Functional β1-Integrins Release the Suppression of Fibronectin Matrix Assembly by Vitronectin

Abstract

β1-null GD25 fibroblasts adherent to vitronectin fail to bind the N-terminal 70-kDa matrix assembly domain of fibronectin or to assemble fibronectin (Sakai, T., Zhang, Q., Fässler, R., and Mosher, D. F. (1998) J. Cell Biol.141, 527–538). We have made four observations that extend this finding. First, the presence of vitronectin on a substrate that otherwise can support fibronectin assembly has a dominant-negative effect on assembly. Second, the dominant-negative effect is lost when active β1A is expressed. Third, β1A containing the extracellular D130A inactivating mutation has a dominant-negative effect on fibronectin assembly. Fourth, β1-null cells adherent to vitronectin are flat and lack filopodia, whereas β1-null cells adherent to fibronectin or β1A-expressing cells adherent to either vitronectin or fibronectin are contracted and exhibit numerous filopodia. These results reveal, therefore, that GD25 cells adherent to vitronectin can only assume a shape suitable for assembly of fibronectin when there is a countervailing signal from functional β1-integrins. β1-null GD25 fibroblasts adherent to vitronectin fail to bind the N-terminal 70-kDa matrix assembly domain of fibronectin or to assemble fibronectin (Sakai, T., Zhang, Q., Fässler, R., and Mosher, D. F. (1998) J. Cell Biol.141, 527–538). We have made four observations that extend this finding. First, the presence of vitronectin on a substrate that otherwise can support fibronectin assembly has a dominant-negative effect on assembly. Second, the dominant-negative effect is lost when active β1A is expressed. Third, β1A containing the extracellular D130A inactivating mutation has a dominant-negative effect on fibronectin assembly. Fourth, β1-null cells adherent to vitronectin are flat and lack filopodia, whereas β1-null cells adherent to fibronectin or β1A-expressing cells adherent to either vitronectin or fibronectin are contracted and exhibit numerous filopodia. These results reveal, therefore, that GD25 cells adherent to vitronectin can only assume a shape suitable for assembly of fibronectin when there is a countervailing signal from functional β1-integrins. Chinese hamster ovary lysophosphatidic acid fluorescein isothiocyanate Dulbecco's modified Eagle's medium. Fibronectin is an extracellular matrix component that is also present as a soluble protein in plasma and other body fluids. The matrix form of fibronectin is believed to support cell adhesion and migration during embryogenesis, tumor growth, wound healing, angiogenesis, and inflammation (1Mosher D.F. Fibronectin. Academic Press, San Diego1989Google Scholar, 2Hynes R.O. Fibronectins. Springer-Verlag, New York1990Crossref Google Scholar, 3George E.L. Georges-Labouesse E.N. Patel-King R.S. Rayburn H. Hynes R.O. Development (Camb.). 1993; 119: 1079-1091PubMed Google Scholar). Assembly of soluble fibronectin into matrix is a multistep process under cellular control (4Mosher D.F. Curr. Opin. Struct. Biol. 1993; 3: 214-222Crossref Scopus (84) Google Scholar). Among the membrane components implicated in fibronectin matrix assembly, integrins have been firmly demonstrated to have a central role (5Wu C. Bauer J.S. Juliano R.L. McDonald J.A. J. Biol. Chem. 1993; 268: 21883-21888Abstract Full Text PDF PubMed Google Scholar, 6Wu C. Keivens V.M. O'Toole T.E. McDonald J.A. Ginsberg M.H. Cell. 1995; 83: 715-724Abstract Full Text PDF PubMed Scopus (294) Google Scholar, 7Wennerberg K. Lohikangas L. Gullberg D. Pfaff M. Johansson S. Fässler R. J. Cell Biol. 1996; 132: 227-238Crossref PubMed Scopus (258) Google Scholar, 8Yang J.T. Hynes R.O. Mol. Biol. Cell. 1996; 7: 1737-1748Crossref PubMed Scopus (113) Google Scholar, 9Sechler J.L. Corbett S.A. 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PubMed Scopus Google Scholar, Mosher D.F. J. Cell Biol. PubMed Scopus Google Scholar). Fibronectin also with Mosher D.F. J. Cell Biol. PubMed Scopus Google Scholar). The proteins in in containing A protein comprising of vitronectin and the of fibronectin N-terminal to the fibronectin and in cells a J. J. Mosher D.F. J. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). and in from by on protein of fibronectin in and 1994; PubMed Scopus Google the expression by into the in the in by or containing the and or two of the the fragment into the 1994; PubMed Scopus Google Scholar), the the and in which to and GD25 and cells by differentiation of the β1-null cell which is in the β1 of of the β1 by R. Pfaff M. J. Johansson S. R. R. J. Cell Biol. 1995; PubMed Scopus Google Scholar). cells with the β1A of by K. Lohikangas L. Gullberg D. Pfaff M. Johansson S. Fässler R. J. Cell Biol. 1996; 132: 227-238Crossref PubMed Scopus (258) Google Scholar). D130A β1A in the expression K. 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Zhang Q. Fässler R. Mosher D.F. J. Cell Biol. 1998; 141: 527-538Crossref PubMed Scopus (95) Google Scholar). β1A with a cytoplasmic domain a extracellular domain is a with a D130A mutation and into GD25 of the in is with and J.C. O'Toole T.E. Ginsberg M.H. Science. 1990; PubMed Scopus Google Scholar). GD25 cells the D130A well to vitronectin or fibronectin not to not cells not bind the 70-kDa fragment when to vitronectin, in the presence of have functional and extracellular domains to support fibronectin matrix assembly by cells on In to GD25 cells β1A T. Zhang Q. Fässler R. Mosher D.F. J. 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The of fibronectin binding and assembly by fibroblasts with in and cell shape Q. Mosher D.F. J. Cell Biol. 1994; PubMed Scopus Google Scholar). We of GD25 and cells on vitronectin or fibronectin with GD25 cells on vitronectin well GD25 cells on fibronectin or cells on either vitronectin or fibronectin contracted and numerous demonstrated that the GD25 cells on vitronectin whereas GD25 cells on fibronectin and cells on fibronectin or vitronectin not These results that role of β1A is to and cell shape and that this signal is when cells are adherent to vitronectin when is present as a on the which the proteins are Hynes R.O. Cell. 1989; Full Text PDF PubMed Scopus Google Scholar). the effect of on the effect of vitronectin on 70-kDa fragment a effect only when of vitronectin in adherent cells to bind the 70-kDa fragment The other also binding of the 70-kDa fragment to GD25 cells as as not with a of vitronectin and fibronectin, the 70-kDa fragment binding of GD25 cells on the protein with 2 vitronectin and of fibronectin, GD25 cells 70-kDa fragment in to the of fibronectin in the of vitronectin during with fibronectin, 2 the 70-kDa fragment binding of GD25 The of fibronectin, 2 from the not and only with for fibronectin to surface by vitronectin the vitronectin the substrate with antibodies vitronectin, the of 70-kDa binding of GD25 cells lost not this effect vitronectin on the surface a dominant-negative effect on the binding of the 70-kDa fragment of fibronectin to GD25 cells when fibronectin is present on the surface to support binding of the 70-kDa vitronectin and fibronectin as with vitronectin, 2 and of fibronectin or GD25 cells on substrate in for and the 70-kDa fragment with cells for binding by of binding the presence of from binding and as of 70-kDa fragment of cellular of the effect of vitronectin in with vitronectin fibronectin or vitronectin fibronectin 2 for and with from vitronectin or GD25 cells on substrate in for and 70-kDa fragment with cells for binding by of binding the presence of from binding and as of 70-kDa fragment of cellular when the 70-kDa The presence of of GD25 cells on vitronectin and fibronectin in assembly of and binding of the 70-kDa fragment present in the that the results are for GD25 an from the The observations for GD25 cells for cells not Fibronectin or the central adhesion domain as an substrate the of the cell to bind the 70-kDa fragment or fibronectin, with GD25 cells the in the of fibronectin, whereas and the presence of the in S. 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