Functional beta 1-Integrins Release the Suppression of Fibronectin Matrix Assembly by Vitronectin*

Qinghong ZhangDagger , Takao SakaiDagger , Julie NowlenDagger , Izumi HayashiDagger , Reinhard Fässler§, and Deane F. MosherDagger

From the Dagger  Departments of Medicine and Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706 and § Department of Experimental Pathology, Lund University, S-221 85 Lund, Sweden

    ABSTRACT
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ABSTRACT
INTRODUCTION
REFERENCES

beta 1-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 beta 1A is expressed. Third, beta 1A containing the extracellular D130A inactivating mutation has a dominant-negative effect on fibronectin assembly. Fourth, beta 1-null cells adherent to vitronectin are flat and lack filopodia, whereas beta 1-null cells adherent to fibronectin or beta 1A-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 beta 1-integrins.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES

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 (1-3). Assembly of soluble fibronectin into matrix is a multistep process under cellular control (4). Among the membrane components implicated in fibronectin matrix assembly, integrins have been firmly demonstrated to have a central role (5-10).

Integrins are a group of cell surface heterdimers of alpha - and beta -glycoprotein subunits that mediate cell adhesion to extracellular matrix proteins such as fibronectin, laminin, vitronectin, and collagen or to countereceptors on other cells (11, 12). The interactions between integrins and their ligands influence a number of cellular processes, including proliferation (13), differentiation (14), survival (15, 16), and migration (17, 18). The extracellular domains of the two subunits are noncovalently associated, forming a ligand-binding pocket, and the cytoplasmic domains interact with cytoskeletal proteins and other cytoplasmic components (12). In addition to mediating adherence, ligation of integrins activates signal transduction pathways (19, 20).

The mechanisms by which integrins modulate fibronectin assembly are not well understood. Transfection of alpha 5-integrin and expression of alpha 5beta 1-integrin by CHO1 cells results in a large increase in fibronectin assembly (5, 21). A chimera comprising the interleukin 2 receptor and the cytoplasmic tail of beta 1, working presumably in a dominant-negative manner, inhibits assembly (22). Monoclonal antibodies to alpha 5 or beta 1 inhibit binding and assembly of fibronectin by fibroblasts and also binding of the N-terminal fibronectin fragment to cell surfaces (23, 24). The 70-kDa N-terminal fragment of fibronectin that mediates binding to assembly sites colocalizes with beta 1-integrin in focal contacts of cycloheximide-treated cells (25). Studies of alpha 5 knock-out mice and cells derived from these mice and also of beta 1 knock-out cells (7, 8, 26) indicate that other molecules can substitute for alpha 5beta 1 in matrix assembly. Expression of activated forms of alpha IIbbeta 3 allows CHO cells to assemble a fibronectin matrix (6, 27). In contrast, overexpression of alpha v, which can pair with beta 1 or beta 3 (28), or alpha 4, which pairs with beta 1 (29), does not confer assembly competency to CHO cells. Transfection of alpha 3 causing overexpression of alpha 3beta 1, an adhesion receptor for entactin and other molecules, however, allows assembly of fibronectin if CHO cells are cultured on an entactin-coated substrate; such assembly is not blocked by antibodies to the cell adhesion domain of fibronectin (30). Thus, expression of several different integrins allows adherent cells to be assembly competent, and the specific integrins specifically required seem dependent on the substrate to which assembling cells are adherent.

We recently found that beta 1-integrin-null GD25 fibroblasts fail to bind the N-terminal 70-kDa matrix assembly domain of fibronectin when the cells are cultured on vitronectin (10). We now show that adhesion to intact vitronectin has a dominant-negative effect on fibronectin matrix assembly by GD25 cells and that expression of functional beta 1A-integrin overcomes the suppression of fibronectin matrix assembly by the vitronectin-coated surface. GD25 cells on vitronectin were well spread, but GD25 cells on fibronectin or GD25-beta 1A cells on either vitronectin or fibronectin were contracted and displayed numerous hairy protrusions. These results indicate that one role of beta 1A-integrin is to facilitate cytoskeleton organization and cell shape and that this signal is missing when beta 1A-deficient cells are adherent to vitronectin.

    EXPERIMENTAL PROCEDURES

Materials-- Lysophosphatidic acid (LPA) was from Avanti Polar Lipids (Birmingham, AL). Fatty acid-free bovine albumin was from Sigma. Vitronectin was purified in its native form (31). Human plasma fibronectin and the 70-kDa N-terminal, gelatin-binding fragment of fibronectin generated by cathepsin D were isolated, iodinated, and reisolated as described previously (32, 33). Fibronectin was also labeled with FITC (33). The labeled proteins were stored in portions at -70 °C in Tris-buffered saline containing 0.1% (w/v) fatty acid-free bovine albumin. A chimeric protein comprising residues 1-53 of vitronectin and the gelatin-binding part of fibronectin (vitronectin portion N-terminal to the fibronectin portion) was constructed and expressed in insect cells using a published strategy (34). Anti-fibronectin and anti-vitronectin antisera were produced in rabbits. IgG was purified from sera by affinity chromatography on protein A.

Recombinant modules III7-III10 of human fibronectin were expressed in bacteria and purified (35) using the expression plasmid kindly provided by Harold Erickson (Duke University). To introduce mutations into the synergistic sequence DRVPHSRN in the III-9 module, in vitro mutagenesis by two-stage polymerase chain reaction was performed using 5' or 3' oligonucleotides containing the base changes and 3' or 5' oligonucleotides outside two convenient BamHI restriction sites. After confirmation of the sequence, the mutated fragment was cloned into the III7-III10-expressing vector (35), replacing the natural BamHI-BamHI fragment. This yielded the mutated polypeptides III7-III10 R1374A, R1374,1379A, and R1379A; in which DRVPHSRN was changed to DAVPHSRN, DAVPHSAN, and DRVPHSAN, respectively.

Cells-- GD25 and GD10 cells were obtained by differentiation of the beta 1-null stem cell clone G201, which is deficient in the integrin beta 1 subunit because of disruption of the beta 1 gene by homologous recombination (36). GD25-beta 1A cells transfected with the beta 1A splice variant of beta 1-integrin were those described by Wennerberg et al. (7). D130A mutant beta 1A was generated in the pBSbeta 1A expression plasmid (7) by oligonucleotide-primed DNA synthesis using the U.S.E. mutagenesis kit (Amersham Pharmacia Biotech). Stably transformed cell lines were cloned after electroporation of the mutant beta 1A cDNA into GD25 cells using puromycin resistance for selection (7, 10). GD25 and GD10 cells were cultured in DMEM (Life Technologies, Inc.) with 10% fetal bovine serum (Intergen, Purchase, NY) (i.e. nonselection medium). The beta 1A-expressing cell lines were cultured in the same medium plus 10 µg/ml puromycin (selection medium) and placed in medium lacking puromycin only for short-term experiments. Cell surface levels of beta 1A, alpha 5, and alpha 6 were monitored by flow cytometry, and clones were chosen in which expression of mutant beta 1A was the same as GD25 cells expressing wild-type beta 1A (10).

Adhesion and Migration Assays-- Cell adhesion and migration assays were performed as described previously (10).

Binding Assay-- Confluent cell layers were usually studied 3 d after sparse seeding in DMEM plus 10% fetal bovine serum. For 2-4-h culture without serum, tissue culture plastic wells were coated overnight with adhesive proteins, 0.02-10 µg/ml, in phosphate-buffered saline at 4 °C, followed by blocking with 1% bovine serum albumin at 37 °C for 1 h. In preparation for experimentation, confluent cells were treated with trypsin and EDTA until cells detached (usually 5 min for GD25-beta 1A and GD25-beta 1A/D130A and 10-15 min for GD25 and GD10), followed by washing twice with DMEM and resuspension at 4 × 105 cells/2-cm2 well in DMEM containing 0.2% bovine serum albumin and 50 µg/ml soybean trypsin inhibitor. Endogenous fibronectin accumulation in cell layers plus medium during the 2-4-h initial incubation time was measured by Western blot. For binding studies, freshly seeded cells on the adhesive substrates or the confluent cell layers were washed with Tris-buffered saline and incubated with 125I-labeled fibronectin or 70-kDa fragment of fibronectin in DMEM containing 0.2% fatty acid-free bovine albumin in the absence or presence of 400 nM LPA for 60 min as described (37).

Microscopy-- Cells cultured on different adhesive substrate-coated glass coverslips were incubated with 400 nM LPA for 60 min at 37 °C. For visualization of bound exogenous fibronectin, cells were incubated with 10 µg/ml FITC-labeled human plasma fibronectin for 60 min at 37 °C. For localization of endogenous fibronectin, cell layers were fixed and incubated with 1:1000 rabbit anti-human fibronectin at 37 °C for 2 h, after the detection of the polyclonal antibody by rhodamine-conjugated goat anti-rabbit IgG. The anti-fibronectin was shown to be cross-reactive with mouse fibronectin at this dilution. Coverslips were mounted with glycerol gel (Sigma), and cells were viewed on an Olympus epifluorescence microscope or a Bio-Rad confocal microscope at the Keck Facility of the University of Wisconsin.

For scanning electron microscopy, cells were washed, prefixed, and post-fixed with 2.5% glutaraldehyde and then 0.1% osmium tetroxide, both in 0.1 M Hepes, pH 7.0. Post-fixed cells were washed, dehydrated with ethanol, and critical point-dried (38). The cells were examined on a Hitachi S-900 high resolution, low voltage, scanning electron microscope at the Integrated Microscopy Resource of the University of Wisconsin.

    RESULTS

The fibronectin matrix of confluent GD25 cells expressing beta 1A integrin is more extensive than the fibronectin matrix of confluent beta 1-null GD25 cells (7, 10). Assembly of fibronectin matrix involves a reversible binding step, which is mediated by the N-terminal region of fibronectin, followed by insolubilization of the fibronectin molecules to form SDS-insoluble multimers (4, 39). We previously found that the N-terminal fragment of fibronectin binds to GD25 cells after adherence to fibronectin but not after adherence to vitronectin (10). To understand how assembly of fibronectin is modulated by vitronectin and beta 1-integrins and to relate our findings to those of Wennerberg et al. (7), we carried out additional studies with GD25 cells lacking beta 1-integrin, GD25-beta 1A cells, which are stably transfected with the cDNA encoding wild-type murine integrin beta 1A, and GD25-beta 1A/D130A cells, which express beta 1A with the inactivating D130A mutation in the extracellular domain.

Fibronectin Assembly by Confluent versus Freshly Seeded GD25 and GD25-beta 1A Cells-- Initial experiments replicated the conditions of Wennerberg et al. (7). Cells were grown to confluence in serum-containing medium and then incubated with the 125I-labeled N-terminal 70-kDa fragment of fibronectin in the absence or presence of LPA, which enhances the binding and assembly of fibronectin (37, 40, 41). In contrast to the quantitative and qualitative decrement in fibronectin staining observed around confluent GD25 cells compared with confluent GD25-beta 1A cells (7, 10), the two cell lines bound the 70-kDa fragment equally well (Fig. 1). Similar results were also found in a 1-h assay of binding of intact fibronectin to confluent cells (data not shown).


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Fig. 1.   Binding of the 70-kDa N-terminal fragment of fibronectin to confluent GD25 and GD25-beta 1A cells. GD25 and GD25-beta 1A cells were grown to confluence in nonselection medium (for GD25) and selection medium (for GD25-beta 1A) for 3 days on tissue culture plastic; the media contained 10% serum. Binding of the 125I-labeled 70-kDa fragment of fibronectin was determined in the absence (-) or presence (+) of 400 nM LPA as described under "Experimental Procedures." The cell-associated 70-kDa fragment in the absence of unlabeled ligand (Total) and in the presence of excess unlabeled 70-kDa fragment (nonspecific binding (NSB)) are presented as mean values ± SD (n = 3). Error bars were omitted when the value was <0.2 ng/mg 70-kDa fragment.

To learn whether decreased fibronectin matrix at confluence is attributable to a deficiency in assembly as GD25 cells are growing to confluence, we studied cells shortly after seeding onto one or the other of the major adhesive proteins in serum; i.e. GD25 and GD25-beta 1A cells were seeded densely on a substrate coated with vitronectin or fibronectin for 2-4 h and tested for binding of FITC-labeled fibronectin or 125I-labeled 70-kDa fragment of fibronectin. Endogenous fibronectin accumulation in cell layers plus medium during the 2-4-h incubation as measured by Western blotting was similar for GD25 cells and GD25-beta 1A cells: 10-50 ng/2-cm2 well. GD25 and GD25-beta 1A cells on both vitronectin and fibronectin substrate had small amounts of cell surface fibronectin staining in punctate or short linear patterns when analyzed by immunofluorescence with antibodies that recognized mouse fibronectin (Fig. 2, a-d). The staining was greatest on GD25-beta 1A cells adherent to fibronectin. GD25 cells on vitronectin did not bind FITC-fibronectin, whereas short linear arrays of FITC-fibronectin binding were abundant when GD25 cells on fibronectin (Fig. 2, A and B). GD25-beta 1A cells adherent on either vitronectin or fibronectin had cell-associated FITC-fibronectin fibrils, especially at areas of cell-cell interaction (Fig. 2, C and D).


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Fig. 2.   Endogenous fibronectin deposition and FITC-fibronectin binding ability of freshly seeded GD25 and GD25-beta 1A cells. GD25 (a, b, A, and B) and GD25-beta 1A (c, d, C, and D) cells were seeded on vitronectin (a, c, A, and C) or fibronectin (b, d, B, and D) in DMEM for 4 h. Cells were incubated without (a-d) or with (A-D) FITC-fibronectin, 10 µg/ml, in the presence of 400 nM LPA for 60 min. The endogenous fibronectin deposition (a-d) was visualized by staining with antibodies against fibronectin, whereas FITC-fibronectin binding (A-D) was visualized on the basis of its intrinsic fluorescence. Bar, 10 µm.

Binding of the 125I-labeled N-terminal 70-kDa fragment of fibronectin to the freshly seeded monolayers correlated with results of fluorescence microscopy. The 70-kDa fragment bound specifically to GD25 cells spread on fibronectin (Fig. 3A) and GD25-beta 1A cells spread on either vitronectin or fibronectin (Fig. 3B). Binding to GD25 cells spread on fibronectin or GD25-beta 1A cells spread on fibronectin or vitronectin was enhanced by LPA treatment (Fig. 3, A and B). For LPA-treated GD25 and GD25-beta 1A cells spread on fibronectin, the Kd values were 7.5 and 4.8 nM, respectively, versus 10.8 and 9.0 nM for the same cells without LPA treatment, and there were 170,000 and 190,000 binding sites per LPA-treated cell, respectively, versus 110,000 and 130,000 sites for untreated cells. GD25-beta 1A cells spread on vitronectin bound less 70-kDa fragment compared with GD25-beta 1A cells spread on fibronectin (Fig. 3B); the Kd was 8.7 nM with LPA versus 14.4 nM without LPA, and there were 80,000 binding sites per LPA-treated cell versus 59,000 sites per untreated cell. GD25 cells spread on vitronectin did not bind the 70-kDa fragment specifically, even after stimulation with LPA (Fig. 3A).


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Fig. 3.   Binding of the 70-kDa N-terminal fragment of fibronectin to freshly seeded GD25 and GD25-beta 1A cells. GD25 (A) and GD25-beta 1A (B) cells were seeded on vitronectin (VN) or fibronectin (FN) in DMEM for 4 h. Increasing amounts of 125I-labeled 70-kDa fragment were incubated with cells in the absence (-LPA) or presence (+LPA) of 400 nM LPA for 60 min. Specific binding was calculated by substraction of nonspecific binding (in the presence of 500 µg/ml unlabeled fibronectin) from total binding and expressed as ng of 70-kDa fragment bound/well. Data represent mean ± SD (n = 3). Error bars are missing when less than the size of symbol.

Effect of beta 1A with the D130A Mutation-- The fact that GD25-beta 1A cells are capable of 70-kDa fragment binding on vitronectin-coated surfaces, whereas GD25 cells are not, indicates that beta 1A-integrins overcome the absent or negative input arising from adhesion of cells to vitronectin. We previously found that expression of beta 1As with inactivating mutations of cytoplasmic residues did not restore the ability of GD25 cells on vitronectin to bind the 70-kDa fragment (10). To test whether beta 1A with a wild-type cytoplasmic domain but a nonactive extracellular domain is restorative, a beta 1A-integrin with a D130A mutation was generated and transfected into GD25 cells. Mutation of the homologous residue in beta 3 is associated with defective platelet alpha IIbbeta 3 function and Glanzmann thrombasthenia (42). GD25 cells expressing the D130A mutant adhered well to vitronectin or fibronectin (Table I) but did not adhere to laminin (data not shown). GD25-beta 1A/D130A cells did not bind the 70-kDa fragment when adhered to vitronectin, even in the presence of LPA (Fig. 4). Therefore, beta 1A-integrin must have both functional intracellular and extracellular domains to support fibronectin matrix assembly by cells cultured on vitronectin. In contrast to GD25 cells lacking beta 1A (10), GD25 cells expressing beta 1A/D130A did not bind the 70-kDa fragment even when cultured on fibronectin (Fig. 4). Furthermore, when D130A cells were studied by immunofluorescence after reaching confluence, no fibronectin extracellular matrix was present (data not shown). These observations indicate that the D130A mutation in beta 1A has a dominant-negative effect on fibronectin matrix assembly. Adherence of GD25 cells expressing beta 1A/D130A to either vitronectin or fibronectin and migration of beta 1A/D130A cells through filters coated with vitronectin or fibronectin was not grossly impaired when compared with GD25 cells or GD25-beta 1A cells (Table I). Therefore, the dominant-negative effect of beta 1A/D130A on cells cultured on fibronectin is specific for fibronectin assembly.

                              
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Table I
Adhesion and migration of GD25, GD25-beta 1A, and GD25-beta 1A/D130A cells
Attachment of GD25, GD25-beta 1A, and GD25-beta 1A/D130A cells to vitronectin or fibronectin or migration through vitronectin- or fibronectin-coated filters in response to platelet-derived growth factor, 30 ng/ml. Results are expressed as a colorimetric assay for the extent of cell attachment to microtiter wells coated with either vitronectin or fibronectin or the number of cells that migrated to the lower surface of the filter. Numbers represent mean ± S.D. (n = 4).


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Fig. 4.   Binding of the 70-kDa N-terminal fragment of fibronectin to freshly seeded GD25, GD25-beta 1A, and GD25-beta 1A/D130A cells. Tissue culture plates were coated with vitronectin (VN) or fibronectin (FN), 2 µg/ml. GD25, GD25-beta 1A, and GD25-beta 1A/D130A cells were seeded on different substrate in DMEM for 4 h. The 125I-labeled 70-kDa fragment was incubated with cells for 60 min. The cell-associated 70-kDa fragment in the absence of unlabeled ligands (Total) and in the presence of excess unlabeled 70-kDa fragment (NSB) are presented as mean values ± SD (n = 3).

Effects of the Central Cell Adhesion Domain of Fibronectin and the Vitronectin Chimera-- One explanation for the differential response of the GD25 cells cultured on vitronectin versus fibronectin is that substratum-bound fibronectin is used as a co-polymerizing molecule (43, 44), in accord with the proposal that a complex of III1, III10, and integrin mediates binding of the N-terminal fragment of fibronectin to cells (45, 46). Therefore, responses of cells adherent to recombinant proteins constituting the central cell adhesion domain of fibronectin were compared with the response to whole fibronectin. GD25 cells spread on III7-III10 were capable of binding of the 70-kDa fragment (Fig. 5). The sequence DRVPHSRN in III9 (residues 1373-1380) has been identified as a synergistic site for integrin functions (47, 48). We mutagenized the synergy sequences to form R1374A with an Ala in place of the Arg1374, R1379A with an Ala replacing Arg1379, and R1374,1379A with Alas replacing both Args. When the mutant proteins were tested, all supported 70-kDa fragment binding to GD25 cells (Fig. 5). To further investigate the determinants for fibronectin matrix assembly on GD25 cells, residues 1-53 of vitronectin containing the RGD sequence (residues 42-44) were expressed as a chimera with the gelatin-binding part of fibronectin. GD25 cells spreading on this chimeric protein bound the 70-kDa fragment specifically (Fig. 5). Of the seven cell adhesion proteins tested, therefore, all were adhesive for GD25 cells, but only intact vitronectin did not support fibronectin matrix assembly.


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Fig. 5.   Comparison of 70-kDa fragment binding to GD25 cells cultured on intact vitronectin, fibronectin, and recombinant molecules containing the cell adhesion domains. Tissue culture plates were coated with vitronectin (VN) or fibronectin (FN), 2 µg/ml, recombinant fibronectin type III module 7-10 wild type (FN7-10), mutant fragments R1374A, R1374,1379A, and R1379A, 10 µg/ml, or a chimeric protein containing residues 1-53 of vitronectin (E53/GE2), 10 µg/ml. GD25 cells were seeded on different substrate in DMEM for 4 h. The 125I-labeled 70-kDa fragment was incubated with cells for 60 min. The cell-associated 70-kDa fragment in the absence of unlabeled ligands (Total) and in the presence of excess unlabeled 70-kDa fragment (NSB) are presented as mean values ± SD (n = 3).

Cell Morphology Correlation-- The LPA stimulation of fibronectin binding and assembly by human fibroblasts correlates with rapid changes in cytoskeleton and cell shape (37). We therefore performed scanning electron microscopy of GD25 and GD25-beta 1A cells on vitronectin or fibronectin after treatment with LPA. GD25 cells on vitronectin were well spread (Fig. 6a). GD25 cells on fibronectin (Fig. 6b) or GD25-beta 1A cells on either vitronectin (Fig. 6c) or fibronectin (Fig. 6d) were contracted and displayed numerous hairy protrusions. Confocal microscopy demonstrated that the GD25 cells on vitronectin were 4-5 µm high, whereas GD25 cells on fibronectin and GD25-beta 1A cells on fibronectin or vitronectin were 9-11 µm high (images not shown). These results suggest that one role of beta 1A integrin is to facilitate cytoskeleton organization and cell shape change and that this signal is missing when beta 1A-deficient cells are adherent to vitronectin even when LPA is present as a costimulant.


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Fig. 6.   Cell morphology of freshly seeded GD25 and GD25-beta 1A cells. GD25 (a and b) and GD25-beta 1A (c and d) cells were seeded on vitronectin (a and c) or fibronectin (b and d) in DMEM for 4 h. Cells on coverslips were incubated with 400 nM LPA for 60 min. The coverslips were washed, processed for scanning electronic microscopy as described under "Experimental Procedures," and photographed. Bar, 10 µm.

Studies of Mixed Substrata-- Integrin-generated signals may depend on the concentration at which the adhesive proteins are coated (49). Therefore, we studied the effect of coating concentration on the effect of vitronectin on 70-kDa fragment binding. Vitronectin had a negative effect only when its coating concentration was >= 2 µg/ml (Fig. 7). Lower coating concentration of vitronectin resulted in adherent cells able to bind the 70-kDa fragment (Fig. 7). The other adhesive substrates tested also supported binding of the 70-kDa fragment to GD25 cells after coating at concentrations as low as 0.02 µg/ml (data not shown). When wells were coated with a mixture of vitronectin and fibronectin, the 70-kDa fragment binding ability of GD25 cells was dependent on the more abundant protein (Fig. 8). After coating with 2 µg/ml vitronectin and increasing concentration of fibronectin, GD25 cells bound more 70-kDa fragment in proportion to the concentration of fibronectin in the initial coating solution. Conversely, increased concentration of vitronectin during co-coating with fibronectin, 2 µg/ml, diminished the 70-kDa fragment binding ability of GD25 cells. The coating efficiency of iodinated fibronectin, 2 µg/ml, from the mixed solutions was measured (data not shown), and only minor competition (<40% decrement compared with control) for fibronectin absorption to plastic surface by vitronectin at the highest vitronectin concentration tested (10 µg/ml) could be demonstrated. When the mixed substrate was preincubated with polyclonal antibodies against vitronectin, the down-regulation of 70-kDa binding of GD25 cells was lost (Fig. 9). Nonimmune IgG did not show this neutralizing effect (Fig. 9). Thus, intact vitronectin on the surface demonstrates a dominant-negative effect on the binding of the 70-kDa fragment of fibronectin to GD25 cells when sufficient fibronectin is present on the surface to support binding of the 70-kDa fragment.


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Fig. 7.   Cellular phenotype as a function of vitronectin concentration during coating. Tissue culture plates were coated with increasing concentrations of vitronectin (VN). GD25 cells were seeded on substrate in DMEM for 4 h, and the 125I-labeled 70-kDa fragment was incubated with cells for 60 min. Specific binding was calculated by substraction of nonspecific binding (in the presence of 500 µg/ml unlabeled fibronectin) from total binding and expressed as ng of 70-kDa fragment bound/mg of cellular protein. Data represent mean ± SD (n = 3).


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Fig. 8.   Mixed vitronectin and fibronectin as substrate. Tissue culture plates were coated with vitronectin, 2 µg/ml, and increasing concentrations of fibronectin (VN + Delta FN) or vice versa (FN + Delta VN). GD25 cells were seeded on adhesive substrate in DMEM for 4 h, and the 125I-labeled 70-kDa fragment was incubated with cells for 60 min. Specific binding was calculated by substraction of nonspecific binding (in the presence of 500 µg/ml unlabeled fibronectin) from total binding and expressed as ng of 70-kDa fragment bound/mg of cellular protein. Data represent mean ± SD (n = 3).


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Fig. 9.   Antibody neutralization of the negative effect of vitronectin in mixed substrate. Tissue culture plates were coated with vitronectin (VN), fibronectin (FN), or vitronectin plus fibronectin (VN + FN), 2 µg/ml for each protein, and then incubated with IgG purified from antiserum against vitronectin (Anti-VN) or nonimmune serum (Nonimmune), 200 µg/ml. GD25 cells were seeded on substrate in DMEM for 4 h, and the 125I-labeled 70-kDa fragment was incubated with cells for 60 min. Specific binding was calculated by substraction of nonspecific binding (in the presence of 500 µg/ml unlabeled fibronectin) from total binding and expressed as ng of 70-kDa fragment bound/mg of cellular protein. Data represent mean ± SD (n = 3). Error bars were omitted when the value was <0.2 ng/mg 70-kDa fragment.

Reproducibility of Results-- The presence of differential responses of GD25 cells on vitronectin and fibronectin in assembly of FITC-fibronectin and binding of the 70-kDa fragment were consistently present in >10 different experiments. To exclude the possibility that the results are unique for GD25 cells, an independent clone, GD10, was derived from the beta 1A-null mouse embryonic stem cells. The observations described above for GD25 cells held true for GD10 cells (data not shown).

    DISCUSSION

Dominant-negative Effect of Vitronectin-- Fibronectin or the recombinant III7-III10 central adhesion domain as an adhesive substrate supported the ability of the cell to bind the 70-kDa fragment or fibronectin, even with GD25 cells lacking beta 1-integrin. alpha vbeta 3-Integrin recognizes the RGD sequence in the III10 module of fibronectin, whereas alpha 5beta 1 and alpha IIbbeta 3 require the presence of the synergy sequence DRVPHSRN in III9 (47, 48, 50, 51). Both the RGD and synergy sites are required for Xenopus gastrulation (52). Recent studies suggest that the one role of the synergy site is to activate alpha 5beta 1 (9), such that alpha 5beta 1 acquires more functions, including support of fibronectin matrix assembly (9) and synergism with the mitogen-activated protein kinase response to growth factor (53). Our initial hypothesis, therefore, was that the effect of substrate-bound fibronectin on beta 1-null cells was positive, e.g. to cause the distinctive cell shape associated with the ability of cells to assemble fibronectin (37), and that failure of GD25 cells to assemble fibronectin when adherent to vitronectin was attributable to lack of the positive signal emanating from ligation of fibronectin. However, when we mutagenized the DRVPHSRV sequence to delete the guanidinium side chains critical for synergy site function, the recombinant III7-III10 fragment retained its ability to support fibronectin assembly. Instead, we found that the negative effect of absorbed vitronectin was dominant, as demonstrated by loss of the negative effect when surface adsorbed with both fibronectin and vitronectin was treated with anti-vitronectin antibodies. The role of fibronectin or the III7-III10 fragment is apparently simply to provide the RGD sequence required for cell adhesion.

The negative determinant(s) of vitronectin are likely located outside the N-terminal RGD-containing region, inasmuch as GD25 cells adherent to a substratum coated with the chimera of the N-terminal sequence of vitronectin and the gelatin-binding part of fibronectin were capable of binding the 70-kDa fragment. Alternatively, the negative signal may arise from a conformation adopted by the RGD sequence when intact vitronectin but not the chimera is adsorbed to the substratum. Vitronectin showed its suppression of 70-kDa fragment binding to beta 1-null GD25 cells only when its coating concentration was >= 2 µg/ml. It is not known whether the density of adsorbed vitronectin is important for the effect or whether vitronectin adsorbed at low concentration adopts a different conformation than vitronectin adsorbed at higher concentration. The cell surface receptor for the negative signal arising as a consequence of adherence to vitronectin is also unknown but is most likely alpha vbeta 3 or alpha vbeta 5, which are expressed by GD25 cells and are both vitronectin receptors (7).

Loss of the Dominant-negative Effect after Expression of beta 1A-- When beta 1A was transfected into GD25 cell, the cells regained the ability to bind the 70-kDa fragment and assemble fibronectin when adherent to vitronectin. Polymerization of fibronectin is dependent on a coordinate series of events thought to involve the actin cytoskeleton, cell surface integrin, and homophilic binding sites within the fibronectin molecule (24, 37, 45, 46, 54-56). Enhanced binding of the 70-kDa fragment of fibronectin induced by LPA treatment or microtubule disruption correlates with changes in cell shape and actin-containing cytoskeleton (37, 56, 57). Rho-dependent actin stress fiber formation and cell contraction induce increase fibronectin binding and represent a rapid, labile way that cells can modulate fibronectin matrix assembly (56, 57). Cells competent for assembly of fibronectin (GD25 on fibronectin and GD25-beta 1A on either vitronectin or fibronectin) have a denser actin microfilament network compared with the incompetent cells (GD25 on vitronectin; Ref. 10). Scanning electron microscopy demonstrated that assembly competent cells also have many more filopodia, which are associated with assembled fibronectin (37).

The results with the D130A mutant indicate that beta 1A rescues the matrix assembly capacity of GD25 cells on vitronectin by forming active integrins that interact with ligands. GD25-beta 1A cells express three beta 1-containing integrins, namely alpha 3beta 1, alpha 5beta 1, and alpha 6beta 1, whereas alpha 1, alpha 2, alpha 4, alpha 9, and alpha v in complex with beta 1 have not been detected (7). The most likely pair of integrin and ligand is alpha 5beta 1 and cellular fibronectin. Cellular fibronectin was synthesized in significant amounts (10-50 ng/2-4 h/4 × 105 cells) by both GD25 and GD25-beta 1A cells. Cycloheximide-treated human foreskin fibroblasts fail to bind the 70-kDa fragment when cultured on vitronectin but do so when cultured on fibronectin or an adhesive fragment of fibronectin (25). The structural determinants in fibronectin for interaction with alpha 5beta 1 are more stringent than the interaction of fibronectin with alpha vbeta 3 (48, 58). CHO cells expressing alpha 5beta 1 do not assemble fibronectin with a mutated synergy region in III9 unless the integrin is activated by manganese (9). Such a result supports a model in which the synergy region activates alpha 5beta 1 to bind fibronectin better, thus concentrating and conformationally altering fibronectin molecules (9, 56). It has been suggested that alpha vbeta 3 also functions in assembly by direct interaction with assembling fibronectin (7). One explanation for our results is that surface-adsorbed vitronectin sequesters alpha v-integrins so that the integrins are unavailable for fibronectin matrix assembly. Such a hypothesis does not explain the differences in cell morphology. We therefore suggest an alternative explanation, namely, that fibronectin assembly by cells on vitronectin requires two events: 1) positive cellular signaling arising from an adhesive interaction involving alpha 5beta 1 that overcomes the negative signaling arising from the adhesive interaction of alpha v-integrins with vitronectin, and 2) strong nanomolar avidity binding of the N-terminal 70-kDa domain of fibronectin to the molecules of large apparent molecular mass that can be identified by crosslinking techniques (41).

When GD25 cells are grown in serum-containing medium, a fibronectin-containing network is present after reaching confluence although diminished when compared with the matrix of cells expressing beta 1A (7, 10). Confluent GD25 cells, however, bound the 70-kDa fragment as well as cells expressing beta 1A. The major adhesive protein for cells immediately after plating in 10% calf serum is vitronectin (59). GD25 cells, therefore, are probably defective in fibronectin matrix assembly immediately after plating but "catch up" with beta 1A-expressing cells as the cells are able to interact with a matrix deposited from endogenous fibronectin and fibronectin present in the culture medium. beta 3 and alpha v colocalize with the fibronectin fibrils in confluent culture of GD25 cells that are able to assemble fibronectin (7). The signal arising from this interaction, which may involve recognition of other components of the matrix as well as the RGD in fibronectin, is enough to overcome the negative signal arising from the interaction with vitronectin.

Endothelial cells and human foreskin fibroblasts bind more fibronectin when cultured on fibronectin than when cultured on vitronectin (44, 60). In the GD25-beta 1A cells, 70-kDa fragment binding was consistantly lower when the cells were cultured on vitronectin than on fibronectin. We tested several fibroblastic cell lines and strains, including human foreskin fibroblasts, and did not observe a reproducible decrement in 70-kDa fragment binding to those cells on substrates of vitronectin, adsorbed either in its native form or after denaturation by urea (data not shown). The differences in our results with foreskin fibroblasts and those previously published (44) may be related to levels of alpha v- or beta 1-integrins as well as of fibronectin produced by the cells under study.

Dominant-negative Effect of the D130A Mutation-- The dominant-negative effect of the D130A mutation was not noted with any of the cytoplasmic mutations of beta 1A that resulted in nonfunctional integrins (10). The mechanism of the dominant-negative effect on fibronectin matrix assembly of expression of the D130A mutant may be similar to the mechanism by which a chimera of the extracellular domain of the interleukin 2 receptor and an intact intracellular domain of beta 1A or beta 3 down-regulates fibronectin assembly (22) and inhibits the activation of integrin (61). The mutant beta 1A subunit and the chimera are molecules with extracellular domains unable to participate in adhesive events but with intracellular domains potentially able to interact with cytoplasmic effector molecules. Overexpression of CD98 has been found to rescue CHO cells expressing chimeras of interleukin 2 and cytoplasmic tails of either beta 1 or beta 3 and thus to allow activation of integrins with wild-type beta 1 cytoplasmic domain (62). It will be interesting to learn whether CD98 can also rescue the D130A cells.

Physiological and Pathophysiological Significance-- Vitronectin is an adhesion-promoting glycoprotein that is involved in the coagulation, fibrinolysis, and complement systems, activities that point to a role in inflammation (59, 63). Vitronectin is present in plasma and serum at concentrations of 200-400 µg/ml (64), and it is also deposited in diseased tissues (59, 63). Vitronectin is synthesized and secreted by tumor-associated fibroblast-like cells (65), suggesting that vitronectin expression can be induced in disease states such as cancer. The adhesion/migration-promoting properties of vitronectin, as well the possibility that vitronectin induces collagenase expression in the tumor stroma (66), suggest potentially important roles for vitronectin in tumorgenesis. Down-regulation of fibronectin matrix assembly by vitronectin and also possibly by other alpha vbeta 3 ligands could be an additional determinant of the migratory malignant phenotype of tumor cells expressing alpha vbeta 3.

    ACKNOWLEDGEMENTS

We thank Doug Annis, Reene Schultz, and Scott Simmons for excellent technical support.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant HL21644 and the Swedish National Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Depts. of Medicine and Biomolecular Chemistry, University of Wisconsin, 1300 University Ave., Madison, WI 53706. Tel.: 608-262-1375; Fax: 608-263-4969; E-mail: dfmosher{at}facstaff.wisc.edu.

    ABBREVIATIONS

The abbreviations used are: CHO, Chinese hamster ovary; LPA, lysophosphatidic acid; FITC, fluorescein isothiocyanate; DMEM, Dulbecco's modified Eagle's medium..

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