Cell surface fibronectin and oncogenic transformation

Fibronectin is a large glycoprotein at the cell surface of many different cell types; a related protein is present in plasma. Fibronectin is a dimer of 230,000-dalton subunits and also occurs in larger aggregates; it forms fibrillar networks at the cell surface, between cells and substrata and between adjacent cells, and it is not a typical membrane protein. Cell surface fibronectin is reduced in amount or absent on transformed cells and in many cases its loss correlates with acquisition of tumorigenicity and, in particular, metastatic ability. Exceptions to the correlations with transformation and tumorigenicity exist. Loss of fibronectin and the resulting reduced adhesion appear to be involved in pleiotrpoic alterations in cell behavior and may be responsible for several aspects of the transformed phenotype in vitro. Fibronectin interacts with other macromolecules (collagen/gelatin, fibrin/fibrinogen, proteoglycans) and is apparently connected to microfilaments inside the cell.     

Thrombospondin inhibits adhesion of endothelial cells

Adsorption of thrombospondin to a substratum inhibits adhesion of endothelial cells to that substratum. Four hours after plating of cells on glass covered with thrombospondin, the number of cells bound per unit area was only 8% of that bound to fibronectin, and 20% of that which could bind to albumin. While on fibronectin cells assumed a well-spread configuration with time in culture, on thrombospondin they stayed completely round. On surfaces constructed by sequential incubation of glass with thrombospondin and fibronectin or other proteins, thrombospondin retained its inhibitory effect on cell adhesion. Fibronectin surfaces treated with thrombospondin lost 50% of their capacity to adhere endothelial cells. Cell spreading was also greatly impaired. These observations indicate that thrombospondin, which is a component of the extracellular matrix, can modulate adhesion of endothelial cells to the matrix.     

Human keratinocytes that have not terminally differentiated synthesize laminin and fibronectin but deposit only fibronectin in the pericellular matrix

Fibronectin and laminin production by human keratinocytes cultured in serum-free, low-calcium medium without a fibroblast feeder layer were examined by several techniques. By indirect immunofluorescence, fibronectin but not laminin appeared as short radial fibrils between the cells and the substratum, and in the pericellular matrix. Synthesis of fibronectin and laminin by 7-day keratinocyte cultures was determined by 18 hr 35S-methionine metabolic labeling followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Fibronectin accounted for 2.9% of total synthesized protein, 26.5% of fluid phase protein secretion, and 4.3% of deposited ECM protein. In contrast, only 0.1% of the total synthesized protein was laminin, little (6.3%) of this product was secreted, and none of this product was deposited in the ECM. Our results indicate that human keratinocytes under culture conditions that prevent terminal differentiation in vitro can synthesize, secrete, and deposit fibronectin in the extracellular matrix. Although these cells synthesize laminin, they secrete very little and deposit no detectable laminin in the matrix under these culture conditions. From these data we believe that fibronectin may play an important role in the interaction of epidermal cells with connective tissue matrix during wound healing or morphogenesis in in vivo situations in which the epidermis is not terminally differentiated.     

Cellular and metabolic specificity in the interaction of adhesion proteins with collagen and with cells

Fibronectin mediates the adhesion of fibroblasts to collagen substrates, binding first to the collagen and then to the cells. We report here that the interaction of the cells with the fibronectin-collagen complex is blocked by specific gangliosides, GD_{1 a} and GT₁, and that the sugar moieties of these gangliosides contain the inhibitory activity. The gangliosides act by binding to fibronectin, suggesting that they may be the cell surface receptor for fibronectin. Evidence is presented that other adhesion proteins or mechanisms of attachment exist for chondrocytes, epidermal cells, and transformed tumorigenic cells, since adhesion of these cells is not stimulated by fibronectin. Chondrocytes adhere via a serum factor that is more temperature-sensitive and less basic than fibronectin. Unlike that of fibroblasts chondrocyte adhesion is stimulated by low levels of gangliosides. Epidermal cells adhere preferentially to type IV (basement membrane) collagen but at a much slower rate than fibroblasts or chondrocytes. This suggests that these epidermal cells synthesize their own specific adhesion factor. Metastatic cells cultured from the T241 fibrosarcoma adhere rapidly to type IV collagen in the absence of fibronectin and do not synthesize significant amounts of collagen or fibronectin. Their growth, in contrast to that of normal fibroblasts, is unaffected by a specific inhibitor of collagen synthesis. These data indicate the importance of specific collagens and adhesion proteins in the adhesion of certain cells and suggest that a reduction in the synthesis of collagen and of fibronectin is related to some of the abnormalities observed in transformed cells.     

Synthesis and retention of fibronectin (LETS protein) by mouse teratocarcinoma cells.

Mouse teratocarcinoma cells in culture were examined for both the synthesis (by metabolic labelling) and surface accumulation (by indirect immunofluorescence) of fibronectin, a glycoprotein with subunits of molecular weight 220000 D known to form part of the extracellular matrix of many cells in vivo. Although lines of both pluripotent and nullipotent embryonal carcinoma cells synthesize the protein and release it into the medium, they do not retain it on their surfaces. Monolayers of the endoderm line PSA5-E both synthesize fibronectin and lay it down in an extracellular network. A line of PYS parietal endoderm cells does not retain surface fibronectin, although it does accumulate other extracellular matrix material. When pluripotent embryonal carcinoma cells differentiate into cystic embryoid bodies, fibronectin accumulates in a basement membrane below the outer endoderm cells (both visceral and parietal-like) and may play a transient role in organizing the inside cells into an epithelial layer.     

Ultrastructural localization of fibronectin in bone marrow of the embryonic chick and its relationship to granulopoiesis

Summary Fibronectin was immunolocated in embryonic chick bone marrow by the use of both a direct peroxidase conjugated antiserum and an indirect Streptavidin bridge technique. Fibronectin is located in the extravascular granulopoietic compartment and, to a lesser extent, in the vascular, erythropoietic compartment. There is no evidence of fibronectin being associated with blood-stromal cell interactions involving either erythropoiesis or thrombopoiesis. However, mature thrombocytes display a substantial surface coat containing fibronectin. Much of the fibronectin appears to be situated on surfaces of those fibroblastic stromal cells which support granulopoiesis. Fibronectin containing extracellular material connects surfaces of developing granulocytes with surfaces of stromal cells. Fibronectin is a surface component of granulocytes as well as nearby stromal cells. However, there appear to be fewer ferritin particles per unit of surface on granulocytic cells. Many of the ferritin particles are not clearly associated with amorphous matrix material at cell surfaces. Immunocytochemical attempts to identify laminin were unsuccessful. These studies indicate that fibronectin is situated at sites where it could mediate adhesive interaction between granulopoietic cells and their stromal cells. Furthermore, cell surface-matrix interaction involving fibronectin could mediate migration of blood cells within the extravascular spaces.     

Basal lamina glycoproteins laminin and type IV collagen are assembled into a fine-fibered matrix in cultures of a teratocarcinoma-derived endodermal cell line

Extracellular matrix glycoproteins synthesized and deposited by a mouse teratocarcinoma-derived endodermal cell line (PYS-2) in culture were analysed by metabolic labelling and immunochemical methods, and the matrix structure was studied by immunofluorescence and electron microscopy. PYS-2 cells secreted two major high-molecular weight glycoproteins, laminin and type IV collagen, which were deposited in apparently unprocessed form under the cells into a lamellar matrix composed of a loose network of fine fibrils and attached dense grains. The cells did not synthesize detectable amounts of fibronectin, but the matrix was found to bind fibronectin from the culture medium. The matrix structure was sensitive to bacterial collagenase indicating a role for type IV collagen in matrix integrity. The PYS-2 matrix which contains defined basal lamina glycoproteins provides possibilities for in vitro studies on the organization of deposited basal lamina components.     

Location and possible function of fibronectin and laminin in clones of chick retinal pigmented epithelial cells

Summary Distribution and organization of the extracellular glycoproteins, fibronectin and laminin, in clonal cultures of chick retinal pigmented epithelial cells have been investigated using indirect immunofluorescence microscopy. Fibronectin is located on the apical and basal cell surfaces and between the cells in the undifferentiated regions of the colony (outer edge and stratified region). It seems to run parallel to intracellular microfilament bundles and to be associated with them across the cell membrane. In the differentiated region of thecolony (center), it is located exclusively on the basal cell surface and seems to be primarily associated with the collagen bundles of the basement membrane. The locations suggest that it may be necessary to stabilizing the sheet of differentiated cells in the colony center. In all regions except the outer edge of the colony, laminin is associated with the basal cell surfaces where it forms a meshwork of short, fine strands. The laminin has a totally different staining pattern from the fibronectin and does not seem to be associated with collagen bundles. The location suggests that laminin may be present in the basal lamina and may be involved in adhesion of the cells to the substratum. This work was supported by Medical Research Council of Canada (MA-6337).     

Synthesis and extracellular deposition of fibronectin in chondrocyte cultures. Response to the removal of extracellular cartilage matrix

Fibronectin, the major cell surface glycoprotein of fibroblasts, is absent from differentiated cartilage matrix and chondrocytes in situ. However, dissociation of embryonic chick sternal cartilage with collagenase and trypsin, followed by inoculation in vitro reinitiates fibronectin synthesis by chondrocytes. Immunofluorescence microscopy with antibodies prepared against plasma fibronectin (cold insoluble globulin [CIG]) reveals fibronectin associated with the chondrocyte surface. Synthesis and secretion of fibronectin into the medium are shown by anabolic labeling with [35S]methionine or [3H]glycine, and identification of the secreted proteins by immunoprecipitation and sodium dodecyl sulfate (SDS)-disc gel electrophoresis. When chondrocytes are plated onto tissue culture dishes, the pattern of surface-associated fibronectin changes from a patchy into a strandlike appearance. Where epithelioid clones of polygonal chondrocytes develop, only short strands of fibronectin appear preferentially at cellular interfaces. This pattern is observed as long as cells continue to produce type II collagen that fails to precipitate as extracellular collagen fibers for some time in culture. Using the immunofluorescence double-labeling technique, we demonstrate that fibroblasts as well as chondrocytes which synthesize type I collagen and deposit this collagen as extracellular fibers show a different pattern of extracellular fibronectin that codistributes in large parts with collagen fibers. Where chondrocytes begin to accumulate extracellular cartilage matrix, fibronectin strands disappear. From these observations, we conclude (a) that chondrocytes synthesize fibronectin only in the absence of extracellular cartilage matrix, and (b) that fibronectin forms only short intercellular "stitches" in the absence of extracellular collagen fibers in vitro.     

Transformed human cells release different fibronectin variants than do normal cells

Fibronectin molecules are dimers composed of subunits whose primary structures may differ. This is due to alternative splicing in at least two regions (ED and IIICS) of the pre-mRNA. Using two monoclonal antibodies specific for two different epitopes of domain 5 (high affinity for heparin), we have quantitatively analyzed the expression of the IIICS sequence in human fibronectins from different sources. The results demonstrated that the percentage of fibronectin subunits containing the IIICS is higher in fibronectins from tumor-derived or simian virus 40-transformed human cells than in fibronectins from human plasma or normal human fibroblasts. Furthermore, we observed that 45-65% of fibronectin subunits from transformed cells or normal embryonic fibroblasts are sialylated on the heparin-binding domain 5, whereas this occurs in only 24-28% of fibronectin subunits from normal adult fibroblasts. On the contrary, no sialylation was observed on domain 5 in fibronectin from human plasma.     

Concomitant loss of cell surface fibronectin and laminin from transformed rat kidney cells

Both fibronectin and laminin were found by immunofluorescence as a matrix at the surface of normal rat kidney cells. These matrices were absent from the surface of virally transformed rat kidney cells. Soluble fibronectin and laminin were detected in the culture media of the transformed as well as the normal cells. Culture supernates of the transformed cells contained even more fibronectin than the supernates of the transformed cells contained even more fibronectin than the supernates of the normal cells while laminin was present in similar amounts in both culture media. This shows that the loss of fibronectin and laminin from the surface of the transformed cells is caused by failure of the cells to deposit these proteins into an insoluble matrix and not caused by inadequate production. Fibronectins isolated from culture media of the normal and transformed cells were similar in SDS polyacrylamide gel electrophresis. Laminin isolated from culture media by affinity chromatography on heparin-Sepharose followed by immunoprecipitation was composed of three main polypeptides, one with a molecular weight of 400,000 and two with a molecular weight close to 200,000 in both cell types. Fibronectins from both cell types were equally active in promoting cell attachment. Rat fibronectin from transformed cells, like normal cells, when applied to culture dishes coated with fibronectin, readily attached and spread on the substratum, requiring approximately the same amount of fibronectin as the normal cells. On the basis of these results it seem that the failure of the transformed cells to incorporate fibronectin into an insoluble cell surface matix is not a consequence of a demonstrable change in the functional characteristics of the fibronectin molecule or in the ability of the cells to interact with fibronectin. It may depend on as yet unidentified interactions of the cell surface. Similar interactions may be needed for the deposition of laminin into the matrix, because laminin was also absent from the surface of transformed cells, despite its being synthesized by these cells.     

Fibronectin in rat heart: a link between cardiac myocytes and collagen

Fibronectin, a glycoprotein that binds to collagen and modifies the adhesion properties and motility of cells in culture, is present in the interstitium of rat hearts. To localize fibronectin more precisely and to assess its relationship to the myocyte and to connective tissue elements, we employed a double antibody technique to label myocardial fibronectin with electron-dense ferritin to permit an ultrastructural analysis. Fibronectin was found to be associated with collagen, and in some cases appeared to link collagen fibers. Fibronectin was also found inserted along the surfaces of cardiac myocytes, connecting these cells to perimyocytic collagen. These ultrastructural relationships imply that fibronectin is a major component of the myocardial interstitium, and may affect myocardial compliance and control the motion of myocytes during the contraction and relaxation of the heart.     

Modulation of protein synthesis and secretion by substratum in primary cultures of rat hepatocytes

Hepatocytes isolated by perfusion of adult rat liver and cultured on substrata consisting of one or more of the major components of the liver biomatrix (fibronectin, laminin, type IV collagen) have been examined for the synthesis of defined proteins. Under these conditions, tyrosine amino transferase, a marker of hepatocyte function, is maintained at similar levels in response to dexamethasone over 5 days in culture on each substratum, and total cellular protein synthesis remains constant. By contrast, there is a rapid decrease in synthesis and secretion of albumin and a 3-7-fold increase in synthesis and secretion of alpha-fetoprotein which are most marked on a laminin substratum, but least evident on type IV collagen, and an increased synthesis of fibronectin and type IV collagen. The newly synthesized matrix proteins are present in the cell layer as well as in cell secretions. The enhanced synthesis of fibronectin is less in cells seeded onto a fibronectin substratum than on laminin or type IV collagen substrata, and its synthesis by hepatocytes seeded onto a mixed substratum of laminin and fibronectin is down-regulated by fibronectin in a dose-related manner. Similarly, type IV collagen synthesis is less when the cells are seeded on the homologous matrix protein substratum than on heterologous substrata. These results indicate that hepatocytes cultured in serum-free medium on substrata composed of components of the liver biomatrix maintain certain functions of the differentiated state (tyrosine amino transferase), lose others (albumin secretion) and switch to increased synthesis of matrix components as well as fetal markers such as alpha-fetoprotein. The magnitude of these effects depends on the substratum on which the hepatocytes are cultured.     

Use of biocarrier beads and flow cytometry for single-cell studies of fibronectin gene regulation in dibutyrl cyclic AMP reverse transformed CHO-K1 cells

The protease sensitivity of a number of cell surface or cytoskeletal components and the relationship of these to the substratum in attached cells has prevented the quantitative measurement of their expression by flow cytometry. Using traditional cell sorting techniques, cells must be treated with a protease to detach them from a substrate in order to produce a single-cell suspension. Unfortunately, proteolytic treatment alters or destroys a number of cellular proteins. Fibronectin either on the cell surface or as part of the substratum laid down by the cell is particularly sensitive to proteases, preventing its quantitative study by flow cytometry. To circumvent these problems and produce a single cell suspension necessary for flow cytometric analysis, CHO-K1, a Chinese hamster ovary cell line, were grown in suspension on specially-treated 25 μm biocarrier beads. The CHO-K1 cell line is composed of transformed epithelial-like cells that have lost the fibronectin deposit around their cell membranes. To restore the typical fibroblastic deposit of fibronectin, the cells attached to beads were induced by dibutyrl cAMP to undergo a reverse transformation reaction to restore fibroblastic morphology and the typical fibroblastic deposite of fibronectin on the cell surface and substratum. The cells attached to beads were then immunofluorescently labeled for the protease-sensitive, extracellular matrix component, fibronectin, and examined on a flow cytometer. Cell surface fibronectin heterogeneity was then examined on a cell-by-cell basis as a function of cell cycle using Hoechst 33342 dye that binds to AT base pairs of cellular DNA. Dual laser measurement and multiparameter list mode data analysis were used to study the relationship between cell surface fibronectin of biocarrier bead attached cells and cell cycle.     

Use of biocarrier beads and flow cytometry for single-cell studies of fibronectin gene regulation in dibutyryl cyclic AMP reverse transformed CHO-K1 cells

The protease sensitivity of a number of cell surface or cytoskeletal components and the relationship of these to the substratum in attached cells has prevented the quantitative measurement of their expression by flow cytometry. Using traditional cell sorting techniques, cells must be treated with a protease to detach them from a substrate in order to produce a single-cell suspension. Unfortunately, proteolytic treatment alters or destroys a number of cellular proteins. Fibronectin either on the cell surface or as part of the substratum laid down by the cell is particularly sensitive to proteases, preventing its quantitative study by flow cytometry. To circumvent these problems and produce a single cell suspension necessary for flow cytometric analysis, CHO-K1, a Chinese hamster ovary cell line, were grown in suspension on specially-treated 25 microns biocarrier beads. The CHO-K1 cell line is composed of transformed epithelial-like cells that have lost the fibronectin deposit around their cell membranes. To restore the typical fibroblastic deposit of fibronectin, the cells attached to beads were induced by dibutyryl cAMP to undergo a reverse transformation reaction to restore fibroblastic morphology and the typical fibroblastic deposite of fibronectin on the cell surface and substratum. The cells attached to beads were then immunofluorescently labeled for the protease-sensitive, extracellular matrix component, fibronectin, and examined on a flow cytometer. Cell surface fibronectin heterogeneity was then examined on a cell-by-cell basis as a function of cell cycle using Hoechst 33342 dye that binds to AT base pairs of cellular DNA. Dual laser measurement and multiparameter list mode data analysis were used to study the relationship between cell surface fibronectin of biocarrier bead attached cells and cell cycle.     

Rat hepatocytes in serum-free primary culture elaborate an extensive extracellular matrix containing fibrin and fibronectin

Adult rat hepatocytes cultured on type IV collagen, fibronectin, or laminin and maintained in serum-free medium were examined by indirect immunofluorescence using polyclonal antibodies against extracellular matrix proteins. An extensive fibrillar matrix containing fibronectin and fibrin was detected in all hepatocyte cultures irrespective of the exogenous matrix substratum used to support cell adhesion. Fibrils radiated from the cell periphery and covered the entire culture substratum. In addition, thicker fibers or bundles of fibers were localized on top of hepatocytes. This matrix did not contain laminin or the major types of collagen found in the liver biomatrix (types I, III, and IV). Isolation of the fibrillar matrix and analysis on polyacrylamide gels under reducing conditions demonstrated a major 58-kD polypeptide, derived from beta-fibrinogen as indicated by immunoblotting and two-dimensional peptide mapping. Plasmin rapidly dissolved the matrix. Deposition of the fibrin matrix in hepatocyte cultures was arrested by hirudin, by specific heparin oligosaccharides that potentiate thrombin inhibition by antithrombin III, and by dermatan sulfate, an activator of heparin cofactor II-mediated inhibition of thrombin. The results indicate that hepatocytes in culture synthesize and activate coagulation zymogens. In the absence of inhibitory and fibrinolytic mechanisms, a fibrin clot is formed by the action of thrombin on fibrinogen. Fibronectin attaches to this fibrin clot but fails to elaborate a fibrillar matrix on its own in the presence of coagulation inhibitors.     

Fibronectin from the ovary of the sea urchin,Pseudocentrotus depressus

Summary Fibronectin, with a subunit molecular weight of 220,000 daltons, was isolated from the ovary of the sea urchin,Pseudocentrotus depressus, using affinity chromatography on heat-denatured mammalian collagen coupled to Sepharose 4B. The distribution of fibronectin in the sea urchin ovary was examined by indirect immunofluorescence using antifibronectin serum. The basement membrane and the connective tissues exhibited strong fluorescence. The fibronectin was localized closely together with collagen bundles in the sea urchin ovary. Biochemical and immunological examinations indicate that sea urchin fibronectin has similar properties as those of mammalian fibronectin.     

Extracellular matrix and embryonal morphogenesis: role of fibronectin in cell migration

The neural crest provides a useful paradigm for cell migration and modulations in cell adhesion during morphogenesis. In the present review, we describe the major findings on the role of the extracellular matrix glycoprotein fibronectin and its corresponding integrin receptor in the locomotory behavior of neural crest cells. In vivo, fibronectin is associated with the migratory routes of neural crest cells and, in some cases, it disappears from the environment of the cells as they stop migrating. In vitro, neural crest cells show a great preference for fibronectin substrates as compared to other matrix molecules. Both in vivo and in vitro, neural crest cell migration can be specifically inhibited by antibodies or peptides that interfere with the binding of fibronectin to its integrin receptor. However, the migratory behavior of neural crest cells cannot result solely from the interaction with fibronectin. Thus, neural crest cells exhibit a particular organization of integrin receptors on their surface and develop a cytoskeletal network which differs from that of non-motile cells. These properties are supposed to permit rapid changes in the shape of cells and to favor a transient adhesion to the substratum. Recent findings have established that different forms of fibronectin may occur, which differ by short sequences along the molecule. The functions of most of these sequences are not known, except for 1 of them which carries a binding site for integrin receptors. We have demonstrated that this site is recognized by neural crest cells and plays a crucial role in their displacement. It is therefore possible that the forms of fibronectin carrying this sequence are not evenly distributed in the embryo, thus allowing migrating neural crest cells to orientate in the embryo. Fibronectin would then not only play a permissive role in embryonic cell motility, but have an instructive function in cell behavior.     

The structure of fibronectin and its role in cellular adhesion

Fibronectin is a large, adhesive glycoprotein which is found in a number of locations, most notably on cell surfaces, in extracellular matrixes, and in blood. Fibronectin has been detected in all vertebrates tested and in many invertebrates. Its presence in sponges is significant because this suggests that fibronectin may have appeared very early in evolution, possibly with the most primitive multicellular organisms. Cellular and plasma fibronectins have many striking similarities. However, the locations of the polypcptide chain differences between these two proteins indicate that plasma fibronectin cannot be derived from cellular fibronectin by means of simple post-translational proteolysis. Instead, these different types of fibronectin may be products of different genes or of differentially spliced messenger RNA molecules. Amniotic fluid fibronectin is possibly a third form of the protein. Cellular and plasma fibronectins are composed of at least six protcaseresistant domains which contain specific binding sites for actin, gelatin, heparin, Staphylococcus aureus, transglutarninase, fibrin, DNA, and a cell surface receptor. The relative locations of these domains have been mapped in the primary structure of fibronectin. The cell surface receptor for fibronectin has not been positively identified, but may be a glycoprotein, a glycolipid, or a complex of the two. Although cell-substratum adhesion is mediated by fibronectin, the locations of the areas of closest approach of the cell to the substratum (the adhesion plaques) and fibronectin are not coincident under conditions of active cell growth. Under conditions of cell growth arrest in low scrum concentrations, some fibronectin may become localized at the adhesion plaques. Models describing the domain structure of fibronectin and the molecular organization of the adhesion plaque area are presented.