Julie Nowlen

The assembly of focal adhesions and actin stress fibers by cells plated on fibronectin depends on adhesion-mediated signals involving both integrins and cell-surface heparan sulfate proteoglycans. These two cell-surface receptors interact with different domains of fibronectin. To attempt to identify the heparan sulfate proteoglycans involved, we used fibronectin-null (FN-/-) mouse fibroblasts to eliminate the contribution of endogenous fibronectin during the analysis. FN-/- fibroblasts plated on the cell-binding domain of fibronectin or on antibodies directed against mouse beta1 integrin chains attach but fail to spread and do not form focal adhesions or actin stress fibers. When such cells are treated with antibodies directed against the ectodomain of mouse syndecan-4, they spread fully and assemble focal adhesions and actin stress fibers indistinguishable from those seen in cells plated on intact fibronectin. These results identify syndecan-4 as a heparan sulfate proteoglycan involved in the assembly process. The antibody-stimulated assembly of focal adhesions and actin stress fibers in cells plated on the cell-binding domain of fibronectin can be blocked with C3 exotransferase, an inhibitor of the small GTP-binding protein Rho. Treatment of cells with lysophosphatidic acid, which activates Rho, results in full spreading and assembly of focal adhesions and actin stress fibers in fibroblasts plated on the cell-binding domain of fibronectin. We conclude that syndecan-4 and integrins can act cooperatively in generating signals for cell spreading and for the assembly of focal adhesions and actin stress fibers. We conclude further that these joint signals are regulated in a Rho-dependent manner.

The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules.

Endothelial cell migration is necessary for the formation of new blood vessels. We investigated the effects of 2 lysophospholipid mediators, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA), on endothelial cell migration. S1P and LPA stimulated migration of fetal bovine heart endothelial cells (FBHEs) in a 3D-modified Boyden chamber assay with concentrations as low as 15 nmol/L stimulating a 2-fold change and concentrations in the 1- to 2-micromol/L range stimulating 14- to 20-fold changes. S1P specifically stimulated the migration of several endothelial cell strains but did not stimulate the migration of tumor cells or smooth muscle cells. LPA stimulated some endothelial and nonendothelial cell types to migrate. For FBHEs, S1P and LPA were mostly chemokinetic in checkerboard assays. S1P and LPA stimulated extracellular signal-regulated kinase 1/2 phosphorylation and enhanced paxillin localization to focal contacts, with no discernible change in the actin cytoskeleton in FBHEs. To characterize responsible receptor-dependent signaling pathways, we investigated the involvement of G(i), Rho, and phosphoinositide 3-OH kinase in S1P- and LPA-stimulated migration. Although perturbation of all 3 signaling molecules resulted in decreased migration, the mechanisms underlying the decreased migration were different. Pertussis toxin treatment, to target G(i), caused endothelial cells to develop dense bundles of F-actin and distribute paxillin staining to the cell periphery in response to S1P or LPA. Modification of Rho with C3 toxin disrupted the actin cytoskeleton. Inhibition of phosphoinositide 3-OH kinase decreased S1P- or LPA-induced endothelial cell migration with only minor disruption of the actin cytoskeleton. Inhibition of extracellular signal-regulated kinase kinase with PD98059 caused a loss of phosphorylation of extracellular signal-regulated kinase 1/2, similar to pertussis toxin, but only a minimal decrease in migration. These results indicate that S1P and, for some cells, LPA stimulate migration of endothelial cells through a mechanism that likely requires a balance between G(i) and Rho signaling to achieve the cytoskeletal remodeling necessary for cell migration.

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. 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Development (Camb.). 1993; 119: Google that other can for in matrix assembly. of of cells to assemble a fibronectin matrix 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, F. L. C. McDonald J.A. Ginsberg M.H. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). In of which can with β1 or K. Bauer J.S. Juliano R.L. Ruoslahti E. J. Cell Biol. 1993; PubMed Scopus Google Scholar), or which with β1 C. McDonald J.A. J. Cell 1995; Google Scholar), not assembly to Transfection of of an adhesion receptor for and other assembly of fibronectin cells are on an such assembly is not by antibodies to the cell adhesion domain of fibronectin C. McDonald J.A. J. Cell 1995; PubMed Google Scholar). expression of integrins adherent cells to assembly and the integrins on the substrate to which cells are We that GD25 fibroblasts fail to bind the N-terminal 70-kDa matrix assembly domain of fibronectin when the cells are on vitronectin T. Zhang Q. Fässler R. Mosher D.F. J. Cell Biol. 1998; 141: 527-538Crossref PubMed Scopus (95) Google Scholar). We that adhesion to vitronectin has a dominant-negative effect on fibronectin matrix assembly by GD25 cells and that expression of functional the of fibronectin matrix assembly by the GD25 cells on vitronectin well GD25 cells on fibronectin or cells on either vitronectin or fibronectin contracted and numerous These results that role of is to and cell shape and that this signal is when cells are adherent to acid from from in form Mosher D.F. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). plasma fibronectin and the 70-kDa fragment of fibronectin by and as Mosher D.F. J. Cell Biol. 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. Lohikangas L. Gullberg D. Pfaff M. Johansson S. Fässler R. J. Cell Biol. 1996; 132: 227-238Crossref PubMed Scopus (258) Google by the cell of the β1A into GD25 cells for K. Lohikangas L. Gullberg D. Pfaff M. Johansson S. Fässler R. J. Cell Biol. 1996; 132: 227-238Crossref PubMed Scopus (258) Google Scholar, 10Sakai T. Zhang Q. Fässler R. Mosher D.F. J. Cell Biol. 1998; 141: 527-538Crossref PubMed Scopus (95) Google Scholar). GD25 and cells in with The β1A-expressing cell in the and in only for Cell surface of and by and in which expression of β1A the as GD25 cells β1A T. Zhang Q. Fässler R. Mosher D.F. J. Cell Biol. 1998; 141: 527-538Crossref PubMed Scopus (95) Google Scholar). Cell adhesion and migration as T. Zhang Q. Fässler R. Mosher D.F. J. Cell Biol. 1998; 141: 527-538Crossref PubMed Scopus (95) Google Scholar). cell in with in by with for In for cells with and cells for and and for GD25 and by with and in containing and fibronectin in cell during the by binding cells on the or the cell with and with fibronectin or 70-kDa fragment of fibronectin in containing in the or presence of for as Q. Mosher D.F. J. Cell Biol. 1994; PubMed Scopus Google Scholar). on with for of fibronectin, cells with plasma fibronectin for of fibronectin, cell and with fibronectin for 2 the of the by The to with fibronectin this with and cells on an or a the of the of cells and with and in cells with and Mosher D.F. J. Cell Biol. 1987; PubMed Scopus Google Scholar). The cells on a the of the of The fibronectin matrix of GD25 cells β1A is the fibronectin matrix of β1-null GD25 cells K. Lohikangas L. Gullberg D. Pfaff M. Johansson S. Fässler R. J. Cell Biol. 1996; 132: 227-238Crossref PubMed Scopus (258) Google Scholar, 10Sakai T. Zhang Q. Fässler R. Mosher D.F. J. Cell Biol. 1998; 141: 527-538Crossref PubMed Scopus (95) Google Scholar). Assembly of fibronectin matrix a binding which is by the N-terminal of fibronectin, by of the fibronectin to form (4Mosher D.F. Curr. Opin. Struct. Biol. 1993; 3: 214-222Crossref Scopus (84) Google Scholar, H. Mosher D.F. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). We that the N-terminal fragment of fibronectin to GD25 cells to fibronectin not to vitronectin T. Zhang Q. Fässler R. Mosher D.F. J. Cell Biol. 1998; 141: 527-538Crossref PubMed Scopus (95) Google Scholar). assembly of fibronectin is by vitronectin and and to to of K. Lohikangas L. Gullberg D. Pfaff M. Johansson S. Fässler R. J. Cell Biol. 1996; 132: 227-238Crossref PubMed Scopus (258) Google Scholar), with GD25 cells which are with the and which β1A with the inactivating D130A mutation in the extracellular the of K. Lohikangas L. Gullberg D. Pfaff M. Johansson S. Fässler R. J. Cell Biol. 1996; 132: 227-238Crossref PubMed Scopus (258) Google Scholar). to in and with N-terminal 70-kDa fragment of fibronectin in the or presence of which the binding and assembly of fibronectin Q. Mosher D.F. J. Cell Biol. 1994; PubMed Scopus Google Scholar, Mosher D.F. 1993; PubMed Google Scholar, Q. Mosher D.F. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). In to the and in fibronectin GD25 cells with cells K. Lohikangas L. Gullberg D. Pfaff M. Johansson S. Fässler R. J. Cell Biol. 1996; 132: 227-238Crossref PubMed Scopus (258) Google Scholar, 10Sakai T. Zhang Q. Fässler R. Mosher D.F. J. Cell Biol. 1998; 141: 527-538Crossref PubMed Scopus (95) Google Scholar), the two cell the 70-kDa fragment well results also in a of binding of fibronectin to cells not fibronectin matrix is to a in assembly as GD25 cells are to cells or the other of the proteins in GD25 and cells on a substrate with vitronectin or fibronectin for and for binding of fibronectin or 70-kDa fragment of fibronectin in cell during the as by for GD25 cells and GD25 and cells on vitronectin and fibronectin substrate of cell surface fibronectin in or when by with antibodies that fibronectin The on cells adherent to GD25 cells on vitronectin not bind whereas of binding when GD25 cells on fibronectin A and cells adherent on either vitronectin or fibronectin of and of the N-terminal 70-kDa fragment of fibronectin to the with results of The 70-kDa fragment to GD25 cells on fibronectin and cells on either vitronectin or fibronectin to GD25 cells on fibronectin or cells on fibronectin or vitronectin by and GD25 and cells on fibronectin, the and and for the cells and there and binding sites and sites for cells on vitronectin 70-kDa fragment with cells on fibronectin the with and there binding sites cell sites GD25 cells on vitronectin not bind the 70-kDa fragment with The that cells are of 70-kDa fragment binding on whereas GD25 cells are that the or from adhesion of cells to We that expression of with inactivating of cytoplasmic not the of GD25 cells on vitronectin to bind the 70-kDa fragment T. 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. Cell Biol. 1998; 141: 527-538Crossref PubMed Scopus (95) Google Scholar), GD25 cells not bind the 70-kDa fragment when on fibronectin when D130A cells by fibronectin extracellular matrix present not These observations that the D130A mutation in β1A has a dominant-negative effect on fibronectin matrix assembly. of GD25 cells to either vitronectin or fibronectin and migration of cells with vitronectin or fibronectin not when with GD25 cells or cells the dominant-negative effect of on cells on fibronectin is for fibronectin and migration of and to vitronectin to fibronectin on vitronectin on fibronectin of and cells to vitronectin or fibronectin or migration or in to are as a for the of cell to with either vitronectin or fibronectin or the number of cells that to the surface of the in a of and cells to vitronectin or fibronectin or migration or in to are as a for the of cell to with either vitronectin or fibronectin or the number of cells that to the surface of the for the of the GD25 cells on vitronectin fibronectin is that fibronectin is as a F. J. Cell Biol. PubMed Scopus Google Scholar, J. Cell 1997; PubMed Google Scholar), in with the that a of and mediates binding of the N-terminal fragment of fibronectin to cells J. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, J. Cell Biol. 1996; PubMed Scopus Google Scholar). of cells adherent to proteins the central cell adhesion domain of fibronectin with the to GD25 cells on of binding of the 70-kDa fragment The in has been as a for S. M. Yamada K.M. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, M. Yamada K.M. Ginsberg M.H. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). We the to form with an in of the with an and with the proteins 70-kDa fragment binding to GD25 cells the for fibronectin matrix assembly on GD25 of vitronectin containing the as a chimera with the of GD25 cells on this protein the 70-kDa fragment the cell adhesion proteins therefore, for GD25 only vitronectin not support fibronectin matrix assembly. 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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To analyze the function of TSP family members, we have expressed and purified mouse TSP1 and TSP3 encoded by recombinant baculoviruses in Spodoptera frugiperda cells and compared these TSPs to mouse TSP2 prepared in a similar way. Yields of purified TSP1 and TSP3 were 5-15 and 2-4 micrograms, respectively, per ml of conditioned medium. Mature, secreted mouse T41 and TSP3 had the previously predicted NH2-terminal sequences of DHVKDTSFDLFSI, and SQDLQVIDLLT, respectively. Analysis by SDS-PAGE and rotary shadowing electron microscopy indicated that TSP1 and TSP2 are disulfide bonded trimers whereas TSP3 is a disulfide-bonded pentamer. Binding assays with 45Ca2+ as ligand and immobilized TSP1, TSP2 and TSP3 demonstrated that all three TSPs are calcium-binding proteins. These results are consistent with previous studies of TSP structure and demonstrate that Spodoptera cells process and secrete TSPs having the same subunit organizations and structure as the native vertebrate molecules.