Codistribution of heparan sulfate proteoglycan, laminin, and fibronectin in the extracellular matrix of normal rat kidney cells and their coordinate absence in transformed cells

We used antibodies raised against both a heparan sulfate proteoglycan purified from a mouse sarcoma and a chondroitin sulfate proteoglycan purified from a rat yolk sac carcinoma to study the appearance and distribution of proteoglycans in cultured cells. Normal rat kidney cells displayed a fibrillar network of immunoreactive material at the cell surface when stained with antibodies to heparan sulfate proteoglycan, while virally transformed rat kidney cells lacked such a surface network. Antibodies to chondroitin sulfate proteoglycan revealed a punctate pattern on the surface of both cell types. The distribution of these two proteoglycans was compared to that of fibronectin by double-labeling immunofluorescent staining. The heparan sulfate proteoglycan was found to codistribute with fibronectin, and fibronectin and laminin gave coincidental stainings. The distribution of chondroitin sulfate proteoglycan was not coincidental with that of fibronectin. Distinct fibers containing fibronectin but lacking chondroitin sulfate proteoglycan were observed. When the transformed cells were cultured in the presence of sodium butyrate, their morphology changed, and fibronectin, laminin, and heparan sulfate proteoglycan appeared at the cell surface in a pattern resembling that of normal cells. These results suggest that fibronectin, laminin, and heparan sulfate proteoglycan may be complexed at the cell surface. The proteoglycan may play a central role in assembly of such complexes since heparan sulfate has been shown to interact with both fibronectin and laminin.     

The interaction of plasminogen activator with a reconstituted basement membrane matrix and extracellular macromolecules produced by cultured epithelial cells

Laminin and fibronectin are glycoproteins that influence cell behavior and mediate cell/substratum adhesion. We have examined the interaction of these macromolecules with the serine protease plasminogen activator (PA) in two types of extracellular matrices; one produced by the murine Engelbreth-Holm-Swarm (EHS) tumor (Matrigel), and another by normal kidney epithelial cells in culture. Matrigel was found to contain significant quantities of tissue-type PA (tPA). Two of the major components of Matrigel, laminin and type IV collagen, were also examined. Tissue-type PA was associated with purified preparations of laminin; however, it was not found associated with type IV collagen. Normal kidney epithelial cells in culture secrete large amounts of urokinase (UK) and deposit a subepithelial matrix containing both laminin and fibronectin. These matrix macromolecules were isolated from the deposited matrix by immunoprecipitation, examined by zymography, and found to contain UK. The potential role of this interaction in the mechanisms of cell migration and matrix remodeling is discussed.     

Similarities and differences between the fibronectins of normal and transformed hamster cells

Fibronectins from normal and virally transformed hamster cells were compared by several criteria. The fibronectin from transformed cells was similar to that from normal cells in being an intact dimeric glycoprotein with the ability to bind to gelatin, activated thiol-Sepharose, and cells. No evidence was found for proteolytic cleavages or abnormalities in disulfide bonding of transformed cell fibronectin. This fibronectin was also shown to be active in promoting cell attachment, elongation, and alignment. Therefore, the fibronectin produced by transformed cells is not defective. However, it was shown that the transformed cells were partially deficient in their capacity to bind fibronectins from either normal or transformed cells. This deficiency has implications for the significance of the loss of fibronectin on oncogenic transformation. Partial proteolysis of the fibronectins from normal and transformed cells gave rise to the same fragments. However, the glycosylated fragments from transformed cell fibronectin appeared somewhat larger than those from normal cell fibronectin. Analysis of fibronectin glycopeptides showed that transformation leads both to more branches per core and to a higher sialylation of the asparagine-linked complex carbohydrate side chains.     

Laminin and fibronectin in normal and malignant neuroectodermal cells

Laminin and fibronectin, the major noncollagenous matrix glycoproteins, were studied in connection with normal brain cells and neuroectodermal cell lines. Laminin, a Mr 900,000 dalton matrix glycoprotein and an essential component of basement membranes, was found to be produced by cultured cells of several malignant cell lines of neuroectodermal origin. In cultured mouse C1300 neuroblastoma line cells laminin was localized, by immunoelectron microscopy, to the rough endoplasmic reticulum and, to sites of cell-to-cell and cell-to-substratum adhesion. Further experiments on the intracellular transport of this glycoprotein in C1300 cells confirmed that laminin is, at least partially, transported through the Golgi pathway. These results favor a role for laminin in attachment and cellular interactions of malignant neuronal cells. Laminin was also found in connection with neurons and glial cells from mammalian brain. In primary cultures from developing rat brain the vast majority of non-neuronal cells (80%) expressed immunoreactivity for the glial fibrillary acidic protein, a cytoskeletal protein specific for astrocytes. During the first week in culture all the glial fibrillary acidic protein-positive cells, with the exception of mature-looking star-shaped astrocytes, exhibited immunoreactivity for laminin. The intracellular laminin disappeared gradually after a few weeks in culture, but an extensive laminin matrix persisted and seemed to be localized on the upper surface of the non-neuronal cells. The neurofilament-positive neurons were negative for laminin. Pretreatment of the cultures with the ionophore monensin, caused accumulation of laminin-immunoreactivity within the Golgi region, which confirmed that laminin is, indeed, produced by cultured astrocytes and secreted through the Golgi complex. No fibronectin immunoreactivity was found in the majority of glial cells. However, under culture conditions where fibronectin was omitted from the culture medium there was, in the primary cultures, a minor population of glial fibrillary acidic protein-positive flat glial cells that exhibited intracytoplasmic immunofluorescence for fibronectin. In the presence of fibronectin in culture medium no fibronectin-positive glial cells could be detected. It thus appears that laminin, and to a minor extent fibronectin, are proteins that normal glial cells are capable of producing under specific conditions. Laminin and fibronectin were localized in adult rat brain in capillary and meningeal structures.(ABSTRACT TRUNCATED AT 400 WORDS)     

The characteristics of actin cytoskeleton structure and its rearrangements by extracellular matrix proteins in normal, immortalized and transformed rat fibroblasts

Comparative analysis of actin cytoskeleton structure in rat embryonic fibroblasts, E1A-immortalized and E1A + cHa-ras-transformed cells has been carried out. A decrease in adhesiveness and the rate of changes in actin cytoskeleton structures was shown to correlate with the level of morphological transformation of cells. E1A + cHa-ras-transformants show the lowest adhesiveness and complete disorganization of actin structures. Cultivation on serum-free media promoted disassembling of actin cytoskeleton structures in a small part of normal fibroblast population, only in a few immortalized cells, but exerted no influence on transformed cells. The influence of immobilized extracellular matrix proteins fibronectin, laminin and collagens type I and III on actin cytoskeleton structure in normal, immortalized and transformed fibroblasts was studied. Transformed cells spread on fibronectin completely restored highly organized actin structures, displayed a lot of stress fibers and focal contacts. The use of laminin revealed differences in locomotion between normal and transformed cells. Normal, immortalized and transformed fibroblasts spread on fibronectin and laminin demonstrate some peculiarities in actin cytoskeleton structures as a result of specificity of ligand-receptor interaction. Cells spread on fibronectin have polygonal shapes, many stress fibers and focal contacts, whereas cells spread on laminin are highly polarized and develop broad lamellae filled with actin microfilament meshwork. Collagens type I and III can affect adhesive properties and actin cytoskeleton structure in all cell lines studied only slightly, in comparison with fibronectin and laminin.     

Fibroblast cellular and plasma fibronectins are similar but not identical

Fibronectins are multimeric, adhesive glycoproteins present on cell surfaces and circulating in blood. Cellular fibronectin produced by fibroblasts in vitro and fibronectin isolated from plasma are known to be very similar immunologically and biochemically. We investigated whether or not they are identifical. Purified chicken and human cell-surface fibronectins are 150-fold more active in hemagglutination of fixed erythrocytes than plasma fibronectins. Cell-surface fibronectin is also 50-fold more active in restoring a more normal morphology to transformed cells originally missing the protein. However, in two other assays that measure cell attachment to collagen and cell spreading, cell-surface and plasma fibronectins have identical specific activities. In sodium dodecyl sulfate polyacrylamide gels, the subunits of human and chicken plasma fibronectins have significantly smaller apparent subunit molecular weights than cellular fibronectins present on cell surfaces or secreted into culture media. These differences are also present in a characteristic large subfragment of both forms of fibronectin after limited proteolysis by trypsin. We conclude that by both biological and biochemical criteria, cellular and plasma fibronectins are similar but not identical.     

Modulation of extracellular-matrix synthesized by cultured stromal cells from normal human breast tissue by epidermal growth factor

A routine, reproducible procedure was developed for the preparation and characterization of stromal cells from normal human breast tissue obtained by reduction mammaplasty. Isolates (n = 15) all exhibited enhanced rates of proliferation, even in the presence of 20% fetal calf serum, when exposed to epidermal growth factor or transforming growth factor a (both 10(-8) M). Cellular responsiveness to these growth factors was consistent with expression of specific surface receptors for epidermal growth factor (approximately 10(4)/cell). In cultures, stromal cells elaborated an extensive, cross-linked, insoluble extracellular matrix which remained firmly associated with the plastic surface of tissue culture ware upon lysis of cells. The insoluble matrix material was analyzed using enzymatic digestion procedures following incorporation of radiolabelled precursors into macromolecular material prior to lysis and preparation. The relative proportion of glycoconjugate (glycopeptides and proteoglycans) and collagenous material present in matrix material was approximately 45% and approximately 55%, respectively, and this was modulated by inclusion of epidermal growth factor into culture medium to approximately 60% and approximately 40%, respectively. Under similar culture conditions stromal cells synthesized twice as much hyaluronate as was produced by control cultures. By use of specific antibody preparations we identified at least four species of glycopeptide present in stromal matrices (namely, fibronectin, laminin, tenascin, and thrombospondin) as well as three types of collagen (types I, III, and IV). The rapid and reproducible procedure for the preparation of radiolabelled insoluble matrix material from normal human breast tissue allows for the study of cellular interaction involving extracellular matrix turnover and degradation.     

Effect of the extracellular matrix molecules fibronectin and laminin on the adhesion and growth of primary renal cortical epithelial cells

Primary tubular epithelial cells were isolated from renal cortex following enzymatic dissociation with collagenase. These cells were then grown in chemically defined media containing insulin, transferrin, selenium, tri-iodothyronine and either fibronectin or laminin. The tubular epithelial cells were studied ultrastructurally and compared to another epithelial cell type present in the renal cortex, the glomerular epithelial cell. In contrast to the constant morphology of glomerular epithelial cells grown in chemically defined media, tubular epithelial cell morphology depended on whether the cells were placed in fibronectin or laminin and on the age of the donor animal used for culture. Primary tubular cells grown in laminin formed colonies; cells grown from young animals were rounded, whereas cells grown from adult animals were flattened. Primary tubular cells grown in fibronectin were flattened regardless of age, but cells from young animals formed colonies while those from adult animals formed a monolayer. Despite these differences in gross morphology, scanning and transmission electron microscopy revealed similar ultrastructural features in primary tubular cells from young and adult animals grown in fibronectin or laminin. Quantitative adhesion studies demonstrated that secondary subcultured tubular cells adhered equally well to dimeric and multimeric forms of fibronectin, but not to laminin. Quantitative colony growth studies of subcultured secondary tubular cells showed that laminin supports colony formation of trypsinized tubular cells, while previous work has demonstrated that fibronectin supports colony formation of glomerular cells. These results are consistent with the hypothesis that different extracellular matrix molecules are involved in colony formation of different cell types, with fibronectin stimulating growth of glomerular cells and laminin supporting growth of tubular cells.     

Mesothelial cells in suspension expose an enriched integrin repertoire capable of capturing soluble fibronectin and laminin

Pleural cavities are lined by a polarized monolayer of mesothelial cells (MC). During pleuritis, MC are shed into effusions, and pleural obstruction may occur. Integrins are cell surface receptors mediating interactions with extracellular matrix (ECM) proteins. The distribution of beta 1-, beta 3-, beta 4-integrins and fibronectin and laminin in normal and chronically inflamed pleura and in/on MC from pleural effusions was examined by immunomorphology and flow cytometry. Adhesion assays of MC to fibronectin and laminin were performed. In situ, resting MC expressed beta 1-, beta 3-, and beta 4-, and alpha v-subunits. Activated MC were beta 1- and alpha v-positive and also expressed alpha 3 and alpha 6; beta 4 was confined to the basal surface of MC; beta 3 was absent. Floating MC from effusions neoexpressed alpha 5 and reexpressed beta 3. In vitro, MC surface expressed beta 1, beta 3, alpha 3, alpha 5, alpha 6, alpha v, and also alpha 1 and alpha 2. In normal pleura, fibronectin and laminin were components of the basement membrane. In pleuritis, the basement membrane was desintegrated. Instead, newly formed fibronectin/laminin containing fibrils extended into the submesothelial connective tissue. Floating MC freshly isolated from effusions carried fibronectin and laminin on their surface and showed specific binding to these ECM proteins. Binding was blocked by anti-beta 1 or anti-alpha 5 and anti-alpha 6 antibodies, respectively. MC incubated with fibronectin showed a clear shift to the S phase, while laminin had no effect. In conclusion, activated and detached MC progressively enrich their integrin repertoire. By capturing soluble fibronectin and laminin and by matrix-mediated bridging, readhering MC may contribute to pleural obstruction. Further, soluble fibronectin bound to alpha 5 beta 1 might be life-sustaining for floating MC by driving cells into cell cycle.     

Colocalization of laminin and fibronectin in bovinelens epithelial cells in vitro

Summary The distribution and organization of the extracellular matrix (ECM) proteins laminin, fibronectin, entactin, and type IV collagen were investigated in primary colonies and secondary cultures of bovine lens epithelial cells using species-specific antisera and indirect immunofluorescence microscopy. Primary cell colonies fixed in formaldehyde and permeabilized with Triton X-100 displayed diffuse clonies. In contrast, thick bundles of laminin and fibronectin were located on the basal cellsurfaces and in between cells in the densely packed center of the colonies, and as “adhesive plaques” and fine extracellular matrix cords in the sparsely populated (migratory) outer edge of the colonies. The distribution of ECM proteins observed in secondary lens epithelial cell cultures was similar to that observed at the periphery of the primary colony. Extraction of the secondary cell cultures with sodium deoxycholate confirmed that laminin and fibronectin were deposited on the basal cell surface. Indeed, the patterns of laminin and fibronectin deposition suggested that these proteins codistribute. These results establish that lens epithelial cells in culture can be used as a model system to study the synthesis and extracellular deposition of the basement membrane proteins, laminin and fibronectin. Supported by Public Health Service grant EY05570 from the National Eye Institute Bethesda, MD.     

Effects of laminin, fibronectin and type IV collagen on liver cell cultures

The effects on mouse liver cells of laminin, fibronectin and type IV collagen, all of which are the main matrix of the basement membrane, were studied. Laminin, a glycoprotein isolated from cultures of rat yolk sac carcinoma cells, promoted the attachment of mouse fetal liver cells to laminin-coated dishes, but did not have a strong influence upon the attachment of normal adult liver cells. On the other hand, fibronectin which was purified from mouse plasma promoted the attachment of adult liver cells but not that of fetal liver cells. The number of neonatal liver cells attached to the surfaces coated was intermediate between those of fetal and adult liver cells in each matrix. DNA synthesis and cell proliferation during the culture of full-term fetal liver cells in laminin-coated dishes were higher than those in fibronectin- or type IV collagen-coated dishes. The amount of alpha-fetoprotein secreted in the laminin-coated dishes was more than in other groups. No differences in secretion of albumin into media, however, were observed in either group. These results suggest that laminin may be necessary for cell growth, tissue organization and cell differentiation during the normal development of liver in vivo.     

Studies of fibronectin matrices in living cells with fluoresceinated gelatin

We have used fluorescein isosthiocyanate-conjugated gelatin (FITC- gelatin) (1 mg/ml) to localize cell surface fibronectin in unfixed live cells in cultures. FITC-gelatin stains the fibronectin matrix on primary cultures of rat and chick embryo fibroblasts as well as untransformed, established cell lines. In live cultured cells, fibronectin in many areas of the extracellular matrix is inaccessible to antibody and cannot be visualized by immunofluorescence staining. In contrast, fibronectin in these areas is fully stainable by FITC- gelatin. At a low concentration (20 micrograms/ml), FITC-gelatin stains the fibronectin matrix of primary cultured cells but not of "untransformed" established cell lines. SEM can detect only the matrix stainable with the low concentration of FITC-gelatin, such as that expressed by primary chick embryo fibroblasts. The binding of fibronectin to the extracellular matrix is very stable and FITC-gelatin remained bound to the matrix for at least 10 d in culture. Radioiodinated gelatin has been used to quantitate the level of cell surface fibronectin in living normal and transformed cells. FITC- gelatin appears to be a useful probe for studying the fibronectin of living cells in culture.     

Laminin B1 expression is required for laminin deposition into the extracellular matrix of PC12 cells.

The extracellular matrix of rat pheochromocytoma PC12 cells was shown by indirect immunofluorescence to consist of a network of fibronectin. The matrix did not contain laminin. The cells synthesized messenger RNA for fibronectin, laminin B2, and s-laminin but not for entactin or the B1 and A chains of laminin. Laminin B2 but not laminin B1 was detectable in the culture medium and in cell lysates. A full-length cDNA clone for the B1 chain of laminin was constructed in the plasmid p-444, which contains the neomycin-resistance marker and human beta-actin promoter. PC12 cells were transfected with this recombinant plasmid, and stable neomycin-resistant clones were isolated and characterized. Clones that synthesized laminin B1 messenger RNA were found to deposit a laminin-containing matrix. In many of these clones the deposition of the fibronectin matrix was greatly diminished. The laminin matrix was predominantly localized in the intercellular spaces forming a honeycomb pattern. The morphology of the laminin-synthesizing transfected cells was markedly different from the parental cells. The cells grew in tight clusters that were resistant to dissociating agents. It is concluded that the B1 chain of laminin contains information that is required for the formation of a stable laminin-containing extracellular matrix network either by interaction with cell surface receptors or other extracellular matrix components. Furthermore, expression of the laminin B1 gene may be a central regulatory point in determining extracellular matrix composition during embryogenesis.     

Extracellular matrix components direct porcine muscle stem cell behavior

In muscle tissue, extracellular matrix proteins, together with the vasculature system, muscle-residence cells and muscle fibers, create the niche for muscle stem cells. The niche is important in controlling proliferation and directing differentiation of muscle stem cells to sustain muscle tissue. Mimicking the extracellular muscle environment improves tools exploring the behavior of primary muscle cells. Optimizing cell culture conditions to maintain muscle commitment is important in stem cell-based studies concerning toxicology screening, ex vivo skeletal muscle tissue engineering and in the enhancement of clinical efficiency. We used the muscle extracellular matrix proteins collagen type I, fibronectin, laminin, and also gelatin and Matrigel as surface coatings of tissue culture plastic to resemble the muscle extracellular matrix. Several important factors that determine myogenic commitment of the primary muscle cells were characterized by quantitative real-time RT-PCR and immunofluorescence. Adhesion of high PAX7 expressing satellite cells was improved if the cells were cultured on fibronectin or laminin coatings. Cells cultured on Matrigel and laminin coatings showed dominant integrin expression levels and exhibited an activated Wnt pathway. Under these conditions both stem cell proliferation and myogenic differentiation capacity were superior if compared to cells cultured on collagen type I, fibronectin and gelatin. In conclusion, Matrigel and laminin are the preferred coatings to sustain the proliferation and myogenic differentiation capacity of the primary porcine muscle stem cells, when cells are removed from their natural environment for in vitro culture.     

Butyrate restores fibronectin at cell surface of transformed cells

Transformed rat kidney cells were found to produce soluble fibronectin in amounts higher than those produced by normal rat kidney cells. In spite of this, they lacked the fibrillar fibronectin network present at the surface of the normal cells. Treatment of the transformed cells with butyrate converted them into extremely flat cells with abundant fibronectin at the cell-substratum interphase. Such cells subsequently grew into monolayers with normal morphology. This was accompanied by a restoration of fibrillar fibronectin network at the cell surface.     

Identification of the heparin-binding determinants within fibronectin repeat III1: role in cell spreading and growth

Fibronectins are high molecular mass glycoproteins that circulate as soluble molecules in the blood, and are also found in an insoluble, multimeric form in extracellular matrices throughout the body. Soluble fibronectins are polymerized into insoluble extracellular matrix (ECM) fibrils via a cell-dependent process. Recent studies indicate that the interaction of cells with the ECM form of fibronectin promotes actin organization and cell contractility, increases cell growth and migration, and enhances the tensile strength of artificial tissue constructs; ligation of integrins alone is insufficient to trigger these responses. Evidence suggests that the effect of ECM fibronectin on cell function is mediated in part by a matricryptic heparin-binding site within the first III1 repeat (FNIII1). In this study, we localized the heparin-binding activity of FNIII1 to a cluster of basic amino acids, Arg613, Trp614, Arg615, and Lys617. Site-directed mutagenesis of a recombinant fibronectin construct engineered to mimic the ECM form of fibronectin demonstrates that these residues are also critical for stimulating cell spreading and increasing cell proliferation. Cell proliferation has been tightly correlated with cell area. Using integrin- and heparin-binding fibronectin mutants, we found a positive correlation between cell spreading and growth when cells were submaximally spread on ECM protein-coated surfaces at the time of treatment. However, cells maximally spread on vitronectin or fibronectin still responded to the fibronectin matrix mimetic with an increase in growth, indicating that an absolute change in cell area is not required for the increase in cell proliferation induced by the matricryptic site of FNIII1.     

Purification, partial characterization of rat kidney hyaluronic acid binding protein and its localization on the cell surface.

Hyaluronic acid binding protein (HABP) has been purified to homogeneity from normal adult rat kidney by hyaluronate Sepharose affinity chromatography, and its apparent molecular mass was found to be 68 kDa. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of HABP under reducing as well as nonreducing conditions revealed a single protein band of 34 kDa, thus indicating that kidney HABP is a homodimer and lacks interchain disulfide bond. Its glycoprotein nature was demonstrated by Con-A binding analysis. The pI value of kidney HABP was 6, indicating its acidic nature. Polyclonal antibodies were raised against it, and the monospecificity of the antibodies towards HABP was confirmed by Western blot analysis of tissue extracts. Immunoblot analysis has elucidated the occurrence of this glycoprotein in various tissues. Moreover, HABP present in these tissues are shown to be structurally and immunologically identical. However, this glycoprotein is antigenically distinct from other well characterized extracellular proteins, e.g., fibronectin, laminin and collagen type IV. With the help of enzyme-linked immunosorbent assay (ELISA) and iodinated [125I]HABP, it has been shown that kidney HABP binds specifically to hyaluronic acid (HA) amongst all the glycosaminoglycans (GAGs), however, HABP can interact with other matrix proteins, e.g., laminin, fibronectin, and collagen type IV. The apparent dissociation constants of HABP for HA, laminin, fibronectin, and collagen type IV were approximately in the range of 10(-9) M, and kinetic analysis showed that these binding interactions were complex and of positive cooperative nature. Indirect immunofluorescence staining demonstrated its localization on human fetus lung fibroblast cell surface. Detection of 34 kDa HABP in the serum-free supernatant culture medium of fibroblasts was further evident by immunoblot analysis, thus confirming the secretory nature of HABP and its occurrence in the extracellular matrix.