses overlapping binding sites for ScuPA, factor XII, vitronectin, and HK (15, 42, 43, 50, 28). Leu¹⁴⁴ to Thr¹⁷³ lies on the surface of uPAR, with the potential to contact ScuPA (Fig. 3A) (3). When the crystal structure of urokinase's NH₂-terminal fragment bound to domain 1 of uPAR is rotated 180° and slightly to the left (Fig. 3B), the NH₂-terminal fragment-binding region of ScuPA in domain 1 does not interact with the domain 2 surface region of uPAR that binds ScuPA and HK (4, 50). If ScuPA-binding regions on domains 1 and 2 do not interact, then peptides from uPAR domain 2 alone or uPAR domain 2 occupying peptides from HK should interfere with ScuPA binding and, thus, cell activation. Therefore, we first asked if peptides from uPAR's domain 2 Leu¹⁴⁴-Thr¹⁷³ regions interfere with ScuPA's induction of pERK1/2 and pAkt (Ser⁴⁷³).

Fig. 3.

Influence of the ScuPA-domain 2 of urokinase plasminogen activator receptor (uPAR) interaction on pERK1/2 or pAkt (Ser⁴⁷³) expression. A: crystal model of domain (D)1 and D2 (in gray) of uPAR with ScuPA's growth factor domain (purple) inserted into D1 of uPAR (gray). This crystal structure is from Ref. 3. The amino acids in yellow represent D2 of uPAR amino acids Leu¹⁴⁴-Tyr¹⁷³ (50). The modeling was performed with the Cn3D4.3 program available from the National Center for Biotechnology Information structure website. HK, high-molecular-weight kininogen. B: the same crystal structure in A rotated 180° on its axis and then to the left. This model shows that the growth factor domain of ScuPA does not interact with the D2 region of uPAR. C–E: the “control” lane represents serum-starved HUVECs. C and D: immunoblots for pERK1/2 and total ERK1/2; E: immunoblot using an antibody to pAkt (Ser⁴⁷³) and total Akt. C: HUVECs were incubated with 16 nM ScuPA in media alone (lane 2) or in media containing PD-98059 (lane 3) or 300 μM uPAR peptide (TKC19, LRG20, YLP20, PGS20, or FHN20, respectively; lanes 4–8) from D2 of uPAR (see Table 1). D: HUVECs were incubated in 10% serum (lane 2) or incubated with PD-98059 (lane 3). HUVECs were also incubated with 16 nM ScuPA alone (lane 4) or in the presence of 300 μM peptides of D5 of HK (HKH20, GGH18, or GKE19, respectively) or 1.34 μM two-chain HK [plasma kallikrein-cleaved HKa (HKa)] (lanes 5–8; see Table 1). E: influence of 16 nM ScuPA alone (lane 2) or 16 nM ScuPA in the presence of wortmannin (lane 3) or 300 μM peptide LRG20, PGS20, TKC19, HVL24, or HKH20, respectively (lanes 4–8; see Table 1). C–E are representative of at least 4 experiments. F: bar graphs showing mean ± SD values for the 4 experiments shown in C and D for pERK1/2. G: bar graphs showing mean ± SD values for the 4 experiments shown in E for pAkt (Ser⁴⁷³). *P ≤ 0.05 compared with ScuPA-stimulated HUVECs alone.

Overlapping peptides (LRG20, YLP20, and PGS20) from uPAR domain 2 (amino acids 144–173) blocked ScuPA-induced expression of pERK1/2 (P < 0.003; Fig. 3, C and F, and Table 1) (50). A corresponding scrambled peptide (PGS20) had no effect. PGS20 itself also did not stimulate pERK1/2 or pAkt (Ser⁴⁷³). Peptide TKC19, which is adjacent to this region in domain 2 (amino acids 174–192), did not block ScuPA-induced pERK1/2 (Fig. 3, C and F). Peptide FHN20, which partially reduced ScuPA-induced pERK1/2 (Fig. 3C) and overlaps with peptide TKC19 at the COOH-terminal region of uPAR domain 2 (amino acids 174–178), did not significantly block (P > 0.08) the induction of pERK1/2 by ScuPA (Fig. 3F and Table 1) (50). These data indicate that peptides derived from uPAR's domain 2 amino acids 144–173, but not those from the COOH-terminal domain 2 region (amino acids 174–192), blocked ScuPA-induced intracellular signaling (50).

We next examined this question from the other side of the proposed partnership by asking if occupying the HK-binding site on uPAR with peptides from domain 5 of HK or with HKa would block the induction of pERK1/2 by ScuPA (Fig. 3, D and F). HKa blocked (P < 0.0027) ScuPA-mediated ERK1/2 phosphorylation (Fig. 3, D and F). Phosphorylation of ERK1/2 by ScuPA was also blocked by peptide HKH20 from the HK domain 5 cell-binding site (amino acids 479–498 on HK) on HUVEC (P < 0.0006; Fig. 3, D and F, and Table 1) (33). This peptide also blocked HK binding to soluble uPAR (50). Although the overlapping peptide GGH18 from HK domain 5 (amino acids 469–486) appeared to exert some inhibition (Fig. 3D), the effect did not reach significance (Fig. 3F). Peptide GGH18 itself also did not stimulate pERK1/2 or pAkt (Ser⁴⁷³). A peptide from the NH₂-terminal region of HK's domain 5, GKE19 (amino acids 402–420), which does not block kininogen binding to soluble uPAR, was not inhibitory (Fig. 3, D and F, and Table 1) (33, 50).

Finally, we examined if ScuPA-induced pAkt (Ser⁴⁷³) also was blocked by these peptides from uPAR and HK. Peptides LRG20 or PGS20 from domain 2 of uPAR inhibited ScuPA-induced pAkt (Ser⁴⁷³) (Fig. 3, E and G). Likewise, peptides to domain 5 of HK (peptides HVL24 or HKH20) also blocked ScuPA-induced pAkt (Ser⁴⁷³) (Fig. 3, E and G). Combined, these investigations indicated that domain 2 on uPAR contains a binding site for ScuPA and HK that stimulated and inhibited, respectively, the induction of pERK1/2 and pAkt (Ser⁴⁷³).

ScuPA-initiated uPAR signaling is mediated through β₁-integrins and VEGFR2.

uPAR acts in trans with specific integrins to mediate cell adhesion and migration (20). Reduced α₅β₁-integrin expression decreased angiogenesis in ECs, and signaling by urokinase leading to lung cancer progression requires β₁-integrins (59, 61). Therefore, we asked if β₁-integrins participate in these ScuPA signaling pathways. Monoclonal antibody 6S6 to β₁-integrin blocked ScuPA-induced pERK1/2 (Fig. 4, A and B). Additionally, antibodies 6S6 and P4C10 (P < 0.0002) and AIIB2 (P < 0.033) to β₁-integrin, P1D6 (P < 0.0002) to α₅-integrin, and ASC-1 (P < 0.0006) to α₃-integrin each blocked ScuPA-induced pAkt (Ser⁴⁷³) (Fig. 4, C and D). Moreover, a peptide from domain 2 of uPAR (IQE13), an integrin-binding region, and one from the β-propeller of the β₁-integrin α-subunit (NLD9), a uPAR-binding domain, blocked (P < 0.0001) the induction of pERK1/2 and pAkt (Ser⁴⁷³) by ScuPA (Fig. 5, A and B) (20, 67). These combined data indicate that β₁-integrins mediate ScuPA-induced pERK1/2 and pAkt but that the induction of pAkt involves additional integrins that contain α₅- and α₃-subunits.

Fig. 4.

Effects of integrin antibodies on ScuPA-induced pERK1/2 and pAkt (Ser⁴⁷³). A: effect of antibodies to integrins on pERK1/2 expression; C: effect of antibodies to integrins on pAkt (Ser⁴⁷³) expression. In A–D, the “control” lanes show serum-starved HUVECs. A: in lanes 2 and 3, HUVECs were incubated with 16 nM ScuPA in the absence or presence of 100 μM PD-98059 and in lanes 4–8, cells were incubated with 16 nM ScuPA in the presence of 188 nM monoclonal antibodies ASC1, HA5, 6S6, and P4C10 (lanes 4–7) or 63 nM AIIB2 (lane 8), respectively. Immunoblots are representative of 3 or more experiments under these conditions. B: graphic representation of all experiments shown in A. C: in lanes 2 and 3, HUVECs were incubated with 16 nM ScuPA in the absence or presence of 30 nM wortmannin, and in lanes 4–8, HUVECs were incubated with 16 nM ScuPA in the presence of 188 nM monoclonal antibodies 6S6, P4C10, P1D6, and ASC1 or 63 nM AIIB2, respectively. The space in the immunoblot in C is the removal of one lane from the immunoblot on the same gel. D: graphic representation of all experiments shown in C. In all experiments shown in B and D, columns with error bars represent results from 3 or more experiments. *P ≤ 0.05.

Fig. 5.

Effects of peptides to uPAR-integrin interaction sites and tyrosine kinase inhibitors on ScuPA-induced pERK1/2 or pAkt (Ser⁴⁷³). A: serum-starved HUVECs were incubated with 16 nM ScuPA alone or in the presence of 50 μM PD-98059, 30 nM wortmannin, 50 nM AG-1478, 300 μM peptide NLD9, or peptide IQE13, respectively, and examined for pERK1/2 or pAkt (Ser⁴⁷³) expression. Shown is a representative experiment of 3 or more experiments performed. B: bar graph showing mean ± SD values of 3 or more experiments shown in A. *P ≤ 0.05 compared with ScuPA-treated HUVECs alone. C and E: influence of PP3 on ScuPA-induced pERK1/2 or pAkt (Ser⁴⁷³). HUVECs were serum starved (control) or stimulated with serum to induce pERK1/2 or pAkt. In other lanes, 16 nM ScuPA was incubated with HUVECs in the absence or presence of 50 μM PD-98059 or 100 μM PP2 or PP3 to measure pERK1/2 or with 30 nM wortmannin to examine pAkt (Ser⁴⁷³). The space in the image in C is the removal of three lanes from the immunoblot on the same gel. The two spaces in the image of E is the removal of one lane on the left side and two lanes on the right side from the immunoblot on the same gel. C and E show single representative experiments; the bar graphs in D and F represent means ± SD or 3 or more experiments. *P ≤ 0.0012 compared with ScuPA-stimulated HUVEC alone.

EGFR (ErbB1, HER1) transduces uPAR signaling in some settings (27, 44). Furthermore, cleaved HK inhibits prostate cancer cell migration by interfering with EGFR downregulation of pERK1/2 and pAkt (48). Therefore, we asked whether ErbB (HER) receptor tyrosine kinase inhibition blocks ScuPA-mediated signaling. The EGFR tyrosine kinase inhibitor AG-1478 (50 nM) blocked ScuPA-induced pERK1/2 and pAkt (Ser⁴⁷³) expression (P < 0.0018; Fig. 5, A and B). The related EGFR tyrosine kinase inhibitor PP3 (100 μM), but not the Src inhibitor PP2 (100 μM), also inhibited both pERK1/2 and pAkt (Ser⁴⁷³) (P < 0.0012; Fig. 5, C–F). However, siRNA knockdown of HER1–HER4 (ErbB1–ErbB4) did not inhibit ScuPA-induced pERK1/2 or pAkt (Ser⁴⁷³) (Fig. 6). Since VEGFR2 (Flt-1/KDR) influences ScuPA activation and activation of VEGFR2 leads to pERK1/2 and pAkt (Ser⁴⁷³) for angiogenesis, we examined its influence on ScuPA-stimulated cells (20, 54). siRNA knockdown of VEGFR2 reduced ScuPA-induced pERK1/2 and pAkt (Ser⁴⁷³) by 37 ± 19% and 69 ± 3%, respectively (means ± SE for 3 experiments; Fig. 7). Additional experiments showed that AG-1478 or PP3 did not prevent VEGF-induced Tyr¹¹⁷⁵ phosphorylation of VEGFR2 (data not shown). These data indicate that both tyrosine kinases associated with EGFR and VEGFR2 participated in the ScuPA stimulatory pathway in ECs.

Fig. 6.

A–D: Influence of HER1–HER4 (ErbB1–ErbB4) gene silencing on ScuPA-induced pERK1/2 and pAkt (Ser⁴⁷³) expression. HMECs were prepared and treated with a 50 nM final concentration of control (siCRTL) or HER1–HER4 (siHER1–siHER4) small interfering (si)RNA as indicated in materials and methods. After treatment, cells were untreated or treated with 16 nM ScuPA in the absence or presence of 50 μM LY-294002 or PD-98059. Samples were then electrophoresed and transferred onto nitrocellulose membranes. The electroblot was sequentially treated with antibodies to each HER receptor (1–4), pERK1/2, pAkt (Ser⁴⁷³), total ERK1/2, and total Akt. Shown is one example of at least 3 experiments performed for each targeted HER.

Fig. 7.

Influence of VEGF receptor 2 (VEGFR2) gene silencing on ScuPA-induced pERK1/2 and pAkt (Ser⁴⁷³) expression. HMECs were prepared and treated with a 50 nM final concentration of control (siCRTL) or VEGRF2 (siVEGFR2) siRNA as indicated in materials and methods. After the knockdown, cells were untreated or treated with 16 nM ScuPA in the absence or presence of 50 μM LY-294002 or PD-98059. Samples were then electrophoresed and transferred onto nitrocellulose membrane. The electroblot was sequentially treated with antibodies to VEGFR2, pERK1/2, pAkt (Ser⁴⁷³), total ERK1/2, and total Akt. Shown is a representative experiment of 3 performed complete experiments.

ScuPA stimulates HUVEC proliferation.

Next, we examined the consequence of ScuPA-induced signaling on EC proliferation. ScuPA stimulated EC proliferation as assessed by cell viability (P < 0.03), which was blocked by the MEK inhibitor PD-98059 (Fig. 8A). Consistent with the signaling experiments, ScuPA-induced EC proliferation also was blocked by peptide LRG20 of uPAR's domain 2 and peptide HVL24 (P < 0.0036) but not by peptide GKE19 (P = 0.54), both from domain 5 of HK. Peptide TKC19 from domain 2 of uPAR exerted much less inhibition (P < 0.032; Fig. 8A). Similarly, ScuPA-stimulated incorporation of BrdU was blocked by PD-98059 (Fig. 8B) and by peptides LRG20 and HVL24 (P < 0.0001) but not by peptides GKE19 or TKC19 (P = 0.06; Fig. 8B). Additional investigations showed that ScuPA-induced cell proliferation and BrdU incorporation were blocked by the PI3K inhibitors wortmannin and LY-294002 and by monoclonal antibody 6S6 to β₁-integrin (P ≤ 0.04) but not by monoclonal antibody ASC1, which did not inhibit signaling (P = 0.61; Fig. 8, C and D). Together, these latter experiments indicated that ScuPA-induced cell proliferation and BrdU incorporation were mediated by the same pathways required to induce the expression of pERK1/2 and pAkt (Ser⁴⁷³).

Fig. 8.

Influence of ScuPA on cell growth. A: cell proliferation. HUVECs were prepared for cell proliferation assays as described in materials and methods. The “control” lane represents the proliferation of untreated HUVECs. ScuPA (64 nM) induced HUVEC proliferation in the absence or presence of 100 μM PD-98059 or 300 μM peptide LRG20, TKC19, HVL24, or GKE19, respectively. Shown are mean ± SD values of 4 independent experiments. B: 5-bromo-2′-deoxyuridine (BrdU) incorporation. The “control” bar represents BrdU incorporation by serum-starved HUVECs alone. BrdU incorporation was stimulated with 64 nM ScuPA in the absence or presence of 100 μM PD-98059 or 300 μM peptides LRG20, TKC19, HVL24, or GKE19, respectively. Shown are mean ± SD values of 4 independent experiments. C and D: cell proliferation and BrdU incorporation, respectively. The “control” lanes represent the level of serum-starved HUVECs. In both C and D, HUVECs were stimulated with ScuPA (64 nM) in the absence or presence of 100 μM PD-98059, 30 nM wortmannin, 50 μM LY-294002, or 188 nM antibody 6S6 or ASC1. Shown are mean ± SD values of 4 independent experiments. *P < 0.05.

The pathway to ScuPA-induced angiogenesis.

We next asked if ScuPA induced aortic sprouting through these signaling pathways. Aortae imbedded in collagen from unstimulated WT or uPAR KO mice developed 4 ± 3 and 7 ± 4 sprouts (means ± SD), respectively (P = 0.49; Fig. 9A). In WT mice, VEGF and ScuPA stimulated 48 ± 5 sprouts (n = 4 experiments) and 60 ± 4 sprouts (n = 3 experiments), respectively (P = 0.11; Fig. 9B). In contrast, VEGF stimulated 85 ± 7 sprouts from aortae from uPAR KO mice (mean ± SD, n = 4 experiments), whereas the addition of ScuPA produced only 1 ± 0.6 sprouts (n = 3 experiments, P < 0.0001; Fig. 9B).

Fig. 9.

A: ability of agonists to stimulate sprouting from the aorta of a wild-type (WT) or uPAR knockout (KO) mouse. Sprouting was stimulated with 0.5 nM VEGF or 32 nM ScuPA. A: representative images of 3 experiments under each of the conditions. B: bar graph showing mean ± SD numbers of sprouts from aortae of WT or uPAR KO mice after each stimulus from 3 independent experiments. C and D: mapping of ScuPA's cellular pathway to aortic sprouts. D: aortae from 6- to 8-wk-old mice were collected and prepared as described in materials and methods. VEGF (0.5 nM), FGF (0.6 nM), or ScuPA (32 nM) stimulated aortic sprouts at 5 days. Simultaneous incubation of ScuPA-induced aortic sprouts with 50 μM PD-98059, 50 μM LY-294002, 50 nM AG-1478, or 128 nM HKa is also shown. D: representative experiment of 2 or more experiments performed. The bar graph in C shows mean ± SD values of 3 or more experiments for control, VEGF, FGF, and ScuPA or the mean of 2 experiments for ScuPA with each of its inhibitors.

We then mapped the signaling pathway of ScuPA-induced sprouting from WT aortae. As shown in Fig. 8D, aortic sprouting induced by 32 nM ScuPA was inhibited by individually adding the MEK inhibitor PD-98059, PI3K inhibitor LY-294002, tyrosine kinase inhibitor AG-1478, or cleaved HK. As shown in Fig. 9C, the number of sprouts generated from untreated WT aortae (4 ± 3 sprouts, mean ± SD, n = 5) did not significantly differ (P ≥ 0.49) from ScuPA-treated WT aortae in two identical experiments when samples were preincubated with PD-98059 (mean of 0.5 sprouts), LY-294002 (mean of 0.5 sprouts), AG-1478 (mean of 5 sprouts), or cleaved HK (mean of 6 sprouts). These latter experiments showed that blockade of MEK, PI3K, or tyrosine kinase or binding by cleaved HK abolished ScuPA-induced angiogenesis in aortic segments.

In vivo angiogenesis experiments showed similar findings. ScuPA-stimulated angiogenesis in Matrigel plugs in the flanks of WT mice was blocked by cotreatment of the Matrigel with LY-294002, cleaved HK, AG-1478, or PD-98059 (Fig. 10A). On microscropic examination, new blood vessels only were observed by CD31 staining in FGF- or ScuPA-treated plugs. The hemoglobin content of the inhibitor-treated ScuPA-stimulated Matrigel plugs was significantly reduced (Fig. 10B). Finally, the specificity of the angiogenic response was shown by indicating that ScuPA induced angiogenesis in plugs in flanks of WT but not uPAR KO mice. These combined in vitro and in vivo data indicate that ScuPA stimulated angiogenesis through a pathway that used MEK, PI3K, and receptor tyrosine kinases and that it is blocked by treatment with cleaved HK.

Fig. 10.

A: photographs of excised Matrigel plugs from the flanks of WT mice (top). Microscopic sections of each Matrigel plug (bottom) stained with CD31 (red) and 4′,6-diamidino-2-phenylindole (DAPI; blue) are shown. These Matrigel plugs were untreated (control) or contained FGF, ScuPA alone, or ScuPA in the presence of LY-294002, HKa, AG-1478, or PD-98059. See materials and methods for the concentrations of each stimulant and inhibitor. B: hemoglobin (Hb) concentration in each matrigel plug/gram of Matrigel for each of the conditions shown in A. By one-way ANOVA, all of the ScuPA-inhibited Matrigel plugs had significantly less Hb than ScuPA alone. The degree of ScuPA-induced angiogenesis alone was significantly less than that induced by FGF in the present assay. *P < 0.05; **P < 0.01; ***P < 0.001. C: comparison of FGF- and ScuPA-induced angiogenesis in Matrigel in WT (top row) and uPAR KO mice (middle row). The bottom row shows microscopic sections of Matrigel plugs from uPAR KO mice stained with CD31 and DAPI.

DISCUSSION

These investigations map a region on domain 2 of uPAR and a signaling pathway in ECs through which ScuPA stimulates proliferation and angiogenesis. The addition of ScuPA to serum-starved HUVECs induces p44/42 MAPK (ERK1/2) and PI3K/Akt (Ser⁴⁷³) phosphorylation. We observed that overlapping peptides from a continuous 29-amino acid region on domain 2 of uPAR block ScuPA-induced pERK1/2 and pAkt (Ser⁴⁷³), cell proliferation, and angiogenesis. Domain 1 of uPAR mediates urokinase's growth factor domain binding and stimulation of pERK1/2, cell proliferation, adhesion, and migration (26, 53, 65). It is not certain that ScuPA binding to domain 2 alone also stimulates signaling. Peptide GGH18 from HK does not stimulate pERK1/2 or pAkt (Ser⁴⁷³), but human ScuPA stimulates murine pERK1/2 in SIN1^−/− cells (37). Regardless, occupation of domain 2 of uPAR with HK peptides or blockade of ScuPA's ability to bind domain 2 with uPAR peptides is sufficient to block all downstream signaling, cell proliferation, and angiogenesis. This urokinase signaling pathway is initiated by nonproteolytic mechanisms as APMSF-treated ScuPA or TcuPA stimulated pERK1/2 expression to the same extent as ScuPA or TcuPA. These data are consistent with the finding that DFP-treated urokinase and noncleavable uPAR (hcr-uPA: R83K, Y87C, R89K, and R19C) stimulate cell proliferation through integrins (20, 38, 52) and uPAR's domain 2 soluble peptide Ser⁸⁸-Arg-Ser-Arg-Tyr⁹² stimulates angiogenesis (7, 20).

We found some cross-talk between the pERK1/2 and pAkt (Ser⁴⁷³) activation pathways in ECs stimulated with ScuPA based on the effects of MEK inhibitors PD-98059 and U-0126 and PI3K inhibitors wortmannin or LY-294002. pERK1/2 regulates pAkt (Ser⁴⁷³) through mTORC2 (37). In all our experiments, PD-98059 or U-0126 inhibited pERK1/2 and LY-294002 inhibited pAkt (Ser⁴⁷³). Wortmannin (30–50 nM) is toxic to cultured HUVECs. Since the IC₅₀ for wortmannin inhibition of PI3K is 5 nM, the findings with HUVECs are not surprising. Additionally, neither wortmannin nor LY-294002 are entirely specific PI3K inhibitors. Wortmannin inhibits PI3K and polo-like kinase 1 with equal affinity (44). LY-294002 inhibits PI3K, mTOR, DNA-dependent protein kinase, casein kinase 2, and Pim-1 (29). In cells of hematopoietic origin, inhibition of PI3K influences pERK1/2 expression through the sequential effects of phospholipase C-γ, Raf-1, and MEK (58). Stimulation of pERK1/2 expression by ScuPA is independent of pAkt (Ser⁴⁷³) because in SIN1^−/− MEFs, p44/42 MAPK is phosphorylated in its absence (37).

The finding that human ScuPA activates pERK1/2 in MEFs was somewhat unexpected. It is well established that the growth factor domain of murine ScuPA does not interact with human uPAR domain 1 (18). Thus, other explanations needed to be sought to explain our findings. Previous work from our laboratory has shown that peptides from domain 2 of uPAR and from HK's domain 5 and ScuPA block HK binding to soluble uPAR (33, 50). Although the present investigations used 300 μM peptide concentrations to demonstrate inhibition of signaling, each of the inhibitory peptides we used have an IC₅₀ of HK binding to soluble uPAR ≤ 20 μM (50). Our data with the peptide inhibitors are consistent with other work that shows that antibodies to domain 2 of uPAR block HK binding (14, 48). Additionally, this region on uPAR participates in the binding of factor XII and vitronectin (28, 43, 43). The crystal models (Fig. 3, A and B) suggest that this region in domain 2 is spacially distinct from urokinase's NH₂-terminal fragment binding site on domain 1 of uPAR. Moreover, this region on domain 2 of uPAR is adjacent to the domain 2 region that interacts with integrins that may be communicating the signal we observed (20, 67).

The initiation of intracellular signal transduction by ScuPA requires interactions between uPAR and specific integrins. The β-propeller of the β₁-integrin α-subunit is involved in the signaling complex with uPAR (67). A uPAR domain 2 peptide that inhibits signal transduction interacts with integrins, which is adjacent to a HK- and ScuPA-binding region (20, 50). Several monoclonal antibodies to β₁-integrins suppress pERK1/2 or pAkt (Ser⁴⁷³) expression induced by ScuPA. The variability among several (e.g., Mab 6S6 vs. AIIB2) antibodies to inhibit signaling must be related to their epitope specificity and their impact on uPAR-β₁-integrin binding (63, 67, 68). ScuPA-induced pERK1/2 is only blocked by the β₁-integrin antibody 6S6. In contrast, ScuPA-induced pAkt (Ser⁴⁷³) is blocked by β₁-integrin antibodies 6S6, P4C10, or AIIB2. pAkt (Ser⁴⁷³) may be influenced by cell spreading independent of signaling through the cell (30). Other integrins also influence ScuPA signaling, since the α₃-integrin antibody ASC-1 is inhibitory. Inhibition with antibodies to integrins only begin to map their role in EC proliferation since α₃-, α₄-, α₅-, and α₉-integrins interact with β₁-integrin and α_vβ₃-integrin influence them as well (54).

EGFR has been proposed to be necessary for ScuPA-induced signaling and cell proliferation (14, 39, 44). It has been proposed that binding of ScuPA to domains 2 and 3 of uPAR communicates through α₅β₁-integrins to associate directly or through additional proteins with the HER (ErbB) receptor(s) (20, 44, 67). ScuPA does not induce pERK1/2 or pAkt (Ser⁴⁷³) in ECs treated with HER1 tyrosine kinase inhibitors AG-1478 and PP3 (5, 12). However, siRNA knockdown of HER1 (EGFR or ErbB1) as well as HER2–HER4 (ErbB2–ErbB4) in these cells does not block ScuPA-induced pERK1/2 and pAkt (Ser⁴⁷³). The reason(s) for this effect is not completely known. It is possible that the degree of HER1 reduction by siRNA was not sufficient to block its Tyr⁸⁴⁵ phosphorylation (39). Further experiments are needed to determine which of the nine tyrosine phosphorylation sites on HER1 are blocked by these inhibitors.

Since ScuPA-induced signaling is associated with angiogenesis, we also determined if siRNA silencing of VEGFR2 decreased ScuPA-induced pERK1/2 and pAkt (Ser⁴⁷³). Unlike the experiments with HER1–HER4, we found that VEGFR2 was directly in the pathway of ScuPA-induced phosphorylation of ERK1/2 and Akt (Ser⁴⁷³) (1, 32, 57). It is of note that siRNA to VEGFR2 has a greater effect on decreasing pAkt (Ser⁴⁷³) than pERK1/2. The reason for this is not completely known and will be the basis for additional investigations. VEGFR2 had been recognized to mediate VEGF-induced MMP-2 production, which proteolytically activates ScuPA when bound to uPAR (54). VEGF, through VEGFR2, also modulates cell migration through uPAR and α₅β₁-integrins (1). Soluble CD146 promotes angiogenesis and neovascularization through upregulation of ScuPA, VEGF, VEGFR2, endothelial nitric oxide synthase, and MMP-2 (32). In cancer, hypoxia upregulates the uPA-uPAR and VEGF-VEGFR2 systems (71). A recent hindlimb ischemia study (36) in C57Bl/6 mice has indicated that at 3 days VEGFR2 substantially decreases and at 10 days VEGFR1, ScuPA, and uPAR markedly increase. Moreover, the cytoplasmic tails of β₃-integrins and VEGFR2 directly interact (69). Further studies are needed to determine if β₁-integrins and VEGFR2 physically interact in a similar fashion. These combined studies suggest that there is an autocrine regulatory system where VEGF-VEGFR2 regulates ScuPA-uPAR and vice versa in angiogenesis-related activities. Finally, it is possible that both EGFR and VEGFR2 are operative in supporting ScuPA-induced cell proliferation and angiogenesis. EGFR inhibitors block in vivo Matrigel plug angiogenesis stimulated by ScuPA, and siRNA to VEGFR2 blocks ScuPA-induced ERK1/2 and Akt (Ser⁴⁷³) phosphorylation. Recent investigations with tumor cells have indicated that a combined mechanism exists whereby EGFR supports cell growth and VEGFR2 influences angiogenesis (13). More investigations are needed to understand how both these growth factor receptor systems contribute to angiogenesis after ScuPA stimulation.

ScuPA stimulates cell proliferation under similar conditions that promote pERK1/2 and pAkt (Ser⁴⁷³) expression. Although there were differences in the density of the cells and the incubation times used for the phosphorylation assays versus the cell proliferation and BrdU incorporation assays, the findings that the same chemical, peptide, and antibody inhibitors interfere with the results in both assays suggest that the intracellular processes are related. In the phosphorylation experiments, 5- to 7-min incubation of ScuPA with serum-starved HUVECs is sufficient to engage signaling events, whereas cell proliferation and BrdU incorporation require 24-h incubations with ScuPA. These findings are not surprising given that cell proliferation activities involve a series of processes that are considerably downstream from the initiating phosphorylation events. The same signaling inhibitors that block cell proliferation also inhibit in vitro and in vivo angiogenesis initiated by ScuPA. Like us, Balsara et al. (2) recognized that tissue from uPAR-deleted mice have a heighten response to exogenous VEGF. The reason for this is not completely known, but considering the postulated autocrine regulatory system between ScuPA-uPAR and VEGF- VEGFR2, we hypothesize that in the absence of uPAR, the VEGF-VEGFR2 axis has the ability to compensate for the uPAR loss either through its overexpression or functional responsiveness.

HKa and peptides from its domain 5 have antiproliferative and antiangiogenic activities, and these functions may be partially mediated through uPAR (11, 16, 46, 70). A recent study (17) has indicated that cleaved HK, like antibody to uPAR, also inhibits EC tube formation and tumor angiogenesis. These data suggest that the effect of HK is mediated in part through effects on the interaction of uPAR with integrins. High local concentrations of plasma kallikrein-formed cleaved HK blocks the ability of ECs to demonstrate ScuPA-induced pERK1/2 or pAkt (Ser⁴⁷³) and permit cell proliferation and migration. The ability of cleaved HK to interfere with ScuPA-mediated signaling through uPAR explains some of its antiproliferation and antiangiogenic activity (11, 16, 46, 70). ScuPA induces angiogenesis by initiating a coordinated interaction between uPAR, specific integrins, and VEGFR2 to induce the expression of pERK1/2 and pAkt (Ser⁴⁷³) (Fig. 11). Inhibition of EGFR also inhibits pERK1/2, pAkt (Ser⁴⁷³), and angiogenesis, but the relationship between VEGFR2 and EGFR (HER1) for ScuPA-induced signaling is presently not known. HK, HKa, and peptides HVL24 and HKH20 block this pathway by interfering with ScuPA binding (Fig. 11) and, perhaps, by initiating an inhibitory pathway mediated by pSrc (19, 47, 48). Together, these data suggest that plasma kallikrein-cleaved HK and ScuPA or HKa interact with ECs in a reciprocal modulating fashion, whereby either form of uPA stimulates cell growth, proliferation, and angiogenesis and HKa inhibits ScuPA's cellular signaling pathway, thereby arresting cell growth and angiogenesis (Fig. 11).

Fig. 11.

Model of ScuPA D2 of uPAR signaling in endothelial cells and the inhibitory role of HKa. ScuPA binds to D1–D3 of uPAR on HUVECs. Interactions with ScuPA with endothelial cells induces uPAR to communicate intracellularly through β₁-integrins. Monoclonal antibody 6S6 to β₁-integrin blocks this pathway. Cell stimulation through uPAR and integrins requires interactions with VEGFR2. Also, tyrosine inhibitors AG-1478 and PP3 to the EGF receptor (EGFR; HER1) block ScuPA signaling. The MEK inhibitor PD-98059 also blocks ScuPA-induced ERK1/2 phosphorylation. LY-294002, a phosphatidylinositol 3-kinase inhibitor, blocks ScuPA-induced Akt phosphorylation. HKa blocks binding of ScuPA to endothelial cells and D2 of uPAR, which leads to inhibition of pERK1/2, pAkt (Ser⁴⁷³), and angiogenesis. Inhibition of any step of the ScuPA signaling pathways blocks cell proliferation and angiogenesis in HUVEC aortic segments in vitro and Matrigel plugs in vivo.

Finally, we have previously described a similar signaling pathway of factor XII through uPAR and β₁-integrins to stimulate cell proliferation and angiogenesis (42). To date, we have not been able to ascertain a major distinguishing feature of the factor XII- or ScuPA-stimulated pathway, although the former has not be studied as extensively as the ScuPA interaction. Considering the constitutive plasma concentration of factor XII (450 nM) versus that of ScuPA (1–2 nM) and the similar affinities of each to bind to uPAR, factor XII at basal levels should be more influential on cell growth and angiogenesis than ScuPA (49). However, upon stimulation where ScuPA production rises dramatically, it may exert major influence after injury. This assessment suggests that both these ligands function to promote vascular homeostasis through uPAR.

GRANTS

This work was supported by National Institutes of Health Grants HL-052775-18 and HL-112666A (to A. H. Schmaier) and HD-057355, HL-090697, and NS-064447 (to D. B. Cines) as well as American Heart Association Scientific Development Grant N004313 (to Z. Shariat-Madar).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: A.H.S. conception and design of research; G.A.L., A.M., F.M., Z.S.-M., and R.G.S performed experiments; G.A.L., A.M., F.M., Z.S.-M., and R.G.S prepared figures; A.H.S. analyzed data; D.B.C. and A.H.S. drafted manuscript.