Fibronectin biosynthesis: influence on fibroblast adhesion during chick embryo development

Eight d (8d) and 16d (16d) chick embryo fibroblasts (CEF) exhibited marked differences in their adhesive capacity on plastic support, but not on fibronectin substratum. This suggests differences in fibronectin (FN) expression and/or FN receptor expression. Both 8d and 16d CEF expressed an identical number of membrane receptors for FN with similar affinity. In contrast, the newly synthesized FN appeared de novo in 30 min in 8d CEF versus 60 min in 16d CEF. This difference is not due to a modification of the polypeptide chain biosynthetic rate. The FN synthesized in 8d CEF became insensitive to endo beta-N-acetyl-glucosaminidase H (endo H) treatment after 20 min, whereas it remained sensitive to endo H until 60 min in 16d CEF. Post-translational modifications of N-linked mannose-rich chains to complex type chain may account for the difference in the expression of cell surface FN and thus for the difference in cell adhesion capacity to plastic.     

Alterations of fibroblast interactions with fibronectin and laminin during chick embryo development

Eight day (8-d CEF) and 16 day old chick embryo fibroblasts (16-d CEF) obtained after a mild trypsin treatment (50 micrograms/ml in Ca2+ and Mg2+-free PBS, plus 10 mM EDTA) for 10 min at 37 degrees C present the same number of fibronectin (FN) binding sites at their surface (approximately 550,000 sites per cell) with a Kd approximately equal to 1.40 microM in both cases. Furthermore, FN interacted with high molecular weight plasma membrane proteins (150,000 and 125,000) insensitive to trypsin treatment. Both 8-d and 16-d CEF adhered and spread to the same extent on a fibronectin coated substratum (80% of the CEF adhered in 60 min). In contrast, 8-d and 16-d CEF behaved differently towards laminin (LM). 8-d CEF exhibited approximately 5500 binding sites per cell with a Kd of 1.5 nM (Codogno P., Doyennette, M.-A. and Aubery M., 1987, Experimental Cell Research, 169, 478-489.) and were highly sensitive to trypsin treatment, whereas 16-d CEF do not express cell surface binding sites for laminin. Differences were also observed in the adhesive capacities of 8-d and 16-d CEF on LM substrata: 8-d CEF adhered and spread on LM in a very specific manner (60% of the cells adhere in 60 min) and 16-d CEF did not adhere to LM even after long periods of incubation exceeding 360 min.     

Concanavalin A-induced impairment of fibroblast spreading on laminin but not on fibronectin

The effect of concanavalin A (Con A) on the adhesion of 8-day-old chick embryo fibroblasts (CEFs) to fibronectin (FN) and laminin (LM) was studied. Con A was shown to inhibit the spreading of CEF on a LM substrate. In contrast, no inhibition of CEF spreading on the FN substrate could be detected when the quantity of FN coated varied from 0.5 to 4 pmoles. The effect induced by Con A was specific, since it was abolished by 100 mM alpha-methylmannopyranoside. The inhibition of CEF spreading was only observed when the lectin was added during the 20 min following cell plating. In addition, the effect of Con A on CEF spreading on the LM substrate was shown to be dependent upon its presence at the cell surface, since under conditions which accelerate the uptake of the lectin, the effect on cell spreading is no longer detectable. Furthermore, the number of CEFs attached to LM was not modified by the lectin. The molecular weight of the isolated Con A binding sites revealed glycoproteins ranging from 30,000 to 72,000. On the other hand, these Con A binding sites did not interact with LM-Sepharose. Only a protein with a molecular weight of 68,000 which did not express affinity for Con A bound tightly to the LM-Sepharose. These data suggested that cell surface Con A binding sites do not interfere with the initial step of CEF adhesion to LM but play a key role during their spreading on this glycoprotein.     

Cell spreading on laminin substrate involves Con A-binding proteins

Concanavalin A (Con A), a tetravalent lectin, was shown to impair 8 chick embryo fibroblast (8 d CEF) spreading on a laminin (LM) substrate but not on a fibronectin substrate (FN), suggesting that cell surface Con A binding proteins could be involved in 8 d CEF spreading on a LM substrate. The interaction of Con A-binding proteins with Con A is dependent upon the carbohydrate moieties of the isolated glycoproteins; since they interact strongly with Con A-Sepharose and are eluted with 0.3 Mol/l alpha-methylmannopyranoside, the isolated Con A binding-proteins inhibit 8 d CEF adhesion to a Con A substrate to the same extent as alpha-methylmannopyranoside. Furthermore, the isolated Con A binding proteins specifically inhibit in a dose-dependent manner 8 d CEF spreading on LM but not on FN.     

A Mr 72K cell surface concanavalin A binding glycoprotein is specifically involved in the spreading of chick embryo fibroblasts onto laminin substrate

In the present study we have identified a 72-kDa cell surface concanavalin A binding glycoprotein (cbg 72) involved in the chick embryo fibroblast (CEF) adhesion onto laminin (LM) substrate. The cbg 72 was shown to interact specifically with immobilized laminin and to be resistant to Triton X-100 extraction when CEF were plated on laminin substrate but not on fibronectin (FN) substrate. This behavior suggested that cbg 72 could interact with cytoskeletal elements during cell spreading onto LM. This assumption is also in good agreement with the partitioning of cbg 72 in Triton X-114. Isolated cbg 72 specifically inhibited CEF spreading onto LM after their initial attachment, whereas cbg 72 did not impair the spreading of CEF onto FN. These data provide a molecular explanation to the inhibition of CEF spreading onto LM observed in the presence of the lectin concanavalin A (P. Codogno, M.-A. Doyennette-Moyne, J. Botti, and M. Aubery, 1988, J. Cell Physiol. 136, 463-470). Moreover, these results provide evidence for the role of a novel LM binding glycoprotein during the adhesion of mesenchymal derived cells. The relationship between cbg 72 and other known cell surface LM binding sites or receptors is discussed.     

Evidence for phosphatidylinositol-linked forms of human and avian fibronectin receptors

Phosphatidylinositol (PI)-linked forms of surface molecules have been hypothesized to mediate the initial stages of cell adhesion or signal transduction. We report evidence for the occurrence of a functional PI-linked subset of cell surface fibronectin receptors (FNR). Treatment of human MG63 osteosarcoma cells or primary chicken embryo fibroblasts (CEF) with PI-specific phospholipase C (PI-PLC) reduced cell surface FNR expression by 30% as detected by immunofluorescence. PI-PLC treatment of cell membranes purified from [35S]methionine-labeled CEF or MG63 cells led to a similar loss of membrane-associated immunoprecipitable FNR from the pelleted membranes, while such treatment led to the appearance of FNR in the supernatant of treated MG63 membranes. Biosynthetic labeling of CEF FNR with [3H]palmitate and [3H]ethanolamine demonstrated the acylation and putative PI linkage of avian FNR subunits. PI-PLC treatment of CEF and MG63 cells also reduced fibronectin-specific adhesion in a short-term in vitro assay, suggesting that the avian and human FNR occur in PI-linked isoforms which appear to contribute to cell adhesion to fibronectin.     

Cell surface glycosaminoglycans (GAGs) of cultured chick fibroblasts : Modifications in relation to the stage of embryo development

We have investigated the changes in glycosaminoglycan (GAG) composition between cultured fibroblasts derived from 8- and 16-day chick embryos. GAG composition has been studied after [3H]glucosamine and [35S]sulfate labeling. Both the 8- and 16-day embryo fibroblasts were found to contain hyaluronic acid (HA), dermatan sulfate (DS), heparan sulfate (HS) and chondroitin sulfates (CS), the latter being the major component in 8- and 16-day cells. These four GAGs were quantified after their separation using cellulose acetate electrophoresis. The amounts of HA and CS were respectively shown to increase 2-fold and 4-fold between the 8th and 16th day of development, whereas the amounts of HS and DS resp. diminished 2.5-fold and 1.2-fold. These results show that the relative proportions of the different GAGs alter during embryo development. The fibroblasts from 8-day-old embryos detached more rapidly from the culture dishes than the cells from 16-day-old embryos when treated with trypsin. However, this difference was not directly related to the different GAG content.     

Peri- and postnatal development of heterogeneity in the amounts of endoplasmic reticulum in mouse hepatocytes

The surface density and area per cell of the endoplasmic reticulum (ER) in periportal and perihepatic hepatocytes from male ddY mice, "17-, 18-, and 19-day-old fetuses," "newborn and 1-, 5-, 10-, and 20-day-old animals," and "adult animals" were analyzed by quantitative electron microscopy. The surface density of rough ER was not significantly different between periportal and perihepatic cells in all age groups examined, except for 19-day-old fetuses in which the value was greater in periportal cells than perihepatic cells. The surface density of smooth ER and total (rough and smooth) ER did not significantly differ between the periportal and perihepatic cells from 17-day-old fetuses to 5-day-old animals. In 10- and 20-day-old and adult animals, the values of smooth and total ER were greater in perihepatic cells than in periportal cells. When the data were expressed as area per cell, the patterns of subacinar distributions hardly differed, but age-related changes differed considerably from the patterns seen in the surface density data. The differences were generally caused by the increase in hepatocyte volume between 20 days of age and adulthood, especially in perihepatic cells, and by the changes in volume during the perinatal period. The results show that differences in the surface density and area per cell of smooth and total ER between periportal and perihepatic hepatocytes evident in adult animals are not present in fetal and newborn animals but arise during postnatal development.     

A cryptic fragment from fibronectin's III1 module localizes to lipid rafts and stimulates cell growth and contractility

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

The role of fibronectin in adhesion of metastatic melanoma cells to endothelial cells and their basal lamina

Vascular endothelial cells synthesize an extracellular matrix or basal lamina composed of collagens, proteoglycans and glycoproteins such as fibronectin (FN). Using affinity-purified anti-FN, we have examined the role of FN in adherence of metastatic B16 melanoma cells to endothelial cell monolayers which lack FN on apical cell surfaces and to their basal lamina which contains FN. B16 melanoma cells, which do not contain significant amounts of FN, attached at much higher rates to endothelial basal lamina and polyvinyl-immobilized FN compared with intact endothelial cell monolayers. Anti-FN failed to inhibit attachment of melanoma sublines of low (B16-F1) or high (B16-F10) metastatic potential to intact endothelial cell monolayers, inhibited slightly B16 cell attachment to basal lamina and completely abolished attachment of B16 cells to polyvinyl-immobilized FN. The antibiotic tunicamycin which inhibits glycosylation of B16 cell surface glycoproteins and blocks experimental metastasis [18] inhibited B16 attachment to endothelial cells, basal lamina and immobilized FN. The results suggest that FN mediates, only in part, the adhesion of B16 melanoma cells to basal lamina through glycoprotein receptors on B16 cells.     

Cell adhesion to fibronectin and tenascin: quantitative measurements of initial binding and subsequent strengthening response

Cell-substratum adhesion strengths have been quantified using fibroblasts and glioma cells binding to two extracellular matrix proteins, fibronectin and tenascin. A centrifugal force-based adhesion assay was used for the adhesive strength measurements, and the corresponding morphology of the adhesions was visualized by interference reflection microscopy. The initial adhesions as measured at 4 degrees C were on the order of 10(-5)dynes/cell and did not involve the cytoskeleton. Adhesion to fibronectin after 15 min at 37 degrees C were more than an order of magnitude stronger; the strengthening response required cytoskeletal involvement. By contrast to the marked strengthening of adhesion to FN, adhesion to TN was unchanged or weakened after 15 min at 37 degrees C. The absolute strength of adhesion achieved varied according to protein and cell type. When a mixed substratum of fibronectin and tenascin was tested, the presence of tenascin was found to reduce the level of the strengthening of cell adhesion normally observed at 37 degrees C on a substratum of fibronectin alone. Parallel analysis of corresponding interference reflection micrographs showed that differences in the area of cell surface within 10-15 nm of the substratum correlated closely with each of the changes in adhesion observed: after incubation for 15 min on fibronectin at 37 degrees C, glioma cells increased their surface area within close contact to the substrate by integral to 125- fold. Cells on tenascin did not increase their surface area of contact. The increased surface area of contact and the inhibitory activity of cytochalasin b suggest that the adhesive "strengthening" in the 15 min after initial binding brings additional adhesion molecules into the adhesive site and couples the actin cytoskeleton to the adhesion complex.     

Modification of the N-linked oligosaccharides in cell surface glycoproteins during chick embryo development. A using lectin affinity and a high resolution chromatography study

Important differences in asparagine-linked glycopeptides were observed in vitro cultured fibroblasts derived from chick embryo at different stages of development. Cells from 8-day and 16-day embryos were labeled metabolically with [3H]mannose. Cell surface glycopeptides obtained after mild trypsin treatment were extensively digested with pronase and then chromatographed on concanavalin-A-Sepharose and other immobilized lectins. The most important changes concerned the complex type chains. The ratio between triantennary plus tetraantennary and biantennary chains increased about 2.5-fold from the 8th to the 16th day of development. In the same way, complex chains with bisecting N-acetylglucosamine increased from 8-day to 16-day cells as shown by Phaseolus-vulgaris-erythroagglutinin--agarose chromatography. In 16-day cells, the majority of triantennary chains (60%) with alpha-linked mannose substituted at C2 and C6 positions and biantennary chains (50%) were shown to contain fucosyl (alpha 1----6)N-acetylglucosaminyl structure in the core region by their ability to bind to a lentil lectin affinity column. Similarly, in 8-day cells, triantennary chains (50%) were more fucosylated than biantennary chains (35%). Thus, complex structures exhibited an increased fucosylation of their invariable core from the 8th to the 16th day of development, except for fucosylated triantennary chains which were retained on Phaseolus vulgaris Leucoagglutin and on lentil lectin. These latter structures were present at the surface of 8-day cells and absent at the surface of 16-day cells. After chromatography on Bio-Gel P6 and treatment with endo-beta-N-acetylglucosaminidase H, the [3H]-mannose-labeled glycopeptides were separated by high resolution chromatography into glycopeptides with complex chains and glycopeptides with high-mannose chains. Analysis of the high-mannose oligosaccharides released after endo-beta-N-acetylglucosaminidase H treatment by chromatography on Bio-Gel P4 indicated that the same type of high-mannose chains were present at the surface of 8-day and 16-day cells. Quantification of mannose, galactose and sialic acid residues using gas liquid chromatography was consistent with a decrease of the relative amount of oligomannose chains and an increase of the relative amount of complex type chains in 16-day cells compared to 8-day cells. Thus N-linked oligosaccharides derived from cell surface glycoproteins undergo changes during embryo development resulting in greater complexity of carbohydrate chains.     

The effect of sphingosine and phorbol ester on the signal transduction enzymes and fibronectin release in cell culture.

In testing the hypothesis that the stimulation of the release of fibronectin (FN) by 12-O-tetradecanoylphorbol 13-acetate (TPA) from human lung fibroblasts in culture is the result of activation of protein kinase C (PKC), we found that the PKC inhibitor sphingosine strongly inhibited FN release in presence and even in absence of TPA. However, a different PKC inhibitor, calphostin C, despite almost complete inhibition of PKC, had no effect on FN release. We concluded that sphingosine is a potent inhibitor of FN release from the cell surface, independent of its inhibition of PKC; and that TPA stimulates release of FN by a pathway other than activation of PKC. We found that the activation of PKC by TPA was accompanied by inhibition of the cAMP-dependent protein kinase (PKA). When PKA was inhibited by an antagonist (H8, a cAMP analogue) at a concentration specific for PKA inhibition, the release of FN was stimulated similar to the stimulation with TPA. Activation of PKA with forskolin resulted in decreased FN release. In conclusion, we have shown that: (1) sphingosine had a robust effect inhibiting the release of FN from fibroblasts, independent of its action on PKC; (2) TPA treatment of these cells resulted in inhibition of PKA; (3) inhibition of PKA stimulated FN release whereas its activation decreased this release. It is possible that PKA, by phosphorylating a protein, may function, directly or indirectly, in keeping FN attached to the cell surface of fibroblasts.     

Effect of ascitic liquid on growth in vitro of embryoid bodies derived from teratocarcinoma.

Embryoid bodies (EB) derived from teratocarcinoma (TC) OTT6050 were cultured with ascitic liquids (AL) from animals carrying 16-, 22- and 35-day evolved EB. At the same time the presence of fibronectin (FN) in AL were analyzed by immunoblotting. Results indicate the probable existence of growth-stimulatory factors for EB, as well as the presence of FN in the 22-day AL.     

Fibronectin, aging and related pathologies]

It could be demonstrated that plasma and tissue fibronectin (FN) increase with age. Some age dependent diseases as diabetes, osteoarthritis and Werner syndrome produce also an increase of tissue fibronectin biosynthesis. Plasma fibronectin decreases in diabetes and in breast cancer. Alternative splicing of the FN gene appears also to vary with age and in some related pathologies. Nutritional status and UV light also influence FN biosynthesis. It appears therefore that the determination of plasma FN and its isoforms as well as the study of tissue FN may be of interest for the study of chronological aging and related pathologies.     

Intracellular transport of membrane glycoproteins: two closely related histocompatibility antigens differ in their rates of transit to the cell surface

The intracellular transport of two closely related membrane glycoproteins was studied in the murine B cell lymphoma line, AKTB-1b. Using pulse-chase radiolabeling, the kinetics of appearance of the class I histocompatibility antigens, H-2Kk and H-2Dk, at the cell surface were compared and found to be remarkably different. Newly synthesized H-2Kk is transported rapidly such that all radiolabeled molecules reach the surface within 1 h. In contrast, the H-2Dk antigen is transported slowly with a half-time of 4-5 h. The rates of surface appearance for the two antigens closely resemble the rates at which their Asn-linked oligosaccharides mature from endoglucosaminidase H (endo H)-sensitive to endo H-resistant forms, a process that occurs in the Golgi apparatus. This suggests that the rate-limiting step in the transport of H-2Dk to the cell surface occurs before the formation of endo H-resistant oligosaccharides in the Golgi apparatus. Subcellular fractionation experiments confirmed this conclusion by identifying the endoplasmic reticulum (ER) as the site where the H-2Dk antigen accumulates. The retention of this glycoprotein in the ER does not appear to be due to a lack of solubility or an inability of the H-2Dk heavy chain to associate with beta 2-microglobulin. Our data is inconsistent with a passive membrane flow mechanism for the intracellular transport of membrane glycoproteins. Rather, it suggests that one or more receptors localized to the ER membrane may mediate the selective transport of membrane glycoproteins out of the ER to the Golgi apparatus. The fact that H-2Kk and H-2Dk are highly homologous (greater than or equal to 80%) indicates that this process can be strongly influenced by limited alterations in protein structure.     

Effects of carbachol on rat Sertoli cell proliferation and muscarinic acetylcholine receptors regulation: an in vitro study

The aim of the present work was to study the effect of muscarinic agonist on cell proliferation and muscarinic acetylcholine receptors (mAChRs) regulation in rat Sertoli cells. Primary cultures of Sertoli cells were obtained from 8-day and 15-day old male Wistar rats. In proliferation assays, [methyl-3H]thymidine incorporation in Sertoli cells from 8-day and 15-day old rats reached a plateau after 60 min of carbachol incubation and decreased after 120 min of agonist incubation. Binding studies with [N-Methyl-3H]scopolamine ([3H]NMS) indicated a rapid loss of cell surface mAChRs when Sertoli cells from 15-day old rats were incubated with carbachol at 35 degrees C for 2 min. This effect was temperature-dependent. When the incubation of the cells was prolonged at 35 degrees C or at 4 degrees C, after the agonist had been washed away, 94% of mAChRs were present in the cell surface after 120 min incubation at 35 degrees C. At 4 degrees C, however, a low percentage of mAChRs was detected in the cell surface. In the presence of cycloheximide, the recycling of mAChRs to the cell surface was not changed, suggesting that the appearance of mAChRs on cell surface was not dependent on de novo receptor synthesis. In conclusion, our studies indicate that the activation of mAChRs may play a role in rat Sertoli cell proliferation. These receptors may be under regulation (internalization and recycling) when cells are exposed to muscarinic cholinergic agonist.     

Altered glycosylation of α(s)β1 integrins from rat colon carcinoma cells decreases their interaction with fibronectin

Malignant cell transformation is generally accompanied by changes in their interactions with environing matrix proteins in a way to facilitate their migration and generate invasion. Our results show the binding of rat colon adenocarcinoma PROb cells to fibronectin strongly reduced when compared to normal rat intestine epithelial cells. This decrease was not due to the level of α(s)β1 integrins expressed at the surface of the cell line. However, β₁- and α_(s)-associated subunits appeared to be structurally altered as shown by immunoprecipitation followed by electrophoresis. Pulse chase experiments using ³⁵S methionine evidenced differences in the biosynthesis of β1- and α (s) associated integrins: normal epithelial IEC18 cells required 16 h for maximal biosynthesis of the completely mature β1 subunit, while PROb cells did it within 4-6 h. Studies using endoglycosidases O, H, D, and N glycanase confirmed that the molecular weight alterations were due to abnormal glycosylation and suggested that α(s)β1 integrins of PROb cells could bear both mature complex and immature high mannose types while IEC18 cells borne only mature complex type oligosaccharidic chains. Treatment of both cell types with castanospermine, an inhibitor of N-glycosylation, reduced the differences observed in their adhesion to the fibronectin without significantly affecting β1 receptors expression at the cell surface. These results strongly suggest a role of the glycosylation of β1 receptors in the adhesion of rat colon adenocarcinoma PROb cells to fibronectin substrata. © 1996 Wiley-Liss, Inc.     

Relationship of heparan sulfate proteoglycans to the cytoskeleton and extracellular matrix of cultured fibroblasts

The distribution of heparan sulfate proteoglycans (HSPG) on cultured fibroblasts was monitored using an antiserum raised against cell surface HSPG from rat liver. After seeding, HSPG was detected by immunofluorescence first on cell surfaces and later in fibrillar deposits of an extracellular matrix. Cell surface HSPG aligned with microfilament bundles of rat embryo fibroblasts seen by phase-contrast microscopy but was diffuse on transformed rat dermal fibroblasts (16C cells) which lack obvious stress fibers. Focal adhesions isolated from either cell type and monitored by interference reflection microscopy showed a concentration of HSPG labeling with respect to the rest of the membrane. Increased labeling in these areas was also seen for fibronectin (FN) by using an antiserum that detects both plasma and cell-derived FN. Double immunofluorescent staining of fully adherent rat embryo fibroblast cells showed some co-distribution of HSPG and FN, and this was confirmed by immunoelectron microscopy, which detected HSPG at localized areas of dorsal and ventral cell membranes, overlapping cell margins, and in the extracellular matrix. During cell shape changes on rounding and spreading, HSPG and FN may not co- distribute. Double labeling for actin and either HSPG or FN showed a closer correlation of actin with HSPG than with FN. The studies are consistent with HSPG being closely involved in a transmembrane cytoskeletal-matrix interaction; the possibility that HSPG coordinates the deposition of FN and other matrix components with cytoskeletal organization is discussed.