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• W1989962141 abstract "Specific association of tissue transglutaminase (tTG) with matrix fibronectin (FN) results in the formation of an extracellular complex (tTG-FN) with distinct adhesive and pro-survival characteristics. tTG-FN supports RGD-independent cell adhesion of different cell types and the formation of distinctive RhoA-dependent focal adhesions following inhibition of integrin function by competitive RGD peptides and function blocking anti-integrin antibodies α5β1. Association of tTG with its binding site on the 70-kDa amino-terminal FN fragment does not support this cell adhesion process, which seems to involve the entire FN molecule. RGD-independent cell adhesion to tTG-FN does not require transamidating activity, is mediated by the binding of tTG to cell-surface heparan sulfate chains, is dependent on the function of protein kinase Cα, and leads to activation of the cell survival focal adhesion kinase. The tTG-FN complex can maintain cell viability of tTG-null mouse dermal fibroblasts when apoptosis is induced by inhibition of RGD-dependent adhesion (anoikis), suggesting an extracellular survival role for tTG. We propose a novel RGD-independent cell adhesion mechanism that promotes cell survival when the anti-apoptotic role mediated by RGD-dependent integrin function is reduced as in tissue injury, which is consistent with the externalization and binding of tTG to fibronectin following cell damage/stress. Specific association of tissue transglutaminase (tTG) with matrix fibronectin (FN) results in the formation of an extracellular complex (tTG-FN) with distinct adhesive and pro-survival characteristics. tTG-FN supports RGD-independent cell adhesion of different cell types and the formation of distinctive RhoA-dependent focal adhesions following inhibition of integrin function by competitive RGD peptides and function blocking anti-integrin antibodies α5β1. Association of tTG with its binding site on the 70-kDa amino-terminal FN fragment does not support this cell adhesion process, which seems to involve the entire FN molecule. RGD-independent cell adhesion to tTG-FN does not require transamidating activity, is mediated by the binding of tTG to cell-surface heparan sulfate chains, is dependent on the function of protein kinase Cα, and leads to activation of the cell survival focal adhesion kinase. The tTG-FN complex can maintain cell viability of tTG-null mouse dermal fibroblasts when apoptosis is induced by inhibition of RGD-dependent adhesion (anoikis), suggesting an extracellular survival role for tTG. We propose a novel RGD-independent cell adhesion mechanism that promotes cell survival when the anti-apoptotic role mediated by RGD-dependent integrin function is reduced as in tissue injury, which is consistent with the externalization and binding of tTG to fibronectin following cell damage/stress. Subtle changes in the extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; FN, fibronectin; HSPG, heparan sulfate proteoglycans; tTG, tissue transglutaminase; PKCα, protein kinase Cα; HOB, human osteoblasts; DMEM, Dulbecco's modified Eagle's medium; MDF, mouse dermal fibroblasts; TCP, tissue culture plastic; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; tet, tetracycline; TdT, terminal deoxynucleotidyl transferase; TUNEL, TdT-mediated dUTP nick end labeling; XTT, 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide; TCP, tissue culture plastic; DTT, dithiothreitol; FAK, focal adhesion kinase.1The abbreviations used are: ECM, extracellular matrix; FN, fibronectin; HSPG, heparan sulfate proteoglycans; tTG, tissue transglutaminase; PKCα, protein kinase Cα; HOB, human osteoblasts; DMEM, Dulbecco's modified Eagle's medium; MDF, mouse dermal fibroblasts; TCP, tissue culture plastic; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; tet, tetracycline; TdT, terminal deoxynucleotidyl transferase; TUNEL, TdT-mediated dUTP nick end labeling; XTT, 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide; TCP, tissue culture plastic; DTT, dithiothreitol; FAK, focal adhesion kinase. complexity/tissue architecture may be crucial for the regulation of the apoptotic machinery leading to anoikis (1Frisch S.M. Screaton R.A. Curr. Opin. Cell Biol. 2001; 13: 555-562Crossref PubMed Scopus (1160) Google Scholar, 2Boudreau N. Sympson C.J. Werb Z. Bissell M.J. Science. 1995; 267: 891-893Crossref PubMed Scopus (1114) Google Scholar). Such a process occurs during tissue injury when the composition and integrity of the ECM are altered in several significant ways (3Davis G.E. Bayless K.J. Davis M.J. Meininger G.A. Am. J. Pathol. 2000; 156: 1489-1498Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). A central component of the ECM, which regulates adhesion-dependent survival signaling, is the adhesive glycoprotein fibronectin (FN) (4Sechler J.L. Schwarzbauer J.E. J. Biol. Chem. 1998; 273: 25533-25536Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). FN binds to cell-surface matrix receptors, primarily the α5β1 integrins, through the Arg-Gly-Asp (RGD) cell-binding site within the Type III10 domain. The importance of the RGD cell-binding domain in adhesion-mediated cell survival has been demonstrated by employing synthetic peptides containing the RGD motif, which induce apoptosis in many cell types, by acting as competitive inhibitors of FN-integrin interaction and activators of caspase 3 (5Hadden H.L. Henke C.A. Am. J. Respir. Crit. Care Med. 2000; 162: 1553-1560Crossref PubMed Scopus (62) Google Scholar, 6Buckley C.D. Pilling D. Henriquez N.V. Parsonage G. Threlfall K. Scheel-Toellner D. Simmons D.L. Akbar A.N. Lord J.M. Salmon M. Nature. 1999; 397: 534-539Crossref PubMed Scopus (404) Google Scholar). A comparable scenario may occur in wounding and inflammatory conditions, whereby fragmentation of FN can lead to detachment-induced apoptosis (5Hadden H.L. Henke C.A. Am. J. Respir. Crit. Care Med. 2000; 162: 1553-1560Crossref PubMed Scopus (62) Google Scholar, 7Jeong J. Han I. Lim Y. Kim J. Parks I. Wood A. Couchman J.R. Oh E.S. Biochem. J. 2001; 356: 231-537Crossref Google Scholar). However, the RGD cell-binding domain of FN is not sufficient in isolation to regulate cell survival, which must be sustained by other critical FN domains such as the C-terminal heparin-binding domain (HepII) (7Jeong J. Han I. Lim Y. Kim J. Parks I. Wood A. Couchman J.R. Oh E.S. Biochem. J. 2001; 356: 231-537Crossref Google Scholar, 8Kapila Y.L. Wang S. Johnson P.W. J. Biol. Chem. 1999; 274: 30906-30913Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), known to synergistically interact with heparan sulfate proteoglycans (HSPG) receptors and integrin α4β1 (9Sharma A. Askari J.A. Humphries M.J. Jones E.Y. Stuart D.I. EMBO J. 1999; 18: 1468-1479Crossref PubMed Google Scholar). Evidence is also accumulating to suggest that changes in the molecular structure and composition of the FN matrix may provide new signals to regulate cell shape, migration, and proliferation. Alterations to the conformation of FN either by multimerization (10Aeschlimann D. Thomazy V. Connect. Tissue Res. 2000; 41: 1-27Crossref PubMed Scopus (289) Google Scholar) or heterotypic association with other matrix molecules (11Pereira M Rybarczyk B.J. Odrljin T.M. Hocking D.C. Sottile J. Simpson-Haidaris P.J. J. Cell Sci. 2002; 115: 609-617Crossref PubMed Google Scholar) could reveal biologically active neo-epitopes, which regulate cell responses via the induction of cytoskeleton assembly (12Hocking D.C. Sottile J. Langenbach K.J. J. Biol. Chem. 2000; 275: 10673-10682Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Modulation of the FN matrix may therefore also be fundamental in the regulation of adhesion-related apoptosis. One protein that binds with high affinity specialized FN domains and modulates the function of FN is tissue-type transglutaminase (tTG, TG-2) (10Aeschlimann D. Thomazy V. Connect. Tissue Res. 2000; 41: 1-27Crossref PubMed Scopus (289) Google Scholar, 13Radek J.T. Jeong J.M. Murthy S.N. Ingham K.C. Lorand L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3152-3156Crossref PubMed Scopus (68) Google Scholar, 14Griffin M. Casadio R. Bergamini C.M. Biochem. J. Rev. 2002; 368: 377-396Crossref PubMed Scopus (0) Google Scholar). tTG is a multifunctional protein implicated in diverse normal and pathological processes (15Fesus L. Piacentini M. Trends Biochem. Sci. 2002; 27: 534-539Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar) but more specifically is regarded as an important component of cell/tissue defense in response to cell damage and stress (16Haroon Z.A. Hettasch J.M. Lai T.S. Dewhirst M.W. Greenberg C.S. FASEB J. 1999; 13: 1787-1795Crossref PubMed Scopus (231) Google Scholar, 17Johnson T.S. Skill N.J. El Nahas A.M. Oldroyd S.D. Thomas G.L. Douthwaite J.A. Haylor J.L. Griffin M. J. Am. Soc. Nephrol. 1999; 10: 2146-2157Crossref PubMed Google Scholar). tTG differs from the other transglutaminases in that the transamidase active site is integrated with a GTP binding/hydrolysis site, which negatively regulates the transamidation activity by structurally blocking the active site (18Liu S. Cerione R.A. Clardy J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2743-2747Crossref PubMed Scopus (277) Google Scholar). Another peculiarity of tTG is its externalization into the ECM via a non-Golgi/endoplasmic reticulum route, through a mechanism that appears to depend on its active-state conformation (19Balklava Z. Verderio E. Collighan R. Gross S. Adams J. Griffin M. J. Biol. Chem. 2002; 277: 16567-16575Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) and an intact FN-binding site in the 28-kDa amino-terminal sandwich region (20Gaudry C.A. Verderio E. Aeschlimann D. Cox A. Smith C. Griffin M. J. Biol. Chem. 1999; 274: 30707-30714Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 21Jeong J.M. Murthy S.N. Radek J.T. Lorand L. J. Biol. Chem. 1995; 10: 5654-5658Abstract Full Text Full Text PDF Scopus (66) Google Scholar). Matrix deposition of tTG increases in situations of tissue damage and cellular stress and results in the immobilization of tTG on matrix FN (16Haroon Z.A. Hettasch J.M. Lai T.S. Dewhirst M.W. Greenberg C.S. FASEB J. 1999; 13: 1787-1795Crossref PubMed Scopus (231) Google Scholar, 17Johnson T.S. Skill N.J. El Nahas A.M. Oldroyd S.D. Thomas G.L. Douthwaite J.A. Haylor J.L. Griffin M. J. Am. Soc. Nephrol. 1999; 10: 2146-2157Crossref PubMed Google Scholar, 20Gaudry C.A. Verderio E. Aeschlimann D. Cox A. Smith C. Griffin M. J. Biol. Chem. 1999; 274: 30707-30714Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 22Verderio E. Nicholas B. Gross S. Griffin M. Exp. Cell Res. 1998; 239: 119-138Crossref PubMed Scopus (140) Google Scholar), which in turn protects tTG from matrix degradation (23Belkin A.M. Akimov S.S. Zaritskaya L.S. Ratnikov B.I. Deryugina E.I. Strongin A.Y. J. Biol. Chem. 2001; 276: 18415-18422Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Consistent with these findings is the observation that guinea pig liver tTG forms specific complexes with human plasma FN, and it appears as a globular protein bound to the N-terminal portion of FN interacting either with the Type I4-I5 motif (24LeMosy E.K. Erickson H.P. Beyer W.F. Radek J.T. Jeong J.M. Murthy S.N. Lorand L. J. Biol. Chem. 1992; 267: 7880-7885Abstract Full Text PDF PubMed Google Scholar) or with a sequence within the gelatin-binding domain of FN (I6-II1-II2-I7-I8-I9) (13Radek J.T. Jeong J.M. Murthy S.N. Ingham K.C. Lorand L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3152-3156Crossref PubMed Scopus (68) Google Scholar). The involvement of tTG in the adhesion of multiple cell types is now consolidated (25Gentile V. Thomazy V. Piacentini M. Fesus L. Davies P.J. J. Cell Biol. 1992; 119: 463-474Crossref PubMed Scopus (229) Google Scholar, 26Jones R.A. Nicholas B. Mian S. Davies P.J. Griffin M. J. Cell Sci. 1997; 110: 2461-2472Crossref PubMed Google Scholar); however, the molecular mechanism and its physiological significance remain controversial. It has been proposed that tTG enhances cell adhesion through matrix remodeling, via protein cross-linking (10Aeschlimann D. Thomazy V. Connect. Tissue Res. 2000; 41: 1-27Crossref PubMed Scopus (289) Google Scholar, 26Jones R.A. Nicholas B. Mian S. Davies P.J. Griffin M. J. Cell Sci. 1997; 110: 2461-2472Crossref PubMed Google Scholar); however, recent findings suggest that tTG involvement in cell-matrix interactions is independent from its transamidation activity (19Balklava Z. Verderio E. Collighan R. Gross S. Adams J. Griffin M. J. Biol. Chem. 2002; 277: 16567-16575Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 27Akimov S.S. Krylov D. Fleischman L.F. Belkin A.M. J. Cell Biol. 2000; 148: 825-838Crossref PubMed Scopus (417) Google Scholar, 28Takahashi H. Isobe T. Horibe S. Takagi J. Yokosaki Y. Sheppard D. Saito Y. J. Biol. Chem. 2000; 275: 23589-23595Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Cell-surface tTG might act as an adhesion co-receptor of integrins β1 and β3 by mediating cell adhesion to the gelatin-binding domain of FN (27Akimov S.S. Krylov D. Fleischman L.F. Belkin A.M. J. Cell Biol. 2000; 148: 825-838Crossref PubMed Scopus (417) Google Scholar) or, conversely, act as an independent adhesion protein by specific binding to α4β1 and α9β1 integrins (28Takahashi H. Isobe T. Horibe S. Takagi J. Yokosaki Y. Sheppard D. Saito Y. J. Biol. Chem. 2000; 275: 23589-23595Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In the current study we have explored the involvement of tTG in FN-mediated cell survival, starting with the hypothesis that the ECM function of tTG is strictly dependent on its association with FN, and that tTG and FN reciprocally modulate each others functions following complex formation. We report that FN-bound tTG supports a novel RGD-independent cell adhesion process, which is mediated by the direct binding of tTG to the cell surface through a mechanism that is critically dependent on cell-surface heparan sulfate and activation of protein kinase Cα (PKCα). We describe that FN-bound tTG, but not FN, can rescue tTG-deficient mouse dermal fibroblasts from apoptosis induced by inhibition of RGD-dependent cell adhesion (anoikis), with maintenance of cell viability. Our findings suggest that matrix FN with bound tTG is functionally distinct from either protein acting in isolation and suggest a novel RGD-independent pathway that may be important in cell survival under conditions of cell damage/stress. Reagents and Antibodies—Mouse monoclonal antibodies included anti-integrin β1 (JB1A) and α5 (PID6) (Chemicon); vinculin and tubulin (Sigma-Aldrich); and tTG (Cub74) (NeoMarkers). Rabbit polyclonal antibodies included anti-human fibronectin (Sigma-Aldrich) and anti-human Tyr(p)397-FAK (Upstate Biotechnology). The tTG inhibitor R283 (19Balklava Z. Verderio E. Collighan R. Gross S. Adams J. Griffin M. J. Biol. Chem. 2002; 277: 16567-16575Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) was synthesized by R. Saint and I. Coutts, Nottingham Trent University. Purified guinea pig liver tTG was either obtained by Sigma-Aldrich or purified according to Leblanc et al. (29Leblanc A. Day N. Menard A. Keillor J.W. Protein Expr. Purif. 1999; 17: 89-95Crossref PubMed Scopus (24) Google Scholar). Human plasma FN and FN proteolytic fragments, GTPγ-S, and synthetic RGD-specific peptides (GRGDTP and GRGDSP) were from Sigma-Aldrich and control RAD peptide (GRADSP) was from Calbiochem. Heparitinase (EC4.2.2.8) was from Sigma-Aldrich, and chondroitinase ABC (protease-free) from Seikagaku Corporation. The PKCα inhibitor GO6976 was from Calbiochem. Cell Lines—Primary human osteoblasts (HOB) were provided by S. Downes (University of Nottingham, Nottingham, UK) and maintained in Dulbecco's modified Eagle's medium (DMEM) as we previously described (30Verderio E. Coombes A. Jones R.A. Li X. Heath D. Downes S. Griffin M. J. Biomed. Mat. Res. 2000; 54: 294-304Crossref Scopus (24) Google Scholar). Swiss 3T3 albino fibroblasts were obtained from American Type Culture Collection and maintained in DMEM supplemented with 10% (v/v) fetal calf serum, 2 mm glutamine, and penicillin/streptomycin (100 units/ml and 100 μg/ml, respectively). Transfected Swiss 3T3 fibroblasts, displaying inducible expression of tTG (clone TG3), were cultured and induced as described by Verderio et al. (22Verderio E. Nicholas B. Gross S. Griffin M. Exp. Cell Res. 1998; 239: 119-138Crossref PubMed Scopus (140) Google Scholar). Primary mouse dermal fibroblasts (MDF) were isolated from the skin of tTG-deficient (MDF-TG-/-) and wild type (MDF-TG+/+) 9-months-old mice and maintained as described by De Laurenzi and Melino (31De Laurenzi V. Melino G. Mol. Cell. Biol. 2001; 21: 148-155Crossref PubMed Scopus (303) Google Scholar). Immobilization of TG on FN and Amino-terminal FN Fragments—96-well plates were coated with human plasma FN (5 μg/ml) or with the 70 kDa (42 μg/ml), 45 kDa (54 μg/ml), and 30 kDa (54 μg/ml) proteolytic fragments of FN in 50 mm Tris-HCl, pH 7.4, 50 μl/well, by incubation at 4 °C for ∼15 h. Concentrations of FN and FN fragments were optimal to saturate tissue culture plastic (TCP), as measured by an ELISA-based assay with polyclonal anti-FN antibody (1/5000) followed by peroxidase-labeled anti-rabbit IgG (1/5000). FN fragments were in a 30-, 60-, and 90-fold stoichiometric excess, respectively, of control FN. For tTG immobilization, the FN solution was removed, the wells were washed once in 50 mm Tris-HCl, pH 7.4, and then incubated with purified guinea pig liver tTG (20 μg/ml) in phosphate-buffered saline (PBS) containing 2 mm EDTA, 100 μl/well. After 1 h at 37 °C, the tTG solution was removed and wells were washed once in 50 mm Tris-HCl, pH 7.4, and once in serum-free culture medium before cell seeding. In some experiments FN-coated plates were blocked with 3% (w/v) lipid milk protein (Marvel) in PBS at 37 °C for 30 min and then washed twice with 50 mm Tris-HCl, pH 7.4, prior to tTG immobilization. The presence of tTG immobilized on FN was confirmed by an ELISA-type assay using Cub74 as we previously described (26Jones R.A. Nicholas B. Mian S. Davies P.J. Griffin M. J. Cell Sci. 1997; 110: 2461-2472Crossref PubMed Google Scholar). The transamidating activity of the immobilized tTG was determined by the incorporation of biotinylated cadaverine into FN as previously described (26Jones R.A. Nicholas B. Mian S. Davies P.J. Griffin M. J. Cell Sci. 1997; 110: 2461-2472Crossref PubMed Google Scholar) and compared with the activity of free tTG standard. Data are expressed as absorbance 450 nm with 5 mm Ca2+ in the reaction buffer minus background absorbance values with 5 mm EDTA. Cell Adhesion Assay—Exponentially growing cells were detached using 0.25% (w/v) trypsin in 5 mm EDTA, collected into medium containing a ∼7% (v/v) fetal calf serum, washed twice with medium without fetal calf serum, and then plated onto 96-well plates (2 × 104 cells/well), coated with FN or FN fragments, with and without immobilized tTG. After a maximum of 20 min incubation (to minimize the secretion of any endogenous protein) at 37 °C in a 5% CO2 atmosphere, cells were fixed in 3.7% (w/v) paraformaldehyde in PBS, permeabilized in 0.1% (v/v) Triton X-100 in PBS, and stained with May Grunwald and Giemsa stain (26Jones R.A. Nicholas B. Mian S. Davies P.J. Griffin M. J. Cell Sci. 1997; 110: 2461-2472Crossref PubMed Google Scholar). In some cases cells were pre-treated for 15 h with 1 mm cycloheximide before plating, to rule out any effects of endogenous secreted adhesion molecules. Digital images of 3 non-overlapping fields covering the central portion of each well were captured using a video digital camera (Olympus DP10) and examined using the Image Analysis program Scion Image (National Institute of Health). At least 9 images of separate fields per sample were examined for a total of at least 400 cells in the FN control. The number of attached cell particles in each field was measured by “thresholding” and “particle analysis” and the spread cells by “density slicing.” Cytoskeletal Staining—Actin stress fibers were visualized using fluorescein isothiocyanate (FITC)-labeled phalloidin and focal adhesions by staining for vinculin. Cells were seeded in 0.79-cm2-wells of chamber slides (8 × 104 cells/well) previously coated with FN and tTG-FN and allowed to adhere for ∼20 min. Cells were fixed using 3.7% (w/v) paraformaldehyde in PBS and permeabilized in 0.1% (v/v) Triton X-100 in PBS. For actin stress fibers, cells were then blocked in PBS buffer supplemented with 5% (w/v) dry milk and then incubated with FITC-labeled phalloidin (20 μg/ml) in blocking buffer. For localization of vinculin, cells were blocked in PBS buffer containing 3% (w/v) bovine serum albumin and then incubated with mouse monoclonal anti-vinculin antibody (1:100) in blocking buffer. Bound antibody was revealed by incubation with rabbit anti-mouse IgG-FITC (1:100) (Dako) in blocking buffer. Coverslips were mounted with Vectashield mountant containing propidium iodide (Vector Laboratories) and examined by laser confocal microscopy using a Leica TCSNT system (Leica Lasertechnik). Consecutive scanning sections (∼2 μm) from the upper to the lower attachment site of cells were overlaid as an extended focus image, and imaged cells (from at least 8 random fields, at least 100 cells in FN control) were scored for actin stress fiber formation with the aid of the Leica TCSNT (version 1.5-451) image processing menu. Inhibition of Integrin-mediated Cell Adhesion—Cells in suspension (2 × 105 cells/ml) were incubated with GRGDTP synthetic peptide (32Dedhar S. Ruoslahti E. Pierschbacher M.D. J. Cell Biol. 1987; 104: 585-593Crossref PubMed Scopus (202) Google Scholar) (50 μg/ml, ∼75 μm, 100 μg/ml ∼150 μm, or 200 μg/ml ∼300 μm). Some experiments were reproduced using the FN-prototype GRGDSP peptide. Alternatively, cells in suspension (2 × 105 cells/ml) were incubated with function blocking anti-integrin antibodies (JB1A, 40 μg/ml; and P1D6, 30 μg/ml). All incubations were performed in serum-free medium at 37 °C for 10 min. Afterwards cells were seeded in the presence of either the competitive RGD peptides or the anti-integrin antibodies. Cell Treatment with C3 Exotranferase—Clostridium botulinum C3 exotransferase (Biomol) (20 μg/ml) was incubated with LipofectAMINE (100 μg/ml) (Invitrogen) in DMEM at 22 °C for 1 h. After that the complex was diluted 10 times in DMEM and added to duplicate 18-h-old cell monolayers (∼80% confluent) in serum-free medium. After 2 h the medium was removed, cells were allowed to recover for ∼30 min in serum-containing DMEM, and then were seeded in 0.79-cm2-wells of chamber slides (45 × 103 cells/well). Quantification of Anoikis and Measurement of Cell Viability—For fluorochrome labeling of DNA strand breaks, 6 × 105 cells were seeded in duplicate in 9.6-cm2-wells pre-coated with FN or tTG-FN in the presence or absence of RGD peptide. After 15 h incubation at 37 °C in a 5% CO2 atmosphere, all cells (adhered and non-adhered) were collected, washed twice in PBS, resuspended at the final concentration of 1.2 × 107 cells/ml, and fixed in suspension by addition of one volume of 4% (w/v) paraformaldehyde in PBS. Cells were then permeabilized in 0.1% (v/v) Triton X-100 in 0.1% (w/v) sodium citrate buffer for 2 min on ice (to minimize loss of fragmented DNA). Cells were labeled with terminal deoxynucleotidyl transferase (TdT) and FITC-dUTP, using a TUNEL kit according to the manufacturer's instructions (Roche). The fluorescence intensity was measured by flow cytometry using a Beckman Coulter EPICS XL. Cells were collected and data stored and analyzed using the software SYSTEM II and WinMDI2.8. DNA fragmentation was also detected by in situ analysis of nuclei following TUNEL, by confocal fluorescent microscopy. Cells found in suspension were fixed and labeled in triplicate on 0.79-cm2-wells of glass slides (∼5 × 104 cells/well). For quantification, the Leica confocal software was used to acquire 3 random images per well for a total of 9 images per experimental sample using a fixed protocol (with constant photo-multiplier tube and section-depth setting). Data are expressed as mean number of apoptotic cells per well. Cell viability was assessed by a colorimetric assay based on the metabolism of the tetrazolium salt XTT (Roche), after an incubation period of 4 h with XTT. Data are expressed as absorbance values at 492 nm after subtraction of values at 690 nm. Statistics—Data are expressed as mean ± S.D. and represent one of at least 3 separate experiments undertaken in triplicate, unless stated otherwise. Differences between data sets were determined by the Student's t test (two-tailed distribution, two-sample equal variance). Differences described as significant in the text correspond to p < 0.05. Tissue Transglutaminase Bound to FN Supports RGD-independent Cell Adhesion of Different Cell Types—Previous work using fluorescent microscopy and immunogold electron microscopy demonstrated a close association of tTG with FN at the cell surface/pericellular matrix (20Gaudry C.A. Verderio E. Aeschlimann D. Cox A. Smith C. Griffin M. J. Biol. Chem. 1999; 274: 30707-30714Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 22Verderio E. Nicholas B. Gross S. Griffin M. Exp. Cell Res. 1998; 239: 119-138Crossref PubMed Scopus (140) Google Scholar), consistent with the in vitro-specific binding of the enzyme with human plasma FN (13Radek J.T. Jeong J.M. Murthy S.N. Ingham K.C. Lorand L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3152-3156Crossref PubMed Scopus (68) Google Scholar, 24LeMosy E.K. Erickson H.P. Beyer W.F. Radek J.T. Jeong J.M. Murthy S.N. Lorand L. J. Biol. Chem. 1992; 267: 7880-7885Abstract Full Text PDF PubMed Google Scholar). To investigate how tTG in complex with FN affects FN cell adhesion, we first bound purified guinea pig liver tTG to human plasma FN coated onto tissue culture plastic (TCP). EDTA was included in the reaction to inhibit tTG transamidating activity. Measurement of binding by an ELISA-type assay showed that FN, immobilized at the saturating concentration of 5 μg/ml, bound a saturating amount of tTG when incubated with 20 μg/ml free tTG (Fig. 1A). Using this initial matrix model of immobilized FN with bound tTG (tTG-FN), the contribution tTG to FN cell adhesion was examined by inhibiting integrin-mediated RGD-dependent cell adhesion with competitive concentrations of soluble RGD peptides. HOBs were selected as the initial cell model because they preferentially adhere on FN in vitro, demonstrate an enhanced spread morphology on biomaterials coated with tTG-FN (33Heath D.J. Christian P. Griffin M. Biomaterials. 2002; 23: 1519-1526Crossref PubMed Scopus (38) Google Scholar), and are characterized by a well defined pattern of integrin cell-surface receptors, consisting mainly of RGD-binding β1 subunit paired with α1, α2, α3, α5, and αV subunits (34Gronthos S. Stewart K. Graves S.E. Hay S. Simmons P.J. J. Bone Miner. Res. 1997; 12: 1189-1197Crossref PubMed Scopus (213) Google Scholar). In the absence of RGD peptide, attachment to tTG-FN was comparable with FN (Fig. 1B, upper panel), although cell spreading appeared to be enhanced on tTG-FN (Fig. 1B, lower panel). At 50 and 100 μg/ml RGD peptide, attachment on FN was significantly reduced (typically to 30-50% of control values on FN) (Fig. 1B, upper panel), but attachment to tTG-FN was not significantly inhibited at these same RGD peptide concentrations. Cell attachment to tTG-FN in the presence of 100 μg/ml RGD peptide was 85-95% of control cell attachment to FN without RGD peptide. Only at 200 μg/ml was cell attachment to tTG-FN significantly lower in comparison to control FN without RGD peptide. At this higher concentration, the RGD peptide may in part act nonspecifically, because the control RAD peptide also led to a small reduction in cell attachment at 200 μg/ml (Fig. 1B, upper inset). Incubation of cells with RGD peptide significantly reduced cell spreading on FN (Fig. 1B, lower panel), typically to 10-50% of control value at 100 μg/ml RGD peptide, but as for cell attachment, cell spreading was only partially reduced on tTG-FN at 50 and 100 μg/ml RGD peptide (usually to 65-85% of control values on FN). Swiss 3T3 fibroblasts displayed a comparable response to HOB cells on the tTG-FN complex. Attachment (Fig. 1C, upper panel) and spreading (Fig. 1C, lower panel) of Swiss 3T3 fibroblasts to FN was significantly decreased with excess RGD peptide, in a more sensitive way than in osteoblasts (typically to 25-35% of control at 100 μg/ml RGD peptide), but was restored to control levels when cells were seeded onto tTG-FN at 50 and 100 μg/ml RGD peptide. An epithelial-like cell line (ECV304) also adhered more efficiently on tTG-FN than FN in the presence of excess RGD peptide (Fig. Suppl. 1). When cells were seeded onto TCP coated with tTG without prior immobilization of FN, cell attachment was found to be negligible at concentrations ranging from 20 to 50 μg/ml of tTG, in the absence or presence of competitive RGD peptide (Fig. 1C). Plates were coated with saturating amounts of FN, and blocking of FN-coated wells with 3% nonfat milk protein prior to tTG immobilization did not affect RGD-independent cell attachment and spreading supported by the tTG-FN matrix formed in the absence of blocking (data not shown). These findings clearly indicate that the complex of tTG bound to FN is the essential component for the RGD-independent cell adhesion to occur. Association of purified tTG to free human plasma FN in solut" @default.
• W1989962141 created "2016-06-24" @default.
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• W1989962141 title "A Novel RGD-independent Cell Adhesion Pathway Mediated by Fibronectin-bound Tissue Transglutaminase Rescues Cells from Anoikis" @default.
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