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• W2010090837 abstract "The role of endogenously synthesized fibronectin (FN) in assembly was studied with cells lacking or expressing FN. Cells were cultured as homogeneous or mixed populations on surfaces coated with different matrix proteins. Compared with FN-expressing cells, FN-null cells poorly assembled exogenous plasma FN (pFN) when adhered to vitronectin or the recombinant cell-binding domain (III7–10) of FN. Vitronectin had a suppressive effect that was overcome by co-adsorbed pFN or laminin-1 but not by soluble FN. In co-cultures of FN-expressing cells and FN-null cells, endogenous FN was preferentially assembled around FN-expressing cells regardless of the adhesive ligand. If the adhesive ligand was vitronectin, exogenous pFN assembled preferentially around cells expressing cellular FN or recombinant EDa- or EDa+ FN. In co-cultures on vitronectin of FN-null cells and β1 integrin subunit-null cells, fibrils of cellular FN and pFN were preferentially deposited by FN-null (β1-expressing) cells immediately adjacent to (FN-secreting) β1-null cells. In co-cultures on vitronectin of FN-null cells and β1-null cells expressing a chimera with the extracellular domain of β1 and the cytoplasmic domain of β3, preferential assembly was by the chimera-expressing cells. These results indicate that the adhesive ligand is a determinant of FN assembly by cells not secreting endogenous FN (suppressive if vitronectin, non-suppressive but non-supportive if III7–10, supportive if pFN or laminin-1) and suggest that efficient interaction of freshly secreted cellular FN with a β1 integrin, presumably α5β1, substitutes for integrin-mediated adherence to a preformed matrix of laminin-1 or pFN to support assembly of FN. The role of endogenously synthesized fibronectin (FN) in assembly was studied with cells lacking or expressing FN. Cells were cultured as homogeneous or mixed populations on surfaces coated with different matrix proteins. Compared with FN-expressing cells, FN-null cells poorly assembled exogenous plasma FN (pFN) when adhered to vitronectin or the recombinant cell-binding domain (III7–10) of FN. Vitronectin had a suppressive effect that was overcome by co-adsorbed pFN or laminin-1 but not by soluble FN. In co-cultures of FN-expressing cells and FN-null cells, endogenous FN was preferentially assembled around FN-expressing cells regardless of the adhesive ligand. If the adhesive ligand was vitronectin, exogenous pFN assembled preferentially around cells expressing cellular FN or recombinant EDa- or EDa+ FN. In co-cultures on vitronectin of FN-null cells and β1 integrin subunit-null cells, fibrils of cellular FN and pFN were preferentially deposited by FN-null (β1-expressing) cells immediately adjacent to (FN-secreting) β1-null cells. In co-cultures on vitronectin of FN-null cells and β1-null cells expressing a chimera with the extracellular domain of β1 and the cytoplasmic domain of β3, preferential assembly was by the chimera-expressing cells. These results indicate that the adhesive ligand is a determinant of FN assembly by cells not secreting endogenous FN (suppressive if vitronectin, non-suppressive but non-supportive if III7–10, supportive if pFN or laminin-1) and suggest that efficient interaction of freshly secreted cellular FN with a β1 integrin, presumably α5β1, substitutes for integrin-mediated adherence to a preformed matrix of laminin-1 or pFN to support assembly of FN. Deposition of fibronectin (FN) 1The abbreviations used are: FN, fibronectin; cFN, cellular fibronectin; EDa, extra domain a; EDb, extra domain b; FAK, focal adhesion kinase; GFP-FN(EDa+), green fluorescent protein-fused fibronectin with EDa; GFP-FN(EDa-), green fluorescent protein-fused fibronectin without EDa; LN, laminin-1; pFN, plasma fibronectin; Rx-pFN, Rhodamine Red™-X-conjugated pFN; VN, vitronectin; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; APC, allophycocyanin.1The abbreviations used are: FN, fibronectin; cFN, cellular fibronectin; EDa, extra domain a; EDb, extra domain b; FAK, focal adhesion kinase; GFP-FN(EDa+), green fluorescent protein-fused fibronectin with EDa; GFP-FN(EDa-), green fluorescent protein-fused fibronectin without EDa; LN, laminin-1; pFN, plasma fibronectin; Rx-pFN, Rhodamine Red™-X-conjugated pFN; VN, vitronectin; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; APC, allophycocyanin. into extracellular matrix is a dynamic process that is tightly regulated and controlled despite the presence of high concentrations of FN in plasma (200–600 μg/ml, 440–1320 nm) and other body fluids (1Hynes R.O. Rich A. Fibronectins, Springer Series in Molecular Biology. Springer-Verlag, New York1990: 24-48Google Scholar, 2Sakai T. Larsen M. Yamada K.M. Nature. 2003; 423: 876-881Crossref PubMed Scopus (406) Google Scholar). FN is a disulfide-linked dimer of 230–250-kDa subunits. Each subunit is comprised mostly of 3 types of repeating modules: 12 type I modules, 2 type II modules, and 15–17 type III modules depending on splicing; and a variable region (V0, V64, V89, V95, and V120) that is not homologous to other parts of FN. There are two general types of FN: plasma FN (pFN), which is secreted by hepatocytes; and cellular FN (cFN), which is expressed and secreted by fibroblasts and other cell types. There are several structural differences between pFN and cFN. Two type III modules (EDa and EDb) are missing completely in pFN, but variably present in cFN. The V region is also completely missing in one of its subunits in pFN, but present in both subunits of cFN (1Hynes R.O. Rich A. Fibronectins, Springer Series in Molecular Biology. Springer-Verlag, New York1990: 24-48Google Scholar, 3Petersen T.E. Skorstengaard K. Vibe-Pedersen K. Mosher D.F. Fibronectin. Academic Press, Inc., San Diego1989: 1-24Google Scholar). Cell adhesion to immobilized FN by α5β1 integrin is mediated by the RGD sequence in the 10th type III module (III10) (4Pierschbacher M.D. Ruoslahti E. Nature. 1984; 309: 30-33Crossref PubMed Scopus (2839) Google Scholar, 5Yamada K. Kennedy D. J. Cell Biol. 1984; 99: 29-36Crossref PubMed Scopus (319) Google Scholar, 6Li F.Y. Redick S.D. Erickson H.P. Moy V.T. Biophys. J. 2003; 84: 1252-1262Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar).FN matrix assembly is a cell-dependent process that takes place at specialized sites on cell surfaces (7Peters D.M.P. Mosher D.F. J. Cell Biol. 1987; 104: 121-130Crossref PubMed Scopus (62) Google Scholar). The N-terminal 70-kDa region of FN binds to these sites with high affinity in a reversible and saturable manner (8McKeown-Longo P.J. Mosher D.F. J. Cell Biol. 1983; 97: 466-472Crossref PubMed Scopus (199) Google Scholar, 9McKeown-Longo P.J. Mosher D.F. J. Cell Biol. 1985; 100: 364-374Crossref PubMed Scopus (247) Google Scholar). Subsequent homophilic interactions among bound FNs are thought to promote polymerization of FN molecules into insoluble matrix (10McKeown-Longo P.J. Mosher D.F. J. Biol. Chem. 1984; 259: 2210-2215Abstract Full Text PDF Google Scholar, 11Chernousov M.A. Fogerty F.J. Koteliansky V.E. Mosher D.F. J. Biol. Chem. 1991; 266: 10851-10858Abstract Full Text PDF PubMed Google Scholar, 12Chen F. Mosher D.F. J. Biol. Chem. 1996; 271: 9084-9089Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 13Sechler J.L. Rao H.W. Cumiskey A.M. Vega-Colon I. Smith M.S. Murata T. Schwarzbauer J.E. J. Cell Biol. 2001; 154: 1081-1088Crossref PubMed Scopus (97) Google Scholar, 14Zhong C.L. Chrzanowska-Wodnicka M. Brown J. Shaub A. Belkin A.M. Burridge K. J. Cell Biol. 1998; 141: 539-551Crossref PubMed Scopus (488) Google Scholar). The receptors for the N-terminal 70-kDa region of FN are poorly characterized. Cross-linking studies with the N-terminal 70-kDa fragment revealed molecules that migrated with apparent sizes of >3000 kDa in SDS gels, suggesting that the receptors for the N-terminal region are either of unprecedented size or resistant to solubilization with SDS (15Zhang Q.H. Mosher D.F. J. Biol. Chem. 1996; 271: 33284-33292Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Integrins are also implicated in FN assembly (16Akiyama S.K. Yamada S.S. Chen W.T. Yamada K.M. J. Cell Biol. 1989; 109: 863-875Crossref PubMed Scopus (504) Google Scholar, 17Fogerty F.J. Akiyama S.K. Yamada K.M. Mosher D.F. J. Cell Biol. 1990; 111: 699-708Crossref PubMed Scopus (188) Google Scholar, 18Giancotti F.G. Ruoslahti E. Cell. 1990; 60: 849-859Abstract Full Text PDF PubMed Scopus (693) Google Scholar, 19Wennerberg K. Lohikangas L. Gullberg D. Pfaff M. Johansson S. Fassler R. J. Cell Biol. 1996; 132: 227-238Crossref PubMed Scopus (260) Google Scholar, 20Wu C.Y. Hughes P.E. Ginsberg M.H. McDonald J.A. Cell Adhes. Commun. 1996; 4: 149-158Crossref PubMed Scopus (87) Google Scholar, 21Wu C.Y. Keivens V.M. Otoole T.E. McDonald J.A. Ginsberg M.H. Cell. 1995; 83: 715-724Abstract Full Text PDF PubMed Scopus (296) Google Scholar, 22Hughes P.E. DiazGonzalez F. Leong L. Wu C.Y. McDonald J.A. Shattil S.J. Ginsberg M.H. J. Biol. Chem. 1996; 271: 6571-6574Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar). Because integrins are key mediators of cell adhesion to immobilized ligands such as FN, however, sorting out the roles of integrins in assembly of FN is complicated.Both pFN and cFN have the potential to be deposited into the extracellular matrix (23Peters D.M.P. Portz L.M. Fullenwider J. Mosher D.F. J. Cell Biol. 1990; 111: 249-256Crossref PubMed Scopus (71) Google Scholar, 24McKeown-Longo P.J. Mosher D.F. Mosher D.F. Fibronectin. Academic Press, Inc., San Diego1989: 163-179Google Scholar). Knockout of FN in the mouse results in embryonic lethality (25George E.L. Georgeslabouesse E.N. Patelking R.S. Rayburn H. Hynes R.O. Development. 1993; 119: 1079-1091Crossref PubMed Google Scholar), indicating that deposition of FN is necessary for early development. Normal skin wound healing and hemostasis, however, were observed in adult mice with a conditional knockout of pFN (26Sakai T. Johnson K.J. Murozono M. Sakai K. Magnuson M.A. Wieloch T. Cronberg T. Isshiki A. Erickson H.P. Fassler R. Nat. Med. 2001; 7: 324-330Crossref PubMed Scopus (279) Google Scholar), suggesting that pFN has a minor role, and cFN is sufficient for physiologically important assembly of FN. A recent study of effects of siRNAs to inhibit FN synthesis in organ culture indicated that expression of cFN by cleft epithelium directs branching morphogenesis of mouse salivary glands by a process that is inhibited by monoclonal antibodies against the α5, α6, or β1 integrin subunit (2Sakai T. Larsen M. Yamada K.M. Nature. 2003; 423: 876-881Crossref PubMed Scopus (406) Google Scholar). When 0.125–8 mg/ml (0.25–16 μm) exogenous pFN was added to organ culture, branching of salivary glands was stimulated (2Sakai T. Larsen M. Yamada K.M. Nature. 2003; 423: 876-881Crossref PubMed Scopus (406) Google Scholar). These results indicated that small amounts of cFN have effects that can be replicated only by larger amounts of pFN.Here, we compare assembly of FN by monolayers of cFN-expressing and FN-null cells studied as homogeneous or mixed cultures on surfaces coated with different matrix proteins. The nature of the surface coating influenced assembly of exogenous FN much more for FN-null cells than for cFN-expressing cells. FN-null cells poorly assembled exogenous FN when adherent to vitronectin (VN) or the recombinant cell-binding domain (III7–10) of FN. VN had a suppressive effect that was overcome by surface-adsorbed pFN or laminin-1 (LN), but not by exogenously added soluble pFN or recombinant EDa+ or EDa-FN, whereas III7–10 was simply unable to support FN assembly by FN-null cells. cFN was preferentially assembled by cFN-expressing cells regardless of the adhesive ligand. If the adhesive ligand was VN, pFN was assembled preferentially by cFN-expressing cells or transfected FN-null cells expressing recombinant EDa+ or EDa-FN. In co-cultures on VN of FN-null cells and β1-null cells or β1-null cells expressing wild-type β1A or a chimeric β integrin subunit with the extracellular domain of β1 and cytoplasmic domain of β3, the deposition pattern of cFN and pFN was dependent upon re-expression of a β integrin subunit with the extracellular domain of β1 in the cFN-expressing β1-null cells. We conclude that secreted FN assembles preferentially around cFN-expressing cells and such locally assembled cFN functions like surface-adsorbed pFN or LN to support assembly of soluble FN. This supporting effect is at least partially because of interaction of secreted FN with integrins containing the extracellular domain of β1.EXPERIMENTAL PROCEDURESCells—The derivation of FN-/-mouse fibroblastic cells (FN-null cells) and FN+/-cells (cFN-expressing cells) from FN-/- or FN+/-mouse embryonic stem cells was described previously (27Saoncella S. Echtermeyer F. Denhez F. Nowlen J.K. Mosher D.F. Robinson S.D. Hynes R.O. Goetinck P.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2805-2810Crossref PubMed Scopus (330) Google Scholar). β1-null GD25 cells and GD10 cells deficient in the integrin β1 subunit had been derived by a similar technique and transfected with the β1A splice variant to give β1-expressing β1A GD25 cells (19Wennerberg K. Lohikangas L. Gullberg D. Pfaff M. Johansson S. Fassler R. J. Cell Biol. 1996; 132: 227-238Crossref PubMed Scopus (260) Google Scholar) or β1A GD10 cells. β1Aβ3 GD10 cells were generated by transfection of GD10 cells by a β1A/β3 chimeric construct in which the cytoplasmic domain of β1A was replaced with the cytoplasmic domain of β3 (28Danen E.H.J. Sonneveld P. Brakebusch C. Fassler R. Sonnenberg A. J. Cell Biol. 2002; 159: 1071-1086Crossref PubMed Scopus (251) Google Scholar). GFP-expressing FN-null cells were generated by transfection of GFP followed by selection of a stable population with puromycin. A plasmid encoding GFP (pEGFP-N1, Clontech Laboratories, Inc., Palo Alto, CA) was digested with NheI and NotI, and the isolated NheI/NotI DNA fragment encoding GFP was then ligated to pIRESpuro2 (Clontech Laboratories, Inc.) double-di-gested with NheI and NotI. FN-null cells were transfected with the selectable GFP expression plasmid by the liposome method (LipofectAmine™, Invitrogen, Carlsbad, CA).Expression of GFP-fused FNs—pFH101 and pFH100 (29Dufour S. Gutman A. Bois F. Lamb N. Thiery J.P. Kornblihtt A.R. Exp. Cell Res. 1991; 193: 331-338Crossref PubMed Scopus (16) Google Scholar), which encode EDa+, EDb-, V89 human FN, and EDa-, EDb-, V89 human FN, respectively, were gift from Dr. Alberto R. Kornblihtt (Buenos Aires, Argentina). The constructs were manipulated so that protein processing is mediated by the native preprosequence of human FN. A HindIII site, which had been made in the leader sequence region during cloning of pFH101, was erased by substituting a DNA fragment generated by RT (reverse transcription)-PCR of total RNA from AH1F human foreskin dermal fibroblasts. The coding sequence of pFH101 was ligated to the NheI and NotI sites in pIRESpuro2 (Clontech Laboratories, Inc.). GFP was introduced between the third and fourth type III modules as pioneered by Ohashi et al. (30Ohashi T. Kiehart D.P. Erickson H.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2153-2158Crossref PubMed Scopus (197) Google Scholar). Site-directed mutagenesis was performed to create a KpnI restriction enzyme site between the third and fourth type III modules into which the cDNA of GFP was inserted after amplification with primers that added KpnI sites at both ends. The sequence at the insertion site in FN is (III3)TTGTMIEQ(gfp)DEFFGT-PRSD(III4). The GFP coding sequence is underlined. The cloning strategy resulted in the insertion of two amino acids (GT) at one end of the GFP. An EcoRI fragment (2530 bp) after EcoRI digestion of the cDNA encoding GFP-FN(EDa+) was replaced with the EcoRI fragment (2260 bps) of pFH100 to construct GFP-FN(EDa-). Plasmids encoding GFP-FN(EDa+) or GFP-FN(EDa-) were transfected into FN-null cells in monolayer culture by the liposome method. About 30 h after transfection, cells were suspended by trypsinization and plated on coverslips. Because of a low transfection efficiency of about ∼1%, most of the cells remained FN-null.Preparations of Insect Cell Medium Containing Human EDa+ or EDa-FN and of AH1F Cell Medium—Recombinant baculovirus was generated by cotransfection of Baculogold-linearized AcNPV viral DNA (BD Biosciences, San Jose, CA) and cDNA encoding mature human EDa+ FN or EDa-FN, which had been cloned in pCOCO transfer vector (31Mosher D.F. Huwiler K.G. Misenheimer T.M. Annis D.S. Adams J. Methods in Cell-Matrix Adhesion. 69. Academic Press, Burlington, MA2002: 69-81Google Scholar). Viruses were cloned and amplified as described (31Mosher D.F. Huwiler K.G. Misenheimer T.M. Annis D.S. Adams J. Methods in Cell-Matrix Adhesion. 69. Academic Press, Burlington, MA2002: 69-81Google Scholar). Human EDa+ FN and EDa-FN were expressed by infecting High Five insect cells (Invitrogen) in SF900II serum-free medium at 27 °C with pass 4 virus. Conditioned medium was collected ∼65-h postinfection, and concentrated to one-eighth of the initial volume using the Amicon® Ultra-Centrifugal filter device (MWCO = 10,000, Millipore, Bedford, MA) after cells were spun down and removed. The concentrated medium was dialyzed against PBS, pH 7.4, and then dialyzed again against DMEM containing 0.2% BSA. For preparation of medium conditioned with cFN of AH1F human dermal fibroblasts, AH1F cells were incubated on VN-coated surface for 24 h in serum-free medium (DMEM + 0.2% BSA), and the medium was centrifuged to save supernatant. FN present in AH1F cell medium and concentrated insect cell medium was quantified by Western blots.Fluorescent Labeling of pFN (Rx-pFN)—Human pFN, purified on DEAE-cellulose as described before (32Mosher D.F. Johnson R.B. J. Biol. Chem. 1983; 258: 6595-6601Abstract Full Text PDF PubMed Google Scholar), was labeled with Rhodamine Red™-X (FluoReporter Rhodamine Red™-X Protein Labeling kit, Molecular Probes, Eugene, OR) according to the manufacturer's instructions with the following slight modifications. Rhodamine Red™-X dissolved in Me2SO was diluted in 0.5 m carbonate buffer (Na2CO3 and NaHCO3, pH 9.5) to 0.5 mg/ml, and pFN was diluted in 0.05 m carbonate buffer (Na2CO3 and NaHCO3, pH 9.5) to 2 mg/ml for the conjugation reaction.Cell Culture—Cells were suspended with 0.05% trypsin, 0.01% EDTA solution for ∼5 min at 37 °C. Trypsin was quenched by washing with 10% fetal bovine serum in DMEM (Cellgro Mediatech, VA), followed by washing with PBS, and cells were cultured at 60∼70% confluence for 4 or 18 h in DMEM supplemented with 0.2% BSA (Sigma) on glass coverslips coated with pFN (3 μg/ml), LN extracted from Engelbreth-Holm-Swarm mouse tumor (10 μg/ml) (BD Biosciences, Bedford, MA), VN (3 μg/ml) (33Bittorf S.V. Williams E.C. Mosher D.F. J. Biol. Chem. 1993; 268: 24838-24846Abstract Full Text PDF PubMed Google Scholar), or III7–10 (3 μg/ml) (34Leahy D.J. Erickson H.P. Aukhil I. Joshi P. Hendrickson W.A. Proteins. 1994; 19: 48-54Crossref PubMed Scopus (74) Google Scholar, 35Zhang Q.H. Sakai T. Nowlen J. Hayashi I. Fassler R. Mosher D.F. J. Biol. Chem. 1999; 274: 368-375Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) unless indicated. The adhesive proteins were diluted to the indicated concentrations in PBS, pH 7.4, and incubated with the coverslips overnight at 4 °C. After blocking with 1% BSA for 30 min at 37 °C and washing with PBS, coverslips were used within 24 h for cell culture. For some experiments, 2 μm lysophosphatidic acid (Avanti Polar Lipids, Alabaster, AL), 100 μg/ml heparin (Sigma), or 15 μg/ml cyclo[Arg-Gly-Asp-d-Phe-Val] (cRGDfV, BIOMOL Research Laboratories Inc., Plymouth Meeting, PA) was added to culture medium.Fluorescence Microscopy—Deposited FN was visualized with rabbit polyclonal antibodies and Rhodamine Red™-X-conjugated AffiniPure donkey anti-rabbit antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). The rabbit polyclonal antibodies, although produced against human pFN, cross-reacted with mouse FN as demonstrated by Western blotting and ELISA. To visualize exogenous pFN only, Rx-pFN was added to the culture medium at 9 μg/ml. To detect EDa+ FN in the presence of pFN, the IST-9 monoclonal antibody to the EDa type III module of human FN (36Carnemolla B. Borsi L. Zardi L. Owens R.J. Baralle F.E. FEBS Lett. 1987; 215: 269-273Crossref PubMed Scopus (76) Google Scholar) (Harlan Sera-lab Limited, UK) and Alexa Fluor R 350 goat anti-mouse IgG (Molecular Probes) were used. Before staining cells with antibodies, cells were fixed with 3.7% paraformaldehyde for 15 min. For staining of intracellular proteins after paraformaldehyde fixation, cells were permeabilized with 0.2% Triton X-100 for 5 min. For staining of focal adhesion kinase (FAK), cells were fixed and permeabilized with methanol for 5 min. Monoclonal antibody against vinculin (hVIN-1) was from Sigma, monoclonal antibody against β1 integrin (MB1.2) was from Chemicon (Temecula, CA), and monoclonal antibodies against paxillin (clone 349), FAK (clone 77), and β3 integrin (2C9.G2) were from BD Pharmingen (San Diego, CA). Secondary antibodies, Rhodamine Red™-X-conjugated AffiniPure donkey anti-mouse IgG, Rhodamine Red™-X-conjugated AffiniPure donkey anti-rat IgG, and Rhodamine Red™-X-conjugated AffiniPure goat anti-Armenian hamster IgG were from Jackson ImmunoResearch Laboratories. After blocking with 1% BSA overnight at 4 °C or for 10 min at 25 °C, cells were stained with ∼10 μg/ml of primary antibodies for 1 h at room temperature, followed by washing with PBS. After staining cells with ∼10 μg/ml of secondary antibodies for 40 min at room temperature and washing with PBS, coverslips were mounted on Vectashield (Vector Laboratories, Inc., Burlingame, CA). Cells were viewed on an Olympus epifluorescence microscope (BX60, Olympus America Inc., Melville, NY). Pictures were taken with an RT Slider digital camera (Spot Diagnostic Instruments, Inc., Sterling Heights, MI) and processed with Spot RT Software v3 and Adobe Photoshop version 5.0 (Adobe System Inc., San Jose, CA) for Mac OS.Flow Cytometry—Cells were harvested and suspended in PBS containing 1% fetal bovine serum. Approximately 1.0 × 106/ml of cells were incubated with ∼0.5 μg/ml of primary antibody, and then incubated at 4 °C with ∼10 μg/ml of allophycocyanin (APC)-conjugated goat anti-rat secondary antibody (BD Pharmingen) or biotin-conjugated mouse anti-Armenian and Syrian hamster IgG monoclonal antibody and streptavidin-APC conjugate (BD Pharmingen). Mouse β1 was detected with MB1.2. Monoclonal antibody MFR5 to mouse α5, H9.2B8 to mouse αV, 2C9.G2 to mouse β3, and GoH3 to mouse α6 were all from BD Pharmingen). For control samples, cells were incubated only with secondary antibody. Cells (at least 8,000 per sample) were analyzed in a Facs-Caliber (BD Biosciences).Assays of LN and FN—Cells (2 × 105) were cultured at 37 °C in 2 ml of DMEM supplemented with 0.2% BSA in 6-well cell culture cluster plates (surface area per well: 10 cm2, Corning Incorporated, Corning, NY) coated with VN (3 μg/ml). After 4 or 18 h, cells were lysed with 300 μl of extraction buffer (1.5% Triton X-100, 0.05 m Tris-Cl, pH 7.5, 0.3 m NaCl, 1 mm phenylmethylsulfonyl fluoride, protease inhibitor mixture (Roche Applied Science)). Protease inhibitor mixture and 1 mm phenylmethylsulfonyl fluoride were added to the harvested culture medium. The cell extracts and culture medium were centrifuged at 12,000 rpm for 15 min at 4 °C, and the supernatant was saved for Western blotting. For detection of FN and LN in Western blots, our anti-FN rabbit antibodies or anti-LN rabbit antibodies (Novus Biologicals, Inc., Littleton, Co) and peroxidase-conjugated AffiniPure donkey anti-rabbit IgG were used.Cell Adhesion Assays—FN, VN, LN, or BSA were coated at 2–10 μg/ml onto wells of a 96-well plate, and the wells were blocked with 1% BSA in PBS. Cells were incubated for 30 min at 37 °C in a suspension of DMEM containing 0.2% BSA with or without 40 μg/ml of GoH3 anti-mouse α6 monoclonal antibody or 15–30 μg/ml cRGDfV. The cells were then allowed to attach to wells for 2 h at 37 °C. Non-adherent cells were removed by washing, and adherent cells were quantified by colorimetric detection at 595 nm using a microplate reader (Model EL340, BIO-TEK Instruments, Inc.), and data were obtained with DELTA Soft II™ (BioMetallics, Inc.).RESULTSFN-null Cells Assemble Exogenously Added pFN When Adherent to pFN or LN but Not When Adherent to VN or a Recombinant FN Protein That Comprises 7th Type III through 10th Type III Repeat—FN-null cells allow experimental analysis of the contributions of the three sources of FN in cell culture (soluble exogenous FN, exogenous FN adherent to the substrate, and endogenous FN) on assembly of FN. We first studied the effects of adhesive proteins coated on the substrate on assembly of exogenously added FN. During a 4-h culture in serum-free medium (DMEM + 0.2% BSA) containing 9 μg/ml (20 nm) Rhodamine Red™-X-conjugated pFN (Rx-pFN), FN-null cells and cFN-expressing cells both assembled exogenously added Rx-pFN when adherent to pFN- or LN-coated coverslips (Fig. 1A). When adherent to VN, however, cFN-expressing cells assembled pFN better than FN-null cells did (Fig. 1A). Comparing substrates, cFN-expressing cells assembled pFN better when adherent to pFN or LN than when adherent to VN, but the difference was not as great as between FN-null cells cultured on the same substrates. These results indicate that substrate-coated VN is poorly supportive for assembly of exogenous pFN by FN-null cells whereas substrate-coated pFN or LN is supportive and that expression of endogenous cFN facilitates assembly of exogenous pFN.The Effect of VN on Assembly of Exogenous pFN by FN-null Cells Is Suppressive and Overcome by Surface-adsorbed pFN or LN—A number of experiments were performed to characterize further the different effects of adhesive proteins on the assembly of pFN by FN-null cells versus cFN-expressing cells. Because α5β1 integrin is known to be strongly supportive for assembly of FN (16Akiyama S.K. Yamada S.S. Chen W.T. Yamada K.M. J. Cell Biol. 1989; 109: 863-875Crossref PubMed Scopus (504) Google Scholar, 17Fogerty F.J. Akiyama S.K. Yamada K.M. Mosher D.F. J. Cell Biol. 1990; 111: 699-708Crossref PubMed Scopus (188) Google Scholar, 18Giancotti F.G. Ruoslahti E. Cell. 1990; 60: 849-859Abstract Full Text PDF PubMed Scopus (693) Google Scholar, 37McDonald J.A. Quade B.J. Broekelmann T.J. Lachance R. Forsman K. Hasegawa E. Akiyama S. J. Biol. Chem. 1987; 262: 2957-2967Abstract Full Text PDF PubMed Google Scholar), we examined whether differences in expression levels of α5 and β1 subunits accounted for defects in FN assembly by FN-null cells on VN. FN-null cells and cFN-expressing cells expressed similar levels of α5 and β1 integrin subunits as assessed by flow cytometry (Fig. 1B). Mean fluorescence intensities varied <1.7-fold. FN-null cells and cFN-expressing cells were also found to express equal amounts of α6 subunit, adhere equally well to LN and respond to the GoH3 anti-mouse α6 monoclonal antibody by inhibited adhesion to LN (results not shown). Finally, FN-null cells and cFN-expressing cells were found to express similar levels of αV and β3 integrin subunits as tested by flow cytometry (results not shown). αVβ3 was the major receptor for adhesion of both FN-null cells and cFN-expressing cells on a VN-coated surface as assessed by inhibited adhesion upon incubation with cRGDfV, which interacts specifically with αVβ3 integrin (38Brooks P.C. Montgomery A.M.P. Rosenfeld M. Reisfeld R.A. Hu T.H. Klier G. Cheresh D.A. Cell. 1994; 79: 1157-1164Abstract Full Text PDF PubMed Scopus (2169) Google Scholar) (results not shown).Although FN and LN are major ligands for α5β1 and α6β1 integrins, respectively, ligation of β1 integrins is not enough to make FN-null cells competent to assemble exogenous pFN. When FN-null cells were cultured on coverslips coated with III7–10, which has the synergistic and RGD sites of FN for interaction with α5β1 (39Leahy D.J. Aukhil I. Erickson H.P. Cell. 1996; 84: 155-164Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar, 40Aota S. Nomizu M. Yamada K.M. J. Biol. Chem. 1994; 269: 24756-24761Abstract Full Text PDF PubMed Google Scholar, 41Mardon H.J. Grant K.E. FEBS Lett. 1994; 340: 197-201Crossref PubMed Scopus (81) Google Scholar), FN-null cells assembled exogenously added Rx-pFN poorly whereas cFN-expressing cells assembled exogenous FN robustly (Fig. 1A).Previously, β1-null GD25 cells were shown to be also defective in initial assembly of pFN when cultured on VN, and co-coating experiments indicated that the defective assembly is caused by a suppressive effect of VN (35Zhang Q.H. Sakai T. Nowlen J. Hayashi I. Fassler R. Mosher D.F. J. Biol. Chem. 1999; 274: 368-375Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Similar co-coating experiments with FN-null cells indicated that VN is also suppressive for FN-null cells. Thus, a co-coat with 6–9 μg/ml pFN overcame the negative effects of a coat of 2 μg/ml VN on FN-null cells (Fig. 2A) as effectively as it did with control β1-null cells (results not shown). Coating with an increasing amount of LN also overcame the suppressive effects of VN, and an increasing amount of VN overcame the facilitating effect of LN (Fig. 2A). Interestingly, whereas a coat of 5 μg/ml LN poorly supported adhesion and assembly of pFN by FN-null cells, addition of an intermediate coat of 3 μg/ml VN enhanced the facilitating effect of LN (Fig. 2A). A coat with 10 μg/ml III7–10 did not overcome the negative effect of 2 μg/ml VN and did not suppress the facilitating eff" @default.
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• W2010090837 title "Assembly of Exogenous Fibronectin by Fibronectin-null Cells Is Dependent on the Adhesive Substrate" @default.
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