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PubMed1982

Fibronectin--mediator between cells and connective tissue.

Fibronectin, previously also termed LETS-protein, is a high-molecular-weight protein (mol. w. ca. 450,000) present in the form of thin fibrils in the pericellular space of fibroblasts and other adherent cells, as well as in distinct areas of the connective tissue. A soluble form, immunologically identical and chemically at least very similar to the cell-attached protein, is found in plasma in a concentration of about 300 micrograms/ml. It is also denominated cold-insoluble globulin. The protein has affinity both to cell surfaces and to various matrix substances such as fibrin and collagen and, therefore, is capable of mediating cell attachment to these substrates. In addition, it serves as an opsonin for the phagocytosis of gelatin-containing compounds and probably is essential for the removal of soluble fibrin from the circulating blood by the reticulo-endothelial system. Bacterial cell walls are also recognized by fibronectin. A conversion of soluble fibronectin to fibrils is achieved by heparin which also enhances the binding of soluble fibronectin to cells. Heparin or, as suggested, the related heparan sulfate present on the surface of various cells, appears to function as a cofactor in the formation of pericellular fibrils. The fibronectin fibrils precipitated with heparin, compared to soluble fibronectin, show a considerably improved affinity to native collagen, especially to type III. Hyaluronic acid has an antagonistic function which, at higher concentrations, prevents the fibronectin fibrils from interacting with collagen and cell surfaces. Masking of fibronectin fibrils was also achieved by sulfated proteoglycans of cartilage. Virus-transformed fibroblasts produce less fibronectin and are less capable of maintaining surface pericellular fibrils. A reasonable explanation is that they have an elevated secretion of hyaluronic acid. The transformed cells attach only weakly to a surface and exhibit a rounded shape in contrast to healthy ones. This phenotype can be corrected to a great extent with fibronectin. It is suggested that fibronectin also influences the formation of connective tissue by accumulating collagen precursors on the surface of fibroblasts and facilitating fibrillogenesis.

Cell Adhesion↗
PubMed1989

A substrate of the cell-attachment sequence of fibronectin (Arg-Gly-Asp-Ser) is sufficient to promote transition of arterial smooth muscle cells from a contractile to a synthetic phenotype.

Extracellular matrix components strongly influence the differentiated properties of isolated rat arterial smooth muscle cells during in vitro cultivation. The attachment and spreading of the cells on a substrate of fibronectin or a 105-kDa cell-binding fragment of fibronectin are accompanied by a structural and functional transformation, referred to as a transition or modulation from a contractile to a synthetic phenotype. Here, the ability of the cell-attachment sequence of fibronectin, Arg-Gly-Asp-Ser (RGDS), to promote this process was studied. The results demonstrate that freshly isolated smooth muscle cells attached to a substrate of the synthetic peptide Gly-Arg-Gly-Asp-Ser-Cys (GRGDSC) in a specific manner and as well as to substrates of fibronectin and the 105-kDa fragment. Subsequent spreading of the cells on the peptide substrate followed the same kinetics and was as extensive as on fibronectin, even if protein synthesis was blocked by treatment of the cultures with cycloheximide. Like fibronectin, the peptide substrate induced formation of actin filament bundles, again without ongoing protein synthesis. Moreover, it was as efficient as fibronectin in supporting the transition of the cells from a contractile to a synthetic phenotype as analyzed by electron microscopy. Antibodies against the beta subunit of the fibronectin receptor interfered with the attachment, spreading, and fine structural reorganization of the cells in a similar manner on substrates of fibronectin, the 105-kDa fragment, and GRGDSC. Taken together, the findings indicate that the cell-attachment sequence (RGDS) mimics intact fibronectin in promoting a change in the differentiated properties of arterial smooth muscle cells and does so by interacting with a cell surface receptor for fibronectin.

Actins↗
PubMed1990

Myonexin: an 80-kDa glycoprotein that binds fibronectin and is located at embryonic myotendinous junctions.

The distribution and function of an 80-kDa glycoprotein located at the surface of skeletal muscle cells and enriched in gelatin-binding fractions of skeletal muscle extracts are examined in the present study. The glycoprotein was purified by concanavalin A affinity chromatography followed by gel filtration and anion exchange chromatography. The purified protein did not display gelatin-binding although the protein bound to fibronectin in several assays. First, the glycoprotein bound to fibronectin-Sepharose and did not elute in high salt buffers although subsequent basic elutions displaced the 80-kDa protein from the column. Second, gel filtration of the 80-kDa glycoprotein in the presence of fibronectin showed separate peaks corresponding to the mass of the 80-kDa glycoprotein and fibronectin as well as a third, higher mass peak shown in immunoblots to contain both fibronectin and the 80-kDa glycoprotein. Third, immunoprecipitation with affinity-purified anti-80-kDa glycoprotein in the presence of the glycoprotein and radioiodinated fibronectin precipitated labeled fibronectin. The quantity of labeled fibronectin precipitated was reduced by the addition of nonradiolabeled fibronectin. Immunofluorescent microscopy using affinity-purified, anti-80-kDa showed this protein located at the myotendinous junctions of frog tadpoles and embryonic chicks. In chicks, it was discernible by immunofluorescence only during the morphogenetic stages that myotendinous junctions were being assembled. Amino acid analysis shows that the 80-kDa glycoprotein has a high concentration of acidic residues. There is only one cysteine per molecule in the 80-kDa glycoprotein and comparisons of reducing and nonreducing gels show that no disulfides are present, indicating that this is not an integrin protein. Amino terminal sequencing reveals that the protein contains marked similarity to the amino terminal of calsequestrin although the protein is distinct from calsequestrin in lacking Ca2(+)-dependent phenyl sepharose affinity and in its molecular weight and distribution. The observations indicate that the 80-kDa glycoprotein is a fibronectin receptor present at chick myotendinous junctions during junction morphogenesis. This apparently novel protein is named "myonexin" to reflect its location and likely function in attaching fibronectin to the surface of muscle cells.

Amino Acid Sequence↗
PubMed1997

Fibronectin-mediated cell migration promotes glioma cell invasion through chemokinetic activity.

In order to investigate the biological role of fibronectin in glioma cell invasion, we studied the relation between migratory responses or adhesiveness of glioma cells to fibronectin and the in vitro invasion in three human malignant glioma cell lines, A172, T98G and U373MG. All these cell lines chemotactically migrated in a dose-dependent manner to fibronectin in concentrations ranging from 0.5 to 10 microg/ml, with A172 cells showing the strongest migration and U373 cells the weakest. Checkerboard analyses demonstrated that A172 and T98G cells showed much stronger chemokinetic responses to fibronectin than U373MG cells. In contrast to the migratory responses, A172 and U373MG cells showed an almost equally high adhesion to fibronectin and T98G cells a low adhesion. The degree of expression of the integrin alpha5 subunit correlated well with the strength of glioma cell adhesion to fibronectin rather than that of migration to the molecule. Furthermore, the cell adhesion to fibronectin was almost completely inhibited by arginine-glycine-aspartic acid (RGD)-containing peptides, but the fibronectin-stimulated cell migration was only partially inhibited. An in vitro invasion assay disclosed that U373MG cells invaded the artificial basement membrane barrier the most and A172 cells the least. However, addition of fibronectin to the glioma cells markedly enhanced the invasive activity of A172 and T98G cells but had little effect on that of U373MG cells. These results indicate that fibronectin-stimulated migration can be one of the factors promoting invasiveness of glioma cells and that the chemokinetic activity of fibronectin may play a crucial role in glioma invasion through conferring motor-driving force on the glioma cells.

Cell Adhesion↗
PubMed1985

Collagen can modulate cell interactions with fibronectin.

We have examined the effects of soluble collagen on the function of fibronectin in baby hamster kidney (BHK) cells. Collagen and its purified alpha1(l) chain noncompetitively inhibited cell spreading on substrates precoated with fibronectin or a 75,000-D cell-binding fragment of fibronectin. Neither preincubation of cells with collagen followed by washing nor the addition of collagen to previously spread cells had any inhibitory effect on cell spreading, which indicates a requirement for the concurrent presence of collagen during the process of spreading. Treatment of collagen or alpha1(l) chain with collagenase abolished the inhibitory effect on fibronectin-mediated cell spreading. However, direct attachment of BHK cells to fibronectin-coated or 75,000-D fragment-coated substrates was not inhibited by collagen or by the alpha1(l) chain. Moreover, the binding of [3H]fibronectin or the 3'-75,000-D fragment to cell surfaces was not inhibited by the presence of soluble collagen, whereas soluble fibronectin inhibited binding. Although the binding of [3H]fibronectin-coated beads to BHK cell surfaces was also not inhibited by collagen, the phagocytosis of such beads was inhibited by the presence of collagen. On the other hand, soluble fibronectin partially inhibited the binding of fibronectin-coated beads but did not inhibit phagocytosis of the beads that did bind. The mechanism of the inhibition of fibronectin function by collagen and the possible interactions of two different kinds of receptors on the cell surface are discussed.

Animals↗
PubMed1987

Selective interaction of peripheral and central nervous system cells with two distinct cell-binding domains of fibronectin.

Mechanisms of cell interaction with fibronectin have been studied with proteolytic fibronectin fragments that have well-defined ligand binding properties. Results of a previous study (Rogers, S. L., J. B. McCarthy, S. L. Palm, L. T. Furcht, and P. C. Letourneau, 1985, J. Neurosci., 5:369-378) demonstrated that (a) central (CNS) and peripheral (PNS) nervous system neurons adhere to, and extend neurites on a 33-kD carboxyl terminal fibronectin fragment that also binds heparin, and (b) neurons from the PNS, but not the CNS, have stable interactions with a 75-kD cell-binding fragment and with intact fibronectin. In the present study domain-specific reagents were used in inhibition assays to further differentiate cell surface interactions with the two fibronectin domains, and to define the significance of these domains to cell interactions with the intact fibronectin molecule. These reagents are (a) a soluble synthetic tetrapeptide Arg-Gly-Asp-Ser (RGDS; Pierschbacher, M. D., and E. Ruoslahti, 1984, Nature (Lond.), 309:30-33) representing a cell-binding determinant in the 75-kD fragment, and (b) an antibody raised against the 33-kD fragment that binds specifically to that fragment. Initial cell attachment to, and neurite extension upon, fibronectin and the two different fragments was evaluated in the presence and absence of the two reagents. Attachment of both PNS and CNS cells to intact fibronectin was reduced in the presence of RGDS, the former more so than the latter. In contrast, the antibody to the 33-kD fragment did not affect attachment of PNS cells to fibronectin, but significantly decreased attachment of CNS cells to the molecule. RGDS inhibited attachment of CNS cells to the molecule. RGDS inhibited attachment of both cell types to the 75-kD fragment to a greater degree than it did attachment to the intact molecule. Cell interaction with the 33-kD fragment was not affected by RGDS. Reduction of neurite lengths (determined after 24 h of culture) by the domain-specific reagents paralleled the reduction in initial adhesion to each substratum. Therefore, it appears that (a) both PNS and CNS cells have receptors for each cell-binding domain of fibronectin, (b) the receptor(s) for the two domains are distinct, with attachment to the 33-kD fragment being independent of RGDS, and (c) the relative importance of each domain to cell interaction with intact fibronectin is different for CNS and PNS cells.

Animals↗
PubMed1999

Synergistic effects of hyaluronan and fibronectin on epithelial migration in rabbit cornea in vitro.

PURPOSE: We investigated the interactions of hyaluronan with extracellular matrix proteins (fibronectin, laminin, and type IV collagen) on corneal epithelial migration by using an organ-culture system of rabbit corneas. METHODS: Rabbit corneal blocks were cultured in the absence or presence of various reagents for a total of 20 h. The corneal blocks were then fixed, dehydrated, embedded in paraffin, and stained by hematoxylineosin, and the length of the path of epithelial migration was measured. RESULTS: The addition of hyaluronan, fibronectin, or type IV collagen alone stimulated epithelial migration in a dose-dependent manner, but laminin did not. When corneal blocks were pretreated with extracellular matrix proteins (fibronectin, laminin, or type IV collagen) for 5 h and then were cultured for 15 h with hyaluronan, only pretreatment with fibronectin demonstrated a synergistic effect with hyaluronan on corneal epithelial migration. In contrast, when corneal blocks were pretreated with hyaluronan for 5 h and then cultured for 15 h with extracellular matrix proteins, the additive effects of hyaluronan with fibronectin or hyaluronan with type IV collagen were observed. The synergistic effects of hyaluronan and fibronectin on corneal epithelial migration were partially inhibited by the addition of antiserum against fibronectin. In contrast, the addition of antiserum against fibronectin receptor completely inhibited the synergistic effects of hyaluronan and fibronectin on epithelial migration. CONCLUSION: These results suggest that hyaluronan and fibronectin have a synergistic effect, with fibronectin pretreatment augmenting hyaluronan-stimulated corneal epithelial migration.

Animals↗
PubMed1989

Fibronectin increases Trypanosoma cruzi amastigote binding to and uptake by murine macrophages and human monocytes.

Trypanosoma cruzi amastigotes present receptors for human fibronectin as indicated by the saturable binding of [125I]fibronectin to this form of the parasite. Scatchard analysis indicates that the number of fibronectin receptors per amastigote was 1.3 x 10(3) with a Kd of approximately 2.3 nM. Addition of physiological concentrations of fibronectin to amastigote-macrophage cocultures significantly increased the binding of amastigotes to murine macrophages. This increase was evidenced in both the number of amastigotes bound to macrophages and the percentage of macrophages containing bound amastigotes. The uptake of amastigotes by either murine macrophages or human blood monocytes was also increased in the presence of exogenous fibronectin. The increase induced by fibronectin was blocked when amastigotes were pretreated with the RGDS tetrapeptide of the fibronectin cell attachment site. Furthermore, the ability of fibronectin to enhance amastigote binding to and uptake by macrophages was inhibited by the F(ab')2 fragment of anti-fibronectin immunoglobulin G (IgG) but not by an irrelevant anti-human IgG F(ab')2 fragment. Pretreatment of either amastigotes or macrophages with fibronectin also resulted in a significant increase in amastigote binding to and uptake by macrophages. These results suggest that fibronectin may play a role in facilitating the attachment and ingestion of T. cruzi amastigotes by macrophages and monocytes in chagasic tissue lesions.

Animals↗
PubMed1998

Inhibition of vascular smooth muscle cell growth by inhibition of fibronectin matrix assembly.

The regulation of vascular smooth muscle cell (VSMC) proliferation by the fibronectin matrix was tested by treating human umbilical artery smooth muscle cells (HUASMCs) with a recombinant fragment of fibronectin (protein III1-C) that has previously been shown to modulate fibronectin matrix assembly. III1-C inhibited HUASMC proliferation by 75% to 90%. The inhibition of growth was time dependent; III1-C had no effect on DNA synthesis after 0 to 5 hours of treatment but did have an effect at 24 hours and beyond. III1-C did not stimulate apoptosis in these cells, indicating that the inhibition of proliferation was not due to an induction of programmed cell death. The effects of III1-C on cell growth were only specific for normal diploid smooth muscle cells. III1-C had no effect on the proliferation of IMR-90 fibroblasts, endothelial cells, NIH 3T3 cells, or the rat aortic smooth muscle cell line A7r5. However, III1-C did inhibit proliferation by primary rat aortic smooth muscle cells. An analysis of HUASMC fibronectin receptor (integrin alpha5beta1) distribution revealed that III1-C did not inhibit alpha5beta1 localization to focal contacts. Moreover, III1-C had no effect on the relative expression levels of seven different integrin subunits on HUASMCs. However, III1-C did inhibit fibronectin matrix assembly by rat aortic smooth muscle cells, HUASMCs, A7r5 cells, IMR-90 cells, and endothelial cells. An analysis of fibronectin synthesis indicated that the inhibition of fibronectin matrix assembly by III1-C was not due solely to a decrease in fibronectin synthesis. Finally, treatment of HUASMCs with anti-fibronectin monoclonal antibody L8 (which is known to inhibit fibronectin matrix assembly) also decreased the rate of HUASMC DNA synthesis. These results demonstrate that III1-C inhibits VSMC proliferation and suggest that this effect may be mediated by the inhibition of fibronectin matrix assembly.

Animals↗
PubMed1989

Synthesis and expression of the fibroblast fibronectin receptor in human monocytes.

Human monocytes adhere to fibronectin, but the receptor (or receptors) mediating this interaction has not been clearly identified. To examine the nature of this receptor, human monocytes were obtained by counter-current elutriation and were found to adhere to immobilized fibronectin but not to vitronectin or von Willebrand factor. Antibodies and peptides were used to determine whether monocyte adherence to immobilized fibronectin was mediated by a receptor similar to that which mediates the adherence of fibroblasts to fibronectin. Antibodies against both the alpha and beta subunits of the fibroblast fibronectin receptor (VLA-5) inhibited monocyte adherence to fibronectin. These antibodies immunoprecipitated two surface proteins from monocytes that migrated at 140 and 120 kD under nonreducing conditions. Surface proteins with identical apparent molecular weights were immunoprecipitated from surface-labeled MG-63 cells, a fibroblast-like cell line. A synthetic peptide containing the Arg-Gly-Asp-Ser (RGDS) sequence also inhibited monocyte adherence to fibronectin. cDNA hybridization experiments revealed the presence of mRNA for both the alpha and beta subunits of the fibroblast fibronectin receptor in human monocytes. We conclude that circulating monocytes express a fibronectin receptor that is structurally and functionally very similar, if not identical, to the fibronectin receptor in adherent fibroblasts, and that this receptor mediates monocyte adherence to immobilized fibronectin.

Antibodies, Monoclonal↗
PubMed1998

Schwann cells use a novel collagen-dependent mechanism for fibronectin fibril assembly.

Cultured rat Schwann cells were stimulated to deposit fibrillar extracellular matrix by treatment with ascorbic acid in the absence of nerve cells. Immunofluoresence staining of the matrix showed that it contains collagens types I and IV, fibronectin and perlecan but not laminin. Collagen type IV, fibronectin and perlecan co-distributed completely in the matrix fibrils, whereas collagen type I was present in only a subset of these fibrils. Time course studies indicated that collagen type I fibrils appear at late stages of matrix formation. Digestion of Schwann cell extracellular matrix with collagenase effectively disrupted most of the matrix including fibronectin fibrils. This was in contrast with fibroblasts, where collagenase treatment removed collagen with no visible effect on fibronectin fibrils. alpha5 integrin was expressed on the cell surface of Schwann cells and partially codistributed with fibronectin-containing fibrils. This suggests that the inability of Schwann cells to deposit fibronectin-containing matrix through a conventional, collagen-independent mechanism was not due to the lack of fibronectin-binding integrins on their cell surface. Polyclonal anti-fibronectin antibodies inhibited the deposition of fibronectin into the matrix fibrils, whereas collagen type IV fibrils were generally unaffected. Growth of Schwann cells on collagen type IV-coated substrate in the absence of ascorbate induced deposition of fine fibronectin fibrils. These results suggest that Schwann cells use an apparently novel, collagen type IV-dependent mechanism for the deposition of fibronectin into their extracellular matrix.

Animals↗
PubMed1991

Detection of fibronectin receptor in sera: its clinical significance as a parameter of hepatic fibrosis.

Pooled sera collected from cirrhotic patients was fractionated by affinity chromatography with a fibronectin receptor monoclonal antibody against the beta-subunit of fibronectin receptor. Eluates were assayed using Western immunoblotting. The relative mobility of the protein reactive with fibronectin receptor antibody was nearly identical to that of the beta-subunit of fibronectin receptor, confirming that fibronectin receptor is present in human serum. Serum levels of the beta-subunit of fibronectin receptor were analyzed by sandwich enzyme-linked immunosorbent assay in patients with various liver diseases. The serum level of fibronectin receptor (micrograms/ml) was significantly higher in patients with chronic hepatitis (inactive, 2.59 +/- 0.04; active, 3.45 +/- 0.13), cirrhosis (4.77 +/- 0.30), alcoholic liver disease (2.96 +/- 0.16) and hepatocellular carcinoma (4.71 +/- 0.49) than in normal subjects (2.11 +/- 0.08). Strong positive correlation was observed between serum levels of fibronectin receptor and histological findings, particularly in the degree of hepatic fibrosis. Immunohistochemical studies with fibronectin receptor antibody revealed that the beta-subunit of fibronectin receptor was present on the plasma membrane of hepatocytes and sinusoidal lining cells in the normal liver and was increased in fibrotic areas and on the plasma membrane of hepatocytes and sinusoidal lining cells of fibrotic liver. The serum level of fibronectin receptor in patients with chronic liver diseases may therefore be a useful marker of hepatic fibrosis.

Adult↗
PubMed1990

Human B lymphocytes define an alternative mechanism of adhesion to fibronectin. The interaction of the alpha 4 beta 1 integrin with the LHGPEILDVPST sequence of the type III connecting segment is sufficient to promote cell attachment.

In this report we have studied the mechanism of human B lymphocyte adhesion to fibronectin and to proteolytic fragments of this protein. B cells adhered to fibronectin and to a 38-kDa fragment, derived from the A chain, containing the Hep II domain and most of the type III connecting segment, IIICS, of fibronectin. Cells did not bind to an 80-kDa fragment containing the RGD adhesive sequence of fibronectin. Attachment to fibronectin or to the 38-kDa fragment was not affected by the 80-kDa fragment, the GRGDSPC synthetic peptide, or by a mAb specific for the alpha chain of the RGD-dependent fibronectin receptor, alpha 5 beta 1. However, B cell adhesion to fibronectin was inhibited by the synthetic peptides CS-1, comprising the first 25 amino acids of IIICS and B12, containing the sequence LHGPEILDVPST of CS-1 (residues 14-25). Moreover, this sequence was shown to be sufficient to induce stable cell adhesion when coated on plastic surfaces. A mAb specific for the alpha-subunit of the alpha 4 beta 1 integrin, completely inhibited B cell attachment to B12, CS-1, 38 kDa, and fibronectin coated substrata. These data clearly indicate that adhesion of B lymphocytes to fibronectin is exclusively mediated by the interaction of alpha 4 beta 1 with residues 14-25 of the IIICS region in fibronectin. Therefore this interaction constitutes an alternative pathway of adhesion to fibronectin, independent of RGD and alpha 5 beta 1.

Amino Acid Sequence↗
PubMed1988

Glycoprotein IIb-IIIa and RGD(S) are not important for fibronectin-dependent platelet adhesion under flow conditions.

Previous studies have indicated that activated blood platelets interact with fibronectin through binding of fibronectin to the glycoprotein IIb-IIIa complex (GPIIb-IIIa). The cell attachment site of fibronectin with its crucial arg-gly-asp(-ser) [RGD(S)]sequence is involved in these bindings. We studied the importance of these interactions for the fibronectin dependence of platelet adhesion under flow conditions. An RGDS-containing hexapeptide (GRGDSP) was compared with a nonreactive control peptide (GRGESP). The GRGDSP-peptide inhibited thrombin-induced aggregation and adhesion under static conditions at 0.1 mmol/L. This concentration had no effect on platelet adhesion to nonfibrillar collagen type I in flow. GRGDSP at 1 mmol/L had a significant inhibitory effect at 1,500 s-1, but not at the lower shear rates of 800 and 300 s-1 where platelet adhesion is also fibronectin dependent. On the matrix of cultured human umbilical vein endothelial cells, 1 mmol/L GRGDSP had no effect on platelet adhesion. The relation between GPIIb-IIIa and fibronectin dependence was investigated with platelets of a patient with Glanzmann's thrombasthenia and monoclonal antibodies to GPIIb-IIIa using endothelial cell matrix (ECM) as a surface. Platelets of normal controls or a patient with Glanzmann's thrombasthenia showed a similar inhibition of adhesion in the presence of fibronectin-free plasma after the ECMs had been preincubated with antifibronectin F(ab')2 fragments. Incubation of platelets with anti-GPIIb-IIIa showed inhibition of platelet adhesion at high shear rates. Dependence on fibronectin for platelet adhesion was still observed even though separate experiments had shown that these anti-GPIIb-IIIa antibodies could block binding of radiolabeled fibronectin to thrombin-activated platelets. These data suggest the existence of another binding system for the interaction of platelets with fibronectin that may only appear when fibronectin is present on a surface.

Amino Acid Sequence↗
PubMed1982

Binding of developing mouse cerebellar cells to fibronectin: a possible mechanism for the formation of the external granular layer.

The role of the matrix glycoprotein fibronectin in the formation of the external granular layer of the developing mouse cerebellum was investigated by in vitro studies of the binding of cerebellar cells to a fibronectin-coated culture substratum and by in vivo immunocytochemical localization of antiplasma fibronectin antiserum in cerebellar tissue. The adhesion of cells dissociated from embryonic and early postnatal mouse cerebellum is developmental stage-specific when the cells are plated on tissue culture substrata derivatized with human plasma fibronectin. Cells dissociated from mouse cerebellum at embryonic day 13 form cellular aggregates on insoluble plasma fibronectin. In contrast, cells dissociated from embryonic day 16 through postnatal day 7 cerebellum form a monolayer. Time-lapse video recordings reveal extensive cell movement of late embryonic and early postnatal cerebellar cells on insoluble plasma fibronectin. Late embryonic and early postnatal cerebellar cells bind to fibronectin but do not degrade the fibronectin substratum. Immunocytochemical studies of the binding of antiplasma fibronectin antisera to cryostat sections of intact embryonic and early postnatal cerebellar tissue reveal a brightly stained region of endogenous fibronectin along the route of granule cell migration from the lateral caudal part of the neuroepithelium lining the fourth ventricle up onto the external surface of the cerebellar anlage. When the formation of the external granular layer is completed, the intense region of fibronectin is no longer visible.

Aging↗
PubMed1984

Association of fibronectin with the platelet cytoskeleton.

Triton-insoluble cytoskeletons prepared from either normal or thrombasthenic platelets were found to contain approximately 1.3 micrograms of fibronectin/10(9) platelets as measured by a radioimmunoassay. Total endogenous platelet fibronectin was quantitatively retained on the platelet cytoskeleton, whereas 70% of exogenously added fibronectin that bound the surface of thrombin-activated platelets was recovered with the Triton-insoluble cytoskeleton. The exogenously added fibronectin specifically bound platelets and cytoskeletons with the same affinity giving an apparent binding constant of 1.47 X 10(-7) M. The possibility that fibrin associated with the platelet cytoskeleton could serve as the fibronectin receptor was investigated by measuring the binding constant of fibronectin for polymerizing fibrin and by measuring the amount of fibronectin associated with cytoskeletons of thrombasthenic platelets which contain 4-fold less fibrin than controls. The binding constant of fibronectin for polymerizing fibrin was 14-fold lower than that for cytoskeletons and cytoskeletons prepared from thrombasthenic platelets contained approximately the same amount of fibronectin as controls. Therefore, it is unlikely that fibrin is the platelet fibronectin receptor. These results support the hypothesis that platelet fibronectin is released from platelet alpha granules upon thrombin stimulation and becomes bound to the platelet surface and cytoskeleton either directly or through some intermediate protein that spans the membrane and interacts both with fibronectin and the internal cell cytoskeleton.

Antigen-Antibody Complex↗
PubMed1998

Transforming growth factor-beta and platelet derived growth factor regulation of fibrillar fibronectin matrix formation by synovial fibroblasts.

OBJECTIVE: To investigate factors regulating fibronectin fibrillar matrix formation by synovial fibroblasts. METHODS: Basal and cytokine stimulated extracellular matrix (ECM) fibronectin produced by synovial fibroblasts was identified by immunofluorescence and Western blot. Alternative mRNA splicing of fibronectin was studied by reverse transcription polymerase chain reaction. The integrin receptor responsible for supporting fibronectin fibrillar matrix was identified by blocking antibodies and receptor levels studied by Western blot. RESULTS: Transforming growth factor-beta (TGF-beta) or platelet derived growth factor (PDGF), but not interleukin 1 or exogenous fibronectin, induced ECM fibronectin. ECM fibronectin was blocked by the addition of antibody to the alpha5beta1 integrin. Cytokines did not significantly change alternative mRNA splicing of fibronectin or levels of alpha5beta1 integrin expression. CONCLUSION: Synovial cell production of a fibrillar fibronectin matrix is induced by growth factors, including TGF-beta and PDGF. This induction is mediated by the alpha5beta1 integrin. Since fibrillar fibronectin formation was not strongly dependent on increased fibronectin or alpha5beta1 integrin levels, this effect may be mediated by growth factor induced changes in receptor affinity.

Alternative Splicing↗
PubMed1982

Distribution of fibronectin in normal and regenerating skeletal muscle.

The distribution of fibronectin in normal and regenerating skeletal muscle (the latter caused by autotransplantation) was investigated by means of indirect immunofluorescent technique. Normal myofibers exhibited a thin, continuous pericellular (endomysium) fibronectin distribution; however, their sarcoplasm was devoid of fibronectin. After autotransplantation, the skeletal muscle fibers underwent a process of degeneration that was followed by regeneration from the premyogenic satellite cells. These cells multiplied, fused to form myotubes, and matured into new myofibers. A decrease and an eventual loss of endomysial fibronectin was seen in the degenerating myofibers. At the same time, fibronectin appeared in the sarcoplasm. No significant fibronectin was expressed in the myogenic zone until the formation of myotubes which possessed a complete, circular fibronectin ring. The sarcoplasm of the myotubes lacked fibronectin. Since fibronectin is a component of basement membrane of several tissues, its disappearance and reappearance can be used to follow the fate of basement membrane. We conclude that fibronectin may not be essential for early myogenesis and that regenerated myotubes form an entirely new or at least certain new molecular components of their basement membrane. The present muscle autotransplantation model can be used to further study the role of fibronectin during myogenesis and cell transformation in vivo.

Animals↗