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• W1964383788 abstract "Thrombomodulin (TM) is an integral membrane glycoprotein that is a potent anticoagulant factor. TM may also possess functions distinct from its anticoagulant activity. Here the influence of TM on cell adhesion was studied in TM-negative melanoma A2058 cells transfected with green fluorescent protein-tagged TM (TMG) or lectin domain-deleted TM (TMG(ΔL)). Confocal microscopy demonstrated that both TMG and TMG(ΔL) were distributed in the plasma membrane. TMG-expressed cells grew as closely clustered colonies, with TM localized prominently in the intercellular boundaries. TMG(ΔL)-expressed cells grew singly. Overexpression of TMG, but not TMG(ΔL), decreased monolayer permeability in vitro and tumor growth in vivo. The cell-to-cell adhesion in TMG-expressed cells was Ca2+-dependent and was inhibited by monoclonal antibody against the lectin-like domain of TM. The effects of TM-mediated cell adhesion were abolished by the addition of mannose, chondroitin sulfate A, or chondroitin sulfate C. In addition, anti-lectin-like domain antibody disrupted the close clustering of the endogenous TM-expressed keratinocyte HaCaT cell line derived from normal human epidermis. Double-labeling immunofluorescence staining revealed similar distributions of TM and actin filament in the cortex region of the TMG-expressed cells. Thus, TM can function as a Ca2+-dependent cell-to-cell adhesion molecule. Binding of specific carbohydrates to the lectin-like domain is essential for this specific function. Thrombomodulin (TM) is an integral membrane glycoprotein that is a potent anticoagulant factor. TM may also possess functions distinct from its anticoagulant activity. Here the influence of TM on cell adhesion was studied in TM-negative melanoma A2058 cells transfected with green fluorescent protein-tagged TM (TMG) or lectin domain-deleted TM (TMG(ΔL)). Confocal microscopy demonstrated that both TMG and TMG(ΔL) were distributed in the plasma membrane. TMG-expressed cells grew as closely clustered colonies, with TM localized prominently in the intercellular boundaries. TMG(ΔL)-expressed cells grew singly. Overexpression of TMG, but not TMG(ΔL), decreased monolayer permeability in vitro and tumor growth in vivo. The cell-to-cell adhesion in TMG-expressed cells was Ca2+-dependent and was inhibited by monoclonal antibody against the lectin-like domain of TM. The effects of TM-mediated cell adhesion were abolished by the addition of mannose, chondroitin sulfate A, or chondroitin sulfate C. In addition, anti-lectin-like domain antibody disrupted the close clustering of the endogenous TM-expressed keratinocyte HaCaT cell line derived from normal human epidermis. Double-labeling immunofluorescence staining revealed similar distributions of TM and actin filament in the cortex region of the TMG-expressed cells. Thus, TM can function as a Ca2+-dependent cell-to-cell adhesion molecule. Binding of specific carbohydrates to the lectin-like domain is essential for this specific function. Thrombomodulin (TM) 1The abbreviations used are: TMthrombomodulinGFPgreen fluorescence proteinTMGTM-GFP fusion proteinTMG(ΔL)lectin-like domain-deleted form of TM-GFP fusion proteinEGFepidermal growth factorPBSphosphate-buffered saline. is a membrane-intercalated glycoprotein, which functions in anticoagulation by virtue of complexation with thrombin. The complex can effectively activate protein C, which in turn catalyzes the proteolytic inactivation of blood coagulation factors Va and VIIIa, leading to down-regulation of the blood coagulation cascade (1Esmon C.T. Esmon N.L. Harris K.W. J. Biol. Chem. 1982; 257: 7944-7947Abstract Full Text PDF PubMed Google Scholar, 2Esmon C.T. FASEB J. 1995; 9: 946-955Crossref PubMed Scopus (361) Google Scholar). TM is constitutively expressed on endothelial cells (1Esmon C.T. Esmon N.L. Harris K.W. J. Biol. Chem. 1982; 257: 7944-7947Abstract Full Text PDF PubMed Google Scholar). As such, it might be one of the factors that localizes the coagulation cascade to sites of vascular injury (2Esmon C.T. FASEB J. 1995; 9: 946-955Crossref PubMed Scopus (361) Google Scholar). thrombomodulin green fluorescence protein TM-GFP fusion protein lectin-like domain-deleted form of TM-GFP fusion protein epidermal growth factor phosphate-buffered saline. The observations from a number of studies support the contention that TM may also play a role in other extravascular activities (3Boffa M.C. Burke B. Haudenschild C.C. J. Histochem. Cytochem. 1987; 35: 1267-1276Crossref PubMed Scopus (70) Google Scholar). Ablation of the TM gene causes early postimplantation embryonic lethality that precedes the establishment of a functional cardiovascular system (4Healy A.M. Rayburn H.B. Rosenberg R.D. Weiler H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 850-854Crossref PubMed Scopus (223) Google Scholar). TM may also have antiscarring properties, by virtue of the modulation of early collagen deposition of normal epidermis (5Raife T.J. Lager D.J. Peterson J.J. Erger R.A. Lentz S.R. J. Investig. Med. 1998; 46: 127-133PubMed Google Scholar). Complete or nearly complete TM deficiency has not been reported in humans (6Lane D.A. Grant P.J. Blood. 2000; 95: 1517-1532Crossref PubMed Google Scholar), which is consistent with the view that a severe reduction of TM function may have more dire consequences than the defects in coagulant or anticoagulant factors. An inverse correlation between TM expression and tumor progression is evident clinically (7Tezuka Y. Yonezswa S. Maruyama I. Matsushita Y. Shimizu T. Obama H. Sagara M. Shirao K. Kusano C. Natsugoe S. Yoshinaka H. Baba M. Fukumoto T. Aikou T. Sato E. Cancer Res. 1995; 55: 4196-4200PubMed Google Scholar, 8Tabata M. Sugihara K. Yonezawa S. Yamashita S. Maruyama I. J. Oral Pathol. Med. 1997; 26: 258-264Crossref PubMed Scopus (29) Google Scholar, 9Suehiro T. Shimada M. Matsumata T. Taketomi A. Yamamoto K. Sugimachi K. Hepatology. 1995; 21: 1285-1290Crossref PubMed Google Scholar). It was demonstrated that TM exerted a growth-suppressing effect independent of its anticoagulant activity but dependent on the lectin-like domain (10Zhang Y. Weiler-Guettler H. Chen J. Wilhelm O. Deng Y. Qiu F. Nakagawa K. Klevesath M. Wilhelm S. Bohrer H. Nakagawa M. Graeff H. Martin E. Stern D.M. Rosenberg R.D. Ziegler R. Nawroth P.P. J. Clin. Invest. 1998; 101: 1301-1309Crossref PubMed Google Scholar). The myriad and diverse possible functions of TM may reflect the glycoprotein structure. TM consists of 557 amino acid residues arranged in five distinct domains: an NH2-terminal lectin-like domain, a domain with six epidermal growth factor (EGF)-like structures that contain thrombin binding sites, an O-glycosylation site-rich domain, a transmembrane domain, and a cytoplasmic tail (11Suzuki K. Kusumoto S. Deyashiki Y. Nishioka J. Maruyama I. Zushi M. Kawahara S. Honda G. Yamamoto S. Horiguchi S. EMBO J. 1987; 6: 1891-1897Crossref PubMed Scopus (303) Google Scholar). The NH2-terminal lectin-like domain has two modules. The first 155-amino acid module, is homologous to Ca2+-dependent lectin (12Petersen T.E. FEBS Lett. 1988; 231: 51-53Crossref PubMed Scopus (49) Google Scholar). The second module, adjacent to the EGF-like domain, is a hydrophobic region of 70 amino acid residues. These lectin-like domains exist in other proteins, where they participate in a wide variety of cell biologic processes, including inflammation and cell-to-cell recognition processes (13Chay C.H. Pienta K.J. J. Cell. Biochem. Suppl. 2000; 35: 123-129Crossref PubMed Google Scholar, 14Gabius H. J. Cancer Invest. 1997; 15: 454-464Crossref PubMed Scopus (106) Google Scholar, 15Mody R. Joshi S. Chaney W. J. Pharmacol. Toxicol. Methods. 1995; 33: 1-10Crossref PubMed Scopus (150) Google Scholar). The TM lectin-like domain is not required for cofactor activity for activating protein C, and its biological function remains mostly unclear. It has been reported that many null mutations in adhesion genes are lethal during embryonic development (16Larue L. Ohsugi M. Hirchenhain J. Kemler R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8263-8267Crossref PubMed Scopus (761) Google Scholar, 17Riethmacher D. Brinkmann V. Birchmeier C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 855-859Crossref PubMed Scopus (407) Google Scholar, 18Hynes R.O. Wagner D.D. J. Clin. Invest. 1996; 98: 2193-2195Crossref PubMed Scopus (58) Google Scholar) and that TM is necessary for embryonic development (4Healy A.M. Rayburn H.B. Rosenberg R.D. Weiler H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 850-854Crossref PubMed Scopus (223) Google Scholar). The lectin-like activity may be influential in a cell-to-cell adhesive interaction (19Ogawa H. Yonezawa S. Maruyama I. Matsushita Y. Tezuka Y. Toyoyama H. Yanagi M. Matsumoto H. Nishijima H. Shimotakahara T. Aikou T. Sato E. Cancer Lett. 2000; 149: 95-103Crossref PubMed Scopus (52) Google Scholar). It is conceivable that TM may function as an additional cellular adhesive molecule. Immunocytochemical studies have localized the TM antigen principally to the intercellular bridges between keratinocytes in stratified squamous epithelium of skin and in various benign or malignant squamous cell carcinomas (7Tezuka Y. Yonezswa S. Maruyama I. Matsushita Y. Shimizu T. Obama H. Sagara M. Shirao K. Kusano C. Natsugoe S. Yoshinaka H. Baba M. Fukumoto T. Aikou T. Sato E. Cancer Res. 1995; 55: 4196-4200PubMed Google Scholar, 20Raife T.J. Lager D.J. Madison K.C. Piette W.W. Howard E.J. Sturm M.T. Chen Y. Lentz S.R. J. Clin. Invest. 1994; 93: 1846-1851Crossref PubMed Scopus (76) Google Scholar, 21Matsushita Y. Yoshiie K. Imamura Y. Ogawa H. Imamura H. Takao S. Yonezawa S. Aikou T. Maruyama I. Sato E. Cancer Lett. 1998; 127: 195-201Crossref PubMed Scopus (27) Google Scholar, 22Larger D.J. Callaghan E.J. Worth S.F. Raife T.J. Lentz S.R. Am. J. Pathol. 1995; 146: 933-943PubMed Google Scholar). Indeed, the levels of both E-cadherin and TM are decreased in metastases of squamouscellcarcinoma(7Tezuka Y. Yonezswa S. Maruyama I. Matsushita Y. Shimizu T. Obama H. Sagara M. Shirao K. Kusano C. Natsugoe S. Yoshinaka H. Baba M. Fukumoto T. Aikou T. Sato E. Cancer Res. 1995; 55: 4196-4200PubMed Google Scholar,23Takeichi M. Science. 1991; 251: 1451-1455Crossref PubMed Scopus (2990) Google Scholar).ItiswellknownthatE-cadherin-dependent cell-to-cell adhesion is important for the maintenance of epithelial structural integrity and that the loss of E-cadherin expression is correlated with increased invasive potential of both carcinoma cell lines and tumor samples (24Conacci-Sorrell M. Zhurinsky J. Ben-Ze'ev A. J. Clin. Invest. 2002; 109: 987-991Crossref PubMed Scopus (525) Google Scholar). The parallel relationship of the expression levels of E-cadherin and TM in tumor progression prompted us to test the adhesion and morphoregulatory activities of TM in comparison with E-cadherin. Although the direct participation of TM in cell-to-cell adhesion is suspected, no supportive experimental evidence has been provided. The present study sought such evidence, through the testing of the hypothesis that TM functions as a cell-to-cell adhesion molecule, and, if so, elucidation of the roles of the participating TM domains. Clones of A2058 melanoma cells that stably expressed green fluorescent protein (GFP)-tagged full-length or lectin-like domain-truncated TM were generated. We report here on our observations that the lectin-like domain of TM prompted the clustering of cells in close proximity with one another by enhancing cell-to-cell adhesiveness through a Ca2+-dependent interaction of TM molecules. This interaction could be involved in limiting cell growth. Materials—Tissue culture dishes and plastic ware were purchased from Corning Life Sciences (Corning, Inc.). Lipofectin and cell culture reagents were from Gibco-BRL (Gaithersburg, MD). Restriction enzymes used in DNA manipulation were purchased from New England Biolabs (Beverly, MA) or Promega Corp. (Madison, WI). The pEGFPN1 vector was from BD Biosciences Clontech (Palo Alto, CA). Monoclonal mouse antibody to the EGF5-EGF6 domain of TM IgG1 antibody and Chromozym PCa were purchased from American Diagnostica Inc. (Greenwich, CT). Human protein C, antithrombin III, G418 (neomycin), and anti-human E-cadherin antibody (clone HECD-1) were from Calbiochem-Novabiochem. Monoclonal anti-lectin-like domain antibody (clone D-3), isotype control IgG antibody, tetramethylrhodamine-conjugated phalloidin, and goat anti-human keratin antibody were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Tetramethylrhodamine goat anti-mouse IgG and tetramethylrhodamine rabbit antigoat antibody were purchased from Molecular Probes, Inc. (Eugene, OR). Supersignal enhanced chemiluminescence reagent was obtained from Pierce. d-Mannose, d-galactose, d-lactose, d-glucose, d-xylose, heparin, low molecular weight heparin, chondroitin sulfate A, chondroitin sulfate B, and chondroitin sulfate C were from Sigma. All other chemicals were of the highest grade commercially available. Construction of Green Fluorescent Protein-tagged Thrombomodulin—Human TM gene in chromosomal DNA was amplified by PCR using a BamHI forward primer, TM719 (5′-CGGGATCCCGGAATGCTTGGGGTCCTGGTCCTTG-3′) and an EcoRI reverse primer (5′-GGAATTCGGAGTCTCTGCGGCGTCCGCT-3′). The 1.7-kb PCR product encoding amino acid residues 1–575 was digested with BamHI and EcoRI. The resulting fragment was ligated to the expression vector pEGFPN1, which had been digested with BglII and EcoRI. This construct was named TMG. The lectin-like module within the NH2-terminal domain of TM was removed by recombinant PCR using the following method. Four primers were designed such that they overlapped and skipped the lectin-like module. Two oligonucleotide primers, TM719 and TM1480 (5′-CATTGCACGCGTGCTCGCAGCCGC-3′), flanked the region from nucleotide 719 to 1480. The other primers were 5′-CACGCTGCAGTCCCAAGCGCCACCCGGCTGCGGCTC-3′ and its reverse complement 5′-GAGCCGCAGCCGGGTGGCGCTTGGGACTGCAGCGTG-3′. Each of the latter two primers was utilized with primer TM719 and TM1480 for PCR amplifications, respectively. The 92- and 66-bp PCR products were purified, denatured, annealed, and amplified using primers TM719 and TM1480. The recombinant 140-bp product was digested with BamHI and MluI and subcloned to replace the wild type NH2-terminal domain of human TM in the expression vector pEGFPN1. The final product was designated TMG(ΔL). Both constructs were confirmed by DNA sequencing. Cell Culture and Transfection of Human Melanoma (A2058) Cells— A2058 cells (ATCC CRL-11147) or HaCaT cells (25Boukamp P. Petrussevska R.T. Breitkreutz D. Hornung J. Markham A. Fusenig N.E. J. Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3493) Google Scholar) were maintained in Dulbecco's modified Eagle's medium supplemented with 0.292 g/liter l-glutamine and 10% fetal bovine serum. A2058 cells grown until 40–60% confluence were transfected with TMG or TMG(ΔL) using Lipofectin reagent. To generate cell lines stably expressing the various constructs, cells were diluted and seeded 2 days after transfection and maintained in Dulbecco's modified Eagle's medium supplemented with 400 μg/ml G418 (neomycin). Clonal expression was examined initially by fluorescence microscopy, and clones for further study were selected and expanded. Thrombomodulin Activity Assay—Cells at a density of 2 × 104/well were split into wells of a 96-well plate and allowed to reattach overnight. The cells were washed in a buffer containing 20 mm Tris (pH 7.4), 0.15 m NaCl, 2.5 mm CaCl2, and 5 mg/ml bovine serum albumin and incubated with 40 μl of reaction mixture (37.5 nm thrombin and 5 μg/ml protein C in the washing buffer) at 37 °C for 30 min. Protein C activation was terminated by adding 40 μl of antithrombin III (6 IU/ml) and heparin (12 IU/ml). The enzymatic activity of activated protein C was measured with the peptide substrate H-d-Lys-Z-Pro-Arg-4-nitroanilidediacetate (Chromozym PCa; 0.5 mm in 20 mm Tris, pH 7.4, 0.15 m NaCl, and 5 mg/ml bovine serum albumin) at 37 °C. The absorbance change at 405 nm was measured with a Thermomax Microplate Reader (Molecular Devices Corp., Sunnyvale, CA). Controls containing thrombin and protein C in the absence of cells were treated similarly. Electrophoresis and Immunoblot Analyses—TM-expressing cells were washed twice with cold phosphate-buffered saline (PBS), lysed in PBS containing 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 5 μg/ml aprotinin, 100 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, and 1 mm EDTA at 4 °C for 20 min and then disrupted with a needle. Total lysates were quantified using a micro-BCA kit (Pierce). Proteins (10 μg) were resolved by SDS-polyacrylamide gel electrophoresis and transferred electrophoretically to a nylon filter. The nylon filter was blocked for 1 h in 5% (v/v) fat-free milk in PBST buffer (PBS with 0.05% Tween 20). After a brief wash in the buffer, the nylon filter was incubated overnight at 4 °C with mouse anti-human TM antiserum diluted in PBST buffer. The antiserum was prepared in our laboratory from BALB/c mice immunized with recombinant TM protein purified from the Pichia pastoris expression system. The primary antibody was removed, and the filter was washed four times in PBST buffer. Subsequent incubation with horseradish peroxidase-labeled goat anti-mouse antibody proceeded at room temperature for 2 h. The filter was washed four times in PBST buffer to remove the secondary antibody, and the blot was visualized with enhanced chemiluminescence reagent. Confocal Microscopy—To examine the distribution of TM, transfected cells were grown on polylysine-coated coverslips overnight. The coverslips were washed three times with cold PBS, and the cells were fixed with a 3.7% (v/v) formaldehyde solution in PBS and mounted with Vectashield mounting medium (Vector Laboratories Inc., Burlingame, CA). Cells were observed using a laser-scanning confocal microscope (Leica model TCS2) with a Leica Mellis-Griot ×63 numerical aperture oil immersion objective, pinhole of 1.5, and electronic zoom 1.5 or 2. GFP was excited using a 488-nm argon/krypton laser and detected with a 515–540-nm band pass filter. Tetramethylrhodamine was excited using a 543-nm argon/krypton laser and detected with a 550–620-nm band pass filter. The images were manipulated with a Leica TCS NT scanner. Immunofluorescence Staining—For immunofluorescence staining, cells were grown on glass coverslips at 37 °C. After being fixed in 3.7% (v/v) formaldehyde in PBS, cells were permeabilized with 0.2% (v/v) Triton X-100 and blocked with 10% fetal bovine serum in PBS. Tetramethylrhodamine-phalloidin, anti-keratin antibody, anti-lectin-like domain antibody, or anti-human TM EGF-like domain antibody was applied to the samples. After three PBS washes, cells were incubated for 1 h at room temperature with tetramethylrhodamine-labeled secondary antibodies. Glass coverslips were washed three times with PBS, mounted, and examined using a confocal microscope. Calcium Switch Methods—Cells were grown overnight on glass coverslips at a constant density (5 × 104 cells/well) in 24-well culture plates. The cells were serum-starved for 8 h, and Ca2+ was removed by incubation with Dulbecco's modified Eagle's medium containing 4 mm EGTA and 1 mm MgCl2 at 37 °C. After 1 h, Dulbecco's modified Eagle's medium containing 1.8 mm Ca2+ was added to replace the Ca2+-free medium. In control experiments, cells received fresh medium in the absence of EGTA. In selected experiments, 10 μg/ml of anti-TM lectin-like antibody, 10 μg/ml isotype-specific control antibody or anti-E-cadherin antibody (20 μg/ml) was added to the Ca2+-containing medium. Determination of Carbohydrate Specificity for TM-mediated Cell Adhesion—A variety of simple carbohydrates (d-mannose, d-galactose, d-lactose, d-glucose, d-xylose), heparin, low molecular weight heparin, chondroitin sulfate A, chondroitin sulfate B, and chondroitin sulfate C were tested to determine their ability to block cell-cell adhesion of the TMG cells. In these experiments, the carbohydrate to be tested was added to the cell monolayer to compare its ability to compete with the TMG. The plates were then incubated at 37 °C overnight and examined by light microscopy (Leica model DM IL). Monolayer Permeability Assay—Horseradish peroxidase flux across A2058 cell monolayers was measured using Transwell cell culture chambers (0.4-μm pore polycarbonate filters; Corning) as previously described (26Herren B. Garton K.J. Coats S. Bowen-Pope D.F. Ross R. Raines E.W. Exp. Cell Res. 2001; 271: 152-160Crossref PubMed Scopus (55) Google Scholar). Briefly, A2058 cells (7.5 × 104) were cultured for 2–3 days in Transwell units (Corning). After reaching confluence, cells were washed, and the medium was replaced with serum-free medium (1.5-ml upper chamber and 2.6-ml lower chamber). Type IV-A horseradish peroxidase (0.1 μm) was added to the upper chamber and incubated at 37 °C. At the indicated time, medium in the lower chambers was assayed for horseradish peroxidase activity using a 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) liquid substrate system according to the manufacturer's instructions (Sigma). Tumor Growth in Vivo—To study the in vivo growth of A2058 cells, BALB/c SCID male mice were used. Cells (106) in 100 μl of PBS were injected subcutaneously into 6–8-week-old male mice. Tumor sizes were recorded every 7 days by measuring the two largest diameters. Expression of TMG and TMG(ΔL) Proteins in A2058 Cells— The cDNA encoding either the full-length human TM or the lectin-like domain truncated TM was cloned from human DNA and ligated to the GFP gene in the mammalian expression vector pEGFPN1 (Fig. 1A). Each recombinant gene was transfected into A2058 cells. Several stable clones expressing TM-GFP fusion proteins were initially screened by the presence of GFP autofluorescence on the cell membrane. These clones (TMG and TMG(ΔL)) were maintained for the experiments described subsequently. The thrombin-interacting domain of TM extended to the outer surface of the cells (as expected in native TM) because the cells expressing TMG or TMG(ΔL) proteins activated protein C in conjunction with thrombin, whereas clones that expressed GFP alone showed no thrombin-dependent protein C activation (Fig. 1B). The TMG and TMG(ΔL) proteins had molecular masses of 110 and 94 kDa, respectively, which were close to the calculated values (Fig. 1C). TM Localization and Cell Morphology—The cell morphology and subcellular localization of GFP-tagged TM protein in A2058 cells were monitored by confocal microscopy. The green fluorescence of the GFP-expressed cells was evident in the cytoplasm, with a higher concentration in the nuclear region (Fig. 2A). Conversely, the green fluorescence of TMG proteins was distributed near the cell surface, particularly at regions of cell-to-cell contact (Fig. 2B). The fluorescence of TMG(ΔL) was evenly distributed on the cell membrane (Fig. 2C). The cells expressing TMG clustered closely together, with strong cell-to-cell adhesion that was distinctly different from parental A2058 cells and cells expressing GFP or TMG(ΔL) (Fig. 2). Phase-contrast images also showed that cells expressing TMG produced compact cell colonies, and cells at the edges of colonies rarely extended membrane protrusions onto the surrounding cell-free surface (Fig. 3A). In contrast, both the clones of the GFP- or TMG(ΔL)-expressed cells were poorly compacted and had a more fibroblastic morphology than TMG-expressed cells (Fig. 3A). Similar results were observed in five stable clones of TMG and seven clones of TMG(ΔL). Lectin-like Domain-mediated Cell-to-cell Adhesion—Because accumulation of TM proteins in the cell-to-cell adhesion sites led to the establishment of the compact clustering morphology, we further explored whether the lectin-like domain of TM played a critical role in cell-to-cell contacts. A monoclonal antibody (clone D-3) directed against the lectin-like domain was used to block the function of lectin-like domain and to test its effect on cell morphology. The antibody bound specifically to TM protein in the TMG cell lysates rather than any proteins in the control or TMG(ΔL) cell lysates, as shown by Western blotting results. When the TMG-expressed cells were incubated with a monoclonal antibody directed against the lectin-like domain, the cell-to-cell contacts were completely inhibited (Fig. 3A). On the other hand, the antibody specific for the EGF-like domain of TM did not cause the TMG-expressed cells to assume the dispersed type colony (Fig. 3B). These results were consistent with a more critical role of the lectin-like domain versus the EGF-like domain in promoting the formation of close cell-to-cell contacts in the cultures of TMG-expressed cells. To further investigate the effect of TM expression on the permeability of the cell-to-cell junction, the infiltration ability of horseradish peroxidase through a monolayer of the cell cultures on the polycarbonate membrane was measured using a Transwell assay system. The permeability of the monolayer of TMG cell cultures was significantly lower compared with the TMG(ΔL) and control cells (Fig. 4). Previous reports have demonstrated that human keratinocytes express TM, which appears to be predominantly localized to the cell membrane and the intercellular bridges (20Raife T.J. Lager D.J. Madison K.C. Piette W.W. Howard E.J. Sturm M.T. Chen Y. Lentz S.R. J. Clin. Invest. 1994; 93: 1846-1851Crossref PubMed Scopus (76) Google Scholar). To further verify the potential physiologic importance of TM in the cell-to-cell adhesion junction, a keratinocyte cell line derived from normal human epidermis (HaCaT) (25Boukamp P. Petrussevska R.T. Breitkreutz D. Hornung J. Markham A. Fusenig N.E. J. Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3493) Google Scholar), which expressed TM endogenously, was used to observe the morphologic changes in the presence of a monoclonal antibody directed against the lectin-like domain of TM. Immunofluorescence microscopy using an anti-TM lectin-like domain antibody showed that a high concentration of TM protein was localized at the intercellular boundary of HaCaT cells (Fig. 5A). The cultured HaCaT cells showed typical compacted sheet-forming colonies. However, HaCaT cells were almost completely dissociated when cultured in the medium containing the anti-lectin-like domain antibody for 24 h. The anti-EGF domain antibody or the isotype control IgG failed to dissociate the compact colonies (Fig. 5, B–D). Ca2+Involvement in TM-mediated Cell-to-cell Adhesion— The NH2 terminus of the TM molecule contains a C-type lectin domain, to which the binding of potential ligand is Ca2+-dependent. The Ca2+ switch method was utilized to investigate whether TM-mediated adhesion junction assembly is Ca2+-dependent. The cell-to-cell contacts of the A2058TMG (Fig. 6A) or HaCaT (Fig. 6F) cells were disrupted when the culture medium was changed to the EGTA-containing medium for 40 min (Fig. 6, B and G). The cell-to-cell adhesion junction was restored in the Ca2+-containing medium for 1 h with the presence of 10 μg/ml control IgG (Fig. 6, C and H). To further verify the TM domain that was directly involved in the Ca2+-modulated adhesion, the functional antibody against the lectin-like domain of TM was added to the Ca2+-containing medium, following the EGTA dissociation of cell-to-cell adhesion. No restoration of cell-to-cell adhesion occurred in the presence of a 10 μg/ml concentration of the antibody (Fig. 6, D and I). Furthermore, TM localization became more uniformly distributed rather than concentrated at the intercellular region (Fig. 6I). Anti-E-cadherin antibody (20 μg/ml) was able to inhibit Ca2+-dependent cell-cell adhesion in HaCaT cells (Fig. 6J) but not in TMG cells (Fig. 6E). TMG Colocalized with Actin Filaments at the Submembrane Cortex—The intracellular domains of adhesion molecules, including cadherins and integrins, interact with the cytoskeleton actin filaments or intermediate filaments through adaptor proteins inside the cell. These interactions provide mechanical continuity from cell to cell (27Kinch M.S. Clark G.J. Der C.J. Burridge K. J. Cell Biol. 1995; 130: 461-471Crossref PubMed Scopus (281) Google Scholar). We examined the colocalization of the TMG proteins and these cytoskeletal elements in TMG-expressed cells by confocal microscopy. The actin and intermediate filaments in the cultured cells were immunohistochemically stained with tetramethylrhodamine-labeled phalloidin or anti-human keratin antibody, respectively. The surface TM molecules and actin filaments were colocalized at the cortex region in cell-cell adhesion sites (Fig. 7A). In contrast, the keratin filaments localized in the cytoplasmic region, and there was little overlap in distribution between the TM and intermediate filaments (Fig. 7B). Influence of Mannose, Chondroitin Sulfate A, or Chondroitin Sulfate C on the Cell-cell Adhesion in TMG Cells—Based on the observation that TMG-expressed A2058 cells formed close clustering colonies, we proposed that the lectin-like domain of TM might mediate the cell-cell adhesion by binding to specific carbohydrate moieties of the neighboring cells. To test the hypothesis, different carbohydrates, including d-mannose, d-galactose, d-glucose, d-xylose, d-lactose, chondroitin sulfate A, B, and C, heparin, and low molecular weight heparin, were tested for their ability to disperse the close clustering morphology of the TMG culture cells. Among these monosaccharides, only mannose was found to be effective in inducing of cell dispersion. Among the sulfate-containing polysaccharides, chondroitin sulfate A (chondroitin 4-sulfate) and chondroitin sulfate C (chondroitin 6-sulfate) also could induce cell dispersion. Heparin showed a minor inhibitory effect on the cell adhesion. On the other ha" @default.
• W1964383788 created "2016-06-24" @default.
• W1964383788 creator A5006541649 @default.
• W1964383788 creator A5040765190 @default.
• W1964383788 creator A5053487335 @default.
• W1964383788 creator A5061009939 @default.
• W1964383788 creator A5064267056 @default.
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• W1964383788 creator A5091264718 @default.
• W1964383788 date "2003-11-01" @default.
• W1964383788 modified "2023-10-18" @default.
• W1964383788 title "Thrombomodulin-mediated Cell Adhesion" @default.
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