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• W2072305777 abstract "We have previously identified angiomotin by its ability to bind to and mediate the anti-angiogenic properties of angiostatin. In vivo and in vitro data indicate an essential role of angiomotin in endothelial cell motility. Here we show that angiostatin binds angiomotin on the cell surface and provide evidence for a transmembrane model for the topology of both p80 and p130 angiomotin isoforms. Immunofluorescence analysis shows that angiomotin co-localized with ZO-1 in cell-cell contacts in endothelial cells in vitro and in angiogenic blood vessels of the postnatal mouse retina in vivo. Transfection of p80 as well as p130 angiomotin in Chinese hamster ovary cells resulted in junctional localization of both isoforms. Furthermore, p130 angiomotin could recruit ZO-1 to actin stress fibers. The p130 but not p80 isoform could be coprecipitated with MAGI-1b, a component of endothelial tight junctions. Paracellular permeability, as measured by diffusion of fluorescein isothiocyanate-dextran, was reduced by p80 and p130 angiomotin expression with 70 and 88%, respectively, compared with control. Angiostatin did not have any effect on cell permeability but inhibited the migration of angiomotin-expressing cells in the Boyden chamber assay. We conclude that angiomotin, in addition to controlling cell motility, may play a role in the assembly of endothelial cell-cell junctions. We have previously identified angiomotin by its ability to bind to and mediate the anti-angiogenic properties of angiostatin. In vivo and in vitro data indicate an essential role of angiomotin in endothelial cell motility. Here we show that angiostatin binds angiomotin on the cell surface and provide evidence for a transmembrane model for the topology of both p80 and p130 angiomotin isoforms. Immunofluorescence analysis shows that angiomotin co-localized with ZO-1 in cell-cell contacts in endothelial cells in vitro and in angiogenic blood vessels of the postnatal mouse retina in vivo. Transfection of p80 as well as p130 angiomotin in Chinese hamster ovary cells resulted in junctional localization of both isoforms. Furthermore, p130 angiomotin could recruit ZO-1 to actin stress fibers. The p130 but not p80 isoform could be coprecipitated with MAGI-1b, a component of endothelial tight junctions. Paracellular permeability, as measured by diffusion of fluorescein isothiocyanate-dextran, was reduced by p80 and p130 angiomotin expression with 70 and 88%, respectively, compared with control. Angiostatin did not have any effect on cell permeability but inhibited the migration of angiomotin-expressing cells in the Boyden chamber assay. We conclude that angiomotin, in addition to controlling cell motility, may play a role in the assembly of endothelial cell-cell junctions. Angiogenesis, the formation of novel blood vessels by a process of sprouting or intussusceptive growth, has attracted considerable interest since it was shown to be crucial for tumor progression (1Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7215) Google Scholar). Sprouting angiogenesis involves several steps: proliferation of endothelial cells, modification of the extracellular matrix, cell migration, and tube morphogenesis. The balance between specific growth factors and inhibitors controls this process (2Hanahan D. Folkman J. Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6086) Google Scholar). One of the identified inhibitors is angiostatin, a 38-kDa proteolytic fragment of plasminogen, which has been shown to potently inhibit angiogenesis and the growth of metastases in mice (3O'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 (3170) Google Scholar). In vitro studies have shown that angiostatin can inhibit endothelial cell proliferation (3O'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 (3170) Google Scholar), migration (4Ji W.R. Castellino F.J. Chang Y. Deford M.E. Gray H. Villarreal X. Kondri M.E. Marti D.N. Llinas M. Schaller J. Kramer R.A. Trail P.A. FASEB J. 1998; 12: 1731-1738Crossref PubMed Scopus (123) Google Scholar), and also induce apoptosis (5Claesson-Welsh L. Welsh M. Ito N. Anand-Apte B. Soker S. Zetter B. O'Reilly M. Folkman J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5579-5583Crossref PubMed Scopus (314) Google Scholar, 6Lucas R. Holmgren L. Garcia I. Jimenez B. Mandriota S.J. Borlat F. Sim B.K. Wu Z. Grau G.E. Shing Y. Soff G.A. Bouck N. Pepper M.S. Blood. 1998; 92: 4730-4741Crossref PubMed Google Scholar). However, angiostatin has a short half-life in vivo and is hard to produce in sufficient amounts, which makes it difficult to utilize as a therapeutic agent (7Bouquet C. Frau E. Opolon P. Connault E. Abitbol M. Griscelli F. Yeh P. Perricaudet M. Mol. Ther. 2003; 7: 174-184Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). We have identified angiomotin by its ability to bind angiostatin in a yeast two-hybrid screen. p80 angiomotin is a protein of 675 residues that is expressed in human endothelium and is a member of a conserved family of proteins that comprises two other proteins in human: angiomotin-like 1/JEAP and angiomotin-like 2 (8Bratt A. Wilson W.J. Troyanovsky B. Aase K. Kessler R. Van Meir E.G. Holmgren L. Gene (Amst.). 2002; 298: 69-77Crossref PubMed Scopus (111) Google Scholar). Recent data suggest transcriptional diversity within this protein family (9Moreau J. Lord M. Boucher M. Belleau P. Fernandes M.J. Gene. 2005; 350: 137-148Crossref PubMed Scopus (13) Google Scholar). The effects of angiomotin on transfected endothelial cells are stimulatory: the cells become more motile and invasive as determined by in vivo and in vitro assays. However, this effect can be blocked by angiostatin (10Troyanovsky B. Levchenko T. Mansson G. Matvijenko O. Holmgren L. J. Cell Biol. 2001; 152: 1247-1254Crossref PubMed Scopus (299) Google Scholar, 11Levchenko T. Aase K. Troyanovsky B. Bratt A. Holmgren L. J. Cell Sci. 2003; 116: 3803-3810Crossref PubMed Scopus (33) Google Scholar, 12Levchenko T. Bratt A. Arbiser J.L. Holmgren L. Oncogene. 2004; 23: 1469-1473Crossref PubMed Scopus (41) Google Scholar). Thus, angiostatin blocks tube formation of angiomotin-transfected cells in the Matrigel tube formation assay as well as migration of angiomotin-transfected cells in the Boyden chamber assay. Also, angiomotin-deficient mouse embryos exhibit impaired migration of the visceral endoderm (13Shimono A. Behringer R.R. Curr. Biol. 2003; 13: 613-617Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). These data indicate that angiomotin may act as a promoter of angiogenesis by enhancing cell motility and migration and that angiostatin is an antagonist of angiomotin. The mechanism by which angiomotin promotes cell motility has not been defined, but in vivo and in vitro evidence shows that the C-terminal PDZ-binding motif is crucial (11Levchenko T. Aase K. Troyanovsky B. Bratt A. Holmgren L. J. Cell Sci. 2003; 116: 3803-3810Crossref PubMed Scopus (33) Google Scholar). p80 angiomotin localizes to lamellipodia of migrating cells (10Troyanovsky B. Levchenko T. Mansson G. Matvijenko O. Holmgren L. J. Cell Biol. 2001; 152: 1247-1254Crossref PubMed Scopus (299) Google Scholar), and angiomotin promotes cell spreading on several different matrices, which indicates that angiomotin can control organization of the actin cytoskeleton. 3A. Bratt, S. Narumiya, and L. Holmgren, manuscript in preparation. Angiomotin also has the ability to stabilize endothelial tubes in the Matrigel in vitro angiogenesis assay (12Levchenko T. Bratt A. Arbiser J.L. Holmgren L. Oncogene. 2004; 23: 1469-1473Crossref PubMed Scopus (41) Google Scholar). Apart from angiomotin, other receptors for angiostatin have been identified, for example ATP synthase (14Moser T.L. Kenan D.J. Ashley T.A. Roy J.A. Goodman M.D. Misra U.K. Cheek D.J. Pizzo S.V. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6656-6661Crossref PubMed Scopus (298) Google Scholar), the integrin αVβ3 (15Tarui T. Miles L.A. Takada Y. J. Biol. Chem. 2001; 276: 39562-39568Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), and the hepatocyte growth factor receptor c-met (16Wajih N. Sane D.C. Blood. 2003; 101: 1857-1863Crossref PubMed Scopus (55) Google Scholar). The exact mechanism by which angiostatin down-regulates neovascularization remains to be determined. Recently, we identified a splice form of angiomotin, p130 angiomotin. 4M. Ernkvist, K. Aase, C. Ukomadu, Y. Zhou, J. Wohlshlegel, R. Blackman, N. Veitonmäki, A. Bratt, S. Fisher, A. Dutta, and L. Holmgren, submitted for publication. This protein differs from p80 angiomotin in that it has an N-terminal extension of 409 amino acids, which mediates the binding of angiomotin to actin stress fibers. Transfection of p130 angiomotin into mouse aortic endothelial (MAE) 5The abbreviations used are: MAE, mouse aortic endothelial cell; ABD, angiostatin binding domain; Amot, angiomotin; TJ, tight junction; AJ, adherens junction; BCE, bovine capillary endothelial cell; bFGF, basic fibroblast growth factor; CHO, Chinese hamster ovary cell; ER, endoplasmic reticulum; HRP, horseradish peroxidase; JAM, junctional adhesion molecule; JEAP, junction-enriched and -associated protein; MAGI, membrane-associated guanylate kinase with inverted domain; P, postnatal day; PBS, phosphate-buffered saline; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate. cells results in increased average cell size and pl60ROCK-dependant stress fiber formation. 4M. Ernkvist, K. Aase, C. Ukomadu, Y. Zhou, J. Wohlshlegel, R. Blackman, N. Veitonmäki, A. Bratt, S. Fisher, A. Dutta, and L. Holmgren, submitted for publication. The formation of mature cell-cell contacts is a crucial step during angiogenesis. Silencing of endothelial adherence junction protein VE-cadherin leads to abnormal tight junctions, malformed vessels, and hemorrhages, and embryos die in utero within 9.5 days from fertilization (18Carmeliet P. Lampugnani M.G. Moons L. Breviario F. Compernolle V. Bono F. Balconi G. Spagnuolo R. Oostuyse B. Dewerchin M. Zanetti A. Angellilo A. Mattot V. Nuyens D. Lutgens E. Clotman F. de Ruiter M.C. Gittenberger-de Groot A. Poelmann R. Lupu F. Herbert J.M. Collen D. Dejana E. Cell. 1999; 98: 147-157Abstract Full Text Full Text PDF PubMed Scopus (1057) Google Scholar). Endothelial tight junctions (TJ) form a seal between cells that isolates the lumen of the blood vessel from the surrounding tissue and restricts the diffusion of solutes from the blood to the surrounding cells. At the molecular level, TJs are formed by homodimerization of the specific tight junction transmembrane proteins occludin, claudin, and JAM. The cytoplasmic domains of these proteins are connected to the actin cytoskeleton through a number of adaptor proteins, such as ZO-1/2/3 and MAGI-1/2/3 (19Dejana E. Nat. Rev. Mol. Cell. Biol. 2004; 5: 261-270Crossref PubMed Scopus (903) Google Scholar). Interestingly, angiomotin-like 1/JEAP is reported to localize to TJs (20Nishimura M. Kakizaki M. Ono Y. Morimoto K. Takeuchi M. Inoue Y. Imai T. Takai Y. J. Biol. Chem. 2002; 277: 5583-5587Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). TJs are structurally important for the cell and are linked to the cytoskeleton both physically and through signaling pathways. For example, the Rho family of GTPases controls both reorganization of the actin cytoskeleton and formation of TJs (21Braga V.M. Curr. Opin. Cell Biol. 2002; 14: 546-556Crossref PubMed Scopus (277) Google Scholar, 22Burridge K. Wennerberg K. Cell. 2004; 116: 167-179Abstract Full Text Full Text PDF PubMed Scopus (1515) Google Scholar, 23Chen X. Macara I.G. Nat. Cell Biol. 2005; 7: 262-269Crossref PubMed Scopus (365) Google Scholar, 24Ozdamar B. Bose R. Barrios-Rodiles M. Wang H.R. Zhang Y. Wrana J.L. Science. 2005; 307: 1603-1609Crossref PubMed Scopus (728) Google Scholar). In epithelial cells, another type of specialized junction, the adherens junction, can be distinguished. It is thought that the main purpose of AJs is to confer adhesion between cells to maintain tissue architecture. AJs are formed by homodimerization of cadherins, with VE-cadherin being the endothelial-specific cadherin. In endothelial cells, it is difficult to distinguish between TJs and AJs, because the two types of structures occur intermingled (25Schulze C. Firth J.A. J. Cell Sci. 1993; 104: 773-782Crossref PubMed Google Scholar, 26Ruffer C. Strey A. Janning A. Kim K.S. Gerke V. Biochemistry. 2004; 43: 5360-5369Crossref PubMed Scopus (43) Google Scholar). Here we show that p80 and p130 angiomotin are membrane proteins involved in control of permeability in cell-cell junctions but that the effect of angiostatin is limited to inhibiting migration of angiomotin-expressing cells. Cell Culture—MAE cells stably expressing p80 and p130 angiomotin (10Troyanovsky B. Levchenko T. Mansson G. Matvijenko O. Holmgren L. J. Cell Biol. 2001; 152: 1247-1254Crossref PubMed Scopus (299) Google Scholar) were cultured in Dulbecco's modified Eagle's medium (Sigma). Chinese hamster ovary (CHO) cells were maintained in Dulbecco's modified Eagle's medium with Ham's F-12 nutrient mixture. CHO cells stably expressing p80 or p130 angiomotin were generated by transfecting CHO cells with pcDNA3 with an insert of either p80 angiomotin or p130 angiomotin using Lipofectamine 2000 (Invitrogen) and selecting clones with G418 at 0.4 mg/ml. Bovine capillary endothelial (BCE) cells were cultured in Dulbecco's modified Eagle's medium supplemented with 2 ng bFGF/ml. All cell culture media were supplemented with 10% fetal calf serum (Invitrogen), 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mm l-glutamine. Plasmid Construction—The cDNA for p80-angiomotin and p130-angiomotin were subcloned into the pENTR-2B vector (Gateway, Invitrogen) and then recombined into the converted Gateway destination vector pcDNA3 (Invitrogen) using the LR recombination reaction (Gateway, Invitrogen). FLAG-tagged MAGI-1b and MAGI-1c were as described (27Dobrosotskaya I.Y. James G.L. Biochem. Biophys. Res. Commun. 2000; 270: 903-909Crossref PubMed Scopus (139) Google Scholar). Antibodies—Three different polyclonal antibodies against the following domains of angiomotin were used: the angiostatin binding domain (B3 antibody) (10Troyanovsky B. Levchenko T. Mansson G. Matvijenko O. Holmgren L. J. Cell Biol. 2001; 152: 1247-1254Crossref PubMed Scopus (299) Google Scholar), the C-terminal (TLE antibody), and N-terminal antibodies of p130 angiomotin 4M. Ernkvist, K. Aase, C. Ukomadu, Y. Zhou, J. Wohlshlegel, R. Blackman, N. Veitonmäki, A. Bratt, S. Fisher, A. Dutta, and L. Holmgren, submitted for publication. (Fig. 3A). The following mouse monoclonal antibodies were used: 9E10 anti-Myc tag (Santa Cruz Biotechnology), M2 anti-FLAG tag (Sigma), AC-15 anti-actin (Sigma), 1A12 anti-ZO-1 (Zymed Laboratories Inc.), C060 anti-caveolin (BD Transduction Laboratories), 349 anti-paxillin (BD Transduction Laboratories), and OC-3F10 anti-occludin (BD Transduction Laboratories). Also rat monoclonal Mec 13.3 anti-CD31/PECAM (BD Pharmingen) and rabbit anti plasminogen (DAKO) were used. Angiostatin—Angiostatin was generated by elastase degradation of plasminogen as previously described (3O'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 (3170) Google Scholar). Western Blot—Proteins were separated on 7.5% Criterion SDS-PAGE gels (Bio-Rad) and transferred to Protran nitrocellulose membranes by semi-dry blotting. Membranes were blocked by incubation with PBS with 5% milk and incubated with primary antibody at 4 °C overnight followed by incubation with HRP-donkey anti-rabbit or HRP-sheep anti mouse (Amersham Biosciences) for 1 h at room temperature. The filters were washed several times in PBS plus 0.05% Tween, and the signal was visualized with Western blotting Luminol Reagent (Santa Cruz Biotechnology). Biotinylation Experiments—∼10 million confluent cells were briefly rinsed twice in PBS and incubated with NHS-Sulfo-LC-Biotin (Pierce, 0.4 mg/ml) in PBS or NHS-LC-biotin (0.4 mg/ml) in Me2SO for 30 min at room temperature. The plates were then rinsed with PBS. Control plates were incubated with PBS alone. One milliliter of lysis buffer (20 mm HEPES, 140 mm KCl, 5 mm MgCl, 10 mm β-glycerophosphate, 3% polyethylene-9-lauryl ether (Thesit), and protease inhibitor mixture, pH 7.4) was added, and the cells were harvested using a rubber policeman. Lysates were spun at 30,000 × g for 25 min, and the supernatants were subjected to immunoprecipitation by incubation with 1 μg of either B3 angiomotin antibody or paxillin antibody and 30 μl of protein G-Sepharose slurry (Pierce). The beads were washed three times in lysis buffer with 1% Thesit. Proteins were eluted with 30 μl of Laemmli buffer, and half of the material was loaded onto a 10% precast Criterion gel (Bio-Rad) and blotted onto a nitrocellulose membrane. Biotinylated proteins were detected with HRP-conjugated streptavidin (Pierce). Trypsin Treatment—Confluent cells grown on 6-cm Petri dishes were washed twice with calcium- and magnesium-free PBS and incubated with 1 ml of sequence grade trypsin (Sigma) at 2 μg/ml or PBS alone at 37 °C for the indicated times. At 80 min a sample of cells was examined for integrity of the membrane using trypan blue, and it was found that 90% of cells had intact cell membranes. The experiment was ended by washing the cells once in PBS and adding 75 μl of Laemmli buffer. Samples were analyzed by Western blot. Triton X-114 Phase Separation—This step was performed as described previously (28Bonifacino J.S. Dasso M. Harford J.B. Lippincott-Schwartz J. Yamada K.M. Current Protocols in Cell Biology. John Wiley & Sons, Inc., NY2005Google Scholar). Angiostatin Binding Assay—Human angiostatin (kringles 1-4) was labeled with 125I by the Iodogen method according to the protocol of the manufacturer (Pierce). The specific activity was estimated at 15,000 cpm/ng of protein. For binding assays HeLa cells stably expressing p80 angiomotin or empty vector were grown to confluency in 12-well plates. The cells were washed with PBS containing 1 mg/ml BSA and were incubated with 10 ng/ml radiolabeled angiostatin for 2 h. Cells were then washed five times with PBS with 1 mg/ml BSA and lysed with 1% Triton X-100 in PBS, and radioactivity was measured in a gamma counter. Cross-linking Experiments—Confluent MAE cells grown on 15-cm plates were incubated with angiostatin (5 μg/ml) in PBS with 0.05% BSA for 100 min at 4 °C when cross-linkers BS 3A. Bratt, S. Narumiya, and L. Holmgren, manuscript in preparation. and Sulfo-EGS (Pierce) were added to a final concentration of 2.5 mm for each. The cells were then incubated for additional 2 h at 4 °C. Cells were washed and lysed in 0.5% Triton X-100, 150 mm NaCl, 50 mm Tris, pH 7.5, and protease inhibitors and centrifuged at 14,000 rpm for 15 min at 4 °C. Immunoprecipitation was carried out from the supernatant with plasminogen antibody bound to protein A beads (Sigma). In Vitro Binding Assay—His-tagged angiostatin binding domain and p80 angiomotin recombinant proteins (described in Ref. 10Troyanovsky B. Levchenko T. Mansson G. Matvijenko O. Holmgren L. J. Cell Biol. 2001; 152: 1247-1254Crossref PubMed Scopus (299) Google Scholar) were supplied by Bioinvent International AB, Lund. His-tagged Endostatin was used as negative control (kindly provided by Thomas Boehm, Children's Hospital, Boston). 2 μg of His-tagged proteins was coupled to nickel-nitrilotriacetic acid-agarose beads (Qiagen) in binding buffer (300 mm NaCl, 50 mm Na2HPO4, and 0.05% Tween) at 4 °C for 2 h. Coupled beads were washed four times in binding buffer, and unspecific binding sites were blocked in binding buffer containing 1% milk. Kringle 4, K1-3, and plasminogen proteins were kindly provided by American Diagnostica, Greenwich, CT. Equimolar amounts of plasminogen and the different kringle fragments were added to the beads and incubated for 3 h at 4 °C. The beads were washed extensively in binding buffer. The beads were resuspended in Laemmli sample buffer and boiled for 5 min. Proteins were resolved by SDS-PAGE using a 12.5% gel, transferred electrophoretically, and visualized using electrochemiluminescence (ECL) using rabbit polyclonal antibodies against plasminogen. Immunofluorescence—Cells plated on chamber slides (Falcon) were rinsed briefly in PBS, fixed in 4% paraformaldehyde for 10 min, and (if not stated otherwise) treated with 0.05% Triton X-100 for 30 s. Cells were then incubated with 5% horse serum for 60 min, incubated with primary antibody diluted in 5% horse serum for 1 h, washed four times in PBS, and incubated with Texas red horse anti-mouse (Vector Laboratories Inc.) or FITC swine anti-rabbit (DAKO) diluted in 5% horse serum for 1 h. F-actin was visualized with Texas red phalloidin (Molecular Probes). For immunofluorescence of mouse retinas, eyes from C57BL6 mice sacrificed at P5 were fixed in 4% paraformaldehyde/PBS at 4 °C for 2-3 h and washed in PBS. Retinas were dissected as previously described (29Chan-Ling T.L. Halasz P. Stone J. Curr. Eye Res. 1990; 9: 459-478Crossref PubMed Scopus (105) Google Scholar) and incubated for 2 h at room temperature in a permeabilization/blocking buffer (PBB, PBS containing 1% BSA, 0.5% Triton X-100, and 5% normal goat serum). Retinas were then incubated at 4 °C overnight with primary antibodies diluted in PBB buffer. After six washes with PBS at room temperature, retinas were incubated 2 h at room temperature in darkness with secondary antibody diluted in PBS plus 0.5% BSA, 0.25% Triton X-100, and 5% normal goat serum. The secondary antibodies were FITC-conjugated swine anti-rabbit (Dako), Alexa Fluor 594-goat anti-mouse (Molecular Probes), and R-phycoerythrin-goat anti-rat IgG mouse (Southern Biotechnology Associates). All specimens were flat-mounted in Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories Inc.). Pictures were captured on a Ziess Axioplan 2 microscope and processed with Zeiss Axiovision software and Adobe Photoshop. Bioinformatics Analysis—Transmembrane helices were predicted with PredictProtein (30Rost B. Fariselli P. Casadio R. Protein Sci. 1996; 5: 1704-1718Crossref PubMed Scopus (533) Google Scholar) and Tmpred (www.ch.embnet.org/software/TMPRED_form.html). Angiomotin Induction Assay—500,000 BCE cells were plated at the indicated densities, and 24 h later cells were rinsed twice with PBS, briefly inverted on tissue paper, and lysed by addition of 100 μl of 2× SDS-PAGE loading buffer. Samples were analyzed by Western blot using the TLE antibody. Sample volume increased with increasing plate size due to residual PBS; therefore, 10% of the volume of the lysates was loaded. Immunoprecipitation with MAGI-1—Two million CHO cells plated on 6-cm Petri dishes 1 day before were transfected with 2 μg of each plasmid DNA using Lipofectamine 2000 reagent (Invitrogen). Cells were harvested 48 h after transfection in a lysis buffer; 150 mm NaCl, 50 mm Tris-HCl, pH 7.4, 0.5% Triton X-100, and protease inhibitors. The cell lysates were rotated end over end at 4 °C for 15 min and centrifuged at 13,000 rpm for 5 min at 4 °C. The supernatants were collected and used for the determination of total protein by the Bradford method. Lysate representing 1.5 mg of total protein were pre-cleared with 20 μl of protein A beads and then incubated with 5 μg of anti-angiomotin or anti-FLAG antibody for each IP sample for 6 h at 4°C under rotation. Afterward 30 μl of protein A beads was added to each IP sample, and the mixture was rotated overnight at 4 °C. After incubation, beads were washed twice with lysis buffer, resuspended in Laemmli sample buffer, boiled, and resolved by 7.5% Criterion precast gel (Bio-Rad). For immunofluorescence staining, 40,000 CHO cells were plated in chamber slides and transfected with 0.35 μg of each plasmid with Lipofectamine 2000 reagent. Aggregation Assay—CHO cells were de-attached by rinsing with Ca2+ and Mg2+ PBS twice and incubation with 0.02% EDTA (Sigma) until de-attached, resuspended in Ca2+- and Mg2+-free Hanks' balanced salt solution (Sigma), washed once in Hanks' balanced salt solution, and resuspended to 100,000 cells/ml in Hanks' balanced salt solution supplemented with 2% fetal bovine serum dialyzed against Ca2+- and Mg2+-free PBS. 50,000 cells were loaded per well in 24-well plates previously coated with 1% BSA. At this time the absolute majority of cells were single cells. CaCl2 (2 mm) and angiostatin (5 μg/ml) was added where indicated. Cells were allowed to aggregate at 37 °C for 60 min during rotation on a platform rotator at 80 rpm. The experiment was stopped by addition of glutaraldehyde to a final concentration of 5%. At least five fields from each well were photographed at 10× magnification and analyzed for cell aggregation. The total number of cells (N0) was counted, and the number of cell aggregates at 60 min (N60) was counted. Aggregation index was calculated as (N0-N60)/N0 as described (31Hirata K. Ishida T. Penta K. Rezaee M. Yang E. Wohlgemuth J. Quertermous T. J. Biol. Chem. 2001; 276: 16223-16231Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Approximately 500 cells were evaluated for each condition. Permeability Assay—The In Vitro Vascular Permeability assay kit from Chemicon Inc., which is based on the diffusion of FITC-labeled dextran across a cell layer grown on a membrane in a 24-well plate format, was used according to the manufacturer's instructions. Briefly, CHO cells were seeded at 12,000 cells per membrane insert and allowed to form a monolayer in 5 days. Triplicate or quadruplicate inserts were used for each condition. Where indicated, angiostatin (5 μg/ml) was added to the well 1 h before the start of the experiment. FITC-dextran was added to the upper chamber, and 100-μl samples were withdrawn at 5, 15, 60, and 120 min from the lower chamber. Fluorescence was measured on a Bio-Tek FL 600 plate reader using an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The background fluorescence of cell culture medium was subtracted. The diffusion of FITC-dextran across a membrane insert without cells was measured in parallel to ensure integrity of cell monolayers. Cell Migration Assay—A Boyden chamber migration assay was performed as described previously (10Troyanovsky B. Levchenko T. Mansson G. Matvijenko O. Holmgren L. J. Cell Biol. 2001; 152: 1247-1254Crossref PubMed Scopus (299) Google Scholar). Briefly, 30,000 cells in serum-free medium were loaded in each well and allowed to migrate toward serum or bFGF 20 (20 ng/ml) for 4 h. Non-migrating cells were removed, and remaining cells were fixed and stained with Giemsa stain. Three fields per well were counted under a microscope. Angiomotin Is Localized on the Cell Surface—We have previously reported that MAE cells transfected with p80 angiomotin respond to angiostatin by inhibited migration and tube formation in vitro. This suggests that p80 angiomotin is a receptor for angiostatin. However, as judged by sequence analysis, angiomotin does not have an obvious signal peptide that could mediate the insertion of the protein into the membrane. To investigate whether p80 angiomotin has any extracellular domains, we incubated MAE cells stably expressing p80 angiomotin (p80 Amot MAE) with sulfo-NHS-LC-biotin, a biotin derivative with a reactive group that conjugates biotin to proteins. Sulfo-NHS-LC-biotin is water-soluble and will not penetrate intact cell membranes. After incubation with sulfo-NHS-LC-biotin we subjected cell lysates to immunoprecipitation against either angiomotin, or, as a negative control, the intracellular protein paxillin. The immunoprecipitates were analyzed by Western blot with HRP-conjugated avidin. As seen in Fig. 1A, p80 angiomotin was biotinylated, whereas paxillin was not. Paxillin could, however, be biotinylated with a hydrophobic, membrane-permeating analogue, NHS-LC-biotin (data not shown). Endogenous p80 angiomotin could also be biotinylated in primary cells when similar experiments were carried out with bovine capillary endothelial (BCE) cells (supplementary Fig. S1). This is of interest, because angiostatin was first identified by its ability to inhibit proliferation of these cells (3O'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 (3170) Google Scholar). To verify that angiomotin has extracellular epitopes we treated MAE cells with trypsin for various times and analyzed cell lysates by Western blot. Trypsin degraded most p80 angiomotin in 80 min, whereas actin, which is intracellular, was not (Fig. 1B). Longer exposure of the blot revealed the appearance over time of a degradation product that likely represents a protected fragment of the C terminus, suggesting that this domain is intracellular. Triton X-114 phase separation can be used to separate hydrophobic integral membrane proteins from hydrophilic soluble proteins. We carried out Triton X-114 fractionation of p80 Amot MAE cells. After phase separation p80 angiomotin could be detected in the aqueous fraction as well the detergent fraction, indicating that angiomotin is a integral membrane protein (Fig. 1C). VE-cadherin, a characterized transmembrane protein, also distributed to both fractions (data not shown). Angiostatin Binds Angiomotin on the Cell Surface—Previously we have shown that angiomotin-transfected HeLa cells can bind and internalize FITC-labeled angiostatin (10Troyanovsky B. Levchenko T. Mansso" @default.
• W2072305777 created "2016-06-24" @default.
• W2072305777 creator A5044134974 @default.
• W2072305777 creator A5047814015 @default.
• W2072305777 creator A5054426500 @default.
• W2072305777 creator A5056059593 @default.
• W2072305777 creator A5072584976 @default.
• W2072305777 creator A5072741407 @default.
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• W2072305777 date "2005-10-01" @default.
• W2072305777 modified "2023-10-01" @default.
• W2072305777 title "Angiomotin Regulates Endothelial Cell-Cell Junctions and Cell Motility" @default.
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