Effect of serum,fibronectin, and laminin on adhesion of rabbit intestinal epithelial cells in culture

Rabbit intestinal epithelial cells, obtained after a limited hyaluronidase digestion, were incubated in medium with or without calf serum, on bacteriological plastic dishes. The dishes, either plain or coated with an air-dried type I collagen film, were pretreated with medium alone or with medium containing purified laminin or purified fibronectin. Cells did not attach in significant numbers to untreated bacteriological plastic, even in the presence of serum. Cells did attach to collagen-coated dishes, and were judged viable on the basis of their incorporation of radiolabeled leucine into cell protein. Cell adhesion to the collagen substrate increased in proportion to the concentration of serum in the medium, with maximal attachment at 5% serum or greater. Pretreatment of plain or collagen-coated dishes with increasing amounts of fibronectin enhanced cell adhesion in a concentration-dependent manner. Either serum, or fibronectin-free serum in the medium enhanced cell attachment to substrates pretreated with cither fibronectin or laminin. Thus, intestinal epithelial cells appear to possess surface receptors for both laminin and fibronectin. The evidence further suggests that calf serum may contain factors, other than fibronectin, capable of enhancing intestinal epithelial cell attachment to collagen substrates.     

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.     

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.     

Expression of fibronectin and laminin by different types of mouse glial cells cultured in a serum-free medium

The expression of fibronectin and laminin by cultured glial cells was studied. The glial culture from neonatal mouse cerebra maintained in a chemically defined, serum-free medium consisted of type-1 astrocytes, oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, oligodendrocytes and type-2 astrocytes. Double-labelling immunofluorescent experiments performed using the mixed glial culture indicated that fibronectin and laminin are expressed in different patterns among the glial subtypes. The staining intensities with anti-fibronectin or anti-laminin antibodies decreased in the order: type-1 astrocytes, O-2A progenitor cells and type-2 astrocytes. Both molecules were deposited in a fibrillar matrix underneath type-1 astrocytes, whereas only intracytoplasmic localization of these molecules was observed with O-2A progenitor cells and type-2 astrocytes. Western blot analysis showed that glial fibronectin has a slightly higher molecular weight than mouse plasma fibronectin (230 kDa) and that glial laminin is a variant with a 220 kDa B chain present and the 400 kDa A chain missing. Using enzyme-linked immunosorbent assays (ELISA), these molecules were detected in the glial extracellular matrix at the concentration of 4 ng/10⁶ cells. A large amount of fibronectin (82 ng/10⁶ cells) was secreted into the culture medium, while secretion of laminin was not detected.     

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).     

HeLa cell adhesion to microcarriers in high shear conditions: evidence for membrane receptors for collagen but not laminin or fibronectin

HeLa-S3 cells were analyzed for their ability to attach and spread on cell culture microcarriers that were made either positively or negatively charged with polymeric plastics or were coated with BSA, gelatin, fibronectin or laminin. The cells stuck to all microcarriers under low shear, i.e. low stirring conditions with similar rates of attachment. Except in the case of gelatin microcarriers where cells fully spread, cells did not or only partially spread on the others. Under high shear, cells attached with the following rates: positive = negative = gelatin = BSA greater than laminin greater than fibronectin. Cells detached from all but the gelatin and BSA coated beads. However, the cells did not fully spread on BSA beads. The observation that cells not only attached but also spread on gelatin beads indicated that gelatin could be a specific substratum adhesion protein while the other surfaces were 'non-specific'. It should be noted that neither antibodies to laminin nor fibronectin interfered with attachment to gelatin. Protein synthesis inhibitors reduced the attachment and spreading on gelatin beads under high but not low shear conditions. With low shear, attachment and spreading appeared normal. We concluded that the density of the cell surface attachment proteins was reduced by the protein synthesis inhibitors and there were not enough present to facilitate attachment under high shear. The results also indicated that protein synthesis was not essential for cell spreading. Proteolysis of the cell surface with low concentrations of trypsin abolished the attachment of cells to gelatin-coated beads. The reappearance of attachment ability took several hours and was inhibited by actinomycin-D.     

Antagonistic effects of laminin and fibronectin on the expression of the myogenic phenotype

Skeletal myoblasts from fetal muscle respond adversely to fibronectin and laminin substrata: when primary mouse skeletal myoblasts are plated onto laminin, more myosin and desmin-positive myoblasts (myo+ cells) develop than on plates coated with fibronectin or collagen. In clonal cultures virtually all cells differentiate into postmitotic, fusion-capable myo + myoblasts on laminin after 3 days. In contrast, on fibronectin, the majority of the cells becomes myosin- and desmin-negative, partially due to proliferation of undifferentiated myoblast precursor cells, partially due to dedifferentiation or modulation of myoblasts into fibroblast-like myo- cells. Loss of the myogenic phenotype on fibronectin was also observed in cloned mouse myoblasts and in cultures of a differentiating mouse satellite cell line, MM14Dy, confirming that the appearance of desmin-negative cells is a result of myoblast modulation and not due simply to overgrowth by muscle fibroblasts. In the light of other effects of laminin on myoblasts, such as the stimulation of migration, differentiation and proliferation, our findings are consistent with the notion that laminin and fibronectin may be counteracting factors in the control of muscle differentiation.     

Possible role of fibronectin in malignancy

Frozen sections of tumors induced by injecting virally transformed cells into animals were stained for fibronectin by immunofluorescence. Many tumor cell lines do not express fibronectin in tumors in situ even though some of them express fibronection in culture. Cell shape and hormones appear to influence the expression of fibronectin in culture; however, it is nuclear how fibronection expression is regulated in vivo.     

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.     

Differential effects of cellular fibronectin and plasma fibronectin on ovarian cancer cell adhesion, migration, and invasion

Summary The main form of fibronectin (FN) encountered by tumor cells in vivo is cellular FN (cFN), which differs structurally and functionally from the commonly used plasma FN (pFN). We compared the effects of cFN and pFN on the ovarian carcinoma lines OVCAR-3 and SKOV-3 and on cultures of normal ovarian surface epithelium, which is the precursor of the epithelial ovarian carcinomas. Ovarian surface epithelial cells and SKOV-3 cells attached and spread faster on cFN than on pFN. On cFN, SKOV-3 migration was enhanced compared with pFN or plastic. In a matrigel transfilter assay, cFN strongly inhibited SKOV-3 invasion, whereas pFN did not. In contrast to SKOV-3, OVCAR-3 cells adhered faster on FN than on plastic but did not discriminate between cFN and pFN, and they did not migrate or invade matrigel either with or without FN. In both carcinoma lines, proliferation was unaffected by either FN. The results show profound differences in the responses to cFN and pFN by two invasive ovarian carcinoma lines. Because cFN is the main type that cancer cells encounter in vivo, extrapolations from culture data to in vivo events should preferably be based on studies using this form of FN.     

The kinetics of fibronectin synthesis and release in normal and tumor promoter-treated human lung fibroblasts

Summary The present study is a detailed kinetic analysis of the synthesis, release and multimerization of fibronectin (FN) in normal and tumor promoter-treated human lung fibroblasts. Pulse/chase and surface labeling experiments were performed to follow the fate of both newly synthesized and pre-existing cell-surface FN over time. The majority of FN (80%) left the intracellular compartment within one hour of synthesis. However, the rate of direct secretion was very low and after one hour, 70% of newly synthesized FN was still at the cell surface. This material was primarily dimeric. Dimeric and multimeric (very high molecular weight) FN was detectable at the cell surface and in the medium 4 hours after synthesis. Pulse-labeled FN multimer levels peaked at 12 hours and declined thereafter. After 24 hours, 85% of pulse-labeled FN had been shed into the medium and the labeled FN remaining at the cell surface was primarily multimeric. Surface labeling experiments confirmed that the majority of FN resides at the cell surface prior to release into the medium.One hour treatment with the phorbol ester tumor promoter, 12-0-tetradecanoyl phorbol-13-acetate (TPA), stimulated a nine-fold increase in release of preexisting, dimeric cell-surface FN (¹²⁵I-labeled). The major effect of longer term TPA treatment up to nine hours was continued depletion of dimeric cell-surface FN. Increased release of cell-surface multimeric FN was also stimulated by TPA, but to a much lesser extent. Release of newly synthesized (pulse-labeled) dimeric FN was also stimulated by TPA though much less than pre-existing FN, and TPA treatment produced a small decrease in the steady-state level of multimeric FN. Thus, preexisting cell-surface IN and newly synthesized FN differ dramatically in their susceptibility to TPA treatment.Abbreviations FN fibronectin - HLF human lung fibroblasts - PMSF phenylmethylsulfonylfluoride - TPA 12-O-tetradecanoyl phorbol-13-acetate - DMSO dimethylsulfoxide - MEM minimal essential medium - ELISA enzyme linked immunosorbent assay - PBS phosphate-buffered saline - BSA bovine serum albumin - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - DOC deoxycholate - DTT dithiotreitol     

A combinatorial approach for directing the amount of fibronectin fibrils assembled by cells that uses surfaces derivatized with mixtures of fibronectin and cell binding domains

Fibrillar fibronectin (FN) has the crucial role of attracting and attaching cells as well as molecules that mediate tissue repair during wound healing. A previous study demonstrated higher extracellular staining of FN fibrils in cells cultured on surfaces tethered with an equimolar mixture of a FN binding domain and FN's cell binding domain, III1-2 and III9-10 respectively, than on surfaces with III9-10 alone. The effect of varying surface amounts of III1-2 and III9-10 on the quantity of FN fibrils formed by NIH-3T3 fibroblasts was examined. GST tagged III1-2 and III9-10 were conjugated to polyurethane surfaces and ELISAs were used to identify the experimental design space or the range of concentrations of GST-III1-2 and GST-III9-10 that demarcated the limits of protein loading on the surface. When GST-III1-2 was fixed and GST-III9-10 varied within the design space, the amount of FN fibrils measured by immunoblotting detergent insoluble cell lysates was dependent on the ratio of III9-10 to III1-2. When the total protein concentration was fixed and the mixture composition of GST-III1-2 and GST-III9-10 varied such that it optimally covered the design space, a parabolic relationship between FN fibril amount and the ratio of III9-10 to III1-2 was obtained. This relationship had a maximum value when the surface was bonded to equal amounts of III1-2 and III9-10 (P<0.05). Thus the ratio of III9-10 to III1-2 can be utilized to direct the quantity of FN fibrils formed on surfaces.     

Role of laminin in axonal extension from olfactory receptor cells

The role of laminin, an extracellular matrix molecule believed to be involved in axon extension, was explored in the outgrowth of olfactory receptor cells and therefore in the maintenance of organization in the olfactory pathway. First, immunocytochemistry was used to examine laminin expression in the olfactory nerve and bulb during development. Laminin immunoreactivity was high in the olfactory nerve and glomerular layers. Although it declined in intensity, laminin expression continued in the nerve and in single glomeruli of adults. Second, the influence of laminin on neurite outgrowth was examined in vitro using olfactory receptor cells harvested from E14 rat embryos. We developed an in vitro assay to quantify the substrate preference of outgrowing neurites. Cells were cultured for 48 h on coverslips coated with either poly-L-lysine alone, or poly-L-lysine overlaid with laminin. On laminin-coated regions of coverslips, the primary neurites of olfactory receptor cells were 52% longer than on the poly-L-lysine control substrates. In addition, the direction of the neurite outgrowth was influenced by laminin. Fifty-six percent of all receptor cells located in a defined area surrounding a laminin zone extended neurites onto laminin. In contrast, only 7% of all receptor cells located in the corresponding laminin zone extended a neurite onto poly-L-lysine. In summary, these data suggest that laminin provides a favorable substrate for the extension of the primary neurite from olfactory receptor cells and the direction of their extension. Therefore, laminin may be a factor underlying continuous olfactory receptor cell axon outgrowth and its pathfinding in the olfactory system. © 1997 John Wiley & Sons, Inc. J Neurobiol 00: 32: 298–310, 1997     

Characterization of a membrane-associated glycoprotein complex implicated in cell adhesion to fibronectin

We have characterized a 140-kDa glycoprotein complex purified by a monoclonal antibody and implicated in cell adhesion to the extracellular molecule fibronectin. Three major polypeptide components were purified by monoclonal antibody JG22E, which had apparent molecular weights of 155,000 (band 1), 135,000 (band 2), and 120,000 (band 3). In two-dimensional gel electrophoresis, each subunit migrated as either a broad band or a series of spots at acidic isoelectric points. After treatment with neuraminidase, the spots became focused around pH 6.2 (band 1), pH 5.6 (band 2), and pH 5.3 (band 3). These three major bands were compared by two-dimensional peptide mapping in a series of pairwise combinations and were found to be distinct proteins. In sucrose gradients, these proteins co-migrated as a complex sedimenting at approximately 8.4 S either before or after affinity purification, whereas separated subunits migrated at 4.7 to 5.8 S. Amino acid analysis revealed no detectable hydroxyproline and a composition characterized by a substantial number of cysteine residues compared to the average protein. Our results suggest that a noncovalent complex of structurally distinct glycoproteins is involved in adhesive interactions of fibronectin with cells.     

33-kDa C-terminal heparin binding fragment of fibronectin promotes attachment and spreading of hepatocytes

The possibility of interaction of hepatocytes with the heparin binding domain of Fibronectin was examined. Rat hepatocytes adhered to coverslips coated with the 33-kDa heparin binding fragment of the C-terminal region of plasma fibronectin. When different concentrations of the heparin binding fragment were used to coat coverslips and used as substratum, cell attachment showed saturation kinetics. Half the maximum attachment was observed at 30–40 min after seeding of cells. The cells became flat after 2–3 h indicating that they spread on the heparin binding domain as they do on intact fibronectin. Among the different glycosaminoglycans tested, maximum inhibition of attachment was observed for heparin. However it was not possible to completely inhibit attachment even at high concentrations. These results indicate that hepatocytes interact with fibronectin not only through the Arg-Gly-Asp-containing cell binding fragment, but also through the heparin binding domain of fibronectin and, further, that there exist heparin-dependent and heparin-independent mechanisms of interaction of cells with the 33-kDa heparin binding fragment of fibronectin     

Consequences of relative cellular positioning on quorum sensing and bacterial cell-to-cell communication

Cell-to-cell bacterial communication via diffusible signals is addressed and the conceptual framework in which quorum sensing is usually described is evaluated. By applying equations ruling the physical diffusion of the autoinducer molecules, one can calculate the gradient profiles that would occur either around a single cell or at the center of volumes of increasing size and increasing cell densities. Water-based matrices at 25 °C and viscous biofilms at colder temperatures are compared. Some basic consequences relevant for the field of microbial signalling arise. As regards induction, gradient-mixing dynamics between as little as two cells lying at a short distance appears to be sufficient for the buildup of a concentration reaching the known thresholds for quorum sensing. A straight line in which the highest concentrations occur is also created as a consequence of the gradient overlap geometry, providing an additional signal information potentially useful for chemotactic responses. In terms of whole population signalling, it is shown how the concentration perceived by a cell in the center is critically dependent not only on the cell density but also on the size of the biofilm itself. Tables and formulas for the practical prediction of N -acyl homoserine lactones concentrations at desired distances in different cell density biofilms are provided.     

Distribution of a major surface-associated glycoprotein,fibronectin, in cultures of adherent cells

Fibronectin was present in media and cell layers of cultures of adherent cells from human skin, kidney, lung, chest wall, liver, and heart. Cell-surface fibronectin, visualized by immunofluorescence, was in dense fibrillar (cultures from lung), discrete fibrillar (e.g., cultures from skin), or punctate (some cultures from kidney) structures. The subunit sizes of cell-surface fibronectin and fibronectin soluble in medium appeared identical in sodium dodecyl sulfate-polyacrylamide gels. To explain the polymorphism of cell-surface fibronectin, there must be chemical differences among the fibronectins synthesized by different cell strains or factors in the cell layer which influence fibronectin binding and aggregation.     

Active sites in the carboxyl-terminal region of the laminin alpha chain in Drosophila neuronal cell spreading

An established Drosophila neuronal cell line (BG2-c6) proved to be useful to analyze laminin-mediated cell spreading and signal transduction [Takagi et al. (2000) Biochem Biophys Res Commun 270:482-487]. Here, we report, in addition to the whole molecule, the truncated alpha chain of Drosophila laminin (containing the entire carboxyl-terminal globular domain) and two dodecapeptides corresponding to the cell-binding sites identified in the alpha1 chain of mouse laminin were also active to stimulate BG2-c6 cell spreading. Our previous study [Takagi et al. (1996) J Biol Chem 271:18074-18081] revealed that these recombinant protein and synthetic peptides promoted neurite outgrowth in the primary cell culture system prepared from Drosophila embryo. Therefore, the similar effects by these proteins and peptides suggest the presence of a common mechanism of laminin and neuronal cell interaction working in both primary and established cells. One of the two active peptides contains the sequence SIKVGV. Its murine counterpart carries the sequence SIKVAV by which the interaction of laminin and cells is mediated. Furthermore, laminin-dependent BG2-c6 cell spreading was inhibited by heparin. This observation suggests that cell surface glycoproteins participate in the interaction of laminin and BG2-c6 cells.     

Identification of active sequences in human laminin α5 G domain

Human laminin‐511 (α5β1γ1) and its truncated protein, laminin‐511 E8 fragment, bind to integrin α6β1 and have been widely used for embryonic stem cell and induced pluripotent stem cell culture under feeder‐free conditions. In this study, we focused on human laminin α5 chain G domain, which is thought to be critical for the biological functions of laminin‐511, and screened its biologically active sequences using a synthetic peptide library. We synthesized 115 peptides (hA5G1‐hA5G115) covering the entire laminin α5 chain G domain and evaluated cell attachment activity using both the peptide‐coated plate and peptide‐chitosan matrix (peptide‐ChtM) assays. Seventeen peptides demonstrated cell attachment activity in the assays. Both hA5G18 and hA5G26‐coated plates and hA5G74‐ChtMs promoted integrin β1‐mediated cell attachment. These findings are useful for the study of molecular mechanisms of laminin‐511, and the active peptides have a potential for use as a molecular probe for cell adhesion receptors.