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• W2089092016 abstract "Endothelial cells express two major cadherins, VE- and N-cadherins, but only the former consistently participates in adherens junction organization. In heart microvascular endothelial cells, we identified a new member of the cadherin superfamily using polymerase chain reaction. The entire putative coding sequence was determined. Similarly to protocadherins, while the extracellular domain presented homology with other members of the cadherin superfamily, the intracellular region was unrelated either to cadherins or to any other known protein. We propose for this new protein the name of vascular endothelial cadherin-2. By Northern blot analysis, the mRNA was present only in cultured endothelial cell lines but not in other cell types such as NIH 3T3, Chinese hamster ovary, or L cells. In addition, mRNA was particularly abundant in highly vascularized organs such as lung or kidney. In endothelial cells and transfectants, this cadherin was unable to bind catenins and presented a weak association with the cytoskeleton. This new molecule shares some functional properties with VE-cadherin and other members of the cadherin family. In Chinese hamster ovary transfectants it promoted homotypic Ca2+ dependent aggregation and adhesion and clustered at intercellular junctions. However, in contrast to VE-cadherin, it did not modify paracellular permeability, cell migration, and density-dependent cell growth. These observations suggest that different cadherins may promote homophilic cell-to-cell adhesion but that the functional consequences of this interaction depend on their binding to specific intracellular signaling/cytoskeletal proteins. Endothelial cells express two major cadherins, VE- and N-cadherins, but only the former consistently participates in adherens junction organization. In heart microvascular endothelial cells, we identified a new member of the cadherin superfamily using polymerase chain reaction. The entire putative coding sequence was determined. Similarly to protocadherins, while the extracellular domain presented homology with other members of the cadherin superfamily, the intracellular region was unrelated either to cadherins or to any other known protein. We propose for this new protein the name of vascular endothelial cadherin-2. By Northern blot analysis, the mRNA was present only in cultured endothelial cell lines but not in other cell types such as NIH 3T3, Chinese hamster ovary, or L cells. In addition, mRNA was particularly abundant in highly vascularized organs such as lung or kidney. In endothelial cells and transfectants, this cadherin was unable to bind catenins and presented a weak association with the cytoskeleton. This new molecule shares some functional properties with VE-cadherin and other members of the cadherin family. In Chinese hamster ovary transfectants it promoted homotypic Ca2+ dependent aggregation and adhesion and clustered at intercellular junctions. However, in contrast to VE-cadherin, it did not modify paracellular permeability, cell migration, and density-dependent cell growth. These observations suggest that different cadherins may promote homophilic cell-to-cell adhesion but that the functional consequences of this interaction depend on their binding to specific intracellular signaling/cytoskeletal proteins. Endothelial permeability to plasma proteins and circulating cells is controlled in part by intercellular junctions. Besides their role in promoting homotypic cell adhesion, emerging evidence suggests that intercellular junctions can transfer cell-cell signals and be responsible for complex cellular responses such as contact inhibition of cell growth and cell polarity. The molecular organization of intercellular junctions in the endothelium has been only partially elucidated in the last few years. At least three types of complex structures have been described: tight junctions, adherens junctions, and complexus adhaerentes. All of these structures are formed by specific transmembrane proteins, which through their extracellular region promote homotypic cell-to-cell adhesion and through the cytoplasmic tail bind to a complex network of cytoskeletal and signaling proteins (1Rubin L.L. Curr. Opin. Cell Biol. 1992; 4: 830-833Crossref PubMed Scopus (84) Google Scholar, 2Dejana E. J. Clin. Invest. 1997; 100: 7-10Google Scholar, 3Lampugnani M.G. Dejana E. Curr. Opin. Cell Biol. 1997; 9: 674-682Crossref PubMed Scopus (199) Google Scholar). Outside of these junctional structures, other adhesive proteins such as PECAM 1The abbreviations used are: PECAM, platelet/endothelial cell adhesion molecule; CHO, Chinese hamster ovary; EC1–EC6 domains, extracellular domains 1–6; PCR, polymerase chain reaction; VE-cad, vascular endothelial cadherin; VE-cad-2, vascular endothelial cadherin 2; PBS, phosphate-buffered saline; mAb, monoclonal antibody; bp, base pair(s); kb, kilobase pair(s). (4DeLisser H.M. Newman P.J. Albelda S.M. Immunol. Today. 1994; 15: 490-495Abstract Full Text PDF PubMed Scopus (285) Google Scholar) or S-Endo-1/Muc-18 (5Bardin N. Frances V. Lesaule G. Horschowski N. George F. Sampol J. Biochem. Biophys. Res. Commun. 1996; 218: 210-216Crossref PubMed Scopus (116) Google Scholar) have been found to be clustered at intercellular contacts. The intracellular molecules that associate with adherens junctions are different from those that associate with tight junctions and from those that link other junctional adhesion proteins such as PECAM, suggesting that a certain specificity in signaling should exist (3Lampugnani M.G. Dejana E. Curr. Opin. Cell Biol. 1997; 9: 674-682Crossref PubMed Scopus (199) Google Scholar). In adherens junctions, the transmembrane proteins responsible for cell-to-cell adhesion belong to the cadherin superfamily of adhesive proteins. In endothelial cells, one of the major cadherins is VE-cadherin or cadherin-5 (VE-cad), which is consistently present at adherens junctions and is cell-specific (2Dejana E. J. Clin. Invest. 1997; 100: 7-10Google Scholar, 3Lampugnani M.G. Dejana E. Curr. Opin. Cell Biol. 1997; 9: 674-682Crossref PubMed Scopus (199) Google Scholar, 6Suzuki S. Sano K. Tanihara H. Cell Regul. 1991; 2: 261-270Crossref PubMed Scopus (317) Google Scholar). Similarly to the other members of the family, the short intracellular tail of VE-cad is linked to three cytoplasmic proteins called catenins: β-catenin, plakoglobin, and p120. β-Catenin and plakoglobin bind α-catenin, which in turn promotes the anchorage of the complex to the actin cytoskeleton. As the other known cadherins, the extracellular domain of VE-cad promotes homotypic, calcium-dependent adhesion. Endothelial cells also express N-cadherin, which in human endothelium does not colocalize with VE-cad at cell contacts but remains diffuse on the cell membrane (7Salomon D. Ayalon O. Patel King R. Hynes R.O. Geiger B. J. Cell Sci. 1992; 102: 7-17PubMed Google Scholar, 8Navarro P. Ruco L. Dejana E. J. Cell Biol. 1998; 140: 1475-1484Crossref PubMed Scopus (245) Google Scholar). Besides these well characterized cadherins, some indirect evidence suggests that other cadherin-like structures may be present in the endothelium (9Ayalon O. Sabanai H. Lampugnani M.G. Dejana E. Geiger B. J. Cell Biol. 1994; 126: 247-258Crossref PubMed Scopus (175) Google Scholar). Therefore, we started an investigation to test this possibility. Using a polymerase chain reaction method previously introduced by Suzuki et al. (6Suzuki S. Sano K. Tanihara H. Cell Regul. 1991; 2: 261-270Crossref PubMed Scopus (317) Google Scholar) and Sano et al.(10Sano K. Tanihara H. Heimark R.L. Obata S. Davidson M. St. John T. Taketani S. Suzuki S. EMBO J. 1993; 12: 2249-2256Crossref PubMed Scopus (333) Google Scholar), we identified a new protein that, on the extracellular domain, presents homology with cadherins. This protein is concentrated at intercellular junctions and expresses adhesive properties, but, in contrast to VE-cad, does not bind to catenins and does not modify paracellular permeability, cell migration, or growth. This indicates that several proteins may participate in the molecular organization of interendothelial junctions, but each molecule may play a specific functional role and possibly transfer different intracellular signals. For its localization in endothelial cells and for its homology with the cadherin and protocadherin family, we propose for this new protein the name of vascular endothelial cadherin-2 (VE-cad-2). All reagents were purchased from Sigma unless indicated otherwise. PCR was performed as described previously by Suzukiet al. (6Suzuki S. Sano K. Tanihara H. Cell Regul. 1991; 2: 261-270Crossref PubMed Scopus (317) Google Scholar) and Sano et al. (10Sano K. Tanihara H. Heimark R.L. Obata S. Davidson M. St. John T. Taketani S. Suzuki S. EMBO J. 1993; 12: 2249-2256Crossref PubMed Scopus (333) Google Scholar). Template cDNA was synthesized using mouse heart microvascular endothelial cell (H5V) (11Garlanda C. Parravicini C. Sironi M. De Rossi M. Wainstok de Calmanovici R. Carozzi F. Bussolino F. Colotta F. Mantovani A. Vecchi A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7291-7295Crossref PubMed Scopus (163) Google Scholar) total RNA according to the protocol of the GeneAmp RNA PCR kit (Perkin-Elmer). Two different sets of degenerated oligonucleotide primers were used in this study. One set corresponds to two well conserved sequences in the cytoplasmic domain of cadherin; the upstream and downstream primers were 5′-GAATTCAC(A/C/G/T)GC(A/C/G/T)CC(A/C/G/T)TA(C/T)GA-3′ and 5′-GAATTCTC(A/C/G/T)GC(A/C/G/T)A(A/G)(C/T)TT(C/T)TT(A/G)AA-3′, respectively. The other set corresponds to two conserved regions in the third (EC3) and in the fourth extracellular domain (EC4) as described previously by Sano et al. (10Sano K. Tanihara H. Heimark R.L. Obata S. Davidson M. St. John T. Taketani S. Suzuki S. EMBO J. 1993; 12: 2249-2256Crossref PubMed Scopus (333) Google Scholar). The upstream and downstream primers were 5′-AA(A/G)(C/G)(C/G)(A/C/G/T)(A/C/G/T)T(A/C/G/T)GA(C/T)T(A/ G)(C/T)GA-3′ and 5′-(A/C/G/T)(A/C/G/T)(A/C/G/T)GG(A/C/G/T)GC(A/ G)TT(A/G)TC(A/G)TT-3′, respectively. The PCR reaction consisted of 33 cycles at 95 °C for 1 min, 45 °C for 2 min, and 72 °C for 3 min usingTaq DNA polymerase obtained from Boehringer Mannheim. The PCR products were subcloned into the pCR II plasmid by use of the TA cloning kit (Invitrogen Co., San Diego, CA) and sequenced according to the dideoxynucleotide chain termination method (12Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4767-4771Crossref PubMed Scopus (1687) Google Scholar). About 8 × 105 plaques of a λgt10 cDNA library from postnatal day 4–8 mouse brain microvasculature (13Schnurch H. Risau W. Development. 1993; 119: 957-968PubMed Google Scholar) were screened for clone 1 and clone 14 by a plaque hybridization method, as described previously (14Breviario F. d'Aniello E.M. Golay J. Peri G. Bottazzi B. Bairoch A. Saccone S. Marzella R. Predazzi V. Rocchi M. Della Valle G. Dejana E. Mantovani A. Introna M. J. Biol. Chem. 1992; 267: 22190-22197Abstract Full Text PDF PubMed Google Scholar), using as probes the cDNAs obtained from PCR experiments. cDNAs were radiolabeled with [32P]α-dCTP (Amersham International, Buckinghamshire, UK), using a random primer DNA labeling kit obtained from Boehringer Mannheim. Plaques showing a strong positive hybridization signal were screened four times to obtain a single phage clone. Phage inserts were subcloned in the pBluescript vector and sequenced by Genomic Express S.A. (Grenoble, France). The nucleotide and the deduced protein sequences were screened against the data bank as described previously (15Breviario F. Caveda L. Corada M. Martin-Padura I. Navarro P. Golay J. Introna M. Gulino D. Lampugnani M.G. Dejana E. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1229-1239Crossref PubMed Scopus (240) Google Scholar). The full-length open reading frame for VE-cad-2 was cut with EcoRI, and the insert was subcloned into the pECE eukaryotic expression vector (16Ellis L. Clauser E. Morgan D.O. Edery M. Roth R.A. Rutter W.J. Cell. 1986; 45: 721-732Abstract Full Text PDF PubMed Scopus (696) Google Scholar) to yield the pECE-VE-cad-2 construct. CHO cells were plated at 5–6 × 105 cells/10-mm Petri dish in Dulbecco's modified Eagle's medium with 10% fetal calf serum. After about 24 h, the cells were transfected by calcium phosphate precipitation with 20 μg of pECE-VE-cad-2 and 2 μg of pCMVneo plasmid. Cells were washed 24 h later with PBS and cultured for another 24–36 h in Dulbecco's modified Eagle's medium with 10% fetal calf serum. They were then cultured in presence of 600 μg/ml G418 (Geneticin; Life Technologies, Inc.). After about 10 days in selective medium, the surviving colonies were ring-cloned. G418-resistant clones were screened for expression by Northern blot analysis and indirect immunofluorescence microscopy. Control cells, CHO cells transfected with pCMVneo were selected, cloned, and cultured in the same way. Total RNA was extracted and purified by use of the Rapid Total RNA isolation kit (5 Prime → 3 Prime, Inc., Boulder, CO) and 20 μg were run in a standard formaldehyde/agarose gel, blotted onto Hybond N membrane (Amersham International), fixed at 80 °C for 2 h, and hybridized at 65 °C in a buffer containing 10% dextran sulfate, 3× SSC, 5× Denhardt's solution, 10% SDS, 100 μg/ml denatured salmon sperm DNA. The membranes were then washed twice with 2× SSC and 0.1% SDS at room temperature for 10 min, twice with 0.5× SSC and 0.1% SDS at 65 °C for 15 min, and once with 0.1× SSC and 0.1% SDS at 65 °C for 10 min and then subjected to autoradiography. CHO, NIH 3T3, and L929 cells were from American Tissue Culture Collection (Rockville, MD). Mouse epithelial cells (PDV), kindly provided by A. Cano (Instituto de Investigaciones Biomedicas, Madrid, Spain) were skin keratinocytes isolated and cultured as described previously (17Fusening N.E. Breitkreutz D. Dzrlieva R.T. Boukamp P. Hezmann E. Bohnert A. Pohlmann J. Rausch C. Schutz S. Hornung J. Cancer Forum. 1982; 6: 209-240Google Scholar). Mouse endothelioma cell lines H5V, T-end, E-end, and B-end were obtained and cultured as described (11Garlanda C. Parravicini C. Sironi M. De Rossi M. Wainstok de Calmanovici R. Carozzi F. Bussolino F. Colotta F. Mantovani A. Vecchi A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7291-7295Crossref PubMed Scopus (163) Google Scholar, 18Williams R.L. Risau W. Zerwes H.G. Drexler H. Aguzzi A. Wagner E.F. Cell. 1989; 57: 1053-1063Abstract Full Text PDF PubMed Scopus (222) Google Scholar, 19Vecchi A. Garlanda C. Lampugnani M.G. Resnati M. Matteucci C. Stoppacciaro A. Schnurch H. Risau W. Ruco L. Mantovani A. Dejana E. Eur. J. Cell Biol. 1994; 63: 247-254PubMed Google Scholar). CHO-VE-cad transfectants were obtained as described previously (15Breviario F. Caveda L. Corada M. Martin-Padura I. Navarro P. Golay J. Introna M. Gulino D. Lampugnani M.G. Dejana E. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1229-1239Crossref PubMed Scopus (240) Google Scholar). Cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum at 37 °C in a 5% CO2atmosphere. Sterile plasticware was from Falcon (Becton Dickinson, Lincoln Park, NJ); both culture medium and serum were from Life Technologies, Inc. A rabbit antiserum was raised against a recombinant fragment spanning the extracellular domain of VE-cad-2 (74–335 amino acids). The fragment was generated by PCR. The primers were designed to create at the 5′-end a BamHI site and at the 3′-end a HindIII restriction site. The cDNA fragment was then subcloned into the BamHI–HindIII site of the expression vector pQE30 (Qiaexpressionist kit; Qiagen, Chatsworth, CA) in the correct reading frame and sequenced to verify that no mutation had arisen during PCR. The resulting pQE30-VE-cad-2 vector was then introduced into M15 (pREP4) cells by a single step transformation method. The fusion protein was induced by the addition of isopropyl-β-d-thiogalactopyranoside and was purified from the extract by nickel-nitrilotriacetic acid resin affinity chromatography, as described by the manufacturer (Qiaexpressionist kit; Qiagen). Polyclonal antibody against VE-cad-2 was produced in rabbit by injecting 0.5 mg of the fusion protein in Freund's complete adjuvant at three subcutaneous sites. Subsequent injections were in Freund's incomplete adjuvant with 0.5 mg of the fusion protein. The fragment antiserum was affinity-purified by affinity chromatography on the corresponding fragment affinity column CN-Br-Sepharose 4B (Pharmacia LKB Biotechnology, Uppsala, Sweden). The antiserum was further characterized for its positive reaction with endothelial cells (H5V) and VE-cad-2 transfectants by enzyme-linked immunosorbent assay, immunoprecipitation, Western blot, and immunofluorescence staining of fixed cell monolayers. Mouse monoclonal antibody to human VE-cad was the clone TEA 1.31 (20Leach L. Clark P. Lampugnani M.G. Arroyo A.G. Dejana E. Firth J.A. J. Cell Sci. 1993; 104: 1073-1081Crossref PubMed Google Scholar). Mouse mAbs against α-catenin and β-catenin were purchased from Transduction Laboratories (Lexington, KY). mAb MEC 13 to CD31 was kindly provided by A. Vecchi (19Vecchi A. Garlanda C. Lampugnani M.G. Resnati M. Matteucci C. Stoppacciaro A. Schnurch H. Risau W. Ruco L. Mantovani A. Dejana E. Eur. J. Cell Biol. 1994; 63: 247-254PubMed Google Scholar). Fluorescein- and rhodamine-conjugated secondary antibodies (reactive with either mouse or rabbit IgG) were purchased from Dakopatts (Glostrup, Denmark). Goat anti-mouse IgG peroxidase-conjugated antibody and protein A peroxidase-conjugated antibody used for immunoblotting detection were from Pierce. Cells were grown on glass coverslips, rinsed in PBS, and fixed in methanol. The cells were then rinsed and incubated for 45 min at 37 °C with the relevant primary antibodies, washed three times with PBS, and incubated for 30 min with the fluorophore-conjugated secondary antibodies. Coverslips were then mounted in Mowiol 4–88 (Calbiochem) and examined with a Zeiss Axiophot microscope. Photographs were taken using T-Max P3200 films. EGTA was used for chelating calcium ions in the culture medium as described previously (21Volberg T. Geiger B. Kartenbeck J. Franke W.W. J. Cell Biol. 1986; 102: 1832-1842Crossref PubMed Scopus (180) Google Scholar). A buffer stock solution of 100 mm EGTA was used to obtain a final concentration of 5 mm. Cells grown to confluence on glass coverslips were incubated with 5 mm EGTA at 37 °C for 30 min, fixed, and processed for indirect immunofluorescence as described above. Cells were cultured to confluence on glass coverslips and treated with 1 μg/ml cytochalasin D in culture medium. 30 min later, cells were fixed with 3% paraformaldehyde, permeabilized with 0.5% Triton X-100, and processed for indirect immunofluorescence as described above. Biotinylation of cell surface proteins was performed as described elsewhere (22Alexander J.S. Blaschuk O.W. Haselton F.R. J. Cell. Physiol. 1993; 156: 610-618Crossref PubMed Scopus (88) Google Scholar) using sulfonitrohydroxysuccinimido-biotin (Pierce). Samples were analyzed by electrophoresis on a 7.5% SDS-polyacrylamide gel and transferred to nitrocellulose membrane. The membranes were blocked with 10% low fat milk and then incubated in fresh blocking solution with horseradish peroxidase-conjugated streptavidin (Biospa Division, Milano, Italy) for 1 h at room temperature. After three washes with PBS containing 0.1% Tween 20, peroxidase-conjugated streptavidin was visualized using the ECL kit as described under “Blot and Immunoprecipitation.” Whole cell extracts were obtained from confluent cells as described previously (23Frachet P. Uzan G. Thevenon D. Denarier E. Prandini M.H. Marguerie G. Mol. Biol. Rep. 1990; 14: 27-33Crossref PubMed Scopus (31) Google Scholar). Detergent solubilization was carried out essentially as reported previously in detail (24Lampugnani M.G. Corada M. Caveda L. Breviario F. Ayalon O. Geiger B. Dejana E. J. Cell Biol. 1995; 129: 203-217Crossref PubMed Scopus (495) Google Scholar). Different cell extracts were adjusted to 1× Laemmli sample buffer and fractionated under reducing conditions on 7.5% SDS-polyacrylamide gels (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207094) Google Scholar). Western blot analyses of the various cell extracts were carried out essentially as described (24Lampugnani M.G. Corada M. Caveda L. Breviario F. Ayalon O. Geiger B. Dejana E. J. Cell Biol. 1995; 129: 203-217Crossref PubMed Scopus (495) Google Scholar). After blocking with 10% low fat milk, the proteins of interest were detected by specific monoclonal or polyclonal antibodies at the optimal dilution in blocking buffer. This was sequentially followed by incubation with goat anti-mouse IgG peroxidase-conjugated antibody (1 mg/ml) for monoclonal antibodies or protein A peroxidase-conjugated antibody (1 mg/ml) (Pierce) for polyclonal antibody and further development of peroxidase activity using an ECL kit (Amersham Biotech Pharmacia International) and autoradiography. Immunoprecipitation of the cadherin-catenin complex was performed using the nonionic detergent-soluble fraction of cells, as previously reported (24Lampugnani M.G. Corada M. Caveda L. Breviario F. Ayalon O. Geiger B. Dejana E. J. Cell Biol. 1995; 129: 203-217Crossref PubMed Scopus (495) Google Scholar) with some modifications. Briefly, cell extracts were precleared by incubation with uncoupled protein G- or protein A-Sepharose CL-4B (Amersham Biotech Pharmacia) for 2 h. After centrifugation, the precleared supernatants were incubated with protein G- or protein A-Sepharose coupled to mAb TEA 1.31 or polyclonal antibody against VE-cad-2 during 1 h. Immunocomplexes were collected by centrifugation; washed five times in a buffer containing 0.5% Triton X-100, 0.1% bovine serum albumin, 50 mmTris-HCl, pH 7.4, 0.1 m NaCl, and 2 mmCaCl2; and finally resuspended in 30 μl of 1× Laemmli sample buffer and boiled for 5 min. Samples were analyzed by electrophoresis, transferred to nitrocellulose membranes, and immunoblotted sequentially with polyclonal antibody to VE-cad-2 or mAb TEA 1.31 to VE-cad or mAbs to α- and β-catenin as described above. Calcium-dependent cell aggregation was done under conditions that preserve, by flow cytometry analysis, VE-cad-2 or VE-cad expression as described previously (15Breviario F. Caveda L. Corada M. Martin-Padura I. Navarro P. Golay J. Introna M. Gulino D. Lampugnani M.G. Dejana E. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1229-1239Crossref PubMed Scopus (240) Google Scholar). When indicated, cytochalasin D was added at 1 μg/ml after the first centrifugation, and the cells were incubated at 37 °C for 30 min. In some experiments, EGTA (5 mm final concentration) was added to the medium. For heterotypic aggregation assays, CHO-VE-cad-2/VE-cad cells were labeled with 2 mm2′,5′-bis(2-carboxyethyl)-5-carboxyfluorescein acetoxymethyl ester and 2′,5′-bis(2-carboxyethyl)-6-carboxyfluorescein acetoxymethyl ester (Molecular Probes, Eugene, OR) in Hanks' balanced salt solution for 10 min at 37 °C and processed as described (26Navarro P. Caveda L. Breviario F. Mandoteanu I. Lampugnani M.-G. Dejana E. J. Biol. Chem. 1995; 270: 30965-30972Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). CHO, CHO-VE-cad-2, or CHO-VE-cad cells were cultured in 96-well plates and grown for 5 days to confluency. All three types of cells were labeled with [125I]iododeoxyuridine (1 mCi/ml) overnight prior to the cell adhesion experiment. 12 h later, the cells were detached as described above and resuspended at 3 × 105 cells/ml in Dulbecco's modified Eagle's medium with 10% fetal calf serum. 100 μl of labeled cell suspension were added to different adherent cell monolayers (CHO, CHO-VE-cad-2, and CHO-VE-cad) and incubated for 1 h at 37 °C. After three washes with PBS containing 10% fetal calf serum, the cells were solubilized with 0.5 m NaOH, 0.1% SDS and counted in a γ-counter. Permeability across cell monolayers was measured in Transwell units (with polycarbonate filter, 0.4-μm pore; Corning Costar Corp., Cambridge, MA) as described previously (15Breviario F. Caveda L. Corada M. Martin-Padura I. Navarro P. Golay J. Introna M. Gulino D. Lampugnani M.G. Dejana E. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1229-1239Crossref PubMed Scopus (240) Google Scholar). Briefly, CHO transfectants were cultured to confluency for 5 days. Then culture medium was replaced with serum-free medium, and horseradish peroxidase conjugated to goat immunoglobulins (8 mg/ml initial concentration in the upper chamber; minimal calculated molecular mass, 200 kDa; specific activity, 28 units/mg) was added to the upper chamber. At 2 h, 100-μl aliquots were collected from the lower compartment and assayed photometrically for the presence of enzymatic activity. In some experiments, EGTA (5 mm, final concentration) was added both to the lower and upper compartments for 2 h at the same time as immunoglobulins. Cell migration was estimated as described previously (15Breviario F. Caveda L. Corada M. Martin-Padura I. Navarro P. Golay J. Introna M. Gulino D. Lampugnani M.G. Dejana E. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1229-1239Crossref PubMed Scopus (240) Google Scholar). Briefly, the cell monolayer was wounded with a plastic tip. Four diameters, regularly distanced by about 45°, were removed. The remaining cells were washed twice with culture medium to remove cell debris and incubated at 37 °C in culture medium. At the indicated time intervals, cells were fixed with Fast Green FCF (0.02% in methanol) and stained with crystal violet (0.5% in a 20:80 mixture of methanol/water). The distance migrated by the cells was measured using a micrograduate scale (Nikon) adapted in the ocular of a Nikon inverted microscope under phase contrasts (magnification × 100). Cell growth was evaluated as described previously (27Caveda L. Martin-Padura I. Navarro P. Breviario F. Corada M. Gulino D. Lampugnani M.G. Dejana E. J. Clin. Invest. 1996; 98: 886-893Crossref PubMed Scopus (160) Google Scholar). Cells were plated at 1 × 104/ml (1 ml/well) in 24-well plates (2 cm2/well). Culture medium was not changed for the duration of the experiment (96 h). Cell number was evaluated after trypsinization of the cells and counting (four replicates) in a Bürker chamber. A PCR method was applied to identify new members of the cadherin superfamily. As primers we first used two degenerated oligonucleotides corresponding to two highly conserved sequences in the cytoplasmic domain of cadherins as previously described by Suzuki et al. (6Suzuki S. Sano K. Tanihara H. Cell Regul. 1991; 2: 261-270Crossref PubMed Scopus (317) Google Scholar). PCR was carried out using cDNA obtained from mouse heart microvascular endothelial cells (H5V). The resulting 160-bp products were then subcloned in the pCRII plasmid and sequenced. Of 35 clones sequenced, three clones encoded the amino acid sequence of N-cadherin, and two clones encoded the amino acid sequence of VE-cad. The other cDNA clones encoded amino acid sequences that did not present homology with the cytoplasmic domain of cadherins. In the second part of the research, we used as primers degenerated oligonucleotides corresponding to two conserved sequences of the extracellular domain of cadherins, as described previously by Sanoet al. (10Sano K. Tanihara H. Heimark R.L. Obata S. Davidson M. St. John T. Taketani S. Suzuki S. EMBO J. 1993; 12: 2249-2256Crossref PubMed Scopus (333) Google Scholar). PCR from the cDNA of the cell line H5V yielded four major bands of 450, 370, 300, and 130 bp in size. The 450- and 130-bp bands correspond to the distance between the two primer sites in classic cadherins and the two primer sites in protocadherins, respectively (10Sano K. Tanihara H. Heimark R.L. Obata S. Davidson M. St. John T. Taketani S. Suzuki S. EMBO J. 1993; 12: 2249-2256Crossref PubMed Scopus (333) Google Scholar). The 370- and 300-bp bands would not be predicted from any of the known members of the cadherin superfamily and were therefore discarded. The 130-bp product was subcloned into the pCR II vector, and 30 clones were isolated and sequenced. Two cDNAs (clones 1 and 14) presented a novel sequence and were considered good candidates to be new putative members of the cadherin superfamily. A cDNA library of postnatal day 4–8 mouse brain capillaries (13Schnurch H. Risau W. Development. 1993; 119: 957-968PubMed Google Scholar) was screened by using clones 1 and 14 as probes. Four clones of 5.8, 4.0, 2.4, and 1.8 kb were obtained by screening with clone 1, while only a clone of 700 bp was obtained using clone 14 as probe. The sequence analysis of the 700-bp clone revealed a partial sequence that did not correspond to any previously identified sequence (data not shown). The sequences of the two cDNA clones of 5.8 and of 4.0 kb obtained by screening with clone 1 overlap and appear to contain the full-length open reading frame of a novel member of the cadherin superfamily. The nucleotide and deduced amino acid sequences of the 4.0-kb clone are shown in Fig. 1. The 3868-bp sequence contains 298 bp of putative 5′-untranslated region, an open reading frame of 3540 nucleotides encoding 1180 amino acids, and a short 3′-untranslated region of 29 bp. At position 299, the cDNA sequence contains a translation initiation site that matches the Kozak criteria (28Kozak M. Nucleic Acids Res. 1984; 12: 857-872Crossref PubMed Scopus (2381) Google Scholar). The polyadenylation signal was not identified. The sequence presents a signal peptide, an extracellular region that can be divided into six domains (EC1–EC6), a transmembrane domain, and a large cytoplasmic region of 443 amino acids. The repeats EC1–EC5 are 104–109 residues long and present homology to the cadherin repeats found in" @default.
• W2089092016 created "2016-06-24" @default.
• W2089092016 creator A5003887782 @default.
• W2089092016 creator A5037522722 @default.
• W2089092016 creator A5052593684 @default.
• W2089092016 creator A5058032428 @default.
• W2089092016 creator A5090485594 @default.
• W2089092016 date "1998-07-01" @default.
• W2089092016 modified "2023-09-27" @default.
• W2089092016 title "Identification of a Novel Cadherin (Vascular Endothelial Cadherin-2) Located at Intercellular Junctions in Endothelial Cells" @default.
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