Plasma fibronectin binds saturably and reversibly to substrate-attached fibroblasts and is subsequently incorporated into the extracellular matrix (McKeown-Longo, P. J., and D. F. Mosher, 1983, J. Cell Biol., 97:466-472). We examined whether fragments of fibronectin are processed in a similar way. The amino-terminal 70,000-mol-wt catheptic D fragment of fibronectin bound reversibly to cell surfaces with the same affinity as intact fibronectin but did not become incorporated into extracellular matrix. The 70,000-mol-wt fragment blocked binding of intact fibronectin to cell surfaces and incorporation of intact fibronectin into extracellular matrix. Binding of the 70,000-mol-wt fragment to cells was partially abolished by cleavage into 27,000-mol-wt heparin-binding and 40,000-mol-wt gelatin-binding fragments and more completely abolished by reduction and alkylation of disulfide bonds. Binding of the 70,000-mol-wt fragment to cells was not blocked by gelatin or heparin. When coated onto plastic, the 70,000-mol-wt fragment did not mediate attachment and spreading of suspended fibroblasts. Conversely, fibronectin fragments that had attachment and spreading activity did not block binding of exogenous fibronectin to substrate-attached cells. These results indicate that there is a cell binding site in the 70,000-mol-wt fragment that is distinct from the previously described cell attachment site and is required for assembly of exogenous fibronectin into extracellular matrix.In vivo, fibronectin is a large, dimeric glycoprotein of plasma and most body fuids, and an insoluble constituent of loose connective tissue and basement membranes. It is synthesized by a wide variety of cell types in culture and is found in culture medium and in the insoluble extracellular matrix around cultured cells (22, 34,47). The fibronectin in the matrix of cultured fibroblasts is found in fibrillar structures that are 5-10 nm in diameter (7,12,19,23,51). Similar structures have been identified in granulation tissue of skin wounds (52). Although there are structural differences between fibronectin purified from plasma and fibronectin synthesized by cultured cells (2, 16), fibronectin of the extracellular matrix is derived from both plasma (or serum) and local cells (l 8, 32, 38). Furthermore, both plasma (32) and cellular (5,8,21, 25, 31,57) flbronectin are present in the matrix as high molecular weight, disulfide-bonded multimers. Multimerization of plasma flbronectin probably occurs by disulfide 364 exchange in the 70,000-mol-wt amino-terminal region of the molecule (33).Fibronectin is believed to mediate cell-matrix adhesion. When coated onto plastic or glass substrata, fibronectin promotes the attachment and spreading of cells. The cell adhesive activity of fibronectin has been localized, first to increasingly smaller regions within the fibronectin subunit (15, 43, 44), and then to a specific tetrapeptide sequence (42). It has been postulated that cells in suspension interact with this region of the fibronectin molecule by means of ...

Tumor invasion and metastasis are the main causes of death from cancer. Epithelial to mesenchymal transition (EMT) is a determining step for a cancer cell to progress from a noninvasive to invasive state. Krüppel-like factor 8 (KLF8) plays a key role in oncogenic transformation and is highly overexpressed in several types of invasive human cancer, including breast cancer. To understand the role of KLF8 in regulating the progression of human breast cancer, we first established stable expression of KLF8 in an immortalized normal human breast epithelial cell line. We found that KLF8 strongly induced EMT and enhanced motility and invasiveness in the cells, by analyzing changes in cell morphology and epithelial and mesenchymal marker proteins, and using cell migration and Matrigel invasion assays. Chromatin immunoprecipitations (ChIP), oligonucleotide precipitations, and promoter-reporter assays showed that KLF8 directly bound and repressed the promoter of E-cadherin independent of E boxes in the promoter and Snail expression. Aberrant elevation of KLF8 expression is highly correlated with the decrease in E-cadherin expression in the invasive human breast cancer. Blocking KLF8 expression by RNA interference restored E-cadherin expression in the cancer cells and strongly inhibited the cell invasiveness. This work identifies KLF8 as a novel EMT-regulating transcription factor that opens a new avenue in EMT research and suggests an important role for KLF8 in human breast cancer invasion and metastasis. [Cancer Res 2007;67(15):7184-93]

Human plasma fibronectin bound to confluent cell layers of cultured human-skin fibroblasts in two distinct pools. Initial binding of fibronectin occurred in a deoxycholatesoluble pool (Pool I). Binding in Pool I was reversible and reached a steady state after 3 h. After longer periods of incubation, fibronectin became bound in a deoxycholate-insoluble pool (Pool II). Binding in Pool II was irreversible and proceeded at a linear rate for 30 h. After 30 h of incubation, a significant proportion of fibronectin bound in Pool II was present as disulfide-bonded multimers. HT1080 cells, a human sarcoma cell line, did not bind fibronectin in either pool. Also, isolated cell matrices prepared by deoxycholate extraction did not bind fibronection. Binding of fibronectin in Pool I of normal fibroblasts occurred via specific, saturable receptors. There were 128,000 binding sites per cell, and KD~ss was 3.6 X 10 -8 M. Fluorescence microscopic localization of fibronectin bound in Pool I and Pool II was performed using fluorescein-conjugated fibronectin. Fluorescent staining in Pool I was present in a punctate pattern and in short, fine fibrils. Pool II fluorescence was exclusively in coarse, dense fibrils. These data indicate that plasma fibronectin may become incorporated into the tissue extracellular matrix via specific cell-surface receptors.

Thrombospondin, a major glycoprotein released from a granules of thrombin-stimulated platelets, is a disulfidebonded trimer of 160-kilodalton subunits. Cultured human foreskin and fetal lung fibroblasts secreted thrombospondin (determined by enzyme-linked immunosorbent assay) into the culture medium in a time-dependent manner (15.7 and 5.8 Thrombospondin is a major platelet a granule glycoprotein that is secreted and then partially bound to platelet membranes when human platelets aggregate in response to thrombin (1)(2)(3)(4)(5)(6)(7)(8). Thrombospondin is a 450-kilodalton (kDal) protein and is composed of three large disulfide-linked subunits (9, 10). During platelet aggregation, thrombin-stimulated platelets develop a membrane-bound lectin-like activity (11-13), which originates from a granules and appears to play a role in mediating platelet aggregation by binding to a specific receptor on other platelets (14,15 3.7 X 10'°Bq). The radioactive postculture medium was removed, centrifuged at 8,000 x g for 2 min to remove cells and debris, and frozen at -35°C. The radioactive cell layers were washed twice, removed with a rubber policeman, pelleted by centrifugation, dissolved by boiling for 5 min in 2% NaDodSO4 containing protease inhibitors as described (20), and frozen until analyzed.In experiments in which the accumulation of thrombospondin antigen was measured by enzyme-linked immunosorbent assay (ELISA), fibroblasts were cultured in minimal essential medium containing 20% rabbit serum in 2-cm2 wells of multiwell plates. When the cells were confluent, the cells were washed, and the medium was replaced with 1 ml of fresh minimal essential medium containing 20% rabbit serum. At various times after the medium change, the postculture medium was removed, centrifuged at 8,000 X g for 2 min, and frozen until assayed. 998The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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.

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.

Thrombospondin was purified from human platelets and labeled with 125I, and its metabolism was quantified in cell cultures of human embryonic lung fibroblasts. 125I-Thrombospondin bound to the cell layer. The binding reached an apparent steady state within 45 min. Trichloroacetic acid-soluble radioactivity was detected in the medium after 30 min of incubation; the rate of degradation of 125I-thrombospondin was linear for several hours thereafter. Degradation of 125I-thrombospondin was saturable. The apparent Km and Vmax for degradation at 37 degrees C were 6 X 10(-8) M and 1.4 X 10(5) molecules per cell per minute, respectively. Degradation was inhibited by chloroquine or by lowering the temperature to 4 degrees C. Experiments in which cultures were incubated with thrombospondin for 45 min and then incubated in medium containing no thrombospondin revealed two fractions of bound thrombospondin. One fraction was localized by indirect immunofluorescence to punctate structures; these structures were lost coincident with the rapid degradation of 50-80% of bound 125I-thrombospondin. The second fraction was localized to a trypsin-sensitive, fibrillar, extracellular matrix. 125I-Thrombospondin in the matrix was slowly degraded over a period of hours. Binding of 125I-thrombospondin to the extracellular matrix was not saturable and indeed was enhanced at thrombospondin concentrations greater than 3 X 10(-8) M. The ability of 125I-thrombospondin to bind to extracellular matrix was diminished tenfold by limited proteolytic cleavage with trypsin. Degradation of trypsinized 125I-thrombospondin was also diminished, although to a lesser extent than matrix binding. Heparin inhibited both degradation and matrix binding. These results suggest that thrombospondin may play a transitory role in matrix formation and/or organization and that specific receptors on the cell surface are responsible for the selective removal of thrombospondin from the extracellular fluid and matrix.

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...