Remodeling of extracellular matrices occurs during development, wound healing, and in a variety of pathological processes including atherosclerosis, ischemic injury, and angiogenesis. Thus, identifying factors that control the balance between matrix deposition and degradation during tissue remodeling is essential for understanding mechanisms that regulate a variety of normal and pathological processes. Using fibronectin-null cells, we found that fibronectin polymerization into the extracellular matrix is required for the deposition of collagen-I and thrombospondin-1 and that the maintenance of extracellular matrix fibronectin fibrils requires the continual polymerization of a fibronectin matrix. Further, integrin ligation alone is not sufficient to maintain extracellular matrix fibronectin in the absence of fibronectin deposition. Our data also demonstrate that the retention of thrombospondin-1 and collagen I into fibrillar structures within the extracellular matrix depends on an intact fibronectin matrix. An intact fibronectin matrix is also critical for maintaining the composition of cell-matrix adhesion sites; in the absence of fibronectin and fibronectin polymerization, neither ␣5␤1 integrin nor tensin localize to fibrillar cell-matrix adhesion sites. These data indicate that fibronectin polymerization is a critical regulator of extracellular matrix organization and stability. The ability of fibronectin polymerization to act as a switch that controls the organization and composition of the extracellular matrix and cell-matrix adhesion sites provides cells with a means of precisely controlling cell-extracellular matrix signaling events that regulate many aspects of cell behavior including cell proliferation, migration, and differentiation. INTRODUCTIONExtracellular matrix remodeling plays an important role during development, wound healing, atherosclerosis, ischemic injury, and angiogenesis. Perturbing matrix remodeling by preventing the turnover of collagen I or by altering the levels of matrix-degrading proteases or protease inhibitors has been shown to result in fibrosis, arthritis, reduced angiogenesis, and developmental abnormalities (Liu et al., 1995;Vu et al., 1998;Holmbeck et al., 1999;Ducharme et al., 2000). In normal adult tissue, some extracellular matrix components such as elastin and fibrillar collagen have half lives of months to years (Krane, 1985;Debelle and Tamburro, 1999). Other extracellular matrix components, such as proteoglycans, thrombospondin-1 and -2, and vitronectin, can be endocytosed and degraded in the lysosomes (McKeownLongo et al., 1984;Yanagishita and Hascall, 1984; MurphyUllrich and Mosher, 1987;Hausser et al., 1992;Godyna et al., 1995a;Pijuan-Thompson and Gladson, 1997;Memmo and McKeown-Longo, 1998). Extracellular matrix molecules can also be degraded extracellularly by proteases such as matrix metalloproteinases (MMPs), plasminogen activators, and plasmin (Hynes, 1990;Marchina and Barlati, 1996;Shapiro, 1998).Recent data indicate that polymerized forms of extracellular matri...

Ligation of integrins with extracellular matrix molecules induces the clustering of actin and actin-binding proteins to focal adhesions, which serves to mechanically couple the matrix with the cytoskeleton. During wound healing and development, matrix deposition and remodeling may impart additional tensile forces that modulate integrin-mediated cell functions, including cell migration and proliferation. We have utilized the ability of cells to contract floating collagen gels to determine the effect of fibronectin polymerization on mechanical tension generation by cells. Our data indicate that fibronectin polymerization promotes cell spreading in collagen gels and stimulates cell contractility by a Rho-dependent mechanism. Fibronectin-stimulated contractility was dependent on integrin ligation; however, integrin ligation by fibronectin fragments was not sufficient to induce either tension generation or cell spreading. Furthermore, treatment of cells with polyvalent RGD peptides or pre-polymerized fibronectin did not stimulate cell contractility. Fibronectin-induced contractility was blocked by agents that inhibit fibronectin polymerization, suggesting that the process of fibronectin polymerization is critical in triggering cytoskeletal tension generation. These data indicate that Rho-mediated cell contractility is regulated by the process of fibronectin polymerization and suggest a novel mechanism by which extracellular matrix fibronectin regulates cytoskeletal organization and cell function.Fibronectin is a high molecular mass, multidomain glycoprotein that circulates in a soluble form in the plasma and is also found in an insoluble, multimeric form within the extracellular matrix (1). The polymerization of soluble fibronectin into insoluble fibrils within the extracellular matrix is a dynamic, celldependent process that is mediated by a series of events involving the actin cytoskeleton and integrin receptors (2). The adhesion of cells to fibronectin via integrin receptors has been shown to be important in the regulation and coordination of such complex processes as cell growth, differentiation, and migration (1). Ligation of integrins with extracellular matrix molecules, including fibronectin, induces the clustering of actin and actin-binding proteins to focal adhesions, which serves to mechanically couple the extracellular matrix with the actin cytoskeleton (3). Recent studies suggest that the interaction of cells with the extracellular matrix form of fibronectin triggers changes in cell cycle progression (4 -7), cell migration (8), and actin filament organization (6, 9) that are distinct from those generated by the interaction of cells with nonpolymerized fibronectin. The mechanism by which the matrix form of fibronectin gives rise to cellular phenotypes distinct from protomeric fibronectin is unknown.Studies aimed at understanding the effect of extracellular matrix on cell function indicate that cells respond to fibronectin-coated beads with a local reinforcement of cytoskeletal links that are proportional t...

The interaction of cells with fibronectin generates a series of complex signaling events that serve to regulate several aspects of cell behavior, including growth, differentiation, adhesion, and motility. The formation of a fibronectin matrix is a dynamic, cell-mediated process that involves both ligation of the α5β1 integrin with the Arg-Gly-Asp (RGD) sequence in fibronectin and binding of the amino terminus of fibronectin to cell surface receptors, termed “matrix assembly sites,” which mediate the assembly of soluble fibronectin into insoluble fibrils. Our data demonstrate that the amino-terminal type I repeats of fibronectin bind to the α5β1 integrin and support cell adhesion. Furthermore, the amino terminus of fibronectin modulates actin assembly, focal contact formation, tyrosine kinase activity, and cell migration. Amino-terminal fibronectin fragments and RGD peptides were able to cross-compete for binding to the α5β1 integrin, suggesting that these two domains of fibronectin cannot bind to the α5β1 integrin simultaneously. Cell adhesion to the amino-terminal domain of fibronectin was enhanced by cytochalasin D, suggesting that the ligand specificity of the α5β1 integrin is regulated by the cytoskeleton. These data suggest a new paradigm for integrin-mediated signaling, where distinct regions within one ligand can modulate outside-in signaling through the same integrin.

The interaction of cells with the extracellular matrix (ECM) form of fibronectin (FN) triggers changes in growth, migration, and cytoskeletal organization that differ from those generated by soluble FN. As cells deposit and remodel their FN matrix, the exposure of new epitopes may serve to initiate responses unique to matrix FN. To determine whether a matricryptic site within the III1 module of FN modulates cell growth or cytoskeletal organization, a recombinant FN with properties of matrix FN was constructed by directly linking the cryptic, heparin-binding COOH-terminal fragment of III1 (III1H) to the integrin-binding III8–10 modules (glutathione-S-transferase [GST]–III1H,8–10). GST–III1H,8–10 specifically stimulated increases in cell growth and contractility; integrin ligation alone was ineffective. A construct lacking the integrin-binding domain (GST–III1H,2–4) retained the ability to stimulate cell contraction, but was unable to stimulate cell growth. Both GST–III1H,2–4 and matrix FN colocalized with caveolin and fractionated with low-density membrane complexes by a mechanism that required heparan sulfate proteoglycans. Disruption of caveolae inhibited the FN- and III1H-mediated increases in cell contraction and growth. These data suggest that a portion of ECM FN partitions into lipid rafts and differentially regulates cytoskeletal organization and growth, in part, through the exposure of a neoepitope within the conformationally labile III1 module.

Abstract. Fibronectin matrix assembly is a cell-dependent process which is upregulated in tissues at various times during development and wound repair to support the functions of cell adhesion, migration, and differentiation. Previous studies have demonstrated that the ot5131 integrin and fibronectin's amino terminus and III-1 module are important in fibronectin polymerization. We have recently shown that fibronectin's III-1 module contains a conformationally sensitive binding site for fibronectin's amino terminus (Hocking, D.C., J. Sottile, and P.J. McKeown-Longo. 1994. J. Biol. Chem. 269: 19183-19191). The present study was undertaken to define the relationship between the ot5131 integrin and fibronectin polymerization. Solid phase binding assays using recombinant III-10 and II1-1 modules of human plasma fibronectin indicated that the III-10 module contains a conformation-dependent binding site for the III-1 module of fibronectin. Unfolded III-10 could support the formation of a ternary complex containing both III-1 and the amino-terminal 70-kD fragment, suggesting that the III-1 module can support the simultaneous binding of III-10 and 70 kD. Both unfolded III-10 and unfolded III-1 could support fibronectin binding, but only III-10 could promote the formation of disulfide-bonded multimers of fibronectin in the absence of cells. III-10-dependent multimer formation was inhibited by both the anti-III-1 monoclonal antibody, 9D2, and amino-terminal fragments of fibronectin. A fragment of III-10, termed III-10/A, was able to block matrix assembly in fibroblast monolayers. Similar resuits were obtained using the III-10A/RGE fragment, in which the RGD site had been mutated to RGE, indicating that III-10/A was blocking matrix assembly by a mechanism distinct from disruption of integrin binding. Texas red-conjugated recombinant 111-1,2 localized to [31-containing sites of focal adhesions on cells plated on fibronectin or the III-9,10 modules of fibronectin. Monoclonal antibodies against the III-1 or the 11I-9,10 modules of fibronectin blocked binding of III-1,2 to cells without disrupting focal adhesions. These data suggest that a role of the tXs~ 1 integrin in matrix assembly is to regulate a series of sequential self-interactions which result in the polymerization of fibronectin.

The deposition of fibronectin into the extracellular matrix is an integrin-dependent, multistep process that is tightly regulated in order to ensure controlled matrix deposition. Reduced fibronectin deposition has been associated with altered embryonic development, tumor cell invasion, and abnormal wound repair. In one of the initial steps of fibronectin matrix assembly, the aminoterminal region of fibronectin binds to cell surface receptors, termed matrix assembly sites. The present study was undertaken to investigate the role of extracellular signals in the regulation of fibronectin deposition. Our data indicate that the interaction of cells with the extracellular glycoprotein, vitronectin, specifically inhibits matrix assembly site expression and fibronectin deposition. The region of vitronectin responsible for the inhibition of fibronectin deposition was localized to the heparin-binding domain. Vitronectin's heparin-binding domain inhibited both ␤ 1 and non-␤ 1 integrin-dependent matrix assembly site expression and could be overcome by treatment of cells with lysophosphatidic acid, an agent that promotes actin polymerization. The interaction of cells with the heparin-binding domain of vitronectin resulted in changes in actin microfilament organization and the subcellular distribution of the actin-associated proteins ␣-actinin and talin. These data suggest a mechanism whereby the heparin-binding domain of vitronectin regulates the deposition of fibronectin into the extracellular matrix through alterations in the organization of the actin cytoskeleton.The deposition of fibronectin into the extracellular matrix is a dynamic, multistep process that is normally tightly regulated in order to ensure controlled matrix deposition. In certain disease states, including pulmonary fibrosis and atherosclerosis, loss of this regulation gives rise to either excess or inappropriate fibronectin deposition (1). In addition, reduced fibronectin deposition has been associated with altered embryonic development, tumor cell invasion, and abnormal wound repair (1). The mechanisms that control the rate and extent of fibronectin deposition are only partially understood.Adherent cells polymerize an insoluble fibronectin matrix by assembling cell-or plasma-derived soluble fibronectin into insoluble fibrils (2). In one of the initial steps of matrix assembly, cell surfaces bind the amino-terminal region of fibronectin in a reversible and saturable manner (3, 4). Subsequent homophilic binding interactions are thought to promote the polymerization of the fibronectin molecule into an insoluble matrix (5-9) and allow for the regeneration of the cell surface amino-terminal binding site (2). The binding of the amino terminus of fibronectin to cell surface receptors, termed matrix assembly sites (3), is mediated by the first five type I repeats of fibronectin (4, 10). The molecule(s) that mediates the binding of the amino terminus of fibronectin to cell surfaces has not been definitively identified. It has been proposed that the III 1 module o...

Abstract-During exercise, local mechanisms in tissues cause arterioles to rapidly dilate to increase blood flow to tissues to meet the metabolic demands of contracting muscle. Despite decades of study, the mechanisms underlying this important aspect of blood flow control are still far from clear. We now report a novel mechanism wherein fibronectin fibrils in connective tissue matrices transduce signals from contracting skeletal muscle to local blood vessels to increase blood flow. Using intravital microscopy, we show that local vasodilation in response to skeletal muscle contraction is specifically inhibited by an antibody that recognizes the matricryptic site in the first type III repeat of fibronectin (FNIII-1). In the absence of skeletal muscle contraction, direct application of FNIII-1-containing fibronectin fragments to cremaster muscle arterioles in situ, triggered a rapid, specific, and reversible local dilation that was mediated by nitric oxide and required the cryptic, heparin-binding sequence of FNIII-1. Furthermore, application of function-blocking FNIII-1 peptides to cremaster muscle arterioles rapidly and specifically decreased their diameter, indicating that the matricryptic site of fibronectin also contributes to resting vascular tone. Alexa fluor 488 -labeled fibronectin, administered intravenously, was rapidly assembled into elongated, branching fibrils in the extracellular matrix of intact cremaster muscle, demonstrating active polymerization of fibronectin in areas adjacent to blood vessels. Together, these data provide the first evidence that a matricryptic, heparin-binding site within fibronectin fibrils of adult connective tissue plays a dynamic role in regulating both vascular responses and vascular tone.

Tissue engineering holds great potential for saving the lives of thousands of organ transplant patients who die each year while waiting for donor organs. However, to successfully fabricate tissues and organs in vitro, methodologies that recreate appropriate extracellular microenvironments to promote tissue regeneration are needed. In this study, we have developed an application of ultrasound standing wave field (USWF) technology to the field of tissue engineering. Acoustic radiation forces associated with USWF were used to non-invasively control the spatial distribution of mammalian cells and cell-bound extracellular matrix proteins within three-dimensional collagen-based engineered tissues. Cells were suspended in unpolymerized collagen solutions and were exposed to a continuous wave USWF, generated using a 1 MHz source, for 15 min at room temperature. Collagen polymerization occurred during USWF exposure resulting in the formation of three-dimensional collagen gels with distinct bands of aggregated cells. The density of cell bands was dependent on both the initial cell concentration and the pressure amplitude of the USWF. Importantly, USWF exposure did not decrease cell viability, but rather enhanced cell function. Alignment of cells into loosely clustered, planar cell bands significantly increased levels of cell-mediated collagen gel contraction and collagen fiber reorganization as compared to sham-exposed samples with a homogeneous cell distribution. Additionally, the extracellular matrix protein, fibronectin, was localized to cell banded areas by binding the protein to the cell surface prior to USWF exposure. By controlling cell and extracellular organization, this application of USWF technology is a promising approach for engineering tissues in vitro.