The N-terminal Domain of Hepatocyte Growth Factor Inhibits the Angiogenic Behavior of Endothelial Cells Independently from Binding to the c-met Receptor
2003; Elsevier BV; Volume: 278; Issue: 39 Linguagem: Inglês
10.1074/jbc.m212768200
ISSN1083-351X
AutoresTatyana Merkulova‐Rainon, Patrick England, Shunli Ding, Corinne Demerens, G Tobelem,
Tópico(s)Pancreatic function and diabetes
ResumoHepatocyte growth factor (HGF) is a pleiotropic factor that plays an important role in complex biological processes such as embryogenesis, tissue regeneration, cancerogenesis, and angiogenesis. HGF promotes cell proliferation, survival, motility, and morphogenesis through binding to its receptor, a transmembrane tyrosine kinase encoded by the MET proto-oncogene (c-met). Structurally speaking, HGF is a polypeptide related to the enzymes of the blood coagulation cascade. Thus, it comprises kringle domains that in some other proteins have been shown to be responsible for the anti-angiogenic activity. To check whether the isolated kringles of HGF were able to inhibit angiogenesis, we produced them as recombinant proteins and compared their biological activity with that of the recombinant HGF N-terminal domain (N). We showed that (i) none of the isolated HGF kringle exhibits an anti-angiogenic activity; (ii) N is a new anti-angiogenic polypeptide; (iii) the inhibitory action of N is not specific toward HGF, because it antagonized the angiogenic activity of other growth factors, such as fibroblast growth factor-2 and vascular endothelial growth factor; and (iv) in contrast with full-length HGF, N does not bind to the c-met receptor in vitro, but fully retains its heparin-binding capacity. Our results suggest that N inhibits angiogenesis not by disrupting the HGF/c-met interaction but rather by interfering with the endothelial glycosaminoglycans, which are the secondary binding sites of HGF. Hepatocyte growth factor (HGF) is a pleiotropic factor that plays an important role in complex biological processes such as embryogenesis, tissue regeneration, cancerogenesis, and angiogenesis. HGF promotes cell proliferation, survival, motility, and morphogenesis through binding to its receptor, a transmembrane tyrosine kinase encoded by the MET proto-oncogene (c-met). Structurally speaking, HGF is a polypeptide related to the enzymes of the blood coagulation cascade. Thus, it comprises kringle domains that in some other proteins have been shown to be responsible for the anti-angiogenic activity. To check whether the isolated kringles of HGF were able to inhibit angiogenesis, we produced them as recombinant proteins and compared their biological activity with that of the recombinant HGF N-terminal domain (N). We showed that (i) none of the isolated HGF kringle exhibits an anti-angiogenic activity; (ii) N is a new anti-angiogenic polypeptide; (iii) the inhibitory action of N is not specific toward HGF, because it antagonized the angiogenic activity of other growth factors, such as fibroblast growth factor-2 and vascular endothelial growth factor; and (iv) in contrast with full-length HGF, N does not bind to the c-met receptor in vitro, but fully retains its heparin-binding capacity. Our results suggest that N inhibits angiogenesis not by disrupting the HGF/c-met interaction but rather by interfering with the endothelial glycosaminoglycans, which are the secondary binding sites of HGF. The hepatocyte growth factor (HGF) 1The abbreviations used are: HGF, hepatocyte growth factor; N, N-terminal domain of HGF; K1, K2, K3, K4, the first, second, third, and fourth kringles of HGF, respectively; NK1, an HGF variant containing N domain and K1; NK2, an HGF variant containing N domain and the first two kringles; IPTG, isopropyl β-d-1-thiogalactopyranoside; NF heparin, non-fractionated heparin; LMW, low molecular weight; NHS, normal human serum; FBS, fetal bovine serum; FGF-2, fibroblast growth factor 2; VEGF, vascular endothelial growth factor; PDGF BB, platelet-derived growth factor BB; c-met/Fc, chimerical protein composed of the HGF receptor (c-met) extracellular domain fused to human IgG1 Fc; HUVEC, human umbilical vein endothelial cell; hASMC, human aortic smooth muscle cell; NHEK, human normal epidermal keratinocyte; SPR, surface plasmon resonance; GAG, glycosaminoglycan; PBS, phosphate-buffered saline. (1Nakamura T. Nishizawa T. Hagiya M. Seki T. Shimonishi M. Sugimura A. Tashiro K. Shimizu S. Nature. 1989; 342: 440-443Crossref PubMed Scopus (1979) Google Scholar), also known as Scatter factor (2Stoker M. Gherardi E. Perryman M. Gray J. Nature. 1987; 327: 239-242Crossref PubMed Scopus (1131) Google Scholar), was originally described as a potent mitogen for mature hepatocytes and as a cytokine capable of inducing the dissociation (scattering) of epithelial cells. HGF has now been proven to be a pleiotropic factor that acts on a wide array of target cell types, including epithelial, endothelial, neuronal, and hematopoietic cells (3Matsumoto K. Nakamura T. CIBA Found. Symp. 1997; 212: 198-214PubMed Google Scholar). HGF promotes cell proliferation, survival, motility, and morphogenesis through binding to its receptor, a transmembrane tyrosine kinase encoded by the MET proto-oncogene (c-met) (4Park M. Dean M. Kaul K. Braun M.J. Gonda M.A. Vande W.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6379-6383Crossref PubMed Scopus (483) Google Scholar, 5Bottaro D.P. Rubin J.S. Faletto D.L. Chan A.M. Kmiecik T.E. Vande W.G. Aaronson S.A. Science. 1991; 251: 802-804Crossref PubMed Scopus (2086) Google Scholar). The signaling cascade triggered by the binding of HGF to c-met contributes to complex biological processes, such as embryogenesis, tissue regeneration, and cancerogenesis (3Matsumoto K. Nakamura T. CIBA Found. Symp. 1997; 212: 198-214PubMed Google Scholar, 6Comoglio P.M. Trusolino L. J. Clin. Invest. 2002; 109: 857-862Crossref PubMed Google Scholar). Studies performed in the recent years have shown that HGF is also a potent angiogenic molecule (7Bussolino F. Di Renzo M.F. Ziche M. Bocchietto E. Olivero M. Naldini L. Gaudino G. Tamagnone L. Coffer A. Comoglio P.M. J. Cell Biol. 1992; 119: 629-641Crossref PubMed Scopus (1202) Google Scholar, 8Silvagno F. Follenzi A. Arese M. Prat M. Giraudo E. Gaudino G. Camussi G. Comoglio P.M. Bussolino F. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1857-1865Crossref PubMed Scopus (86) Google Scholar, 9Rosen E.M. Lamszus K. Laterra J. Polverini P.J. Rubin J.S. Goldberg I.D. CIBA Found. Symp. 1997; 212: 215-229PubMed Google Scholar, 10Van Belle E. Witzenbichler B. Chen D. Silver M. Chang L. Schwall R. Isner J.M. Circulation. 1998; 97: 381-390Crossref PubMed Scopus (410) Google Scholar). HGF is a polypeptide structurally related to the enzymes of the blood coagulation cascade. In the cell it is synthesized as a biologically inactive single chain precursor that is then cleaved by specific serine proteases yielding a fully active disulfide-linked heterodimer composed of α- and β-chains (11Naldini L. Tamagnone L. Vigna E. Sachs M. Hartmann G. Birchmeier W. Daikuhara Y. Tsubouchi H. Blasi F. Comoglio P.M. EMBO J. 1992; 11: 4825-4833Crossref PubMed Scopus (523) Google Scholar, 12Miyazawa K. Shimomura T. Kitamura A. Kondo J. Morimoto Y. Kitamura N. J. Biol. Chem. 1993; 268: 10024-10028Abstract Full Text PDF PubMed Google Scholar). The α-chain consists of an N-terminal domain (N) followed by four kringle modules and mediates the binding of HGF to the c-met receptor (13Hartmann G. Naldini L. Weidner K.M. Sachs M. Vigna E. Comoglio P.M. Birchmeier W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11574-11578Crossref PubMed Scopus (192) Google Scholar, 14Lokker N.A. Mark M.R. Luis E.A. Bennett G.L. Robbins K.A. Baker J.B. Godowski P.J. EMBO J. 1992; 11: 2503-2510Crossref PubMed Scopus (239) Google Scholar). Additional structure-function studies indicated that the N-terminal domain and first kringle (K1) are primarily involved in this interaction (15Lokker N.A. Godowski P.J. J. Biol. Chem. 1993; 268: 17145-17150Abstract Full Text PDF PubMed Google Scholar). The α-chain is also involved in the high affinity binding of HGF to heparin. Based on deletion analysis, the hairpin loop in the N-terminal domain and the second kringle (K2) were shown to be implicated in heparin binding (16Mizuno K. Inoue H. Hagiya M. Shimizu S. Nose T. Shimohigashi Y. Nakamura T. J. Biol. Chem. 1994; 269: 1131-1136Abstract Full Text PDF PubMed Google Scholar, 17Zhou H. Mazzulla M.J. Kaufman J.D. Stahl S.J. Wingfield P.T. Rubin J.S. Bottaro D.P. Byrd R.A. Structure. 1998; 6: 109-116Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Recently x-ray crystal structures were determined for the complexes of heparin with NK1, a naturally occurring HGF variant containing N domain and K1, which suggested that some heparin binding activity is also located within K1 (18Lietha D. Chirgadze D.Y. Mulloy B. Blundell T.L. Gherardi E. EMBO J. 2001; 20: 5543-5555Crossref PubMed Scopus (101) Google Scholar). Up to date, no discernible function has been demonstrated for the third (K3) and fourth (K4) kringles of HGF. The kringle domain is an 80-amino acid triple-loop structure maintained by three intramolecular disulfide bonds highly conserved between different kringle containing proteins (19Ikeo K. Takahashi K. Gojobori T. J. Mol. Evol. 1995; 40: 331-336Crossref PubMed Scopus (25) Google Scholar). Kringles are thought to play an important role in regulating the nature and strength of protein-protein interactions. Several reports have documented the anti-angiogenic activity of isolated kringles of different origin. The most known example is angiostatin, the three- to four-kringle containing fragment of human plasminogen (20O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W.S. Cao Y. Sage E.H. Folkman J. Cell. 1994; 79: 315-328Abstract Full Text PDF PubMed Scopus (3172) Google Scholar, 21Cao Y. Ji R.W. Davidson D. Schaller J. Marti D. Sohndel S. McCance S.G. O'Reilly M.S. Llinas M. Folkman J. J. Biol. Chem. 1996; 271: 29461-29467Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar), and plasminogen kringle 5 (22Cao Y. Chen A. An S.S. Ji R.W. Davidson D. Llinas M. J. Biol. Chem. 1997; 272: 22924-22928Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). More recently the prothrombin kringle-2 domain has been shown to inhibit endothelial cell growth and angiogenesis in the chorioallantoic membrane of chick embryos (23Lee T.H. Rhim T. Kim S.S. J. Biol. Chem. 1998; 273: 28805-28812Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar); a recombinant apolipoprotein (a) containing 18 kringle repeats has been implicated in the reduction of tumor microvessel density in transgenic mice (24Trieu V.N. Uckun F.M. Biochem. Biophys. Res. Commun. 1999; 257: 714-718Crossref PubMed Scopus (38) Google Scholar). The molecular mechanism by which the kringles suppress blood vessel formation is still unknown. We hypothesized that one or more of the kringles of HGF might inhibit angiogenesis and that the mechanism of the anti-angiogenic activity of a kringle domain could be related to its ability to antagonize the HGF binding to c-met. To verify this hypothesis, we produced each of the five HGF α-chain individual modules as recombinant proteins and analyzed their binding properties and biological activity in different assays related to angiogenesis. Materials—The bacterial expression vectors pET15b and pET21b(+), the Escherichia coli expression strain BL21(DE3), and tetracycline hydrochloride were purchased from Novagen (VWR International, Fontenay-sous-Bois, France). EZMix 2×YT microbial medium, carbenicillin, chloramphenicol, IPTG, non-fractionated heparin (catalog number H 3393) and low molecular weight heparin (average molecular weight 3000, catalog number H 3400), a peroxidase conjugated antibody against goat IgG, protein A from Staphylococcus aureus, and 0.2% gelatin were all purchased from Sigma (St. Louis, MO). Pwo DNA polymerase was purchased from Roche Diagnostics (Mannheim, Germany). TALON Superflow metal affinity resin was obtained from Clontech (Palo Alto, CA). The Ultra-free-4 and Ultrafree-15 Centrifugal Filter Device supplied with Bio-Max-5K filters and 0.22-μm Millex-GV filter units were obtained from Millipore (Bedford, MA). The BCA protein assay reagent, the IODO-GEN Pre-Coated Iodination Tubes, and EZ-Link biotin-LC-hydrazide were all obtained from Pierce (Perbio Sciences, Bezons, France). Nitrocellulose Hybond C Extra membranes, ECL Plus Western blotting detection reagent, Sephadex G-25M prepacked PD-10 columns, and [methyl-3H]thymidine (74 GBq/mmol) were all purchased from Amersham Biosciences (Buckinghamshire, UK). Iodine-125 (3.7 GBq/ml) was purchased from ICN Biomedicals (Costa Mesa, CA). All cell culture reagents were purchased from Invitrogen (Cergy Pontoise, France) unless otherwise specified. Normal human AB male serum (NHS) and fetal bovine serum (FBS) were obtained from BioWest (Cholet, France). NSO-expressed recombinant human HGF, recombinant human FGF-2, VEGF165, VEGF121, PDGF BB, c-met/Fc chimera (soluble c-met), and human HGF-specific polyclonal goat IgG were all purchased from R&D Systems (Minneapolis, MN). Type I rat tail collagen was purchased from BD Biosciences (Bedford, MA). Research grade CM5 sensor chips (carboxymethylated dextran matrix), SA sensor chips, amine coupling kit (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide), and surfactant P20 were all obtained from BIACORE (Uppsala, Sweden). Recombinant Protein Expression and Purification—The isolated HGF α-chain domains were produced using the pET-based bacterial expression system (Novagen). The DNA encoding different HGF polypeptides was generated by PCR using Pwo DNA polymerase and the pGEM/HGF-TM construct as a template (a generous gift of Dr. Pascale Briand at Institut Cochin de Génétique Moléculaire) (25Nguyen T.H. Pages J.C. Farge D. Briand P. Weber A. Hum. Gene Ther. 1998; 9: 2469-2479Crossref PubMed Google Scholar). This construct encodes for an HGF splice variant lacking a pentapeptide FLPSS in the first kringle domain (the amino acids from 162 to 166, according to the sequence deposited in the Swiss-Prot data base under accession number P14210) but which preserves all the biological activity of full-length variant (26Shima N. Tsuda E. Goto M. Yano K. Hayasaka H. Ueda M. Higashio K. Biochem. Biophys. Res. Commun. 1994; 200: 808-815Crossref PubMed Scopus (80) Google Scholar, 27Lokker N.A. Presta L.G. Godowski P.J. Protein Eng. 1994; 7: 895-903Crossref PubMed Scopus (61) Google Scholar). The primers used for PCR were: N forward, 5′-ATATCATATGCAAAGGAAAAGAAGAAATACAA-3′ and N reverse, 5′-ATATGGATCCTAGTTTCTAATGTAGTCTTTGTTTT-3′;K1 forward, 5′-ATATCATATGAACTGCATCATTGGTAAAGGA-3′ and K1 reverse, 5′-ATATGTCGACTTCTGAACACTGAGGAATGTC-3′; K2 forward, 5′-ATATGGATCCAGTTGAATGCATGACCTGCA-3′ and K2 reverse, 5′-ATATGTCGACGTCAGCGCATGTTTTAATTGC-3′; K3 forward, 5′-ATATGGATCCAACTGAATGCATCCAAGGTC-3′ and K3 reverse, 5′-ATATGTCGACCATATCACAGTTTGGAATTTG-3′; and K4 forward, 5′-ATATGGATCCAGATTGTTATCGTGGGAATGG-3′ and K4 reverse, 5′-ATATGTCGACACCTTCACAACGAGAAATAGG-3′. The purified PCR fragment encoding the HGF N-terminal domain (amino acids from 32 to 127) was cloned into NdeI and BamHI sites of pET15b, in-frame with N-terminal His Tag. The DNA for K1 (amino acids from 127 to 208) was cloned into NdeI and SalI sites of pET21b(+), in-frame with C-terminal His Tag, and the DNA for K2 (amino acids from 209 to 290), K3 (amino acids from 303 to 385), and K4 (amino acids from 390 to 471) were all cloned into BamHI and SalI sites of pET21b(+), inframe with N-terminal T7 Tag and C-terminal His Tag. All PCR-generated constructs were checked for polymerase fidelity by sequencing (Génome Express, Grenoble, France). All plasmids encoding HGF kringles, or the plasmid encoding the HGF N domain were electroporated, respectively, into E. coli strain BL21(DE3), or into E. coli BL21(DE3) based strain pRI (kindly provided by Dr. Janne L. Simonsen at Arhus University) carrying two additional plasmids: one encoding a tRNAIle specific for the rare isoleucine codon AUA and another for the LacZ repressor RP4. Bacteria were grown at 30 °C in EZMix 2× YT medium containing 1 m glucose and appropriate antibiotics. Protein expression was induced with 1 mm IPTG for 4 h at 25 °C. Bacterial cells were harvested by centrifugation and stored at –20 °C. Bacterial pellets (5 g wet weight) were resuspended in 100 ml of lysis buffer (50 mm Tris-HCl, pH 7.4, containing 0.2 m NaCl, 10 mm imidazole, 10% glycerol, 1% Nonidet P-40, 2 mm β-mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride, and 0.2 mg/ml lysozyme) and incubated for 20 min at room temperature with rotation. The suspensions were sonicated and centrifuged at 10,000 × g for 30 min. All HGF kringles were recovered in the supernatants and purified by cobalt-chelate affinity chromatography on a 2-ml TALON column under non-denaturing conditions. The pellet containing insoluble N was resuspended in 30 ml of isolation buffer (20 mm Tris-HCl, pH 8, containing 0.5 m NaCl, 2 m urea, and 2% Triton X-100) and subjected to three successive rounds of brief sonication, centrifugation (10,000 × g for 10 min), and resuspension. The supernatants from all three steps were pooled and applied to a TALON column equilibrated with isolation buffer. To achieve the N domain renaturation, the column was washed with a 2 to 0 m gradient of urea in IMAC buffer (20 mm Tris-HCl, pH 8, and 0.5 m NaCl) containing 10 mm imidazole. Renatured N was eluted by IMAC buffer containing 0.5 m imidazole. Further steps were all performed at 4 °C. Selected column fractions corresponding to the peaks of purified N, K1, K2, K3, or K4 were pooled and diluted with 10 volumes of refolding buffer (PBS containing 10% glycerol, 1 mm reduced glutathione, and 0.2 mm oxidized glutathione). Following an overnight incubation with stirring, the protein solutions were concentrated using Ultrafree-15 Centrifugal Filter Devices and dialyzed against PBS containing 10% glycerol for 2 days with at least three changes of buffer. The final protein preparations were sterilized using 0.22-μm Millex-GV filter units and stored at –80 °C until required. The protein concentration was determined using the BCA protein assay. The purified recombinant HGF-derived proteins were analyzed by SDS-PAGE and by circular dichroism as described previously (28Bulteau A.L. Verbeke P. Petropoulos I. Chaffotte A.F. Friguet B. J. Biol. Chem. 2001; 276: 45662-45668Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Cells—Human umbilical vein endothelial cells (HUVECs) were isolated by collagenase (Roche Diagnostics) digestion (29Jaffe E.A. Nachman R.L. Becker C.G. Minick C.R. J. Clin. Invest. 1973; 52: 2745-2756Crossref PubMed Scopus (6011) Google Scholar). Cells were routinely grown in flasks coated with gelatin, in M199 medium containing 2 mm glutamine, 50 units/ml penicillin, 50 μg/ml streptomycin, 2.5 μg/ml amphotericin B, 7.5% NHS, and 7.5% FBS (regular medium). HUVECs from the second or third passage were used. The human colorectal carcinoma cell line DLD-1, the human osteogenic sarcoma cell line TE 85, and the human pancreatic adenocarcinoma cell line Capan-1 were obtained from the American Type Culture Collection and were cultured in RPMI supplemented with 10% FBS. Human aortic smooth muscle cells (hASMCs) and the corresponding growth medium (SmGM-2), and the human normal epidermal keratinocytes (NHEK) and the corresponding growth medium (KGM) were purchased from Clonetics (BioWhittaker France). Proliferation Assay—Cells that had been grown until confluence were harvested by trypsinization, and their proliferation was measured by use of a [3H]thymidine incorporation assay. Cells were plated in 24-well plates: HUVECs at a density of 2 × 104 cells/well in M199 medium supplemented with 2.5% FBS, hASMCs at a density of 2 × 104 cells/well in SmGM-2 supplemented with 1% FBS, NHEKs at a density of 104 cells/well in KGM supplemented with 3 mg/ml AlbuMAX (Invitrogen), TE 85 cells at a density 5 × 103 cells/well, DLD-1 and Capan-1 cells at a density of 104 cells/well in RPMI supplemented with 3 mg/ml AlbuMAX. Cells were allowed to attach for 4 h at 37 °C, and proliferation was induced by adding 10% FBS or 10 ng/ml of FGF-2, VEGF165, HGF, or PDGF BB. The rising concentrations (10–5000 nm) of purified recombinant HGF-derived proteins were added to some wells, and HUVECs were incubated for a further 24 h. [3H]Thymidine (1 μCi/well) was added during the last 18 h of incubation. Cells were washed three times with PBS and treated with ice-cold 10% (w/v) trichloroacetic acid for 30 min. The resulting precipitates were solubilized with 0.3 n NaOH (0.5 ml/well), and the incorporated radioactivity was measured in a LS-6500 multipurpose scintillation counter (Beckman Coulter, Fullerton, CA). Migration Assay—HUVEC migration was evaluated in a modified Boyden chamber assay. Transwell cell culture chamber inserts (BD Biosciences) with porous polycarbonate filters (8-μm pore size) were coated with 0.2% gelatin in PBS. HUVECs suspended in M199 medium supplemented with 2.5% FBS were added to the inserts at 4 × 104 cells per well. The inserts were placed over chambers containing a chemotactic stimulus (10 ng/ml HGF, FGF-2, or VEGF165), and cells were allowed to migrate for 5 h at 37 °C in a CO2 incubator. For inhibition experiments one of the purified HGF α-chain-derived polypeptides (each at 1 μm) was added to the lower chamber. The filters were then rinsed with PBS, fixed with 1% (w/v) paraformaldehyde, and stained with hematoxylin of Harris. The upper surface of the filters was scraped with a cotton swab to remove the non-migrant cells. The number of cells per high power field (×200) was recorded. Each experimental point was performed in triplicate, and 10 fields per filter were analyzed. "In Gel" Three-dimensional Collagen Culture—Type I rat tail collagen composed gels were formed according to the manufacturer's recommendations. Briefly, an ice-cold collagen solution was neutralized by addition of 1 n NaOH and mixed with 10× PBS in the ratio 9:1. HUVECs (passage 2) isolated by trypsin-EDTA treatment and suspended in the M199 medium supplemented with 2.5% FBS, were added at a final concentration of 5 × 105 cells/ml in a 1 mg/ml collagen solution. 0.4 ml of this suspension was poured into wells of a 24-well plate (Nunc, Roskilde, Denmark), and the gels were allowed to form at 37 °C for 40 min. The gels were then overlaid with 0.5 ml of M199 medium supplemented with 2.5% fetal bovine serum and 10 ng/ml of the growth factors. When indicated, purified recombinant HGF domains (1 μm) were added at the same time as the growth factors to the overlaying medium. Cultures were incubated for 48 h at 37 °C in a humidified atmosphere containing 5% CO2. Digital images were captured using a Kappa CF11DSP charge-coupled device camera (KAPPA opto-electronics GmbH, Gleichen, Germany). Radiolabeled Ligand Binding and Displacement Experiments—HGF (5 μg), VEGF165 (10 μg), FGF-2 (10 μg), or recombinant HGF N-terminal domain (25 μg) were iodinated with 1 mCi of [125I]NaI using the IODO-GEN Pre-Coated Iodination Tubes according to the manufacturer's instructions. Iodinated proteins were purified by chromatography on PD-10 columns equilibrated with 25 mm Tris-HCl, pH 7.4, containing 0.4 m NaCl, 5 mm EDTA, and 0.25% AlbuMAX and concentrated using the Ultrafree-4 Centrifugal Filter Device. The specific activities determined by trichloroacetic acid precipitation were 18 × 106 cpm/pmol for 125I-HGF, 8 × 106 cpm/pmol for 125I-VEGF165, 9 × 105 cpm/pmol for 125I-FGF-2, and 5 × 105 cpm/pmol for 125I-N. Binding and displacement studies with the iodinated proteins were carried out on confluent HUVECs in 24-well dishes, at 4 °C. Cells were washed with PBS and preincubated for 30 min with 0.5 ml of binding medium (M199 medium containing 0.1% AlbuMAX). To determine the binding parameters for N, increasing amounts of radiolabeled N were added to HUVECs, with or without a 100-fold molar excess of unlabeled N, and incubated for 6 h. For displacement experiments, constant amounts of radiolabeled HGF (1.5 nm), VEGF165 (1.2 nm), or FGF-2 (1 nm) were added to HUVECs in the presence of increasing concentrations of unlabeled N for 4 h. At the end of incubation, cells were washed three times with binding medium and solubilized with 0.3 n NaOH. HUVEC-associated radioactivity was determined in a γ-counter, and the results were analyzed using the program Ligand included in the KELL package (Biosoft, Cambridge, UK). All experiments were done in triplicate. The Kd values represent the means ± S.D. obtained from three or four experiments. In Vitro Binding Studies by Surface Plasmon Resonance—The binding studies were performed on a BIACORE 2000 instrument. The analytes were routinely diluted in BIACORE running buffer (PBS, pH 7.5, containing 0.005% P-20) completed with 0.2 mg/ml BSA, and injected at 20 μl/min. Between binding cycles the coated surfaces were regenerated by two injections of 1 m NaCl. In each case the first flow channel of the sensor chip did not contain any immobilized ligand and served as a reference surface. In one set of experiments, a non-fractionated heparin (NF heparin) was biotinylated with EZ-Link Biotin-LC-Hydrazide as described in a previous study (30Yu Q. Toole B.P. BioTechniques. 1995; 19: 122-129PubMed Google Scholar) and about 600 RU were captured on the surface of streptavidin-coated SA sensor chips. The c-met/Fc chimera was captured on the surface of a CM5 sensor chip with immobilized protein A (600 RU). HGF and purified HGF domains were injected over immobilized heparin and c-met. In a second set of experiments, HGF and its domains were immobilized on the CM5 sensor chip surface (∼1000 RU) using the amine coupling kit according to the manufacturer's instructions. NF heparin, low molecular weight heparin (LMW heparin), and soluble c-met, were used as analytes. Binding curves were obtained over a range of analyte concentrations for each of the analyte/ligand pairs. The data were analyzed with the non-linear least squares algorithm implemented in the BIAevaluation 3.1 software package. Statistics—Results were expressed as mean ± S.E. Statistical significance was evaluated by analysis of variance followed by Bonferroni/Dunn analysis. Production and Purification of Recombinant HGF α-Chain Structural Modules—The HGF kringles were all expressed in E. coli in a soluble form. Recombinant N-terminal domain was mainly accumulated in insoluble inclusion bodies and was easily solubilized with 2 m urea containing 2% Triton X-100. The proteins were purified to homogeneity using a simple one-step procedure on TALON metal affinity column (Fig. 1). They were subsequently subjected to oxidative folding using the glutathione-based oxido-shuffling system. The final protein preparations were analyzed in SDS-PAGE under reducing and non-reducing conditions to ensure the presence of disulfide bonds (Fig. 1). We observed very little or no difference in the mobility of the non-reduced and reduced N, although this protein possesses two disulfide bonds. In contrast, the non-reduced K1 exhibited a substantial mobility shift compared with the reduced sample. Similar results had been previously reported for N and K1 produced in E. coli (31Stahl S.J. Wingfield P.T. Kaufman J.D. Pannell L.K. Cioce V. Sakata H. Taylor W.G. Rubin J.S. Bottaro D.P. Biochem. J. 1997; 326: 763-772Crossref PubMed Scopus (48) Google Scholar). K2, K3, and K4 also demonstrated an increased mobility under non-reducing conditions. These results indicate that our HGF kringle preparations have a compact conformation under non-reducing conditions and that the disulfide bonds were formed during the refolding procedure. The circular dichroism analysis of N, K1, K2, K3, and K4 demonstrated that these proteins were folded. The far-UV CD spectra (180–260 nm) of N, K1, K2, K3, and K4 are all indicative of a high content in secondary structure. A broad band of positive ellipticity can be seen in the 230- to 260-nm region: it most probably originates from oxidized disulfides and aromatic residues involved in the characteristic hydrophobic core of the kringle-type folding. The near-UV CD spectra (240–340 nm) show bands in the 275- to 290-nm region, which are characteristic of an asymmetric orientation of aromatic residues (Trp and Tyr). These signals are typical for the folded tertiary structure of globular proteins (data not shown). Effect of Isolated HGF Domains on Cell Proliferation—Angiogenic response involves a surge in endothelial cell proliferation, which could be induced by several growth factors (32Kumar R. Yoneda J. Bucana C.D. Fidler I.J. Int. J. Oncol. 1998; 12: 749-757PubMed Google Scholar). Under our experimental conditions, the highest capacity of induction of HUVEC proliferation was demonstrated for FGF-2, whereas HGF was a very poor mitogen (about 750% stimulation against 25%; Fig. 2, A and B, respectively). Therefore, we first analyzed the effect of isolated HGF domains on the proliferation of HUVECs stimulated with 10 ng/ml FGF-2 (Fig. 2A). In these experiments none of HGF-derived kringles exhibited any activity, whereas N inhibited HUVEC proliferation in a concentration-dependent manner. The influence of N on HUVEC proliferation could be detected at a concentration of 100 nm (about 10% of inhibition). Depending on the umbilical cord specimen, a maximum inhibition effect (up to 100%) was observed for a concentration of N of 1–5 μm. Because our hypothesis was that kringles could modulate angiogenesis by competitive binding to the c-met receptor, we also studied the effect of recombinant HGF domains on the proliferation of HUVECs induced by HGF. The kringles of HGF had no effect on the proliferation of HUVECs in the presence of HGF (Fig. 2B). In contrast, N significantly inhibited the proliferation of HUVECs induced not only by HGF but also by two isoforms of VEGF: VEGF165 and VEGF121 (Fig. 2C). Thus, the anti-proliferative effect of N was not specific toward HGF. We further tested the specificity of the anti-proliferative effect of N toward the endothelial cell type. In the prel
Referência(s)